Synthesis, characterization, theoretical, molecular docking and in vitro biological activity studies of Ru(II) (η⁶-p-cymene) complexes with novel aniline substituted aroyl selenoureas.

Journal of Biomolecular Structure and Dynamics ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20 Synthesis, characterization, theoretical, molecular docking and in vitro biological activity studies of 6 Ru(II) (η -p-cymene) complexes with novel aniline substituted aroyl selenoureas Moideen Musthafa, Ramaiah Konakanchi, Rakesh Ganguly, Chandrasekar Balachandran, Shin Aoki & Anandaram Sreekanth To cite this article: Moideen Musthafa, Ramaiah Konakanchi, Rakesh Ganguly, Chandrasekar Balachandran, Shin Aoki & Anandaram Sreekanth (2020): Synthesis, characterization, theoretical, 6 molecular docking and in vitro biological activity studies of Ru(II) (η -p-cymene) complexes with novel aniline substituted aroyl selenoureas, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2020.1778531 To link to this article: https://doi.org/10.1080/07391102.2020.1778531 View supplementary material Published online: 29 Jun 2020. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tbsd20 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS https://doi.org/10.1080/07391102.2020.1778531 Synthesis, characterization, theoretical, molecular docking and in vitro biological activity studies of Ru(II) (g6-p-cymene) complexes with novel aniline substituted aroyl selenoureas Moideen Musthafaa, Ramaiah Konakanchib, Rakesh Gangulyc, Chandrasekar Balachandrand, Shin Aokid,e and Anandaram Sreekantha a Department of Chemistry, National Institute of Technology, Tiruchirappalli, Tamil Nadu, India; bChemistry Division, H&S Department, Malla Reddy Engineering College for Women (Autonomous Institution), Hyderabad, Telangana, India; cDivision of Chemistry & Biological Chemistry, Nanyang Technological University, Singapore, Singapore; dFaculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Japan; eResearch Institute for Science and Technology, Tokyo University of Science, Yamazaki, Noda, Japan Communicated by Ramaswamy H. Sarma ABSTRACT A sequence of aroyl selenourea ligands (L1–L3) substituted by aniline and their Ru(II) (g6-p-cymene) complexes (1-3), [Ru(II) (g6-p-cymene) L] (L ¼ monodentate aroyl selenourea ligand) have been synthesized and characterized the composition of the ligands and their metal complexes. The molecular structures of ligand L1 and complex 3 were also confirmed by single XRD crystal method. The singlecrystal XRD study showed that aroyl selenourea ligand coordinates with Ru via Se novel neutral monodentate atom. In vitro DNA interaction studies were investigated by Fluorescence and UV-Visible spectroscopic methods which showed that the intercalative mode of binding is in the order of 1 > 2 > 3 with Ru(II) (g6-p-cymene) complexes. Spectroscopic methods have been used for measuring the binding affinity of bovine serum albumin to complex. Moreover, the cytotoxic study of complexes (1–3) were evaluated against HeLa S3, A549, and IMR90 cells, resulting in complexes 1 and 2 showed promising cytotoxic activity against HeLa S3 cell with IC50 values of 24 and 26 mM, respectively. Also, the morphological changes of HeLa S3 and A549 cells were confirmed by fluorescence microscope in the presence of complexes 1 and 2 using AO (acridine orange, 200 mM) and EB (ethidium bromide, 100 mM). In addition, the docking results strongly support the protein binding studies of the complexes. 1. Introduction Metal complexes are used to combat cancer and have a massive effect on cancer treatment. According to its specific structure and mechanical growth, magnetic, thermodynamic, intrinsic and kinetic properties, metal complexes are used to produce extremely effective anticancer, DNA and cleaving medicines (Guerriero et al., 2017). Cisplatin has proven to be a drug for anticancer, and several researchers have found other potential metal medicines with increased pharmacological activity since they have high-level neuro, hepato and nephrotoxic side effects (Bruijnincx & Sadler, 2008; Hill & Speer, 1982; Rosenberg, 1978). In order to overcome these problems, scientists concentrated on alternative metal-based bio-compatible medicines (Adeyemo et al., 2018; Muggia, 2009; Swaminathan et al., 2019). Ruthenium compounds emerged out to be the most promising candidate as anticancer agents. Ruthenium based complexes with a high pharmacological effect are reported to be target-specific and less toxic (Reedijk, 2008; Song et al., 2019). Many ruthenium complexes have been investigated ARTICLE HISTORY Received 2 April 2020 Accepted 26 May 2020 KEYWORDS Cytotoxicity; crystal structures; selenourea; Ru(II) (g6-p-cymene; docking studies and reported as anticancer agents; ruthenium-arene complexes find their own significance. The main reasons for the increasing design of ruthenium-arene based anticancer drugs are the amphiphilic nature of the arene ruthenium unit, provided by the hydrophobic arene ligand counterbalanced by the hydrophilic metal center, and the synthetic diversity of the arene ligand, which is an excellent framework for the coupling of organic segments for targeted chemotherapy. Ru-p-cymene complexes like [RuCl2(g6-p-cymene)(pta)] (RAPTA-C)and [RuCl2(g6-benzene)(pta)] (RAPTA-B) where pta ¼ 1,3,5-triaza-7-phosphaadamantane, have recently attracted considerable attention due to the very promising in vivo activities on the inhibition of metastasis growth, together with a high selectivity and low general toxicity. There are also few reports dealing with RuCl2(g6-p-cymene) type complexes showing enhanced biological activity on the replacement of pta with other suitable ligand (Figure 1).The structural backbone of the complexes described in this paper is similar to that of RAPTA-C; but pta ligand has been replaced by aroyl selenourea ligand. Moreover, there are a considerable number of reports on the biological applications of [RuCl2(g6-p- CONTACT Anandaram Sreekanth sreekanth@nitt.edu Department of Chemistry, National Institute of Technology, Tiruchirappalli, Tamil Nadu, India Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2020.1778531 ß 2020 Informa UK Limited, trading as Taylor & Francis Group 2 M. MUSTHAFA ET AL. cymene) L] where L is a N or P donor monodentate ligand. Surprisingly, no such complex with Se donor ligand is explored for its biological applications. Ruthenium based complexes are reported as target-specific and less toxic with high pharmacological effects. In particular, NAMI-A, or trans-[RuCl4(DMSO)(Im)]ImH (where Im is imidazole), is generally nontoxic but is anti-metastatic and prevents cancer spread. KP1019 or trans-[tetrachlorobis(1Hindazole)ruthenate(III)], NKP-1339 or Sodium trans[tetrachloridobis(1H-indazole)ruthenate(III)], NKP-1339 (IT-139; KP-1339) is the first-in-class ruthenium-based anticancer agent in development against solid cancer with limited side effects. (Bratsos et al., 2007). In determining the pharmacological properties of complexes, the structure of ligands plays an important key role. Due to the diverse coordination and biological interventions of a wide range, including anticancer, antifungal, anti-tuberculosis, insecticide and other diseases, we were aiming at working on aroyl selenourea ligands (Alcolea et al., 2016; Barbosa et al., 2018; Campos Jr. et al., 2018; Hussain et al., 2014; Musthafa et al., 2019; Olsen et al., 2016). Selenium is known primarily for its antioxidant function and its healing, chemopreventive, anti-inflammatory, and anti-virus properties (Ganther, 1999; Holben & Smith, 1999). Recently, selenoureas have emerged as free radical scavengers, enzyme inhibitors, anticancer agents with biological aspects. (Alcolea et al., 2016; Hussain et al., 2015; Olsen et al., 2016). We describe the in vitro cytotoxicity of Ru p-cymene complexes with aroyl selenourea ligands. Our monitoring complexes are identical to the RAPTAB backbone (Coverdale et al., 2019), with the replacement of pta ligand with aroyl selenourea ligand. In addition, there are only a few Ru p-cymene complexes for their action against cancer has been reported and this is the first study evaluated Figure 1. Structure of a) RAPTA-C and b) [RuCl2(g6-p-cymene)L] (L ¼ sugar based phosphite analog). Scheme 1. Synthesis of the aroyl selenourea ligands (L1-L3) for their biological applications on Ru(II) (g6-p-cymene) complexes which contain aroyl selenourea ligands. 2. Results and discussion 2.1. Synthesis of the ligands and complexes Benzoyl chloride, or thiophene-2-carbonyl chloride, or furan2-carbonyl chloride are synthesized as aniline replaced aroyl selenourea ligands (L1-L3), potassium selenocyanate, and aniline in dry acetone (Scheme 1) (Musthafa et al., 2020). These aroyl selenourea ligands were allowed to react with the Ru(II) (g6-p-cymene) precursor [RuCl2 (g6-(p-cymene) (L1L3)])]2. In toluene 2:1 molar ratio and the new general formulation complexes, Ru(II) (g6-p-cymene) (L ¼ aniline substituted aroyl selenourea ligands) (Scheme 2) (Jeyalakshmi et al., 2016), high yields are obtained. Elemental analysis, single crystal XRD, UV-Visible, FT-IR, 1H, 13C NMR, and mass spectroscopy experiments are confirming the structures of the ligands and Ru(II) complexes. The complexes are soluble in DMSO, DMF, CH3OH, CHCl3, and CH2Cl2. 2.2. Characterization of the ligands and their complexes Electronic spectrum of ligands (L1–L3) display two strong absorption bands observed approximately at 260–283 and 309–336 nm, respectively, allocated to p ! p and n ! p transitions. Electronic spectra of Ru(II) complexes (1-3) were observed at 267–289 nm and according to the selenourea moiety, LMCT bands appear approximately at 334–382 nm. The moderately intense band is assigned to d ! d transitions in the region of 430–453 nm. The FT-IR spectra of the ligands showed bands in the regions of 3225–3207 cm1 indicate amide N  H, the peak at 3120–3155 cm1 indicate selenourea N  H. The stretching frequencies observed in the range of 1663–1675 cm1 for C ¼ O and 1262–1274 cm1 for C ¼ Se, respectively (Musthafa et al., 2019, 2020). In the complexes N  H and C ¼ O bands remained unchanged, while the m(C ¼ Se) (1262–1274 cm1) decreased, indicating that only selenium (neutral monodentate) is coordinated with the Ru ion. The FTIR spectra of ligands and complexes were included in the Supplementary information (SI) (Figures S1–S6). 1H NMR spectra of all the ligands of aroyl selenourea (L1-L3) showed that carbonyl and N  H attached selenocarbonyl are observed as singlets and the values appeared in the range of JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 3 Scheme 2. Synthesis of the RuCl2 (g6-p-cymene) complexes (1-3) Figure 2. Thermal ellipsoid plot of ligand L1. 12.82–13.17 ppm and the peaks at 11.19–11.50 ppm corresponding to selenocarbonyl bound N  H. The signals were observed at 6.55–7.77 ppm indicating all the other aromatic protons (phenyl, thiophene and furan). The 13C NMR spectra of the ligands displayed the signals for C ¼ O and C ¼ Se, respectively at 178.39–178.79 and 158.84–168.67 ppm. Signals were shown to match aromatic carbon in the ligands between 112.0 and 146.1 ppm. The 13C NMR spectra of the complexes showed no significant shifts. The new peaks observed at 79.54–82.33 ppm represent the confirmation of p-cymene. The 1 H and 13C NMR spectra of the ligands and their metal complexes were shown in the SI (Figures S7–S18) (Jeyalakshmi et al., 2016). The mass spectra of the ligands and their complexes were also confirming the structure of the compounds (Shown in ESI Figures S19–S24, Supporting Information). 2.3. X-ray crystallographic analysis The ligand L1 and complex 3 molecular structures were confirmed by single-crystal X-ray diffraction studies as shown in Figures 2–4, respectively. Crystal and selected inter-atomic bond lengths and bond angles are summarized in Tables 1–3. The ligand L1 has shown monoclinic crystal system and space group P121/c1, and the complex 3 showed monoclinic crystal system and space group P121/n1. The crystal structures of the ligand and its complex were observed that there is a correlation between selenocarbonyl and carbonyl oxygen with intra-molecular hydrogen. The furan ring was oriented in the structures of complex 3 in two opposite directions. Slightly elongated thermal parameters of the furan groups (C18–C21, S2) indicate that a possible disorder, which was successfully modeled between two positions. 2.4. DNA binding studies Understanding the binding nature of synthesized complexes (1-3) towards DNA is a significant step in the development of the anticancer drug, synthetic restriction enzymes and so forth (Mahadevan & Palaniandavar, 1998; Nikoli
c et al., 2015; Ramakrishnan & Palaniandavar, 2008). Consequently, the 4 M. MUSTHAFA ET AL. Figure 3. Thermal ellipsoid plot of complex 3 with atomic labeling. Figure 4. Crystal packing of ligand L1 and complex 3. ability and the trend for the binding of complexes (1-3) to CT-DNA were examined with various techniques. 2.4.1. Electronic absorption spectral titration Electronic spectral experiments have tested the association of complexes (1-3) with CT-DNA. Complexes (1-3) exhibited a band at 287–320 nm which was used for further studies (Balakrishnan et al., 2019; Jeyalakshmi et al., 2017). It is understood that an intercalated compound bound to DNA demonstrates a hypochromism with a bathochromic difference in the rate of absorption due to the combination of the chromophore with the base DNA pair. The observed spectral changes were plotted by taking [DNA]/ (eaef) in Y-axis and [DNA] in X-axis, according to the equation [DNA]/(eaef) ¼ [DNA]/(ebef) þ 1/Kb (ebef) where [DNA] is the concentration of DNA in base pairs, ea is the apparent extinction coefficient value found by calculating A (observed)/[complex], ef is the extinction coefficient for the free compound, and eb is the extinction coefficient for the compound in the fully bound form (Rohini, Haribabu, et al., 2018). The intrinsic binding JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 5 Table 1. Crystallographic data and refinement parameters for ligand L1 and complex 3. Identification code L1 3 Empirical formula Formula weight (g/mol) Temperature (K) Wavelength (Å) Crystal system Space group Unit cell dimensions a (Å) b(Å) c (Å) a ( ) C14H12N2OSe 303.22 100(2) 0.71073 Monoclinic P121/c1 C22H24Cl2N2O2RuSe 599.36 100(2) 0.71073 Monoclinic P121/n1 13.1366(15) 4.8877(6) 19.895(2) 90 14.3983(5) 9.4258(5) 16.8343(9) 90 b ( ) c ( ) 103.284(4) 90 90.7652(15) 90 Volume (Å3) 1243.2(3) 2284.47(19) 4 4 Z Density (calculated) Mg/m3 Absorption coefficient (mm–1) F(000) Crystal size (mm3) Theta range for data collection ( ) Index ranges 1.620 3.008 608 0.040  0.060  0.220 2.33 to 27.22 16  h  16, 6  k  6, 25  l  25 15,894 2760 0.0841 99.4 Multi-Scan 0.8890 and 0.5570 Full-matrix least-squares on F2 2760/0/163 1.159 R1 ¼ 0.0471, wR2 ¼ 0.0991 R1 ¼ 0.0871, wR2 ¼ 0.1172 n/a 0.863 and –0.937 Reflections collected Independent reflections [R(int)] Completeness to theta Absorption correction Max. and min. transmission Refinement method Data/restraints/parameters Goodness-of-fit on F2 Final R indices [I > 2sigma(I)] R indices (all data) Extinction coefficient Largest diff. peak and hole (e.Å–3) 1.743 2.535 1192 0.020  0.030  0.100 2.42 to 27.10 –18  h  18, 12  k 12, 18  l  21 22,874 5021 0.0940 99.7 Multi-Scan 0.9510 and 0.7860 Full-matrix least-squares on F2 5021/64/302 1.020 R1 ¼ 0.0411, wR2 ¼ 0.0717 R1 ¼ 0.0790, wR2 ¼ 0.0866 n/a 0.743 and –0.862 Table 2. Selected bond lengths (Å) and bond angles ( ) for ligand L1. Table 3. Selected bond lengths (Å) and bond angles ( ) for complex 3. Bond lengths (Å) Bond lengths (Å) Ru1-C1 Ru1-C3 Ru1-C5 Ru1-Cl1 Ru1-Se1 Se1-C17 C18-O1 Bond angles ( ) C1-Ru1-Se1 C3-Ru1-Se1 C5-Ru1-Se1 Cl1-Ru1-Se1 N1-C17-Se1 Se1-C8 C1-N1 C8-N1 Bond angles ( ) O1-C1-N1 N1-C1-C2 N2-C8-Se1 C14-C9-N2 C1-N1-C8 1.829(5) 1.390(6) 1.403(6) C1-O1 C8-N2 C9-N2 1.224(6) 1.325(6) 1.407(7) 122.3(5) 116.2(4) 128.6(4) 126.5(5) 127.2(4) O1-C1-C2 N2-C8-N1 N1-C8-Se1 C10-C9-N2 C8-N2-C9 121.4(5) 114.9(4) 116.5(4) 113.9(5) 133.6(4) constant Kb was calculated from the ratio of the slope and the intercept (Aneesrahman et al., 2018). In all the cases, similar trends were observed as can be seen from Figures 5(a) and S25 (Supporting Information). Among the complexes, complex 1 exhibits a maximum shift. Figure 5(b) shows the magnitudes of binding constants (Kb) and the values are tabulated in Table 4. The Kb values were found to be in the range of 5.718  105 – 3.991  105 M1. The binding energy of complex 1 is more than that of complex 2 and 3 comparatively. Binding constant values of complex 1 when studied by molecular docking (1BNA) is more than the other 2 complexes (2 and 3) (Pursuwani et al., 2020). 2.188(5) 2.178(4) 2.199(5) 2.431(3) 2.5275(13) 1.858(5) 1.219(5) Ru1-C2 Ru1-C4 Ru1-C6 Ru1-Cl2 C11-N1 C18-N2 2.148(5) 2.225(5) 2.143(5) 2.428(3) 1.449(5) 1.376(6) 95.30(14) 161.84(14) 99.80(13) 91.84(12) 121.4(3) C2-Ru1-Se1 C4-Ru1-Se1 C6-Ru1-Se1 Cl2-Ru1-Se1 N2-C17-Se1 129.76(16) 136.10(15) 82.42(12) 91.33(14) 120.0(3) 2.4.2. Fluorescence spectroscopic studies Fluorescence spectroscopy was used for further confirmation of the binding modes of the complexes (1-3) to DNA. Since all the complexes (1-3) did not show any fluorescence in solution, the Ethidium bromide was used as a fluorescence probe (Rohini, Ramaiahet al., 2018). In the Tris buffer solution at room temperature, EB shows a weak luminescence, but in the presence of CT-DNA, the rapid intercalation of DNA base pairs has shown strong luminescence. If the complex under investigation has the intercalation abilities, the intensity of fluorescence starts to decrease as it replaces the EB from 6 M. MUSTHAFA ET AL. Figure 5. (a) Complex 1 absorption spectra in the Tris-HCl buffer with CT-DNA addition, [DNA] ¼ 0-40 lM, [complex] ¼ 25 lM.The arrow indicates that as the CTDNA concentration increases the absorption intensity decreases. (b) The plot of titrating complexes with CT-DNA [DNA]/(ea  ef ) versus [DNA]. (c) In the presence of complex 1 fluorescence curves EB bound with DNA. [DNA] ¼ 5 mM, [EB] ¼ 5 mM and [complex] ¼ 0–50 mM. (d) The fluorescence titrations of the complexes with CT-DNA are from Stern-Volmer equation. Table 4. Binding constant of DNA (Kb), Stern-volumer constant (Kq) and apparent binding constant (Kapp) values of complexes (1-3). Complex 1 2 3  Kb (M–1) DG (kJ mol1) Kq (M–1) Kapp (M–1) 5.718  10 ± 0.09 4.811  105 ± 0.06 3.991  105 ± 0.03 –27.32 –26.89 –26.42 1.956  10 ± 0.03 1.845  104 ± 0.04 1.883  104 ± 0.08 1.310  106 ± 0.05 1.210  106 ± 0.03 1.110  106 ± 0.02 5 DNA, the EB displacement assay, thereby providing indirect proof for the DNA binding mode. In addition to the CT-DNAEB solution with complexes (1-3) (0–50 lM), a decrease in fluorescence intensity was observed up to 59.4, 55.4, and 54.3% with a small redshift (8, 3, and 2 nm) (Figure 4(c) and S26, Supporting Information). The quantitative assessment of the interaction of the complexes with CT DNA was given by Stern-Volmer equation, Fo/F ¼ 1 þ Kq [Q] where Fo and F are the fluorescence intensities in the absence and presence of complex respectively, Kq is a linear Stern-Volmer quenching constant, and [Q] is the concentration of complex. The slope of the plot of Fo/F versus [Q] gave Kq (Figure 5(c,d)). The apparent DNA binding constant (Kapp) values were calculated by using the equation KEB [EB] ¼ Kapp [complex], where [complex] is the complex concentration at 50% reduction in 4 the fluorescence intensity of EB (Muralisankar et al., 2016). The Kq and Kapp quenching constant is 1 > 2 > 3, as described in Table 4. The free energies of complexes 1, 2, and 3 were evaluated as negative values and are –27.32, 26.89, and –26.42 kJ mol1. These values indicate that these complexes with DNA interaction spontaneity. 2.5. Protein interaction studies 2.5.1. UV-Visible absorption spectra In order to determine the quenching mechanism, the BSA UV-Visible absorption spectrum in the presence and absence of complexes (1-3) were studied. Quenching usually occurs either by dynamic or static mode. Dynamic quenching is a process in which the fluorophore and the quencher come JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 7 Figure 6. (a) BSA (10 lM) and complexes (1-3) (4 lM) BSA absorption spectra, (b) In the absence and presence of complex 1 fluorescence quenching curves of BSA. [BSA] ¼ 1 mM and [complex] ¼ 0–20 mM. (c) The Stern-Volmer diagram of the titrations of fluorescent complexes to BSA. (d) Fluorescence titration of complexes with BSA from Scatchard equation. into contact during the transient existence in the excited state. On the other hand, static quenching refers to the formation of a fluorophore-quencher complex in the ground state. UV-Visible absorption spectroscopy is the tool to determine the type of quenching involved. The addition of the complexes to BSA leads to an increase in BSA absorption intensity without affecting the position of the absorption band (Figure 6(a)). It showed the existence of static interaction between BSA and the complexes (1-3). The complexes (1-3) indicated a static type of quenching mechanism as reported (Krishnamoorthy et al., 2012; Lakowicz & Weber, 1973; Ramachandran et al., 2012; Sathyadevi et al., 2012). 2.5.2. Fluorescence spectra Protein fluorescent properties are largely attributable to residual tryptophan, tyrosine and phenylalanine. Fluorescence changes reflect conformational changes in the protein (Burstein et al., 1973; Mukhopadhyay et al., 2015; Selvakumaran et al., 2014). The interactions of BSA (bovine serum albumin) with complexes (1–3) were studied using fluorescent spectroscopy. BSA solution (1 lM) with complexes (1–3) (0–20 lM) and changes in fluorescence spectra in 290–500 nm (kex 280 nm, Figure 6(b) and S26, Supporting Information) were reported. A fluorescence intensity reduction of BSA was due to the addition complexes (1–3) (74.6%, 2 nm, complex 1; 75.2%, 1 nm, complex 2; 69.5%, 1 nm, complex 3, respectively). The quenching of BSA during the addition of complexes (1-3) indicates the interaction of the molecules and the consequent conformational changes. Figure 6(c,d) have measured the binding constant and number of binding sites. Table 5 shows the results of Kq, Kb, and n. The Kq values of complexes (1–3) showed a higher binding potential with BSA in complex 2. The binding sequence observed is 1 > 2 > 3. From the Scatchard equation, n values obtained for all molecules were 1, which suggests that the number of binding sites of BSA for each molecule is one. The Kb values also reflect the higher binding potential of complex 1, compared with BSA complexes 2 and 3. The free energies of complexes 1, 2, and 3 were evaluated as negative values and are –27.42, 26.96, and 25.70 kJ mol1. These values indicate complexes with BSA interaction spontaneity. 8 M. MUSTHAFA ET AL. 2.6. Frontier molecular analysis (FMO) and molecular electrostatic surfaces (MESP) 2.6.1. FMO analysis FMO helps to identify electronic transitions and kinetic stability, electric and optical characteristics (Bhat et al., 2019; Li et al., 2013; Konakanchi et al., 2019). FMOs were determined by using BL3YP; LANL2DZ theory rate of all three complexes (1–3) as shown in Figure 7. The HOMO-LUMO energy gap of complexes (1–3) are 3.03, 3.02, 2.98 eV, respectively (Table S1, Supporting Information). The small difference in the HOMO–LUMO energies of the complexes depicts their similar reactivity nature. In order to assess the important chemical reactivity descriptors such as softness, hardness, electro negativity, chemical potential, and electrostatic and ionization energy, this HOMO–LUMO energy gap was investigated. Chemical hardness (g) and softness is basically the measurement of chemical reactivity which stabilizes the system and chemical potential l by applying a charge which gives an idea about the transfer of charge from higher to lower Table 5. Constant Protein Binding (Kb), Constant Stern-Volumer (Kq) and number of binding sites (n) values of complexes (1–3). Complex 1 2 3  Kb (M–1) DG (kJ mol1) Kq (M–1) n 5.966  10 ± 0.04 4.956  104 ± 0.06 2.989  104 ± 0.07 –27.42 –26.96 –25.70 7.864  104 ± 0.09 4.988  104 ± 0.04 3.711  104 ± 0.07 0.945 0.923 0.977 4 potential (Figure 7). HOMO–LUMO energy of the complexes (1–3) are simulated by using theory level BL3YP/LANL2DZ (Bhat Lone, Ali, et al.,2018,; Bhat, Lone, & Srivastava, 2018). Electro negativity (v) represents the tendency to attract electrons. These properties have been defined as follows: g ¼ (I – A)/2 l ¼ – (I –A)/2 v ¼ (I þ A)/2 In which I and A represents the ionizing potential and electron affinity of the complexes obtained by HOMO and LUMO energies in the form of I¼ –EHOMO and A ¼ –ELUMO by Janak theorem and Perdew et al. With the use of these equations these descriptors were calculated. It has been observed that the hardness (g) is directly associated with stability is 1.76, 1.66 1.76 e.V for complexes (1–3), respectively. The chemical potential l is primarily the tendency of the electrons to escape from an equilibrium system and it was found to be –3.36, –3.64, and –3.52 for complexes (1–3), respectively. The global index of electrophilicity (x) is related to the chemical hardness and chemical potential and initially developed by Parr et al. This represents the measure of energy stabilization achieved when an electronic charge from the environment is obtained from the system and is given by the x ¼ l2/g. For complexes (1–3), corresponding values are 6.39, 7.98, 7.04 e.V. All of this was measured using BL3YP/LANL2DZ basis set for the target complexes and is shown in Table S1 (Supporting Information). From Table S1 (Supporting Information), it is clear that the energy gap of HOMO–LUMO Figure 7. The energy level complexes (1-3) of HOMO and LUMO of complexes simulated by DFT; theory level BL3YP/LANL2DZ. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 9 Figure 8. The molecular electrostatic surfaces of complexes (1–3). Table 6. Molecular docking parameters (B-DNA) of complexes (1-3). Complex 1 2 3 No. of rotational bonds 9 9 9 Binding constant of DNA (Kb) 5.718  104 ± 0.09 4.811  104 ± 0.06 3.991  104 ± 0.03 Best binding energy (kcal/mol) –4.15 –4.00 –3.04 Nucleotide residues involved in the interaction with complexes DA 17, DA 18 DT 7, DA 18 DG 4, DA 5, DA 17 and reactivity descriptors like hardness, potential and global electrophilicity of each three complexes is more or less similar, suggesting their almost similar behavior in the study of the interaction between enzymes and proteins. molecular docking process using AutoDock 4.2 molecular docking software and were displayed with Maestro €dinger software (Gopalakrishnan et al., 2017; Konakanchi Schro et al., 2018; Lone et al., 2018; Morris et al., 2009). 2.6.2. MESP analysis 2.7.1. Molecular docking with DNA This is important to predict measures of molecular reactions (Costa et al., 2017; Lone et al., 2018; Ramaiah, Srishailam, et al., 2019). The molecular reactivity to charged reactants is measured and hydrogen binding interactions are shown. The molecular electrostatic surfaces show fundamental properties, such as electron density scale, shape and variance, while correlating them with dipole moment, partial charges, electro negativity and chemical reactivity in the molecule (Ramaiah et al., 2020). MESP for analysis of drug-receptor and enzymesubstrate interactions together with H-bonding interactions has been studied by the computational organic chemists. A contrasting view of the molecular electrostatic maps is shown in Figure 8. Complexes (1–3) MESP determined with the basis set BL3YP; LANL2DZ has a similar or more similar environment. The photo representation of electrostatic potential in the rainbow color scheme is between – 6.138 e2, þ 6.138 e2, –6.315 e2, and þ 6.315 e2 between –6.018 e2 and þ6.108 e2, for complexes (1-3), respectively. The brightest red region represents the high electron density region (negative potential), and the darkest blue region shows the weak electron density region (positive potential). The experiments carried out by MESPs have shown the three complexes areas most vulnerable to high electron density electrophilic attack involving carbonyl oxygen. Molecular docking is an important in-silico computational tool for the rational design of new chemotherapeutic drugs, which predicts non-covalent interaction between the drug molecules and the nucleic acids of DNA. Confirmations of docked complexes were analyzed in terms of energy (kcal/mol). From the docking scores, the binding energy of the complexes were calculated and details are shown in Table 6. Molecular docking experiment reveals that the docked complexes fit into the DNA comfortably, without disrupting the double-helical structure of DNA, resulting in the binding energy between –3.04 and –4.15 kcal/mol. Nucleotide residues involved in the interaction with complexes (1-3) is DA 17, DA 18; DT 7, DA 18 and DG 4, DA 5, DA 17, respectively. Complex 1 showed higher binding energy of –4.15 kcal/mol and binding constant of DNA (Kb) of 5.718  104 ± 0.09 when compared to complexes 2 and 3. The molecular docking model with DNA (PDB ID: 1BNA) of complex 1 is shown in Figure 9. 2.7. Molecular docking study DNA (PDB ID: 1BNA), BSA (PDB ID: 3V03) and human aromatase enzyme (PDB ID: 3EQM) were subjected to the 2.7.2. Molecular docking with BSA The molecular docking technique is an attractive scaffold to understand the ligand-protein interactions which can substantiate the experimental results. Conformations of docked complexes were analyzed in terms of binding energy and hydrophobic interaction between complexes and BSA. The molecular docking experiment reveals that the docked complexes fit into the BSA by Van der Waals interaction. The binding energy of the individual complexes has been tabulated and the highest binding energy for each of the 10 M. MUSTHAFA ET AL. Figure 9. Molecular docking model of complex 1 with DNA (PDB ID: 1BNA). Table 7. Molecular docking parameters of complexes (1–3) with BSA (PDB ID: 3V03). No. of Rotational bonds Number of binding sites (n) Binding constant of BSA (Kb) Best binding energy (kcal/mol) 1 9 0.945 5.966  104 ± 0.04 –12.91 2 9 0.923 4.956  104 ± 0.06 –10.48 3 9 0.977 2.989  104 ± 0.07 –3.91 Complex complexes has shown in Table 7. Complex 1 has the highest binding energy and the value of –12.91 kcal/mol and binding constant of BSA (Kb) of 5.966  104 ± 0.04 when compared to complexes 2 and 3. Interaction of complex 1 with BSA (PDB ID: 3V03) molecular docked model is shown in Figure 10. The binding force in terms of Van der Waals interactions of complex 1 is GLU A:29, LYS A:439, THR A:190, LYS A:294, VAL A:342, ASP A:450, ARG A: A217, complex 2 is HIS A:145, LEU A:189 and complex 3 is ILE A:455, ARG A:458. Type of binding interaction of complex 1 is pi-cation: ARG A:194 mixed pi/alkyl hydrophobic: TYR A:451, CYS A:447: complex 2 is pication: ARG A:458, GLU A:424 Pi-alkyl: LEU A:454, ILE A:455, ALA A:193 and complex 3 is mixed pi/alkyl hydrophobic: ALA A:193, LEU A:189, HIS A:145 pi-cation: LYS A:431, LEU A:454. 2.7.3. Molecular docking with the human aromatase enzyme All the complexes (1–3) were subjected to molecular docking with human aromatase enzyme using the AutoDock Tools Binding forces Van der Waals: GLU A:29, LYS A:439, THR A:190, LYS A:294, VAL A:342, ASP A:450, ARG A: A217 Van der Waals: HIS A:145, LEU A:189 Van der Waals: ILE A:455, ARG A:458 Type of binding interaction Pi-cation: ARG A:194 Mixed pi/alkyl hydrophobic: TYR A:451, CYS A:447 Pi-Cation: ARG A:458, GLU A:424Pi-Alkyl: LEU A:454, ILE A:455, ALA A:193 Mixed pi/alkyl hydrophobic: ALA A:193, LEU A:189, HIS A:145 Pi-cation: LYS A:431, LEU A:454 (ADT) version 1.5.6 and AutoDock version 4.2 molecular docking software and were displayed with Maestro €dinger software. The X-ray crystallographic structure of Schro the human aromatase enzyme (PDB ID: 3EQM) was retrieved from Protein Data Bank. Docked ligand conformation was analyzed in terms of energy, hydrogen bonding, and hydrophobic interaction between the ligand and human aromatase enzyme. The binding energy of the individual complexes has been tabulated and the highest binding energy for each of the complexes has shown in Table 8. Complex 1 has the highest binding energy of -6.48 kcal/mol and inhibition constant (Ki) of 38.19 when compared to complexes 2 and 3. The binding force in terms of Van der Waals interactions of complex 1 is ASN A:571, GLU A:489, GLY A:488, PRO A:666, ASN A:631, ASP A:644, GLY A:490, VAL A:495, ASP A:626, LYS A:517,complex 2 is ASP A:626, PHE A:492, GLY A:490, GLU A:489, ALA A:491, ASN A:571 and complex 3 is GLY A:488, GLY A:490, ASN A:631, LYS A:517, LEU A:633, ASP A:644. Types of binding interaction seen in complexes (1–3) are pipi interactions, hydrogen bond, pi-cation interaction, pi-alkyl JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 11 Figure 10. Molecular docking model of complex 1 with BSA (PDB ID: 3V03). Table 8. Molecular docking parameters of complexes (1-3) against human aromatase enzyme (PDB ID: 3EQM). No. of Rotational bonds 9 Best binding energy (kcal/mol) –6.48 Inhibition constant (Ki) lM 38.19 2 9 –2.11 15.16 3 9 –1.93 28.33 Complex 1 Binding forces Van der waals: ASN A:571, GLU A:489, GLY A:488, PRO A:666, ASN A:631, ASP A:644, GLY A:490, VAL A:495, ASP A:626, LYS A:517 Van der waals: ASP A:626, PHE A:492, GLY A:490, GLU A:489, ALA A:491, ASN A:571 Van der waals: GLY A:488, GLY A:490, ASN A:631, LYS A:517, LEU A:633, ASP A:644 Type of binding interaction Pi-Pi interactions: LEU A:633, ARG A:630 Hydrogen bonds: ALA A:491 Pi-cation interactions: ARG A:630 Pi-alkyl: PRO A:666 Mixed pi/alkyl hydrophobic: ARG A:630, PHE A:492, ALA A:491, VAL A:495 Figure 11. Molecular docking model of complex 1 with human aromatase enzyme (PDB ID: 3EQM). interaction and mixed pi/alkyl hydrophobic interaction. Complex 1 interaction with BSA (PDB ID: 3V03) molecular docked model is shown in Figure 11. Based on the docking result of DNA, BSA interaction and in vitro experimental studies (Harshitharaj et al., 2016; Kumar et al., 2011), the target molecules were chosen to 12 M. MUSTHAFA ET AL. demonstrate the effects of the docking parameters (Tables 6–8), and the binding positions were shown in Figures 9–11 and S27–S32 (Supporting Information)). The docking finding showed the higher bonding energy in complex 1 for DNA (–4.65 kcal/mol), BSA (–12.91 kcal/mol), and human aromatase enzyme (–6.48 kcal/mol) than complexes 2 and 3 suggesting enhanced biological activity. 2.8. Cytotoxicity study of complexes (1–3) against HeLa S3, A549, and IMR90 cells Cytotoxic activity of complexes (1-3) were assessed against human cancer HeLa S3, A549, and normal IMR90 cells by MTT assay (Balachandran et al., 2018; Konakanchi et al., 2018; Ramaiah et al., 2019). The cytotoxicity results revealed that complexes 1 and 2 showed promising cytotoxic activity against HeLa S3 cell with 50% inhibition of cell proliferation was observed at 24 and 26 mM, respectively, as summarized in Table 9 and Figure 12. At the same time, complexes 1 and 2 showed moderate cytotoxic activity against A549 cells with 50% inhibition of cell proliferation was observed at 64 and 69 mM, respectively. Moreover, complex 3 showed weak and least cytotoxic activity against both tested HeLa S3 and A549 cells. The cytotoxicity study is then carried out with complexes (1-3) against normal IMR90 cells (Table 9 and Figure S33 in the Supporting Information), resulting no toxicity was observed up to 100 mM, suggest that complexes 1 and 2 as a promising anticancer agent against HeLa S3 cells than A549 cells with less toxicity. The Ru-p-cymene complexes (1–3) showed higher activity than the recently reported ruthenium complexes (Moideen et al., 2020). In addition, the cytotoxic activity also compared with well-known anticancer agent cisplatin. Table 9. IC50 values of complexes (1-3) against A549, HeLa S3, and IMR-90 cell lines. Complex A549 (mM) HeLa S3 (mM) IMR-90 (mM) 1 2 3 Cisplatin 64 69 83 23 24 26 56 26 >100 >100 >100 – 2.9. Fluorescence microscopic study of complexes 1 and 2 Morphological changes of HeLa S3 and A549 cells were assessed by a fluorescence microscope in the presence of complexes 1 and 2 using AO (acridine orange, 200 mM) and EB (ethidium bromide, 100 mM) (Jeyalakshmi et al., 2019). AO is an indicator of live cells and EB is an indicator of dead cells. HeLa S3 and A549 cells were treated with complex 1 (50 mM, 100 mM) and complex 2 (50 mM, 100 mM) for 24 h and then co-stained with AO/EB for 15 min at dark condition. The images were taken immediately at the fluorescence microscope (Biorevo, BZ-9000, Keyence). The results revealed that the dramatic morphological changes were observed when the exposure of complexes 1 and 2 in HeLa S3 and A549 cells is summarized in Figures 13 and 14. We speculated that complexes 1 and 2 induce apoptosis mediated cell death in cancer cells. 3. Conclusion Spectroscopic techniques like FTIR, UV-Visible, 1H, 13C NMR and mass spectra were used to characterize and confirm the structure of aniline substituted aroyl selenourea derivatives. The molecular structures were evaluated using single-crystal X-ray diffraction. Ligand L1 and complex 3 belongs to monoclinic crystal systems, with space groups P121/c1 and P121/ n1, containing four and two molecules per unit cell, respectively. In vitro anticancer activity of the complexes 1 and 2 have shown promising activity against HeLa S3 with IC50 values of 24 and 26 mM, respectively compared with standard drug cisplatin with IC50 value of 24 mM. Finally, these encouraging results are helpful in the in vivo biological applications in future endeavors. 4. Experimental 4.1. Synthesis of aniline substituted aroyl selenourea ligands (L1-L3) The ligands (L1-L3) were prepared by the following method. A solution of benzoyl chloride (0.6 mL, 5 mmol) or thiophene2-carbonyl chloride (0.6 mL, 5 mmol) or furan -2-carbonyl Figure 12. Cytotoxic study of complexes (1-3) against HeLa S3 and A549 cells. Three independent experiments were used to measure the results of mean ± SD. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 13 Figure 13. Cancer cell death and morphological changes of HeLa S3 cells were examined by fluorescence microscope (Biorevo, BZ-9000, Keyence, 20) using fluorescence dyes such as AO (acridine orange, 200 mM) and EB (ethidium bromide, 100 mM) after the treatment of complexes 1 and 2 for 24 h. Figure 14. Cancer cell death and morphological changes of A549 cells were examined by fluorescence microscope (Biorevo, BZ-9000, Keyence, 20) using fluorescence dyes such as AO (acridine orange, 200 mM) and EB (ethidium bromide, 100 mM) after the treatment of complexes 1 and 2 for 24 h. chloride (0.5 mL, 5 mmol) in dry acetone (25 mL) was added dropwise to potassium selenocyanate (0.485 g, 5 mmol) in dry acetone (25 mL). The reaction mixture was stirred for one hour at room temperature. After cooling, aniline (1 g, 5 mmol) dissolved in acetone (30 mL) was added to it dropwise, and the resulting mixture was refluxed for 2 h at 65  C. 14 M. MUSTHAFA ET AL. The reaction mixture was poured into hydrochloric acid (0.1 N, 200 mL) and the resulting pale yellow/brown substance was filtered off. Recrystallisation purified the solid product from a chloroform/ethanol mixture (1/2). 4.1.1 N-(phenylcarbamoselenoyl)benzamide (L1) Yield: 1.25 g, 74%. Yellow solid, m.p.: 154  C. Anal. Calc. for C14H12N2OSe (%): C, 55.46; H, 3.99; N, 9.24. Found: C, 55.38; H, 4.05; N, 9.20. UV–Vis (DMF): kmax, nm 260, 309. FT-IR: V, cm1 3344, 3260 (N  H), 3027 (¼C  H), 1667 (C ¼ O), 1262 (C ¼ Se). 1H NMR (500 MHz, CDCl3): d, ppm 12.98 (s, 1H, NH), 11.80 (s, 1H, NH), 8.00-7.98 (d, J ¼ 4 Hz, 2H), 7.69-7.68 (d, J ¼ 4 Hz, 3H), 7.58-7.56 (d, J ¼ 8 Hz, 2H), 7.55-7.47 (t, J ¼ 6 Hz, 2H), 7.45–7.34 (m, 1H). 13C NMR (125 MHz, CDCl3): d, ppm 180.96 (C ¼ Se), 168.61 (C ¼ O), 139.47, 133.71, 132.43, 129.26, 129.18, 128.92, 127.39, 125.62. LC-MS ¼ 303.02 [M – H]–. 4.1.2 N-(phenylcarbamoselenoyl)thiophene-2-carboxamide (L2) Yield: 1.30 g, 72%. Yellow solid, m.p.: 160  C. Anal. Calc. for C21H16N2OSeS (%): C, 46.61; H, 3.26; N, 9.06; S, 10.37. Found: C, 46.51; H, 3.29; N, 9.04; S, 10.12. UV-Vis (DMF): kmax, nm 276, 314. FT-IR: V, cm1 3389, 3270 (N  H), 3069 (¼C  H), 1668 (C ¼ O), 1269 (C ¼ Se). 1H NMR (500 MHz, CDCl3): d, ppm 11.80 (s, 1H, NH), 9.14 (s, 1H, NH), 7.74–7.72 (d, J ¼ 4 Hz, 1H), 7.62–7.60 (d, J ¼ 4 Hz, 1H), 7.48–7.46 (d, J ¼ 8 Hz, 2H), 7.43–7.42 (t, J ¼ 6 Hz, 2H), 7.39–7.21 (m, 2H). 13C NMR (125 MHz, CDCl3): d, ppm 179.72 (C ¼ Se), 161.11 (C ¼ O), 138.43, 135.62, 134.73, 131.04, 129.06, 128.67, 127.65, 124.72. LC-MS ¼ 308.96 [M – H]–. 4.1.3 N-(phenylcarbamoselenoyl)furan-2-carboxamide (L3) Yield: 1.06 g, 71%. Pale yellow solid, m.p.: 145  C. Anal. Calc. for C21H16N2O2Se (%): C, 49.01; H, 3.47; N, 9.52. Found: C, 49.82; H, 3.53; N, 9.56; UV–Vis (DMF): kmax, nm 283, 336. FTIR: V, cm1 3275, 3118 (N  H), 3045 (¼C  H), 1670 (C ¼ O), 1279 (C ¼ Se). 1H NMR (500 MHz, CDCl3): d, ppm 12.66 (s, 1H, NH), 9.48 (s, 1H, NH), 7.63–7.62 (d, J ¼ 4 Hz, 1H), 7.58–7.38 (d, J ¼ 4 Hz, 1H), 7.36–7.35 (d, J ¼ 8 Hz, 2H), 7.28–7.27 (t, J ¼ 6 Hz, 2H), 7.25–6.58 (m, 2H). 13C NMR (125 MHz, CDCl3): d, ppm 179.72 (C ¼ Se), 156.61 (C ¼ O), 146.73, 144.70, 138.47, 129.02, 127.58, 124.69, 119.35, 113.54. LC-MS ¼ 293.05 [M – H]–. 4.2. Synthesis of complexes cymene phenyl-H), 2.88–2.81 (m, 1H, p-cymene CH(CH3)2), 2.14 (s, 3H, p-cymene CH3), 1.82–1.75 (d, J ¼ 10.0 Hz, 6H, pcymene CH(CH3)2). 13C NMR (125 MHz, CDCl3): d, ppm 178.39 (C ¼ Se), 168.67 (C ¼ O), 135.6, 133.5, 133.5, 131.0, 130.9, 129.5, 128.8, 128.4, 128.1, 85.5 (benzene carbon). LC-MS ¼ 611.41 [M] þ. 4.2.2 [Ru(II)(g6-p-cymene) L2] (2) [RuCl2(g6- p-cymene)]2 (0.22 g, 0.44 mmol) and L2 (0.317 g, 0.88 mmol) were used. Yield: 83%. Yellowish orange solid, m.p.: 210  C. Anal. Calc. for C27H22Cl2N2O2RuSe: C, 42.93; H, 3.93; N, 4.55; S, 5.21. Found: C, 42.52; H, 3.35; N, 4.44; S, 5.15. UV-Vis (DMF): kmax, nm 275, 347, 436. FT-IR: V, cm1 3219, 3155 (N–H), 3155 (¼C–H), 1657 (C ¼ O), 1274 (C ¼ Se). 1H NMR (500 MHz, CDCl3): d, ppm 12.92 (s, 1H, NH), 11.32 (s, 1H, NH), 8.64 (s, 1H), 8.60 (d, J ¼ 8.7 Hz, 2H), 8.01 (s, 1H), 7.97 (dd, J ¼ 14.0, 7.5 Hz, 4H), 7.65 (t, J ¼ 7.4 Hz, 2H), 7.43 (s, 2H), 7.14 (s, 1H), 5.34–5.27 (d, J ¼ 10.0 Hz, 2H, p-cymene phenyl-H), 5.26-5.25 (d, J ¼ 5.0 Hz, 2H, p-cymene phenyl-H), 2.95–2.92 (m, 1H, p-cymene CH(CH3)2), 2.88 (s, 3H, p-cymene CH3), 2.23-1.69 (d, J ¼ 10.0 Hz, 6H, p-cymene CH(CH3)2). 13C NMR (125 MHz, CDCl3): d, ppm 178.59 (C ¼ Se), 163.52 (C ¼ O), 137.22, 136.42, 135.50, 134.99, 129.37, 128.61, 126.43 (aromatic carbons), 102.83, 83.22, 82.39, 81.33, 80.57 (aromatic carbons of p-cymene), 30.55, 22.30, 18.41 (aliphatic carbons). LC-MS ¼ 616.42 [M] þ. 4.2.3 [Ru(II)(g6-p-cymene) L3] (3) [RuCl2 (g6- p-cymene)]2 (0.22 g, 0.44 mmol) and L3 (0.331 g, 0.88 mmol) were used. Yield: 75%. Orange solid, m.p.: 205  C. Anal. Calc. for C27H22Cl2N2ORuSeS: C, 44.09; H, 4.04; N, 4.67. Found: C, 43.09; H, 4.08; N, 4.39. UV–Vis (DMF): kmax, nm 287, 382, 453. FT-IR: V, cm1 3225, 3149 (N–H), 3040 (¼C–H), 1675 (C ¼ O), 1268 (C ¼ Se). 1H NMR (500 MHz, CDCl3): d, ppm 12.82 (s, 1H, NH), 11.19 (s, 1H, NH), 5.36–5.23 (d, J ¼ 10.0 Hz, 2H, p-cymene phenyl-H), 5.26–5.25 (d, J ¼ 5.0 Hz, 2H, p-cymene phenyl-H), 2.94-2.23 (m, 1H, p-cymene CH(CH3)2), 2.86 (s, 3H, p-cymene CH3), 1.65–1.29 (d, J ¼ 10.0 Hz, 6H, p-cymene CH(CH3)2). 13C NMR (125 MHz, CDCl3): d, ppm 178.59 (C ¼ Se), 158.84 (C ¼ O), 137.17, 129.38, 128.66, 126.47, 122.82 (aromatic carbons), 112.91, 102.88, 98.90, 83.23, 82.33 (aromatic carbons of p-cymene), 31.25, 29.70, 18.42 (aliphatic carbons). LC-MS ¼ 600.42 [M] þ. 4.2.1 [Ru(II)(g6-p-cymene) L1] (1) [Ru(II)(g6-p-cymene)]2 (0.22 g, 0.44 mmol) and L1 (0.326 g, 0.88 mmol) were used. Yield: 71%. Orange solid, m.p.: 206  C. Anal. Calc. for C29H24Cl2N2ORuSe: C, 47.30; H, 4.30; N, 4.60. Found: C, 47.15; H, 4.42; N, 4.56. UV–Vis (DMF): kmax, nm 267, 334, 430. FT-IR: V, cm1 3207, 3120 (N–H), 3022 (¼C–H), 1663 (C ¼ O), 1262 (C ¼ Se). 1H NMR (500 MHz, CDCl3): d, ppm 13.17 (s, 1H, NH), 11.50 (s, 1H, NH), 8.25–8.24 (d, J ¼ 5.0 Hz, 1H), 7.92–7.84 (d, J ¼ 5.0 Hz, 1H), 7.82–7.61 (m, 4H), 7.39–7.60 (m, 1H), 7.43–7.08 (t, J ¼ 5.0 Hz, 1H), 5.39–5.27 (d, J ¼ 10.0 Hz, 2H, p-cymene phenyl-H), 5.14–5.20 (d, J ¼ 5.0 Hz, 2H, p- Acknowledgments The author Moideen Musthafa is thankful to MHRD, Govt. of India for providing senior research fellowship and Ramaiah Konakanchi, thanks the Malla Reddy Engineering College for Women (Autonomous Institution), Hyderabad, India, for support and encouragement during this research work. 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