Photophysical properties and in vitro cytotoxicity studies of new Ru(II) carbonyl complexes and mixed geometrical Ru(II)–Ni(II) complex in HS-DNA/BSA protein and human lung (A549) and liver (HepG2) cells

Abstract

Two new ruthenium(II) carbonyl complexes have been synthesized from [RuHCl(CO)(PPh₃)₃] and 2-hydroxy-1-naphthaldehydethiosemicarbazone [H₂-(Nap-tsc)]. The stoichiometric reaction afforded two different complexes (1 and 2) exhibiting different structural motifs. In complex 1, the ligand coordinated through the N(2) nitrogen and thiolate sulphur by forming a strained four member chelate ring by leaving three donor sites (phenolic oxygen, N1 nitrogen and terminal nitrogen) uncoordinated. However, in complex 2, it coordinated as an ONS tridentate with the formation of a six member and a five member ring. In order to utilize the three unutilized donor sites in complex 1, it was reacted further with [NiCl₂(PPh₃)₂], which resulted in the formation of a new hetero bimetallic complex 3 wherein all the donor atoms of the ligand were utilized. The complexes have been characterized analytically and spectroscopically and X-ray diffraction (1, 3) and have also been evaluated for their binding affinity with HS-DNA. The electrostatic binding of the complexes with DNA was evident from absorption and fluorescence titration experiments. The binding interaction between the complexes 1–3 and bovine serum albumin (BSA) was studied by absorption, fluorescence and synchronous spectra at room temperature. From the results, it is inferred that complex 2 had a better binding ability with the tryptophan residues of BSA. The mechanism of complex interaction was found as static quenching. The in vitro cytotoxicity of metal complexes (1–3) has been evaluated by colorimetric assay (MTT assay).

---

1. Introduction

Thiosemicarbazones belong to nitrogen–sulphur derivatives of thiourea which have received considerable interest due to the fascinating chemical, biological, structural and electronic properties of their metal complexes.^1–8 In recent years anticancer metallodrug research has been expanded to several transition metals.^9–11 A large number of the thiosemicarbazone complexes have found wide medicinal applications due to their potentially beneficial biological properties. In addition, metal complexes that reveal the capacity to bind with nucleobases, DNA fragments, amino acids, peptides, and proteins are currently receiving special attention mainly due to the clinical use of transition metal complexes as antitumor drugs.^12–14 The currently used anticancer drugs, such as cisplatin, carboplatin (paraplatin), and oxaliplatin (eloxatin), are still hindered by clinical problems, including acquired or intrinsic resistance, a limited spectrum of activity, and high toxicity leading to side effects.^15,16 The search for anticancer agents with improved properties has focused on the synthesis of a new generation of metallo drugs.^17–22 In addition to platinum, ruthenium has acquired special attention due to three chemical properties, i.e., (i) stable oxidation states (+1 to +8 and −2) among which +2 and +3 are easily accessible in aqueous solution^23,24 (ii) slow ligand exchange rate similar to Pt(II) and Pt(IV) and (iii) the iron-mimicking property of ruthenium with selective binding affinity to transferrin and albumin^10,25,26 and it can be exploited for multifaceted applications such as (a) radiodiagnostic imaging agents (isotopes ⁹⁷Ru and ¹⁰⁷Ru), (b) antimicrobials, e.g., [RuCl₂(chloroquine)]₂, (c) immunosuppressants,e.g., cis-[Ru(NH₃)₄(Him)₂]³⁺, (d) NO-scavenger or delivery tools, e.g., Ru(III) polyaminocarboxylates (AMD6245 and AMD1226) for the medication of diabetes, arthritis, epilepsy, septic shock and stroke, (e) antibiotics, e.g. Ru(III) thiosemicarbazone, and (f) anticancer agents, e.g. NAMI-A and KP1019.²⁷ NAMI-A entered clinical trials, being the first ruthenium drug, and KP1019 followed thereafter.^28–32 More recently, a number of heterometallic cytotoxic complexes³³ have been reported, which show a synergic effect of two different metals with known antitumor properties. In continuation of our ongoing interest in the synthesis of new ruthenium thiosemicarbazone complexes,^8g,8i,8m herein, we report the synthesis, structural characterization, DNA/protein interaction and cytotoxic study of new ruthenium(II) and heterobinuclear thiosemicarbazone complexes (1–3).

2. Experimental section

Materials and methods

The ligand [H₂-(Nap-tsc)],³⁴ the metallic ancestors [RuHCl(CO)(PPh₃)₃]³⁵ and [NiCl₂(PPh₃)₂]³⁶ were synthesized according to the standard procedures. All the reagents used in this study were analar grade. Solvents were purified and dried according to the standard procedure.³⁷ HS-DNA, ethidium bromide (EB) and BSA were obtained from Sigma Aldrich and used as received. Infrared spectra were measured as KBr pellets on a Nicolet Avatar Model FT-IR spectrophotometer in 400–4000 cm⁻¹ range. Elemental analysis of carbon, hydrogen, nitrogen, and sulphur were determined by using Vario EL III CHNS at the Department of Chemistry, Bharathiar University, Coimbatore, India. The electronic spectra of the complexes were recorded in dichloromethane using a Systronics 119 Spectrophotometer in the 800–200 nm range. ¹H NMR spectra were recorded in DMSO at room temperature with a Bruker 400 MHz instrument, chemical shift relative to tetramethylsilane. Melting points were recorded in a Lab India apparatus.

Preparation of new ruthenium(II) complexes

The ligand [H₂-(Nap-tsc)] (0.0259 g; 0.1 mmol) was added to a solution of [RuHCl(CO)(PPh₃)₃] (0.095 g; 0.1 mmol) in dry benzene (20 cm³). The above mixture was heated under reflux for 5 h. The resulting yellow solution obtained was concentrated to about 3 cm³. The reaction mixture was then subjected to thin layer chromatography where two spots were identified. They were isolated by silica gel column chromatography by ethyl acetate–petroleum ether (60–80 °C) and benzene–methanol as solvent mixture. The first compound (1) was isolated from a greenish yellow band eluted by using 1 : 19 ethyl acetate–petroleum ether. Followed by first compound, the second dark yellow band (2) was eluted by using 2 : 18 methanol–benzene.

[Ru(H-Nap-tsc)(CO)Cl(PPh₃)₂] (1). Yield: 41%. M.p. 245 °C. Anal. calcd for C₄₉H₄₀N₃O₂S₁ClRuP₂: C, 63.05; H, 4.32; N, 4.50; S, 3.44. Found: C, 63.10; H, 4.36; N, 4.54; S, 3.47%. FT-IR (cm⁻¹) in KBr: 3383 (ν_O–H), 1587 (ν_C=N), 1311 (ν_C–O), 746 (ν_C–S), 1931 (ν_C≡O), 1430, 1090, 694 cm⁻¹ (for PPh₃); UV-Vis (CH₂Cl₂), λ_max: 250 nm (28 250), (dm³ mol⁻¹ cm⁻¹) (intra-ligand transition); 333 (14 780), 366 (13 240) and 389 (9370), nm (dm³ mol⁻¹ cm⁻¹) LMCT (s → d); 407 (9013), nm (dm³ mol⁻¹ cm⁻¹) (MLCT); ¹H NMR (DMSO-d₆, ppm): δ 11.12 (s, –OH), δ 8.64 (s, 1H, CH=N), δ 6.97–7.71 (m, aromatic protons), δ 5.74 (s, –NH imine) and δ 6.61 (s, –NH₂).

[Ru(Nap-tsc)(CO)(PPh₃)₂] (2). Yield: 50%. M.p. 250 °C. Anal. calcd for C₄₉H₃₉N₃O₂S₁RuP₂: C, 65.62; H, 4.38; N, 4.68; S, 3.58. Found: C, 65.68; H, 4.39; N, 4.71; S, 3.61%. FT-IR (cm⁻¹) in KBr: 1613 (ν_C=N), 1392 (ν_C–O), 741 (ν_C–S), 1935 (ν_C≡O), 1428, 1089, 694 cm⁻¹ (for PPh₃); UV-Vis (CH₂Cl₂), λ_max: 268 (36 810), nm (dm³ mol⁻¹ cm⁻¹) (intra-ligand transition); 342 (13 800), 370 (13 500) and 380 (12 160), nm (dm³ mol⁻¹ cm⁻¹) LMCT; 453 (10 720) and 474 (9840), nm (dm³ mol⁻¹ cm⁻¹) forbidden (d → d) transitions; ¹H NMR (DMSO-d₆, ppm): δ 8.14 (s, 1H, CH=N), δ 6.60–7.60 (m, aromatic protons) and δ 5.27 (s, –NH₂).

Preparation of [RuNi(Nap-tsc)(CO)Cl(PPh₃)₃] (3)

[Ru(H-Nap-tsc)(CO)Cl(PPh₃)₂] (1) (0.060 g; 0.0642 mmol) in 10 cm³ of ethanol was added to [NiCl₂(PPh₃)₂] (0.042 g; 0.0642 mmol) in ethanol (10 cm³) and the reaction mixture was refluxed for 5 h. A red solution obtained was chromatographed and a dark red band was eluted by using 1 : 19 methanol–benzene mixture as eluent. The red solid obtained was then recrystallized from DMF to yield needle shaped red crystals. Yield: 52%. M.p. >250 °C. Anal. calcd for C₆₇H₅₃N₃ClNiO₂S₁RuP₃: C, 64.26; H, 4.27; N, 3.36; S, 2.56. Found: C, 64.28; H, 4.29; N, 3.40; S, 2.59%. FT-IR (cm⁻¹) in KBr: 1608 (ν_C=N), 1513 (ν_C=N), 1367 (ν_C–O), 744 (ν_C–S), 1929 (ν_C≡O), 1431, 1092, 694 cm⁻¹ (for PPh₃); UV-Vis (CH₂Cl₂), λ_max: 243 (35 050) nm (dm³ mol⁻¹ cm⁻¹) (intra-ligand transition); 370 (29 430) and 385 (24 230) nm (dm³ mol⁻¹ cm⁻¹) LMCT; 404 (22 130) and 439 (18 430) nm (dm³ mol⁻¹ cm⁻¹) forbidden (d → d) transitions; ¹H NMR (DMSO-d₆, ppm): δ 8.80 (d, 1H, CH=N), δ 6.80–7.80 (m, aromatic protons) and δ 6.41 (d, (J = 10.4 Hz), –NH₂).

Single crystal X-ray crystallography

Single crystals of [Ru(H-Nap-tsc)(CO)Cl(PPh₃)₂]·DMF (1a), [Ru(H-Nap-tsc)(CO)Cl(PPh₃)₂]·CH₂Cl₂ (1b) and [RuNi(Nap-tsc)(CO)Cl(PPh₃)₃] (3) were obtained from CH₃CN–DMF (1), CH₂Cl₂–CH₃CN and DMF (3), respectively. The crystallographic data sets for the single-crystal X-ray studies for the new complexes were collected with Mo Kα (λ = 0.71073 Å) radiation on a Bruker SMART 1000 CCD diffractometer. All calculations were performed using the SHELXTL and SHELXL-97 programmes.^38,39

DNA binding study

All the experiments involving the binding of the complexes 1–3 with herring sperm HS-DNA were carried out in deionised water with tris(hydroxymethyl)-aminomethane (Tris, 5 mM) and sodium chloride (50 mM) and pH was adjusted to 7.2 with hydrochloric acid at room temperature. A solution of HS-DNA (0.01 g) in Tris–HCl buffer (10 ml) gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.7–1.8 : 1 indicating that the DNA was sufficiently free of protein. The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M⁻¹ cm⁻¹) at 260 nm. The complexes 1–3 were dissolved in a mixed solvent of 5% DMSO and 95% Tris–HCl buffer. Absorption spectra were recorded after equilibrium at 20 °C for 10 min. Absorption titration experiments were performed with a fixed concentration of the complexes (10 μM) with the gradual increase of DNA concentration (0–50 μM). While measuring the absorption spectra, an equal amount of DNA was added to both the test solution and the reference solution to eliminate the absorbance of DNA itself. The intrinsic binding constant K_b was determined by using Stern–Volmer eqn (1).^40,41

[DNA]/[ε_a − ε_f]) = [DNA]/[ε_b − ε_f] + 1/K_b[ε_b − ε_f] (1)

The absorption coefficients ε_a, ε_f, and ε_b correspond to A_obsd/[DNA], the extinction coefficient for the free complex and the extinction coefficient for the complex in the fully bound form, respectively. The slope and the intercept of the linear fit of the plot of [DNA]/[ε_a − ε_f] versus [DNA] give 1/[ε_a − ε_f] and 1/K_b[ε_b − ε_f], respectively. The intrinsic binding constant K_b can be obtained from the ratio of the slope to the intercept (Table 4).⁴⁰ Emission measurements were carried out by using a JASCO FP- 6500 spectrofluorometer. Tris-buffer was used as a blank to make preliminary adjustments. The excitation wavelength was fixed and the emission range was adjusted before measurements. All measurements were made at 20 °C. For emission spectral titrations, complex concentration was maintained constant as 10 μM and the concentration of DNA was varied from 0 to 50 μM. The emission enhancement factors were measured by comparing the intensities at the emission spectral maxima under similar conditions. In order to know the mode of attachment of HS-DNA fluorescence quenching experiments of ethidium bromide (EB) DNA were carried out by adding 0–40 μM ruthenium(II) complexes with the samples containing 10 μM EB, 10 μM DNA and Tris-buffer (pH = 7.2). Before measurements, the system was shook and incubated at room temperature for ∼5 min. The emission was recorded at 530–750 nm.

Bovine serum albumin binding study

The binding ability of the complexes 1–3 with bovine serum albumin (BSA) was studied from the fluorescence spectra recorded with an excitation wavelength at 280 nm and the corresponding emission at 346 nm assignable to BSA. The excitation and emission slit widths and scan rates were maintained constant for all of the experiments. A stock solution of BSA was prepared in 50 mM phosphate buffer (pH = 7.2) and stored in the dark at 4 °C for further use. A concentrated stock solution of the compounds was prepared as mentioned for the DNA binding experiments, except that the phosphate buffer was used instead of a Tris–HCl buffer for all of the experiments. Titrations were manually done by using a micropipette for the addition of the complexes. The possible quenching mechanism has been interpreted by using Stern–Volmer eqn (3), the ratio of the fluorescence intensity in the absence of (I₀) and in the presence of (I_corr-corrected fluorescence intensity) the quencher is related to the concentration of the quencher [Q] by a coefficient K_SV.

I₀/I_corr = 1 + K_SV[Q] (2)

In order to correct the inner filter effect the following eqn (3), is used.

I_corr = I_obs × 10^{(A_exc + A_em)/2} (3)

where I_corr is the corrected fluorescence value, I_obs the measured fluorescence value, A_exc the absorption value at the excitation wavelength, and A_em the absorption value at the emission wavelength.⁴²

The binding constant (K_b) and the number of binding sites (n) can be determined according to the method⁴³ using the Scatchard eqn (4):

log[(I₀ − I)/I] = log K_b + n log[Q] (4)

Where, K_b is the binding constant for the complex–protein interaction and n is the number of binding sites per albumin molecule, which can be determined by the slope and the intercept of the double logarithm regression curve of log[(I₀ − I)/I] versus log[Q].

For synchronous fluorescence spectral studies also, the same concentration of BSA and complexes were used. The spectra obtained from 300 to 400 nm when Δλ = 60 nm and from 290 to 500 nm when Δλ = 15 nm. The excitation and emission slit widths were 5 and 6 nm, respectively. Fluorescence and synchronous measurements were performed using a 1 cm quartz cell on JASCO F 6500 spectrofluorimeter.

Cytotoxicity

Effect of the complexes (1 and 2) on the viability of human lung (A549) and liver cancer cells (HepG2) were assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.⁴⁴ The cells were seeded at a density of 10 000 cells per well, in 200 μl RPMI 1640 medium and were allowed to attach overnight in a CO₂ incubator and then the complexes dissolved in DMSO were added to the cells at a final concentration of 1, 10, 25 and 50 μM in the cell culture media. After 48 hours, the wells were treated with 20 μl MTT (5 mg ml⁻¹ PBS) and incubated at 37 °C for 4 hours. The purple formazan crystals formed were dissolved in 200 μl DMSO and read at 570 nm in a micro plate reader.

3. Results and discussion

Reaction of [RuHCl(CO)(PPh₃)₃] with an equimolar amount of [H₂-(Nap-tsc)] resulted in the formation of two new ruthenium(II) complexes (1 and 2). They were isolated by column chromatography using 1 : 19 ethyl acetate–petroleum ether and 2 : 18 methanol–benzene solvent mixture respectively. The elemental analyses of the complexes 1 and 2 agreed with the proposed molecular formulae. The structure of the first complex was confirmed by X-ray crystallographic study. Repeated attempts were made to grow single crystals suitable for X-ray work in various organic solvents and were unsuccessful for the second complex 2 (Scheme 1).

Scheme 1 Preparation of new ruthenium(II) carbonyl complexes 1 and 2.

Though [H₂-(Nap-tsc)] ligand has five potential donor atoms (two hydrazinic nitrogen (N1 and N2), one amide nitrogen (N3), one oxygen and one sulphur atom), it has utilized only two donor sites in 1 while the other three potential donor sites (phenolate oxygen, the imine nitrogen (N1) and the amide nitrogen(N3)) have remained unutilized. In order to utilize the three remaining donor sites in the complex 1, a reaction was carried out to utilize all the coordination sites with [NiCl₂(PPh₃)₂](Scheme 2).

Scheme 2 Preparation of new hetero bimetallic complex 3.

The reaction of the complex 1 with an equimolar amount of [NiCl₂(PPh₃)₂] resulted in the formation of a new hetero bimetallic complex, [RuNi(Nap-tsc)(CO)Cl(PPh₃)₃] (3). Recrystallisation of the product from DMF leads to form red crystals. Structure determination of this new complex showed that the coordinated [H₂-(Nap-tsc)] of [Ru(H-Nap-tsc)(CO)Cl(PPh₃)₂] has lost two more protons, viz. the phenolic proton and one proton from the NH₂ group, and coordinated to Ni as a tridentate NNO manner. The triphenylphosphine occupies the fourth coordination site of Ni. It is an interesting to note that in this binuclear complex, all the available donor sites of the [H₂-(Nap-tsc)] ligand is involved in bond formation.

IR spectra

The IR spectra the complexes and the ligand were recorded as KBr pellets gave preliminary information about the coordination of the ligand to ruthenium metal. The ligand showed a sharp band at 1609 cm⁻¹ corresponding to the azomethine group and was observed at 1587 cm⁻¹ and 1613 cm⁻¹ in the complexes 1 and 2 respectively indicate the interaction of azomethine nitrogen to metal.⁸ⁱ A sharp band at 817 cm⁻¹ corresponding to the thione sulphur atom was completely disappeared in the complexes and a new band at 746 and 741 cm⁻¹ indicating the participation of thiolate sulphur atom through the enolisation and subsequent deprotonation.^8a A band at 3446 cm⁻¹ corresponding to the phenolic OH in the ligand completely disappeared in complex 2 indicating the participation of phenolic oxygen with ruthenium.⁸ⁱ This is further supported by the increase in phenolic C–O stretching frequency from 1278 cm⁻¹ to 1392 cm⁻¹. However, there was no appreciable change in theses frequencies in complex 1 indicating the non participation of phenolic oxygen in bonding.^8b This may be due to the formation of intra molecular hydrogen bonding between phenolic OH and the N1 nitrogen of the ligand. The IR spectrum of the obtained red solid (complex 3) from the equimolar reaction of complex 1 and [NiCl₂(PPh₃)₂] exhibited a broad signal at 1608 cm⁻¹ indicating the coordination of azomethine nitrogen to nickel and the broadening may due to the mixing of ruthenium azomethine band with nickel azomethine band. The phenolic OH stretching at 3446 cm⁻¹ in complex 1 completely disappeared in 3 indicating the participation of phenolic oxygen in bonding with nickel. This is further evident from the increase in phenolic C–O stretching frequency from 1311 cm⁻¹ to 1367 cm⁻¹. In all the three complexes characteristic absorption was found at 1929–1935 cm⁻¹ indicating the presence of terminal carbonyl group in the complex.^8l

Electronic spectra

Electronic spectra of the complexes recorded in dichloromethane showed intense absorptions in the ultraviolet and visible regions. [Ru(H-Nap-tsc)(CO)Cl(PPh₃)₂] (1) and [Ru(Nap-tsc)(CO)(PPh₃)₂] (2) displayed six bands in the region around 250–474 nm. The bands appeared at 250–268 nm have been assigned to intra ligand transition. The bands appeared at 333–407 nm were assigned to charge transfer transition⁴⁵ and the shoulder at 453 and 474 nm were assigned to forbidden (d → d) transition. [RuNi(Nap-tsc)(CO)Cl(PPh₃)₃] (3) displayed five bands in the region 370–439 nm. The bands at 370 and 385 were assigned to ligand to metal charge transfer and the shoulder at 404–439 nm band due to forbidden (d → d) transition.

¹H-NMR spectra

The ¹H-NMR spectra of the ligand and the complexes have been taken in DMSO (Fig. S1–S4†). The spectra of the ligand and all the complexes showed a complex overlap of signals as multiplet at δ 6.6–7.9 ppm range corresponding to aromatic protons of the ligand and triphenylphosphine.^8a The spectrum of [H₂-(Nap-tsc)] showed four singlets at δ 10.7, 11.5, 9.2 and 8.1 ppm corresponding to –OH, –N(2)H–C=S, –NH₂ and –CH=N groups respectively. Complex 1 exhibited a singlet at δ 11.12 ppm corresponding to phenolic –OH group.⁴⁶ In addition, there is a new sharp singlet observed at δ 5.74 ppm in complex 1 which may be due to the presence of imine –NH group that was formed from the intramolecular hydrogen bonding between phenolic –OH and N1 nitrogen atom^8g whereas the singlet corresponding to –OH group completely disappeared in complex 2 and 3 confirming the involvement of phenolic oxygen in coordination.^8e In addition, 1 and 2 exhibited a sharp singlet at δ 8.84 and 8.14 ppm corresponding to azomethine proton respectively and the same functionality has been observed as a doublet for complex 3 at δ 8.80 ppm. The terminal NH₂ protons of the Schiff base appeared as a singlet in the range δ 6.61–10.49 ppm in all the complexes except 3.^8a A doublet observed at δ 6.41 ppm corresponding to N3H proton of the complex 3 may be due to the restricted rotation on C–N bond of the ligand.^8e

Description of the crystal structures

The structures of 1 and 3 have been determined by single crystal X-ray diffraction. Crystal structures of the complexes along with their numbering schemes and packing diagrams are given in Fig. 1, 2, 3 and S5–S7.† The crystallographic data and bond parameters are given in Tables 1 and 2. Complex 1 was crystallized in two different solvent mixtures CH₃CN–DMF and CH₃CN–CH₂Cl₂. In both cases complex 1 crystallized in monoclinic space group P2₁/n. In CH₃CN–DMF medium, the crystal was a solvate containing disordered DMF molecule (1a). The solvent contribution to the structure factors was removed using the SQUEEZE procedure, amounting to 3.75 DMF molecules per Ru complex. Similarly in CH₃CN–CH₂Cl₂ medium, the crystal was a solvate containing disordered dichloromethane molecule (1b). It is interesting to note that in both the cases acetonitrile (CH₃CN) moiety was selectively omitted from solvent inclusion into the crystal lattice. In the complexes 1a and 1b, the ruthenium atom is in a distorted octahedral environment with trans triphenylphosphine ligands (P1–Ru1–P2 175.44(3)° for 1a and 175.52(3)° for 1b). The chloride is trans to the sulphur atom (S1–Ru1–Cl1 163.40(4)° for 1a and 163.62(3)° for 1b) and the carbonyl group lies trans to the coordinated N2 atom C49–Ru1–N2 with an angle of 162.3(1)° and 162.8(1)° for 1a and 1b respectively. The trans angles deviate from linearity and N2, S chelation (four member ring) leads to small N2–Ru1–S1 bite angle 65.44(8)° for 1a and 65.53(7)° for 1b. The Ru–ligand distances, namely Ru1–C49 1.844(4) Å for 1a and 1.836(3) Å for 1b, Ru1–P1 2.3962(9) Å for 1a and 2.396(9) Å for 1b, Ru1–P2 2.3784(1) Å for 1a and 2.3732(9) Å for 1b, Ru1–S1 2.404(1) Å for 1a and 2.4049(9) Å for 1b and Ru1–Cl1 2.423(1) Å for 1a and 2.4287(9) Å for 1b are comparable with distances found in other ruthenium complexes.^8g,47 Ru1–N2 bond distance 2.217(3) Å for 1a and 2.211(3) Å for 1b is little bit longer and this may be due to the trans influence of carbonyl group.^47c,48 In the structure of complexes 1a and 1b, one weak intramolecular O(2)–H(2)⋯N(1) hydrogen bond (D⋯A distance 2.536 Å and 2.546 Å respectively and D–H⋯A 152.99° and 152.85°) can be found (Table S1†).

Fig. 1 ORTEP diagram of (1) with hydrogen bonding.

Fig. 2 ORTEP diagram of (1) with hydrogen bonding.

Fig. 3 ORTEP diagram of (3).

Table 1 Crystallographic data of new thiosemicarbazone complexes 1a, 1b and 3

	[Ru(H-Nap-tsc)(CO)Cl(PPh3)2]·(CH3)2NCHO (1a)	[Ru(H-Nap-tsc)(CO)Cl(PPh3)2]·CH2Cl2 (1b)	[RuNi(Nap-tsc)(CO)Cl(PPh3)3] (3)
Empirical formula	C52H47ClN4O3P2Ru1S1	C50H42Cl3N3O2P2Ru1S1	C67H55ClN3NiO3P3RuS
Formula weight	1006.46	1018.29	1270.34
Crystal system	Monoclinic	Monoclinic	Orthorhombic
Space group	P21/n	P21/n	Iba2
Wavelength	0.71073 Å	0.71073 Å	0.71073 Å
Temperature	293 K	293 K	293 K
a	14.3378(2) Å	14.3713(2) Å	22.4615(3) Å
b	16.5494(3) Å	16.5083(3) Å	22.8701(4) Å
c	19.5910(3) Å	19.6494(3) Å	24.3408(4) Å
α	90°	90°	90°
β	95.898(1)°	96.112(1)°	90°
γ	90°	90°	90°
V	4623.98(13) Å^3	4635.24(13) Å^3	12503.8 (3) Å^3
Crystal size	0.18 × 0.22 × 0.22 mm	0.16 × 0.2 × 0.2 mm	0.05 × 0.08 × 0.08 mm
Z value	4	4	8
Limiting indices	−19 ≤ h ≤ 18, −21 ≤ k ≤ 20, −24 ≤ l ≤ 24	−19 ≤ h ≤18, −21 ≤ k ≤ 19, −24 ≤ l ≤ 24	−28 ≤ h ≤ 29, −28 ≤ k ≤ 27, −27 ≤ l ≤ 31
Dcalc	1.463 Mg m^−3	1.459 Mg m^−3	1.35 Mg m^−3
Reflections collected	43 472/6905 [R(int) = 0.0992]	40 219/7082 [R(int) = 0.099]	46 168/7244 [R(int) = 0.2472]
Theta range for data collection	1.61 to 28.23°	1.61 to 28.24°	2.44 to 28.24°
F000	2080	2080	5216
Goodness-of-fit on F^2	0.987	1.039	0.988
Refinement method	Full-matrix least-squares on F^2	Full-matrix least-squares on F^2	Full-matrix least-squares on F^2
μ (Mo Kα)	0.671 mm^−1	0.669 mm^−1	0.743 mm^−1
Completeness to theta 2θmax	28.23°	28.24°	28.24°
Data/restraints/parameters	10 913/0/532	10 902/7/568	14 675/0/712
Final R indices [I > 2sigma(I)]	R1 = 0.0535, wR2 = 0.1348	R1 = 0.0506, wR2 = 0.1051	R1 = 0.083, wR2 = 0.1427
R Indices (all data)	R1 = 0.0985, wR2 = 0.1476	R1 = 0.0995, wR2 = 0.1189	R1 = 0.1911, wR2 = 0.172
Largest diff. peak and hole	1.026 and −0.693 e Å^−3	0.708 and −0.644 e Å^−3	0.726 and −0.866 e Å^−3

---

Table 2 Selected bond lengths (Å) and angels (°) for complexes 1a, 1b and 3

[Ru((H-Nap-tsc)(CO)Cl(PPh3)2)]·(CH3)2NCHO (1a)		[Ru(H-Nap-tsc)(CO)Cl(PPh3)2)]·CH2Cl2 (1b)		[RuNi(Nap-tsc)(CO)Cl(PPh3)3)](3)
Bond lengths
				Ru1–C67	1.84(1)
Ru1–C49	1.844(4)	Ru1–C49	1.836(3)	Ni1–N1	1.843(7)
Ru1–N2	2.217(3)	Ru1–N2	2.211(3)	Ru1–N2	2.150(7)
Ru1–P1	2.3962(9)	Ru1–P1	2.396(9)	Ni1–N3	1.838(7)
Ru1–P2	2.3784(9)	Ru1–P2	2.3732(9)	Ni1–O1	1.839(7)
Ru1–S1	2.404(1)	Ru1–S1	2.4049(9)	Ni1–P1	2.205(3)
Ru1–Cl1	2.423(1)	Ru1–Cl1	2.4287(9)	Ru1–P2	2.387(2)
				Ru1–P3	2.379(2)
				Ru1–Cl1	2.433(2)
				Ru1–S1	2.417(2)
Bond angles
C49–Ru1–N2	162.3(1)	C49–Ru1–N2	162.8(1)	N1–Ni1–N3	83.5(3)
C49–Ru1–P1	91.5(1)	C49–Ru1–PP1	91.6(1)	N1–Ni1–O1	93.3(3)
C49–Ru1–P2	90.9(1)	C49–Ru1–P2	90.6(1)	N1–Ni1–P1	178.1(2)
C49–Ru1–S1	96.9(1)	C49–Ru1–S1	97.3(1)	N3–Ni1–O1	176.7(3)
C49–Ru1–Cl1	99.7(1)	C49–Ru1–Cl1	99.0(1)	N3–Ni1–P1	95.4(3)
N2–Ru1–P1	88.85(8)	N2–Ru1–P1	88.79(7)	O1–Ni1–P1	87.8(2)
N2–Ru1–P2	90.07(8)	N2–Ru1–P2	90.18(7)	C67–Ru1–N2	165.9(3)
N2–Ru1–S1	65.44(8)	N2–Ru1–S1	65.53(7)	C67–Ru1–P2	91.7(3)
N2–Ru1–Cl1	98.01(8)	N2–Ru1–Cl1	98.13(7)	C67–Ru1–P3	91.1(3)
P1–Ru1–P2	175.44(3)	P1–Ru1–P2	175.52(3)	C67–Ru1–Cl1	98.3(3)
P1–Ru1–S1	92.43(3)	P1–Ru1–S1	92.16(3)	C67–Ru1–S1	99.5(3)
P1–Ru1–Cl1	88.25(3)	P1–Ru1–Cl1	88.35(3)	N2–Ru1–P2	90.1(2)
P2–Ru1–S1	91.15(3)	P2–Ru1–S1	91.37(3)	N2–Ru1–P3	88.1(2)
P2–Ru1–Cl1	87.51(3)	P2–Ru1–Cl1	87.49(3)	N2–Ru1–Cl1	95.7(2)
S1–Ru1–Cl1	163.40(4)	S1–Ru1–Cl1	163.62(3)	N2–Ru1–S1	66.5(2)
				P2–Ru1–P3	175.38(9)
				P2–Ru1–Cl1	90.13(8)
				P2–Ru1–S1	89.28(8)
				P3–Ru1–Cl1	85.83(8)
				P3–Ru1–S1	93.88(8)
				Cl1–Ru1–S1	162.14(8)

---

The complex 3 crystallized in orthorhombic space group Iba2. The thiosemicarbazone moiety is coordinated to Ru(II) and Ni(II) as N2, S, O, N1, N3 chelating ligand, forming a four member ring along with five and six member rings. The geometry around the ruthenium atom is distorted octahedral with trans triphenylphosphine ligands (P2–Ru1–P3 175.38(9)°). Two 0.5H₂O molecules are present in the crystal lattice. The chloride is in a trans position to the sulphur atom (Cl1–Ru1–S1 162.14(8)°) and the carbonyl group lies trans to the coordinated N2 atom with an angle of 165.9(3)°.The trans angles deviate from linearity and N2, S1 chelation (four member ring) leads to small N2–Ru1–S1 bite angle 66.5(2)°. The Ru–ligand distances, namely Ru1–C67 1.84(1) Å, Ru1–P2 2.387(2) Å, Ru1–S1 2.417(2) Å and Ru1–Cl1 2.433(1) Å are comparable with distances found in other ruthenium complexes.^8g,47 The geometry around the nickel atom is distorted square planar environment with trans angles N3–Ni1–P1 and N3–Ni1–O1{178.1(2)° and 176.7(3)°}. The trans angles deviate from linearity and N1, N3 chelation (five member ring) leads to small N1–Ni1–N3 bite angle 83.5(3)°. The Ni–ligand distances, namely Ni1–O1 1.839(7) Å, Ni1–P1 2.205(3) Å, Ni1–N1 1.843(7) Å and Ni1–N3 1.838(7) Å are comparable with literature values.⁸ⁿ

Electrochemistry

Electrochemical studies of new ruthenium(II) complexes 1–3 have been done by using platinum wire working electrode and platinum disc counter electrode. All the potentials were referenced to Ag/AgCl electrode. Ferrocene was used as external standard. Voltammograms and voltammetric data are presented in Fig. S8† and Table 3. The complex [Ru(H-Nap-tsc)(CO)Cl(PPh₃)₂] (1) showed quasi-reversible one electron oxidation at 0.845 V with the peak to peak separation of 470 mV and quasi reversible reduction at −0.380 V with the peak to peak separation of 241 mV. The complex [Ru(Nap-tsc)(CO)(PPh₃)₂] (2) exhibited both quasi irreversible oxidation and reduction at 0.785 V (with the peak to peak separation of 390 mV) and at 0.490 V (with a peak to peak separation of −140 mV) respectively. In addition, a quasi-reversible ligand reduction at −1.469 V with a peak to peak separation of 302 mV and an irreversible ligand oxidation at 1.510 V were also observed.⁸ⁱ [RuNi(Nap-tsc)(CO)Cl(PPh₃)₃)] (3) showed two successive quasi reversible one electron oxidations at the potentials 0.578 V and 0.190 V with a peak to peak separation of 508 mV and 190 mV corresponding to Ru(II)/Ru(III) and Ni(II)/Ni(III) processes respectively.^8g In addition, a reversible reduction at −1.618 V with peak to peak separation of 98 mV corresponding to ligand reduction was also observed.

Table 3 Electrochemical data of the new complexes (1–3)

Complex	Oxidation				Reduction					Ligand reduction
	Epa (V)	Epc (V)	E1/2 (V)	ΔEp (mV)	Epa (V)	Epc (V)	E1/2 (V)	ΔEp (mV)	Epa (V)	Epc (V)	E1/2 (V)	ΔEp (mV)
(1)	1.080	0.610	0.845	470	−0.260	−0.501	−0.380	241	—	—	—	—
(2)	0.590	0.980	0.785	390	−0.420	−0.560	−0.490	−140	−1.318	−1.620	−1.469	302
(3)	0.324	0.832	0.578	508	—	—	—	—	−1.520	−1.618	−1.569	98
	0.270	0.080	0.175	190

---

DNA binding studies

Electronic absorption titration. The electronic absorption spectroscopy is one of the most useful techniques to evaluate the DNA binding ability of the new complexes 1–3. The absorption spectra of the three ruthenium(II) complexes (10 μM) in the absence and presence of HS-DNA (0–50 μM) are recorded, and the absorption spectra of the three compounds are given in Fig. 4. The absorption spectra of complex 1 showed intra ligand (IL) transition centered at 251 nm. As the DNA concentration is increased, the hyperchromism of 81.24% with a red shift of 12 nm was observed in the IL band. The binding behaviour of complex 2 is quite different. It exhibited hyperchromism of 89.91% at 267 nm with 5 nm blue shift. The absorption spectra of complex 3 mainly consist of four resolved bands [intra ligand (IL) and charge transfer (CT) transitions] centered at 243 nm (IL), 341 nm, 417 nm and 440 nm (CT). As the DNA concentration is increased, the hyperchromism of 87.01% with a red shift of 19 nm was observed in the IL band. The CT band at 341 nm showed 15.30% hyperchromism. In contrast, the remaining two CT bands at 417 nm and 440 nm showed 2.32% and 2.35% hypochromism respectively with negligible shifts in the wavelength. The observed hyper chromic effect with red/blue shift suggested that the new complexes bind to CT-DNA by external contact, possibly due to electrostatic binding.⁴⁹ The intrinsic binding constant K_b is determined by monitoring the changes in the absorbance in the IL band at the corresponding λ_max with increasing concentration of DNA (Table 4) and is given by the ratio of slope to the Y intercept in plots of [DNA]/(ε_a − ε_f) versus [DNA] (insets in Fig. 4). From the binding constant values, it is inferred that the complex 2 exhibited better binding than 1 and 3. In the emission spectra, complexes 1–3 had fluorescence at 360, 389 and 409 nm respectively (Fig. 5). Addition of CT DNA to the complex solution resulted in the hyperchromism of 27.25%, 43.69% and 31.82% without any shift in wavelengths. The enhanced fluorescence intensity observed for the complexes 1–3 represents electrostatic binding mode of HS-DNA.

Fig. 4 Absorption titration spectra of 1–3 with increasing concentrations (0–50 μM) of HS-DNA (Tris–HCl buffer, pH 7.2); the inset shows binding isotherms with HS-DNA.

Table 4 Binding constant for interaction of complexes (1–3) with HS-DNA

System	Kb (×10^5 M^−1)
HS-DNA + 1	3.5741 ± 0.19
HS-DNA + 2	3.6873 ± 0.28
HS-DNA + 3	3.6643 ± 0.22

---

Fig. 5 Changes in the emission spectra of 1–3, with increasing concentrations (0–50 μM) of HS-DNA (Tris–HCl buffer, pH 7.2).

Competitive studies with ethidium bromide. The competitive EB binding studies were carried out in order to examine the binding of each complex with HS-DNA. Competitive binding experiments were carried out in the buffer by keeping [DNA]/[EB] = 1 and increasing the concentration of the complexes 1–3. The fluorescence spectra of EB were measured using an excitation wavelength of 620 nm, and the emission range was set between 550 and 750 nm. The enhanced fluorescence of EB in the presence of DNA can be quenched by the addition of a second molecule (complexes) by either replacing the EB and/or by accepting the excited-state electron of the EB through a photoelectron transfer mechanism. The extent of fluorescence quenching of EB bound to HS-DNA can be used to determine the extent of binding between the second molecule and HS-DNA. But in this case, the fluorescence emission intensities of EB bound to HS-DNA at 620 nm showed increasing trend with increasing concentration of the complexes (1–3) showed in Fig. 6, indicating that they cannot displace EB from the DNA–EB complex. This observation is often considered that they can bind weakly to the DNA probably by electrostatic interaction.

Fig. 6 The emission spectra of the DNA–EB system (λ_exc = 515 nm, λ_em = 530–750 nm), in the presence of complexes 1–3. [DNA] = 10 μM, [EB] = 10 μM. The arrow shows the emission intensity changes upon increasing the concentration of complex.

Quenching mechanism of BSA by complexes

UV absorption spectra of BSA in the presence of complexes (1–3). UV absorption spectrum is used to explore the structural change and to know the complex formation in solution. This is useful to distinguish the type of quenching exist i.e., static or dynamic quenching.⁵⁰ The UV absorption spectra of BSA in the presence of three complexes (1–3) (Fig. 7) show that the absorption intensity of BSA was enhanced with the addition of these complexes from 277 to 280 nm. There was a red shift of 1 nm for complex–BSA spectrum of 1 and 3. In addition, there was a blue shift of 2 nm for complex–BSA spectrum of 2. This phenomenon indicates the interaction of BSA with the compounds.⁵¹ The mechanisms of quenching are usually classified as either dynamic quenching or static quenching. Static quenching refers to fluorophore–quencher complex formation and the dynamic quenching refers to a process that the fluorophore and the quencher come into contact during the transient existence of the excited state. However, the formation of non-fluorescence ground-state complex induced the change in absorption spectrum of fluorophore. Thus, possible quenching mechanism of BSA by 1, 2 and 3 was a static quenching process.⁵²

Fig. 7 Absorption spectra of absence and presence of complexes with BSA (1 × 10⁻⁵ M).

Fluorescence quenching studies of BSA. The possible binding interactions of the ruthenium(II)–2-hydroxy 1-naphthaldehyde complexes (1–3) with BSA have been investigated by emission-titration experiments at room temperature. A solution of BSA (10 μM) was titrated with various concentrations of the complexes (0–50 μM). Fluorescence spectra were recorded in the range of 290–450 nm upon excitation at 280 nm. The changes observed on the fluorescence emission spectra of a solution of BSA on the addition of the ruthenium complexes (1–3) are shown in Fig. 8. Upon the addition of complexes 1–3 to BSA, a significant decrease of the fluorescence intensity of BSA at 346 nm from the initial fluorescence intensity was observed accompanied by a red shift of 2, 10 and 4 nm, respectively. The observed quenching may be attributed to possible changes in secondary structure of protein leading to changes in the tryptophan environment of BSA which indicates the binding of each complex to BSA.⁵³ The Stern–Volmer⁵⁴ and Scatchard equations and graphs may be often used in order to study the interaction of a quencher with BSA. The Stern–Volmer quenching constant K_q obtained from the plot of I₀/I vs. [Q] was found to be 8.2 × 10⁴ M⁻¹, 3.5 × 10⁵ M⁻¹ and 8.8 × 10⁴ M⁻¹ corresponding to complexes 1, 2 and 3 respectively. The observed linearity in the plots (Fig. 9) indicated the ability of the complexes to quench the emission intensity of BSA, as understood from K_q values, follows the order 2 > 3 > 1. The strong protein-binding ability of 2 with enhanced hydrophobicity is consistent with its strong DNA binding affinity.

Fig. 8 The emission spectrum of BSA (10 μM; λ_exc = 280 nm; λ_emi = 346 nm) in the presence of increasing amounts of complexes 1–3 (0–50 μM). The arrow shows the emission intensity changes upon increasing complex concentration.

Fig. 9 Plot of I₀/I vs. [Q].

Binding constants and the number of binding sites. For the static quenching interaction, if it is assumed that there are similar and independent binding sites in the biomolecule, the binding constant (K_b) and the number of binding sites (n) can be determined according to the method⁵⁵ using the Scatchard equation: log[(I₀ − I)/I] = log K_b + n log[Q] where, in the present case, K_b is the binding constant for the complex–protein interaction and n is the number of binding sites per albumin molecule, which can be determined by the slope and the intercept of the double logarithm regression curve of log[(I₀ − I)/I] versus log[Q] (Fig. 10 and Table 4). The values of “n” at room temperature are approximately equal to 1, which indicates that there is just one single binding site in BSA for the complexes 1, 2 and 3.

Fig. 10 Plot of log[(I₀ − I)/I] vs. log[Q].

Synchronous fluorescence spectroscopic studies of BSA. Synchronous fluorescence spectral study was used to obtain information about the molecular environment in the vicinity of the fluorophore moieties of BSA.⁵⁶ When the difference (Δλ) between the excitation and emission wavelengths is fixed at 15 and 60 nm, and the amount of complexes (1–3) added to BSA (10 μM) is increased, a large decrease in fluorescence intensity with a red-shift in the tryptophan emission maximum is observed (Fig. 11). In contrast, the emission intensity of tyrosine residue increases with red shift in the emission maximum (Fig. 12). The observation indicated that complexes mainly bind to tryptophan residues of BSA. It indicates that the hydrophobicity of microenvironment around tryptophan residues decreases in the presence of the complex.⁵⁷

Fig. 11 Synchronous spectra of BSA (10 μM) in the presence of increasing amounts of complexes 1–3 (0–50 μM) for a wavelength difference of Δλ = 60 nm. The arrow shows the emission intensity changes upon increasing concentration of complex.

Fig. 12 Synchronous spectra of BSA (10 μM) in the presence of increasing amounts of complexes 1–3 (0–50 μM) for a wavelength difference of Δλ = 15 nm. The arrow shows the emission intensity changes upon increasing concentration of complex.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cytotoxic effect of the new complexes 1–3 and the ligand on the proliferation of human lung cancer and liver cancer cell lines A549 and HepG2 were assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). From the results, it is found that complex 2 exhibited higher antiproliferative activity than 1 and 3 (Fig. 13). The IC₅₀ values were shown in Table 5. In comparison with the conventional standard cisplatin, complexes 1–3 were found to have a lower IC₅₀ value on A549 cell line. However, for HepG2, all the complexes have higher IC₅₀ values than the standard cisplatin. From the studies, it is found that the complexes exhibited significant activity on A549 and HepG2 cell lines. However, by comparing the activity of the complexes with their analogous compounds, 2-hydroxy-1-naphthaldehyde 4(N)-ethyl thiosemicarbazone complexes of ruthenium, It is found that the activity of the complex [Ru(H-Nap-etsc)Cl(CO)(PPh₃)₂] was almost similar to the complex 1 whereas, the activity [Ru(Nap-etsc)(CO)(PPh3)2]·Cl was very high as compare with all the three analogs and also cisplatin.^8m Further, comparison with salicylaldehyde–4(N)-ethylthiosemicarbazone complexes, there were no appreciable variation in the activity in both the cell lines (Table 6).^8o

Fig. 13 The IC₅₀ value (50% inhibition of cell growth for 48 hours) for the ligand, complexes 1–3 along with cisplatin are depicted.

Table 5 Quenching constant (K_q), binding constant (K_bin) and number of binding sites (n) for the interactions of complexes 1–3 with BSA

Complex	Kq/M^−1	Kbin/M^−1	n
1	8.2 ± 0.17 × 10^4	9.24 ± 0.16 × 10^5	1.25
2	3.5 ± 0.23 × 10^5	2.7 ± 0.64 × 10^6	1.28
3	8.8 ± 0.18 × 10^4	1.6 ± 0.57 × 10^6	1.31

---

Table 6 In vitro cytotoxicity of complexes 1–3 for human tumour cell lines A549 and HepG2^a

Complexes	IC50 values (μM)
	A549	HepG2
a Cells were exposed to different concentrations of complexes for 48 h and cell viability was assessed using MTT assay. The IC50 values were calculated and tabulated. Results shown are mean ± SEM, which are three separate experiments performed in triplicate.
[H2-(Nap-tsc)]	22 ± 0.21	20 ± 0.18
1	20 ± 0.19	18 ± 0.15
2	17 ± 0.13	15 ± 0.13
3	18 ± 0.15	17 ± 0.14
Cisplatin	25 ± 0.23	9 ± 0.09

---

4. Conclusion

The reaction of 2-hydroxy-1-naphthaldehydethiosemicarbazone [H₂-(Nap-tsc)] with octahedral [RuHCl(CO)(PPh₃)₃] afforded two different complexes exhibiting different structural features and they were characterized by the analytical, spectral and X-ray crystallography. Even though, the ligand [H₂-(Nap-tsc)] has five potential donor atoms, (three nitrogen, one oxygen and one sulphur atom) among them only two donor atoms were utilized in complex 1, resulting a four member chelate (1) formation. Whereas in complex 2, it acted as tridentate donor by forming one six and five member chelate (2) rings. With the intention of utilizing the three remaining donor sites in 1, it was further reacted with [NiCl₂(PPh₃)₂] which results new heterobinuclear complex (3) wherein all the five donor atoms of the ligand were binded with metal cations. Further, an attempt was made to compare their nuclearity with their potential biological activities such as DNA/protein binding and cytotoxic studies. Mode of DNA interaction with the complexes (1–3) has been determined by absorption/emission titration and competitive ethidium bromide measurements. The results revealed that all the complexes can electrostatically interact with HS-DNA. The interaction between complexes (1–3) and BSA has been investigated by employing absorption, emission and synchronous spectral techniques. Synchronous fluorescence studies indicate that the binding of complexes (1–3) with BSA mostly changes the polarity around tryptophan residues rather than tyrosine residues. These results are consistent with the DNA binding results and the order of activity of the complexes was found as 2 > 3 > 1. The in vitro cytotoxicity of the complexes (1–3) were tested by MTT assays against human lung adenocarcinoma cell A549 and human liver carcinoma cell HepG2, suggesting that all the complexes are active and exhibited significant cytotoxic effects against selected tumor cell lines.

Acknowledgements

The authors gratefully acknowledge Council of Science and Industrial research (CSIR), New Delhi, India and Department of Science and Technology (DST), New Delhi, India for the financial support. Dr V. Vijaya Padma and Dr P. Poornima, Department of Biotechnology is acknowledged for their help in carrying out cytotoxicity studies.

References

1. M. J. M. Campbell, Coord. Chem. Rev., 1975, 15, 279.
2. S. Padhye and G. B. Kauffman, Coord. Chem. Rev., 1985, 63, 127.
3. D. X. West, S. B. Padhye and P. B. Sonowane, Struct. Bonding, 1991, 76, 1.
4. D. X. West, A. E. Liberta, S. B. Chikate, P. B. Sonowane, A. S. Kumbhar and R. G. Yerande, Coord. Chem. Rev., 1993, 123, 49.
5. T. S. Lobana, R. Sharma, G. Bawa and S. Khanna, Coord. Chem. Rev., 2009, 253, 977.
6. S. K. Chattopadhyay, Ruthenium: Properties, Production and Applications, Nova Publishers, 2011, vol. 293.
7. D. H. Petering, Bioinorg. Chem., 1972, 1, 255.
8. (a) R. Prabhakaran, S. V. Renukadevi, R. Karvembu, R. Huang, J. Mautz, G. Huttner, R. Subashkumar and K. Natarajan, Eur. J. Med. Chem., 2008, 43, 268; (b) R. Prabhakaran, P. Kalaivani, R. Jayakumar, M. Zeller, A. D. Hunter, S. V. Renukadevi, E. Ramachandran and K. Natarajan, Metallomics, 2011, 3, 42; (c) R. Prabhakaran, R. Sivasamy, J. Angayarkanni, R. Huang, P. Kalaivani, R. Karvembu, F. Dallemer and K. Natarajan, Inorg. Chim. Acta, 2011, 374, 647; (d) P. Kalaivani, R. Prabhakaran, E. Ramachandran, F. Dallemer, G. Paramaguru, R. Renganathan, P. Poornima, V. Vijaya Padma and K. Natarajan, Dalton Trans., 2012, 4, 2486; (e) P. Kalaivani, R. Prabhakaran, F. Dallemer, P. Poornima, E. Vaishnavi, E. Ramachandran, V. Vijaya Padma, R. Renganathan and K. Natarajan, Metallomics, 2012, 4, 101; (f) R. Prabhakaran, P. Kalaivani, P. Poornima, F. Dallemer, G. Paramaguru, V. Vijaya Padma, R. Renganathan and K. Natarajan, Dalton Trans., 2012, 41, 9323; (g) R. Prabhakaran, P. Kalaivani, R. Huang, M. Sieger, W. Kaim, P. Viswanathamurthi, F. Dallemer and K. Natarajan, Inorg. Chim. Acta, 2011, 376, 317; (h) R. Prabhakaran, P. Kalaivani, S. V. Renukadevi, R. Huang, K. Senthilkumar, R. Karvembu and K. Natarajan, Inorg. Chem., 2012, 51, 3525; (i) P. Kalaivani, R. Prabhakaran, P. Poornima, F. Dallemer, K. Vijayalakshmi, V. Vijaya Padma and K. Natarajan, Organometallics, 2012, 31, 8323; (j) R. Prabhakaran, P. Kalaivani, P. Poornima, R. Huang, V. Vijaya Padma and K. Natarajan, JBIC, J. Biol. Inorg. Chem., 2013, 18, 233; (k) P. Kalaivani, R. Prabhakaran, M. V. Kaveri, R. Huang, R. J. Staples and K. Natarajan, Inorg. Chim. Acta, 2013, 405, 415; (l) R. Prabhakaran, P. Kalaivani, R. Huang, P. Poornima, V. Vijaya Padma and K. Natarajan, Bioorg. Med. Chem., 2013, 21, 6742; (m) P. Kalaivani, R. Prabhakaran, P. Poornima, R. Huang, H. Virginie, F. Dallemer, V. Vijaya Padma and K. Natarajan, RSC Adv., 2013, 3, 20363; (n) R. Prabhakaran, R. Karvembu, T. Hashimoto, K. Shimizu and K. Natarajan, Inorg. Chim. Acta, 2005, 358, 2093; (o) P. Kalaivani, R. Prabhakaran, F. Dallemer, E. Vaishnavi, P. Poornima, V. Vijaya Padma, R. Renganathan and K. Natarajan, J. Organomet. Chem., 2014, 762, 67.
9. P. C. A. Bruijnincx and P. J. Sadler, Curr. Opin. Chem. Biol., 2008, 12, 197.
10. J. Reedijk, Platinum Met. Rev., 2008, 52, 2.
11. J. Reedijk, Eur. J. Inorg. Chem., 2009, 1303.
12. P. C. A. Bruijnincx and P. J. Sadler, Adv. Inorg. Chem., 2009, 61, 1.
13. P. J. Dyson and G. Sava, Dalton Trans., 2006, 1929.
14. C. S. Allardyce, A. Dorcier, C. Scolaro and P. J. Dyson, Appl. Organomet. Chem., 2005, 19, 1.
15. A. M. Thayer, Chem. Eng. News, 2010, 88, 24.
16. L. Kelland, Nat. Rev. Cancer, 2007, 7, 573.
17. W. H. Ang, A. Casini, G. Sava and P. J. Dyson, J. Organomet. Chem., 2011, 696, 989 and references cited therein.
18. (a) S. Nobili, E. Mini, I. Landini, C. Gabbiani, A. Casini and L. Messori, Med. Res. Rev., 2010, 30, 550; (b) R. W. Y. Sun and C. M. Che, Coord. Chem. Rev., 2009, 253, 1682.
19. K. Strohfeldt and M. Tacke, Chem. Soc. Rev., 2008, 37, 11.
20. S. D. Shyder, Y. Fu, A. Habtemariam, S. H. Van Rijt, P. A. Coopr, P. M. Loadman and P. J. Sadler, MedChemComm, 2011, 2, 666 , and references therein.
21. (a) F. Hackenberg, L. Oehninger, H. Alborzinia, S. Can, I. Kitanovic, Y. Geldmacher, M. Kokoschka, S. Woelfl, I. Ott and W. S. Sheldrick, J. Inorg. Biochem., 2011, 105, 991; (b) Y. Geldmacher, I. Kitanovic, H. Alborzinia, K. Bergerhoff, R. Rubbiani, P. Wefelmeier, A. Prokop, R. Gust, I. Ott and S. Woelfl, ChemMedChem, 2011, 6, 429; (c) S. Top, I. Efremenko, M. N. Rager, A. Vessieres, P. Yaswen, G. Jaouen and R. H. Fish, Inorg. Chem., 2011, 50, 271.
22. (a) A. Kastl, A. Wilbuer, A. L. Merkel, L. Feng, P. Di Fazio, M. Ocker and E. Meggers, Chem. Commun., 2012, 48, 1863; (b) P. K. Lee, W. H.-T. Law, L. Hua-Wei and K.-W. Kenneth, Inorg. Chem., 2011, 50, 8570.
23. K. R. Seddon and E. A. Seddon, The Chemistry of Ruthenium, Elseviers Science Publishers, Amsterdam, 1983, vol. 19.
24. M. J. Clarke, F. C. Zhu and D. R. Frasca, Chem. Rev., 1999, 99, 2511.
25. C. S. Allardyce and P. J. Dyson, Platinum Met. Rev., 2001, 45, 62.
26. D. T. Richens, Chem. Rev., 2005, 105, 1961.
27. O. Lentzen, C. Moucheron and A. Kirsch-De Mesmaeker, Metallotherapeutic drugs and metal-based diagnostic agents: The use of metals in medicine, ed.M. Gielen and E. R. T. Tiekink, John Wiley & Sons Ltd., Chichester, 2005.
28. C. A. Vock, W. H. Ang, C. Scolaro, A. D. Phillips, L. Lagopoulos, L. J. Jeanneret, G. Sava, R. Scopelliti and P. J. Dyson, J. Med. Chem., 2007, 50, 2166.
29. I. Kostova, Curr. Med. Chem., 2006, 13, 1085.
30. C. G. Hartinger, S. Z. Seifried, M. A. Jakupec, B. Kynast, H. Zorbas and B. K. Keppler, J. Inorg. Biochem., 2006, 100, 891.
31. S. Kapitza, M. Pongratz, M. A. Jakupec, P. Heffeter, W. Berger, L. Lackinger, B. K. Keppler and B. Marian, J. Cancer Res. Clin. Oncol., 2005, 131, 101.
32. L. Bratsos, S. Jedner, T. Gianferrara and E. Alessio, Chimia, 2007, 61, 692.
33. (a) D. Nieto, A. M. G. Vadillo, S. Bruna, C. J. Pastor, C. R. Luci, L. G. León, J. M. Padrón, C. N. Ranninger and I. Cuadrado, Dalton Trans., 2012, 41, 432; (b) J. F. G. Pantoja, M. Stern, A. A. Jarzecki, E. Royo, E. R. Escajeda, A. V. Ramírez, R. J. Aguilera and M. Contel, Inorg. Chem., 2011, 50, 11099; (c) M. Wenzel, B. Bertrand, M. J. Eymin, V. Comte, J. A. Harvey, P. Richard, M. Groessl, O. Zava, H. Amrouche, P. D. Harvey, P. Le Gendre, M. Picquet and A. Casini, Inorg. Chem., 2011, 50, 9472; (d) F. Pelletier, V. Comte, A. Massard, M. Wenzel, S. Toulot, P. Richard, M. Picquet, P. Le Gendre, O. Zava, F. Edafe, A. Casini and P. J. Dyson, J. Med. Chem., 2010, 53, 6923.
34. D. L. Klayman, J. F. Brtosevich, T. S. Griffin, C. J. Mason and J. P. Scovill, J. Med. Chem., 1979, 22, 855.
35. N. Ahmed, J. J. Levision, S. D. Robinson and M. F. Uttley, Inorg. Synth., 1974, 15, 48.
36. J. Venanzi, J. Chem. Soc., 1958, 43, 719.
37. A. I. Vogel, Text Book of Practical Organic Chemistry, Longman, London, Vth edn, 1989, p. 268.
38. G. M. Sheldrick, SHELXTL Version 5.1, An Integrated System for Solving, Refining and Displaying Crystal Structures from Diffraction Data, Siemens Analytical X-ray Instruments, Madison, WI, 1990.
39. G. M. Sheldrick, SHELXL-97, A Program for Crystal Structure Refinement Release 97-2, Institut fur Anorganische Chemie der Universitat Gottingen, Tammanstrasse 4, D-3400, Gottingen, Germany, 1998.
40. A. Wolfe, G. H. Shimer and T. Meehan, Biochemistry, 1987, 26, 6392.
41. G. Cohen and H. Eisenberg, Biopolymers, 1969, 8, 45.
42. M. Van de Weert and L. Stella, J. Fluoresc., 2010, 20, 625.
43. M. Jiang, M. X. Xie, D. Zheng, Y. Liu, X. Y. Li and X. Chen, J. Mol. Struct., 2004, 692, 71.
44. T. Mossman, J. Immunol. Methods, 1983, 65, 55.
45. J. G. Tojal, J. L. Dizarro, A. G. Orad, A. R. P. Sanz, M. Ugalda, A. A. Diaz, J. L. Serra, M. I. Arriortua and T. Rojo, J. Inorg. Biochem., 2001, 86, 627.
46. K. Natarajan and U. Agarwala, Inorg. Nucl. Chem. Lett., 1978, 14, 7.
47. (a) F. Basuli, M. Ruf, C. G. Pierpont and S. Bhattacharya, Inorg. Chem., 1998, 37, 6113; (b) F. Basuli, S.-M. Peng and S. Bhattacharya, Inorg. Chem., 2000,39, 1120; (c) F. Basuli, S.-M. Peng and S. Bhattacharya, Inorg. Chem., 2001, 40, 1126; (d) F. Basuli, S. M. Peng and S. Bhattacharya, Inorg. Chem., 1997, 36, 5645.
48. D. M. Boghaei and S. Mohebi, J. Chem. Res., 2001, 224.
49. E. C. Long and J. K. Barton, Acc. Chem. Res., 1990, 23, 271; R. F. Pasternack, E. J. Gibbs and J. J. Villafranca, Biochemistry, 1983, 22, 251.
50. Y. J. Hu, Y. Liu, J. B. Wang, X. H. Xiao and S. S. Qu, J. Pharm. Biomed. Anal., 2004, 36, 915.
51. Y. Y. Yue, X. G. Chen, J. Qin and X. J. Yao, Dyes Pigm., 2008, 79, 176.
52. H. Y. Liu, Z. H. Xu, X. H. Liu, P. X. XI and Z. Z. Zeng, Chem. Pharm. Bull., 2009, 57, 1237.
53. Y. Wang, H. Zhang, G. Zhang, W. Tao and S. Tang, J. Lumin., 2007, 126, 211.
54. F. Dimiza, F. Perdih, V. Tangoulis, I. Turel, D. P. Kessissoglou and G. Psomas, J. Inorg. Biochem., 2011, 105, 476.
55. M. Jiang, M. X. Xie, D. Zheng, Y. Liu, X. Y. Li and X. Chen, J. Mol. Struct., 2004, 692, 71.
56. N. Wang, L. Ye, B. Q. Zhao and J. X. Yu, Braz. J. Med. Biol. Res., 2008, 41, 589.
57. B. Liu, Y. Guo, J. Wang, R. Xu, X. Wang, D. Wang, L. Q. Zhang and Y. N. Xu, J. Lumin., 2010, 130, 1036.

---

Footnote

† Electronic supplementary information (ESI) available: Crystallographic data for [Ru(H-Nap-tsc)(CO)Cl(PPh₃)₂]·(CH₃)₂NCHO (1a), [Ru(H-Nap-tsc)(CO)Cl(PPh₃)₂]·CH₂Cl₂ (1b), [RuNi(Nap-tsc)(CO)Cl(PPh₃)₃] (3) have been deposited. ¹H-NMR spectra of the ligand and complexes (Fig. S1–S4); packing diagram of the unit cell for complex 1a, (Fig. S5); packing diagram of the unit cell for complex 1b (Fig. S6); packing diagram of the unit cell for complex 2 (Fig. S7); cyclic voltammograms of complex (1–3); hydrogen bonding table 1a and 1b (Table S1). CCDC 821289, 957349 and 821290. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra08492f

---