Structural, spectroscopic, radical scavenging activity, molecular docking and DFT studies of a synthesized Schiff base compound

Accepted Manuscript Structural, Spectroscopic, Radical Scavenging Activity, Molecular Docking and DFT Studies of a Synthesized Schiff Base Compound

Halil Gökce, Yelda Bingöl Alpaslan, Celal Tuğrul Zeyrek, Erbil Ağar, Aytaç Güder, Namık Özdemir, Gökhan Alpaslan PII:

S0022-2860(18)31309-7

DOI:

10.1016/j.molstruc.2018.11.005

Reference:

MOLSTR 25834

To appear in:

Journal of Molecular Structure

Received Date:

27 June 2018

Accepted Date:

03 November 2018

Please cite this article as: Halil Gökce, Yelda Bingöl Alpaslan, Celal Tuğrul Zeyrek, Erbil Ağar, Aytaç Güder, Namık Özdemir, Gökhan Alpaslan, Structural, Spectroscopic, Radical Scavenging Activity, Molecular Docking and DFT Studies of a Synthesized Schiff Base Compound, Journal of Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.11.005

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Graphical Abstract

ACCEPTED MANUSCRIPT Structural, Spectroscopic, Radical Scavenging Activity, Molecular Docking and DFT Studies of a Synthesized Schiff Base Compound Halil Gökcea, Yelda Bingöl Alpaslanb, Celal Tuğrul Zeyrekc, Erbil Ağard, Aytaç Güdera, Namık Özdemire, Gökhan Alpaslan a,* aDepartment

of Medical Services and Techniques, Vocational School of Health Services, Giresun University, Giresun, Turkey

bDepartment cDepartment

of Physics, Faculty of Arts & Science, Giresun University, Giresun, Turkey

of Training and Application, Ankara Nuclear Research and Training Center, Turkish Atomic Energy Authority, Ankara, Turkey

dDepartment eDepartment

of Chemistry, Faculty of Arts & Science, Ondokuz Mayıs University, Samsun, Turkey

of Mathematics and Science Education, Faculty of Education, Ondokuz Mayıs University, Samsun, Turkey

Abstract - A synthesized Schiff base, (E)-4-nitro-2-[(o-tolylimino)methyl]phenol (L), was prepared and characterized by FT-IR, single crystal X-Ray diffraction, NMR chemical shift and UV-Vis. spectroscopic techniques. Theoretical calculations of L were performed by using density functional theory (DFT) calculations with the B3LYP/6-311++G(d,p) level in the ground state. The obtained calculation geometry was found to be in well consistent with the experimental geometry. X-ray diffraction investigation shows that the L crystallized in phenol-imine form with O-H⋯N intramolecular hydrogen bond. X-ray diffraction and Hirshfeld surface analysis were done to examine the contribution of intermolecular contacts in crystal packing of L. Frontier molecular orbitals, electronic absorption wavelengths and nonlinear optical features of the L were investigated with molecular modeling methods. It worthy of note that, the radical scavenging activities of L were examined by using ABTS•+, DMPD•+ and DPPH• assays. According to acquired results, L shows effective DPPH• (SC50 10.99 ± 0.12 μg/mL), DMPD•+ (SC50 11.51 ± 0.16 μg/mL), and ABTS•+ (SC50 8.55 ± 0.17 μg/mL) scavenging activities compared with standard antioxidant compounds such as Trolox (TRO), rutin (RUT) and butylated hydroxy anisole (BHA). Molecular docking study was carried out to predict the potency of inhibition of L against mitochondrial Ubiquinol-Cytochrome C Reductase binding (UQCRB) protein. The molecular docking results showed that the L exhibited a good activity with -7.80 kcal/mol value of binding affinity energy. Keywords: Spectroscopy; Molecular modeling; Radical Scavenging Activity; Mitochondrial UQCRB protein *

Corresponding author. E-mail address: [email protected] (G. Alpaslan)

1

ACCEPTED MANUSCRIPT 1. Introduction Schiff base ligands derived from Salicylideneaniline (SA) have drawn considerable attention because of their tautomerism and chemical properties, as well as antiallergic and antioxidant activities [1-4]. This groups (SA) of Schiff base are great interest due to the formation of O-H…N (phenol-imine) and O…H-N (keto-amine) type hydrogen bonds, which result in tautomerization between phenol-imine and keto-amine forms [5,6]. This tautomerism bring about change of electronic structure and proton transfer [7,8]. Recently, nonlinear optical (NLO) materials have been investigated for signal and optical data processing, optical switching, sensor protection and various other photonic technologies [9-11]. The NLO properties depend on highly delocalized -electron states in the molecular structure [12,13]. Therefore, tautomer forms of Schiff base can be employed as anion sensors and design of various molecular electronic devices such as optical switches and optical applications [14-17]. Ubiquinol-cytochrome c reductase binding protein (UQCRB) is a component of the mitochondrial complex III. Mitochondrial UQCRB is an integral membrane complex involved in energy transduction in a wide range of organisms [18]. In relation to angiogenesis, mitochondria has a very important biological role to regulate as in vascular homeostasis such as mitochondrial reactive oxygen species (mROS) production [19], calcium loading [20], or mitochondrial enzyme activation [21]. In conformity with the Prediction of Activity Spectra for Substances (PASS) analysis results for the prepared compound (E)-4-nitro-2-((otolylimino)methyl)phenol (L) [22], the molecular docking study was performed to evaluate the inhibitory nature of L against mitochondrial UQCRB protein. Syntheses and characterizations of some metal(II) complexes of Schiff base (E)-4nitro-2-[(o-tolylimino)methyl]phenol (L) performed by most researchers in the literature [2326]. Likewise, synthesis of pure L were also given in these studies [23-26]. However, a detailed structural, spectroscopic, electronic, NLO, molecular docking and antioxidant activity investigations on pure (E)-4-nitro-2-[(o-tolylimino)methyl]phenol (L) were not included in previous these studies. Therefore, aim of this study is on determination of experimental characterization features, molecular electronic structure computations, molecular docking study and radical scavenging activities of pure (E)-4-nitro-2-[(o-tolylimino)methyl]phenol (L) (Scheme 1). In this connection, the molecular electronic structure computations or quantum chemical computations (molecular geometry structure optimization, vibrational analysis, UVVis. spectral features, HOMO-LUMO studies and NLO feature) of the compound were done by DFT/B3LYP method at the 6-311++G(d,p) basis set to support and confirm experimental records. Furthermore, the radical scavenging activities of L were determined in order to demonstrate the biochemical characteristics. Therefore, the stable radical ie. 2,2′-azino-bis(32

ACCEPTED MANUSCRIPT ethylbenzthiazoline-6-sulfonic acid) (ABTS•+), N,N-dimethyl-p-phenylenediamine (DMPD•+), 1,1-diphenyl-2-picryl-hydrazyl (DPPH•) scavenging activity analysis were performed via spectrophotometric methods. The molecular docking study is revealed to the interaction of interaction of L with mitochondrial UQCRB protein. 2. Experimental Details 2.1. Synthesis The 2-methylaniline (0.055 mmol, 5.9 mg) and 2-hydroxy-5-nitrobenzaldehyde (0.055 mmol, 9.2 mg) were separately dissolved in 15 ml of ethanol. Then, these two solutions were mixed by using magnetic stirrer and the final mixture was refluxed for 5 hours. The product was obtained as powder. Single crystals of L were formed via slow evaporation of an absolute ethanol (Yield 75 %; m.p. : 439-440 K). 1H NMR (400 MHz; DMSO-d6, ppm): δ 14.88 (1H, OH); 10.28 (1H, -CH=N-); 7.12-9.16 (7H, aromatic protons); 2.38 (3H, -CH3). 13C NMR (400 MHz; DMSO-d6, ppm): δ 168.13 (Cphenyl-OH, phenol); 161.96 (-CH=N-, azomethine); 114.38-145.28 (11 aromatic carbons); 18.14 (-CH3). 2.2. Spectroscopic equipments Shimadzu FTIR-8900 and Unicam UV-vis spectrophotometers were used to record FT-IR and UV-Vis. spectra, respectively. FT-IR spectrum was taken by using KBr pellet technique at the interval of 400-4000 cm-1 in the solid phase of the sample. UV-Vis. spectrum of the compound dissolved in ethanol was taken in the region of 200-600 nm spectral with bandwidth of 2 nm by using 10 mm quartz cell. 2.3. X-ray crystallography Data collection were done at 298 K by the ω-scan technique using monochromated MoKα radiation (λ=0.71073 Å). Unit Cell parameters were obtained using X-AREA software [27]. Absorption correction was found by the integration method via X-RED32 software [27]. The crystallographic data were solved and refined by using SHELXT-2014 [28] and SHELXL-2017 [28], respectively. The molecular figures were prepared by the help of ORTEP-3 and Mercury program packages [29,30]. Crystallographic results were given in Table 1. 2.4. Determination of free radical scavenging activities 2.4.1. DPPH• radical scavenging activity assay 3

ACCEPTED MANUSCRIPT The DPPH• scavenging capacity of L and standards were carried out in keeping with Blois method [31] with minor modifications. For this reason, prepared L and standard solutions (200 µL) at the different concentrations (1-25 μg/mL) was added to DPPH• solution (2.8 mL, 0.2 mM) in absolute ethanol in the test tubes. Then, all tubes were vortexed and incubated at the room temperature for 30 min. Absorbance values were recorded at 517 nm (Optizen, Korea). The results were given as SC50 (μg/mL) by using linear regression analysis. SC50 value is the scavenging concentration for elimination of DPPH radical by 50%. 2.4.2. DMPD•+ radical scavenging activity assay DMPD•+ radical cation scavenging activity assays were carried out according to spectrophotometric method practiced by Fogliano et al. [32]. Deionized water was used for preparation of DMPD radical cation solution (100 mM). 1 mL of prepared solution was mixed with acetate buffer (100 mL, 0.1 M, pH 5.25). And then, the DMPD•+ (colored radical cation) was attained after addition 0.2 mL ferric chloride solution (0.05 M) (the final concentration of DMPD•+ was 0.01 mM). The final solution was used as control tube and its absorbance value was recorded at 505 nm. L and standard solutions (15 μL) at the different concentrations (125 μg/mL) were mixed with DMPD•+ solution (210 μL). Then, all tubes were vortexed and waited at the room temperature for 10 min. After incubation process, degradation of absorbance value was recorded at 505 nm. For these measurements, the acetate buffer was used as a blank sample. The results were computed as SC50 by linear regression analysis like DPPH radical scavenging activity assay. 2.4.3. ABTS•+ radical scavenging activity assay ABTS•+ scavenging capacities of L and standards were investigated following by Re et al. method [33]. In this experimental method, 2.0 mmol/L ABTS and 2.45 mmol/L potassium persulfate was mixed for formation of ABTS•+. This mixture was incubated at room temperature in the dark for 16 h. The generated ABTS•+ solution was stable during 2 days. In order to calibrate of ABTS•+ solution absorbance, PBS (0.1 M pH 7.4) was used for dilution. The final absorbance was arranged as 0.750 ± 0.020 at the 30°C and 734 nm. The all the assays were carried out at the same condition. The diluted ABTS•+ solution (1.0 mL) was mixed with the L or standard solutions (3.0 mL) at different concentrations (1-10 µg/mL in PBS). The results were computed as SC50 by linear regression analysis like DPPH radical scavenging activity assay. 2.5. Statistical analysis 4

ACCEPTED MANUSCRIPT The obtained results were expressed as mean ± S.D. of the three measurements. Variance analyses were carried out by using ANOVA procedures. Duncan’s Multiple Range tests were used to determine significant differences of mean values. P value of < 0.05 was assumed as significant. SPSS were used for these analyses (version 15.0.0; SPSS Inc., Chicago, IL, USA) for windows. 3. Computational Details 3.1. Quantum chemical computations All computations and molecular visualizations were done by using Gaussian 09W and Gauss-View5 software packages, respectively [34,35]. Initial structure for calculation of the molecular geometry was formed using the atomic coordinates obtained from experimental crystallographic

study.

Molecular

electronic

structure

computations

for

geometry

optimization, vibrational frequencies, NLO parameters, HOMO-LUMO simulations and UVVis. spectral features of the molecule was carried out by using B3LYP functional [36] in DFT method at 6-311++G(d,p) basis set [37]. Since there is not any imaginary frequency value in the calculated vibrational frequencies, the molecular structural geometry computation is in an acceptable form. To compare experimental vibrational frequency values with the computed vibrational wavenumbers, we were used dual scaling factor. To scale the calculated vibrational frequencies, the vibrational wavenumbers were multiplied by 0.983 (between 01700 cm-1) and 0.958 (between 1700-4000 cm-1) for B3LYP/6-311++G(d,p) computational level [38]. VEDA 4 software was used to perform in terms of potential energy distribution (PED) of vibrational assignments of computed wavenumbers [39]. The calculations of UV-Vis. spectroscopic parameters (oscillator strengths, wavelengths and excitation energies) of L were performed by using TD-DFT method in gas phase and ethanol solvent [40]. In additional, energy values and shapes of the frontier molecular orbitals (HOMO and LUMO) were calculated and simulated by using the same basis set. Non-linear optical parameters of L were computed by DFT method. 3.2. Molecular docking computations We have performed molecular docking calculations to examine the state of mitochondrial Ubiquinol-Cytochrome C Reductase binding (UQCRB) protein settlement of investigated (E)-4-nitro-2-((o-tolylimino)methyl)phenol (L) compound. Firstly, the PASS program allows predicting the probable profile of biological activity of a drug-like organic compound based on its structural formula [22]. It has been used to predict the general biological potential of synthesized L [22]. PASS analysis of L predicted inhibition of 5

ACCEPTED MANUSCRIPT UQCRB with activities which values of Pa (probability to be active) and Pi (probability to be inactive) were 0.899 and 0.005. The results of PASS analysis for the investigated compound L is given in Table 2. The PASS analysis shows that the interaction of L with UbiquinolCytochrome C Reductase protein has the highest Pa value. Thus for the molecular docking study, 3D molecular structures of mitochondrial Ubiquinol-Cytochrome C Reductase binding (UQCRB) protein which PDB ID number is 1NTK was taken from the protein data bank [41]. The molecular docking calculations were performed by using the AutoDock-Vina software and AutoDockTools (ADT) [42]. The water molecules were omited by using Discover Studio Visualizer 4.0 software [43] while the polar hydrogens and Kollman atomic charges were added to the target UQCRB (PDB ID: 1NTK) protein by using ADT graphical tools. The active site of the UQCRB protein was defined to include residues of active site within the grid size of 40x40x40 Å by using ADT graphical tools. The all of the interactions with title compound L and mitochondrial UQCRB protein were illustrated with PyMol and Discover Studio Visualizer 4.0 software [43,44]. 4. Results and Discussion 4.1. Molecular structure analysis Single crystal X-ray diffraction analysis shows that molecular structure of L is in phenol-imine form. The most important indicator of this form is the C-N and C-O bond lengths and O1-H1···N2 intramolecular hydrogen bond observed in the molecule (Fig.1.). While the O1-C1 bond length is found at 1.3225(17) Å, the N2-C7 bond length is found as 1.2794(18) Å. These bond lengths are consistent with related compounds previously studied [4,45,46]. The dihedral angle between the C1/C6 and C8/C13 rings is 8.18(5) and molecular structure is almost planar. Oxygen atoms of Nitrogen dioxide are twisted slightly out of the plane of L. These torsion angles are -8.3(2) for [O3-N1-C4-C5], -8.3(3) for [O2-N1-C4C3], 172.38(17) for [O3-N1-C4-C3] and 170.99(17) for [O2-N1-C4-C5]. In the crystal structure, molecules are linked into a zig-zag C(10) chain along the c-axis by intermolecular C9-H9···O3 hydrogen bonds (Fig. 2). The parameters of the hydrogen bonds are given in Table 3. The DFT/B3LYP/6-311++G(d,p) computational level was used to optimize molecular geometry structure of the compound. Some structural parameters of the optimized molecular geometry were summarized in Table 4 and they were compared with the experimental crystallographic data of L. The biggest differences between experimental and calculated bond parameters were found as 0.024 Å in C11-C12 bond distance and 1.40 in O2-N1-O3 bond 6

ACCEPTED MANUSCRIPT angle, respectively. By using root mean square error method (RMSE), bond lengths and bond angles of experimental and theoretical results were compared and the calculated RMSE for bond lengths and bond angles are 0.012 Å and 0.609, respectively. In theoretical structure, the dihedral angle between the rings (C1-C6 and C8-C13) of L is 40.91°. The same dihedral angle in the experimental structure is 8.18 (5). The differences of bond parameters between theoretical and the experimental structure can be attributed to the fact that the experimental structure has an intermolecular hydrogen bond. The acceptability of structural geometry can be supported by superimposing molecular geometric structures obtained with theoretical (B3LYP/6-311++G(d,p)) and experimental (single crystal X-Ray diffraction) methods (Fig.3). As a result of this comparison, RMSE value is obtained as 0.380 Å. These results show that obtained optimized structure for L can be a good model for other theoretical calculations of L. 4.2. HOMA analysis In Schiff base compounds, the aromaticity state of the rings of the compounds is an important indication for tautomerism form. If the molecule is in phenol-imine form, the phenol ring must be aromatic [47]. Therefore, to support phenol-imine form of L, we calculated the Harmonic Oscillator Model of Aromaticity (HOMA) indices for C1/C6 and C8/C13 rings by using following equation [48,49];

[∑ 𝑛

]

1 2 𝛼 (𝑅 ‒ 𝑅𝑜𝑝𝑡) 𝐻𝑂𝑀𝐴 = 1 ‒ 𝑛𝑖 = 1 𝑖 𝑖

(1)

where n is the number of bonds, Ri (normalization constant) is 257.7 and Ropt for C-C bond distances is 1.388 Å. If the HOMA index is 1, the molecular system under investigation is purely aromatic. However, it is zero for non-aromatic systems [50]. While the calculated index of the C1/C6 ring is 0.915 for the compound, index of the C8/C13 ring is 0.963 for the compound. This result indicates that crystal structure of L have both aromatic character and phenol-imine form. 4.3. Hirshfeld surface analysis Hirshfeld surface is mostly used to determine intermolecular interactions in crystal packing of a molecular system. The Hirshfeld surface of L was depicted to monitorize the intermolecular distances and to defined the interaction points via help of Crystal Explorer software by using .cif file generated with the experimental single crystal X-Ray diffraction method [51,52]. The Hirshfeld surface was plotted over dnorm function which is normalized contact distance and it is defined by the following equation. 7

ACCEPTED MANUSCRIPT

𝑑𝑛𝑜𝑟𝑚 =

𝑉𝐷𝑊 𝑖

𝑑𝑖 - 𝑟

𝑉𝐷𝑊 𝑖

𝑟

+

𝑉𝐷𝑊 𝑒

𝑑𝑒 - 𝑟

𝑉𝐷𝑊 𝑒

𝑟

,

(1)

where, di and de distance from a point on the surface to the nearest nucleus outside and inside 𝑉𝐷𝑊 𝑖

the Hirshfeld surface, respectively. The 𝑟

𝑉𝐷𝑊 𝑒

and 𝑟

are the Van der Waals radius of the di

and de atoms. 3D Hirshfeld mapping can be degraded to 2D fingerprint histograms by using di and de distance values [53]. Electrostatic potentials for molecules can easily be mapped onto their Hirshfeld surfaces and imaged within a crystal packing diagram. The positive (blue regions) and negative (red regions) electrostatic potentials on Hirshfeld surface point out hydrogen bond donors and acceptors, respectively [53,54]. Likewise, the red and blue regions present contact points shorter and longer than sum of Van der Waals radius with negative and positive dnorm, respectively. The white regions symbolize the intermolecular distances close to Van der Waals radii with dnorm equal to zero [53,54]. The 3D Hirshfeld surface mapping and 2D fingerprint histograms plots of L were depicted in Figure 4. Figure 4 has been shown that red regions are placed on the phenolic oxygen atom and nitro group. This state indicates that the intermolecular hydrogen bonds were formed at values of 2.357 Å and 2.473 Å of the C-H...O interactions between aromatic rings and nitro group. Similarly, there is C-H...O intermolecular hydrogen bond interaction between methyl hydrogen and nitro group at 2.505 Å. Additionally, the C-H...O interaction which is with phenolic oxygen of aromatic ring is recorded as 2.567 Å. From 2D fingerprint histogram of L, the contributions to Hirshfeld surface of the contact distances for the CC, CH/HC, OH/HO and HH interactions are obtained as 9.1%, 15.1%, 27.3% and 37.7%, respectively. 4.4. Vibrational analysis The experimental and computed IR vibrational frequencies and their vibrational assignments were presented in Table 5. The measured and simulated IR spectra of L were depicted in Fig. 5. The free OH stretching vibrational that are in absence of intra- and intermolecular hydrogen bonding interactions can be observed as sharp bands in the region of 3450-3550 cm-1 [55-58]. However, the OH stretching vibrational bands under effect of intraand inter-molecular hydrogen bonding interactions can be observed as mixed with aromatic and aliphatic CH stretching bands in the region of 2800-3200 cm-1 [55-58]. The OH stretching mode of the compound was computed at 2969 cm-1 with PED contribution of 91%. The OH 8

ACCEPTED MANUSCRIPT in-plane bending modes were obtained between 1420 (cal. with PED contribution of 16%), 1497 (cal. with PED contribution of 14%) and 1544 (exp.)/1550 (cal. with PED contribution of 15%) cm-1 as combined with other vibrational bands, whereas the OH out-of-plane bending mode was found at as an individual band 869 (exp.)/888 (cal. with 88% contribution of PED) cm-1. The CH stretching modes in aromatic rings of L were computed at the interval of 30293087 cm-1 and they were observed at 3063 cm-1. The CH in-plane and out-of-plane bending vibrational modes of aromatic groups appear as strong-medium peaks in the ranges of 10001600 cm-1 and 650-1000 cm-1, respectively [55-58]. The asymmetric stretching bands for methyl group in the compound were observed at 2956 cm-1, while they were computed at 2978 (87%) and 2949 (97%) cm-1. Similarly, the symmetric stretching vibration was observed and computed at 2857 cm-1 and 2903 (91%) cm-1, respectively. The scissoring, symmetric bending and rocking modes for methyl group were obtained at 1475 (65%) and 1458 (94%) cm-1, 1384 (exp.)-1394 (85%) cm-1 and 1041 (exp.)-1042 (67%) and 990 (51%) cm-1, respectively. Additionally, the recorded band at 2920 cm-1 was assigned to C7H7 stretching band and it was computed at 2921 cm-1 with 96% contribution of PED. The in-plane (δHCN) and out-of-plane (τHCNC) bending modes for this C7H7 group were found at 1358 (56%) cm1

and 958 (59%) cm-1, respectively. The C=N stretching modes of azomethine groups in the o-hydroxy Schiff base

derivatives can occur to bands at the interval of 1500-1700 cm-1 depending on O-H...N intramolecular hydrogen bond interaction [59,60]. In our study, the C=N stretching vibrational band was obtained at 1611 (exp.)-1640 (cal. with PED contribution of 38%) cm-1. The bands in the 1400-1625 cm-1 region assign to CC skeletal vibration in aromatic rings [59,60]. However, the CC stretching modes in aromatic groups can be also observed in region below 1400 cm-1. In this connection, the measured and computed vibrational wavenumbers at 1581 (exp.)-1589 (33%), 1086 (exp.)-1089 (33%) and 1053 (62%) cm-1 for the title compound were corresponded to aromatic CC stretching modes. The compounds containing nitro group exhibit two extremely strong absorption bands in the 1650-1500 cm-1 and 1350-1250 cm-1 regions due to asymmetric and symmetric stretching vibrations of NO2 group, respectively. [56]. In our study, two bands obtained at 1544 (exp.)-1556 (68%) cm-1 and 1333 (exp.)-1335 (60%) cm-1 were corresponded to asymmetric and symmetric stretching vibrations of NO2 group in the compound. The found band at 824 (exp.)-831 (38%) cm-1 was assigned to scissoring or bending mode (δONO) of nitro group, whereas out-of-plane (γOCON) mode of NO2 group was obtained at 707 (exp.)-716 (63%) cm-1. The C-OH stretching vibrational mode was found at 1292 (exp.)/1303 (cal. 32% with contribution of PED) cm-1 for L as combined with vibrational bands of other molecular groups in the title compound [59]. 9

ACCEPTED MANUSCRIPT 4.5. UV and HOMO-LUMO analyses The UV-Vis. spectra recorded and simulated in ethanol of (E)-4-nitro-2-[(otolylimino)methyl]phenol were given in Fig. 6. The computations for six electronic states of UV-Vis. spectral parameters of L were obtained at the TD-DFT/B3LYP/6-311++G(d,p) level with IEFPCM solvent model using the optimized molecular geometry in ethanol. The measured and computed electronic absorption wavelengths, computed oscillator strengths and electronic transitions were listed in Table 6. The major contributions of electronic transitions corresponding to simulated electronic absorption wavelengths were computed via help of GaussSum 3.0 software [61]. It can be observed π→π* and n→π* electronic transitions in UV spectrum of Schiff base compound (E)-4-nitro-2-[(o-tolylimino)methyl]phenol due to presence of azomethine and aromatic ring groups [62]. The recorded bands at 312 and 282 nm can be ascribed to π→π* electronic transition resulted from azomethine and aromatic ring groups, while the electronic absorption band (shoulder) at 430 nm can be corresponded to the n→π* transition resulted from azomethine group. The six computed electronic absorption wavelengths were found at 390.07, 347.65, 328.01, 323.63, 320.43 and 305.02 nm with TD-DFT/B3LYP/6311++G(d,p) level. The oscillator strengths and major contributions of electronic transitions corresponded to these computed wavelengths 0.0460 for H→L (97%) transition, 0.5557 for H→L+1 (94%) one, 0.0428 for H-1→L (97%) one, 0.0009 for H-5→L (94%) one, 0.2090 for H-2→L (94%) one and 0.0361 for H-1→L+1 (84%) one, respectively. The frontier molecule orbitals (FMOs), which are known as the highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs), have an important key role in chemical reactions [63]. The HOMOs and LUMOs are donor groups occupied by electrons and acceptor groups unoccupied by electrons, respectively [64]. They can be useful for explanation of intramolecular charge transfers, molecular electronic transitions and many molecular electronic features. The molecular orbital simulations (or HOMOs (H, H-1, H-2 and H-5) and LUMOs (L and L+1) plots) in electronic transitions corresponded to UV electronic absorption wavelengths were obtained by using optimized molecular structure in ethanol at the B3LYP/6-311++G(d,p) computational level. The HOMO and LUMO analyses were theoretically performed to interpret intra-molecular charge transfers and molecular electronic transitions of the compound. The HOMOs and LUMOs visualizations and their energy values of the title sample were simulated in Fig. 7. The HOMO and LUMO energy values and HOMO-LUMO energy band gap for the L were computed as -6.577 eV, -2.981 eV and 3.596 eV, respectively. As it can be seen from Figure 10

ACCEPTED MANUSCRIPT 7, the HOMO, HOMO-1 and HOMO-2 were mostly formed from π-type bonding molecular orbitals of aromatic groups in the compound, whereas the L and L+1 were mainly occurred from π-type anti-bonding molecular orbitals of aromatic groups. Additionally, the HOMO-5 is localized on lone pair π orbitals of oxygen atoms of nitro group. Moreover, the HOMO and HOMO-2 were also contained π-type bonding and lone pair π-type molecular orbitals of azomethine groups. These localizations on HOMOs and LUMOs of the title compound can be confirmed by π→π* and n→π* electronic transitions that are resulted from azomethine and aromatic groups. 4.6. Non-linear optical effects The non-linear optical properties of the organic molecules result from movement of delocalized  electrons. If the number of delocalize  electrons increases on organic molecules, non-linear optical features also increase [12,65]. The theoretical methods are quite useful in examining non-linear optical properties [66-68]. For this purpose, the total dipole moment (μ), the mean polarizability (α) and the first-order hyperpolarizability () values of L were calculated at the B3LYP/6-311++G(d,p) level. There is necessary information to compute NLO values the obtained with keyword polar=enonly in the literature [12]. The Gaussian software computes the NLO parameters in terms of atomic units (a.u.). These parameters can be transformed by using 1 a.u = 0.1482×10-24 electrostatic unit (esu) for α values and 1 a.u = 8.6393×10-33 esu for  values. The calculated the mean polarizability (α) and the first hyperpolarizability () for L are 32.6001x10-24, and 199.5244×10-31 esu, respectively. When the calculated NLO values are compared with NLO values of urea (the obtained with same basis set), α (4.9067×10-24 esu) and  (7.8782×10-31 esu) values of L are approximately 6.4 and 25.3 times greater than NLO values of urea. These high values show that L has useful NLO properties. The aromatic character of L provided by HOMA analysis indicates the high number of π electrons and depending on these the high NLO feature of L is prominent. 4.7. Molecular docking studies of L with mitochondrial UQCRB protein The protein was chosen because of its highest Pa value and its biological importance. UQCRB is responsible for the transfer of electrons across the mitochondrial inner membrane. It has important role in stability of complex III which is a multisubunit transmembrane protein encoded by both the mitochondrial (cytochrome b) and the nuclear genomes. Mitochondrial complex III deficiency could be affected several parts of the body, including the liver, brain, 11

ACCEPTED MANUSCRIPT heart, kidneys, and the muscles used for movement [18-21]. Recent research showed that a component (UQCRB) of the mitochondrial complex is responsible for embryonic development and regeneration of tissue [69]. The research result also demonstrates that UQCRB could be applied as a target agent in new approaches for mitochondria-related disease and cancer of human [70]. Thus to evaluate the inhibitory nature of (E)-4-nitro-2-((otolylimino)methyl)phenol (L) against UQCRB protein the molecular docking studies were implemented. The ligand-protein binding sites were predicted in nine different conformations modes. The calculated bonding energy as a result of molecular docking and root-mean-square deviation (RMSD) values for each of conformation modes are presented in Table 7. If the RMSD values is less than 2 Å, the docking results is considered in valid [71]. RMSD value shows the deviation of the ligand form the active site with which it interacts, and it is the most important parameter used for docking results. The other parameter is the bonding energy because of the structure may give low bonding energy outside the active site as well. The best conformation and binding site of ligand (L) inside protein (UQCRB) receptor and the interactions with amino acids which bound to the ligand were examined and visualized with ADT, PyMol and Discover Studio Visualizer 4.0 software. Most favorable docked structure obtained from the rigid molecular docking of the L with the target UQCRB (PDB ID: 1NTK) protein is shown in Fig. 8. The docking results showed that the investigated compound L is bonded at the active location of the target UQCRB by non-covalent interactions. Those with H-bonding, -, -alkyl and C-H bond are most prominent of the non-covalent interactions. The non-covalent interactions of L with mitochondrial UQCRB protein using the molecular docking study are shown in Fig. 9. The predicted binding interactions of (E)-4-nitro-2-((otolylimino)methyl)phenol (L) with Ubiquinol-Cytochrome C Reductase binding protein (UQCRB) are given in Table 8. Between nitrogen atoms of ASN3, ARG5 amino acids and O3 atom of L H-bond with distances of 2.983, 2.804 and 2.124 Å are presented. PHE336 and PHE442 form weak π-π interactions with two benzene rings of L. As calculated by HOMA analysis, the presence of these weak pi-pi interactions between protein and ligand can be confirmed by molecular docking analysis. TRP443 also forms π-alkyl interaction with methyl group (C14) of L while LYS6 form carbon hydrogen bond interaction with O3 atom of L. Binding free energy) of -7.8 kcal/mol as calculated by AutoDock-Vina refers good binding affinity between the investigated ligand L and the target mitochondrial UQCRB protein macromolecule. Especially, it is evident that the O3 atom of L is crucial for binding. 4.8. DPPH• radical scavenging activity 12

ACCEPTED MANUSCRIPT DPPH radical scavenging activities of the antioxidant compounds originate their hydrogen or electron donating abilities to DPPH radical. This stable radical (DPPH•) transforms DPPH molecule when it takes hydrogen radical or electron from the antioxidant compounds. At the final situation, maximum absorbance value of DPPH• at 517 nm decreases because of the color change from purple to yellow [72]. For this reason, DPPH radical scavenging assay is commonly preferred as a molecule in order to determine the antioxidative activity [73]. DPPH radical scavenging activity (in terms of SC50 values (µg/mL)) of L d and standards decreased in that order: TRO (25.75 ± 0.42) > RUT (17.36 ± 0.34) > C14H12N2O3 (10.99 ± 0.12) > BHA (8.34 ± 0.04) (P < 0.05) (Table 9). 4.9. DMPD•+ radical scavenging activity When DMPD radical cation solution reacts with the antioxidant compound as electron or hydrogen donors, dark color of DMPD radical cation changes the lighter so absorbance value of DMPD radical cation solution decreases at 505 nm. This radical cation has the maximum absorbance at 505 nm [74]. Absorbance value of the solution mixed with compound decreased depend on the compound concentration (1-10 μg/mL). SC50 value (μg/mL) of L was found as 11.51 ± 0.16 μg/mL (Fig. 10). In addition, SC50 values of TRO, BHA and RUT were determined as 27.70 ± 0.16, 14.02 ± 0.27 and 10.91 ± 0.09, respectively. (P < 0.05) 4.10. ABTS•+ radical scavenging activity ABTS radical cation assay has been comprehensively used for determination of the antioxidant activity. In this test method, blue-green color of the ABTS radical cation has the maximum absorbance at 734 nm. As it is treated with antioxidant compounds, absorbance value of the radical cation decreases so we can measure the absorbance value easily. To prepare the ABTS radical cation, different oxidants can be used for example K2S2O8, MnO2, etc. We generated the ABTS•+ by using a K2S2O8 [75]. The SC50 values (μg/mL) of L and standards are reported in Figure 10 and Table 9. According to obtained results, we can say that ABTS•+ scavenging activity of L has an effective scavenging activity as good as standards especially BHA and rutin. SC50 values of L, RUT, BHA and TRO were investigated as 8.55 ± 0.17, 16.77 ± 0.06, 7.88 ± 0.09 and 4.13 ± 0.18 (P < 0.01). 5. Conclusion The structural, spectral and electronic features were studied by using both experimental (X-Ray, FT-IR, NMR and UV spectroscopies) and theoretical (DFT/B3LYP/613

ACCEPTED MANUSCRIPT 311++G(d,p)

computational

level)

methods

for

Schiff

base

(E)-4-nitro-2-[(o-

tolylimino)methyl]phenol (L) molecule. The experimental single crystal X-Ray diffraction indicated enol-imine form and presence of intramolecular O-H...N and intermolecular C-H...O interactions. Additionally, intermolecular C-H...O interactions were investigated via 3D Hirshfeld surface analysis and its 2D fingerprint plots. This form was supported by positions of OH (at 2800-3200 (exp.)/2969 (cal.) cm-1) and C=N (at 1611 (exp.)/1640 (cal.) cm-1) stretching bands in vibrational analysis. Additionally, the experimental NMR chemical shift values of hydrogen and carbon atoms in -OH (14.88 ppm) and -CH=N- (10.28 ppm for H and 161.69 ppm for C) groups indicated to enol-imine form of L. The intramolecular charge transfers and electronic transitions in L were confirmed with HOMO, LUMO and UV examinations. The L can be served as a useful NLO material with 32.6001×10-24 esu (for α) and 199.5244×10-31 esu (for ) values. The molecular docking process of the investigated compound L was used to obtain most possible bonding types and confirmations. The L is bonded at the active sites of the target mitochondrial UQCRB protein macromolecule by weak non-covalent interactions. Those with H-bonding, -, -alkyl and C-H bond are most prominent. The docking analysis also indicated that the most probable site for the electrophilic attack is O3. These results draw us to the conclusion that L will be the mitochondrial Ubiquinol-Cytochrome C Reductase inhibitor activity. But, the prediction results should confirmed by biological testing to validate the computational results. Moreover, this effective antioxidant activity of L can be arisen from the electron donating groups on the phenyl ring of the molecule. With regard to attained results, this molecule can be used several sectors such as drug, medicine and food industries. For this reason, the other in vivo or in vitro tests (toxicology, anticancer, anti-inflammatory etc.) will be studied in the future. As a result of

aforementioned,

the

phenol-imine

form

of

Schiff

base

(E)-4-nitro-2-[(o-

tolylimino)methyl]phenol (L) has been supported by FT-IR analysis (νC-O, νC=N, νOH and δHOC vibrations), NMR chemical shifts (-OH and -CH=N-), UV-Vis. wavelengths (π→π* and n→π* electronic transitions of azomethine and aromatic groups), HOMA analysis (0.915 for C1/C6 ring and 0.963 for C8/C13 ring) and molecular docking study (weak pi-pi interactions between protein and ligand). Acknowledgment The authors are grateful to the Scientific Research Project Office of Giresun University, Turkey, for a research grant. (Project no: FEN-BAP-A-220413-61) References 14

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ACCEPTED MANUSCRIPT [68] Ü. Ceylan, G. Özdemir Tarı, H. Gökce, E. Ağar, Spectroscopic (FT–IR and UV–Vis) and theoretical (HF and DFT) investigation of 2-Ethyl-N-[(5-nitrothiophene-2-yl) methylidene] aniline, J. Mol. Struct. 110 (2016) 1-10. [69] J. Chang, H. Jin Jung, H.-J. Park, S.-W. Cho, S.-K. Lee, H. J. Kwon, Cell-permeable mitochondrial ubiquinol–cytochrome c reductase binding protein induces angiogenesis in vitro and in vivo, Cancer Letters 366 (2015) 52-60. [70] D.S. Rosenblatt, Methylenetetrahydrofolate reductase, Clin. Invest. Med. 24 (2001) 5659. [71] B. Kramer, M. Rarey, T. Lengauer, Evaluation of the FLEXX incremental construction algorithm for protein-ligand docking, Proteins 37 (2) (1999) 228-241. [72] A. Güder, H. Korkmaz, Evaluation of in-vitro Antioxidant Properties of Hydroalcoholic Solution Extracts Urtica dioica L., Malva neglecta Wallr. and Their Mixture, Iran J. Pharm. Res. 11 (2012) 913-923. [73] C. Alaşalvar, M.S. Soylu, A. Güder, Ç. Albayrak, G. Apaydın, N. Dilek, Molecular structure, quantum mechanical calculation and radical scavenging activities of (E)-4,6dibromo-2- [(3,5-dimethylphenylimino)methyl]-3-methoxyphenol and (E)-4,6-dibromo2-[(2,6-dimethylphenylimino)methyl]-3-methoxyphenol compounds, Spectrochim. Acta Part A 130 (2014) 357-366. [74] M. Gür, H. Muğlu, M.S. Çavus, A. Güder, H.S. Sayıner, F. Kandemirli, Synthesis, characterization, quantum chemical calculations and evaluation of antioxidant properties of 1,3,4-thiadiazole derivatives including 2- and 3-methoxy cinnamic acids, J. Mol. Struct. 1134 (2017) 40-50. [75] Ç.A. Kaştaş, G. Kaştaş, A. Güder, M. Gür, H. Muğlu, O. Büyükgüngör, Investigation of two o-hydroxy Schiff bases in terms of prototropy and radical scavenging activity, J. Mol. Struct. 1130 (2017) 623-632. Figures Captions Scheme 1. Synthesis of (E)-4-nitro-2-[(o-tolylimino)methyl]phenol (L). Figure 1. ORTEP-3 drawing of L with displacement ellipsoids plotted at 30% probability level; intra-molecular hydrogen bonds are represented by dashed lines. Figure 2. The C-H···O intermolecular hydrogen bonds of L. Figure 3. A molecular fit of the experimental and calculated structures shown in black and red, respectively. H atoms have been omitted for clarity. Figure 4. The 3D Hirshfeld surface and 2D fingerprint plotsof the compound (L). 20

ACCEPTED MANUSCRIPT Figure 5. The experimental (top) and simulated (bottom) IR spectra of (E)-4-nitro-2-[(otolylimino)methyl]phenol (L). Figure 6. The experimental and simulated UV-Vis. spectra of (E)-4-nitro-2-[(otolylimino)methyl]phenol (L). Figure 7. HOMOs and LUMOs simulations of (E)-4-nitro-2-[(o-tolylimino)methyl]phenol (L). Figure 8. Representation of docking results of L embedded into the UQCRB (For interpretation of the references to color in this figure legend, the reader is referred to web version of this article). Figure 9. (a) The ligand binds at the active site of UQCRB. (b) (E)-4-nitro-2-((otolylimino)methyl)phenol (L) and UQCRB interactions (3D). (c) (E)-4-nitro-2-((otolylimino)methyl)phenol (L) and UQCRB interactions (2D). (For interpretation of the references to color in this figure legend, the reader is referred to web version of this article). Figure 10. Comparison of the DPPH•, ABTS•+, and DMPD•+ radical scavenging activities of L and standards. CMPD: Compound, BHA: Butylated hydroxyanisole, RUT: Rutin, TRO: Trolox. Table captions Table 1. Crystal data and refinement of L. Table 2. PASS prediction for the activity spectrum of the title compound (L). Pa:Probability to be active. Pi:Probability to be inactive. Table 3. Hydrogen-bond geometry (Å, ο). Table 4. Selected molecular structure parameters of the compound (L). Table 5.

The selected experimental and computed vibrational wavenumbers and their

assignments of L. Table 6. The experimental and computed UV parameters in ethanol of L. Table 7. Binding affinity of different poses of the investigated ligand (L) as calculated with Autodock Vina. Table 8.

Binding interactions of (E)-4-nitro-2-((o-tolylimino)methyl)phenol (L) with

Ubiquinol-Cytochrome C Reductase binding protein (UQCRB) Table 9. SC50 (µg/mL) values of L and standards for DPPH•, ABTS•+, and DMPD•+ radical scavenging activities.

21

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Scheme 1.

Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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Figure 7.

E(LUMO+1)= -2.407 eV

E(LUMO)= -2.981 eV

E(HOMO)= -6.577 eV

E(HOMO-1)= -7.124 eV

E(HOMO-2)= -7.230 eV

E(HOMO-5)= -8.533 eV

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Figure 8.

Fig. 9.

Figure 10.

ACCEPTED MANUSCRIPT Highlights



(E)-4-nitro-2-[(o-tolylimino)methyl]phenol was prepared and characterized.



Molecular geometry and theoretical spectroscopy were determined by DFT method.



Molecular docking properties of the compound were investigated.



Antioxidant properties were determined by using different tests.

ACCEPTED MANUSCRIPT Table 1. Crystal data and refinement of L. Crystal Data CCDC number Chemical formula Crystal shape/color Formula weight Crystal system Space group Unit cell parameters

Volume Z Dx(Mg cm-3)  (mm-1) F000 Crystal size (mm3) Data collection Diffractometer/meas.meth Absorption correction Tmin; Tmax No. of measured, independent and observed reflections Criterion for observed reflections Rint Refinement R[F2>2σ (F2)], wR, S No. of reflection No. of parameters Weighting scheme ∆max, ∆min (e Å-3)

1431039 C14H12N2O3 Prism/Orange 256.26 Monoclinic P21/c a = 7.5438(6) Å b = 11.3558(7) Å c = 14.2645(12) Å  = 95.115(7)o 1217.11(16) Å3 4 1.398 0.10 536 0.61  0.38  0.18 Stoe IPDS-II diffractometer/ωscans integration 0.963, 0.984 7515, 3073, 1782 I > 2σ(I) 0.031 0.047, 0.124, 0.91 3073 173 w=1/[σ2(F02)+(0.0684P)2]; P=( F02+2Fc2)/3 0.17, -0.28

ACCEPTED MANUSCRIPT Table 2. PASS prediction for the activity spectrum of the title compound (L). Pa:Probability to be active. Pi:Probability to be inactive. Pa0.6 0.899 0.888 0.868 0.799 0.777 0.738 0.722 0.720 0.714 0.742 0.742 0.742 0.709 0.704 0.689 0.685 0.689 0.706 0.678 0.671 0.684 0.662 0.660 0.664 0.658 0.659 0.663 0.646 0.635 0.660 0.639 0.652 0.637 0.619 0.632 0.609 0.621 0.630 0.610 0.628

Pi 0.005 0.004 0.003 0.005 0.005 0.004 0.005 0.004 0.004 0.034 0.034 0.034 0.004 0.005 0.003 0.005 0.013 0.038 0.010 0.006 0.024 0.005 0.004 0.012 0.007 0.017 0.021 0.009 0.005 0.032 0.011 0.029 0.018 0.005 0.019 0.006 0.020 0.036 0.037 0.057

Name of inhibitor Ubiquinol-cytochrome-c reductase inhibitor Glucan endo-1.6-beta-glucosidase inhibitor Laccase inhibitor HMGCS2 expression enhancer Phosphatidylserine decarboxylase inhibitor UGT2B12 substrate Pyruvate decarboxylase inhibitor Acaricide Antituberculosic Acrocylindropepsin inhibitor Chymosin inhibitor Saccharopepsin inhibitor Hydroxylamine reductase (NADH) inhibitor N-hydroxyarylamine O-acetyltransferase inhibitor Arylformamidase inhibitor Hyponitrite reductase inhibitor Glucan endo-1.3-beta-D-glucosidase inhibitor Polyporopepsin inhibitor Urethanase inhibitor Antimycobacterial Protein-glutamate methylesterase inhibitor Thiol protease inhibitor PfA-M1 aminopeptidase inhibitor Aryl-acylamidase inhibitor Antiparasitic Arylsulfatesulfotransferase inhibitor Alkane 1-monooxygenase inhibitor 3-Phytase inhibitor Plastoquinol-plastocyaninreductase inhibitor Fusarinine-C ornithinesterase inhibitor Acetylcholine neuromuscular blocking agent Dehydro-L-gulonate decarboxylase inhibitor Bisphosphoglycerate phosphatase inhibitor 3-Hydroxybenzoate 4-monooxygenase inhibitor Insulysin inhibitor Chitosanase inhibitor Arylacetonitrilase inhibitor Lysase inhibitor GST A substrate Fibrinolytic

ACCEPTED MANUSCRIPT Table 3.Hydrogen-bond geometry (Å, ο). D–H···A

D–H

H···A

D···A

D–H···A

O1–H1···N2

0.82

1.85

2.5876 (16)

149

C9–H9···O3i

0.93

2.48

3.302(2)

147

Symmetry code: (i) -x+1, y-1/2, -z+1/2.

ACCEPTED MANUSCRIPT Table 4.Selected molecular structure parameters of the compound (L). Parameters

Experimental

B3LYP

Bond lengths (Å) C1–C6

1.417 (2)

1.424

C1–O1

1.3225 (17)

1.3311

C3–C4

1.387 (2)

1.399

C4–N1

1.4537 (19)

1.4667

C7–N2

1.2794 (18)

1.2861

C8–C9

1.388 (2)

1.401

C8–N2

1.4171 (18)

1.4113

C11–C12

1.370 (2)

1.394

N1–O2

1.2181 (18)

1.2266

N1–O3

1.2214 (19)

1.2275

C13–C14

1.502 (2)

1.507

C9–C10

1.372 (2)

1.390

Max. Dif.

0.024

RMSE

0.012

Bond Angles () O1–C1–C2

119.60 (13)

118.71

O1–C1–C6

120.96 (13)

121.54

C2–C1–C6

119.44 (13)

119.73

C5–C4–N1

118.83 (14)

119.26

O2–N1–O3

123.02 (14)

124.42

C7–N2–C8

122.36 (12)

121.28

C9–C8–N2

122.77 (13)

121.47

C13–C8–N2

117.41 (13)

118.21

N2–C7–C6

121.81 (13)

121.88

O3–N1–C4

118.35 (14)

117.85

Max. Dif.

1.40

RMSE

0.60

Torsion Angles () O1–C1–C2–C3

178.26 (18)

-179.74

C1–C6–C7–N2

-1.6 (3)

0.3

C6–C7–N2–C8

-178.93 (16)

-176.65

C5–C4–N1–O2

170.99 (17)

179.77

ACCEPTED MANUSCRIPT Table 5. The selected experimental and computed vibrational wavenumbers and their assignments of L. The calculated Scaled IR IIR τCCCC(24) in ring+γC14CCC(22) 468 463 8.26 δONC(15)+δNCC(11) 547 547 3.80 δphenyl(42) 593 591 7.85 δphenyl(30)+νCN(11)+δONO(10) 638 640 11.11 δphenyl(48) 693 685 17.17 γOCON(63) 707 716 8.70 τHCCC(48) in ring+γN2CCC(14) 754 756 39.82 δONO(38)+δphenyl(14) 824 831 16.52 τHCCC(70) in ring 834 46.76 τHCCC(71) in ring 856 7.10 τHOCC(88) 869 888 46.02 τHCCC(57) in ring 916 13.51 τHCCC(57) in ring 937 936 14.08 τHCCC(77) in ring 976 974 0.10 τHCNC(59)+τHCCC(18) in ring 985 12.47 ρCH3(51) 990 1.32 ρCH3(67) 1041 1042 3.11 νCC(62) in ring 1053 7.60 [νCC(33)+δHCC(31)] in ring+νCN(15) 1086 1089 147.91 τHCCC(61) in ring 1126 8.80 τHCCC(71) in ring 1166 0.35 [δHCC(33)+νCC(13)] in ring+δHCN(11) 1236 1240 2.17 νCO(32)+[δHCC(18)+νCC(12)] in ring 1292 1303 102.64 νsNO2(60)+δONO(10) 1333 1335 531.93 δHCN(56) 1358 13.80 δsCH3(85) (symmetric bending) 1383 1394 2.56 νCC(46) in ring+δHOC(16) 1420 14.84 δsCH3(94) (scissoring) 1458 11.49 δsCH3(65) (scissoring) 1475 29.04 δHOC(14)+νCC(13) in ring+νasNO2(13) 1497 56.14 νasNO2(68)+δHOC(15) 1544 1556 129.01 νCC(33) in ring 1581 1589 37.32 νC=N(38) 1611 1640 49.77 νsCH3(91) 2857 2903 17.70 νCH(96) 2920 2921 79.66 νasCH3(97) 2956 2949 13.17 νOH(91) 2800-3200 2969 711.70 νasCH3(87) 2978 14.41 νCH(96) in ring 3063 3066 2.59 s, symmetric; as, asymmetric; ν, stretching; δ, in-plane bending; τ, torsion; γ, out-ofplane bending; δs, scissoring and symmetric bending; ρ, rocking; IIR, IR intensity (km/mol); PED, potential energy distribution. Assignments (PED%)

Exp. IR (cm-1)

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Table 6.The experimental and computed UV parameters in ethanol of L. Exp. λ (nm)

Transition

Cal. λ (nm)

430 -

n→π* -

312

π →π*

282

π →π*

390.07 347.65 328.01 323.63 320.43 305.02

Oscillator strength 0.0460 0.5557 0.0428 0.0009 0.2090 0.0361

Excitation energy (eV) 3.1785 3.5664 3.7799 3.8310 3.8693 4.0648

Major contributions H→L (97%) H→L+1 (94%) H-1→L (97%) H-5→L (94%) H-2→L (94%) H-1→L+1 (84%)

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Table 7. Binding affinity of different poses of the investigated ligand (L) as calculated with AutodockVina (Compound- inhibitor (L-UQCRB)).

Mode 1 2 3 4 5 6 7 8 9

Affinity Distance from best mode (kcal/mol) RMSD l.b RMSD u.b. -7.8 0.000 0.000 -7.5 12.874 13.795 -7.4 1.476 1.798 -7.2 21.774 23.072 -7.2 3.861 6.988 -7.2 1.232 2.036 -7.0 3.387 7.100 -6.9 18.464 19.655 -6.9 18.089 19.478

L: (E)-4-nitro-2-((o-tolylimino)methyl)phenol UQCRB: Ubiquinol–Cytochrome C Reductase binding protein

Table 8. Binding interactions of (E)-4-nitro-2-((o-tolylimino)methyl)phenol (L) with Ubiquinol–Cytochrome C Reductase binding protein (UQCRB). Name

Distance (Å)

Bonding types

C:ASN3:ND2 :UNK0:O3 C:ARG5:NH2 :UNK0:O3 :UNK0:H1 A:GLN339:OE1 C:LYS6:CE :UNK0:O3 A:PHE336 - :UNK0

2.98257

A:PHE442 - :UNK0

5.65347

A:TRP443 :UNK0:C14

4.67833

Conventional hydrogen bond Conventional hydrogen bond Conventional hydrogen bond Carbon hydrogen bond - (T-shaped, hydrophobic) - (T-shaped, hydrophobic)  –Alkyl (hydrophobic)

2.80361 2.12359 3.60963 5.45028

From biding site of protein C:ASN3:ND2

Binding mode protein  (from) H-Donor

To binding site of ligand :UNK0:O3

 (to) binding mode ligand H-Acceptor

C:ARG5:NH2

H-Donor

:UNK0:O3

H-Acceptor

:UNK0:H1

H-Donor

A:GLN339:OE1

H-Acceptor

C:LYS6:CE

H-Donor

:UNK0:O3

H-Acceptor

A:PHE336

-Orbitals

:UNK0

-Orbitals

A:PHE442

-Orbitals

:UNK0

-Orbitals

A:TRP443

-Orbitals

:UNK0:C14

Alkyl

Table9. SC50 (µg/mL) values of L and standards for DPPH•, ABTS•+, and DMPD•+ radical scavenging activities. Parameter CMPD BHA RUT TRO • DPPH 10.99 ± 0.12 8.34 ± 0.04 17.36 ± 0.34 25.75 ± 0.42 DMPD•+ 11.51 ± 0.16 14.02 ± 0.27 10.91 ± 0.09 27.70 ± 0.16 ABTS•+ 8.55 ± 0.17 7.88 ± 0.09 16.77 ± 0.06 4.13 ± 0.18 CMPD: Compound, BHA: Butylated hydroxyanisole, RUT: Rutin, TRO: Trolox