Experimental and theoretical investigation of a pyridine containing Schiff base: Hirshfeld analysis of crystal structure, interaction with biomolecules and cytotoxicity

Accepted Manuscript Experimental and theoretical investigation of a pyridine containing Schiff base: Hirshfeld analysis of crystal structure, interaction with biomolecules and cytotoxicity S. Chithiraikumar, M.A. Neelakantan PII:

S0022-2860(15)30551-2

DOI:

10.1016/j.molstruc.2015.12.063

Reference:

MOLSTR 22097

To appear in:

Journal of Molecular Structure

Received Date: 9 November 2015 Revised Date:

19 December 2015

Accepted Date: 21 December 2015

Please cite this article as: S. Chithiraikumar, M.A. Neelakantan, Experimental and theoretical investigation of a pyridine containing Schiff base: Hirshfeld analysis of crystal structure, interaction with biomolecules and cytotoxicity, Journal of Molecular Structure (2016), doi: 10.1016/ j.molstruc.2015.12.063. 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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Experimental and theoretical investigation of a pyridine containing Schiff base: Hirshfeld analysis of crystal structure, interaction with biomolecules and cytotoxicity

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S. Chithiraikumar, M.A. Neelakantan* Chemistry Research Centre, National Engineering College, K. R. Nagar, Kovilpatti- 628503,

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Thoothukudi District, Tamil Nadu, India.

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*Corresponding Author Tel: +91 94425 05839, Fax: +91 4632 232749 E-mail Address: [email protected] [email protected]

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Abstract A pyridine containing Schiff base (E)-2-methoxy-6-(((pyridin-2-ylmethyl)imino)methyl) phenol (L) was isolated in single crystals. The molecular structure of L was studied by FT-IR, NMR, UV-Vis techniques, single crystal XRD analysis and computationally by DFT method. L

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prefers enol form in the solid state. Electronic spectrum of L was recorded in different organic solvents to investigate the dependence of tautomerism on solvent types. The polar solvents facilitate the proton transfer by decreasing the activation energy needed for transition state. Potential energy curve for the intramolecular proton transfer in the ground state is generated in

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gas and solution phases. The 3D Hirshfeld surfaces and the associated 2D fingerprint plots were investigated. The percentages of various interactions are analyzed by fingerprint plots of

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Hirshfeld surface. The interaction of L with CT DNA was investigated under physiological conditions using UV–Vis spectroscopy, fluorescence quenching and molecular docking methods. Molecular docking studies reveal that binding of L to the groove of B-DNA is through hydrogen bonding and hydrophobic interactions. The in vitro cytotoxicity of L was carried out in two different human tumor cell lines, MCF 7 and MIA-Pa-Ca-2 exhibits moderate activity.

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Molecular docking

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Keywords: Pyridine containing Schiff base; DFT studies; Hirshfeld surfaces; DNA binding;

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1.

Introduction Schiff base compounds have received considerable scrutiny from both theoretical and

experimental standpoints [1-4]. These compounds are used in diverse fields of chemistry and biochemistry. Schiff bases are acting as ligands to metal ions because of their multiple ligation

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sites. Pyridine derivatives have occupied a unique position in the field of medicinal chemistry. Many naturally occurring compounds having pyridine moiety exhibit interesting biological and pharmacological activities. Pyridine derivatives have been used as herbicides, for regulation of arterial pressure and cholesterol levels in blood [5,6]. Some of them constitute an important class

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of antitumor compounds [7]. 2-hydroxy Schiff base compounds received considerable attention mainly due to the presence of strong hydrogen bonds (O–H· ··N) and (O···H–N) and

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tautomerism between phenol–imines and keto–amine forms [8-10]. In crystalline materials, intermolecular interactions play crucial role in the packing of molecules [11]. This molecular arrangement leads to physical properties of the compound. These interactions play a vital role in specific biological reactions associated with supramolecular chemistry, in particular, drug– receptor interactions, enzyme inhibition and protein folding [12, 13]. They play a role in managing protein and DNA structure and enzyme–substrate binding. Thus, the investigation and

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understanding of these interactions have become important. Hirshfeld surface [14-16] and associated 2D fingerprint plots [17] are simple visualization tool for the analysis of intermolecular interactions. Density functional theory calculations have been used extensively for calculating a wide variety of molecular properties such as equilibrium structure, charge

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distribution, FT-IR and NMR spectra, and provided reliable results which are in agreement with experimental data. The charge density data has been used to understand the properties of molecular systems.

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In continuation of our interest on Schiff bases [18, 19], we report herein a pyridine

containing Schiff base, (E)-2-methoxy-6-(((pyridin-2-ylmethyl)imino)methyl)phenol (L). The compound was prepared and characterized by UV, FT-IR, NMR and single crystal XRD analysis. Density Functional Theory (DFT) with B3LYP was used to perform theoretical calculations on the structure [20, 21]. The IR and NMR spectra were computed at this level and compared with the experimental results. This paper describes the tautomeric effect of L in different solvents in the UV–visible spectra. Potential energy curve for the intramolecular proton transfer in the ground state is generated in gas phase and methanol solution. Molecular 3

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interactions were studied by 3D Hirshfeld surface analysis and the associated 2D fingerprint plots. Electronic absorption and emission spectral studies were used to study the binding of L with CT-DNA. The molecular docking was done to identify the interaction of L with B-DNA. In vitro anticancer activity against human pancreatic cancer (MIA-PA-CA-2) and human breast

2.

Experimental

2.1.

Materials and instrumentation

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cancer (MCF-7) cell lines was also evaluated.

Picolylamine and o-vanillin were, USA and used without further purification. All the

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solvents used were of UV spectral grade. Doubly distilled deionized water was used throughout the experiments. CT-DNA was purchased from Genei, Bangalore and used without purification.

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Trizma base was purchased from Sigma Aldrich. Tris-HCl and ethidium bromide were obtained from HiMedia. Elemental analysis was carried out using a Thermo Finnigan Flash EA 1112 series CHN analyzer. FT-IR spectra were recorded on a Shimadzu 8400S spectrophotometer with KBr pellets in the range of 400–4000 cm-1. Electronic absorption spectra were recorded at room temperature using a Shimadzu UV-2450 spectrophotometer. The fluorescence spectra were recorded on a Jasco FP-8300 spectrofluorophotometer. 1H NMR and

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C NMR measurements

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were performed in CDCl3 on a Bruker Avance III 400 MHz spectrometer. Single crystal X-ray diffraction was recorded in Bruker Kappa Apex II diffractometer. 2.2.

Synthesis of (E)-2-methoxy-6-(((pyridin-2-ylmethyl)imino)methyl)phenol (L) The Schiff base (L) was reported in literature [22, 23]. But, in the present investigation

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we were able to isolate the single crystals. The L was prepared by stirring a mixture of a solution containing o-vanillin (0.5 g, 3 mmol) in 20 mL methanol and picolylamine (0.28 g, 3 mmol) in 20 mL methanol. The reaction mixture was stirred for 2-3 h (Scheme S1). Yellow colored crystal

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obtained by the slow evaporation of reaction mixture is suitable for X-ray analysis (yield 84%). M.p. 98 °C, Found: C, 69.41; H, 5.82; N, 11.56; O, 13.21; calc. for C14H14N2O2; C, 69.28; H, 5.78; N, 11.52. O, 13.42. FTIR (KBr): cm-1 3421 (O-H), 1639 (C=N)azomethine, 1589 (C=N)py, 1074 (C−O). 1H NMR (400 MHz, CDCl3): δ (ppm) 3.90 (s, 3H, −O−CH3), 4.95 (s, 2H, −CH2), 8.56 (s, 1H, =N–CH–), 8.52 (d, 1H, (CH=N)py), 7.20−6.81 (m, 6H, Arom-H).

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C NMR (400

MHz, CDCl3) 166.87 (C=N), 56.15 (−O−CH3), 101−108(aromatic carbons), 122−158(pyridine ring carbons) 2.3.

Crystallography 4

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Single crystal X-ray diffraction experiment was performed on a Bruker Kappa Apex II diffractometer using MoKa radiation; k = 0.71073 Å at 296(2) K. A yellow prism of L with dimensions of 0.25 mm × 0.20 mm × 0.20 mm was used. The structure was solved by direct method procedure using SHELXS-97 program [24]. The refinement was carried out using Full

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Matrix Least Square method on F2, which is in correspondence with 288 parameters. All the non-hydrogen atoms were refined anisotropically. The hydrogen atoms bonded to carbon were inserted at calculated positions using a riding model. Hydrogen atoms bonded to oxygen were located from difference map and allowed to refine with temperature factors riding on those of the

2.4.

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carrier atoms. The geometrical parameters were obtained using PARST [25] and SHELXL-97. Computational procedures

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All the computational calculations were performed with Gaussian 03W program using Density Functional Theory (DFT) with Becke’s three-parameter exchange and Lee–Yang–Parr correlation functional (B3LYP) with a combination of 6-311G basis set [26]. Gauss View program has been used for the molecular visualization of computed structures [27]. The harmonic vibrational frequencies of the studied structures were calculated at the same level to characterize the potential energy surface (PES). NMR signals of the studied structure were

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computed at the corresponding optimized geometry using the same theory level. The minimum energy structures are ensured by the absence of any imaginary frequency. In solution phase, the geometry optimization of the studied structure is performed at the same level with polarizable continuum model (PCM) [28].

DNA binding measurements

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2.5

2.5.1. Electronic absorption titration study Electronic absorption spectral titration was used to study the binding of L with CT-DNA.

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The binding experiments were performed in Tris–HCl/NaCl buffer (50 mM Tris HCl/NaCl buffer, pH-7.2). The concentration of CT-DNA was determined from the absorption intensity at 260 nm with λmax value 6600 M-1 cm-1. Stock solution of DNA was stored at 4°C and used within seven days. Absorption titration experiments were done using fixed concentration of L (40 µM) and varying the concentration of CT-DNA (10–50 µM). While measuring the spectra, an equal amount of DNA was added to both the compound and reference solutions to eliminate the absorbance of DNA itself. From the absorption data, the intrinsic binding constant Kb was determined using the equation [29]. 5

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[ DNA] [ DNA] 1 = + (ε a − ε f ) (ε b − ε f ) K b (ε b − ε f ) where εa, εf and εb are the molar extinction coefficients of the apparent, free and bound

ratio of the slope and the intercept gives the binding constant (Kb). 2.5.2. Fluorescence titration study

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compounds, respectively. A plot of [DNA]/(εa−εf) vs [DNA] gives slope and an intercept. The

The interaction of L with CT-DNA was studied by fluorescence spectral method using EB-bound CT-DNA in Tris–HCl/NaCl buffer solution (pH 7.2). The excitation wavelength was

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fixed at 420 nm and the emission range was adjusted before the measurements. Changes in the fluorescence intensities at 610 nm of EB (25 µM) bound CT-DNA (10 µM) were measured with

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respect to different concentrations of L (10–100 µM). The magnitude of the binding strength of L with CT-DNA can be calculated using the linear Stern–Volmer equation [30], I/Io = 1 + Ksv[Q]

where I0 and I represents the fluorescence intensities of EB-DNA in the absence and presence of the quencher, respectively. Q is the concentration of the Schiff base. Ksv is the linear Stern– Volmer quenching constant. The relative binding tendency of L to CT-DNA was determined by

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comparing slope of the line in the fluorescence intensity versus compound concentration. The apparent binding constant (Kapp) was calculated using the equation Kapp = KEB [EB]/[L]

where [L] is the concentration of the ligand at which there is 50% reduction in the fluorescence

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intensity of EB. KEB = 1.0 × 107 M-1 and [EB] = 25 µM. Molecular docking analysis

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Molecular docking analysis was carried out by using AutoDock 4.2 program [31]. The PDB format of L was obtained from its CIF file using Mercury software. The crystal data of BDNA were obtained from Protein Data Bank (PDB) identifier (http://www.rcsb.org) [32]. The water molecules and other unsupported elements (e.g., Na, K, Hg, etc.,) were removed from the 1BNA (B-DNA). Gasteiger charges were added to the compound (L) by AutoDock Tools (ADT) before subjecting to docking analysis. Ligand docking calculations were carried out using Lamarckian genetic algorithm (LGA) [33]. The binding area was focused on the macromolecules (DNA/L) with a grid box size of 40×40×40 was created along the x, y and z axis, i.e., blind

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docking was performed. The output of the AutoDock was further analyzed with PyMOL software package [34]. 2.7.

Hirshfeld surface analysis Hirshfeld surfaces and the associated 2D-fingerprint plots were calculated using Crystal

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Explorer [35], which accepted a structure input file in CIF format. Bond lengths to hydrogen atoms were set to standard values. For each point on the Hirshfeld isosurface, two distances

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the distance from the point to the nearest nucleus external to the surface and di, the distance to the nearest nucleus internal to the surface, were defined. The normalized contact distance

and

and

was given by

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where

) based on

are the vander Waals radii of the atoms. The value of

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(

was negative

or positive depending on intermolecular contacts, being shorter or longer than the van der Waals displayed a surface with a red-white-blue color scheme, where

separations. The parameter

bright red spots highlighted shorter contacts, white areas represented contacts around the van der Waals separation, and blue regions were devoid of close contacts. For a given crystal structure and set of spherical atomic electron densities, the Hirshfeld surface was unique [36] and it was

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this property that suggested the possibility of gaining additional insight into the intermolecular interaction of molecular crystals. 2.8.

Cytotoxic studies

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Sulphorhodamine B (SRB) is a suitable and responsive assay to determine the drug induced cytotoxicity and cell proliferation for large scale drug screening applications [37]. The cell lines were grown-up in RPMI 1640 medium containing 10% fetal bovine serum and 2 mM

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L-glutamine. For screening test, the cells were inoculated into 96 well microtiter plates in 90 µL at plating densities depending on the repetition time of individual cell lines. After the inoculation of cells, the microtiter plates were incubated at 36°C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of experimental drugs. After 24 h, each plate of every cell line was fixed insitu with trichloroacetic acid (TCA), to represent a measurement of the cell line at the time of drug addition. The synthesized compound (L) was dissolved in a 97% (v/v) waterethanol mixture and stored frozen prior to use. At the time of drug addition, an aliquot of drugs was thawed and diluted 10 times to the desired test concentration with complete medium

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containing test article at a concentration of 100, 200, 400 and 800 µg/ml. Further, a serious of dilutions was prepared to give a total of five drug concentrations plus control. Aliquots of 10 µL of these different drug dilutions were added to the suitable microtiter wells already containing 90 µL of medium, resulting in the required final drug concentrations. After the addition of

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compound, plates were incubated at standard conditions for 48 h and assay was terminated by adding cold TCA. Cells were fixed insitu by the mild addition of 50 µL of cold 30 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded and the plates were washed five times with phosphate buffer saline and air dried.

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Sulphorhodamine B (SRB) solution (50 µL) at 0.4 % (w/v) in 1 % acetic acid was added to each of the wells and plates were incubated for 20 minutes at room temperature. After staining, the

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remaining dye was removed by washing five times with 1 % acetic acid. The plates were air dried. Bound stain was then eluted with 10 mM trizma base (2-Amino-2-(hydroxymethyl)-1,3propanediol) and the absorbance was examined on an Elisa plate reader at a wavelength of 540 nm with 690 nm reference wavelength.

The growth inhibition percentage was calculated as,

[(Ti−Tz)/(C−Tz)] × 100 for concentrations for which Ti>/=Tz (Ti−Tz) positive or zero

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[(Ti−Tz)/Tz] × 100 for concentrations for which Ti
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2.9. Statistical analysis

All data were expressed as the mean of three experiments ± SD (standard deviation). Statistical significance (P<0.05) was performed by one-way ANOVA followed by an assessment

3. 3.1.

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of differences using SPSS 16.0 software. Results and discussion

Crystal structure and optimized geometry of L Crystallographic data and refinement details for the compound L is given in Table 1. The

compound L was crystallized into a monoclinic crystal lattice system with the space group of P21/c. The selected bond lengths and bond angles are given in Table S1. The ORTEP diagram of L with the atom numbering scheme is shown in figure 1. L consists of one aromatic ring (C2– C7), one pyridine ring (C10–C14), and an azomethine frame (N1–C8). The unit cell packing 8

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diagram of L, viewed along the b axis is shown in figure S1. The important hydrogen bond is O2-H1…N1 (1.681 Å). In general, the keto-enol tautomerism appears in o-hydroxy Schiff bases due to the intramolecular proton transfer from oxygen atom to nitrogen atom [38]. In the solid state, o-hydroxy Schiff bases can exist in one or both of these forms [39]. Single crystal X-ray

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diffraction study shows that L prefers the enol form in the solid state (Fig. 1). A significant intramolecular interaction is involved between phenolic oxygen atom O2 and nitrogen atom N1 and constitutes a six-membered ring S(6) [40]. The C–O bond is of single bond length in enolimine tautomer, whereas it is of double bond in keto-amine tautomer. In addition, C–N bond is of

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double bond length in enol-imine tautomer and of single bond length in keto-amine tautomer. The C8–N1 double bond and C7–O2 single bond distances in compound L are consistent with

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the distances of the C–N double bond and the C–O single bond reported for related compounds [41]. The C8–N1 bond length of 1.279 Å and C8–O1 bond length of 1.344 Å show that the molecule exists in enol-imine form rather than in keto-amine form in the solid state. The geometric parameters obtained experimentally and their corresponding values in the optimized structure (Fig. S2) are given in Table 2. The bond lengths and bond angles obtained for enol form of L compare favorably with the experimental crystal data. The small discrepancies

3.2.

FT-IR spectra

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observed between the experimental and theoretical values are due to change in phase.

The FT-IR spectrum of L is given in Fig. S3. The azomethine (−C=N) absorption band was observed at 1639 cm-1 for L. The −OH stretching vibration is very sensitive to inter- and

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intra-molecular hydrogen bonds and lies in the region 3000–3500 cm-1. The absorption band observed at 1254 cm−1 for L is assigned to C–O stretching mode of vibration of phenol group.

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Vibration peak at 1589 cm-1 corresponds to (−C=N) stretching vibration of pyridine ring. The C−H asymmetric and symmetric stretching vibrations were observed at 2961and 2846 cm-1, respectively [42]. In L, the absence of C=O and NH absorption bands shows the preference of enol form of L in the solid state. The vibrational frequencies of L were also calculated by using B3LYP/6-311G method (Fig. S4). The calculated results correlate well with the experimental values (Table 3). As shown in Fig. S5a, good linear relationship between experimental and theoretical vibrational frequencies are observed. 3.3.

1

H and 13C NMR spectra

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The formation of Schiff base, L is confirmed by 1H and

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C NMR spectra recorded in

CDCl3. The 1H NMR signal at 8.55 ppm can be attributed to the azomethine proton confirmed the formation of L (Fig. 2). The signal at 4.96 ppm can be attributed to the CH2 protons linked to the pyridine ring. The methoxy protons show singlet at 3.91 ppm. The aromatic protons exhibit 13

pyridine ring protons. The

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multiplets in the range of 6.81−7.2 ppm, and the multiplets at 7.3−7.69 ppm is due to the C NMR spectrum of L is shown in the Fig. 3. The azomethine

carbon resonance is observed at 166.87 ppm. The signal at 64.76 ppm for L is due to the methylene carbon linked to the pyridine ring. The methoxy carbon shows signal at 56.15 ppm.

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The signal at 148 ppm is due to the aromatic carbon, which is linked to the methoxy group. Signals at 101−118 ppm are due to the aromatic signals and 122−158 ppm are due to the pyridine

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carbon resonances. The computed 1H and 13C NMR chemical shifts at B3LYP/6-311G levels of theory along with experimental data are given in Tables S2 and S3. It is clear that the results are in good agreement with experimental values. As shown in Fig. S5b, there is good linear relationship between experimental and theoretical chemical shifts. 3.4.

UV–visible spectroscopy

The UV-visible spectra of L were recorded in polar protic (methanol, ethanol, isopropanol,

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and tert-butanol), polar aprotic (acetonitrile, DMF and DMSO) and non-polar (dichloromethane, THF and chloroform) solvents (Fig. 4). The spectra of L in polar aprotic and nonpolar solvents show two bands in the range 253–277 nm and 330–334 nm, whereas three bands in the range 264-266 nm, 332-334nm and 420-423nm in polar protic solvents (Table 4). The absorption band above 400 nm

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belongs to the keto–amine form of L (Scheme S2). This may be due to the proton transfer through a transition state with minimum activation barrier in polar solvents [43]. The bands correspond to

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253–277 nm are due to π–π* electronic transition and depends on the nature of the solvents. Table 4 reveals that the absorption maxima of L was affected by solvent type and has a maximum shift of ∆λ = 25 nm. Examination of the data given in Table 4 indicates that the absorption maxima for the compound L in aprotic solvents appear at higher wavelengths than in protic solvents is due to the interaction of the compound and solvent (Fig. 4). This clearly demonstrates that solvation effect has greater influence on L in aprotic solvents. 3.5.

Quantitative solvent spectral relationship

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Kamlet-Taft equation is a linear solvation energy relationship (LSERs) that correlate the effects of solvent polarity on spectral features of solute [44]. Using multiple linear regression analysis, the Kamlet-Taft parameters were estimated. λmax = C0 + C1 f (n) + C2 f (ε)+C3β + C4 (α)

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Kamlet-Taft equation is where λmax is the maximum absorption band, f(n)=(n2−1)/(n2+1) is the refractive index function, f(ε)=(ε−1)/(ε+2) is the dielectric function, and β and α are Kamlet–Taft parameters. The C1 explains the orientation induction interaction between solute/solvent molecules and C2 represents the contributions from hydrogen bond acceptor/donor capacity.

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dispersion polarization interactions between them. The C3 and C4 coefficients represent the

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Table 5 showed LSER coefficients and statistical parameters for π–π* electronic transition of L. The parameter C1 coefficient has a negative sign demonstrates that the first band of L in UV spectra experiences bathochromic effect according to the solvent polarity and depends on dispersion–polarization forces. The │C1│>│C2│value indicates that the refractive index function has much more effect on transition in comparison to the dielectric function. The │C4│>│C3│indicates that the π – π* electronic transition occur by the effect of H–bond acceptor 3.6.

Energy and stability

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ability of the solvents [45].

To investigate the tautomeric stability, optimization calculations at B3LYP/6-311G level were performed for the enol and keto forms of L. Physicochemical properties such as total

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energy, HOMO and LUMO energy and chemical hardness were calculated with the same level of theory (Table 6). The chemical hardness is quite useful to rationalize the relative stability and reactivity of chemical species. Hard species having large HOMO–LUMO gap will be more

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stable and less reactive than soft species having small HOMO–LUMO gap (Fig.5) [46, 47]. The energy of enol form in apolar solvent and gas phase is less than that of keto form, whereas chemical hardness of the enol form is greater than the keto one, which indicates that the enol form is preferred by the L (Table 6). The energy of keto form in methanol, ethanol and DMSO is less than that of enol form demonstrate that the keto form is the most stable tautomeric form in polar solvent. This means that the tautomerization in polar solvent occurs more easily than in apolar solvent. In addition, the energy gap between enol and keto form decreases from the gas

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phase to solution phase. Hence the effect of solvation on the stabilization of keto form is more than that in enol form. Mulliken charge density on L also supports the keto–enol tautomerism (Fig.6). The charge density on L is computed by using the Mulliken charge scheme at DFT level. The more

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negative charge density on N atom of C=N group (-0.47543) compared with the O atom of –OH group (-0.1835) demonstrates that the proton transfer takes place from –OH group to –C=N group of L. 3.7.

Potential energy curves (PECs) for L

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In this study, we have generated the PECs for L by varying O(2)–H(2) distance from 1.01 to 1.90 Å in gas phase and from 1.01 to 1.93 Å in the presence of MeOH solvent. Each time the

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O(2)–H(2) distance is increased by 0.03 Å and the remaining geometrical parameters are allowed in fixed distance. The PECs are plotted using the relative energies (with respect to the lowest energy) as a function of O–H distances (Å) (Fig.7). The potential energy diagram for L in gas phase shows two shallow minima, one at stable O-H distance (1.068 Å) for enol form and the other at 1.808 Å for keto form. In gaseous state the enol form is more stable than the keto form [48]. But the energy barrier in going from enol to keto form is 6.82 kcal/mol. The methanol

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solvent phase also shows two distinct energy minima corresponding to enol and keto forms. Here, the keto form is more stable than the enol form. So, the solvent molecules reduce the energy barrier and accelerate the enol to keto conversion process in L. 3.8.

Hirshfeld surface and finger print analysis

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Hirshfeld surfaces (HSs) and their associated 2D fingerprint plots have been used to quantify the various intermolecular interactions in the molecule [49]. The Hirshfeld surfaces mapped over dnorm (range of −0.078−1.282 Å) are displayed in figure 8. The Hirshfeld surfaces

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mapped over shape index and curvedness (range of −1.0 to 1.0 Å and −4.0 to 4.0 Å) are shown in figures 8 and S6. The surfaces are shown as transparent to allow visualization of the molecular moiety, in a similar orientation for both structures, around which they are calculated. The dominant interaction between oxygen (O) and hydrogen (H) atoms can be observed in the Hirshfeld surface as the red areas (Fig. 8). Other visible spots in the Hirshfeld surfaces correspond to C…H and H…H contacts. The curvedness surface indicates the electron density surface curves around the molecular interactions. The proportions of H⋅⋅⋅O/O⋅⋅⋅H interactions comprise 8% of the Hirshfeld surfaces for L. The O⋅⋅⋅H interaction is represented by a spike (di = 12

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1.427, de = 1.127 Å) in the bottom left (donor) area of the fingerprint plot (Fig. 9). The H⋅⋅⋅O interaction is also represented by another spike (de = 1.427, di = 1.127 Å) in the bottom right (acceptor) region of the fingerprint plot. The proportions of N⋅⋅⋅H/N⋅⋅⋅H interactions comprise 4.1%, of the Hirshfeld surfaces for L. The N⋅⋅⋅H interaction is represented by a spike (di = 1.442,

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de = 1.143 Å) in the bottom left (donor) area of the fingerprint plot (Fig. 9). The H⋅⋅⋅N interaction is also represented by another spike (de = 1.442, di = 1.143 Å) in the bottom right (acceptor) region of the fingerprint plot. The proportions of H⋅⋅⋅C/C⋅⋅⋅H interactions comprise 14% of the Hirshfeld surfaces for L. The C⋅⋅⋅H interaction is represented by a spike (di = 1.625,

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de = 1.102 A) in the bottom left (donor) area of the fingerprint plot (Fig. 9). The H⋅⋅⋅C interaction is also represented by another spike (de = 1.625, di = 0.1.102 A) in the bottom right (acceptor)

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region of the fingerprint plot. The numbers of interactions in terms of percentage are represented in a pie chart (Fig. S7). 3.9.

Molecular docking

Molecular docking study was performed with AUTODOCK 4.2 to identify the interaction mechanism between L with B-DNA. The bonding interaction, types of bonding and bonding distance of L with B-DNA are given in Table 7. Most favorable orientation of L with 1BNA (B-

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DNA) are depicted in figure 10. The results show that the binding of L with targeted DNA is groove binding. Compound L binds at the active site of B-DNA with docking energy of -8.48 kcal/mole. Compound L forms hydrogen bonds with A27, C28, and T12 with distances of 3.14388 Å, 2.92038 Å, and 2.0979 Å respectively, in addition to the hydrophobic interactions

3.10.

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with G11, A27 and C28.

DNA binding studies

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3.10.1. Electronic spectral studies

The absorption spectrum of L in the absence and presence of CT-DNA is shown in figure

11. Binding of L with DNA bases is expected to bring about marked changes in its electronic spectrum. A strong hyper chromic effect without any noticeable shift in the position of maximum absorption peak was observed for the L upon addition of incremental amount of DNA (0-50µM). The spectral changes observed are clearly indicative of grove binding of the L to DNA. In order to determine quantitatively the binding affinity of L to CT-DNA, the intrinsic binding constant Kb of the L was determined and is found to be 1.2 × 104 M-1. 3.10.2. Fluorescence titration studies 13

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The fluorescence spectral method is used to study the relative binding of L to DNA. The emission spectra of ethidium bromide bound DNA at 617 nm were recorded in the absence and presence of increasing amounts of L (Fig.12). Ethidium bromide is a conjugate planar molecule with very weak fluorescence intensity due to fluorescence quenching of the free ethidium

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bromide by solvent molecules. But, it is greatly enhanced when ethidium bromide is specifically intercalated into the adjacent base pairs of double stranded DNA. The enhanced fluorescence can be quenched upon the addition of L. On addition of increasing concentration of L to CT-DNA pretreated with ethidium bromide a significant reduction in the emission intensity (Fig. 12) was

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observed, indicating that L is interacting with DNA. The extent of quenching of the emission intensity gives a measure of the binding property of L. The binding strength of L with CT-DNA is determined from the Ksv value using linear Stern–Volmer equation. The Ksv value obtained is

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5.7×103 M-1. The apparent DNA binding constant for L determined is 0.91×105 M-1. The observed value is comparable with the Kb value determined from the absorption spectral techniques suggesting that L bind to CT-DNA through groove binding. 3.11.

Cytotoxicity

The cytotoxicity responses of L towards MCF7 (human breast cancer cell) and MIA-Pa-

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Ca-2 (human pancreatic cancer cell) cells have been studied by using sulphorhodamine B (SRB) assay under identical conditions. Results were expressed as IC50 values and were compared with Adriamycin (positive control) [IC50 < 0.1 µM for MCF7 and MIA-Pa-Ca-2]. The results indicate that compound L show moderate cytotoxic activity against MCF7 and MIA-Pa-Ca-2 cell lines.

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The IC50 values of L for MCF 7 and MIA-Pa-Ca-2 cell lines are 90 ± 0.18 and 98 ± 0.18 µM, respectively. Growth curve of MIA-Pa-Ca-2 and MCF-7 cell lines are shown in Fig. S8. Microscopic images of control cancer cells and apoptotic morphological changes in MCF7 and

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MIA-Pa-Ca-2 cancer cell lines treated with compound L are shown in figure 13. The cells shrinkages noticed in the morphology may be due to the cytotoxic activity of the compound by targeting the cellular membrane. The rapid loss of cellular membrane potential results in rupture of the plasma membrane, and cytolysis [50, 51]. 4.

Conclusions The compound L has been synthesized and characterized by spectral studies and single

crystal XRD. The L exits in the enol form with monoclinic crystal lattice system and P21/c space group in the solid state. The proton transfer process studied in various organic solvents of 14

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different polarities show the transformation of enol into keto form in polar solvents. The potential energy studies also show enol form in gas phase and keto form in polar solvents. Hirshfeld surfaces and fingerprint plots indicate that the structures are stabilized by H⋯H, H⋅⋅⋅O/O⋅⋅⋅H, N⋅⋅⋅H/N⋅⋅⋅H and H⋅⋅⋅C/C⋅⋅⋅H intermolecular interactions. The DNA binding results

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show that the L binds to DNA in a groove binding mode. The molecular docking studies demonstrate that L fits tightly into the spiral line of the DNA target in the major groove. L exhibits moderate cytotoxicity against MCF7 and MIA-Pa-Ca-2 cell lines. Acknowledgements

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M.A.N. gratefully acknowledges the financial support from the Department of Science and Technology (DST), New Delhi, India (EMR-II/2014/000081) and Board of Research in Nuclear

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Sciences (BRNS), DAE-BARC, Mumbai, India (No.35/14/03/2014). STIC, Cochin is acknowledged for performing the NMR and SCXRD analysis. Also we thank anticancer drug screening facility (ACDSF) at ACTREC, Tata Memorial Centre, Navi Mumbai for cytotoxicity studies. Supplementary

Spectra, crystal packing and computational analysis of L are embedded in the supplementary

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material. Detailed crystallographic data for the structural analyses have been deposited with the Cambridge Crystallographic Data Centre, CCDC with deposition number 1432844. Copies of this information can be obtained from The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK (fax: +44-1223-336033; e-mail: [email protected] or

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Table 1. Crystal Data of L L

Empirical formula

C14H14N2O2

Formula weight

242.27

Temperature

296(2) K

Wavelength

0.71073 A

Crystal system, space group

Monoclinic, P21/c

Unit cell dimensions

a = 9.0780(8) Å alpha

= 90 deg.

b = 5.6522(4) Å beta

= 92.637(4) deg.

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Parameters

c = 24.216(2) Å gamma = 90 deg. 1241.20(17) A3

Z, Calculated density Absorption coefficient F(000)

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Volume

4, 1.296 mg/m3

0.088 mm−1 512

Crystal size

0.35 x 0.30 x 0.30 mm

Limiting indices

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Theta range for data collection

1.68 to 28.31 deg.

-12<=h<=11, -7<=k<=6, -24<=l<=32 9481 / 3061 [R(int) = 0.0334]

Completeness to theta = 28.31

98.8 %

Absorption correction

Semi-empirical from equivalents

Max. and min. transmission

0.9740 and 0.9698

Refinement method

Full-matrix least-squares on F2

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Reflections collected / unique

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Data / restraints / parameters Goodness-of-fit on F

2

3061 / 0 / 165 0.870

Final R indices [I>2sigma(I)]

R1 = 0.0477, wR2 = 0.1297

R indices (all data)

R1 = 0.1135, wR2 = 0.1819

Extinction coefficient

0.015(3)

Largest diff. peak and hole

0.152 and -0.230 e.A−3

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Table 2. Selected bond lengths (Å), angles (◦) and bond angles of L in comparison with calculated values. DFT/ B3LYP 1.450 1.387 1.364 1.462 1.354 1.348 1.011 1.295 1.391 1.407

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O(1)-C(2)-C(3) C2-C3-C4 O1-C2-C3 C3-C4-C5 C6-C7-O2 C7-O2-H2 C6-C8-N1 C8-N1-C9 N1-C9-C10 C9-C10-N2

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C(1)-O(1) C(2)-O(1) C(7)-O(2) C(9)-N(1) C(10)-N(2) N(2)-C(14) O(2)-H(2) C(8)-N(1) C(3)-C(2) C(3)-C(4)

X-ray analysis Bond length (Å) 1.422(3) 1.362(2) 1.344(2) 1.454(3) 1.335(3) 1.331(3) 0.8200 1.279(3) 1.372(3) 1.390(3) Bond angles (°) 125.4(2) 120.7(2) 125.4(2) 120.0(2) 122.1(2) 109.5 122.3(2) 122.3(2) 113.82(18) 114.22(19) Torsion angle (°) 0.3(3) -179.7(2) -178.5(2) 2.0(3) -0.7(3) 179.7(2) -0.4(4)

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Parameters

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C(2)-C(3)-C(4)-C(5) C(4)-C(3)-C(2)-O(1) N(1)-C(9)-C(10)-N(2) N(1)-C(9)-C(10)-C(11) C(11)-C(10)-N(2)-C(14) C(9)-C(10)-N(2)-C(14) C(10)-N(2)-C(14)-C(13)

124.81 120.75 124.81 119.95 121.66 108.08 122.36 120.86 113.02 114.24 0.139 -179.9 176.07 -4.25 -0.584 179.61 0.026

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Table 3. The experimental and computed vibrational frequencies of L (cm-1). Assignments

L DFT/ B3LYP 3437

ν (CH) aromatic

3042

3203

ν (CH) pyridine

3088

ν (C=N)

1639

ν (C=N) pyridine

1589

ν (CH) (CH2)

1482

ν (CH) (CH2)

1431

1347

ν (COC)

1074

1082

ν (C-O) phenolic

1254

1231

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3234

1667

1592

1378

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ν (OH)

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Experimental 3421

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Table 4. Absorption maxima (λmax) of L in different organic solvents.

Solvent

D

λmax (nm)

n

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Sl. No. 1

Methanol

32.66

2

Ethanol

24.55

3

Isopropanol

18

4

tert-Butanol

12

5

Acetonitrile

35.94

1.34

270

6

DMSO

46.45

1.48

277

7

DMF

36.71

1.43

276

8

Chloroform

4.89

1.45

268

9

DCM

8.93

1.46

265

10

THF

7.51

1.40

253

264

1.36

264

1.37

267

1.38

266

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Table 5. LSER coefficients and statistical parameter for π–π* electronic transition of L.

C1

C2

C3

C4

R2

L

-562.875

18.47438

-13.3385

28.90832

0.88

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CHCl3 Enol Keto form form

DCM Enol form Keto form

EtOH Enol form Keto form

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Gaseous phase Enol Keto form form

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Table 6. Calculated total energies and hardness (∆E) of L at DFT/B3LYP/6-311G level

MeOH DMSO Enol Keto form Enol form Keto form form

-801.462

-801.378

-801.889

-801.458

-801.462

-801.935

-801.461

-801.935

-801.892

-801.939

-801.461

-801.937

∆E (eV)

4.78679

3.2184

4.8202

3.2863

4.805

3.300

4.7925

3.3131

4.7903

3.3142

4.7884

3.3165

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Table 7. Bonding interactions, bonding type and bonding distances of L with B-DNA.

2.92038

Hydrogen Bond

2.0979

Hydrogen Bond

5.48987 4.47902 5.38411

Hydrophobic Hydrophobic Hydrophobic

Binding site of ligand

Conventional Hydrogen Bond Conventional Hydrogen Bond Conventional Hydrogen Bond Pi-Pi T-shaped Pi-Pi T-shaped Pi-Pi T-shaped

B:DA27:N6

L:O26

H-Donor

H-Acceptor

B:DC28:N4

L:N6

H-Donor

H-Acceptor

A:DT12:O4

L:H27

H-Donor

H-Acceptor

A:DG11 B:DA27 B:DC28

L L L

Pi-Orbitals Pi-Orbitals Pi-Orbitals

Pi-Orbitals Pi-Orbitals Pi-Orbitals

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Hydrogen Bond

Binding site of DNA

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4 5 6

3.14388

Bonding types

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Category

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B:DA27:N6 :UNK1:O26 B:DC28:N4 :UNK1:N6 :UNK1:H27 A:DT12:O4 A:DG11 - :UNK1 B:DA27 - :UNK1 B:DC28- :UNK1

Distance

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Name

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S. No

Binding mode DNA Ligand

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Fig. 1. ORTEP diagram of L with thermal ellipsoids at 50% probability (CCDC No. 1432844).

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Fig. 2. 1H NMR Spectrum of L in CDCl3.

13

C NMR Spectrum of L in CDCl3.

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Fig. 3.

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(b)

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Fig. 4. Electronic absorption spectra of L in (a) polar aprotic solvents; (b) non-polar solvents; (c) polar protic solvents.

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Fig. 5. Frontier molecular orbital of L at DFT/B3LYP/6-311G level.

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Fig. 6. Histogramic representation of Mulliken charge distribution on different atoms of L.

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(a)

(b)

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Fig. 7. Potential Energy curve of L in (a) gaseous phase; (b) methanol.

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dnorm

di form

Curvedness form

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Fig. 8. Hirshfeld surfaces mapped over dnorm, dnorm, de and curvedness form for L

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O...H (8%)

C...H (14%)

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Fig. 9. Fingerprint plots of L: full (top left) and resolved into H⋅⋅⋅O/O⋅⋅⋅H (top right), H⋅⋅⋅N/N⋅⋅⋅H (bottom left) and H⋅⋅⋅C/C⋅⋅⋅H (bottom right) contacts showing the percentage of contact contributed to the total Hirshfeld surface area of the molecule.

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Fig. 10. Docked pose of L bound to the major groove of B-DNA.

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Fig. 11. Electronic absorption spectra of L bounded CT-DNA. Insert: Plot of [DNA]/(εa−εf) vs [DNA]

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Fig. 12. Emission spectrum of L bounded CT-DNA. Insert: Plot of I/Io vs [Q]

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Fig.13. Digital images of cancer cell lines were treated with L. (a) MCF-7 cancer cells (control); (b) MCF-

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treated with L.

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7 cancer cells treated with L; (c) MIA-PA-CA-2 cancer cells (control); (d) MIA-PA-CA-2 cancer cells

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Highlights Single crystal X-ray structure of a pyridine containing Schiff base (L)




Optimized geometry, IR, NMR spectra and potential energy curves




Hirshfeld analysis of intermolecular interactions




Binding of L with CT-DNA and molecular docking




Cytotoxicity of L with MCF-7 and MIA-PA-CA-2 cancer cell line

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