An investigation on new ruthenium(II) hydrazone complexes as anticancer agents and their interaction with biomolecules.

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An investigation on new ruthenium(II) hydrazone complexes as anticancer agents and their interaction with biomolecules† Mani Alagesan,a Nattamai S. P. Bhuvaneshb and Nallasamy Dharmaraj*a A new set of ruthenium(II) hydrazone complexes [Ru(H)(CO)(PPh3)2(L)] (1) and [RuCl2(DMSO)2(HL)] (2), with triphenyl phosphine or DMSO as co-ligands was synthesized by reacting benzoyl pyridine furoic acid hydrazone (HL) with [Ru(H)(Cl)(CO)(PPh3)3] and [RuCl2(DMSO)4]. The single crystal X-ray data of complexes 1 and 2 revealed an octahedral geometry around the ruthenium ion in which the hydrazone is coordinated through ON and NN atoms in complexes 1 and 2 respectively. The interaction of the compounds with calf thymus DNA (CT-DNA) has been estimated by absorption and emission titration methods which indicated that the ligand and the complexes interacted with CT-DNA through intercalation. In addition, the DNA cleavage ability of these newly synthesized ruthenium complexes assessed by an agarose gel electrophoresis method demonstrated that complex 2 has a higher DNA cleavage activity than that of complex 1. The binding properties of the free ligand and its complexes with bovine serum albumin (BSA) protein have been investigated using UV-visible, fluorescence and synchronous

Received 18th July 2013, Accepted 22nd December 2013 DOI: 10.1039/c3dt51949j www.rsc.org/dalton

fluorescence spectroscopic methods which indicated the stronger binding nature of the ruthenium complexes to BSA than the free hydrazone ligand. Furthermore, the cytotoxicity of the compounds examined in vitro on a human cervical cancer cell line (HeLa) and a normal mouse embryonic fibroblasts cell line (NIH 3T3) revealed that complex 2 exhibited a superior cytotoxicity than complex 1 to the cancer cells but was less toxic to the normal mouse embryonic fibroblasts under identical conditions.

Introduction The drug resistance developed by cancer cells to cisplatin and the severe side effects of platinum have led to a new line of investigation that focuses on the potential of ruthenium metallo-pharmaceuticals in the field of chemotherapy.1 In general, the drug discovery approach targets DNA extensively. The coordination of the ruthenium atom to the nucleic bases is seen to be enhanced through H-bonding interactions or

a Inorganic & Nanomaterials Research Laboratory, Department of Chemistry, Bharathiar University, Coimbatore 641 046, India. E-mail: [email protected]; Fax: +91 4222422387; Tel: +91 4222428319 b Department of Chemistry, Texas A&M University, College Station, TX 77843, USA † Electronic supplementary information (ESI) available: a packing diagram of the Ru(II) complex with intermolecular hydrogen bonding (Fig. S1), the UV-visible absorption spectra of complexes 1 and 2 in aqueous PBS buffer (Fig. S2), plots of [DNA]/(εa – εf ) versus [DNA] (Fig. S3), Stern–Volmer plots for the fluorescence titrations of the ligand and complexes (Fig. S4), electronic absorption spectra of BSA (Fig. S5), Scatchard plots (A) Stern–Volmer plots (B) for compounds (Fig. S6), synchronous spectra of BSA in the presence of compounds (Fig. S7 and S8), cell survival curves (Fig. S9). CCDC numbers 917920 and 917921. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c3dt51949j

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weakened because of steric interactions, suggesting the possibility to design compounds to target specific nucleotides. Intercalating molecules in particular have been targets of many studies because intercalation distorts the helical shape of DNA, causing the inhibition of replication enzymes. In the burgeoning area of DNA intercalation studies, ruthenium metal intercalators have been imperative since their redox and photophysical properties make it possible to utilize multiple techniques to study intercalation processes.2–6 The binding modes and affinities are not only related to the structures of the ruthenium complexes but also to the structures of DNA.7 Therefore, to evaluate and understand more clearly the factors that determine the DNA-binding behaviours, the preparation of complexes with different shapes and electronic properties are necessary. Varying the substituent group or co-ligand or subsistent position in the intercalative ligand can create some interesting differences in the space configuration and the electron density distribution of the complexes, which will result in some differences in the spectral properties and the DNAbinding behaviours of the complexes, and will be more helpful to clearly understand the binding mechanism of Ru(II) complexes to DNA.8 A variety of ruthenium metallointercalators containing a triphenyl phosphine/DMSO etc. co-ligand have

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now been synthesized to ameliorate cisplatin activity, particularly on resistant tumors, or to reduce host toxicity at active doses.9,10 Among all of the ruthenium-based anticancer agents, ruthenium–DMSO complexes are believed to show the greatest potential as anticancer agents due to their selectivity for solid tumor metastases and low toxicity in vivo.11 A lot of work has been carried out with ruthenium, however there is no report which correlates exactly the co-ligand triphenyl phosphine and DMSO precursor complexes of Ru(II).12,13 Hence, the main thrust of this paper is to study the upshot of different complexes of Ru with triphenyl phosphene and DMSO as the co-ligands and their structure, binding aspects and anticancer activities. On the other hand, reagents that react with protein chains are extremely useful in biochemistry and biology.10 The magnitude of the albumin interactions with the drug is essential since it plays a dominant role in drug disposition and efficacy. The bound drug can act as a depot while the unbound drug produces the desired pharmacological effect. Further interactions of the drug with serum albumin determines the pharmacology and pharmacodynamics of the drug, and explains the relationship between the structure and function of the drug as they greatly influence the absorption, distribution, metabolism, and excretion properties of typical drugs.14,15 In this respect, we have synthesized two new Ru hydrazone complexes with different co-ligands and assessed their interaction with DNA and BSA. The cytotoxicity of the complexes was assessed using cancer and normal cell lines under in vitro conditions.

Experimental General All of the chemicals used for the preparation of the compounds and buffer solution are of analytical grade or chemically pure grade. RuCl3·3H2O, purchased from Himedia was used without further purification unless otherwise mentioned. The starting materials, benzoyl pyridine furoic acid hydrazone (HL)16 [Ru(H)(Cl)(CO)(PPh3)3]17 and [RuCl2(DMSO)4]18 were prepared according to reported methods. Protein free calfthymus DNA (CT-DNA), obtained from Sigma-Aldrich chemicals was stored at 0–4 °C and its purity was checked by measuring its optical density before use. Doubly distilled water was used to prepare Tris–HCl buffer (5 mM Tris–HCl, 50 mM NaCl, pH 7.2, Tris–Tris(hydroxymethyl)methylamine). DNA stock solutions were freshly prepared before study using this buffer solution. Agarose and ethidium bromide were purchased from Aldrich and used without further purification. The PBR322 supercoiled DNA and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from SigmaAldrich and used as received. Methods Elemental analyses (C, H and N) were performed on a Vario EL III Elemental analyzer instrument. The IR spectra

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(4000–400 cm−1) obtained with KBr disks were recorded with a Nicolet Avatar Model FT-IR spectrophotometer. The melting points were determined with a Lab India instrument. The electronic absorption spectra of the samples were recorded using a Jasco V-630 spectrophotometer. The emission spectra were measured using a Jasco FP 6600 spectrofluorometer. All other chemicals and reagents used for the biological studies were of high quality and procured commercially from the reputed suppliers. The circular dichroism spectra were recorded using a JASCO J-810 spectropolarimeter with a PMT detector in a DMSO-buffer solution. The synthesis of the complexes The synthesis of [Ru(H)(CO)(PPh3)2(L)] (1). A warm methanolic solution (20 mL) containing [Ru(H)(Cl)(CO)(PPh3)3] was added to a methanolic solution of HL and refluxed for 12 h. Upon the slow evaporation of the solvent, brown crystals of complex 1 suitable for X-ray studies were obtained over a period of three months. Yield: 52%. Melting point: >255 °C. Elemental analysis: found (calculated) (%) for C54H43N3O3P2Ru: C, 68.61 (68.63); H, 4.55 (4.58); N, 4.42 (4.44). UV-visible (solvent: Tris–HCl buffer, nm): (ε, M−1 cm−1): 273 (21 037), 309 (10 390). Selected IR bands (KBr, ν in cm−1): 1583, 1478 (>CvN– NvC192 °C. Elemental analysis: found (calculated) (%) for C21H25Cl2N3O4RuS2: C, 40.5 (40.7); H, 3.99 (4.06); N, 6.72 (6.78). UV-visible (solvent: 5% DMSO and 95% Tris–HCl buffer nm): (ε, M−1 cm−1) 270 (29 204), 325 (31 768). IR (KBr, ν cm−1): 3063 (NH), 1597 (CvN), 426 pyridine (CvN), 1096 (CvS). 1H NMR (DMSO-d6, δ ppm) 2.96–3.14 (s, 12H), 6.28–6.32 (m, 2H), 7.62–7.91 (m, 8H), 7.98–8.04 (m, 1H), 8.42 (br s, 1H). ESI-MS: calcd for C21H25Cl2N3O4RuS2 is 618.9707; found [M + H]−: 619.9845. Single crystal X-ray diffraction studies. The single crystal X-ray diffraction data of the complexes were collected on a BRUKER APEX 2 X-ray (three-circle) diffractometer. The integrated intensity information for each reflection was obtained by a reduction of the data frames with the program APEX2.19 The integration method employed a three dimensional profiling algorithm and all of the data were corrected for Lorentz and polarization factors, as well as for crystal decay effects. Finally the data were merged and scaled to produce a suitable data set. A solution was obtained readily using SHELXTL (XS).20 The solvent molecules (observed from the difference Fourier maps) could not be identified because of significant


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Table 1

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The experimental data for crystallographic analysis

Empirical formula Formula weight Temperature (K) Wavelength (Å) Crystal system Space group Unit cell dimensions a (Å) b (Å) c (Å) α (°) β (°) γ (°) Volume (Å3) Z Density (calculated) (Mg m−3) Abs. coefficient (mm−1) F(000) Crystal size (mm3) Reflections collected Independent reflections Goodness-of-fit on F2 Final R indices [I > 2σ(I)] R indices (all data)

1

2

C54H43N3O3P2Ru 944.92 296(2) 0.71073 Monoclinic P2(1)/c

C21H25Cl2N3O4RuS2 619.53 150(2) K 0.71073 Monoclinic P2(1)/c

10.4162(4) 21.7789(9) 20.2709(9) 90 97.932(3) 90 4554.5(3) 4 1.378 0.462 1944 0.06 × 0.05 × 0.04 27 127 4047 [R(int) = 0.0899] 1.059 R1 = 0.0527, wR2 = 0.1281 R1 = 0.0899, wR2 = 0.1506

15.779(2) 12.7157(16) 14.1337(18) 90 98.491(2) 90 2804.8(6) 4 1.467 0.928 1256 0.24 × 0.12 × 0.11 52 979 2146 [R(int) = 0.0640] 1.319 R1 = 0.0431, wR2 = 0.0950 R1 = 0.0456, wR2 = 0.0957

disorder and partial occupancy; they were squeezed out using PLATON.21 Accordingly, the density and the formula reported in the CIF file and the table does not account for the solvation; CH3OH was used as the solvent. The hydrogen atoms were placed in idealized positions and were set riding on the respective parent atoms. All non-hydrogen atoms were refined with anisotropic thermal parameters. The structure was refined (weighted least squares refinement on F2) to convergence.20,22 The relevant details concerning the data collection and the details of the structure refinement are summarized in Table 1. Stability studies The stabilities of complexes 1 and 2 were checked by recording the UV-visible spectrum of them by dissolving in a minimum amount of DMSO (1 × 10−3 M), and then diluted with PBS buffer. The hydrolysis profiles of these complexes were recorded by monitoring the electronic spectra for the resulting mixture over 24 h.

DNA binding experiments Absorption titration In order to investigate the possible binding modes of the ligand and complexes with CT-DNA a UV-visible titration method has been used. In absorption titration experiments, the spectra of CT-DNA in the presence and absence of each compound have been recorded by varying the nucleotide concentration in diverse [compound]/[CT-DNA] mixing ratios. All experiments involving calf-thymus DNA (CT-DNA) were performed with a Tris–HCl buffer solution ( pH = 7.2). The


concentration of CT-DNA was determined by UV absorbance at 260 nm. Solutions of CT-DNA in Tris–HCl buffer gave a ratio of UV absorbance at 260 and 280 nm, A260/A280, of approximately 1.9 indicating the protein free nature of DNA.22 The molar absorption coefficient, ε260, was taken as 6600 M−1 cm−1.23 A stock solution of CT-DNA was stored at 277 K and used after no more than 4 days. The absorption titrations of the Ru(II) complexes in buffer were done using a fixed concentration of ruthenium complexes to which increments of the DNA stock solution were added. An equal concentration solution of CT-DNA was added to the Ru(II) complex solution and reference solution to eliminate the absorbance of CT-DNA itself. Control experiments with DMSO were performed and no changes in the spectra of CT-DNA were observed. The magnitude of the binding strength of the compounds with CT-DNA can be estimated through the binding constant Kb, which can be obtained by monitoring the changes in the absorbance of the corresponding λmax with increasing concentrations of CT-DNA and is given by the equation ½DNA=½εa εf ¼ ½DNA=½εb εf þ 1=K b ½εb εf through a plot of [DNA]/[εa − εf ] versus [DNA], where [DNA] is the concentration of DNA in the base pairs. The apparent absorption coefficients εa, εf and εb correspond to Aobsd/[Ru], the extinction coefficient for the free compounds and in the fully bound form respectively. The slope and Y intercept of the linear fit of [DNA]/[εa − εf ] versus [DNA] give 1/[εa − εf ] and 1/Kb [εb − εf ] respectively. The intrinsic binding constant Kb can be obtained from the ratio of the slope to the Y intercept.24 Circular dichroic experiments were carried out by keeping the concentration of CT-DNA constant but varying the concentration of the compounds, by monitoring the changes in the positive peak and negative peak of CT-DNA to confirm the mode of binding. Luminescence titration in the presence of ethidium bromide (EB) The competitive DNA binding studies of the new complexes with EB was investigated with fluorescence spectroscopy in order to inspect whether the compound could displace boundEB from the DNA–EB complex by the addition of the solution of the respective complexes to the Tris–HCl buffer of the DNA– EB mixture. Before measurements were taken, the mixture was shaken up and recorded. The fluorescence spectra of DNA bound EB were obtained in the excitation and the emission wavelengths of 515 and 602 nm, respectively. The luminescence titration quenching experiments were conducted by keeping the concentration of DNA in buffer constant and adding small aliquots of the Ru(II) complex solutions. The resulting solution was allowed to equilibrate for 5–10 min at room temperature. The Stern–Volmer constant, KSV is used to evaluate the quenching efficiency of each complex,25 I 0 =I ¼ K q ½Q þ 1; where I0 is the emission intensity in the absence of the quencher, I is the emission intensity in the presence of the

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quencher, Kq is the quenching constant and [Q] is the quencher concentration. The Kq value is obtained as a slope from the plot of I0/I versus [Q].


DNA cleavage experiment The cleavage of DNA was monitored using agarose gel electrophoresis. Reactions using supercoiled pBR322 plasmid DNA in Tris–HCl buffer (50 mM) with 18 mM NaCl ( pH, 7.2) were treated with the ruthenium complexes (2.5–10 µM) followed by dilution with the Tris–HCl buffer to a total volume of 20 µL. The samples were incubated for 0.5 h at 37 °C. A loading buffer containing 25% bromophenol blue, 0.25% xylene cyanol and 30% glycerol was added and electrophoresis was performed at 40 V for 3 h in a Tris–Acetate–EDTA (TAE) buffer using 1% agarose gel containing 1.0 µg mL−1 ethidium bromide. The agarose gel electrophoresis of the plasmid DNA was visualized by photographing the fluorescence of intercalated ethidium bromide under a UV illuminator.26 The cleavage efficiency was measured by determining the ability of the complex to convert the supercoiled DNA (SC) to the nicked circular form (NC).

Protein binding studies The protein binding study was performed by tryptophan fluorescence quenching experiments using bovine serum albumin (BSA). The excitation wavelength of BSA at 280 nm and the quenching of the emission intensity of the tryptophan residues of BSA at 345 nm was monitored using the ligand or complexes as quenchers with increasing concentration.27 The excitation and emission slit widths and scan rates were kept constant for all of the experiments. A stock solution of BSA was prepared using 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. The titrations were manually done using a micropipette for the addition of the compounds. For the synchronous fluorescence spectra, the same concentrations of BSA and the compounds were used, and the spectra were obtained by scanning simultaneously the excitation and emission monochromator. The wavelength interval (Δλ) is fixed individually at 15 and 60 nm, at which the spectrum only shows the spectroscopic behaviour of the tyrosine and tryptophan residues of BSA, respectively. The Stern–Volmer and Scatchard graphs were used to study the interaction of a quencher with serum albumins (SAs). The fluorescence quenching was analysed according to the Stern–Volmer quenching equation,28 I 0 =I ¼ 1 þ K q τ0 ½Q ¼ 1 þ K SV ½Q where I0 = the initial tryptophan fluorescence intensity of SA, I = the tryptophan fluorescence intensity of SA after the addition

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of the quencher, Kq = the quenching rate constant of SA, KSV = the dynamic quenching constant, τ0 = the average lifetime of SA without the quencher and [Q] = the concentration of the quencher.

Cytotoxicity studies The growth inhibitory effect towards tumour cell lines was evaluated by means of an MTT (tetrazolium salt reduction) assay on human cervical cancer cells (HeLa) and normal NIH 3T3 (mouse embryonic fibroblasts) cells which were obtained from the National Centre for Cell Science, Pune, India.29,30 The cancer cells were grown in Eagles minimum essential medium containing 10% fetal bovine serum (FBS). For the screening experiment, the cells were seeded into 96-well plates in 100 μL of the respective medium containing 10% FBS, at a plating density of 10 000 cells per well and incubated at 37 °C, under conditions of 5% CO2, 95% air and 100% relative humidity for 24 h prior to the addition of compounds. The compounds were dissolved in DMSO and diluted in the respective medium containing 1% FBS. After 24 h, the medium was replaced with the respective medium with 1% FBS containing the compounds at various concentrations and incubated at 37 °C under conditions of 5% CO2, 95% air and 100% relative humidity for 48 h. Triplicate cultures were established for each treatment, and the medium not containing the compounds served as the control. After 48 h, 10 μL of MTT (5 mg mL−1) in phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for 4 h. The medium with MTT was then flicked off and the formed formazan crystals were dissolved in 100 μL of DMSO. Mean absorbance for each drug dose was expressed as a percentage of the control untreated well absorbance and plotted vs. drug concentration. The IC50 values represent the drug concentrations that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells % inhibition ¼ ½mean OD of untreated cells ðcontrolÞ= mean OD of treated cells 100 and a graph was plotted with the percentage of cell inhibition versus concentration. From this, the IC50 value was calculated.

Results and discussion Synthesis and characterization The synthesis of new anticancer drugs that would be more efficient, with less toxicity and less sensitivity to resistance mechanisms remains a fundamental challenge. Ruthenium complexes witnessed a spectacular development during the last decade.31,32 Many works have been carried out on an assortment of ruthenium complexes to evaluate their pharmacological properties. As a part of our continuous interest on the cytotoxicity and other biological potentials of transition metal complexes of hydrazone ligands, in this study, we


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Scheme 1

The synthetic route of the ruthenium(II) complexes.

compared the biological activity of two different Ru(II) complexes with co-ligands such as PPh3 and DMSO. The straightforward reaction of the precursor, [Ru(H)(Cl)(CO)(PPh3)3] or cis-[RuCl2(DMSO)4] with the hydrazone ligand (HL) as portrayed in Scheme 1, yielded complexes [Ru(H)(CO)(PPh3)2(L)] (1) and [RuCl2(DMSO)2(HL)] (2), respectively. Both complexes are non-hygroscopic, brown in colour and found to be air and light stable at room temperature and were soluble in organic solvents such as methanol, ethanol, DMSO and DMF. Complex 2 is soluble in water at room temperature. The complexes were analytically pure as their micro analytical data conform to the proposed molecular formula (data given in the Experimental section). The IR spectral data of the complexes suggested the formation of the complexes as given in Scheme 1. The strong band that appeared around 1960 cm−1 along with a sharp and less intense band around 2010 cm−1 were assigned to the coordinated terminal carbonyl group and Ru–H in complex 1. Furthermore, in the IR spectrum of the precursor complex cis-[RuCl2(DMSO)4], the SvO stretching frequency of the S-bonded DMSO appears at a higher frequency (1090 cm−1) in comparison with that of the O-bonded DMSO (915 cm−1) due to the increase in the SvO bond order in the former case. The desertion and retention of the bands around 915 and 1090 cm−1 in the new complex 2 indicate the replacement of the O-bonded DMSO and S-bonded DMSO cis to it by the hydrazone ligand, as reported earlier.33 The IR spectrum of the ligand (HL) exhibited a very strong band around 1600 cm−1 and a sharp band around 3200 cm−1 due to the carbonyl group (CvO) and NH stretching, respectively. However, complex 1 showed a negative shift in the position of the absorption due to the carbonyl functionality owing to its coordination to the metal through the oxygen atom along with the disappearance of the absorption of NH stretching suggesting that enolisation of the ligand followed by deprotonation occurred prior to coordination with the ruthenium ion. In the case of complex 2, a similar shift in the


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former band of the ligand HL was observed but without any change in the latter. In addition, a band observed at 426 cm−1 in the spectrum of complex 2 was assigned as the Ru–N bond of the pyridine moiety in the benzoyl pyridine segment of the hydrazone. These facts did confirm that the selected ligand (HL) behaves as a uninegative bidentate chelating agent coordinated through the carbonyl oxygen and the imine nitrogen in complex 1, but as neutral bidentate in complex 2 with the pyridine nitrogen and imine nitrogen as donors. The electronic absorption spectra of the diamagnetic Ru(II) complexes (1) and (2) were recorded in DMSO–Tris–HCl buffer solutions in the range of 800–200 nm using a quartz cuvette of 1 cm path length. Based on the position and nature of the peaks, all the bands are assigned to either intraligand n → π* or π → π* transitions and MLCT or LMCT. The single crystals of the new Ru(II) complexes were isolated by the slow evaporation of the reaction mixture over a period of 2–3 months. The structures of the new complexes were finally confirmed by the singlecrystal X-ray diffraction studies.

Crystal structure description of the new Ru(II) complexes The Ru(II) ion, designated as a borderline acid on the Pearson scale, has a large number of electrons with many closely spaced energy levels (each with a slightly different electronic configuration) and therefore, can accommodate a wider variety of coordination environments.34 The molecular structures of newly synthesized complexes 1 and 2 have been determined by the single crystal X-ray diffraction method and the ORTEP drawings are shown in Fig. 1. Selected bond distances and bond angles with geometrical parameters that are essential for discussion are given in Table 2. The structure shows that the hydrazone ligand is coordinated to Ru through the imine nitrogen, phenolic oxygen and pyridine nitrogen atom, forming a five membered ring by ON and NN chelation with bite angles N3–Ru1–O1 (77.1) and N1–Ru1–N2 (77.81). In complex 1, the ruthenium(II) ion is in an octahedral environment equatorially coordinated by an ON chelation of the imine nitrogen, the phenolic oxygen of the hydrazone ligand and two triphenyl phosphine, hydride and carbon monoxide atoms of the starting 1. From the single crystal X-ray structures of 2, it is clear that the chelating ligand has coordinated to ruthenium by NN chelate neutrally through the pyridine nitrogen and imine nitrogen by replacing the co-ligand DMSO that is trans to the S-bonded DMSO from the precursor and a pair of chlorine atoms at the axial position completes the octahedral geometry. The unit cell dimensions show that the crystals belong to the monoclinic system with the P21/c space group. The molecular structure of complex 2 shows that the coordination sphere around Ru(II) is S2Cl2NN leaving the geometry of the complex unchanged forming an octahedral complex as in the case of complex 1. The coordination polyhedron of the ruthenium ion can be described as a distorted octahedron. The distortion in the complex from the ideal octahedral geometry is due to the small bite angle of the NN chelate of the hydrazone ligand and the bending of the chloride ligands towards the

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Fig. 1

Table 2

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An ORTEP view of the Ru(II) complexes 1 and 2 with the atom numbering scheme.

Selected bond lengths (Å) and angles (°) for the complexes

1

2

Ru(1)–H(1) Ru(1)–P(1) Ru(1)–P(2) Ru(1) –O(1) Ru(1)–N(3) Ru(1)–C(18)

1.3002(7) 2.385(2) 2.260(3) 2.092(7) 2.218(8) 1.80(1)

Ru(1)–N(1) Ru(1)–N(2) Ru(1)–Cl(1) Ru(1)–Cl(2) Ru(1)–S(1) Ru(1)–S(2)

H(1)–Ru(1)–P(1) H(1)–Ru(1)–P(2) H(1)–Ru(1)–O1) H(1)–Ru(1)–N(3) H(1)–Ru(1)–C(18) P(1)–Ru(1)–P(2) P(1)–Ru(1)–O(1) P(1)–Ru(1) –N(3) P(1)–Ru(1)–C(18) P(2)–Ru(1)–O(1) P(2)–Ru(1)–O(1) P(2)–Ru(1)–O(1) P(2)–Ru(1)–O(1) P(2)–Ru(1)–O(1) P(2)–Ru(1)–O(1)

99.8(1) 92.8(1) 159.1(1) 91.2(1) 98.51(9) 99.9(1) 79.3(1) 167.7(1) 80(1) 93.7(1) 93.7(1) 93.7(1) 93.7(1) 93.7(1) 93.7(1)

N(1)–Ru(1)–S(2) N(1)–Ru(1)–S(1) N(2)–Ru(1)–S(1) S(2)–Ru(1)–S(1) N(1)–Ru(1)–Cl(2) N(2)–Ru(1)–Cl(2) S(2)–Ru(1)–Cl(2) S(1)–Ru(1)–Cl(2) S(2)–Ru(1)–Cl(1) S(1)–Ru(1)–Cl(1) Cl(2)–Ru(1)–Cl(1)

2.038(3) 2.078(3) 2.428(8) 2.406(1) 2.284(1) 2.245(2) 90.16(8) 97.98(8) 172.14(9) 93.46(3) 171.01(9) 94.33(9) 94.67(4) 89.31(4) 174.87(3) 89.40(3) 89.62(3)

chelate which is evident from the angles N1–Ru1–N2 = 77.81 (12) and Cl1–Ru1–Cl2 = 89.62(3). Complex 2 is stabilized by intramolecular NHO (azomethine nitrogen N3 is hydrogen bonded to the oxygen atom of the DMSO) type hydrogen bonds whereas complex 1 has no intra or intermolecular H bonding interactions (Fig. S1†). The angles of the O-bonded DMSO in one of the precursors are relatively narrow compared with those of the S-bonded DMSO ligands. This is due to the different electronic situation of the two types of sulfur atom and can be attributed to the compression effect of the bulkier lone pair of the pyramidal sulphur atoms on the bonding pairs.35 The overall geometries of complexes 1 and 2 are very similar.

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Stability studies Aquation is widely accepted as an important step for the function of Ru-DMSO compounds in biological systems.36,37 Thus, the solution chemistry of complexes 1 and 2 is analyzed by UVvisible absorption spectroscopy. Compound 2 is soluble in water whereas, 1 is insoluble and both are stable in DMSO. The complexes are first dissolved in DMSO, and the concentrated DMSO solutions were diluted using aqueous PBS buffer ( phosphate buffered saline solution), at pH 7.4, to a final concentration of 1 × 10−3 M. The UV-visible absorption spectrum of these complexes are recorded every 1 h over 24 h at room temperature. The resulting spectral profiles for complexes 1 and 2 are given in Fig. S2.† The spectrum of the freshly prepared buffer solution of complexes 1 and 2 displayed two absorptions in the visible region at 282 nm and another less intense maximum located at 430 nm. The lower energy band is attributed to the Ru/ligand MLCT (metal to ligand charge transfer) transition, and the remaining is assigned to intraligand transitions. These absorption bands remain unchanged over 24 h, and also no new absorptions were observed during the experimental period indicating the substantial stability of complexes 1 and 2 in aqueous PBS buffer. Furthermore, the stability of complexes 1 and 2 in aqueous solution was assessed using ESI-MS analysis after 24 h. The molecular ion peaks for complexes 1 and 2 were found at m/z 944.1765 and at 619.9845, respectively. These results confirmed that the Ru(II) complexes 1 and 2 are very stable in an aqueous solution up to 24 h.

DNA binding studies Electronic absorption studies UV-visible absorption spectroscopy is a versatile and normally engaged method to determine the binding characteristics of


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Fig. 2 Changes in the electronic absorption spectra of the ligand HL (c) and complexes 1 (a) and 2 (b) (25 μM) with increasing concentrations of CT-DNA (0–40 μM).

metal complexes with DNA.38 In general, hyperchromism and hypochromism are the spectral features of DNA concerning changes of its double helix structure. Additionally, the existence of a red shift is indicative of the stabilization of the DNA duplex.39,40 The interaction of complexes 1 and 2 and the corresponding ligand with CT-DNA was followed by recording the UV-visible spectra of the system (Fig. 2). The experiment was carried out keeping the concentration of the ruthenium(II) complexes constant and varying the concentration of CT-DNA. Fig. 2 shows the electronic absorption spectra of complexes 1 and 2 with the corresponding hydrazone ligand in the absence and presence of CT-DNA. The ligand centered transitions at 273 nm and 321 nm for complexes 1 and 2 respectively, were considered for the corresponding absorptivity changes upon the incremental addition of DNA. The band around 321 nm shows hypochromism by about 31.2% and a red shift of 2 nm. For complex 1 under the same conditions, upon the addition of DNA, the IL band at about 273 nm exhibits hypochromism of 23.9% with a 2 nm red shift. The changes in the spectrum indicate that the complex possesses a strong affinity for CT-DNA. In order to compare quantitatively the binding strength of the two complexes, the intrinsic binding constant, Kb of the two complexes with DNA was calculated by monitoring the changes in the absorbance at 273 nm for complex 1 and 321 nm for complex 2 with an increasing concentration of DNA. The magnitudes of the intrinsic binding constants (Kb) were calculated to be 1.24 ± 0.06 × 104 M−1 for the ligand, (HL) and 4.11 ± 0.51 × 104 M−1 and 9.90 ± 0.71 × 104 M−1 for complexes 1 and 2, respectively. The observed values of Kb revealed that the ligand and the Ru(II) complexes bind to DNA via an intercalative mode.41 These results are similar to those reported earlier for the intercalative mode of various metallointercalators.10,42,43 From the results obtained, it has been found that both the ruthenium complexes exhibit a good binding affinity to DNA, greater than that of the corresponding free ligand. This is mainly due to the chelation of the ruthenium metal with the ligand. It is to be noted that complex 2 exhibited a relatively higher binding constant compared to complex 1. The highest binding constant is exhibited by


complex 2, which may be due to the hydrophobic nature of the DMSO complexes.44,45 The obtained data in our experiment indicates that the size and the shape of the intercalated ligand has a significant effect on the strength of the DNA binding, and the most suitable intercalating ligand leads to the highest affinity of complexes with DNA. The different DNA-binding properties of the ruthenium complexes 1 and 2 are due to the difference in the co-ligands. When compare with the Ru-PPh3 complex, the Ru-DMSO has a great planarity and hydrophobicity, which leads to a greater binding affinity to DNA. Furthermore, the binding mode needs to be proved through some more experiments. EB-bound DNA studies Ruthenium based intercalators have been the subject of many studies since their redox and photophysical properties make it possible to utilize multiple techniques to study DNA intercalation processes.14,46 In order to confirm the intercalative mode of binding between the compounds chosen in this work and CT-DNA, an ethidium bromide (EB) fluorescence displacement experiment was carried out. EB forms soluble complexes with nucleic acids and emits intense fluorescence in the presence of CT-DNA due to the intercalation of the planar phenanthridine ring between adjacent base pairs on the double helix.47 The changes observed in the spectra of EB on its binding to CT-DNA are often used for the interaction study between DNA and other compounds, such as metal complexes.48,49 The quenching is due to the reduction of the number of binding sites on the DNA that are available to EB. The fluorescence emission intensity of the DNA–EB system in the absence and presence of compounds added to DNA pre-treated with EB, causes an appreciable reduction in the emission intensity upon increasing the amounts of every ligand and the Ru(II) complex causes 35.4%, 59.9% and 11% for 1, 2 and HL, respectively (Fig. 3). This indicates that both the complexes and the ligand could compete with EB in binding to DNA and complex 2 binds to DNA stronger than complex 1. Furthermore, the quenching data were analyzed according to the Stern–Volmer equation and the Kq value is obtained as a slope from the plot

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Fig. 3 The fluorescence quenching curves of ethidium bromide bound to DNA: complexes 1 (a) and 2 (b) and ligand HL (c). [DNA] = 7.5 μM, [EB] = 7.5 μM and [compounds] = 0–16 μM.

of I0/I versus [Q]50 (Fig. S4†). The quenching plots illustrate that the quenching of EB bound to CT-DNA by free ligand and the complexes are in good agreement with the linear Stern–Volmer equation. The Kq values for HL, 1 and 2 were found to be 1.73 ± 0.04 × 102 M−1, 1.55 ± 0.15 × 103 M−1 and 8.26 ± 0.28 × 103 M−1, respectively. Furthermore, the binding constant (Kapp) values are obtained for the compounds using the following equation, K EB ½EB ¼ K app ½compound The apparent binding constants are found be 7.2 ± 0.03 × 104 M−1, 2.7 ± 0.11 × 105 M−1 and 5.6 ± 0.28 × 105 M−1 for HL, 1 and 2, respectively. These results are analogous to our earlier reports.36 The Stern–Volmer constant K (K is a liner Stern– Volmer quenching constant dependent on the ratio of the bound concentration of ethidium bromide to the concentration of DNA) of the complexes is consistent with the UV-vis titration results. These results suggest that complex 2 intercalated more strongly than complex 1. The binding activities of the Ru(II) complexes may be better than the ligand. However, their pharmacodynamical, pharmacological and toxicological properties should be further studied in vivo. Circular dichroism studies The CD spectral analysis gives valuable information on the binding mode of metal complexes with DNA.51 In Fig. 4, the CD spectrum of free DNA has a positive peak at approximately 278 nm and a negative peak at 247 nm which corresponds to B-DNA. These bands are caused by stacking interactions between the bases and the helical supra structure of the polynucleotide that provides an asymmetric environment for the bases.52 Simple groove binding and electrostatic interaction of the molecules show less or no perturbation on the base stacking and helicity, while intercalation increases the intensities of both the positive and negative bands.53,54 In our experiment, the respective addition of free ligand and complexes 1 and 2 to the solution of DNA increased the intensity of both the positive and negative bands of free DNA due to the intercalation of the test compounds between the base pairs of DNA.

6094 | Dalton Trans., 2014, 43, 6087–6099

Fig. 4 The circular dichroism spectra of free CT-DNA (10 µm) and after the addition of the ligand and complexes 1 and 2 (10 µm).

DNA cleavage studies Compounds that show enhanced cleavage activity in a microenvironment are rare55 but offer a unique mechanism to target tumor cells that are often resistant to radiotherapy56,57 and chemotherapy58,59 and the most susceptible toward metastasis,60,61 a particularly attractive chemotherapeutic target. The study on the cleavage capacity of transition metal complexes to DNA is considerably interesting as it can contribute to understanding the toxicity mechanism of them and to develop novel artificial nucleases. The degree to which the ruthenium complex could function as a DNA cleavage agent was examined using supercoiled (SC) pBR322 DNA as the target. The efficiency of the cleavage of the molecule was probed using agarose gel electrophoresis. When circular plasmid DNA is conducted by electrophoresis, the fastest migration will be observed for the supercoiled form (Form I). If one strand is cleaved, the supercoil will relax to produce a slower-moving nicked circular form (Form II). If both strands are cleaved, a linear form (Form III) will be generated that migrates in between. The cleaved amount was enhanced with the increase of the concentration of the Ru(II) complex, showing the


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Fig. 5 The cleavage of supercoiled pBR322 DNA by the copper(II) complexes in a buffer containing 50 mM Tris–HCl and 50 mM NaCl at 37 °C. Lane 1, DNA control; lane 2, DNA + (2.5 µM) complex 1; lane 3, DNA + complex 1 (5 µM); lane 4, DNA + (10 µM) complex 1; lane 5, DNA + (2.5 µM) complex 2; lane 6, DNA + (5 µM) complex 2; lane 7, DNA + (10 µM) complex 2. Forms I and II are supercoiled and nicked circular DNA, respectively.

potential chemical nuclease activity of the complexes. The ruthenium complexes were found to promote the cleavage of pBR322 DNA from the supercoiled Form (I) to the nicked Form (II) by varying the concentration (2.5–10 μM) in the agarose gels which provides a measure of the extent of hydrolysis of the phosphodiester bonds in each DNA (Fig. 5). The complexes can induce the obvious cleavage of the DNA at the concentration of 10 μM. The results showed that complex 2 has more cleaving capacity than complex 1. Moreover, the complexes did not require any addition of external agents to effect the DNA cleavage activity. This indicates that the cleavage of DNA probably follows a hydrolytic cleavage mechanism which is unaffected by external free radicals. Moreover, the inhibition or promotion of DNA cleavage is not observed appreciably under aerobic and anaerobic conditions suggesting that oxidative cleavage is not a factor. Hence, the DNA cleavage observed here is expected to occur through a hydrolytic process.62,63

Protein binding studies The quenching of BSA fluorescence by HL and its Ru(II) complexes The interaction of the complexes with BSA has been studied from tryptophan emission quenching experiments. Albumin is the most abundant serum protein in the blood and its

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interaction is of high interest because non-covalent conjugation of albumin to a number of drugs and has been shown to carry active molecules in the blood.64 Serum albumins are proteins that are, amongst others, involved in the transport of metal ions and metal complexes with drugs through the blood stream. Serum albumin can bind reversibly to a large number of endogenous and exogenous compounds65 and the changes in the emission spectra of tryptophan are common in response to a change in the protein conformation, subunit association and substrate binding or denaturation.66 As the major soluble protein constituents of the circulatory system, they contribute to colloid osmotic blood pressure and are chiefly responsible for the maintenance of blood pH.67 A useful feature of the intrinsic fluorescence of proteins is the high sensitivity of tryptophan and its local environment. Therefore, the intrinsic fluorescence of proteins can provide considerable information on their structure and dynamics often utilized in the study of protein folding and association reactions. A solution of BSA (1 μM) was titrated with various concentrations of the compounds (0–14 μM). The effect of the compounds on the fluorescence emission spectrum of BSA is shown in Fig. 6. The addition of the above compounds to the solution of BSA resulted in a significant decrease in the fluorescence intensity of BSA at 346 nm, up to 42.7%, 63.7% and 83.8% of the initial fluorescence intensity of BSA accompanied by a blue shift of 2 nm for the ligand and the Ru(II) complex, respectively. The observed blue shift is mainly due to the fact that the active site in the protein is buried in a hydrophobic environment. This result recommended a distinct interaction of the compounds with the BSA protein. As is well known, the dynamic quenching only affected the excited state of fluorophores and did not change the absorption spectrum. However, the formation of a nonfluorescence ground state complex induced a change in the absorption spectrum of the fluorophores. The UV-vis spectra of BSA in the presence of the compounds (Fig. S5†) show that the absorption intensity of BSA was enhanced as the complexes and ligand were added, and there was a little blue shift. This reveals that there exists a static interaction between BSA and the added compounds due to the formation of a ground state complex of the type BSA-compound as has been reported previously (Fig. S5†).68,69

Fig. 6 The emission spectrum of bovine serum albumin (BSA) (1 µM; λex = 280 nm, λem = 345 nm) in the presence of increasing amounts of complexes 1 (a) and 2 (b) and the ligand HL (c) (0–12 μM). The arrow shows that the emission intensity decreases upon the increase in the concentration of the compounds.


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Dalton Transactions A comparison of the interaction between the test compounds and DNA/protein

DNA binding Compounds


HL Complex 1 Complex 2 RuC51H43N3O2P210 RuC50H41N3O2P210 RuC44H33NO6P242

−1

Protein binding −1

K (M )

Kq (M )

Kapp (M )

KSV (M−1)

Kbin (M−1)

n

1.2 ± 0.06 × 104 4.1 ± 0.51 × 104 2.2135 × 105 — — 1.3 × 104

1.7 ± 0.04 × 102 1.5 ± 0.15 × 103 8.2 ± 0.28 × 103 1.4 × 104 8.9 × 103 1.2 ± 0.1 × 103

7.2 ± 0.03 × 104 2.7 ± 0.11 × 105 5.6 ± 0.28 × 105 6.5 × 105 4.2 × 105 —

2.6 ± 0.8 × 105 2.5 ± 0.32 × 106 4.3 ± 1.8 × 107 7.3 × 105 6.20 × 105 —

4.4 ± 0.25 × 103 2.02 ± 0.1 × 104 3.4 ± 0.39 × 105 1 × 105 1.02 × 104 —

0.93 0.98 1.03 0.84 0.69 —

To study the quenching process further, the fluorescence quenching data were analyzed with the Stern–Volmer equation and Scatchard equation. The quenching constant (Kq) is calculated using the plot of I0/I versus [Q]. If it is assumed that the binding of the compounds with BSA occurs at equilibrium, the equilibrium binding constant can be analyzed according to the Scatchard equation ðΔI=I 0 Þ=½Q ¼ nK KðΔI=I 0 Þ where n is the number of binding sites per albumin, K is the association binding constant calculated from the plot of (ΔI/I0)/[Q] versus ΔI/I0 (Fig. S6B†) and n is given by the ratio of the y intercept to the slope. The quenching constants (Kq = 2.50 ± 0.32 × 106 M−1, 4.36 ± 1.8 × 107 (1 and 2), 2.63 ± 0.8 × 105 M−1 (HL)) have been calculated from the plot of I0/I versus [Q] (Fig. S6A†). The binding constants (Kbin) calculated from the Scatchard plot (Fig. S6B†) were found to be 2.02 ± 0.1 × 104 M−1, 3.4 ± 0.39 × 105 M−1 and 4.4 ± 0.25 × 103 M−1, which correspond to the free ligand (HL) and the ruthenium complexes 1 and 2. From Table 3, it is clear that the constants such as Kb, Kq, Kapp and K obtained in our experiments are comparable with few other ruthenium complexes.10,42 The higher magnitude of Kbin and Kq in the case of complexes 1 and 2 compared to that of free ligand (HL) indicated a strong interaction between the BSA protein and the complexes over the ligand used in this study. Among the tested complexes 1 and 2, the latter with DMSO as a co-ligand showed a higher affinity towards protein due to a better hydrophobic interaction than the former. Synchronous spectral studies Synchronous fluorescence spectra provide information on the molecular microenvironment, particularly in the vicinity of the fluorophore functional groups.70 The fluorescence of protein is due to the presence of tyrosine and tryptophan residues. Among them, tryptophan is the most dominant fluorophore, located at the substrate binding sites. Most of the drugs bind to the protein in the active binding sites. Hence, synchronous method is usually applied to find out the conformational changes in the active binding sites of the protein that is around the tryptophan and tyrosine region. To investigate the structural changes that occurred in BSA upon the addition of our compounds, the synchronous fluorescence spectra of BSA were measured before and after the addition of test compounds to get valuable information on the molecular

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−1

microenvironment. In the synchronous fluorescence spectroscopy, according to Miller,71 the difference between the excitation and emission wavelength indicates the type of chromophores. A higher Δλ value such as 60 nm is indicative of the characteristics of the tryptophan residue while a lower Δλ value such as 15 nm is characteristic of the tyrosine residue.72 The synchronous fluorescence spectra of BSA with various concentrations of the test compounds were recorded at Δλ = 15 nm and Δλ = 60 nm and are shown in Fig. S7 and S8† respectively. In the synchronous fluorescence spectra of BSA at Δλ = 15, the addition of the compounds to the solution of BSA resulted in a small decrease in the fluorescence intensity of BSA at 302 nm, up to 32.8, 75 and 10.8% of the initial fluorescence intensity of BSA for the ligand and the complex, respectively, with no shift in their emission wavelength maxima. But, at the same time, in the case of the synchronous fluorescence spectra of BSA at Δλ = 60, the addition of the compounds to the solution of BSA resulted in a significant decrease in the fluorescence intensity of BSA at 342 nm, up to 61.7, 82.8 and 43.9% of the initial fluorescence intensity of BSA accompanied with a blue shift of 1–2 nm for the ligand and the complex, respectively. The synchronous fluorescence spectral studies clearly suggested that the fluorescence intensity of tyrosine was not affected much by the increasing concentration of the ligand but with complex 2, a significant decrease along with a blue shift of the fluorescence intensity of tryptophan has been observed. These results suggest that the interaction of the ligand and the complex with BSA affects the conformation of tryptophan only and does not have much effect on the tyrosine micro-region. Overall, the results of the BSA protein binding experiments with our compounds revealed that the binding of the compounds with BSA is mainly due to hydrophobic and electrostatic interactions. The binding strength of the Ru(II) complex with BSA is significantly higher than that of the ligand, which can be explained by the fact that the hydrophobicity of the complex is greater than that of the ligand. So, the strong interaction between the compounds and BSA suggested that these compounds can easily be stored in protein and can be released in desired target areas. Hence, it will be interesting to study the cytotoxicity properties of these compounds. Cytotoxicity Chemotherapy is a major therapeutic approach for both localized and metastasized cancers.73 The effect of the newly


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Table 4 The cytotoxic activity of the compounds

IC50 values (µM) Compound


HL Complex 1 Complex 2 Ru1C50H41N3O2P2(10) Ru1C51H43N3O2P2(10) Ru1C56H45N3O2P2(10) Cis-platin

HeLa

NIH 3T3

308 ± 0.39 31.22 ± 0.99 3.68 ± 0.99 122 109 94 35.7 ± 0.950

>100 78.1 ± 0.18 >100 >100 >100 >100 >100

synthesized ruthenium complexes were evaluated for their cytotoxicity against the human tumor cell line, which is the cervix carcinoma cell line (HeLa), and the normal cell line (NIH 3T3) by an MTT assay method. The MTT assay is a colorimetric determination of cell viability widely applied to examine in vitro cytotoxicity. The metabolic activity of the cells was assessed by their ability to cleave the tetrazolium rings of the pale yellow MTT and form a dark blue water-insoluble formazan crystal. The assay was based on the fact that only live cells reduce yellow MTT to blue formazan products. The assay, developed as an initial stage of drug screening, measures the amount of MTT reduction by mitochondrial dehydrogenase and assumes that cell viability (corresponding to the reductive activity) is proportional to the production of blue formazan that is measured spectrophotometrically. The results of the MTT assay (Table 4) show that both complexes inhibit the growth of the cells in a concentration dependent manner. A low IC50 value implies cytotoxicity at low drug concentrations. Additionally, the current study represents the first thorough correlation of potential ruthenium metal-based complexes as novel therapeutic agents for the treatment of cancer. Table 3 gives the IC50 values (the concentration of the complex required to achieve 50% cell death) of our complexes for the inhibition of cell growth. The ruthenium dimethyl sulphoxide hydrazone complex shows a better performance than the complex containing triphenyl phosphene which is analogous to our earlier report.10 However, the complexes are able to interact directly with DNA without further activation steps.

Conclusion The synthesis and structural analysis of two new Ru(II) hydrazone complexes with triphenyl phosphine or dimethyl sulphoxide as a co-ligand is presented along with studies on their binding with DNA/BSA and their cytotoxicity against tumor cell lines. An intercalative mode of binding between the octahedral ruthenium(II) complexes and CT-DNA was identified by UVvisible absorption and fluorescence emission measurements. Both the complexes cleaved supercoiled-DNA (SC) into the nicked circular (NC) form in a dose dependent manner with complex 2 displaying a higher efficiency than complex 1. From the protein binding studies, the mechanism of quenching of BSA was found to be a static one indicating that the


compounds did bind to BSA via a hydrophobic interaction, and particular complex 2 shows more affinity. In addition, the in vitro cytotoxicity assay conducted versus a cancer cell line and normal cell line demonstrated that both the complexes are active against the HeLa cancer cell line but to the normal cell line, complex 2 showed less toxicity than complex 1. The outcome of this study would be helpful to understand the mechanism of interactions of Ru(II) hydrazone complexes with serum albumin and nucleic acid and also in the development of potential probes for BSA and DNA structure and conformation or new chemotherapeutic agents.

Acknowledgements The corresponding author of the manuscript (N. D.) acknowledges the Council of Scientific and Industrial Research (CSIR), Ministry of Human Resources Development (MHRD), Government of India, New Delhi, for the financial support in the form of a major research project (CSIR Sanction letter No. 01 (2684)/12/EMR-II dated 03.10.2012) and the authorities of Bharathiar University, Coimbatore, India, for the award of University Research Fellowship (URF) to one of the authors (M. A.).

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Paper


Dalton Trans., 2014, 43, 6087–6099 | 6099

Synthesis and in vitro evaluation of new nitro-substituted thiazolyl hydrazone derivatives as anticandidal and anticancer agents.

Underestimated potential of organometallic rhenium complexes as anticancer agents.

Anticancer activity of new coumarin substituted hydrazide-hydrazone derivatives.

Bacteriocins as Potential Anticancer Agents.

Sirtuin inhibitors as anticancer agents.

Phosphorescent iridium(III)-bis-N-heterocyclic carbene complexes as mitochondria-targeted theranostic and photodynamic anticancer agents.

Carboxylate derivatives of tributyltin (IV) complexes as anticancer and antileishmanial agents.

Rhodium, iridium, and ruthenium half-sandwich picolinamide complexes as anticancer agents.

Luminescent iminophosphorane gold, palladium and platinum complexes as potential anticancer agents.

Chemistry and biology of two novel gold(I) carbene complexes as prospective anticancer agents.

Investigation of podophyllotoxin esters as potential anticancer agents: synthesis, biological studies and tubulin inhibition properties.

Metal Zn(II), Cu(II), Ni (II) complexes of ursodeoxycholic acid as putative anticancer agents.

Ruthenium(II) polypyridyl complexes as mitochondria-targeted two-photon photodynamic anticancer agents.

Validation data supporting the characterization of novel copper complexes as anticancer agents.

Benzylidine pregnenolones and their oximes as potential anticancer agents: synthesis and biological evaluation.

Lipophilicity-dependent ruthenium N-heterocyclic carbene complexes as potential anticancer agents.

Structure-activity relationship for Fe(III)-salen-like complexes as potent anticancer agents.

Essential oils and their constituents as anticancer agents: a mechanistic view.

Plant antimicrobial peptides as potential anticancer agents.

Signal Transduction Inhibitors as Promising Anticancer Agents.

Ferrocene incorporated selenoureas as anticancer agents.

Natural compounds as anticancer agents: Experimental evidence.

Synthesis and luminescence properties of salicylaldehyde isonicotinoyl hydrazone derivatives and their europium complexes.

Synthesis and biological evaluation of thiazoline derivatives as new antimicrobial and anticancer agents.