Structural, spectral, DFT and biological studies on macrocyclic mononuclear ruthenium (II) complexes

Accepted Manuscript Structural, spectral, DFT and biological studies on macrocyclic mononuclear ruthenium (II) complexes M. Muthukkumar, C. Kamal, G. Venkatesh, C. Kaya, S. Kaya, Israel V.M.V. Enoch, P. Vennila, R. Rajavel PII:

S0022-2860(17)30913-4

DOI:

10.1016/j.molstruc.2017.06.132

Reference:

MOLSTR 24018

To appear in:

Journal of Molecular Structure

Received Date: 19 February 2017 Revised Date:

28 June 2017

Accepted Date: 29 June 2017

Please cite this article as: M. Muthukkumar, C. Kamal, G. Venkatesh, C. Kaya, S. Kaya, I.V.M.V. Enoch, P. Vennila, R. Rajavel, Structural, spectral, DFT and biological studies on macrocyclic mononuclear ruthenium (II) complexes, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.06.132. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract

ACCEPTED MANUSCRIPT Structural, spectral, DFT and biological studies on Macrocyclic mononuclear

Ruthenium (II) complexes M. Muthukkumar1, C.Kamal2, G.Venkatesh3, C. Kaya4, S.Kaya5, Israel V.M.V Enoch6, P.vennila7, R. Rajavel8 Department of Chemistry, Selvam Arts and Science College, Namakkal, Tamilnadu, India. 2, 3

Department of chemistry VSA Group of Institutions salem, Tamilnadu, India. 4,5

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Department of chemistry, Cumhuriyet University Sivas 58140.Turkey.

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1

Department of Chemistry,School of Karunya University,Coimbatore,TamilNadu,India

Department of Chemistry, Thiruvalluvar Government Arts College, Rasipuram– 637 401, India. 8

Department of chemistry, Periyar University, Salem- 636011, Tamilnadu, India.

Phone No.: +91 98650 94324

Abstract

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Corresponding author E-mail: [email protected]

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Macrocyclic mononuclear ruthenium (II) complexes have been synthesized by condensation method [Ru (L1, L2, L3) Cl2] L1 = (C36 H31 N9), L2= (C42H36N8), L3= (C32H32 N8)]. These ruthenium complexes have been established by elemental analyses and spectroscopic techniques (Fourier transform infrared spectroscopy (FT-IR), 1H- nuclear magnetic resonance (NMR),

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C- NMR and

Electrospray ionisation mass spectrometry (ESI-MS)). The coordination mode of the ligand has been

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confirmed and the octahedral geometry around the ruthenium ion has been revealed. Binding affinity and binding mode of ruthenium (II) complexes with Bovine serum Albumin (BSA) have been characterized by Emission spectra analysis. UV-Visible and fluorescence spectroscopic techniques have also been utilized to examine the interaction between ligand and its complexes L1, L2, & L3 with BSA.

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Chemical parameters and molecular structure of Ru (II) complexes L1H, L2H, & L3H have been determined by DFT coupled with B3LYP/6-311G** functional in both the gaseous and aqueous phases.

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Key words: Mononuclear ruthenium, Bovind serum Albumin, Density Functional Theory, IR, NMR. 1.0 Introduction

The challenge faced by the Pharmaceutical chemistry is numerous which includes the improvement of clinical performance of metal complex drugs for cancer cure viz, cisplatin, carboplatin and oxaliplath [1]. Also potency of other metal complexes for the treatment of a variety of pathologies, especially cancer is thoroughly researched. Enlargement of application of drugs and side effects caused by the existing drugs have opened-up the new way for the search of other metal complexes [2, 3]. Among other metal based complexes, ruthenium metal complexes have promising benefits since they have antiproliferation activity and of very low toxicity than platinum drugs. Enormous data were

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reported earlier on the use of Ru complexes as drug molecules and revealed have tumor seeking properties since they interact with serum proteins such as albumin and transferring [4, 5]. These complexes have been activated by the formation of toxic Ru (II) species generated by the intercellular reduction [6, 7]. From the literature, it has been found that, Ru (II) complexes have a tendency to bind with serum albumin proteins such as BSA and HSA (Human serum albumin), beside

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binding to the target DNA. As these drugs are transported as protein complexes it is important to examine the drug protein interaction. Biological activities of compounds having an amine and a system with carbonyl groups were evaluated for their antimicrobial and anti-HIV activities [8-11].

Amino heterocyclic compounds containing two (or) more potential donor centers play an

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important role in the study of comparative reactivity of bidentate ligand system [12]. Important physiological functions viz., cell nourishment, exchange of normal liquid, acidity of blood

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maintenance, colloid osmotic pressure sustenance, binding all sizes of exogenous and endogenous waters like fatty acids, amino acids, enzymes, hormones, drugs, billirubin, metabolic metallic ions, etc and maintain storage and transportation are governed by serum albumins which are the major soluble protein constituents of the circulatory system [12-14]. Molecular interactions of the drug molecules with serum albumins and the understanding of their clinical effects along with their metabolism will lead to the discovery of new medicines [14-16]. Further, the study of BSA and its binding features, help

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us to define its structures and functions. BSA is a major component of Plasma protein and is a single chain 582 amino acid globular nonglycoprotin cross linked with 17 cysteine residues (eight disulfide bonds and one free thiol). It has a linear and distinct structure [17-20]. Three novel Schiff base macrocyclic Ruthenium(II) complexes comprising Isatin, Benzil and 2,3-butadione based 4-

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aminoantipyrine and o-Phenylenediamine shown as L1H, L2H, & L3H respectively have been completely analysed for their structure and electronic properties in the present study. Further, their

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binding nature with the BSA has also been investigated using absorption and fluorescence spectroscopy.

2.0 Experimental 2.1 Materials

All the chemicals used in this study have been obtained in AR grade and were used without further purification. The solvents used were purified following the standard procedures. The buffer solutions were prepared using double distilled water. Crystalline BSA, 2-[4-(2-hydroxyethyl) piperazin1-yl] ethanesulfonic acid (HEPES) buffer were obtained from was been purchased from Sigma-Aldrich [15-17].

Hi-media India, whereas RuCl3.3H20

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2.2 General method

Elemental analyses (C, H, N and O) were recorded on a Vario EL III CHNS analysis at IISC, Bangalore. Infrared spectra of the complexes were recorded on a Perkin-Elemer 783 FT-IR spectrometer in the range 4000-400 cm-1 using KBr pellets. 1H NMR &13C NMR spectra were recorded on a Bruker using MeoD as solvent and TMS as an internal standard. Mass spectra for the ligand and

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complexes were recorded on Q-TOF microTM mass spectrometer using an electrospray ionization probe. Using cuvettes with 1 cm path lengths absorption spectra were recorded using a Jasco V-630, double beam UV-Visible spectrophotometer (Tokyo,Japan). A Jasco FP-750 Spectrofluorimeter fitted

nm was set for both emission and excitation. 2.3 Synthetic procedure for new ruthenium (II) complex

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2.3.1 Synthesis of Schiff bases (L1, L2 & L3)

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with a 120 W xenon lamp for excitation was used to record fluorescence spectra. A band width of 5

All the three Schiff bases viz., L1, L2 & L3 were obtained by mixing 4-aminoantipyrine (0.4 g, 0.2 mmol) & Isatin (1.46 g, 0.10 mmol), 4-aminoantipyrine (0.40 g 0.2 mmol) & Benzil (0.10 g, 0.10 mmol) and 4-aminoantipyrine (0.40 g, 0.2 mmol) & 2, 3-Butadion (0.4 g, 0.10 mmol), respectively in methanolic solution and were kept over magnetic stirrer and stirred for more than 3 h. All the three

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solids were obtained separately and are in a light brown color, then, they were filtered and vaccuo dried. The solids thus obtained were refluxed with o-phenylenediamine (0.4 g, 0.10 mmol) dissolved in 40 ml ethanol for 30 h separately in order to complete the synthesis. The Schiff bases L1, L2 & L3 were of light yellow, brown and brown in color were filtered off and then dried over CaCl2 (Fig.1 and

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Scheme 1, 2 & 3). Further, the obtained Schiff bases were recrystallized using methanol.

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2.3.2 Synthesis of Complex (L1H, L2H & L3H) The methanolic solutions of all the three synthesized Schiff bases were refluxed with the methanolic solution of metal (II) chloride (0.20 mmol.) for 6h. The volume of the solution was then reduced to one third of its initial volume by keeping it on a hot plate. Further, the precipitated solid complex was washed thoroughly with methanol and then dried in vaccuo. 2.3.3 Characterization data: L1H Yield: 70% (0.50 mg); Color: yellow; MP: 230-240 ⁰C; micro analytical data: C36 H31 N9 RuCl2 required: C, 56.77; H, 4.10; N, 16.36. found: C, 56.29; H, 3.90; N, 16.36; IR (KBr pellet, cm-1): 1608 ν(C=N); 1490 N(C=C); 442 ν(M-N); 316 ν(M-Cl); UV-vis (CHCl3), λmax (nm): 250, 261, 265. 1H

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NMR (300.13 MHz, CDCl3, ppm): 8.4 (Ar c-NH); 8.14 (-C=N); 6.78 – 7.78 (m, 14H); 3.33 (s, 6H); 13

C NMR (300.13 MHz, CDCl3, ppm): 152 (-C=N); 128 (Ar c); 48 (N-CH3); 36(C-CH3); ESI-MS

(Calcd, found, M/Z) = 761.00, 761.40 (M-Cl)+. L2H Yield: 60% (0.50 mg); Color: brown; MP: 235-240 0C; micro analytical data: C42H36N8 RuCl2

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required: C, 61.16; H, 4.40; N, 13.59. Found: C, 60.68; H, 4.20; N, 13.30; IR (KBr pellet, cm-1): 1614 ν(C=N); 1508 ν(C=C); 1228 ν(C-N); 450 ν(M-N); 321 ν(M-Cl); UV-vis (CHCl3), λmax (nm): 253, 264, 268. 1H NMR (300.13 MHz, CDCl3, ppm): 8.18 (-C=N); 7.35-7.90 (m, 24H). 3.33 (s, 6H); 13C NMR (300.13 MHz, CDCl3, ppm): 153.69 (-C=N); 128 (Ar c); 48.11 (N-CH3); ESI-MS (calcd, found, M/z) =

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824.07; 824.12 (M-Cl)+. L3H

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Yield: 65% (0.55 mg); Color: brown; MP: 240-2450C; Micro analytical data: C32H32N8 RuCl2 required: C, 54.86; H, 4.60; N, 15.99; Found: C, 53.81; H, 3.78; N, 14.08; IR (KBr pellet, cm-1): 1606 ν(C=N); 1491 ν(C=C); 1197 ν(C-N); 450 ν(M-N); 318 ν(M-Cl); UV-vis (CHCl3), λmax (nm): 245,264, 270. 1H NMR (300.13 MHz CDCl3, ppm): 8.08 (-C=N); 7.85-7.87 (m,14H); 3.33 (s,6H); 2.83 (s,6H); 13

C NMR (300.13 MHz, CDCl3, ppm): 154.28(-C=N); 126.59 (Ar c); 48.12 (N-CH3); ESI-MS (calcd;

2.4 Computational details

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found, M/Z) = 700,701 (M-Cl).

Structural parameters were calculated using Gaussian 09W software package and the optimized molecular structures were visualized by Gauss view 5.0 programs [21]. The optimized molecular

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structure of Schiff bases complexes L1H, L2H and L3H have been computed using DFT/B3LYP/6311G+ (d, p) basis set. The energy level diagram of HOMO and LUMO (L1H, L2H and L3H) were determined by DFT calculations. The optimized molecular Structure, bond length, bond angle, dihedral

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angle and chemical reactivity L1H, L2H & L3H have been established using DFT method. 3.0 Results and Discussions 3.1 Spectroscopies studies

In order to explain the co-ordination of C=N group, the IR spectra of free ligand were compared with newly synthesized ruthenium (II) complexes, where L1H, L2H, & L3H exhibited intense bands at 1647 cm-1, 1645 cm-1 and 1650 cm-1, respectively [21-23]. However the complexes showed sharp bands at 1604 cm-1, 1600 cm-1 and 1600 cm-1 which corresponding to the azomethine. A slight deviation in frequencies by 40-30 cm-1 in the spectrum of the complex indicated the coordination of azomethine group to ruthenium atom. The absence of strong absorption band in complex L2H near 1700 cm-1,

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indicated the absence of the >C=O group of benzil and confirmed the condensation of oxygen atom the carboxyl group of benzil and the nitrogen atom of the amino group of the o-phenylenediamine [23, 24]. The band at 450-470 cm-1 could be due to (M-N) and showed the coordination of the azomehine nitrogen to the central metal atom [25-28]. In all the complexes the bands corresponding to (M-N) appeared at 310-343 cm-1. UV-Vis Spectra of all the three complexes recorded in methanol showed the four intense

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absorption bands in the region around 250-430 nm. In the electronic spectra of (L1H, L2H, & L3H) complexes two bands appeared in the region around 250-261 nm which have been assigned to the intra – ligand transition. These bands absorption maxima located in the 340-420 nm range may be assigned to a N(Pπ) Ru (dπ) LMCT transition caused by the promotion of electrons from the completely HOMO

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of the ligand of primary nitrogen to the empty LUMO of ruthenium dπ character [29]. The other high intensity bands at 262-291 nm might be assigned to the CT transition arising from the promotion of an

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electrons from the metal t2g level to LUMO derived from π* level of the ligand, in accordance with the assignments made for other similar octahedral ruthenium(II) complexes [30, 31] as shown in Fig. 2A, B & C. The emission spectra of complexes (L1H, L2H, & L3H) showed a shoulder peak at 380 nm and excited peak at 340 nm in each case respectively. From this absorption it has been confirmed that there is of the impurity in the complexes. Therefore, there was maximum in absorptivity. Magnetic, conductance analysis all the macrocyclic Ru(II) Schiff base complexes were found to

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have diamagnetic character assigning +2 oxidation state to ruthenium. The molar Conductivity measurement of all the complexes in 10-3 M DMSO solution indicated the non-electrolyte (below 2 S cm2mol-1).

H NMR spectra of complexes (L1H, L2H, & L3H) were recorded in MeoD shown in Fig. 3A,

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B & C. The 1H NMR spectra of the ligand showed a singlet at 8.14 to 8.2 ppm which correspond C=N

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group. However, this signal is shifted to an upfiled region of 8.1-8.4 ppm in the case of macrocylic Ru(II) complexes [32]. This could be due to the coordination of nitrogen atom with Ru(II) ion. The sharp singlet observed in the region of 8.4 ppm corresponding to the NH group in both free ligand and complex L1H is indicating the non- participation of NH in coordination [33] (Fig.3A). The 1H NMR for the multiple protons of the aromatic ring moiety of the ligands and metal complexes were observed as multiples in the range of 6.78-778 ppm. 13

C NMR spectra complexes (L1H, L2H, & L3H) were recorded in the MeoD shown in Fig. 4A

B & C. The

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C NMR Spectra showed the signal at 140.79 ppm, which colud be assigned

uncoordinated C=N group. In the case of complexes the down field shift was observed around 153.69, 154.28 ppm indicating the coordinated C=N group. The other signals at 128.94, 130.08 ppm for carbon

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atoms of phenyl group, 48.13 for N-CH3 and 35.94 ppm for C-CH3 are quite agreed well with the literature [34, 35]. The ESI–MS analyses of complexes L1H, L2H & L3H showed most abundant peak at M/z 761.00, 824.07 and 700.00 respectively, for [M-C]+ ions which revealed that, the identity of the complexes was retained in solution. The observed isotopic distributions and their simulation patterns

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are in good agreement with the assigned formulations which are shown in Fig.5A, B & C. 3.2 Molecular geometry

Ground-state electronic structure calculations of all complexes (gaseous phase as well as aqueous phase) under investigation have been done using density functional theory (DFT) method

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along with the GAUSSIAN 09W software package. The functional used throughout this study is the B3LYP, consisting of a hybrid exchange functional as defined by Becke’s three parameter equation and

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the Lee–Yang–Parr correlation functional. The ground state geometries were obtained in both the gaseous phase and aqueous phase by full geometry optimization, starting from structural data. The optimum structures located as stationary points on the potential energy surfaces were represented by the absence of imaginary frequencies [36-40]. Molecular geometry optimized structures and numbering of Ru complexes (L1H, L2H, & L3H) is shown Fig. 6 respectively. Bond angles, bond lengths and dihedral angles of studied complexes are presented in Table 2, 3 & 4. The bond angle, length and

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dihedral angle are slightly varied from experimental values. The calculated bond length complexes of Ru1-Cl3 (2.45 Å), Ru1-Cl2 (2.42 Å) Ruthenium (II) complexes (L1H), Ru46-Cl47 (2.41 Å), Ru46Cl48 (2.46 Å) complexes (L2H), Ru1-Cl3 (2.41 Å) and Ru1-Cl2 (2.45 Å) complexes (L3H) are slightly

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deviated from the experimental bond length of Ru-Cl (2.49 Å). The electrophilic and nucleophilic Molecular Electrostatic Potential (MEP) surface of L1H, L2H, & L3H is shown (Fig.6) in dissimilar colour grades [38-40]. In MEP map of the electrophilic (positive) interior is pointed out in red colour

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where as nucleophilic (negative) interior pointed out in blue colour. The MEP diagram reveal the physic chemical properties viz., molecular size, shape, negative, positive and neutral electrostatic potential. The nucleophilic regions are frequently associated with the lone pair of electronegative atom. 3.3 Global and Local Reactivity descriptors. It is known that, DFT is one of the important tools used to predict the chemical properties of atomic and molecular systems. The stability or reactivity of chemical systems can be analysed easily with the help of DFT. DFT of chemical reactivity is known as Conceptual Density Functional Theory (CDFT). Concentrating on the interpretation of the Lagrangian multiplier, µ, given in the Euler equation derived with the help of Hohenberg and Kohn theorems, Parr [35] and co-workers introduced

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the CDFT. In the light of CDFT, quantum chemical descriptors such as chemical potential (µ), electronegativity (χ) and chemical hardness (η) are defined via the following equations as the partial derivatives of the total electronic energy (E) with respect to number of electrons (N) at a fixed external potential. It should be noted that the electronegativity is considered the negative of the chemical

µ = −χ = (

∂E ) − − − − − − − (1) ∂N ν ( r )

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potential (χ=-µ).

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1 ∂2E 1 ∂µ η= ( ) = ( ) − − − − − − − (2) ( r ) ν 2 ∂N 2 2 ∂N ν (r )

In 1960s, Pearson who introduced the Hard and Soft Acid Base (HSAB) principle that states which “hard acids prefer to coordinate to hard bases and soft acids prefer to coordinate to soft bases”

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defined the chemical hardness as the resistance towards electron cloud polarization or deformation of atom, ion or molecules. Many experimental studies showed that HSAB is generally valid in complex formation reactions. For that reason, in many studies related to synthesis of transition metal complexes, mentioned principle has been took into consideration. Softness (σ) that is a measure of the polarizability of chemical systems is given as the multiplicative inverse of chemical hardness. 1

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σ = ( ) − − − − − − − (3) η

To get rid of the complexity of Eq. 1 & Eq. 2 with a view to calculate the chemical hardness,

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electronegativity and chemical potential of chemical species with the help of their ionization energy (I) and electron affinity (A) values, Pearson and Parr obtained [35, 37] and used the following equations for the prediction of aforementioned quantum chemical parameters applying the finite difference

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method to Eq.1 and Eq. 2.

χ = −µ = ( η =(

I+A ) − − − − − − − ( 4) 2

I−A ) − − − − − − − (5) 2

To predict the ionization energy and electron affinity values of molecules, Koopmans proposed an alternative method that is known as Koopman’s theorem [41]. According to this theorem, ionization energy and electron affinity of a molecule are approximately equal to negative values of its HOMO and

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LUMO orbital energies, respectively. Within the framework of mentioned theorem and using Eq. 3 and Eq. 4, the equation can be written as,

η=

- E HOMO − E LUMO − − − − − − − (6 ) 2

E HOMO − E LUMO − − − − − − − (7) 2

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χ = −µ =

One of the quantum chemical descriptors used to discuss the electron donating or accepting abilities of molecules is electrophilicity. According to an electrophilicity index introduced by Parr,

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electrophilic power of a chemical compound is associated with its electronegativity and chemical hardness (Eq.7). In addition, nucleophilicity is given as the multiplicative inverse of the electrophilicity

ω=

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with a similar logic to the correlation between hardness and softness.

µ2 χ2 = − − − − − − − (8) 2η 2η

ε=

1

ω

− − − − − − − (9 )

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There is a remarkable correlation between proton affinity (PA) and gas phase basicity. Molecules including heteroatom’s like O and N exhibit high tendency to protonation process. Although some theoretical models proposed to calculate the proton affinities of molecules, in general, in such

equation is used.

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studies including the use of computational chemistry programs like Gaussian 0.9, the following

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PA = E ( pro ) − ( E ( non − pro ) + E ( H + ) ) − − − − − −(10)

where, Enon-pro and Epro are the energies of the non-protonated and protonated inhibitors, respectively. EH+ is the energy of H+ ion and was calculated as:

E H + = E ( H O ) + − E ( H 2O ) − − − − − −(11) 3

In the section about DFT analysis of synthesized Ru complexes, Gaussian 0.9 program has been used. Geometries of all the studied compounds were fully optimized based on density functional theory (DFT) with functional B3LYP [35, 42-44] which has become very popular in recent times.The calculations in both gas phase and aqueous phase were performed considering B3LYP/3-21G, B3LYP/LANL2DZ and B3LYP/STO-3G calculation levels. In the Fig. 6, optimized structures,

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HOMOs, LUMOs and electrostatic potential structures of studied Ru complexes (L1, L2 and L3) are given. In the tables of supplementary material S1 and S2, calculated quantum chemical parameters like EHOMO, ELUMO, HOMO-LUMO energy gap, hardness, softness, electronegativity, proton affinity, electrophilicity, nucleophilicity and total electronic energy for mentioned complexes in the gas phase and aqueous phase are reported, respectively.

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As is known, more stable molecules have the lower total energy values compared to others. Namely, the energy is an important tool in terms of the analysis of the stabilities of molecules. Another parameter considered as stability criteria is the chemical hardness. According to Maximum Hardness Principle, “there seems to be a rule of nature that molecules arrange themselves so as to be as hard as possible, namely chemical hardness can be considered as measure of the stability”. It is apparent from

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complex with low energy and high hardness values is L3H.

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the data given in both the Tables S1 and S2 of supplementary material indicated the most stable

3.4 BSA interaction studies (BSA binding using fluorescence spectra) The complexation was achieved by adding complexes o L1H, L2H & L3H of the concentration of 3.0 ×10-5 mol dm-3 with BSA of about 3.0×10-5 mol dm-3 concentration in complexes titration. The observed spectra data are listed in Table 1. The addition of complexes L1H, L2H, & L3H shifted the absorption bands to the longer wavelength which was centered at 338-340 and 341nm

and is

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corresponded to n → π* transition of tryptophan residues in BSA and this could be due to the binding of complexes L1H, L2H, & L3H. A hyperchromic shift was seen in the absorbatice. Fig 7A to C showed the fluoresescence spectra of BSA with different concentrations. Addition of complexes to

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BSA results in red shift in fluorescence [45-47]. There was the observation of quenching of florescence after the addition of L1H, L2H, & L3H to aliquot. This quenching of fluorescence was caused by the binding of complexes L1H, L2H & L3H with BSA. Fig.8A shows the stren-volmer plot for the

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quenching of tryptophan. The observation of linear plot with the linear regression of about very close (0.9928 & 0.9942 calculated using straight line equation) to unity suggested the static quenching. The Ksv value was calculated from the plot as 1.002×105 mol-1 dm3. Fo / F = 1 + KSV [Q ] − − − − − −(12)

Where, F0 and F are the fluorescence intensities of BSA in the absence and presence of the quencher, respectively [Q] is the concentration of the quencher and Ksv is the stren-volmer quencher constant. The binding constant and number of binding sites were calculated from Eq.3. The Ksv value was calculated from the plot was 1.062x105mol=1 dm3.

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LogFo − F / F = n log K A − n log(1 /[ Dt ] − ( Fo − F )[ Pt ] / Fo) − − − − − −(13) Where, Fo is the initial intensity of fluorescence before the addition of the quencher and F is the fluorescence intensity of BSA at each addition of complex L1H, L2H, & L3H. [Dt] and [Pt] refer to the concentration of the complexes L1H, L2H, & L3H the concentration of protein (BSA), respectively. The plot of log (1/[Dt]-(FO-F) / [Pt] [Fo] vs. (Fo-F) / F is shown in Fig.8A & 8B for all the complexes sites was 1.72 (approximately 2). 3.5 Binding mode of L1H complex with topisomerase II

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L1H, L2H, & L3H. The calculated binding constant is 1.594x105 mol-1 dm3 and the number of binding

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L1H complex was subjected to antimicrobial study against two gram positive and two gram negative bacteria. The result of the study revealed that, the complex has potent activity than the free ligand [48]. Hence an attempt has been made to study the antibacterial activity through molecular

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docking for the ruthenium complex of the L1H, L2H, & L3H. Studies were carried out using GLIDE extra precision (XP) Schrödinger 9.5 software. The 3D crystal structure of the targeted protein topisomerase II was retrieved from the protein data bank.

Hydrogen bonding interaction plays a pivotal role in the action of both and antagonists both through hydrogen bonds involve a conformational change in the protein with resulted action. The

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molecular energy value is a indicative of the nature of hydrogen bond. It could be, i) Weak when the value is about < 20-50KJ/mole and ii) Moderately weak is the value is 80-150KJ/mole. The weak hydrogen bond may be existed between as D...H...A (D- Donor, A- Accept). Where, the hydrogen atom

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forms a bond between two structural modes.

Docking simulation [49] of L1H within the active site of topisomerase II has been analyzed. The Glide energy value for (1) was observed – 6.86 kcal/mol upon examination of docking feature

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between (I) and target protein has been observed that there is an existence two hydrogen bonds interactions. The back bone oxygen atom of the Asp has been well interacted with hydrogen atom of L1H complex. Further ARG, 518 ALA, 431 GLU 28 THR and TYR 77 and number of hydrophobic interactions are formed between IPBA and target. Further Docking studies have been carried out on the same protein against cefixime, a standard drug. This has glide energy value of -157.87 against Asp. These results showed that the compound (L1H) has least score against topisomerase (II) 3.6 Binding mode of L2H complex with topisomerase II Docking stimulation [49, 50] of L2H complex with the active site of topisomerase (II) has been analyzed. The results of this study observed the glide energy value as – 4.32 k Cal/mol. It could be

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due to because of the interaction of the backbone oxygen atom of Asp with compound (L2H). It has three hydrogen bond interactions. For the same protein, docking study was performed with standard drug amoxicillin. The results of the study revealed that, the stansdard drug interacted well interactions with the target protein and whose glide energy value is found as 144.54 kcal/mol. 3.7 Binding mode of L3H complex with topisomerase II

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Docking stimulation of (L3H) with topisomerase II showed that the glide energy value found on 3.48 kCal/mole. This is due to the interaction between the back bone oxygen atom of Asp and the compound (3). Among the three compounds the order of reactivity is as follows L1H>L2H>L3H.

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3.8 Binding mode of L1H complex with BSA and Naproxen

Docking study was performed [49, 50] for the compound L1H complex with BSA protein. The

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examination of the interaction between L1H complex and BSA suggested the existence hydrogen bond interaction. The formation H-bond has been further confirmed from glide energy values. For the same target protein, dockings study was carried out using a naproxen (anti-inflammatory) drug. The results of this study clearly revealed the interaction of the donor atom Asp with acceptor N atom of the compound.

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4.0 Conclusion

Three new macrocyclic ruthenium (II) tetradentate Schiff base complexes from three different ligands L1, L2 & L3 have been synthesized and duly characterized using various spectral techniques (IR, UV-Vis, NMR and ESI-MS). The octahedral geometry of all the studied complexes has been

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confirmed from the results of spectral data. The results of emission spectra revealed the binding nature of studied complexes (L1H, L2H & L3H) with BSA which was evidenced from the quenching of

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fluorescence. Further, it is supported from the observation of decrease in absorption of the impurity with increasing photon energy. The physico-chemical parameters of all the three complexes have been calculated using DFT. From the results obtained, it is clear that, the complex L3H has been found to be most stable (low energy and high hardness values) among all the three complexes examined in the present study. The results of Molecular Docking study suggested the order of reactivity of all the studied complexes as follows L1H>L2H>L3H. Further, it is clear from the present study that, the interaction of the studied complexes with BSA is existed between donor atom Asp and acceptor N atom of the compound.

ACCEPTED MANUSCRIPT

5.0 References

[1] N.P. E. Barry, P. J. Sadler, Chem. Commun., 2013,49, 5106-5131 [2] B.W. Harper, A.M. Krause-Heuer, M. P. Grant, M. Manohar, K.B. Garbutcheon Singh, J.R. Aldrich Wright, Chem. – Eur. J, 2010, 16, 7064-7077. [3] N. J. Wheate, S.Walker, G. E. Craig, R. Oun, Dalton Trans, 2010, 39, 8113-8127.

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[4] L. Breydo, V. N. Uversky, Metallomics, 2011, 3, 1163-1180. [5] N. P. E. Barry, P. J. Sadler, Chem.Commun., 2013, 49, 5106-5131.

[6] P. C. A. Bruijninex, P. J. Sadler, Curr. Opin. Chem. Biol., 2008, 12, 197-206. [7] G. Suess-Fink, Dalton Trans., 2010, 39, 1673-1688;

[8] A. C. G. Hotze, B.M. Kariuki, M. J. Hannon, Angew. Chem., Int. Ed., 2006, 118, 4957-4960.

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[9] W. H. Ang, P. J. Dyson, Eur. J. Inorg. Chem., 2006, 20, 4003-4018.

[10] R. Atchudan, T.N. Jebakumar Immanuel Edison, D. Chakradhar, S. Perumal, J. Jin Shim, Y.R.

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Lee, Sens. Actuators, B, 2017, 246, 497-509.

[11] T.N. Jebakumar Immanuel Edison, R. Atchudan, J. Jin Shim, S. Kalimuthu, B. Cheol Ahn, Y.R. Lee, J. Photochem. Photobiol. B, 2016, 158, 235-242.

[12] D. Wang, S. J. Lippard, Nat. Rev. Drug Discovery, 2005, 4, 307-320. [13] P. J. Dyson, Chimia, 2007, 61, 698-703.

[14] E. R. Jamieson, S. J. Lippard, Chem. Rev., 1999, 99, 2467-2498.

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[15] A. R. Timerbaev, C. G. Hartinger, S. S. Aleksenko, B. K. Keppler, Chem. Rev., 2006, 106, 2224-2248.

[16] M.B. Ferrari, C. Pelizzi, G. Pelosi, M.C. Rodrigue, Polyhedron, 2002, 21, 2593.

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[17] D.P. Singh, R. Kumarm, P.Tyagi, Transition Met. Chem. 2006, 31, 970. [18] A. Chaudhary, R .Swaroop, R. Singh, Bol. Soc. Chil. Quim. 2002, 47, 203. [19] D. A. Garnovskii, M. F. C. Guedes da Sliva, M. N. Kopylovich, A.D. Granovski, J. J. R.

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Frausto da Sliva, A. J. L. Pomberio, polyhedron., 2003, 22, 264. [20] S. M. T. Shaikh, J. Seetharamappa, P. B. Kandagal, D. H. Manjunatha, Int. J. Bio. Macromolecu., 2007, 41, 81-86. [21] L.N. Obazi, G.U. Kaior, L. Rhyman, Ibrahim A. Alswaidan, Hoong-Kun Fun, P. Ramasami, J. Mol. Struct., 2016, 1120, 180–186. [22] S. B. Rosso, M. Gonzalez, L. A. Bagatolli, R. O Duffard, G. D. Fidelio, Life Sciences., 1998, 63, 2343- 2351. [23] D. Romanini, G. Avalle, B. Farruggia, B. Nerli, G. Pico, Chemico-Biological Interactions 1998, 115, 247-260.

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[24] D. Carter, J.B. Chang, Ho, JJ. X.; Keeling, K. Krishnasami, Z. Preliminary, European Journal of Biochemistry. 1994, 226, 1049-1052. [25] J. R. Brown, P. Shockley, Lipid-Protein Interaction, 1982, 1, 25-68. [26] T. Peters, Adv Protein Chem. 1985, 37, 161-245. [27] A. K., Singh, R. Singh, P. Saxena, Transition Met. Chem. 2004, 29, 867-869. [28] L. K. Gupta, S. Chandra, Transition Met. Chem. 2006, 31, 368-373.

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[29] S.S. Nivasan, P. Athappan , Transition Met. Chem. 2001, 26, 588-593.

[30] M. Shakir , K.S. Islam, A.K. Mohamed, Transition Met. Chem., 1999, 24, 577-580.

[31] A.B.P. Lever, Inorganic Electronic Spectroscopy, 2nd ed., Elsevier, New York, 1984. [32] M.G. Bhowon, H.L.K. Wah, R. Narain, Polyhedron, 1999, 18, 341-345.

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[33] J. F. M. Da Silva, S. J. Garden, A.C Pinto, J. Braz. Chem. Soc. 2001, 12, 1-105.

[34] V.B. Rana, D.P. Singh, P. Singh, M.P. Teotia, Transition Met. Chem. 1982, 7, 174-177.

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[35] M. I. Bruce, J. Howard, I. W. Nowell, G. Shaw, P. Woodward, J. Chem. Soc., Chem. Commun.,

[36] R. G. Parr, L.V. Szentpaly, S. Liu, J. Am. Chem. Soc. 1999, 121, 1922-1924. [37] A. D. Becke, Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 1993, 98, 5648–5652.

[38] R.G. Pearson, J. Am. Chem. Soc. 1963, 85, 3533-3559.

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[39] G. Venkatesh, M.Govindaraju, P. Vennia, Indian. J. Chem., 2016, 55, 413-422. [40] P. Vennila, M. Govindaraju, G. Venkatesh, C.Kamal, J. Mol. Struct, 2016, 1111, 151-156. [41] G.Venkatesh, M. Govindaraju, P. Vennila, C. Kamal, J. Theor. Comput. Chem, 2016, 15, 1650007.

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[42] T. Koopmans, Physica, 1933, 1, 104–113.

[43] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B., 1988, 37, 785-789.

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[44] S. Kaya, C. Kaya, J. Phys. Theor. Chem., 2015, 11, 155-163. [45] G. Venkatesh, M. Govidaraju, C. Kamal, P. Vennila, S. Kaya., RSC Adv., 2017, 7, 1401-1412. [46] Yongnian Ni, R. Zhu, S. Kokot, Analyst, 2011,136, 4794-4801. [47] X. Li, G. Wang, D. Chen, Y. Lu, RSC Adv., 2014, 4, 7301-7312. [48] F. Ahmad, Y. Zhou, Z. Ling, Q. Xiang, X. Zhou, RSC Adv., 2016, 6, 35719-35730. [49] N. Lihi, D. Sanna, I. Bányai, K. Várnagy, I. Sóvágó, New J. Chem., 2017,41, 1372-1379 [50] C.F. Sousa, J. T. S. Coimbra, I. Gomes, R. Franco, P.A. Fernandes, P. Gameiro., RSC Adv., 2017,7, 10009-10019. [51] Y. Zhang, S. Zhang, G. Xu, H. Yan, Y. Pu, Z. Zuo, Mol. BioSyst., 2016,12, 3734-3742.

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Table.1 Absorption and fluorescence spectral data for the binding complex L1H,

Concentration of complexes 1, 2& 3 mol dm-3

Absorption maximum number

Absorbance, a.u

RI PT

L2H &L3H with BSA

Fluorescence maxima, nm

2 338 338 338

3 341 341 343

1 0.99 0.98 1

2 1.0038 1.0045 1.0058

3 1.0015 1.002 1.0029

334 336 337

315 316 317

320 322 322

7.5×10-6

341

339

343

1

1.0058

1.0058

337

317

323

-5

1.0×10

341

340

344

1.5×10-5

343

341

345

-5

345

342

346

-5

2.5×10

346

344

347

3.0×10-5

347

344

347

M AN U 1.0029

1.0066

1.0058

338

318

323

1.005

1.0012

1.0087

338

318

325

1.0088

1.0014

1.0146

349

319

326

1.0118

1.0017

1.0176

340

321

327

1.0177

1.0024

1.0186

340

321

327

AC C

EP

TE D

2.0×10

SC

0 2.5×10-6 5.0×10-6

1 340 340 341

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Table.2 Optimized geometrical parameters of L1H obtained by B3LYP/6–311+G** density functional calculations. Dihedral angle

RI PT

Aqueous phase (⁰) 108.62 125.61 124.84 108.48 97.59 101.83 106.12 106.17 112.21 119.15 133.12 107.11 112.29 91.35 88.42 100.49 80.42 88.12 106.75 78.12

C11-N1-C2-N3 C11-N1-C2-C4 H49-N1-C2-N3 N1-C2-N3-Ru46 C4-C2-N2-C12 C4-C2-N3-Ru46 Ru46-N3-C12-C13 C2-N3-Ru46-N5 C2-N3-Ru46-N14 C2-N3-Ru46-N5 C12-N3-Ru46-Cl47 C12-N3-Ru46-C48 C4-N5-Ru46-N28 C4-N5-Ru46-Cl47 C4-N5-Ru46-Cl48 C26-N5-Ru46-N3 C26-N5-Ru46-N28 C26-N5-Ru46-Cl47 C26-N5-Ru46-Cl48 C27-N28-Ru46-N5

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C2-N1-C11 C2-N1-H49 N1-C2-N3 N1-C2-C4 C2-N3-Ru46 C12-N3-Ru46 C4-N5-Ru46 C13-N14-Ru46 C40-N14-Ru46 C26-C27-N28 C27-N28-C41 C27-N28-Ru46 C41-N28-Ru46 N3-Ru46-N5 N3-Ru46-N14 N3-Ru46-Cl47 N3-Ru46-Cl48 N5-Ru46-N28 N5-Ru46-Cl47 N5-Ru46-Cl48

Gaseous phase (⁰) 108.59 125.59 124.84 108.45 97.59 101.8 106.12 106.17 112.18 119.15 133.03 107.11 112.29 91.35 88.39 100.49 80.42 88.02 106.75 78.03

M AN U

Bond angle

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Aqueous phase (Å) 1.42 1.41 1.06 1.42 1.44 1.41 2.15 1.42 1.94 1.43 1.45 1.47 1.51 1.45 1.43 2.08 1.46 2.24 2.36

EP

N1-C2 N1-C11 N1-H49 C2-N3 C2-C4 N3-C12 N3-Ru46 N5-C26 N14-Ru46 N14-C40 N15-N16 N15-C20 N16-C17 C27-N29 N28-C41 N14-Ru46 N29-C30 Ru46-Cl47 Ru46-Cl48

Gaseous phase (Å) 1.41 1.41 1.03 1.41 1.44 1.41 2.13 1.42 1.96 1.43 1.46 1.46 1.49 1.45 1.43 2.06 1.46 2.21 2.35

AC C

Bond length

Gaseous phase (⁰) -163.81 2.8 16.74 -179.66 125.35 15.94 -10.31 -11.25 139.68 -137.31 115.37 -59.69 -153.07 107.51 -73.78 145.38 -13.82 -113.24 65.47 10.03

Aqueous phase (⁰) -163.92 2.9 16.74 -179.72 125.35 15.98 -10.31 -11.31 139.58 -137.31 115.42 -59.69 -153.07 107.41 -73.78 145.42 -13.92 -113.24 65.52 10.12

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Aqueous

phase

phase

(⁰)

(⁰)

Ru1-Cl2-C3

88.93

88.98

2.29

Ru1-N5-Cl2

101.37

2.01

2.12

Ru1-N7-Cl2

80.34

Ru1-N7

2.12

2.14

Ru1-N10-Cl2

Ru1-N10

2.14

2.15

Ru1-N2-Cl2

Ru1-N24

2.11

2.11

Cl3-Ru1-N5

C4-N5

1.43

1.44

Cl3-Ru1-N7

N5-C8

1.41

1.41

Cl3-Ru1-N10

C9-N10

1.31

1.33

C9-N11

1.45

N10-C36

Gaseous

Aqueous

phase

phase

(Å)

(Å)

Ru1-Cl2

2.36

2.38

Ru1-Cl3

2.31

Ru1-N5

Bond

angle

Dihedral angle

N7-Ru1-N5-C8

Gaseous Aqueous phase

phase

(⁰)

(⁰)

-124.36

-124.75

N10-Ru1-N5-C4

128.84

128.84

80.34

N10-Ru1-N5-C8

-14.34

-14.26

171.76

171.78

N24-Ru1-N5-C4

73.83

73.83

107.12

107.12

N24-Ru1-N5-C8

-69.28

-69.27

123.34

123.36

Cl2-Ru1-N7-C6

78.15

78.15

158.34

158.34

Cl2-Ru1-N7-C22

-91.15

-91.05

83.04

83.23

Cl3-Ru1-N7-C6

139.49

139.49

Cl3-Ru1-N24

87.25

87.28

Cl2-Ru1-N7-C22

-29.7

-29.7

1.45

N5-Ru1-N7

77.52

77.52

N5-Ru1-N7-C6

-25.89

-25.89

1.42

1.42

N5-Ru1-N10

81.63

81.32

N5-Ru1-N7-C22

164.91

164.94

N11-N12

1.46

1.46

N5-Ru1-N24

138.42

138.62

N10-Ru1-N7-C6

-102.71

-102.71

N24-C37

1.44

1.46

N7-Ru1-N10

107.86

107.86

N10-Ru1-N7-C22

88.09

88.09

N25-N26

1.47

1.47

N7-Ru1-N24

78.14

78.14

N24-Ru1-N7-C6

-171.82

-171.86

N25-C30

1.46

1.48

N10-Ru1-N24

74.48

74.53

N24-Ru1-N7-C22

18.88

18.92

-

-

-

C23-N24-N25

128.17

128.17

Cl2-Ru1-N10-C9

-99.79

-99.29

-

-

-

C23-N25-N26

107.02

107.32

Cl2-Ru1-N10-C9

73.19

73.23

AC C

TE D

M AN U

101.47

EP

length

Bond

RI PT

Gaseous

SC

Table.3 Optimized geometrical parameters of L2H obtained by B3LYP/6–311+G** density functional calculations.

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-

-

C23-N24-C30

119.66

119.66

Cl3-Ru1-N10-C9

-112.67

-112.43

-

-

-

N26-N25-C30

111.34

111.45

-

-

-

-

-

-

N25-N26-C27

109.23

109.63

-

-

-

AC C

EP

TE D

M AN U

SC

RI PT

-

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Table.4 Optimized geometrical parameters of L3H obtained by B3LYP/6–311+G** density functional calculations.

Bond angle

Gaseous phase (⁰)

Ru1-Cl3 Ru1-N5 Ru1-N7 Ru1-N10 Ru-N24 N10-C36 N11-N12 N11-C16 N12-C13 C23-N24 C23-N25 N24-C37 N25-N26 N25-C30 -

2.45 1.94 2.08 2.07 2.05 1.43 1.46 1.46 1.50 1.33 1.43 1.43 1.46 1.46 -

2.48 1.96 2.12 2.16 2.12 1.45 1.46 1.46 1.48 1.28 1.45 1.44 1.45 1.42 -

Cl2-Ru1-N7 Cl2-Ru1-N10 Cl2-Ru1-N24 Cl3-Ru1-N5 Cl3-Ru1-N7 N5-Ru1-N10 N7-Ru1-N24 N10-Ru1-N24 Ru1-N5-C4 Ru1-N5-C8 N10-C9-N11 Ru1-N10-C9 RU1-N10-C36 C9-N10-C36 C9-N11-N12 C22-C23-N24 C22-C23-N25 C24-C23-N25 Ru1-N24-C23 Ru1-N24-C37

100.92 100.18 100.7 78.05 81.18 89.19 91.12 81.75 108.53 108.03 133.45 106.41 112.67 135.19 106.93 120.85 107.42 131.12 104.51 113.23

TE D

EP

AC C

Aqueous phase (⁰) 100.86 100.24 100.74 78.12 81.18 89.23 91.12 81.75 108.53 108.23 133.45 106.35 112.67 135.22 106.93 120.91 107.42 131.12 104.55 113.25

Dihedral angle

Gaseous phase (⁰)

Aqueous phase (⁰)

Cl2-Ru1-N5-C8 Cl3-Ru1-N5-C4 Cl3-Ru1-N5-C8 N7-Ru1-N5-C4 N7-Ru1-N5-C8 C4-N5-N24-C37 C8-N5-N24-C23 C8-N5-N24-C37 Cl2-Ru1-N7-C6 Cl2-Ru1-N7-C22 Cl3-Ru1-N7-C6 Cl3-Ru1-N7-C22 N5-Ru1-N7-C6 N5-Ru1-N7-C22 N24-Ru1-N7-C6 N5-Ru1-N10-C36 N24-Ru1-N10-C9 N24-Ru1-N10-C36 Cl2-Ru1-N10-C23 Cl3-Ru1-N24-C24

-104.06 -67.50 73.78 14.05 155.32 -145.31 137.20 -11.85 -125.03 107.75 61.36 -65.86 -16.95 -144.18 133.85 161.50 -148.23 9.37 -111.71 70.04

-104.16 -67.55 73.82 14.23 155.36 -145.31 137.23 -11.85 -125.12 107.75 61.36 -65.86 -16.98 -144.18 133.85 161.51 -148.25 9.39 -111.74 70.12

RI PT

Aqueous phase (Å)

SC

Gaseous phase (Å)

M AN U

Bond length

ACCEPTED MANUSCRIPT Figure .1

NH H N

NH2 +

N

O+

O

NH2 MeOH 6-8 hrs NH2

O 4-aminoantipyrine

Isatin

o-phenylenediamine

N N

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Scheme. 1. Synthesis of Isatin Schiff base ligand.

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Scheme. 2. Synthesis of Benzil Schiff base ligand.

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N

N

N

N

N

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N

Scheme. 3. Synthesis of Biacetyl Schiff base ligand

N

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H3C

NH N

Cl

N

N

Ru

N N

Cl

N

N

N Cl

N

Ru

N N

N

Cl

N

Ru

N

N

N

N

N

N

Cl

N N

RI PT

N

N

Cl

CH3

SC

Synthetic strategies of the complexes L1, L2 & L3

AC C

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Figure. 2 Absorbance titration of the compounds L1H, L2H & L3H with BSA.

N

AC C

EP

TE D

M AN U

SC

RI PT

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H NMR Spectra of L1H, L2H & L3H complexes.

Fig. 3B

AC C

EP

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M AN U

Fig. 3A

SC

RI PT

Figure.3

Fig. 3C

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Fig. 4B

AC C

EP

TE D

M AN U

Fig. 4A

SC

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Figure 4. 13C NMR Spectra of L1H, L2H & L3H complexes.

Fig. 4C

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Fig. 5A

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Figure.5 Mass Spectra of L1H, L2H & L3H complexes

Fig. 5B

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Fig. 5C

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Figure.6 The optimized structures, HOMOs, LUMOs and electrostatic potential structures of studied L1H, L2H & L3H complexes

ACCEPTED MANUSCRIPT Figure.7 Fluorescence titrations of the compounds [Ru-L1H, L2H & L3H] (0-50µM) with BSA(1µM). The arrows show the diminution of the emission intensity with increasing

AC C

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M AN U

SC

RI PT

concentration of compounds..

M AN U

SC

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Figure. 8 (A) Stern-volmer plot for the quenching of fluorescence of BSA by complexes L1H, L2H & L3H (B) Plot of log (F0 – F/ F ) vs. log (1/(Dt)-(FO-F) / (Pt) (Fo)

ACCEPTED MANUSCRIPT

HIGHLIGHTS

Template synthesis of macrocyclic Ru (II) complexes.

•

Molecular geometry optimized using DFT in Gaseous as well as Aqueous Phases.

•

Molecular modeling & spectroscopic studies carried out.

•

Screening for biological activity was done.

AC C

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•