Benzothiazole and benzoxazole promoted cleavage of Ru − C(aryl) bond in a four-membered ortho-metalated ruthenium(II) organometallics

The four-membered ruthenium(II) organometallics Ru(η2-RL)(PPh3)2(CO)(Cl) (1) where η2-RL = C6H2O-2-CHNHC6H4R(p)-3-Me-5 and R = CH3 reacts with 2-(2-hydroxyphenyl)benzothiazole (Hhpbt) and 2-(2-hydroxyphenyl)benzoxazole (Hhpbo) in refluxing ethanol to afford Ru(PPh3)2(CO)(hpbt)Cl (2) and Ru(PPh3)2(CO)(hpbo)Cl (3) respectively in excellent yield. In the course of these reactions, the Ru − C(aryl) bond in 1 is cleaved, and the RL ligand is no longer coordinated with the metal center in the products. The spectral (UV–vis, IR, 1H NMR) and electrochemical data of the complexes are reported. The identity of complex 2 has been established by single-crystal X-ray structure determination. The electronic structure and the absorption spectra of the complexes are scrutinized by DFT and TD-DFT analyses. The complexes were also tested for their ability to exhibit DNA-binding activity.


Introduction
Benzothiazole, benzoxazole, and their derivatives are active pharmacophore and important heterocyclic moieties in medicinal chemistry [1].They are also found in various biomolecules in the living system [2].Due to their various biological activity and industrial applications, they are attracting the interest of different research groups.They exhibit a wide range of biological properties like anti-cancer, anti-tumor, anti-microbial, anti-fungal, antiinflammatory, anti-allergic, anti-diabetic, anti-convulsant, anti-tubercular, and anti-HIV activities [3].The coordination chemistry of those ligands is also successfully explored, and nowadays complexes of almost any transition metal and rare-earth are known with those ligands [4].Their metal derivatives have attracted the attention of the researchers due to their catalytic, optical, and luminescent properties [5].
Organometallic complexes incor porating the metal − carbon σ-bond have been attracting the attention of different research groups because they are found to serve as the intermediates in different organic catalytic reactions [6].It has been found that the cleavage of the metal − carbon bonds by electrophilic reagents is the key step in many of these catalytic reactions.Despite of their importance, the cleavage of the metal − carbon σ-bonds has not been studied extensively.The cleavage of iron −, ruthenium −, cobalt −, platinum −, palladium −, rhenium −, rhodium −, iridium −, and mercury − carbon bonds are documented in the literature [7].There are examples of the cleavage of Ru − C(aliphatic) and Ru − CO bonds in the literature [8], but the cleavage of the Ru − C(aryl) bond is scanty [9].
Ruthenium(II) organometallics of type Ru(η 2 -RL) (PPh 3 ) 2 (CO)(Cl) (1) were discovered almost three decades ago by the decarbonylative orthometallation of 4-methyl-2,6-diformylphenol by Ru(PPh 3 ) 3 Cl 2 in presence of primary amines [10].Organometallics of type 1 incorporates the zwitterionic iminium-phenolato motif.The reactivity of 1 has also been investigated.Several monoanionic σ-donor ligands such as carboxylate [11], nitrate, nitrite [12], xanthate [13], acetylacetonate [14], and pyridine-2-thiolate [15] react with 1 to afford new ruthenium(II) organometallics via displacement of Ru − O and Ru − Cl bonds.The reactions of 1 with 2,2bipyridine and 1,10-phenanthroline have also been documented [16].Complex 1 also undergoes facile insertion of alkynes into the Ru − C(aryl) bond [17] and isonitrile into the Ru − O bond [18].2-Mercaptopyrimidine and pyridine-2,6-dicarboxylate ligands have also been found to react with 1 furnishing new ruthenium organometallics [19].These results led us to explore further the reactivity of 1 towards other chelating ligands.Here in this report, we have examined the reactions of 2-(2hydroxyphenyl)benzothiazole and 2-(2-hydroxyphenyl) benzoxazole with 1 in order to assess the stability of Ru − C(aryl), Ru − O, and Ru − Cl bonds to ligands of these type.In the course of this work, we have discovered that under refluxing condition in ethanol, 1 reacts with 2-(2-hydroxyphenyl)benzothiazole and 2-(2-hydroxyphenyl)benzoxazole (Chart 1) to afford the Ruthenium(II) complexes Ru(PPh 3 ) 2 (CO)(hpbt)Cl (2) and Ru(PPh 3 ) 2 (CO)(hpbo)Cl (3), respectively.In both the reactions, Ru − C(aryl) bond in 1 is cleaved, and the RL ligand is no longer coordinated with the ruthenium center in the products 2 and 3. Herein, we are reporting the synthetic procedures, structure, spectroscopic, and electrochemical properties of the resulting complexes.The UV-vis absorption titration and competitive DNA binding fluorescence experiments were carried out to study the interaction of the complexes 2 and 3 with calf-thymus DNA (CT-DNA).Density functional theory (DFT) and time-dependent density functional theory (TD − DFT) studies have also been performed to get better insight into the electronic structure, optical, and electrochemical properties of these complexes.

Materials and methods
The compound Ru(η 2 -RL)(PPh 3 ) 2 (CO)Cl (1) was prepared by the literature method [10].2-(2-Hydroxyphenyl)benzothiazole was purchased from Sigma Aldrich, India.2-(2-Hydroxyphenyl) benzoxazole was prepared by the literature method [20].All other reagents were obtained from commercial sources and were used as received.Solvent dichloromethane for cyclic voltammetric study was obtained as HPLC grade from Sigma-Aldrich, India, and distilled over phosphorus pentoxide (P 2 O 5 ) under a nitrogen atmosphere prior to use.Infrared spectra were recorded on a Perkin-Elmer L120-00A FT-IR spectrometer as a KBr pellet.Electronic spectra were recorded on a Shimadzu UV-1800 PC spectrophotometer. 1 H NMR spectra were collected on a Bruker DPX-400 spectrometer in CDCl 3 .Microanalyses were performed using a Perkin-Elmer 2400 series-II elemental analyzer.All electrochemical measurements were performed under a nitrogen atmosphere using CHI 600D electrochemistry system.The supporting electrolyte was tetrabutylammonium perchlorate, and potentials are referenced to Ag/AgCl electrode.

X-ray crystallography
Block-shaped yellow crystals of the compound Ru(PPh 3 ) 2 (CO)(hpbt)Cl•CH 2 Cl 2 (2)•CH 2 Cl 2 were obtained in a week at room temperature after adding a layer of 15 mL of hexanes to a 5 mL solution of the compound in dichloromethane (0.01 M) in a 25-mL Schlenk tube.A crystal measuring 0.32 × 0.21 × 0.18 mm 3 was directly harvested from the Schlenk tube without any manipulation and was mounted on a Bruker AXS SMART APEX CCD diffractometer (Mo-K α, λ = 0.71073 Å).The data were reduced in SAINTPLUS [21], and empirical absorption correction was applied using the SADABS package [21].The metal atom was located by the Patterson method, and the rest of the non-hydrogen atoms were emerged from successive Fourier synthesis.Hydrogen atoms were placed in idealized positions.The structure was refined by a full matrix least-squares procedure on F 2 .All non-hydrogen atoms were refined anisotropically.All calculations were performed using the SHELXTL V6.14 program package [22].Molecular structure plot is drawn using the Oak Ridge thermal ellipsoid plot ORTEP-32 [23].Relevant crystal data are given in Table 1.
The largest difference peaks are located near the Ru atom and are likely not from any unaccounted atoms.

Computational study
Unrestricted Kohn-Sham geometry optimization and TD − DFT calculations on those optimized geometries were carried out on 2 and 3 through the ORCA 2.9.1 electronic structure package [24].The geometry of the complexes was fully optimized in the gas phase without imposing any symmetry constraints.The single crystal X-ray coordinates of 2·CH 2 Cl 2 have been used as the initial input in the calculation.Geometry optimizations for the complexes were converged to the S = 0 spin and employed the Becke-Perdew (BP86) functional [25] and the SV(P) (Ahlrichs split valence polarized) basis with the SV/C auxiliary basis for all atoms except for nitrogen, oxygen, sulfur, phosphorus, and chlorine, where the larger TZVP (Ahlrichs triple-valence polarized) basis in conjunction with the TZV/J auxiliary basis were used [26].
For ruthenium atom, the scaled-ZORA (zeroth-order regular approximation) Hamiltonian was used to take account of the relativistic effect in the calculations [27].These calculations employed the resolution of identity (RI) approximation developed by Neese [28].The coordinates of all DFT energy minimized model of the complexes presented in this study are included in the Supporting Information.For single-point calculations, all calculations were optimized with a Grid4 optimization grid and tight SCF convergence criteria.These calculations employed the B3LYP functional [29] and ZORA (Ru), TZVP (N, O, P, S, and Cl), and SVP (C and H) basis sets.On the basis of the optimized ground state geometry, the absorption spectral properties were calculated by the timedependent density functional theory (TD-DFT) approach [30].Forty excited states were calculated by including one electron excitations within an energy window of ± 3 hartree with respect to the HOMO/LUMO energies.Isosurface plots of molecular orbitals and electron density difference maps (EDDMs) were generated using the gOpenMol program using isodensity values of 0.04 and 0.003 b −2 respectively.

General remarks
Ru(η 2 -RL)(PPh 3 ) 2 (CO)Cl (1) reacts with excess of Hhpbt and Hhpbo in refluxing ethanol furnishing yellow microcrystalline solids of 2 and 3 respectively in excellent yield.The synthetic reaction is shown in Scheme 1.The type 2 and 3 organometallics are diamagnetic as the oxidation state of the ruthenium center is + 2 (idealized t 2g 6 ).They also behave as nonelectrolyte in common organic solvents.It has been found that the complex 1 reacts with different monoanionic/neutral bidentate ligands to furnish new ruthenium organometallics in which the Ru − C(aryl) bond of 1 is retained in the product, but the reaction of 1 with Hhpbt and Hhpbo is different.It was anticipated that during the course of the reaction, Ru − O and Ru − Cl bonds of 1 will be cleaved by the Hhpbt and Hhpbo ligands, and the Schiff base ligand (RL) will change its η 2 -hapticity to η 1 .In the present case, cleavage of Ru − C(aryl) bond of 1 is occurred, and the Schiff base ligand is no longer coordinated with the ruthenium center in the products.We failed to isolate the departed R(H)L ligand from the reaction mixture.

Spectroscopic and electrochemical studies
The electronic spectra of 2 and 3 were collected in dichloromethane solution at room temperature and display well-defined peaks in the region 500 − 300 nm.In Scheme 1 Synthesis of the complexes 2 and 3 dichloromethane solution 2 displays three characteristic allowed absorption bands near 454 nm, 357 nm, and 325 nm, whereas 3 exhibits absorption bands near 436 nm, 415 nm, 347 nm, and 306 nm.The absorption bands of complex 3 are blue shifted with respect to those of 2. This is possibly due to fact that the LUMOs of complex 3 are destabilized in comparison with the complex 2 (vide infra).Electronic spectra of the complexes are shown in Fig. 1.In IR spectra of the complexes, CO stretch appears near 1945 cm −1 which is significantly higher than that in 1 (∼1905 cm −1 ).
In the proton NMR spectrum of 2 in CDCl 3 , two triplets and three doublets for the protons of hpbt ligand are clearly seen at 6.96 ppm, 6.15 ppm, 8.07 ppm, 6.86 ppm, and 6.70 ppm, respectively.The other protons of the hpbt ligand were not resolved as they were hidden under the multiplet of the two PPh 3 ligands which appears in the regions 7.05-7.29 ppm and 7.57-7.61ppm.For 3, the hpbo protons appear as two doublets at 7.48 ppm and 6.83 ppm and as two triplets at 6.97 ppm and 6.22 ppm.The rest of the hpbo protons along with the phenyl protons of the PPh 3 groups appear as multiplet in the regions 7.01-7.21ppm and 7.67-7.70ppm.
Complexes 2 and 3 are electroactive in dichloromethane solution and exhibit two successive quasi-reversible one electron oxidation processes which are assignable to the Ru 3+ /Ru 2+ and Ru 4+ /Ru 3+ couples.The cyclic voltammograms of the complexes are shown in Fig. 2. Complex 2 displays two successive oxidation processes at 1.13 V and 1.27 V, whereas complex 3 displays the oxidation processes at 1.16 V and 1.31 V vs. Ag/AgCl.The redox potentials of 3 shift to higher potential because of the presence of more electronegative oxygen atom in the ligand frame.The redox potentials of 2 and 3 are higher than the precursor complex 1 indicating poor stabilization of the ruthenium high valence state in 2 and 3 compared with 1.The better stabilization of the Ru 3+ state in 1 seems to be favored by the strong σ-donor property of the electron rich η 2 -RL ligand.For both 2 and 3, DFT results (vide infra) predict that the compositions of HOMO and HOMO-1 are metal-ligand mixed centered which means that the oxidations are mixed metal-ligand centered.

Crystal structure
The structural identity of Ru(PPh 3 ) 2 (CO)(hpbt)Cl (2) was established by single crystal X-ray structure analysis of 2•CH 2 Cl 2 .Compound 2•CH 2 Cl 2 crystallizes in monoclinic space group P2 1 /c.All atoms of the structure are in general position.The lattice dichloromethane molecule of 2•CH 2 Cl 2 was refined with full occupancy about a general position.Molecular view is shown in Fig. 3, and selected bond parameters are listed in Table S1 (Supporting Information).The RuCNOClP 2 coordination sphere of 2•CH 2 Cl 2 has distorted octahedral geometry as can be seen from the angles at the metal center.The Ru1, C50, N1, O1, and Cl1 atoms define an equatorial plane with mean deviation of 0.0045 Å.The carbonyl and the chloride groups lie trans to the O1 and N1 atoms, respectively.The coordination of hpbt ligand to Ru1 center results in the distortion of the equatorial angles around the central ruthenium atom in such a way that the four equatorial angles differ considerably from the mean value of 90°.The largest deviation is that of C50-Ru1-N1 [100.23(14)°].The sum of the four equatorial angles around Ru1 is 360.01°indicating small deviation from planarity.The bite angle of the chelating hpbt ligand is 88.89°.The PPh 3 ligands lie in trans position (P1─Ru1─P2, 173.94(3)°).The Ru1─N1 and Ru1─O1 distances in 2 correspond well with that observed in other structurally characterized benzothiazole containing Ru(II) complexes [4c, 31].Ru-Cl1 and Ru-C50 bond distances fall in the range observed in other structurally characterized ruthenium complexes [19,32].

Computational studies
The geometry optimization for both the complexes 2 and 3 was performed in gas phase assuming S = 0 spin state to get an insight into the ground state geometry, electronic structure, and nature of FMOs of the complexes.The geometry used for the ground state optimization is based on the crystal structure parameters of the complex 2 without any ligand simplification.The optimized geometrical models of the complexes are shown in Fig. 4, and the significant metrical parameters are listed in Table 2.
The isodensity plots of frontier molecular orbitals from HOMO − 5 to LUMO + 5 for complexes 2 and 3 are shown in Figs.S5 and S6 (Supporting Information).The partial frontier molecular orbital compositions and energy levels of 2 and 3 are listed in Tables S2 and S3, respectively.DFT calculation shows that for both the complexes, HOMO is mainly composed of ruthenium d-orbitals and ligand π-orbitals, whereas LUMO is primarily composed of ligand π* orbitals.
We have also calculated the HOMO − LUMO energy gaps (Supporting Information, Fig. S7) of the complexes.It has been found that the gaps are 3.42 eV and 3.55 eV for 2 and 3, respectively.The LUMO of complex 3 is destabilized due to the presence of more electronegative oxygen atom in the ligand frame in comparison with the complex 2 which carries less electronegative sulfur atom.The HOMO − LUMO energy gaps are consistent with the similar spectral and electrochemical behaviors of the complexes.Mulliken atomic charges for the complexes have been included in Table S4.The calculated charges on ruthenium atoms are significantly lower than the formal charge of + 2 due to the considerable charge donation from P, N, O, and Cl donors.
The most relevant calculated absorption energies associated with their oscillator strength, the main configuration, their assignments, and electron density difference maps (EDDMs) as well as the experimental results of 2 and 3 are given in Table 3 and Table 4.The corresponding simulated UV-vis absorption spectra of complexes 2 and 3 are shown in the Supporting Information (Fig. S8).In the experimental absorption spectrum, complex 2 displays broad absorption band near 454 nm.This feature corresponds to the calculated absorptions at 433 nm

CT-DNA binding studies
A combined approach of UV-vis and fluorescence spectroscopic techniques has been used to investigate the binding characteristic of the complexes 2 and 3 with calfthymus(CT) DNA.CT-DNA binding studies with the complexes were carried out by following a literature procedure [33].The experiments were performed in Tris-HCl buffer (30 mM, pH 7.4).UV absorbance at 260 nm and 280 nm of about 1.8:1 confirmed that the CT-DNA was free from protein contamination.Concentration of CT-DNA solution was determined by absorption spectroscopy using the molar extinction coefficient of 13,600 M −1 cm −1 at 260 nm [34].Stock solution of the DNA was stored at 4 °C.The titration experiment was carried out by taking fixed concentration of the complexes [2, 1.06 × 10 −4 M; 3, 1.179 × 10 −4 M] with varying concentration of CT-DNA [0-40 μM] into a rectangular quartz cuvette having the path length of 1 cm.Equal amounts of CT-DNA solution were added to the complex solution and the reference solution to eliminate the absorbance of DNA itself.The reference solution was the same Tris-HCl buffer solution.After addition of DNA to the complex in Tris-HCl buffer, the resulting solution was allowed to equilibrate for 5 min at room temperature.Then the solution was scanned in the specified wavelength window.There is usually a change in the absorption intensity and shift in wavelength in the UV-vis spectra whenever DNA interacts with metal complex.Intercalation of metal complexes between DNA base pair generally results a small red/blue shift causing a hypochromic effect, whereas non-intercalating/electrostatic or groove binding interaction gives rise to hyperchromic effect [35].The electronic spectra obtained during the titration of fixed concentration of the complexes 2 and 3 in the presence of increasing concentration of CT-DNA are given in Figs. 5 and 6.Upon addition of increasing concentrations of CT-DNA to the complexes 2 and 3, the absorption shows "hypochromic" with red shift of ~ 2 nm of the absorption maxima of the complexes.This hypochromic red shift is believed to be generated through active interaction of the complexes with DNA base pair.
Quantitative affinity of the complexes for CT-DNA was evaluated from the intrinsic binding constant (K b ) values evaluated by monitoring the changes in absorbances at 434 nm for 2 and 412 nm for 3 with increasing concentration of CT-DNA using Eq. ( 1) where [DNA] is the concentration of CT-DNA, ɛ a is the ratio of the absorbance/[complex], ɛ f is the extinction coefficient of free 2 and 3, and ɛ b is the extinction coefficient of metal complex in the fully bound form.The values of K b evaluated from the ratio of the slope to the intercept in the plot of We have also investigated the competitive binding of ethidium bromide (EB) vs. the complexes 2 and 3 with CT-DNA using fluorescence spectroscopy.The competitive binding experiments were performed by titrating EBbound CT-DNA solution with increasing amounts of the complexes 2 and 3.The emission spectra of the EB-DNA adduct in the absence and presence of the complexes are shown in Fig. 7.The fluorescence intensity of the EB-DNA adduct decreases significantly in the presence of increasing concentration of the complexes which is due to the displacement of EB from CT-DNA.The relevant binding constant (K b ) values were calculated using the Scatchard equation and were found to be 1.17 × 10 3 M −1 for complex 2 and 1.02 × 10 3 M −1 for complex 3 (Fig. 8).

Conclusions
The results of this study demonstrate that Ru(η 2 -RL) (PPh 3 ) 2 (CO)Cl (1) reacts with 2-(2-hydroxyphenyl)benzothiazole (Hhpbt) and 2-(2-hydroxyphenyl)benzoxazole (Hhpbo) in refluxing ethanol affording Ru(PPh 3 ) 2 (CO)(hpbt)Cl (2) and Ru(PPh 3 ) 2 (CO)(hpbo)Cl (3), respectively.In the course of the reaction, cleavage of Ru − O and most importantly Ru − C(aryl) bonds occurred, and the RL ligand is no longer coordinated with the ruthenium center in 2 and 3.The complexes are characterized by different spectroscopic techniques and by elemental analysis.The identity of complex 2 has been established by single crystal X-ray structure determination.Density functional theory (DFT) and time-dependent density functional theory calculations were also performed.This provides a detailed assignment of the significant spectral features of the complexes.For both the complexes, HOMO is mainly composed of ruthenium d-orbitals and ligand π-orbitals, whereas LUMO is primarily composed of ligand π* orbitals.The LUMO of complex 2 is stabilized in comparison with the complex 3.The calculations also predict that the bands which appear in the experimental electronic spectra of 2 and 3 are due to the d → d, MLCT, LMCT, ILCT, and LLCT transitions.

Fig. 3
Fig. 3 Molecular structure of the complex 2·CH 2 Cl 2 drawn at 30% probability level.Hydrogen atoms and solvent dichloromethane molecule have been omitted for clarity

[
DNA]/( ɛ a − ɛ f ) vs [DNA] were found to be 8.175 × 10 3 M −1 for 2 and 8.25 × 10 3 M −1 for 3. Binding affinity measurements for CT-DNA with the ligands Hhpbt and Hhpbo did not show any significant interactions.The standard Gibb's free energy for DNA binding was calculated from the following relation[36] ΔG b 0 = − RT lnK b and was found to be − 22.32 kJ/M for complex 2 and − 22.34 kJ/M for complex 3.

Table 2
Selected DFT optimized bond lengths (Å) and angles (°) of the complexes in the ground state

Table 3
Main calculated optical transitions for 2 with composition in terms of molecular orbital contribution of the transition, computed vertical excitation energies, and oscillator strength

Table 4
Main calculated optical transitions for 3 with composition in terms of molecular orbital contribution of the transition, computed vertical excitation energies, and oscillator strength