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J Biol Inorg ChemDOI 10.1007/s00775-015-1249-3

ORIGINAL PAPER

Synthesis and evaluation of new salicylaldehyde‑2‑picolinylhydrazone Schiff base compounds of Ru(II), Rh(III) and Ir(III) as in vitro antitumor, antibacterial and fluorescence imaging agents

Narasinga Rao Palepu · S. L. Nongbri · J. Richard Premkumar · Akalesh Kumar Verma · Kaushik Bhattacharjee · S. R. Joshi · Scott Forbes · Yurij Mozharivskyj · Romita Thounaojam · K. Aguan · Mohan Rao Kollipara

Received: 10 December 2014 / Accepted: 14 February 2015 © SBIC 2015

5–8 with plasmid circular DNA (pcDNA) and HeLa RNA demonstrated their fluorescence imaging property upon binding with nucleic acids. The docking study with some key enzymes associated with the propagation of cancer such as ribonucleotide reductase, thymidylate synthase, thymidylate phosphorylase and topoisomerase II revealed strong interactions between proteins and compounds 5–8. Conformational analysis by density functional theory (DFT) study has corroborated our experimental observa-tion of the N, N binding mode of ligand. Compounds 5–8 exhibited a HOMO (highest occupied molecular orbital)–LUMO (lowest unoccupied molecular orbital) energy gap 2.99–3.04 eV.Graphical Abstract Half-sandwich ruthenium, rhodium and iridium compounds were obtained by treatment of metal precursors with salicylaldehyde-2-picolinylhydra-zone (HL) by in situ metal-mediated deprotonation of the ligand. Compounds under investigation have shown potential antitumor, antibacterial and fluorescence imag-ing properties. Arene ruthenium compounds exhibited higher activity compared to that of Cp*Rh/Cp*Ir in inhibit-ing the cancer cells growth and pathogenic bacteria. At a

Abstract Reaction of salicylaldehyde-2-picolinylhy-drazone (HL) Schiff base ligand with precursor com-pounds [{(p-cymene)RuCl2}2] 1, [{(C6H6)RuCl2}2] 2, [{Cp*RhCl2}2] 3 and [{Cp*IrCl2}2] 4 yielded the corre-sponding neutral mononuclear compounds 5–8, respec-tively. The in vitro antitumor evaluation of the compounds 1–8 against Dalton’s ascites lymphoma (DL) cells by fluo-rescence-based apoptosis study and by their half-maximal inhibitory concentration (IC50) values revealed the high antitumor activity of compounds 3, 4, 5 and 6. Compounds 1–8 render comparatively lower apoptotic effect than that of cisplatin on model non-tumor cells, i.e., peripheral blood mononuclear cells (PBMC). The antibacterial evaluation of compounds 5–8 by agar well-diffusion method revealed that compound 6 is significantly effective against all the eight bacterial species considered with zone of inhibition up to 35 mm. Fluorescence imaging study of compounds

Electronic supplementary material The online version of this article (doi:10.1007/s00775-015-1249-3) contains supplementary material, which is available to authorized users.

N. R. Palepu · S. L. Nongbri · M. R. Kollipara (*) Centre for Advanced Studies in Chemistry, North Eastern Hill University, Shillong 793 022, Indiae-mail: mohanrao59@gmail.com

J. R. Premkumar Centre for Molecular Modeling, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India

A. K. Verma Department of Molecular Oncology, Cachar Cancer Hospital and Research Centre, Meherpur, Silchar, Cachar 788015, Assam, India

K. Bhattacharjee · S. R. Joshi Department of Biotechnology and Bioinformatics, Microbiology Laboratory, North Eastern Hill University, Shillong 793 022, India

S. Forbes · Y. Mozharivskyj Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4M1, Canada

R. Thounaojam · K. Aguan Department of Biotechnology and Bioinformatics, North Eastern Hill University, Shillong 793 022, India

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concentration 100 µg/mL, the apoptosis activity of arene ruthenium compounds, 5 and 6 (~30 %) is double to that of Cp*Rh/Cp*Ir compounds, 7 and 8 (~12 %). Among the four new compounds 5–8, the benzene ruthenium com-pound, i.e., compound 6 is significantly effective against the pathogenic bacteria under investigation.

Keywords Half-sandwich compounds · Apoptosis · Antibacterial · Bioimaging · DFT

AbbreviationsAO Acridine orangeDFT Density functional theoryDL Dalton’s ascites lymphomaDMSO Dimethyl sulfoxideEtBr Ethidium bromideFBS Fetal serum albuminHOMO Highest occupied molecular orbitalLUMO Lowest unoccupied molecular orbitalPBS Phosphate-buffered salinepcDNA Plasmid circular DNARPMI Roswell park memorial institute mediumTAE Tris-acetate EDTATDDFT Time-dependant density functional theory

Introduction

Cisplatin has been widely used as anti-metastatic drug for treating ovarian and testicular cancers since its discovery over 40 years ago [1]. Dose-limiting side effects and the development of resistance after repeated use in treatment

have limited the use of the platinum diammine compounds, cisplatin and carboplatin [2]. The mode of action of cispl-atin is mediated by apoptosis, which is the basic mecha-nism of classical chemotherapy, failure of which results in catastrophic health hazards [3, 4]. There is an immense

necessity for the discovery of metallodrugs capable of inducing apoptosis with less or no side effects in the host.

Compounds of ruthenium got their own prominence in the field of metallodrugs since the discovery of Ru(III) ammine complexes like [RuCl3(NH3)3] and it is increased with the inception of NAMI-A and KP1019 which are currently in clinical trials [5–8]. Organometallic arene ruthenium(II) compounds could be highly promising anti-cancer agents to overcome the disadvantages of the existing platinum drugs [9]. The less toxicity of ruthenium is attrib-uted by its ability to mimic iron in oxidation states and pos-sessing similar ligand exchange kinetics to platinum(II) compounds [10]. Organometallic Rh(III) and Ir(III) com-pounds of lapacoles and C∩N donor ligands are observed as potent anticancer agents [11, 12]. Arene ruthenium and Cp*Ir compounds of picolinamide derived ligands have been found to be active against both HT-29 and MCF-7 cell lines [13]. The ligand under study has a hydrazone Schiff base active pharmacophore unit (–CONH–N=C–) which shows good anticancer bioactivities [14].

Dalton’s lymphoma (DL) is a transplantable, poorly differentiated malignant tumor that appeared originally as lymphocytes in mouse that grows both in solid and ascitic forms [15]. The possible anticancer role of half-sandwich ruthenium, rhodium and iridium compounds against DL

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with thiazole-derived ligands has been investigated [16]. The antibacterial activity of precursor compound, benzene ruthenium dimer against Staphylococccus aureus, Escheri‑chia coli and Candida albicans has been investigated by using the disk diffusion and the pour plate methods [17]. Our efforts to find the cytotoxicity of arene metal com-pounds 5–8 against various bacteria have given positive results.

Transition metal compounds containing d6, d8 and d10 electronic configuration possess large stokes shifts, less self-quenching and high photostability. In addition, exchangeable ancillary ligands to tune emission energies, relatively long phosphorescence lifetimes, high quantum yields and good aqueous solubility of their complexes have made them to exploit as better bio-molecular and cellular probes than organic fluorophores [18, 19]. Ruthe-nium polypyridine compounds containing indole moieties act as orange red luminescent probes for binding proteins [20]. The inorganic bio-labeling, live cell imaging and in vivo tumor imaging property of rhodium and iridium com-pounds containing polypyridine ligands and cyclometalated C∩N donor ligands with d6 electronic structure have been investigated [21–26].

DFT calculations are useful to understand the biologi-cal activity of drugs by exploiting the structural and elec-tronic properties [27, 28]. Electronic properties of metal compounds and their excited state characteristics have been explored using DFT and time-dependent DFT (TDDFT) level calculations [29–32]. DFT calculations play a signifi-cant role in elucidating photochemical reactions of metal compounds [33–35]. The investigation of electronic prop-erties of cyclometalated ruthenium polypyridyl compounds and azoimidazole compounds of rhodium and iridium by DFT and TDDFT studies has been in practice for many years [36].

In the present study, the synthesis and characterization of Ru(II), Rh(III) and Ir(III) compounds with the ligand (HL) are described. All the compounds synthesized have been evaluated for their in vitro antitumor activity on Dal-ton’s ascites lymphoma (DL) cells, antibacterial activity on various pathogenic bacteria and fluorescence imaging prop-erties with plasmid circular DNA and HeLa RNA. The anti-tumor activity of precursor compounds is also evaluated. DFT/TDDFT calculations and docking analyses have been performed to pursue the structural and electronic prop-erties of the compounds and their interactions with some key enzymes, which are associated with the propagation of cancer. The poor water solubility and having side effects of the contemporary anticancer drugs have prompted us to develop new water-soluble metallodrugs with no/less side effects. The scope for the better use of the compounds under study is their aqueous solubility and having less cytotoxicity on normal cells.

Experimental section

Materials and methods

All the reactions were carried out without using any inert conditions. MCl3·3H2O (M=Ru, Rh and Ir) were pur-chased from Arora Matthey Ltd. 2-methyl picolinate (Sigma-Aldrich), hydrazine hydrate (Sd fine), salicylalde-hyde (Sisco), α-Phellandrene (Merck), 1,4-cyclohexadiene (Sigma-Aldrich), Pentamethylcyclopentadiene (Sigma-Aldrich) were used as received. All the solvents used for synthesis were dried and distilled prior to use according to the standard procedures and stored over activated molecular sieves [37]. Precursor compounds [{(p-cymene)RuCl2}2] 1, [{(C6H6)RuCl2}2] 2, [{Cp*RhCl2}2] 3 and [{Cp*IrCl2}2] 4 and the ligand salicylaldehyde-2-picolinylhydrazone (HL) were prepared according to the literature methods [38–40]. Agarose and ethidium bromide (EtBr) (10 mg/mL) were purchased from Himedia, pcDNA 3.4 and HeLa RNA were extracted using Qiagen Plasmid extraction Kit, and 6× DNA loading buffer was purchased from USB Corp. Com-position of 10× TAE buffer: pH-8, 400 mM Tris, 11.4 mL glacial acetic acid and 0.5 M EDTA. For agarose gel elec-trophoresis study, 1× TAE was used. For carrying out fluo-rescence spectral study, the ultrapure form of deoxyribonu-cleic acid from calf thymus (CT-DNA), cat no. 14375 was purchased from USB Corp. and used directly. The A.R. grade crystalline form of tris(hydroxymethyl)aminometh-ane (tris buffer) was used for making the working buffer solution as received from Himedia. For apoptosis study, inbred Swiss albino mice colony was maintained under conventional laboratory conditions at room temperature (20 ± 2 °C) with free access to food pellets (Amrut labo-ratory, New Delhi) and water ad libitum. Dalton’s ascites lymphoma (DL) was maintained in vivo in 10–12-week-old mice by serial intraperitoneal (i.p) transplantation of 1 × 107 viable cells per animal (0.25 mL phosphate-buff-ered saline (PBS), pH-7.4). Dalton’s lymphoma (DL) cells harvested from Dalton’s lymphoma-bearing mice were cultured in RPMI-1640 medium supplemented with 10 % FBS and antibiotic solution (penicillin 1000 IU and strep-tomycin 10 mg/mL) in 5 % CO2 incubator at 37 °C. The ethical evaluation of experimental protocols was reviewed and approved by the Institutional Animal Use and Care Committee of North Eastern Hill University, Shillong, India. The care, welfare and use of the animals were in accordance with the National Institutes of Health (NIH) guidelines.

Physical measurements

Elemental analysis was performed on a Perkin-Elmer-2400 CHN analyzer. Infrared (IR) spectra were recorded on a

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Perkin-Elmer 983 spectrophotometer by dispersing the compounds as KBr discs. 1H and 13C NMR spectra were recorded with Bruker Avance II 400 MHz spectrometer and their chemical shifts were reported using tetramethyl silane (TMS) as the internal standard. The analytical grade type II water used for making the buffer solution as well as quantum yield calculation was obtained from Elix 10 water purification system (Millipore India Pvt. Ltd.). Steady-state absorption spectra were recorded on a Perkin-Elmer model Lambda 25 absorption spectrophotometer. Fluorescence spectra were taken in Quanta Master (QM-40) steady-state apparatus obtained from Photon Technology International (PTI) and all the spectra were corrected for the instrument response function. Quartz cuvettes of 10 mm optical path length received from PerkinElmer, United States, (part no. B0831009) and Hellma, Germany, (type 111-QS) were used for measuring absorption and fluorescence spectra, respectively. Single crystals picked up from the samples were analyzed on a STOE IPDSII diffractometer using Mo-Kα radiation (λ = 0.71073 Å) in the whole recipro-cal sphere. A numerical absorption correction was based on the crystal shape originally determined by optical face indexing but later optimized against equivalent reflections using STOE X-shape software [41]. Crystal structures were determined and solved using the SHELX software [42]. The non-hydrogen atoms were refined anisotropically using weighted full-matrix least-square on F2. ORTEP dia-grams in Fig. 1a–c were drawn with ORTEP-3 [43] and Fig. S1a–d were drawn with MERCURY 3.5 [44]. The gel stained with compounds 5–8 was visualized under UV light using Gel Imaging system (DNR Bio-imaging system Mini Lumi).

Syntheses of compounds (5–8)

Synthesis of compound [(p-cymene)RuL(N,N)Cl] (5)

Precursor compound [{(p-cymene)RuCl2}2] 1 (50 mg, 0.08 mmol) and ligand (HL) (40 mg, 0.16 mmol) were dis-solved in acetonitrile (20 mL) and stirred. Yellow precipitate was obtained after 1 h and the reaction was continued for another 10 h to ensure the completion of the reaction. The precipitate was washed with diethyl ether (3 × 10 mL) and dried in vacuum. The compound was obtained as yellow solid. Yield: 74 %. Anal. Calcd (%) for C23H24ClN3O2Ru: C, 54.06; H, 4.73; N, 8.22. Found (%): C, 54.26; H, 4.93; N, 8.32. FTIR [KBr, cm−1]: 3433 (m) ν(O–H), 2966 (s) ν(C–

H), 1622 (vs) ν(C=O), 1600 (vs) ν(C=N). 1H NMR (400 MHz,

DMSO-d6, δ, ppm): 12.52 (s, H10, 1H), 8.83 (s, H5, 1H), 8.71 (d, H1, 1H, 2JHH = 4 Hz), 8.34 (t, H2, 1H), 8.14 (d, H4, 1H, 3JHH = 8 Hz), 8.07 (t, H3, 1H), 7.47 (d, H6, 1H, 7JHH = 8 Hz), 7.29 (t, H7, 1H), 6.94–6.89 (m, 2H, H8, H9),

6.10 (d, 2H, Ph(p-cym), JHH = 8 Hz), 5.76 (d, 2H, Ph(p-cym), JHH = 8 Hz), 2.82–2.79 (m, 1H, isopropyl(p-cym)), 2.48 (s, 3H, CH3(p-cym)), 1.16 (d, 6H, J = 4 Hz, (CH3)2(p-cym)). UV–Vis spectrum [Water, 298 K, 2 × 10−5 M] λmax, nm [Absorbance]: 300 [0.22], 380 [0.15].

Synthesis of compound [(C6H6)RuL(N,N)Cl] (6)

Compound 6 was prepared by following the similar procedure for 5 by using precursor compound [{(C6H6)RuCl2}2] 2 (50 mg, 0.09 mmol) and ligand (HL) (48 mg, 0.18 mmol). Single crystals suitable for XRD were obtained by solvent diffusion method by diffus-ing diethyl ether into a solution of acetonitrile. Yield: 65 %. Anal. Calcd (%) for C19H16ClN3O2Ru: C, 50.17; H, 3.55; N, 9.24. Found (%): C, 50.35; H, 3.73; N, 9.38. FTIR [KBr, cm−1]: 3447 (m) ν(O–H), 1622 (vs) ν(C=O), 1549 (vs) ν(C=N);

1H NMR (400 MHz, DMSO-d6, δ, ppm): 9.47 (d, H1, 1H, 2JHH = 4 Hz), 8.75 (s, H5, 1H), 8.14 (t, H2, 1H), 7.97 (d, H4, 1H, 3JHH = 8 Hz), 7.77 (d, H9, 1H, 8JHH = 4 Hz), 7.72 (t, H3, 1H), 7.29 (t, H8, 1H), 7.97–7.95 (m, H6, H7, 2H), 6.06 (s, 6H, benzene), O–H singlet was not found. 13C NMR (400 MHz, DMSO-d6, δ, ppm): 166.73 (1C, C6), 158.64 (1C, C5), 155.95 (1C, C1), 153.21 (1C, C12), 150.96 (1C, C7), 139.95 (1C, C2), 131.69 (1C, C4), 131.34 (1C, C9), 127.89 (1C, C3), 125.34 (1C, C10), 119.31 (1C, C11), 116.67 (1C, C13), 85.92 (6C benzene). UV–Vis spectrum [Water, 298 K, 2 × 10−5 M] λmax, nm [Absorbance]: 322 [0.25], 380 [0.12].

Synthesis of compound [Cp*RhL(N,N)Cl] (7)

Precursor compound [{Cp*RhCl2}2] 3 (50 mg, 0.08 mmol) and ligand (HL) (40 mg, 0.16 mmol) were stirred in meth-anol (20 mL) for 10 h. The solvent was evaporated and the residue was dissolved in dichloromethane and filtered through Celite. The volume was reduced to 1 mL and pre-cipitated and washed with diethyl ether (3 × 10 mL) and dried in vacuum. The compound was obtained as an orange solid. Single crystals suitable for XRD were obtained by solvent diffusion method by diffusing hexane into a satu-rated solution of chloroform. Yield: 81 %. Anal. Calcd (%) for C23H25ClN3O2Rh: C, 54.76; H, 4.90; N, 8.18. Found (%): C, 54.95; H, 5.05; N, 8.30. FT IR [KBr, cm−1]: 3414 (m) ν(O–H), 2965 (m) ν(C–H), 1619 (vs) ν(C=O), 1598 (vs) ν(C=N);

1H NMR (400 MHz, DMSO-d6, δ, ppm): 12.52 (s, H10, 1H), 8.86 (d, H1, 1H, 2JHH = 4 Hz), 8.71 (d, H4, 1H, 3JHH = 4 Hz), 8.54 (s, H5, 1H), 8.19 (t, H2, 1H), 8.07–8.01 (m, H3, 1H), 7.79–7.76 (m, H6, 1H), 7.46 (dd, H9, 1H), 7.32–7.25 (m, H7, 1H), 6.93 (t, H8, 1H), 1.60 (s, 15H, Cp*). UV–Vis spectrum [Water, 298 K, 2 × 10−5 M] λmax, nm [Absorbance]: 325 [0.26], 380 [0.15].

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Fig. 1 a Molecular structure of compound 6 with atom number-ing scheme. Thermal ellipsoids are depicted with 50 % prob-ability level. Solvent molecule and hydrogen atoms are omitted for clarity except on N3 and O2. b Molecular structure of com-pound 7 with atom numbering scheme. Thermal ellipsoids are depicted with 50 % probability level. Hydrogen atoms are omit-ted for clarity except on O2. c Molecular structure of com-pound 8 with atom numbering scheme. Thermal ellipsoids are depicted with 50 % probability level. Hydrogen atoms are omit-ted for clarity except on O2

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Synthesis of compound [Cp*IrL(N,N)Cl] (8)

Compound 8 was prepared by following the same pro-cedure for compound 7 by using precursor compound [{Cp*IrCl2}2] 4 (50 mg, 0.06 mmol) and ligand (HL) (30 mg, 0.12 mmol). The compound was obtained as yel-low solid. Single crystals suitable for XRD were obtained by solvent diffusion method by diffusing hexane into a saturated solution of acetone. Yield: 65 %. Anal. Calcd (%) for C23H25ClN3O2Ir: C, 45.80; H, 4.18; N, 6.97. Found (%): C, 45.95; H, 4.30; N, 7.15. FTIR [KBr, cm−1]: 3434 (m) ν(O–H), 2922 (m) ν(C–H), 1631 (vs) ν(C=O), 1600 (vs) ν(C=N);

1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.81 (d, H1, 1H, 2JHH = 4 Hz), 8.53 (s, H5, 1H), 8.16 (t, H2, 1H), 8.02 (d, H4, 1H, 3JHH = 8 Hz), 7.74 (t, H3, 1H), 7.39 (d, H6, 1H, 7JHH = 4 Hz), 7.27 (t, H7, 1H), 6.95–6.92 (m, 2H), 1.58 (s, 15H, Cp*), O–H singlet was not found. 13C NMR (400 MHz, CDCl3, δ, ppm): 166.59 (1C, C6), 159.43 (1C, C5), 155.51 (1C, C1), 154.07 (1C, C12), 150.15 (1C, C7), 139.15 (1C, C2), 130.97 (1C, C4), 130.66 (1C, C9), 127.68 (1C, C3), 126.01 (1C, C10), 118.90 (1C, C11), 117.03 (1C, C13), 87.43 (5C, Cp*), 9.22 (5C, CH3, Cp*). UV–Vis spec-trum [Water, 298 K, 2 × 10−5 M] λmax, nm [Absorbance]: 325 [0.18], 375 [0.11].

UV–visible and fluorescence spectral studies

UV–visible and fluorescence spectral studies were car-ried out to study the absorption and fluorescence prop-erties. UV–visible spectra of compounds 5–8 were recorded in water at concentration 20 μM. Fluorescence spectra of compounds 5–8 were recorded in water at con-centration 20 μM for calculating fluorescence quantum yield. To study the effect of CT-DNA on the fluorescence of compounds 5–8 the experiments were carried out in 0.05 M tris buffer of physiological condition of pH-7.4. The compounds were taken at very low concentration (20 μM) to avoid any aggregation and kept constant dur-ing spectral measurements. Fluorescence quantum yields (ϕf) were calculated in water by comparing the total fluo-rescence intensity under the whole fluorescence spectral range with that of a standard (quinine bisulfate in 0.5 M H2SO4 solution, ϕs

f = 0.546) [45] with the following equation using adequate correction for water refractive index (n) [46].

where, Ai and As are the optical density of the sample and standard, respectively, and ni is the refractive index of water at 293 K. The relative experimental error of the measured quantum yield was estimated within ±10 %.

(1)φif = φs

f ×

Fi

Fs×

(1 − 10−Ai)

(1 − 10−As)

×

(

ni

ns

)2

,

For fluorescence emission, the samples were excited at the high-energy absorption band for the correspond-ing compounds. For collecting the fluorescence emission, 3.0/3.0 and 1.5/0.5, band pass was used in the excitation and emission sides, respectively. All the measured spectra were subtracted off from the corresponding sample blanks measured under identical conditions. The corresponding steady-state data obtained were analyzed using Origin 6.0 (Microcal Software, Inc.,).

Fluorescence-based apoptosis study

Apoptosis study in DL cells

Fluorescence-based apoptotic cell death was determined in DL cells after treatment with precursor compounds 1–4 and new synthesized compounds 5–8 under in vitro condi-tions by using acridine orange and ethidium bromide (AO/EtBr) staining method as described earlier [47]. In this method, the apoptotic index and cell membrane integrity can be assessed simultaneously and there is no cell fixa-tion step, thus avoiding a number of potential artifacts. In brief, for in vitro treatment, five different doses (20, 40, 60, 80 and 100 μg/mL) of compounds 1–8 were selected and treatment was given for 12 h in RPMI-1640 medium. After 12 h, the DL cells were thoroughly washed in PBS fol-lowed by staining with AO/EtBr and examined under fluo-rescence microscope and photographed. For counting per-cent of apoptotic cells, twenty microscopic fields (~1000 cells) were randomly selected per slide and the apoptotic nuclei (red/orange) were counted with 40× magnification using the following formula.

Both viable and apoptotic cells took up acridine orange. They showed green fluorescence when bound to double-strand DNA in viable cells and red/orange when bound to single-strand DNA where the latter case appeared predomi-nantly in apoptotic cells.

Apoptosis study in normal cells: peripheral blood mononuclear cells (PBMC)

Apoptosis-inducing effect of compounds 1–8 was exam-ined on the normal PBMC cells. PBMC cells were col-lected according to the method mentioned by Takayama et al. [48]. In brief, fresh blood was collected from healthy normal mice (N = 5) in a heparinised syringe and was transferred into a glass tube. Pure lymphocytes were iso-lated from blood by the Ficoll density gradient technique.

Percentage of apoptotic cells

=

Total number of red/orange nuclei

Total number of green and red/orange nuclei× 100.

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The tested dose for each compound was 100 μg/mL and for 12 h incubation. Apoptosis study was performed using AO/EtBr staining method as mentioned above.

Antibacterial activity

Antibacterial activity of the compounds 5–8 was deter-mined against eight different bacterial representatives that include three Gram-positive bacteria (Staphylococ‑cus aureus MTCC 96, Staphylococcus epidermidis MTCC 3615, Proteus mirabilis MTCC 425) and five Gram-nega-tive bacteria (Escherichia coli MTCC 739, Pseudomonas aeruginosa MTCC 2453, Vibrio parahaemolyticus MTCC451, Klebsiella pneumoniae MTCC 2653 and Pro‑teus vulgaris MTCC 426) according to the agar well-diffu-sion method [49].

The stock solutions for all compounds were prepared freshly in Milli-Q water (0.5 mg/5 mL) just before use. The different concentrations of samples were prepared by the addition of aliquots of the stock solutions to Milli-Q water. All the test organisms were inoculated in Mueller–Hinton broth (pH-7.4) and incubated with agitation for 24 h at 37 °C. All the test organisms were seeded on Mueller–Hinton agar plates by using sterilized cotton swabs after maintaining the optical density of the culture suspension at 0.5 McFarland Nephelometer standards. Agar surface was bored by using sterilized cork borer to make wells (6 mm diameter) and filled with 50 µL of the test sample solutions into separate wells. The standard antibiotic amoxicillin as positive control and Milli-Q water as negative control were also maintained. Plates were incubated at 37 °C for 24 h and inhibition zones formed around the wells were meas-ured (mm) using a Vernier caliper scale. Triplicate plates were maintained for each organism and the results were recorded as the mean diameter of the zones of inhibition.

Fluorescence bioimaging of DNA and RNA by agarose gel electrophoresis

The stock solutions of compounds 5–7 were made in deion-ised water and compound 8 was made in DMSO and water (3:1) to a concentration 1 mg/mL. Different dilutions of the compounds 5–8 were made in distilled water. For DNA imaging experiment, about 0.3 µg of plasmid circular DNA was mixed with 1 µL of diluted compounds (1 µg/µL) and made up to 10 µL volume with distilled water. The mix-tures were kept at room temperature for 10 min before loading. Samples were loaded with 2 µL gel loading dye in 0.8 % agarose gel. The gel was run at 100 V for 50 min. HeLa RNA was used for RNA imaging experiment. The experimental procedure for RNA imaging is similar to that of DNA.

Computational details

DFT calculations

The structures of 5–8 and the various conformations of the ligand (HL) were optimized at B3LYP level [50–52] using the mixed basis set, i.e., 6–31G (d) for nonmetals and LANL2DZ for metal atoms. The absence of imaginary fre-quencies ensured that the structures considered in this study were minima on the potential energy surface (PES). Time-dependent density functional theory (TDDFT) calculations of the compounds 5–8 were carried out to understand the low-lying electronic transitions. Singlet and triplet excita-tion energies have been obtained with the TDDFT based on S0 geometries. All the computations were performed with the help of Gaussian 09 program package [53]. Gauss Sum 2.2 [54] was used for UV–Vis spectral analysis, oscillator strengths, transitions between various states and the frac-tional contributions of various groups to each molecular orbital.

Molecular docking

Ligand docking simulations were performed using Mole-gro Virtual Docker software (MVD—2010, 4.0) for Win-dows, which is based on a new heuristic search algorithm that combines differential evolution with a cavity predic-tion algorithm [55, 56]. The binding sites were automati-cally detected by software itself. The search space of the simulation exploited in the docking studies was defined as a subset region of 15 Å around the active site cleft. Ten runs were performed and five poses returned. The best interactions were selected based on the lowest interac-tion energy. The three-dimensional coordinates of key enzymes (associated with cancer progression) such as ribonucleotide reductase (PDB ID: 4R1R), thymidylate synthase (PDB ID: 2G8D), thymidylate phosphorylase (PDB ID: 1UOU) and topoisomerase II (PDB ID: 1QZR) were obtained from the Protein Data Bank (PDB) at the Research Collaboratory for Structural Bioinformatics (RCSB). The PM3 structures of the proteins were taken for the docking studies.

Statistical analysis

In the apoptosis study, the statistical results were expressed as the mean ± standard deviation obtained from triplicates of each independent experiment. To determine the effect of different drugs and the concen-tration of the compounds the one-way ANOVA test was performed. A probability of *p ≤ 0.05 was considered significant.

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Results and discussion

Synthesis

Reactions of the dinuclear precursor compounds 1–4 with the ligand salicylaldehyde-2-picolinylhydrazone (HL) have yielded the neutral and mononuclear compounds 5–8 (Scheme 1). These compounds are non-hygroscopic and sta-ble in air as well as in solution. Compounds 5, 6 and 8 are yellow in color while compound 7 is orange. Compounds 5, 7 and 8 are very soluble (1 mg/mL) in polar solvents like dichloromethane, chloroform, acetone and acetonitrile. Compound 6 is insoluble in dichloromethane and chloro-form but very soluble in acetone and acetonitrile. In water, compounds 5, 7 and 8 are very soluble and compound 8 is soluble (1–10 mg/mL). All the compounds 5–8 are insoluble in non-polar solvents like hexane, diethyl ether and petro-leum ether. The compounds were obtained by deprotonation of N–H proton of the ligand by metal (Ru/Rh/Ir). In these compounds, metal binds to two nitrogen atoms in chelating N, N mode by removing proton from one of the two nitro-gen atoms. In case of ruthenium compounds, deprotonation has taken place in acetonitrile medium where as in rho-dium and iridium compounds it has happened in methanol. In general, ruthenium, rhodium and iridium follow similar chemistry but they differ little in the case of deprotonation of N–H proton. When the reaction is carried out in methanol for ruthenium compounds, the product is decomposed.

Characterization by spectral studies

The infrared spectra of free ligand exhibit carbonyl stretch-ing frequency at 1679 cm−1 where as in the compounds 5–8 this band has shifted to 1622, 1622, 1619 and 1631 cm−1, respectively. The decrease in carbonyl stretching frequency from ligand to metal compounds is probably due to back donation by metal. The O–H stretching frequency in com-pounds 5–8 is found at 3433, 3447, 3414 and 3434 cm−1, respectively. The stretching frequencies at 2966, 2965 and 2922 cm−1 correspond to methyl groups of p-cymene in compound 5, Cp* of compounds 7 and 8, respectively.

The 1H NMR spectra of compounds 5–8 exhibit a down-field shift in protons of ligand after forming complexes, which is because of the deshielding effect exerted by metal on the ligand. Compounds 5 and 7 show a singlet at δ 12.52 corresponding to O–H proton of ligand. In compounds 6 and 8, the O–H singlet is not found in 1H NMR but the crystal structures have confirmed the presence of the O–H proton. The 1H NMR spectrum of compound 5 displays a doublet at δ 1.16 for methyl protons of isopropyl group, a singlet at δ 2.48 for methyl group and a septet at δ 2.82 for one proton of isopropyl group and two doublets at δ 5.76 and δ 6.10 due to aromatic protons of p-cymene. The 1H NMR spectrum of compound 6 shows a singlet of six pro-tons at δ 6.06 corresponding to benzene ring. In compounds 7 and 8, methyl groups of Cp* ligand exhibit a singlet at δ 1.60 and δ 1.58, respectively. In compound 5, two doublets

Scheme 1 The schematic representation of the synthesis of compounds 5–8 with ligand HL in binding mode 1

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at δ 8.71 and δ 8.14 and two triplets at δ 8.34 and δ 8.07 correspond to pyridine ring and one doublet at δ 7.47, one triplet at δ 7.29 and a multiplet of two protons at δ 6.94 cor-respond to phenyl ring. In compound 6, two doublets at δ 9.47 and δ 7.97, two triplets at δ 8.14 and δ 7.72 correspond to pyridine ring and one doublet at δ 7.77, one triplet at δ 7.29 and a multiplet of two protons at δ 7.97 correspond to four protons of phenyl ring of the ligand. In compound 7, two doublets at δ 8.86 and δ 8.71, one triplet and one multiplet at δ 8.19 and δ 8.07 correspond to pyridine ring and two multiplets at δ 7.79 and δ 7.32, a doublet of dou-blet at δ 7.46 and one triplet at δ 6.93 correspond to four protons of the phenyl ring. In compound 8, two doublets at δ 8.81 and δ 8.02, two triplets at δ 8.16 and δ 7.74 corre-spond to pyridine ring and one doublet at δ 7.39, one triplet at δ 7.27 and one multiplet of two protons at δ 6.95 cor-respond to phenyl ring of the ligand. The proton NMR data indicate the formation of compounds 5–8. All these com-pounds formed after deprotonation of hydrogen on N2 (see Fig. 1a–c). Absence of NH proton peak in NMR spectra supports the deprotonation and formation of neutral com-pounds. The 1H NMR spectra and 13C NMR spectra of two representative compounds 6 and 8 are given in Figs. S2–S5. These compounds exhibit single resonances at δ 85.92 for benzene in compound 6, δ 9.22 for methyl carbon of Cp* and δ 87.43 for Cp* ring carbons in compound 8. Carbonyl carbon resonates at δ 166.73 in compound 6 and δ 166.59 in compound 8. Olefin carbon resonates at δ 150.96 and δ 150.15 in compounds 6 and 8, respectively.

Single crystal X‑ray study

The molecular structures of compounds 6, 7 and 8 are determined by X-ray structural analysis and their crystallo-graphic details are summarized in Table S1. Selected bond lengths and bond angles are summarized in Table 1. These compounds adopt the piano-stool geometry that is com-mon for ruthenium, rhodium and iridium half-sandwich compounds [57, 58]. The metal center is coordinated to the arene and pentamethylcyclopentadienyl ligand, which occupies one face of the octahedron, a terminal chloride and the chelating ligand. The metal atom has an octahe-dral arrangement with two cis nitrogen atoms (N1, N2) of ligand in bidentate chelating fashion. The aromatic ring occupies three coordination sites in these compounds to complete the octahedral geometry around the metal center.

Compound 6 is triclinic with P-1 space group and compounds 7 and 8 are orthorhombic with Pna21 and Pbca space groups, respectively. In compounds 6, 7 and 8, Ru-bencentroid, Cp*Rhcentroid and Cp*Ircentroid distances are 1.682, 1.788 and 1.798 Å, respectively. In compound 6, the bond angle at N(1)–Ru(1)–Cl(1) is 87.82(14), N(2)–Ru(1)–Cl(1) is 85.04(13) and N(1)–Ru(1)–N(2) is

75.41(17) which indicates a slight distortion from the octa-hedral bond angle which is because of the strain existing around the metal with ligand. The bond lengths of Ru(1)–N(1), Ru(1)–N(2) and Ru(1)–Cl(1) are 2.108(4), 2.100(4) and 2.4024(17) Å, respectively. In compound 7, the bond angle at N(1)–Rh(1)–Cl(1) is 89.66(11), N(2)–Rh(1)–Cl(1) is 90.62(13) and N(1)–Rh(1)–N(2) is 75.07(17) which indicates a slight distortion from the octahedral bond angle which is because of the strain existing around the metal with ligand. The bond lengths of Rh(1)–N(1), Rh(1)–N(2) and Rh(1)–Cl(1) are 2.132(4), 2.084(4) and 2.386(16) Å, respectively. In compound 8, Ir(1)–N(1) and Ir(1)–N(2) distances are 2.089(3) and 2.094(3) Å, respec-tively. The distance between Ir(1)–Cl(1) is 2.403(11) Å which is longer than Rh(1)–Cl(1). The bond angles around the metal viz., N(1)–Ir(1)–N(2) is 75.71(14), N(1)–Ir(1)–Cl(1) is 85.77(9) and N(2)–Ir(1)–Cl(1) is 86.56(9) show a deviation from octahedral geometry. The crystal structures of compounds 6, 7 and 8 show that the intramolecular hydrogen bond is found between H(2) and Cl(2) in com-pound 6 and H(2) and N(3) in compounds 7 and 8 with a distance of 2.047, 1.880 and 1.863 Å, respectively (Fig. S1a–c). In compound 6, the bond length of C(12)–O(1) is 1.236(6) in compounds 7 and 8 the bond lengths of C(16)–O(1) are 1.223(6) and 1.232(5) Å, respectively, which correspond to C=O group and supporting the existence

Table 1 Selected bond lengths (Å) and bond angles (°) in com-pounds 6, 7 and 8

*M(1) = Ru in compound 6, Rh in compound 7 and Ir in compound 8

Parameters Compound 6 Compound 7 Compound 8

Interatomic distances (Å)

M(1)–N(1) 2.108 (4) 2.132 (4) 2.089 (3)

M(1)–N(2) 2.100 (4) 2.084 (4) 2.094 (3)

M(1)–Cl(1) 2.4024 (17) 2.3866 (16) 2.4037 (11)

M(1)–Cp*/arene (centroid)

1.682 1.788 1.798

N(1)–C(11) 1.354 (6) 1.331 (7) 1.337 (5)

N(1)–C(15) – 1.343 (6) 1.486 (6)

C(15)–C(16) – 1.493 (7) 1.486 (6)

N(2)–C(16) – 1.346 (6) 1.338 (5)

N(2)–N(3) 1.370 (5) 1.390 (5) 1.378 (4)

N(2)–C(12) 1.339 (6) – –

C(12)–O(1) 1.236 (6) – –

C(16)–O(1) 1.223 (6) 1.232 (5)

Bond angles (°)

N(1)–M(1)–N(2) 75.41 (17) 75.07 (17) 75.71 (14)

N(1)–M(1)–Cl(1) 87.82 (14) 89.66 (11) 85.77 (9)

N(2)–M(1)–Cl(1) 85.04 (13) 90.62 (13) 86.56 (9)

C(11)–C(12)–N(2) 112.4 (5) – –

C(15)–C(16)–N(2) – 110.4 (4) 111.5 (4)

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of compounds in keto form. Compound 6 has crystallized with one acetonitrile solvent molecule. One chloride Cl(2) is also found in crystal structure forming hydrogen bond with H(2). The protonation on N(3) results in N(3)H(3) ionic species which is balanced by Cl(2) subsequently making the compound neutral. This chloride has no con-tribution in balancing charge of the ruthenium metal. The crystal packing of compounds 7 and 8 shows a serpentine network in view along c-axis (Fig. S1d). The crystal struc-tures of compounds 6, 7 and 8 have supported the predic-tion of the anionic nature of the ligand. The ligand (HL) has become anionic after removal of the N–H proton while forming compounds 5–8. This is clearly corroborated by their crystal structures. In compounds 5 and 6, the Ru metal is in +2 oxidation state which is balanced by one chloride and one anionic nitrogen atom. In compounds 7 and 8, the Rh and Ir metals are in +3 oxidation state which is balanced by one chloride, Cp* and one anionic nitrogen atom of the ligand. The metal chloride bond lengths, the C–C bond lengths within the Cp* ring C–Me distances and metal to centroid distances are usual and are consistent with the values reported previously [59, 60].

UV–visible and fluorescence spectral studies

In aqueous medium, the electronic spectra of the com-pounds 5–8 display a single band at ~300–330 nm. The band at this wavelength is attributed to intra ligand π–π* transitions. The medium intensity bands of these com-pounds at 380–400 nm are assignable to n–π* transitions due to the presence of carbonyl group in the ligand. Com-pounds 5–8 have exhibited fluorescence after exciting at high energy bands at wavelengths 300, 322, 325 and 325 nm, respectively (Fig. 2). All the compounds show emission at ~412 nm. The quantum yield calculations have revealed that compound 5 shows the highest quantum yield which is 1.65 times to that of compound 6 and 4.5 times to that of compound 7 and 13 times to that of compound 8 (Table 2).

The change in fluorescence after binding with DNA has been observed in compounds 5–8. In compounds 5–7, a significant change is observed. The binding of compounds 5–7 to DNA has brought significant increase in fluorescence with increased intensity (Fig. 3). This has clearly supported the binding of DNA and consequent exhibition of fluores-cence. This has corroborated our observation of fluores-cence when the compounds bound on gel run with DNA.

Apoptosis analysis in DL and normal PBMC cells

The nuclei of control DL cells are round in shape with uni-form green fluorescence without any membrane deformities prior to treatment with compounds under study. Compounds 1–8 have exhibited relatively lower in vitro cytotoxicity than cisplatin against the DL cell lines. After treatment at lower dose 20 and 40 μg/mL with compounds 3, 4, 5 and 6, membrane folding, blebbing and chromatin condensation are observed. At higher doses 60, 80 and 100 μg/mL with the compounds 3, 4, 5 and 6, cell membrane abnormality with fragmented nucleus, nucleolus pyknosis and cytoplas-mic vacuoles are noticed (Fig. 4). Treated cells displayed a phenomenon of early apoptosis as shown by the emitted red fluorescence in more or less all cases. At higher doses 60, 80 and 100 μg/mL, compounds 1, 2, 7 and 8 have shown moderate apoptotic effect (Fig. 5). After treatment with com-pounds 1–8, apoptotic cell death in normal PBMC cells is found to be lesser as compared to that of cisplatin (reference drug) (Fig. 6). At a concentration of 100 μg/mL, compounds

Fig. 2 a Steady-state absorp-tion and b fluorescence emis-sion spectra for the compounds 5–8 in water

Table 2 Summary of steady-state absorption and fluorescence spec-tra of compounds 5–8 in aqueous medium

Sl. no. Compound λabs/nm λem/nm ϕf/10−2

1 5 300 412 2.82

2 6 322 412 1.70

3 7 325 412 0.63

4 8 325 412 0.22

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5 and 6, which demonstrate high cytotoxicity showed 2 and 7 % apoptotic cell death, respectively, with PBMC cells. Whereas their activity against DL cells is 30 and 28 %, respectively, which clearly demonstrate their less toxicity on normal cells. The IC50 values for compounds 1–8 are calcu-lated using dose–response curve. The IC50 values of com-pounds 1–8 are found as 0.74, 0.83, 0.42, 0.24, 0.33, 0.42, 0.88 and 0.69 mM, respectively. The IC50 value of cisplatin is found to be 0.29 mM. This has clearly demonstrated and supported the high cytotoxicity of compounds 3, 4, 5 and 6.

Antibacterial activity

The antibacterial potential of the four metal compounds 5–8 is evaluated according to their zone of inhibition against var-ious test organisms. The results in terms of zone of inhibition are compared with the activity of the standards. There are no zones of inhibition obtained around the negative control wells on agar. Whereas, in the wells containing compounds under investigation 5, 6 and 7 zones of inhibition are found. In case of compound 5, zone of inhibition is observed at con-centrations 20 and 30 µg/mL for S. epidermidis. The results have revealed that only compound 6 is potent antibacterial against all the bacteria studied. It has exhibited zone of inhi-bitions up to 35 mm against all the bacterial species consid-ered. In case of compound 7, zone of inhibition is observed

only for S. epidermidis at a concentration 30 µg/mL. Com-pound 8 has not exhibited cytotoxicity with any bacterial species. A direct correlation is found between the concen-tration of compound and size of inhibitory zones since the diameter of inhibitory zones increased with increase of con-centration of compounds (Fig. 7). The inhibitory actions of these compounds vary between different species of the same genera. This may be the reason for different inhibitory activ-ity against S. aureus and S. epidermis. Surprisingly, only benzene ruthenium compound, i.e., compound 6 is signifi-cantly antagonistic against all the bacteria. The nature of the arene ring also plays a crucial in the cytotoxicity as described by Sadler et al. [61]. We predict that benzene ring is highly responsible for its activity against bacterial species. An in-depth understanding of the mechanism of action can further help in understanding this and which is under way.

Fluorescence bioimaging of DNA and RNA by agarose gel electrophoresis

Visualization of DNA/RNA under UV light is normally not feasible in the absence of detecting dyes such as ethidium bromide or SYBR Green I [62]. The dye intercalates with the DNA/RNA strands and shifts the higher frequency UV light to lower frequency visible light such as red and green, respectively, and thereby allowing us to visualize them on

Fig. 3 Fluorescence emission spectra in buffer with CT-DNA and without CT-DNA for com-pound 5 (a) compound 6 (b) and compound 7 (c)

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the agarose gel. The DNA samples which are treated with compounds 5–8 were clearly visible upon UV illumination and the color is appeared to be reddish orange. This dem-onstrates that the compounds 5–8 bind to DNA and their emission spectra are within the visible region. Compound 5 is found to have the highest binding affinity to DNA as it shows best fluorescence and visibility of the DNA band in the same concentration range that were used for other compounds (Fig. 8a). This has been further supported by the fluorescence spectra of compounds 5–8.

In case of RNA imaging with compounds 5–8, all com-pounds have shown appreciable fluorescence upon binding with RNA. The binding of compounds 6 and 8 seems to be comparatively higher than that of compounds 5 and 7 based on the intensity of fluorescence appeared on the gel (Fig. 8b).

In case of compounds 5–8, the visibility of the bands has not changed in all the dilutions that we have used, i.e., 1/20, 1/10, 1/5, 1/2 and 1 μg. In case of compound 5, although only these dilutions are shown in the picture (Fig. 8a), the visibility has not changed much in dilutions 1/100 and 1/500 μg that are tested. In general, after washing for sev-eral times in the buffer, the intercalated ethidium bromide detaches from the DNA and fluorescence decreases conse-quently subsiding their visibility of DNA on the gel. How-ever, in case of compounds 5–8 which bound to DNA the visibility remained the same even after overnight washing. This suggests the strong binding of the compounds 5–8 to DNA. We anticipate that some strong interactions are exist-ing other than intercalation between compounds 5–8 and DNA. We are investigating for the possibility of ligand

Fig. 4 Morphological features of apoptotic and viable Dalton’s lym-phoma (DL) cells stained with acridine orange and ethidium bromide. a Control DL cells are green and more or less rounded in shape indi-cating viable cells. b DL cells treated with cisplatin for 12 h showed many apoptotic features with membrane blebbing and fragmented

nuclei. Compounds 1–8 (c–j) showed moderate to high apoptotic fea-tures, which include membrane blebbing, swelling, chromatin con-densation and cell membrane abnormality with fragmented nuclei. Green nuclei are viable cells and red/orange nuclei indicate apoptotic cells. Scale bar 50 µm

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exchange of chloride by aquation and consequent binding to any of the nitrogen bases of the DNA similar to cisplatin.

Theoretical studies

Docking analysis

The observed apoptotic cell death after treatment with different compounds 1–8 in DL cells has prompted us to

perform molecular docking study to understand the pos-sible interactions between proteins and compounds under study. Four protein structures, ribonucleotide reductase, thymidylate synthase, thymidylate phosphorylase and topoisomerase II, that are associated with cancer pro-gression have been considered. The mechanistic chemis-try of these key enzymes is highly expressed and closely involved at various stages of cell multiplication and hence responsible for the propagation of cancer. Moreover, their inhibition by various chemotherapeutic agents is associ-ated with the inhibition of cancer growth and invasion. Therefore, in the present study, these enzymes are used for molecular docking to investigate the possible binding of newly synthesized compounds [63, 64]. The docking studies have revealed that all the compounds 1–8 interact with enzymes at various sites and their hydrogen bonding interactions are given in Table 3. Molecular docking study has shown that compound 5 could dock with topoisomer-ase II and show four hydrogen bond interactions with four different amino acids viz., Tyr-144B, Tyr-128A, Thr-27A, Thr-27B in the piperazinedione (CDX)-binding site. Com-pound 5 exhibits strong interaction with ribonucleotide reductase and renders six hydrogen bonds with Ser-625, Thr-209, Glu-441 and Cys-225. Compound 6 has been masked inside the active site of thymidylate synthase by four hydrogen bonds with Asn-229, Glu-60 and Ser-219. Compound 7 shows six hydrogen bond interactions with thymidine phosphorylase by Thr-154, Lys-115, Ser-117, Ser-144, Gly-145 in CMU 5-Chloro-6-(1-(2-Iminopyr-rolidinyl)) methyl-binding site. Compound 8 also shows appreciable interactions with five different amino acids viz., Thr-154, Lys-115, Ser-117, Ser-144 and Gly-145 in the active site of thymidine phosphorylase (Fig. 9a–d; Table 3).

Density functional theory (DFT) calculations

The ligand (HL) is a multi-coordinating species with N–N linkage connecting two coordination sites. Four possible binding modes have been considered viz., mode 1 and mode 2 as N, N donor-forming five-membered rings, mode 3 and mode 4 as N, O donor-forming five-membered rings (Fig. 10a). The mononuclear compounds are isolated in the bonding mode 1 of the ligand where ligand exists in conformation 1. The compound 7, which is experimentally isolated with only one of the various conformations, is subjected to theoretical investigation using DFT methods. It is found that the least energetic and the most stable conformer C1 (in mode 1) of the ligand is forming compounds. The ligand is flexible and has nitrogen and oxygen donor atoms, which can bind to metal through (N, N) and (N, O) fashion forming five- or six-membered rings. Arene metal compounds with (N, O)

Fig. 5 Quantitative analysis of apoptotic cell death, a time-dependent increase in apoptotic nuclei was observed after treatment with syn-thetic compound 1–8. Compounds 3, 4, 5 and 6 showed higher apop-totic effect in DL cells. Cisplatin is used as positive reference drug. Results are expressed as mean ± SD, n = 3

Fig. 6 Apoptotic cell death in normal peripheral blood mononuclear cells (PBMC) of normal mice in response to eight compounds with 12 h incubation period. Results are expressed as mean ± SD, n = 3, one-way ANOVA, *p ≤ 0.05 as compared with cisplatin

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donor ligands like beta diketones forming five-membered rings have been reported [65]. Keeping it in view, we have expected the ligands to form compounds in (N, N) and (N, O) modes of bonding, but in compounds 5–8 the ligand binds only through the two nitrogen atoms in (N, N) mode. We have carried out DFT study to explore the reason for the preferential mode of bonding taking com-pound 7 under consideration.

Structural features

Four possible conformations of the ligand (HL), confor-mations C1–C4 that represent the four binding modes are considered (Fig. 10b). In C1, the ligand possesses hydrogen bonding at two sites, between H5 and N2 (O–H…N) at a distance of 1.803 Å and between H11 and N1 (N–H…N) at a distance of 2.191 Å forming a six- and five-membered rings, respectively. This has been observed as the most stable conformation among others. C2 possesses only one H-bond between H9 and N1 (N–H…N) with a distance of 2.174 Å and relative energy of this conformation is found as 11.15 kcal/mol. C3 possesses no hydrogen bond and its rela-tive energy is 21.68 kcal/mol. C4 exhibits one hydro-gen bond between H11 and N1 (O–H…N) with a dis-tance of 1.811 Å and its relative energy is found to be 10.66 kcal/mol.

Based on the conformational analysis, we have justi-fied our experimental data of formation of compound 7 with ligand in (N, N) mode of bonding [Cp*RhL(N,N)Cl)]

forming five-membered ring in C1 because of its highest stability.

Electronic properties

In all the new compounds 5–8, the majority of HOMO is present on ‘imp’ (imp: iminomethyl phenol part of the ligand) viz., 44 % in compound 5, 47 % in compound 6 and compound 7 and 43 % in compound 8. Majority of LUMO is present on ‘Pa’ (picolinamide part of the ligand) viz., 82 % in compound 5, 75 % in compound 6, 90 % in compound 7 and 88 % in compound 8. HOMO and LUMO energy gaps in compounds 5–8 are found as 3.04, 3.02, 2.98 and 2.99 eV, respectively (Table 4). In compounds 5–8 HOMO–HOMO−1 energy gaps are found as 0.72, 0.71, 0.72 and 0.72 eV, respectively (Table 4). The energy differ-ence between singlet and triplet (ΔEST) reflects the amount of the MLCT character of the corresponding wave func-tions. Singlet and triplet energy difference in compounds 5–8 are 0.36 eV, 0.34 eV, 0.30 eV and 0.50 eV, respectively (Table 4). In compound 5, the major electronic transition is HOMO to LUMO+1 with 47 % in triplet (T1) state with LMCT and LLCT character. In compound 6, the major electronic transition is HOMO–LUMO+1 with 40 % in singlet (S1) state with MLCT and LLCT character. In compound 7, the major electronic transition is HOMO to LUMO+1 with 55 % in singlet (S1) state with MLCT and LLCT character. In compound 8, the major electronic tran-sition is HOMO to LUMO with 86 % in singlet (S1) state MLCT and LLCT character (Table 5).

Fig. 7 a Zone of inhibition around the wells with com-pounds 5–8. b Antagonistic activity of compounds 5–8 against test organisms

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Conclusions

Metal-mediated deprotonation is responsible for formation of neutral compounds [(p-cymene)RuL(N,N)Cl] 5, [(C6H6)RuL(N,N)Cl] 6, [Cp*RhL(N,N)Cl] 7 and [Cp*IrL(N,N)Cl] 8. Compounds 3, 4, 5 and 6 have exhibited high antitumor activity and could be tried as potential antitumor metallod-rugs. The antitumor activity of arene ruthenium compounds

(5 and 6) is appreciably better than the corresponding pre-cursor compounds (1 and 2). In case of Rh and Ir, the anti-tumor activity of compounds (7 and 8) was found to be lesser than that of corresponding precursor compounds (3 and 4). The order of the percent apoptotic DL cells treated with compounds 1–8 at 100 µg/mL for 12 h is as follows: compound 5 (~30 %) > compound 6 (~28 %) = com-pound 4 (~28 %) > compound 3 (~18 %) > compound 7

Fig. 8 a Agarose gel electrophoresis analysis of plasmid circular DNA (pcDNA 3.4) with compounds 5–8 as detecting dyes at different dilutions. 0.3 µg of plasmid DNA was mixed with a serial dilution of compounds 5–8 as shown on each lane and agarose gel electrophore-sis was carried out 100 V for 50 min. b Agarose gel electrophoresis

analysis of HeLa RNA with compounds 5–8 as detecting dyes at dif-ferent dilutions. 0.3 µg of HeLa RNA was mixed with a serial dilution of compounds 5–8 as shown on each lane and agarose gel electropho-resis was carried out 100 V for 50 min

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Table 3 Molecular docking results of compounds 1–8 with DNA topoisomerase II, thymidine phosphorylase, thymidylate synthase and ribonu-cleotide reductase

CDX (S)-4, 4′-(1-methyl-1, 2-ethanediyl) bis-2, 6- piperazinedione), CMU 5-chloro-6-(1-(2-iminopyrrolidinyl) methyl), UMP 2′-deoxyuridine 5′-monophosphate, GDP guanosine-5′-diphosphate were used as reference compounds and docking was performed with all compounds 1–8 in respective binding site of reference compound in the key enzymes

Sl. No PDB code and name Compound Mol dock score Rerank score H-bond Interactions Interacting amino acids

1. 1QZRDNA Topoisomerase II

CDX −118.12 −88.32 −7.88 5 Asn142A, Gln365A, Thr27A, Gln365B(2)

1 −54.11 209.21 0 – –

2 −105.12 −25.71 0 – –

3 −57.23 492.32 0 – –

4 −56.12 367.11 0 – –

5 −89.42 221.22 −4.23 4 Tyr144B, Tyr128A, Thr27A, Thr27B

6 −102.21 94.62 −3.74 3 Tyr144A, Tyr28B,Thr27A

7 −70.33 310.11 −4.14 4 Tyr28B,Tyr144A, Thr27A, Thr27B

8 −81.19 268.03 −3.62 3 Tyr144A, Tyr28B, Thr27A

2. 1UOUThymidine Phosphorylase

CMU −108.15 −91.06 −9.10 7 Arg202(2), Ser217, Lys221, His116, Ser117, Leu148

1 −111.26 −43.33 0 –

2 −102.47 −48.96 0 –

3 −96.41 64.62 0 –

4 −97.46 62.50 0 –

5 −120.24 −34.34 −1.12 2 His116, Thr151

6 −123.40 −14.01 −2.00 3 His116, Lys221, Thr151

7 −109.32 165.83 −8.99 6 Thr154, Lys115, Ser117, Ser144, Gly145(2)

8 −112.14 170.37 −8.58 5 Thr154, Lys115, Ser117, Ser144, Gly145

3. 2G8DThymidylate synthase

UMP −103.84 −75.49 −16.19 12 Arg23(2), Arg218, Ser 219, Trp261(2), Asp221(2), Asn229, Gln217(2), Cys198

1 −116.14 −70.39 0 –

2 −82.89 −63.66 0 –

3 −115.45 −75.14 0 –

4 −112.19 −43.22 0 –

5 −145.19 −93.40 −2.65 1 Asn229

6 −120.34 −85.46 −4.50 4 Asn229(2), Glu60, Ser219

7 −134.57 −96.69 −2.52 1 Leu224

8 −132.30 −86.85 −2.66 1 Asn229

4. 4R1RRibonucleotide reductase

GDP −174.77 −124.90 −10.92 12 Ser625(2), Thr209(2), Thr624,Glu623, Glu441, Cys225, Asn437(2), Thr209(2)

1 −107.24 −79.65 0 –

2 −86.12 −65.65 0 –

3 104.695 −70.04 0 –

4 104.793 −69.44 0 –

5 −145.58 −104.80 −6.94 6 Ser625(2), Thr209(2), Glu441, Cys225

6 134.648 −104.57 −3.84 3 Ser213(3)

7 −140.04 −96.35 −6.85 3 Thr209(2), Arg251

8 −140.15 −96.59 −6.65 3 Thr209(2), Arg251

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(~12 %) = compound 8 > compound 2 (~10 %) > com-pound 1 (~8 %). The IC50 values obtained from dose–response curve has supported the apoptosis data in showing high activity for compounds 3, 4, 5 and 6. Compound 6 has shown appreciable antibacterial activity against eight bac-terial species with zone of inhibition up to 35 mm. Com-pound 6 could be a potential antimicrobial agent for a wide range of bacteria. Compounds 5–8 showed strong binding

to the DNA and RNA giving a clear vision of the bands on the agarose gel. These compounds could be implemented as detecting dyes similar to ethidium bromide. The ligand has bound to the metal in mode 1 with N, N bonding type because of its highest relative stability. In compounds 5–8, HOMO is mostly located on iminomethyl phenol part of the ligand and LUMO is located mostly on picolinamide part of the ligand and show MLCT, LLCT and LMCT transitions.

Fig. 9 a Docking structure of thymidylate synthase (2G8D) enzyme. Here, a and b show interaction with reference ligand, i.e., 2′-deoxyu-ridine 5′-monophosphate, c and d show interaction with compound 6. Hydrogen atoms are omitted for clarity. Encircled region shows ligand interaction site. Dotted lines indicate hydrogen bonds. b Docking structure of topoisomerase II (1QZR). Here, a and b show interaction with reference ligand, i.e., CDX: (S)-4, 4′-(1-methyl-1, 2-ethanediyl) bis-2, 6- piperazinedione), c and d show interaction with compound 5. Hydrogen atoms are omitted for clarity. Encircled region shows ligand interaction site. Dotted lines indicate hydrogen

bonds. c Docking structure of ribonucleotide reductase (4R1R). Here, a and b show interaction with reference ligand, i.e., GDP: Guanosine-5′-diphosphate, c and d show interaction with compound 5. Hydrogen atoms are omitted for clarity. Encircled region shows ligand interac-tion site. Dotted lines indicate hydrogen bonds. d Docking structure of thymidylate phosphorylase (1UOU). Here, a and b show interac-tion with reference ligand, i.e., CMU: 5-chloro-6-(1-(2-iminopyrro-lidinyl) methyl), c and d show interaction with compound 7. Hydro-gen atoms are omitted for clarity. Encircled region shows ligand interaction site. Dotted lines indicate hydrogen bonds

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Fig. 10 a Four anticipated possible binding modes of ligand. b Relative energies of four conformations of ligand

Table 4 DFT‑computed HOMO–LUMO, HOMO–HOMO−1 and LUMO–LUMO+1 energy gaps and singlet–triplet splitting (ΔES1–T1) found in compounds 5–8

Compound HOMO LUMO ΔE HOMO–LUMO (eV) ΔEHOMO–HOMO–1 (eV) ΔE LUMO–LUMO+1 (eV) (ΔES1–T1) (eV)

5 −4.92 −1.88 3.04 0.72 0.18 0.36

6 −4.99 −1.97 3.02 0.71 0.16 0.34

7 4.98 −2.00 2.98 0.72 0.08 0.30

8 −4.91 −1.92 2.99 0.72 0.55 0.50

Table 5 TDDFT-computed excitation energies (Eth in eV), wavelength (λ in nm) and oscillator strength (f) and transition analyses of com-pounds 5–8

Compound State Eth λcal f Excitation (%) Character

5 S1 2.12 582.84 0.002 H−2–L+1 (23 %), H–L+2 (29 %) MLCT/LLCT

T1 1.76 702.29 0.000 H−3–L+1 (13 %), H−1–L+1 (15 %) H–L+1 (47 %) LMCT/LLCT

6 S1 2.13 580.88 0.001 H–L (24 %), H–L+1 (47 %) MLCT/LLCT

T1 1.79 691.94 0.000 H−1–L+1 (12 %), H–L (13 %), H–L+1 (40 %) MLCT/LLCT

7 S1 2.03 609.13 0.003 H–L+1 (55 %), H–L (22 %) LMCT

T1 1.72 720.49 0.000 H–L+1 (56 %), H−1–L+1 (10 %) LMCT

8 S1 2.43 510.09 0.053 H–L (86 %) MLCT, LLCT

T1 1.92 643.53 0.000 H–L (84 %) MLCT, LLCT

J Biol Inorg Chem

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Acknowledgments K. M. Rao gratefully acknowledges financial support from the CSIR, New Delhi, through the Research Grants No. 01(2493)/11/EMR-II. P. N. Rao thanks UGC, New Delhi for provid-ing fellowship in the form of JRF and SRF. We thank SAIF and DST-PURSE SCXRD of NEHU for collecting NMR and X-ray analysis data. We thank Dr. G. Narahari Sastry, IICT, Hyderabad, for provid-ing facility to carry out DFT calculations, Mr. Veeranjaneylulu, IISc Bangalore for his support in various analyses, Dr. S. Mitra and Mr. Mullahmuhai for their help in fluorescence studies.

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