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Journal of Molecular Structure 1141 (2017) 495e511

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Journal of Molecular Structure

journal homepage: ht tp: / /www.elsevier .com/locate/molstruc

Modification of benzoxazole derivative by bromine-spectroscopic,antibacterial and reactivity study using experimental and theoreticalprocedures

V.V. Aswathy a, Sabiha Alper-Hayta b, G€ozde Yalcin c, Y. Sheena Mary a, *,C. Yohannan Panicker a, P.J. Jojo a, Fatma Kaynak-Onurdag d, Stevan Armakovi�c e,Sanja J. Armakovi�c f, Ilkay Yildiz g, C. Van Alsenoy h

a Department of Physics, Fatima Mata National College, Kollam, Kerala, Indiab Republic of Turkey Ministry of Health, Turkish Medicines and Medical Devices Agency, 2176 Str. S€o�güt€ozü, 06520 Çankaya, Ankara, Turkeyc Ankara University, Biotechnology Institute, Tandogan 06100, Ankara, Turkeyd Trakya University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 22030 Balkan Yerleskesi, Edirne, Turkeye University of Novi Sad, Faculty of Sciences, Department of Physics, Trg D. Obradovi�ca 4, 21000 Novi Sad, Serbiaf University of Novi Sad, Faculty of Sciences, Department of Chemistry, Biochemistry and Environmental Protection, Trg D. Obradovi�ca 3, 21000 Novi Sad,Serbiag Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Tandogan, Ankara, Turkeyh Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium

a r t i c l e i n f o

Article history:Received 29 September 2016Received in revised form1 April 2017Accepted 3 April 2017Available online 6 April 2017

Keywords:BenzoxazoleALIERDFBDEMoleculardockingAntibacterial activity

* Corresponding author.E-mail address: sypanicker@rediffmail.com (Y.S. M

http://dx.doi.org/10.1016/j.molstruc.2017.04.0100022-2860/© 2017 Elsevier B.V. All rights reserved.

a b s t r a c t

N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide (NBBPA) was synthesized in this studyas an original compound in order to evaluate its antibacterial activity against representative Gram-negative and Gram-positive bacteria, with their drug-resistant clinical isolate. Microbiological resultsshowed that this compound had moderate antibacterial activity. Study also encompassed detailed FT-IR,FT-Raman and NMR experimental and theoretical spectroscopic characterization and assignation of thering breathing modes of the mono-, ortho- and tri-substituted phenyl rings is in agreement with theliterature data. DFT calculations were also used to identify specific reactivity properties of NBBPAmolecule based on the molecular orbital, charge distribution and electron density analysis, whichindicated the reactive importance of carbonyl and NH2 groups, together with bromine atom. DFT cal-culations were also used for investigation of sensitivity of the NBBPA molecules towards the autoxidationmechanism, while molecular dynamics (MD) simulations were used to investigate the influence of water.The molecular docking results suggest that the compound might exhibit inhibitory activity against GyrBcomplex.

© 2017 Elsevier B.V. All rights reserved.

1. Introduction

Benzoxazole ring is one of the most common heterocycles inmedicinal chemistry. Previous reports revealed that substitutedbenzoxazoles possess diverse chemotherapeutic activitiesincluding antimicrobial [1e5], antiviral [6], topoisomerase I and IIinhibitors [7] and antitumor activities [8,9]. Bacterial resistance toantibacterial agents or antibiotics is of grave concern in the medicalcommunity, as many species of bacteria have evolved resistance to

ary).

certain antibiotics and synthetic agents. Therefore, there could be arapidly growing global crisis in the clinical management of life-threatening infectious diseases caused by multidrug-resistantstrains of the Gram-positive pathogens like Streptococcus,Enterococcus, and Staphylococcus, and Gram-negative pathogenslike Escherichia, Salmonella, and certain Pseudomonas strains.Especially the emergence of multidrug-resistant strains of Gram-positive bacterial pathogens such as methicillin-resistant Staphy-lococcus aureus and Staphylococcus epidermis and vancomycin-resistant Enterococcus is an alarming problem of ever increasingsignificance [10e12]. To meet this crisis successfully, many re-searchers across the globe are working to unearth new compounds

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511496

which can selectively attack novel targets in microorganisms.Hence, the development of novel, potent, and unique antibacterialagents is the preeminent way to overcome bacterial resistance anddevelop effective therapies [13]. In previous studies, we synthe-sized some compounds which bearing a hydrogen, chlorine,methyl, nitro, an amine, ester, and amide substitution at the 5thposition on the benzoxazole ring and examined for their in vitroantimicrobial activity against some Gram-positive, Gram-negativebacteria and Candida albicans [1e3,14,15]. On the basis of theseconsiderations, we synthesized N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide (NBBPA) as antimicrobialagent reported in this work, choosing a bromine atom at the 2ndposition of phenyl of second carbon of benzoxazole ring. Thestrategy employedwas to examine the effect of the bromine againstsome Gram-positive, Gram-negative bacteria and their drug-resistant isolates.

One of the main characteristics of biologically active moleculesis their overall stability, especially in aquatic mediums. Theseorganic molecules are synthesized to be highly stable, whichhardens their removal from the water and soil [16]. Since phar-maceutical care products are frequently used and improperlydumped into the environment their active components accumulatein water, which is very harmful because it has been shown thataforementioned molecules exhibit toxic effects towards aquaticorganisms [16,17]. The fact that biologically active molecules thatserve as active components of pharmaceutical products have beendetected in all types of water is particularly upsetting, since con-ventional methods for the removal of these molecules are ineffec-tive [18,19]. Removal of these molecules based on forceddegradation by advanced oxidation processes could be fine alter-native [17,18,20,21] and it also might serve as a basis for the studiesof their toxic effects [22e24]. Rationalization of studies based onforced degradation can be done by application of DFT calculationsand MD simulations [25e27]. Principles of molecular modelingenable prediction of reactive properties of investigated molecules,further influencing the improvement and development of proced-ures for the purification of water. Bearing in mind the usefulness oftheoretical analysis, in this work we have investigated reactivity inorder to gain an insight into the possible degradation properties ofNBBPA molecule.

2. Experimental section

2.1. Synthesis of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide

The firstly synthesis of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide was obtained in two step procedures asgiven below (Scheme 1):

First step: 5-Amino-2-(2-bromophenyl) benzoxazole was syn-thesized by heating 0.01 mol 2,4-diaminophenol.2HCl with0.01 mol 2-bromobenzoic acid in 12.5 g polyphosphoric acid (PPA)and stirring at 200 �C for 4 h. At the end of the reaction period, theresidue was poured into ice water mixture and neutralized withexcess of 10 M NaOH solution extracted with benzene and then thissolution was dried over anhydrous sodium sulphate and evapo-rated under diminished pressure. The residue was boiled with200 mg charcoal in ethanol and filtered. After the evaporation ofsolvent in vacuo, the crude product was obtained and recrystallizedfrom ethanol.

Second step: Phenylacetic acid (0.5 mmol) and thionyl chloride(1.5 ml) were refluxed in benzene (5 ml) at 80 �C for 3 h. Excessthionyl chloride was removed in vacuo. The residue was dissolvedin ether (10 ml) and this solution was added during 1 h to a stirred,ice-cold mixture of 5-amino-2-(2-bromophenyl)benzoxazole

(0.5 mmol), sodium bicarbonate (0.5 mmol), diethyl ether (10 ml)and water (10 ml). The mixture was kept stirred overnight at roomtemperature and filtered. The precipitate was washed with water,2 N HCl and water and finally with ether to give N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide. Theproduct was recrystallized from ethanol-water as needles, whichwas dried in vacuo. The chemical, physical and spectral data of thecompound are reported below; C21H15BrN2O2.1,75H2O, yield:61,42%, mp: 145e147 �C. MS (70 eV)m/z: 429 (MþþHþ23(Na)), 431(MþþHþ2 þ 23(Na)). Elemental Analysis: Calculated: C: 57.48, H:4.25, N:6.38; Found: C: 57.19, H: 3.92, N: 6.32.

The chemicals were purchased from the commercial vendersand were used without purification. The reactions were monitoredand the purity of the products was checked by thin layer chroma-tography TLC. Kieselgel HF 254 chromatoplates (0.3 mm) was usedfor TLC and the solvent system was ethylacetate:n-hexane (2:1).The melting point was taken on a Buchi SMP 20 capillary apparatusand is uncorrected. 1H NMR spectra was obtained with a Varian400 MHz spectrometer in dimethylsulfoxide-d6 (DMSO-d6) andtetramethylsilane (TMS) was used as an internal standard. Massanalyses was carried out with aWaters Micromass ZQ by using ESIþ

method. Elemental analysis was performed on LECO 932 CHNS(Leco 932, St. Joseph, MI, USA) instrument and was within 0.4% ofthe theoretical values. All chemicals and solvents were purchasedfrom Aldrich Chemical Co. or Fischer Scientific.

The FT-IR spectrum (Fig. 1) was recorded using KBr pellets on aDR/Jasco FT-IR 6300 spectrometer. The FT-Raman spectrum (Fig. 2)was obtained on a Bruker RFS 100/s, Germany. For excitation of thespectrum the emission of Nd:YAG laser was used, excitationwavelength 1064 nm, maximal power 150 mW, measurement onsolid sample. The spectral resolution after apodization was 2 cm�1.

2.2. Microbiology

Microorganisms Pseudomonasaeruginosa isolate (gentamicin-resistant), Escherichiacoli isolate, which has an extended spectrumbeta lactamase enzyme (ESBL), Staphylococcus aureus isolate(meticilline-resistant (MRSA)), P. aeruginosa ATCC 27853 (AmericanType Culture Collection), E. coli ATCC 25922, S. aureus ATCC 29213,Enterococcus faecalis ATCC 29212, E. faecalis isolate (vancomycin-resistant enterococci). Methods Standard strains of P. aeruginosaATCC 25853, E. coli ATCC 25922, S. aureus ATCC 25923, E. faecalisATCC 29212 and clinical isolates of these microorganisms resistantto various antimicrobial agents were included in the study. Resis-tance was determined by Kirby Bauer Disk Diffusion method ac-cording to the guidelines of Clinical and Laboratory StandardsInstitute (CLSI) [28] in the clinical isolates. Standard powders ofampicillin trihydrate, gentamycin sulphate, ofloxacin were ob-tained from the manufacturers. Stock solutions were dissolved indimethylsulphoxide (ofloxacin), pH 8 phosphate buffer saline (PBS)(ampicillin trihydrate) and distilled water (gentamicin sulphate).Newly synthesized compound was dissolved in 80% DMSO-20%EtOH. Bacterial isolates were subcultured in Mueller Hinton Agar(MHA) plates and incubated over night at 37 �C for 24e48 h. Themicroorganisms were passaged at least twice to ensure purity andviability. The solution of the newly synthesized compound andstandard drugs were prepared at 400, 200, 100, 50, 25, 12.5, 6.25,3.125, 1.562, 0.78, 0.39, 0.19, 0.095, 0.047, 0.024 mg/ml concentra-tions, in the wells of microplates by diluting in Mueller HintonBroth (MHB). Bacterial susceptibility testing was performed ac-cording to the guidelines of CLSI M100-S16 [29].

The bacterial suspensions used for inoculationwere prepared at105 cfu/ml by diluting fresh cultures at MacFarland 0.5 density(107 cfu/ml). Suspensions of the bacteria at 105 cfu/ml concentra-tion were inoculated to the twofold diluted solution of the

Scheme 1. Synthetic pathway of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide.

Fig. 1. FT-IR spectrum of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide.

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511 497

compounds. There were 104 cfu/ml bacteria in the wells after in-oculations. MHB was used for diluting the bacterial suspension andfor twofold dilution of the compound. 80% DMSO-20% EtOH,methanol, DMSO, PBS, pure microorganisms and pure media wereused as control wells. A 10 ml bacteria inoculum was added to eachwell of the microdilution trays. The trays were incubated at 37 �Cand MIC endpoints were read after 24 h of incubation. All organ-isms were tested in triplicate in each run of the experiments. Thelowest concentration of the compound that completely inhibitsmacroscopic growth was determined and minimum inhibitoryconcentrations (MICs) were reported in Table 1.

3. Computational details

Calculations of the wavenumbers, NMR chemical shifts, MEPand NBO analysis of the NBBPA molecule are carried out withGaussian09 program [30] using B3LYP/6-311þþG (5D, 7F) quantum

chemical calculations method. A scaling factor of 0.9613 is used toscale the theoretically predicted wavenumbers [31]. The assign-ments of the wavenumbers are done by using GaussView [32] andGAR2PED [33] software. The theoretically predicted geometricalparameters (Fig. 3) are given in Table 2.

In this work Schr€odinger Materials Science Suite 2015-4 hasbeen also employed for the DFT calculations and MD simulations.Desmond program was used for MD simulations [34e37], whileJaguar 9.0 program [38] was used for DFT calculations. Calculationsperformedwith Jaguarwere donewith B3LYP exchange-correlationfunctional [39], together with 6e311þþG(d,p), 6-31 þ G(d,p) and6-311G(d,p) basis sets for the calculations of ALIE, Fukui functionsand BDEs, respectively. OPLS 2005 force field [40] was used in caseof MD simulations with Desmond program. Simulation time wasset to 10 ns, within isothermaleisobaric (NPT) ensemble class. Tomodel the interactions with water one NBBPA molecule was placedin cubic box with ~3000 water molecules. Temperature was set to

Fig. 2. FT-Raman spectrum of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acetamide.

Table 1The microbiological result of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide.

Compounds Microorganisms (MIC value, mg/ml)

Gram-negative Gram-positive

E.c E.c* P.a. P.a.* S.a. S.a.* E.f. E.f.*

Title compound 128 128 64 128 128 64 32 128Ampicillin 2 >1024 e e 0.5 e 0.5 0.5Ofloxacine 0.015 16 1 1 0.125 0.5 1 4Gentamycin 0.25 256 1 64 0.5 128 8 8

Abbreviations:E.c:Escherichia coliATCC 25922, E.c*:Escherichiacoliisolate, which hasan extended spectrum beta lactamase enzyme (ESBL), P.a: Pseudomonas aeruginosaATCC 27853 (American Type Culture Collection),P.a*: Pseudomonas aeruginosaiso-late (gentamicin-resistant), S.a: Staphylococcus. aureus ATCC 29213,S.a*:Staphylo-coccus aureus isolate [meticilline-resistant (MRSA)], E.f: Enterococcus faecalis ATCC29212, E.f*: Enterecoccus faecalis isolate (vancomycin-resistant enterococci).

Fig. 3. Optimized geometry of N-[2-(2-bromophen

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300 K, pressure set to 1.0325 bar, cut off radius to 12 Å. Simple pointcharge (SPC) model [41] was used for the treatment of solvent.Noncovalent interactions in Jaguar program are treated by themethod developed by Johnson et al. [42,43]. Maestro GUI [44] wasused for the preparation of input files and analysis of results whenSchr€odinger Materials Science Suite 2015e4 was used.

4. Results and discussion

4.1. Chemistry

For the synthesis of NBBPA firstly, 5-amino-2-(p-bromophenyl)benzoxazole was obtained by heating 2-bromobenzoic acid with2,4-diaminophenol in PPA. N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide was obtained from 5-amino-2-(p-bro-mophenyl)benzoxazole with phenylacetylchloride obtained by

yl)-1,3-benzoxazol-5-yl]-2-phenyl acetamide.

Table 2Optimized geometrical parameters.

Bond lengths (Ǻ)C1eC2 1.3987 C1eC6 1.3830 C1eH7 1.0799C2eC3 1.4168 C2eH8 1.0765 C3eC4 1.4042C3eN24 1.4198 C4eC5 1.3906 C4eH9 1.0818C5eC6 1.4004 C5eN10 1.4103 C6eO22 1.3988N10eC11 1.3080 C11eC12 1.4558 C11eO22 1.4273C12eC13 1.4102 C12eC14 1.4145 C13eC15 1.3956C13eBr23 1.9469 C14eC16 1.3889 C14eH17 1.0788C15eC18 1.3942 C15eH19 1.0795 C16eC18 1.3972C16eH20 1.0813 C18eH21 1.0817 N24eH25 1.0102N24eC26 1.3708 C26eO27 1.2509 C26eC39 1.5352H28eC29 1.0835 C29eC30 1.4055 C29eC31 1.3978C30eC32 1.4055 C30eC39 1.5130 C31eC33 1.3982C31eH34 1.0822 C32eC35 1.3978 C32eH36 1.0835C33eC35 1.3982 C33eH37 1.0819 C35eH38 1.0822C39eH40 1.0924 C39eH41 1.0923Bond angles (�)C2eC1eC6 116.9 C2eC1eH7 120.9 C6eC1eH7 122.2C1eC2eC3 121.0 C1eC2eH8 120.3 C3eC2eH8 118.7C2eC3eC4 120.9 C2eC3eN24 122.4 C4eC3eN24 116.7C3eC4eC5 117.8 C3eC4eH9 121.7 C5eC4eH9 120.5C4eC5eC6 120.4 C4eC5eN10 130.5 C6eC5eN10 109.1C1eC6eC5 123.0 C1eC6eO22 129.5 C5eC6eO22 107.5C5eN10eC11 106.1 N10eC11eC12 131.5 N10eC11eO22 113.0C12eC11eO22 115.5 C11eC12eC13 125.2 C11eC12eC14 117.4C13eC12eC14 117.4 C12eC13eC5 120.7 C12eC13eBr23 123.7C15eC13eBr23 115.6 C12eC14eC16 121.8 C12eC14eH17 118.1C16eC14eH17 120.1 C13eC15eC18 120.7 C13eC15eH19 119.0C18eC15eH19 120.3 C14eC16eC18 119.7 C14eC16eH20 119.8C18eC16eH20 120.5 C15eC18eC16 119.6 C15eC18eH21 119.6C16eC18eH21 120.7 C6eO22eC11 104.3 C3eN24eH25 115.1C3eN24eC26 129.3 H25eN24eC26 115.6 N24eC26eO27 124.1N24eC26eC39 116.4 O27eC26eC39 119.5 H28eC29eC30 119.4H28eC29eC31 119.7 C30eC29eC31 120.8 C29eC30eC32 118.4C29eC30eC39 120.8 C32eC30eC39 120.8 C29eC31eC33 120.1C29eC31eH34 119.8 C33eC31eH34 120.1 C30eC32eC35 120.8C30eC32eH36 119.4 C35eC32eH36 119.7 C31eC33eC35 119.7C31eC33eH37 120.2 C35eC33eH37 120.2 C32eC35eC33 120.1C32eC35eH38 119.8 C33eC35eH38 120.1 C26eC39eC30 117.7C26eC39eH40 105.7 C26eC39eH41 105.7 C30eC39eH40 110.5C30eC39eH41 110.5 H40eC39eH41 105.9Dihedral angles (�)C6eC1eC2C3 0.0 C6eC1eC2eH8 �180.0 H7eC1eC2eC3 180.0H7eC1eC2eH8 0.0 C2eC1eC6eC5 0.0 C2eC1eC6eO22 180.0H7eC1eC6eC5 �180.0 H7eC1eC6eO22 0.0 C1eC2eC3eC4 0.0C1eC2eC3eN24 180.0 H8eC2eC3eC4 180.0 H8eC2eC3eN24 �0.0C2eC3eC4eC5 0.0 C2eC3eC4eH9 �180.0 N24eC3eC4eC5 �180.0N24eC3eC4eH9 0.0 C2eC3eN24eH25 �180.0 C2eC3eN24eC26 0.0C4eC3eN24eH25 0.0 C4eC3eN24eC26 �180.0 C3eC4eC5eC6 0.0C3eC4eC5eN10 180.0 H9eC4eC5eC6 180.0 H9eC4eC5eN10 0.0C4eC5eC6eC1 0.0 C4eC5eC6eO22 �180.0 N10eC5eC6eC1 180.0N10eC5eC6eO22 0.0 C4eC5eN10eC11 180.0 C6eC5eN10eC11 0.0C1eC6eO22eC11 �180.0 C5eC6eO22eC11 0.0 C5eN10eC11eC12 �180.0C5eN10eC11eC22 0.0 N10eC11eC12eC13 �0.0 N10eC11eC12eC14 180.0O22eC11eC12eC13 180.0 O22eC11eC12eC14 �0.0 N10eC11eO22eC6 0.0C12eC11eO22eC6 180.0 C11eC12eC13eC15 �180.0 C11eC12eC13eBr23 0.0C14eC12eC13eC15 0.0 C14eC12eC13eBr23 180.0 C11eC12eC14eC16 180.0C11eC12eC14eH17 0.0 C13eC12eC14eC16 �0.0 C13eC12eC14eH17 �180.0C12eC13eC15eC18 0.0 C12eC13eC15eH19 180.0 Br23eC13eC15eC18 �180.0Br23eC13eC15eH19 0.0 C12eC14eC16eC18 �0.0 C12eC14eC16eH20 180.0H17eC14eC16eC18 �180.1 H17eC14eC16eH20 0.0 C13eC15eC18eC16 �0.0C13eC15eC18eH21 180.0 H19eC15eC18eC16 �180.0 H19eC15eC18eH21 0.0C14eC16eC18eC15 0.0 C14eC16eC18eH21 �180.0 H20eC16eC18eC15 180.0H20eC16eC18eH21 �0.0 C3eN24eC26eO27 0.0 C3eN24eC26eC39 �180.0H25eN24eC26eO27 �180.0 H25eN24eC26eC39 0.0 N24eC26eC39eC30 �0.1N24eC26eC39eH40 123.9 N24eC26eC39eH41 �124.1 O27eC26eC39eC30 179.9O27eC26eC39eH40 �56.1 O27eC26eC39eH41 55.9 H28eC29eC30eC32 �179.3H28eC29eC30eC39 1.1 C31eC29eC30eC32 0.5 C31eC29eC30eC39 �179.1H28eC29eC31eC33 179.6 H28eC29eC31eH34 0.0 C30eC29eC31eC33 �0.2C30eC29eC31eH34 �179.8 C29eC30eC32eC35 �0.5 C29eC30eC32eH36 179.3C39eC30eC32eC35 179.1 C39eC30eC32eH36 �1.1 C29eC30eC39eC26 89.8C29eC30eC39eH40 �31.7 C29eC30eC39eH41 �148.6 C32eC30eC39eC26 �89.7C32eC30eC39eH40 148.7 C32eC30eC39eH41 31.9 C29eC31eC33eC35 �0.1C29eC31eC33eH37 �179.8 H34eC31eC33eC35 179.5 H34eC31eC33eH37 �0.2C30eC32eC35eC33 0.2 C30eC32eC35eH38 179.8 H36eC32eC35eC33 �179.6H36eC32eC35eH38 �0.0 C31eC33eC35eC32 0.1 C31eC33eC35eH38 �179.5H37eC33eC35eC32 179.8 H37eC33eC35eH38 0.2

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V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511500

treating phenylaceticacid with thionylchloride. The compound wasprepared as a new product. The structure of title compound wassupported by spectral data. The 1H NMR, mass spectra andelemental analysis results are in agreement with the proposedstructure.

4.2. Antimicrobialactivities of the NBBPA

The newly synthesized was evaluated for their antibacterialactivity against S. aureus, E. faecalis as Gram-positive, E. coli, P.aeruginosa as Gram-negative bacteria and their drug-resistantclinical isolate. All the microbiological result were shown inTable 1. According to antibacterial results NBBPA showed moderateactivity against the tested microorganisms. The compound indi-cated more effect against drug-resistant S. aureus (64 mg/ml) andE. coli (128 mg/ml) than standard drug Gentamycin (128 mg/ml,256 mg/ml, respectively). Moreover, this molecule found to be moreactive against E. Faecalis than tested the other microorganisms.

4.3. Computational and spectral studies

The mono-, tri-, 1,2-substituted phenyl rings and benzoxazolering are designated as PhI, PhII, PhIII and PhIV in the followingdiscussions.

4.3.1. Geometrical parametersThe CeC bond lengths in the phenyl ring lie in the range

1.3978e1.4055 Å for PhI phenyl ring, 1.3830e1.4168 Å for PhIIphenyl ring and 1.3889e1.4145 Å for PhIII phenyl ring and forbenzene the CeC bond length is 1.3993 Å [45]. The CeN bondlengths of the NBBPA, C3eN24 ¼ 1.4198 Å, C5eN10 ¼ 1.4103 Å andC26eN24 ¼ 1.3708 Å are less than the normal CeN bond (1.48 Å)shows some resonance in this section of the molecule [46]. Thebond lengths, C6eO22 ¼ 1.3988 Å, C11eO22 ¼ 1.4273 Å and C11]

N10¼ 1.3080Å are in agreement with that of similar derivative [47].The bond length C26]O27 ¼ 1.2509 Å is in agreement with thereported values [48]. In the present case, the CeBr bond length is1.9469 Åwhich is in agreement with literature [49]. At C13 position,the bond angles are,C12eC13eC15 ¼ 120.7�, C12eC13eBr23 ¼ 123.7�

and C15eC13eBr23 ¼ 115.6� and this asymmetry shows the repul-sion between bromine and benzoxazle ring. Similarly at C12 and C11positions, the angles, C13eC12eC14 ¼ 117.4�, C13eC12eC11 ¼ 125.2�,C14eC12eC11 ¼ 117.4� and N10eC11eC12 ¼ 131.5�,N10eC11eO22 ¼ 113.0�, C12eC11eO22 ¼ 115.5� which shows theinteraction between O22 and H17 atoms. The interaction betweenNH and neighbouring units is revealed by the bond angles aroundC26 and N24 which are respectively, C39eC26eN24 ¼ 116.4�,C39eC26eO27 ¼ 119.5�, N24eC26eO27 ¼ 124.1� andC26eN24eC3¼129.3�, C26eN24eH25¼115.6�, C3eN24eH25¼115.1�.

4.3.2. IR and Raman spectraThe observed IR, Raman bands are vibratioal assignments

together with calculated scaled wavenumbers are given in Table 3.The C]O stretching vibration is observed in the range

1800e1550 cm�1 [50], and for NBBPA the band observed at1670 cm�1 experimentally is assigned as the C]O stretching modewhich agrees with the computed value at 1673 cm�1 with a PED of73% and high IR intensity of 398.27 and Raman activity of 59.03. Insubstituted benzenes, stretching vibrations of CeBr is expected inthe range 635 ± 85 cm�1 [51] and for the NBBPA, this vibration isassigned at 687 cm�1 theoretically with PED 32% and this is inagreement with the reported values [49]. According to literature[52] the C]N stretching modes are expected in the region1650e1500 cm�1 and for the NBBPA, this mode is assigned at1511 cm�1 theoretically with a PED of 39% while the reported

values are at 1520 cm�1 [53] and at 1536 cm�1 [54]. In the presentcasae, the CeN stretching modes are assigned at 1190, 1098 cm�1

(IR), 1192 cm�1 (Raman) and at 1222, 1188, 1100 cm�1 theoreticallywith PED contribution 34, 35, 40% as expected [52,55]. The CeOeCstretching modes of the title compound are assigned at 1137 and956 cm�1 theoretically with PEDs 35 and 40%, with low IR in-tensities and the reported the CeOeC stretching modes are 1191,1187, 988 and 993 cm�1 [53,56].

The NeH modes are normally expected in the regions,3400 ± 40 cm�1 (stretching mode), 1510e1500, 1400e1300,740e730 cm�1 (deformationmodes) according to literature [50,51].For NBBPA, the PED analysis gives the NH modes at 3450, 1524,1262, 671 cm�1 theoretically with 99% PED for stretching mode,45e37% PEDs for deformation modes and the deformation modes1524 and 1262 cm�1 possess high Raman activity. Experimentallybands are observed at 3455, 3262, 1527, 1260, 670 cm�1 in the IRspectrum and at 1526 cm�1 in the Raman spectrum. The splitting inthe IR spectrum and red shift from the computed value of the NHstretching vibration indicates the weakening of the NH bond asreported in literature [57].

Normally the CH2 vibrational modes appear in the ranges,2950e2850 cm�1 (stretching), 1470e900 cm�1 (deformationsmodes) [51,52,55] and the bands at 2936, 1422, 1165 cm�1 (IR),2975, 2936, 1423, 1166 cm�1 (Raman) and 2969, 2934, 1425, 1285,1167, 900 cm�1 (DFT) are assigned as the stretching and deforma-tion modes of the CH2 group of NBBPA.

The phenyl ring CH stretching modes are assigned at 3033 cm�1

(IR), 3071, 3050, 3020 cm�1 (Raman) for PhI and 3082, 3065 cm�1

(IR) for PhIII rings. The DFT calculations give these modes in theranges, 3068e3036, 3118e3058 and 3094e3052 cm�1 for PhI, PhIIand PhIII rings, respectively as expected [51]. The phenyl ringstretching modes are assigned at 1470, 1320 cm�1 (IR), 1588 cm�1

(Raman), 1585e1317 cm�1 (DFT) for PhI, 1577, 1541, 1391 cm�1 (IR),1543, 1310 cm�1 (Raman), 1579e1309 cm�1 (DFT) for PhII and1447 cm�1 (IR), 1605, 1567 cm�1 (Raman), 1597e1275 cm�1 (DFT)for PhIII rings which are in agreement with literature [51]. All thephenyl ring stretching modes possess a PED contribution equal orgreater than 40%. In the case of tri-substituted phenyl rings withmixed substituents, the ring breathing mode is expected between600 and 750 cm�1 [51,58] and in the present case the mode at749 cm�1 (DFT) is assigned as the ring breathing mode of the tri-substituted benzene and the reported values are at 764 cm�1 (IR),766 cm�1 (Raman) and at769 cm�1 (DFT) [59]. The ring breathingmode at 749 cm�1 has a PED of 50% with a high IR intensity and lowRaman activity. For the NBBPA compound, the DFT calculations givethe ring breathing mode of ortho substituted phenyl ring at1111 cm�1 with a PED of 46% which is in agreement with literature[58] and the reported value is at 1087 cm�1 [60]. The band at978 cm�1 with a PED of 40% is assigned as the ring breathing modeof the mono substituted phenyl ring of the NBBPA compound asexpected [51,58]. Both the above two ring breathing modes havelow IR intensities and experimentally no bands are seen in the IRspectrum. The in-plane CH bending modes of the phenyl rings areobserved at 1282, 1070, 1017 cm�1 (IR), 1283, 1018 cm�1 (Raman)for PhI, 1129 cm�1 (IR), 1128 cm�1 (Raman) for PhII and at1044 cm�1 (IR), 1246, 1045 cm�1 (Raman) for PhIII. The corre-sponding theoretical CH deformationmodes of the phenyl rings arein the ranges,1281e1013 cm�1 for PhI,1235e1126 cm�1 for PhII and1243e1043 cm�1 for PhIII [51]. The out-of-plane CH deformationmodes of the phenyl rings are assigned at 968, 910, 725 cm�1 in theIR spectrum, 971, 726 cm�1in the Raman spectrum for PhI, 951, 851,824 cm�1 in the IR spectrum, 846 cm�1 in the Raman spectrum forPhII and no bands are seen experimentally for PhIII. The PEDanalysis gives these modes in the ranges, 969e726 cm�1 for PhI,951e826 cm�1 for PhII and 960e741 cm�1 for PhIII rings,

Table 3Calculated (scaled) wavenumbers, observed IR, Raman bands and vibrational assignments of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide.

B3LYP/6-311þþG (5D, 7F) IR Raman Assignmentsa

y(cm�1) IRI RA y(cm�1) y(cm�1) e

3450 85.42 322.96 3455, 3226 e yNH(99)3118 18.71 82.17 e e yCHII(96)3094 1.99 94.67 e e yCHIII(97)3081 4.45 190.46 3082 e yCHIII(95)3076 3.04 115.34 e e yCHII(96)3068 11.19 282.61 e 3071 yCHI(93)3066 11.71 226.15 3065 e yCHIII(95)3058 6.44 48.33 e e yCHII(99)3057 21.11 43.31 e e yCHI(100)3052 1.26 58.43 e e yCHIII(93)3049 3.90 114.49 e 3050 yCHI(99)3037 2.48 76.34 e e yCHI(99)3036 5.57 14.31 3033 3020 yCHI(99)2969 2.99 72.03 e 2975 yCH2(99)2934 11.05 237.43 2936 2936 yCH2(99)1673 398.27 59.93 1670 1670 yC]O (73)1597 32.48 404.23 e 1605 dNH(18),

yC]O(12), yPhIII(47)1585 7.60 706.00 e 1588 yPhI(45),

yPhII(18)1579 6.91 49.75 1577 e yC]O(21),

yPhII(46), yPhI(12)1566 7.58 1062.04 e 1567 yPhIII(51),

dCHIII(23)1558 0.97 8.00 e e yPhI(66),

dCHI(21)1539 35.14 450.64 1541 1543 yPhII(58),

dCHIII(21)1524 3.23 2622.68 1527 1526 dNH(44),

yC]N(17)1511 592.34 145.75 e e yC]N(39),

dNH(12)1467 11.15 2.32 1470 e dCHI(14),

yPhI(50)1443 51.14 31.39 1447 e dCHIII(13),

yPhIII(49)1440 49.03 88.51 e e yPhII(46),

dCH2(15), dCHII(13)1425 5.96 0.85 1422 1423 dCH2(77)1409 0.45 41.06 e e dCHI(24),

yPhI(42)1399 17.82 10.57 e e dCHIII(23),

yPhIII(40)1396 139.44 232.47 1391 e yPhII(48),

dCHII(18)1317 196.56 501.12 1320 e yPhI(65),

dCHII(10)1309 0.36 0.16 e 1310 yPhII(50),

yCN(15)1285 0.01 1.91 e e dCH2(56),

dCHI(20)1281 5.76 314.97 1282 1283 dCHI(56),

yCN(17)1275 1.60 24.13 e e yPhIII(77),

dCHI(10)1262 32.38 694.12 1260 e dNH(45),

yCN(14)1243 14.04 5.13 e 1246 dCHIII(62),

yCN(25)1235 41.57 430.58 e e yPhII(18),

dCHII(43), yCN(15)1222 9.38 570.34 e e yCN(34),

yCC(13), yPhIII(22)1188 87.80 18.62 1190 1192 yCC(33), yCN(35)1175 1.41 41.66 e e yCC(31),

yCN(18), dCH2(20)1167 109.86 251.05 1165 1166 dCH2(62),

yPhI(18)1159 0.15 2.62 e e dCHI(72),

yPhI(20)1158 0.24 3.30 e e dCHIII(83),

yPhII(11)

(continued on next page)

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511 501

Table 3 (continued )

B3LYP/6-311þþG (5D, 7F) IR Raman Assignmentsa

y(cm�1) IRI RA y(cm�1) y(cm�1) e

1146 3.66 43.49 e e dCHI(67),yPhI(16)

1137 0.01 2.53 e e yCO(35),dCHII(33)

1126 4.91 131.33 1129 1128 dCHII(46),yCN(11), yPhII(17)

1111 10.42 5.20 e e dCHIII(41),yPhIII(46)

1100 6.88 32.78 1098 e yCN(40),dCHII(21)

1060 6.85 0.13 1070 e yPhI(12),dCHI(45)

1043 3.21 5.56 1044 1045 yCC(17),dPhIII(18), dCHIII(43)

1013 27.21 104.08 1017 1018 dCHI(52),yPhI(16)

1010 3.86 13.75 e 1010 dCHIII(39),tPhIII(18)

990 85.01 79.26 e e yPhIII(46),yPhI(16), yCBr(11)

978 0.30 43.70 e e dPhI(13), yPhI(40)969 0.13 0.31 968 971 gCHI(74),

tPhI(14)960 0.01 0.60 e e gCHIII(94)956 7.20 16.03 e e dPhIII(22),

yCO(40), dPhIV(27)951 0.01 0.01 951 e gCHII(88)938 0.34 0.39 e e gCHI(92)935 1.52 0.06 932 933 yCC(16),

yPhII(21), dPhII(19)904 7.89 151.17 910 e gCHI(77)900 6.28 13.71 e e dCH2(68),

gC]O(15)898 3.91 14.55 e e gCHIII(70),

tPhIII(10)893 0.88 0.57 e e dCN(19),

dPhII(12), yCC(31), yPhII(10)847 0.01 0.10 851 846 gCHII(78),

tPhII(10)836 28.34 0.03 e e gCHIII(68),

dPhIV(26)828 11.60 17.79 e e gCHI(98)826 0.69 0.29 824 e gCHII(90)808 23.75 2.13 e e dCH2(13),

dCN(11), gCHI(11), yPhI(10)806 27.47 0.06 804 807 yCO(14),

dCN(20), dPhII(32)789 2.16 10.52 e e tPhIII(33),

gCC(31), gCHIII(29)749 33.83 2.83 748 e yPhII(50),

dCN(10)741 15.02 0.81 e e gCHIII(72),

tPhIII(17)726 2.46 0.14 725 726 tPhI(22),

gCHI(47), gCC(10)710 32.41 1.42 e e dPhIII(48),

yCBr(10), dCH2(17)697 11.38 5.79 695 e tPhI(56),

gCHI(37)687 53.20 0.75 e e yCBr(32),

tPhII(13), tPhIV(18), gCN(11)671 2.64 0.26 670 e gNH(37),

tPhIV(20), gCC(11), tPhIII(10), tCN(16)668 4.26 3.35 670 e tPhIV(32),

tPhIII(18), gCC(18), gNH(14)633 5.73 12.01 633 640 dPhIV(20),

dPhI(19), dPhII(14)617 4.39 0.58 616 e dPhIII(71)611 0.70 3.57 e 612 dPhI(87)605 7.87 0.03 e e dPhI(28),

tPhI(11), dCH2(23)602 4.72 0.97 e e gCN(23),

tPhII(23), tPhIV(12), tPhI(14)582 11.10 15.88 e e dPhIII(40),

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511502

Table 3 (continued )

B3LYP/6-311þþG (5D, 7F) IR Raman Assignmentsa

y(cm�1) IRI RA y(cm�1) y(cm�1) e

dPhIV(12)543 21.96 0.47 e e gC]O(60),

dCH2(13)536 2.57 3.57 537 e dCC(37),

dPhII(19)509 37.75 3.92 512 514 tPhIII(56),

gCC(11), tPhIV(11)500 0.01 0.11 e e tPhI(23),

gCC(17), dPhII(23)439 6.40 0.10 441 e tPhIII(54),

gCBr(22)429 1.06 1.12 429 e tPhI(37),

gCC(11), dC]O(10)419 3.21 e 417 417 tPhII(61),

tPhIII(10)402 8.33 1.46 402 e tPhI(85)398 0.02 0.01 e e dCN(26),

dCC(19), dPhII(16)369 0.01 0.36 e e tPhIV(39),

tPhII(13), tPhIII(10), gCN(12)360 9.14 6.42 e 355 dCBr(26),

dC]O(19), dCN(22)328 0.24 4.28 e e dCBr(15),

tPhI(14), dCN(16), dCH2(19)314 0.61 0.23 e e dCC(68),

dCH2(10)295 1.97 8.07 e e tPhIII(27),

tPhII(28), tPhIV(13)294 0.15 0.87 e e dCBr(30),

dCN(22)228 4.10 1.40 e e tPhI(24),

dCN(18), dCBr(10), dPhIII(11)217 0.11 2.20 e e tPhII(49),

tPhIV(22)200 3.38 1.72 e e dCBr(20),

dCN(18), dCC(10)185 0.14 0.69 e e tPhII(14),

tPhIII(13), gCBr(12), tPhIV(11), tCN(10), gCC(14)164 0.39 2.85 e 162 dCBr(18),

tPhI(16), dPhII(28)118 2.15 2.00 e 124 tPhIII(50),

tPhIV(10), gCBr(10)117 0.47 2.42 e e dCBr(15),

dCN(27), gCC(13), dCC(13)72 0.01 1.32 e e tCN(18),

gCC(25), gNH(18), tCC(11)61 0.18 1.31 e e dCC(43),

gCC(16), dCH2(15)51 6.59 0.04 e e tNH(26),

tCC(21), tCH2(16), tCN(19)35 1.09 4.85 e e tCC(51), tNH(17)30 0.32 2.37 e e tCH2(36),

tCC(22), tNH(20)29 0.67 5.08 e e dCH2(20),

gCC(16), dCN(37)17 0.04 2.63 e e tCH2(42),

tCN(13), tCC(23)6 0.71 2.10 e e tCC(61),

tCH2(31), tNH(10)

a y-stretching; d-in-plane deformation; g-out-of-plane deformation; t-torsion; PhI-mono substituted phenyl ring; PhII-tri-substituted phenyl ring; PhIII-di-substitutedphenyl ring; PhIV-benzoxazole ring; potential energy distribution (%) is given in brackets in the assignment column.

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511 503

respectively [51].

4.3.3. NMR spectraThe absolute isotropic chemical shielding of the NBBPA mole-

cule was calculated using B3LYP/GIAO model and the results havebeen shown in Table 4. The 1H NMR experimental values are: 1HNMR (ppm): 3.60 (CH2, 2H, s); 7.08e7.78 (phenyl protons, 9H, m);7.60e7.64 (C1 of benzoxazole, 1H, d, J7,6: 9.2 Hz), 7.90e7.95 (C2 of

benzoxazole, 1H, dd, J6,4: 1.6 Hz, J6,7:7.8 Hz), 8.06e8.09 (C4 of ben-zoxazole, 1H, d, J4,6: 2.4 Hz), 10.30 (NH, 1H, s). The protons of thephenyl rings, PhI, PhII and PhIII resonate in the ranges,7.7804e7.9211, 6.7663e9.39 and 7.6219e8.8452 ppm theoretically.The chemical shift of hydrogen atom associated with the NH groupis 6.2093 ppm and for the hydrogen atoms of the CH2 groups is4.124 and 4.1239 ppm. The predicted shifts of carbon atoms are127.7619e134.8546 ppm for PhI, 108.7313e145.5238 ppm for PhII

Table 4Calculated NMR parameters (with respect to TMS).

Protons sTMS scalc dcalc ¼ sTMS�scalc

H7 32.7711 25.0983 7.6728H8 32.7711 23.3811 9.3900H9 32.7711 26.0048 6.7663H17 32.7711 23.9259 8.8452H19 32.7711 24.8866 7.8845H20 32.7711 25.0698 7.7013H21 32.7711 25.1492 7.6219H25 32.7711 26.5618 6.2093H28 32.7711 24.9907 7.7804H34 32.7711 24.85 7.9211H36 32.7711 24.9906 7.7805H37 32.7711 24.9097 7.8614H38 32.7711 24.8501 7.9210H40 32.7711 28.6471 4.1240H41 32.7711 28.6472 4.1239Carbon atomsC1 196.852 85.7999 111.0521C2 196.852 79.2258 117.6262C3 196.852 59.3433 137.5087C4 196.852 88.1207 108.7313C5 196.852 53.9699 142.8821C6 196.852 51.3282 145.5238C11 196.852 37.2263 159.6257C12 196.852 67.762 129.0900C13 196.852 52.7907 144.0613C14 196.852 64.9735 131.8785C15 196.852 60.8552 135.9968C16 196.852 70.5372 126.3148C18 196.852 67.3127 129.5393C26 196.852 33.7502 163.1018C29 196.852 66.3824 130.4696C30 196.852 61.9974 134.8546C31 196.852 67.7182 129.1338C32 196.852 66.3833 130.4687C33 196.852 69.0901 127.7619C35 196.852 67.718 129.1340C39 196.852 144.4808 52.37120

Fig. 4. HOMO-LUMO plots of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide.

Fig. 5. MEP plot of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acetamide.

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511504

and 126.3148e144.0613 ppm for PhIII rings. For the carbon atoms,C39, C26 and C11, the predicted shifts are 52.3712, 163.1018 and159.6257 ppm respectively.

4.3.4. Nonlinear optical studiesThe interaction of electromagnetic fields in various media to

produce new physical properties, produce nonlinear optical effects[61]. For the title compound, the polarizability, first hyper-polarizability and second hyperpolarizability are respectively,4.4539� 10�23, 6.8293 � 10�30 and�36.898� 10�37 esu and thesevalues of the investigated molecule clearly reveal that they havenonlinear optical behavior with non-zero values. The reportedvalues of the first hyperpolarizability of similar derivates are2.66 � 10�30 esu [62] and 1.37 � 10�30 esu [47] and the firsthyperpolarizability of the title compound is 52.53 times that of thestandard NLO material urea [63]. The CeN bond lengths in the titlecompound are intermediate between a single and double bondwhich produces an extended p-electron delocalization over themolecular system [64]which is responsible for the nonlinearity ofthe system.

4.3.5. Frontier molecular orbitalsThe site selectivity and chemical reactivity such as the electronic

chemical potential, m, hardness, h, global electrophilicity, m, can bedefined successfully by DFT methods [65,66]. The HOMO-LUMOplots are shown in Fig. 4 and HOMO is localized over the wholemolecule except the mono-substituted phenyl ring and CH2 groupwhile the LUMO is over the whole molecule except over the mono-substituted phenyl ring, CH2 and acetamide group. For the NBBPA

compound, EHOMO ¼ �8.131 eV and ELUMO ¼ �5.318 eV and theenergy gap¼ 2.813 eV. According to the literature data [67e71], thechemical potential m ¼ �(I þ A)/2 where I ¼ �EHOMO andA ¼ �ELUMO are ionization potential and electron affinity; harness,h ¼ (I-A)/2 and electrophilicity index, u ¼ m2/2h. The differentdescriptors are: m ¼ 6.7245, h ¼ 1.4065 and u ¼ 16.075.

4.3.6. Molecular electrostatic potentialUsing the molecular electrostatic potential map the charge

distribution of a molecule can be pictured which is importantbecause, these values determine how molecules interact with oneanother as they point out the reactive sites in the molecule. In theMEP plot (Fig. 5) blue regions are the stronger positive regions andred regions are the lower electrostatic potential regions. From theMEP plot it is very clear that carbonyl and NH groups are the lowerelectrostatic potential sites favorable of electrophilic reactive re-gions and other parts of the NBBPA molecule are favorable fornucleophilic reactive regions.

Fig. 6. ALIE surface of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acet-amide molecule.

Fig. 7. Intramolecular noncovalent interactions and their strengths (in electron/bohr3)of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acetamide molecule.

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511 505

4.3.7. ALIE surface, Fukui functions and non-covalent interactionsIn order to investigate reactive properties of organic molecules

Sjoberg et al. have introduced average local ionization energy(ALIE) [72,73]. The definition of this quantum-molecular descriptoris based on sum of orbital energies weighted by the orbital den-sities according to the following equation:

IðrÞ ¼Xi

rið r!Þjεijrð r!Þ : (1)

where rið r!Þ denotes electronic density of the i-th molecular orbitalat the point r!, εi denotes orbital energy, while rð r!Þ denotes totalelectronic density function. ALIE indicates the energy necessary forthe removal of electron from the certain point around the mole-cules. This quantity can be represented as maximal and minimalvalue for each atom, or its values can be mapped to the electrondensity surface. The later mentioned method, applied in this work,enables visualization of molecule locations prone to the electro-philic attacks, Fig. 6.

Inspection of ALIE surface presented in Fig. 6 indicates that thereare two specific molecule sites prone to electrophilic attacks(designated by the red color). In this regard near vicinity of bromineatom Br23 and near vicinity of central benzene ring are charac-terized by ALIE values which indicate that here electrons are leasttightly bound and therefore the most easily removed. Thesemolecule sites are characterized by the ALIE value of around196 kcal/mol. On the other side the highest ALIE values are around338 kcal/mol and are localized in the near vicinities of hydrogenatoms.

Charge density analysis in the case of NBBPA molecule revealedthe existence of two intramolecular noncovalent interactions, Fig. 7.These noncovalent interactions are located between bromine andnitrogen atoms (Br23eN10) and between oxygen and hydrogenatoms (O27eH8). Both of these noncovalent interactions are strong,with the one between oxygen and hydrogen atom being signifi-cantly stronger.

Further investigation of local reactivity properties encompassedthe calculations of Fukui functions, which are calculated in Jaguarprogram by finite difference approach according to the followingequations:

fþ ¼�rNþdðrÞ � rNðrÞ

�

d; (2)

f� ¼�rN�dðrÞ � rNðrÞ

�

d: (3)

In equations (2) and (3), N stands for the number of electrons inreference state of the molecule, while d stands for the fraction ofelectron which default value is set to be 0.01 [74]. Mapping of theFukui functions to the electron density surface enables visualiza-tion of molecule sites where electron density increase or decreaseas a consequence of charge addition or removal, Fig. 8. In the case ofFukui fþ function it is necessary to track the positive color (purplecolor in Fig. 8a), which indicates the molecule sites where electrondensity increased after the addition of charge. In this regard it canbe seen that after charge addition electron density increased in thenear vicinity of atoms of benzene ring with bromine atom and inthe near vicinity of carbon atom C11, indicating that these moleculesites act as electrophiles. In the case of Fukui fe function it isnecessary to track the negative color (red color in Fig. 8b), whichshows the molecule sites where electron density decreased afterthe removal of charge. It can be seen in Fig. 8b that electron densitydecreased in the near vicinity of terminal benzene ring, showingthat this part of the molecule could act as nucleophile.

4.3.8. Natural bond orbital analysisThe natural bond orbitals (NBO) calculations were performed

using NBO 3.1 program [75] as implemented in the Gaussian09package at the DFT/B3LYP level and the results are tabulated inTables 5 and 6. The strong inter-molecular hyper-conjugativeinteraction are: C11eC12 from N10 of n1(N10)/s*(C11eC12), N10eC11from O22 of n2(O22)/p*(N10eC11), C12eC13 from Br23 of n3(Br23)/p*(C12eC13), C26eO27 from N24 of n1(N24)/p*(C26eO27), N24eC26from O27 of n2(O27)/s*(N24eC26) having electron densities,0.03329, 0.30325, 0.44545, 0.29909, 0.07226e and stabilizationenergies of 14.28, 28.72, 10.08, 64.93, 23.26 kJ/mol. The naturalhybrid orbital with low occupation numbers, higher energies andconsiderable p-character are: n2(O22), n3(Br23), n2(O27) withenergies, �0.3278, �0.28331, �0.23752a.u and p-characters, 100.0,100.0, 99.95% and low occupation numbers, 1.74929, 1.92825,1.86624 while the orbital with high occupation number, lowerenergies are: n1(O22), n2(Br23), n1(O27) withenergies, �0.60252, �0.28482, �0.67917a.u. and p-characters,58.72, 99.34, 38.97% and occupation numbers, 1.97241, 1.97414,

Fig. 8. Fukui functions a) fþ and b) fe of the N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acetamide molecule.

Table 5Second-order perturbation theory analysis of Fock matrix in NBO basis corresponding to the intra-molecular bonds of the title compound.

Donor(i) Type ED/e Acceptor(j) Type ED/e E(2)a E(j)-E(i)b F(i,j)c

C3eC4 p 1.68597 C1eC2 p* 0.33296 17.93 0.29 0.065C3eC4 p 1.68597 C5eC6 p* 0.46084 20.70 0.28 0.071C5eC6 p 1.60101 C1eC2 p* 0.33296 19.33 0.29 0.068C5eC6 p 1.60101 C3eC4 p* 0.38314 18.67 0.28 0.065C5eC6 p 1.60101 N10eC11 p* 0.30325 10.97 0.26 0.049N10eC11 p 1.86715 C5eC6 p* 0.46084 14.53 0.34 0.069LP N10 s 1.90927 C11eC12 s* 0.03329 14.28 0.64 0.086LP O22 p 1.74929 C5eC6 p* 0.46084 21.58 0.35 0.082LP O22 p 1.74929 N10eC11 p* 0.30325 28.72 0.33 0.089LP Br23 n 1.92825 C12eC13 p* 0.44545 10.08 0.29 0.053LP N24 s 1.65272 C3eC4 p* 0.38314 31.93 0.29 0.086LP N24 s 1.65272 C26eO27 p* 0.29909 64.93 0.27 0.119LP O27 p 1.86624 N24eC26 s* 0.07226 23.26 0.70 0.116LP O27 p 1.86624 C26eC39 s* 0.06509 18.66 0.60 0.096

a E(2) means energy of hyper-conjugative interactions (stabilization energy kJ/mol).b Energy difference between donor and acceptor i and j NBO orbitals.c F(i,j) is the Fock matrix element between i and j NBO orbitals.

Table 6NBO results showing the formation of Lewis and non Lewis orbitals.

Bond (A-B) ED/ea EDA% EDB% NBO s% p%

pC3-C4 1.68597 49.21 50.79 0.7015 (sp1.00)C 0.00 100.00

e �0.25489 e e þ0.7126 (sp1.00)C 0.00 100.00

pC5-C6 1.60101 49.78 50.22 0.7055 (sp1.00)C 0.00 100.00

e �0.25756 e e þ0.7087 (sp1.00)C 0.00 100.00

pN10-C11 1.86715 58.40 41.60 0.7642 (sp1.00)N 0.00 100.00

e �0.31882 e e þ0.6450 (sp1.00)C 0.00 100.00

n1 N10 1.90927 e e sp2.02 33.07 66.93

e �0.36852 e e e e e

n1 O22 1.97241 e e sp1.43 41.18 58.72

e �0.60252 e e e e e

n2 O22 1.74929 e e sp1.00 0.00 100.00

e �0.32784 e e e e e

n1 Br23 1.99512 e e sp0.15 86.85 13.15

e �0.93118 e e e e e

n2 Br23 1.97414 e e sp99.99 0.66 99.34

e �0.28482 e e e e e

n3 Br23 1.92825 e e sp1.00 0.00 100.00

e �0.28331 e e e e e

n1 N24 1.65272 e e sp1.00 0.00 100.00

e �0.26022 e e e e e

n1 O27 1.97473 e e sp0.64 61.03 38.97

e �0.67917 e e e e e

n2 O27 1.86624 e e sp99.99 0.05 99.95

e �0.23752 e e e e e

a ED/e is expressed in a.u.

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511506

1.97473.

4.3.9. Reactive and degradation properties based on autoxidationand hydrolysis

Computational analysis of molecules based on the combinationof DFT calculations and MD simulations allows inexpensive initialprediction of degradation properties. In this regard it is useful tonote that bond dissociation energy (BDE) for hydrogen abstractionindicate to what extent is some molecule prone to autoxidationmechanism, while BDE values for the rest of the single acyclicbonds can serve for the classification of bonds according to theirstrengths. These results are important for the rationalization andoptimization of procedures related to forced degradation studies[76e79]. Analysis of BDEs for hydrogen abstraction can indicatewhether some molecule is prone to autoxidation mechanism ornot. According to the Wright et al. [80], for investigated pharma-ceutical molecule can be concluded that it is vulnerable towardsautoxidation mechanism if the BDE ranges from 70 to 85 kcal/mol.Gryn'ova et al. [81] agrees with this statement, but also states thatin this regard CeH dissociation is questionable for the BDE values inthe range between 85 and 90 kcal/mol, meaning that BDE values forhydrogen abstraction in this range could also be important for theautoxidation mechanism. When BDE values for hydrogen abstrac-tion are lower than 70 kcal/mol, formed radicals are resistant forthe O2insertion [25,80e82] and these values are not suitable forautoxidation mechanism.

In Fig. 9 all BDE values have been provided. Values provided inred color corresponds to the BDE values for hydrogen abstraction,while values provided in blue corresponds to the BDE values for the

Fig. 10. RDFs of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acetamide'satoms with significant interactions with water molecules.

Fig. 11. Receptor ligand interactions which were determined by Autodock. The weaknoncovalent interactions with N45, E49, I77, F103, E104, K109, V117 amino acidresidues.

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511 507

rest of the single acyclic bonds. Results presented in Fig. 9 indicatethat NBBPAmolecule is very stable in open air and in the presence ofoxygen since there are no BDE values for hydrogen abstraction inthe range from 70 to 85 kcal/mol. The lowest BDE value forhydrogen abstraction is 91.53 kcal/mol, which is higher even thanupper limit defined by Wright and Gryn'ova. On the other side thelowest BDE values for the rest of the single acyclic bonds have beencalculated for bonds denoted with numbers 13 and 15, indicatingthat degradation could start by detaching of bromine atom or ter-minal benzene ring.

Comparison of atoms of NBBPAmolecule with regard to theirinteractions with water molecules have been performed by calcu-lations of radial distribution functions (RDFs) after MD simulations.RDF, g(r), indicates the probability of finding a particle in the dis-tance r from another particle [83]. RDFs that resemble the mostimportant interactions with water molecules are presented inFig. 10. According to the results presented in Fig. 10, six atoms ofNBBPAmolecule have pronounced interactions with water. g(r)curves of carbon atoms C1, C4 and C26 are very similar in terms ofprofile and peak distance (around 3.5 Å), with atom C4 havingsomewhat lower maximal g(r) value comparing with C1 and C26.g(r) curve of bromine atom Br23 is also similar with carbons, but it'smaximal g(r) value is the highest of all atoms with significant in-teractions with water molecules. However, the most importantinteractions with water molecules according to RDFs can be seenfor oxygen atom O27 and hydrogen atom H25. The maximal g(r)value for O27 is located at distance of around 2.6 Å, while in thecase of H25maximal g(r) value is located at around 1.8Å, indicatingstrong interactions with water in both cases.

4.3.10. Molecular docking studiesDNA gyrase basically consisted of two subunits (A and B) and

functions by dimer form of these subunits. The ATP binding pocket,which is founded highly conserved between species is located insubunit B of DNA gyrase, and defined by N45, E49, D72, R75, I77,K102, V117, and T164 residues in T. thermophilus and E. coli DNAgyrase subunit B (GyrB). This region is thought to be a drug targetfor antibacterial agents [84e86]. High resolution crystal structureof DNA gyrase B form complex was downloaded from the proteindata bank website (PDB ID: 1KIJ [87]. All molecular docking cal-culations were performed on Auto Dock Vina software [88]. Re-ceptor was in dimer form so firstly one of the subunit B was

Fig. 9. BDEs of all single acyclic bonds of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acetamidemolecule

Fig. 12. Molecular surface model of Autodock analyzing.

Table 7The binding affinity values of different poses of the title compound predicted byAutodockVina

Mode Affinity (kcal/mol) Distance from best mode (Å)

e e RMSD l.b. RMSD u.b.

1 �9.1 0.000 0.0002 �8.4 1.661 2.4643 �7.9 2.733 2.9314 �7.8 6.935 10.3255 �7.3 5.849 12.4566 �6.9 5.934 9.3037 �6.8 6.121 12.734

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511508

removed and then the receptor was prepared by the cleaning of allheteroatom's (i.e., nonreceptor atoms such as water, ions, co-crystallized ligand, etc.). The Auto Dock Tools (ADT) graphicaluser interface was used to calculate Gasteiger charges and polarhydrogen's. Ligand and receptor input files were arranged in PDBQTformat. The docking area was defined around the ATP binding site,which was determined in crystal structure, by a grid box of40 Å � 40 Å � 40 Å using 0.375 Å grid point spacing in AutoGrid.The docking conformations of ligands in the binding sites of thereceptor were searched with Lamarkian genetic Algorithm (LGA) inAutodock. The cocrystallized inhibitor was extracted from receptorand docked again for the affordance of the docking protocol. Thedocking protocol predicted the same conformation as was presentin the crystal structure with RMSD value well within the reliablerange of 2 Å [89]. Amongst the docked conformations, one which

Fig. 13. Receptor ligand interactions which were determined by Discovery Studio Client 3.5.cation bond with the other benzene rings of compound.

binded well the active site was analyzed for detailed interactionswith ADT and Discover Studio Visualizer 3.5 software [90]. Theligand binds at the active site of the substrate (Figs. 11e13) by weaknoncovalent interactions with N45, E49, I77, F103, E104, K109, V117amino acid residues. Discover Studio Visualizer was also predictedelectrostatic interactions with N45, D48, E104, K109, V117 and vander Waals interactions with E49, I77, F103, Q105, T166. E104 formspe2 bond with benzene ring which attaches to bromine and K109also forms p-cation bond with the other benzene rings of com-pound. The docked ligand title compound forms a stable complexwith GyrB complex and gives a binding affinity (DG in kcal/mol)value of �9.1 (Table 7). These preliminary results suggest that thecompound might exhibit inhibitory activity against GyrB complex.

5. Conclusion

Microbiological results against Gram-negative (E. coli, P. aeru-ginosa) and Gram-positive (S. aureus, E. faecalis) bacteria showedthat N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide compound exhibit higher activity against E. coliand S. aureus, with MIC values of 128 mg/ml and 64 mg/ml respec-tively, than the standard drug Gentamycin. Additionally, it wasfound that the activity against E. faecalis was more potent than to

pe2 bond between benzene ring which bounds bromine and e104. K109 also forms p-

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511 509

other tested microorganisms. The red shift and splitting of NHstretching mode shows the weakening of the NH bond, while thefirst hyperpolarizability of the title compound is 52.53 times that ofthe standard NLO material e urea. It was also concluded that CeNbond lengths an extended p-electron delocalization over the mo-lecular system, which is responsible for the nonlinearity of themolecule. The carbonyl and NH groups are favorable sites forelectrophilic attacks, while ALIE surface indicated the significanceof bromine atom in terms of electrophilic attacks. HOMO is spreadover the whole molecule, except the mono-substituted phenyl ringand CH2 group, while LUMO is spread over the whole molecule,except over the mono-substituted phenyl ring, CH2 and acetamidegroup. HOMO and LUMO visualization indicates charge transferwithin the molecule. Fukui functions show that benzene ring withbromine atom and terminal benzenering could have significantimportance with the addition and removal of charge. Analysis ofelectron density determined that there are two intramolecularnoncovalent interactions. BDE values for hydrogen abstractionindicate high stability of the title molecule in the open air or in thepresence of oxygen, while BDE values of the rest of the singleacyclic bonds indicate that degradation could start with thedetaching bromine atom. RDFs obtained after MD simulationsindicate that there are six atoms of NBBPA molecule with signifi-cant interactions with water molecules among which the mostimportant are hydrogen atom H25 and oxygen atom O27. Thedocked title compound forms a stable complex with GyrB complexand gives a binding affinity value of �9.1 kcal/mol and mightexhibit inhibitory activity against GyrB complex. Obtained resultsconcerning antimicrobial activity emphasize the potential of NBBPAmolecule for further studies, while other results encompassed bythis study provide important insights into the reactivity of the titlecompound.

Acknowledgments

Part of this work has been performed thanks to the supportreceived from Schr€odinger Inc. Part of this study was conductedwithin the projects supported by the Ministry of Education, Scienceand Technological Development of Serbia, grant numbers OI 171039and TR 34019. Additionally, this study is supported by a grant(Project Number:16H0237002) from Scientific Research ProjectsCommittee of Ankara University.

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