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

Article abstract of DOI:10.1016/j.molstruc.2017.04.010

N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide (NBBPA) was synthesized in this study as an original compound in order to evaluate its antibacterial activity against representative Gram-negative and Gram-positive bacteria, with their drug-resi

Full text of DOI:10.1016/j.molstruc.2017.04.010

Journal of Molecular Structure 1141 (2017) 495e511
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Modification of benzoxazole derivative by bromine-spectroscopic,
antibacterial and reactivity study using experimental and theoretical
procedures
a
b
c
a, *
€
V.V. Aswathy , Sabiha Alper-Hayta , Gozde Yalcin , Y. Sheena Mary
,
a
a
d
ꢀ e
C. Yohannan Panicker , P.J. Jojo , Fatma Kaynak-Onurdag , Stevan Armakovic ,
Sanja J. Armakovic , Ilkay Yildiz , C. Van Alsenoy
a Department of Physics, Fatima Mata National College, Kollam, Kerala, India
g
h
ꢀ f
b
€ꢁ
€
Republic of Turkey Ministry of Health, Turkish Medicines and Medical Devices Agency, 2176 Str. Sogütozü, 06520 Çankaya, Ankara, Turkey
c Ankara University, Biotechnology Institute, Tandogan 06100, Ankara, Turkey
d Trakya University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 22030 Balkan Yerleskesi, Edirne, Turkey
e
ꢀ
University of Novi Sad, Faculty of Sciences, Department of Physics, Trg D. Obradovica 4, 21000 Novi Sad, Serbia
University of Novi Sad, Faculty of Sciences, Department of Chemistry, Biochemistry and Environmental Protection, Trg D. Obradovica 3, 21000 Novi Sad,
f
ꢀ
Serbia
g Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Tandogan, Ankara, Turkey
h Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenylacetamide (NBBPA) was synthesized in this study
as 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 results
showed 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 the
ring breathing modes of the mono-, ortho- and tri-substituted phenyl rings is in agreement with the
literature data. DFT calculations were also used to identify specific reactivity properties of NBBPA
molecule based on the molecular orbital, charge distribution and electron density analysis, which
indicated 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 autoxidation
mechanism, 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 GyrB
complex.
Received 29 September 2016
Received in revised form
1 April 2017
Accepted 3 April 2017
Available online 6 April 2017
Keywords:
Benzoxazole
ALIE
RDF
BDE
Moleculardocking
Antibacterial activity
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
certain antibiotics and synthetic agents. Therefore, there could be a
rapidly growing global crisis in the clinical management of life-
Benzoxazole ring is one of the most common heterocycles in
medicinal chemistry. Previous reports revealed that substituted
benzoxazoles possess diverse chemotherapeutic activities
including antimicrobial [1e5], antiviral [6], topoisomerase I and II
inhibitors [7] and antitumor activities [8,9]. Bacterial resistance to
antibacterial agents or antibiotics is of grave concern in the medical
community, as many species of bacteria have evolved resistance to
threatening infectious diseases caused by multidrug-resistant
strains of the Gram-positive pathogens like Streptococcus,
Enterococcus, and Staphylococcus, and Gram-negative pathogens
like 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 increasing
significance [10e12]. To meet this crisis successfully, many re-
searchers across the globe are working to unearth new compounds
* Corresponding author.
E-mail address: sypanicker@rediffmail.com (Y.S. Mary).
http://dx.doi.org/10.1016/j.molstruc.2017.04.010
0022-2860/© 2017 Elsevier B.V. All rights reserved.

496
V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
which can selectively attack novel targets in microorganisms.
Hence, the development of novel, potent, and unique antibacterial
agents is the preeminent way to overcome bacterial resistance and
develop 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 5th
position on the benzoxazole ring and examined for their in vitro
antimicrobial activity against some Gram-positive, Gram-negative
bacteria and Candida albicans [1e3,14,15]. On the basis of these
considerations, we synthesized N-[2-(2-bromophenyl)-1,3-
benzoxazol-5-yl]-2-phenylacetamide (NBBPA) as antimicrobial
agent reported in this work, choosing a bromine atom at the 2nd
position of phenyl of second carbon of benzoxazole ring. The
strategy employed was to examine the effect of the bromine against
some Gram-positive, Gram-negative bacteria and their drug-
resistant isolates.
One of the main characteristics of biologically active molecules
is their overall stability, especially in aquatic mediums. These
organic molecules are synthesized to be highly stable, which
hardens their removal from the water and soil [16]. Since phar-
maceutical care products are frequently used and improperly
dumped into the environment their active components accumulate
in water, which is very harmful because it has been shown that
aforementioned molecules exhibit toxic effects towards aquatic
organisms [16,17]. The fact that biologically active molecules that
serve as active components of pharmaceutical products have been
detected 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 forced
degradation by advanced oxidation processes could be fine alter-
native [17,18,20,21] and it also might serve as a basis for the studies
of their toxic effects [22e24]. Rationalization of studies based on
forced degradation can be done by application of DFT calculations
and MD simulations [25e27]. Principles of molecular modeling
enable 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 of
theoretical analysis, in this work we have investigated reactivity in
order to gain an insight into the possible degradation properties of
NBBPA molecule.
(0.5 mmol), sodium bicarbonate (0.5 mmol), diethyl ether (10 ml)
and water (10 ml). The mixture was kept stirred overnight at room
temperature 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.
The
product was recrystallized from ethanol-water as needles, which
was dried in vacuo. The chemical, physical and spectral data of the
compound 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 venders
and were used without purification. The reactions were monitored
and the purity of the products was checked by thin layer chroma-
tography TLC. Kieselgel HF 254 chromatoplates (0.3 mm) was used
for TLC and the solvent system was ethylacetate:n-hexane (2:1).
The melting point was taken on a Buchi SMP 20 capillary apparatus
and is uncorrected. 1H NMR spectra was obtained with a Varian
400 MHz spectrometer in dimethylsulfoxide-d6 (DMSO-d6) and
tetramethylsilane (TMS) was used as an internal standard. Mass
analyses was carried out with a Waters 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% of
the theoretical values. All chemicals and solvents were purchased
from Aldrich Chemical Co. or Fischer Scientific.
The FT-IR spectrum (Fig. 1) was recorded using KBr pellets on a
DR/Jasco FT-IR 6300 spectrometer. The FT-Raman spectrum (Fig. 2)
was obtained on a Bruker RFS 100/s, Germany. For excitation of the
spectrum the emission of Nd:YAG laser was used, excitation
wavelength 1064 nm, maximal power 150 mW, measurement on
solid sample. The spectral resolution after apodization was 2 cmꢁ1
.
2.2. Microbiology
Microorganisms Pseudomonasaeruginosa isolate (gentamicin-
resistant), Escherichiacoli isolate, which has an extended spectrum
beta lactamase enzyme (ESBL), Staphylococcus aureus isolate
(meticilline-resistant (MRSA)), P. aeruginosa ATCC 27853 (American
Type 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. aeruginosa
ATCC 25853, E. coli ATCC 25922, S. aureus ATCC 25923, E. faecalis
ATCC 29212 and clinical isolates of these microorganisms resistant
to 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 Standards
Institute (CLSI) [28] in the clinical isolates. Standard powders of
ampicillin trihydrate, gentamycin sulphate, ofloxacin were ob-
tained from the manufacturers. Stock solutions were dissolved in
dimethylsulphoxide (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. The
microorganisms were passaged at least twice to ensure purity and
viability. The solution of the newly synthesized compound and
standard drugs were prepared at 400, 200, 100, 50, 25, 12.5, 6.25,
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 as
given below (Scheme 1):
First step: 5-Amino-2-(2-bromophenyl) benzoxazole was syn-
thesized by heating 0.01 mol 2,4-diaminophenol.2HCl with
0.01 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, the
residue was poured into ice water mixture and neutralized with
excess of 10 M NaOH solution extracted with benzene and then this
solution was dried over anhydrous sodium sulphate and evapo-
rated under diminished pressure. The residue was boiled with
200 mg charcoal in ethanol and filtered. After the evaporation of
solvent in vacuo, the crude product was obtained and recrystallized
from 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. Excess
thionyl chloride was removed in vacuo. The residue was dissolved
in ether (10 ml) and this solution was added during 1 h to a stirred,
ice-cold mixture of 5-amino-2-(2-bromophenyl)benzoxazole
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 Hinton
Broth (MHB). Bacterial susceptibility testing was performed ac-
cording to the guidelines of CLSI M100-S16 [29].
The bacterial suspensions used for inoculation were prepared at
105 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

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
497
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.
compounds. There were 104 cfu/ml bacteria in the wells after in-
oculations. MHB was used for diluting the bacterial suspension and
for twofold dilution of the compound. 80% DMSO-20% EtOH,
methanol, DMSO, PBS, pure microorganisms and pure media were
chemical calculations method. A scaling factor of 0.9613 is used to
scale the theoretically predicted wavenumbers [31]. The assign-
ments of the wavenumbers are done by using GaussView [32] and
GAR2PED [33] software. The theoretically predicted geometrical
parameters (Fig. 3) are given in Table 2.
used as control wells. A 10 ml bacteria inoculum was added to each
well of the microdilution trays. The trays were incubated at 37 ꢀC
and MIC endpoints were read after 24 h of incubation. All organ-
isms were tested in triplicate in each run of the experiments. The
lowest concentration of the compound that completely inhibits
macroscopic growth was determined and minimum inhibitory
concentrations (MICs) were reported in Table 1.
In this work Schrodinger Materials Science Suite 2015-4 has
€
been also employed for the DFT calculations and MD simulations.
Desmond program was used for MD simulations [34e37], while
Jaguar 9.0 program [38] was used for DFT calculations. Calculations
performed with Jaguar were done with B3LYP exchange-correlation
functional [39], together with 6e311þþG(d,p), 6-31 þ G(d,p) and
6-311G(d,p) basis sets for the calculations of ALIE, Fukui functions
and BDEs, respectively. OPLS 2005 force field [40] was used in case
of MD simulations with Desmond program. Simulation time was
set to 10 ns, within isothermaleisobaric (NPT) ensemble class. To
model the interactions with water one NBBPA molecule was placed
in cubic box with ~3000 water molecules. Temperature was set to
3. Computational details
Calculations of the wavenumbers, NMR chemical shifts, MEP
and NBO analysis of the NBBPA molecule are carried out with
Gaussian09 program [30] using B3LYP/6-311þþG (5D, 7F) quantum

498
V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
Fig. 2. FT-Raman spectrum of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acetamide.
300 K, pressure set to 1.0325 bar, cut off radius to 12 Å. Simple point
charge (SPC) model [41] was used for the treatment of solvent.
Noncovalent interactions in Jaguar program are treated by the
method developed by Johnson et al. [42,43]. Maestro GUI [44] was
used for the preparation of input files and analysis of results when
Table 1
The microbiological result of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-
phenylacetamide.
Compounds
Microorganisms (MIC value,
Gram-negative
m
g/ml)
Gram-positive
€
Schrodinger Materials Science Suite 2015e4 was used.
E.c
E.c*
P.a.
P.a.*
S.a.
S.a.*
E.f.
E.f.*
Title compound
Ampicillin
Ofloxacine
128
2
0.015
0.25
128
>1024
16
64
e
1
128
e
128
0.5
0.125
0.5
64
e
32
0.5
1
128
0.5
4
4. Results and discussion
1
64
0.5
128
Gentamycin
256
1
8
8
4.1. Chemistry
Abbreviations:E.c:Escherichia coliATCC 25922, E.c*:Escherichiacoliisolate, which has
an extended spectrum beta lactamase enzyme (ESBL), P.a: Pseudomonas aeruginosa
ATCC 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 ATCC
29212, E.f*: Enterecoccus faecalis isolate (vancomycin-resistant enterococci).
For the synthesis of NBBPA firstly, 5-amino-2-(p-bromophenyl)
benzoxazole was obtained by heating 2-bromobenzoic acid with
2,4-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
Fig. 3. Optimized geometry of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acetamide.

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
499
Table 2
Optimized geometrical parameters.
Bond lengths (Ǻ)
C1eC2
C2eC3
C3eN24
C5eC6
N10eC11
C12eC13
C13eBr23
C15eC18
C16eH20
N24eC26
H28eC29
C30eC32
C31eH34
C33eC35
C39eH40
Bond angles (ꢀ)
C2eC1eC6
1.3987
1.4168
1.4198
1.4004
1.3080
1.4102
1.9469
1.3942
1.0813
1.3708
1.0835
1.4055
1.0822
1.3982
1.0924
C1eC6
C2eH8
C4eC5
1.3830
1.0765
1.3906
1.4103
1.4558
1.4145
1.3889
1.0795
1.0817
1.2509
1.4055
1.5130
1.3978
1.0819
1.0923
C1eH7
C3eC4
C4eH9
1.0799
1.4042
1.0818
1.3988
1.4273
1.3956
1.0788
1.3972
1.0102
1.5352
1.3978
1.3982
1.0835
1.0822
C5eN10
C11eC12
C12eC14
C14eC16
C15eH19
C18eH21
C26eO27
C29eC30
C30eC39
C32eC35
C33eH37
C39eH41
C6eO22
C11eO22
C13eC15
C14eH17
C16eC18
N24eH25
C26eC39
C29eC31
C31eC33
C32eH36
C35eH38
116.9
121.0
120.9
117.8
120.4
123.0
106.1
115.5
117.4
115.6
120.1
120.3
120.5
120.7
129.3
116.4
119.7
120.8
119.8
119.4
120.2
119.8
105.7
110.5
C2eC1eH7
120.9
120.3
122.4
121.7
130.5
129.5
131.5
125.2
120.7
121.8
120.7
119.7
119.6
104.3
115.6
119.5
120.8
120.8
120.1
119.7
120.2
120.1
105.7
105.9
C6eC1eH7
122.2
118.7
116.7
120.5
109.1
107.5
113.0
117.4
123.7
118.1
119.0
119.8
119.6
115.1
124.1
119.4
118.4
120.1
120.8
119.7
120.1
117.7
110.5
C1eC2eC3
C2eC3eC4
C3eC4eC5
C4eC5eC6
C1eC6eC5
C5eN10eC11
C1eC2eH8
C2eC3eN24
C3eC4eH9
C4eC5eN10
C3eC2eH8
C4eC3eN24
C5eC4eH9
C6eC5eN10
C1eC6eO22
C5eC6eO22
N10eC11eC12
C11eC12eC13
C12eC13eC5
C12eC14eC16
C13eC15eC18
C14eC16eC18
C15eC18eC16
C6eO22eC11
H25eN24eC26
O27eC26eC39
C30eC29eC31
C32eC30eC39
C33eC31eH34
C35eC32eH36
C35eC33eH37
C33eC35eH38
C26eC39eH41
H40eC39eH41
N10eC11eO22
C11eC12eC14
C12eC13eBr23
C12eC14eH17
C13eC15eH19
C14eC16eH20
C15eC18eH21
C3eN24eH25
N24eC26eO27
H28eC29eC30
C29eC30eC32
C29eC31eC33
C30eC32eC35
C31eC33eC35
C32eC35eC33
C26eC39eC30
C30eC39eH40
C12eC11eO22
C13eC12eC14
C15eC13eBr23
C16eC14eH17
C18eC15eH19
C18eC16eH20
C16eC18eH21
C3eN24eC26
N24eC26eC39
H28eC29eC31
C29eC30eC39
C29eC31eH34
C30eC32eH36
C31eC33eH37
C32eC35eH38
C26eC39eH40
C30eC39eH41
Dihedral angles (ꢀ)
C6eC1eC2C3
H7eC1eC2eH8
H7eC1eC6eC5
C1eC2eC3eN24
C2eC3eC4eC5
N24eC3eC4eH9
C4eC3eN24eH25
C3eC4eC5eN10
C4eC5eC6eC1
N10eC5eC6eO22
C1eC6eO22eC11
C5eN10eC11eC22
O22eC11eC12eC13
C12eC11eO22eC6
C14eC12eC13eC15
C11eC12eC14eH17
C12eC13eC15eC18
Br23eC13eC15eH19
H17eC14eC16eC18
C13eC15eC18eH21
C14eC16eC18eC15
H20eC16eC18eH21
H25eN24eC26eO27
N24eC26eC39eH40
O27eC26eC39eH40
H28eC29eC30eC39
H28eC29eC31eC33
C30eC29eC31eH34
C39eC30eC32eC35
C29eC30eC39eH40
C32eC30eC39eH40
C29eC31eC33eH37
C30eC32eC35eC33
H36eC32eC35eH38
H37eC33eC35eC32
0.0
0.0
ꢁ180.0
180.0
0.0
0.0
0.0
180.0
0.0
0.0
C6eC1eC2eH8
C2eC1eC6eC5
H7eC1eC6eO22
H8eC2eC3eC4
ꢁ180.0
0.0
0.0
H7eC1eC2eC3
C2eC1eC6eO22
C1eC2eC3eC4
H8eC2eC3eN24
N24eC3eC4eC5
C2eC3eN24eC26
C3eC4eC5eC6
H9eC4eC5eN10
N10eC5eC6eC1
180.0
180.0
0.0
180.0
ꢁ180.0
ꢁ180.0
ꢁ180.0
180.0
ꢁ180.0
180.0
0.0
ꢁ0.0
ꢁ0.0
ꢁ180.0
180.0
ꢁ0.0
180.0
ꢁ0.0
0.0
ꢁ0.0
C2eC3eC4eH9
ꢁ180.0
C2eC3eN24eH25
C4eC3eN24eC26
H9eC4eC5eC6
0.0
0.0
0.0
C4eC5eC6eO22
180.0
0.0
ꢁ180.0
180.0
0.0
C4eC5eN10eC11
C5eC6eO22eC11
N10eC11eC12eC13
O22eC11eC12eC14
C11eC12eC13eC15
C14eC12eC13eBr23
C13eC12eC14eC16
C12eC13eC15eH19
C12eC14eC16eC18
H17eC14eC16eH20
H19eC15eC18eC16
C14eC16eC18eH21
C3eN24eC26eO27
H25eN24eC26eC39
N24eC26eC39eH41
O27eC26eC39eH41
C31eC29eC30eC32
H28eC29eC31eH34
C29eC30eC32eC35
C39eC30eC32eH36
C29eC30eC39eH41
C32eC30eC39eH41
H34eC31eC33eC35
C30eC32eC35eH38
C31eC33eC35eC32
H37eC33eC35eH38
C6eC5eN10eC11
C5eN10eC11eC12
N10eC11eC12eC14
N10eC11eO22eC6
C11eC12eC13eBr23
C11eC12eC14eC16
C13eC12eC14eH17
Br23eC13eC15eC18
C12eC14eC16eH20
C13eC15eC18eC16
H19eC15eC18eH21
H20eC16eC18eC15
C3eN24eC26eC39
N24eC26eC39eC30
O27eC26eC39eC30
H28eC29eC30eC32
C31eC29eC30eC39
C30eC29eC31eC33
C29eC30eC32eH36
C29eC30eC39eC26
C32eC30eC39eC26
C29eC31eC33eC35
H34eC31eC33eH37
H36eC32eC35eC33
C31eC33eC35eH38
ꢁ180.0
0.0
180.0
180.0
0.0
0.0
0.0
0.0
180.0
ꢁ180.0
ꢁ180.0
180.0
ꢁ0.0
0.0
ꢁ180.1
180.0
0.0
ꢁ0.0
ꢁ180.0
123.9
ꢁ56.1
1.1
179.6
ꢁ179.8
179.1
ꢁ31.7
148.7
ꢁ179.8
0.2
ꢁ180.0
ꢁ180.0
0.0
0.0
180.0
ꢁ180.0
ꢁ0.1
0.0
ꢁ124.1
179.9
ꢁ179.3
ꢁ179.1
ꢁ0.2
55.9
0.5
0.0
ꢁ0.5
ꢁ1.1
ꢁ148.6
31.9
179.5
179.8
0.1
179.3
89.8
ꢁ89.7
ꢁ0.1
ꢁ0.2
ꢁ179.6
ꢁ179.5
ꢁ0.0
179.8
0.2

500
V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
treating phenylaceticacid with thionylchloride. The compound was
prepared as a new product. The structure of title compound was
supported by spectral data. The 1H NMR, mass spectra and
elemental analysis results are in agreement with the proposed
structure.
values are at 1520 cmꢁ1 [53] and at 1536 cmꢁ1 [54]. In the present
casae, the CeN stretching modes are assigned at 1190, 1098 cmꢁ1
(IR), 1192 cmꢁ1 (Raman) and at 1222, 1188, 1100 cmꢁ1 theoretically
with PED contribution 34, 35, 40% as expected [52,55]. The CeOeC
stretching modes of the title compound are assigned at 1137 and
956 cmꢁ1 theoretically with PEDs 35 and 40%, with low IR in-
tensities and the reported the CeOeC stretching modes are 1191,
1187, 988 and 993 cmꢁ1 [53,56].
4.2. Antimicrobialactivities of the NBBPA
The newly synthesized was evaluated for their antibacterial
activity against S. aureus, E. faecalis as Gram-positive, E. coli, P.
aeruginosa as Gram-negative bacteria and their drug-resistant
clinical isolate. All the microbiological result were shown in
Table 1. According to antibacterial results NBBPA showed moderate
activity against the tested microorganisms. The compound indi-
The NeH modes are normally expected in the regions,
3400
40 cmꢁ1 (stretching mode), 1510e1500, 1400e1300,
740e730 cmꢁ1 (deformation modes) according to literature [50,51].
For NBBPA, the PED analysis gives the NH modes at 3450, 1524,
1262, 671 cmꢁ1 theoretically with 99% PED for stretching mode,
45e37% PEDs for deformation modes and the deformation modes
1524 and 1262 cmꢁ1 possess high Raman activity. Experimentally
bands are observed at 3455, 3262, 1527, 1260, 670 cmꢁ1 in the IR
spectrum and at 1526 cmꢁ1 in the Raman spectrum. The splitting in
the IR spectrum and red shift from the computed value of the NH
stretching vibration indicates the weakening of the NH bond as
reported in literature [57].
cated more effect against drug-resistant S. aureus (64
E. coli (128 g/ml) than standard drug Gentamycin (128
256 g/ml, respectively). Moreover, this molecule found to be more
mg/ml) and
m
mg/ml,
m
active against E. Faecalis than tested the other microorganisms.
4.3. Computational and spectral studies
Normally the CH2 vibrational modes appear in the ranges,
2950e2850 cmꢁ1 (stretching), 1470e900 cmꢁ1 (deformations
modes) [51,52,55] and the bands at 2936, 1422, 1165 cmꢁ1 (IR),
2975, 2936, 1423, 1166 cmꢁ1 (Raman) and 2969, 2934, 1425, 1285,
1167, 900 cmꢁ1 (DFT) are assigned as the stretching and deforma-
tion modes of the CH2 group of NBBPA.
The mono-, tri-, 1,2-substituted phenyl rings and benzoxazole
ring are designated as PhI, PhII, PhIII and PhIV in the following
discussions.
4.3.1. Geometrical parameters
The CeC bond lengths in the phenyl ring lie in the range
1.3978e1.4055 Å for PhI phenyl ring, 1.3830e1.4168 Å for PhII
phenyl ring and 1.3889e1.4145 Å for PhIII phenyl ring and for
benzene the CeC bond length is 1.3993 Å [45]. The CeN bond
lengths of the NBBPA, C3eN24 ¼ 1.4198 Å, C5eN10 ¼ 1.4103 Å and
The phenyl ring CH stretching modes are assigned at 3033 cmꢁ1
(IR), 3071, 3050, 3020 cmꢁ1 (Raman) for PhI and 3082, 3065 cmꢁ1
(IR) for PhIII rings. The DFT calculations give these modes in the
ranges, 3068e3036, 3118e3058 and 3094e3052 cmꢁ1 for PhI, PhII
and PhIII rings, respectively as expected [51]. The phenyl ring
stretching modes are assigned at 1470, 1320 cmꢁ1 (IR), 1588 cmꢁ1
(Raman), 1585e1317 cmꢁ1 (DFT) for PhI, 1577, 1541, 1391 cmꢁ1 (IR),
1543, 1310 cmꢁ1 (Raman), 1579e1309 cmꢁ1 (DFT) for PhII and
1447 cmꢁ1 (IR), 1605, 1567 cmꢁ1 (Raman), 1597e1275 cmꢁ1 (DFT)
for PhIII rings which are in agreement with literature [51]. All the
phenyl ring stretching modes possess a PED contribution equal or
greater than 40%. In the case of tri-substituted phenyl rings with
mixed substituents, the ring breathing mode is expected between
600 and 750 cmꢁ1 [51,58] and in the present case the mode at
749 cmꢁ1 (DFT) is assigned as the ring breathing mode of the tri-
substituted benzene and the reported values are at 764 cmꢁ1 (IR),
766 cmꢁ1 (Raman) and at769 cmꢁ1 (DFT) [59]. The ring breathing
mode at 749 cmꢁ1 has a PED of 50% with a high IR intensity and low
Raman activity. For the NBBPA compound, the DFT calculations give
the ring breathing mode of ortho substituted phenyl ring at
1111 cmꢁ1 with a PED of 46% which is in agreement with literature
[58] and the reported value is at 1087 cmꢁ1 [60]. The band at
978 cmꢁ1 with a PED of 40% is assigned as the ring breathing mode
of the mono substituted phenyl ring of the NBBPA compound as
expected [51,58]. Both the above two ring breathing modes have
low IR intensities and experimentally no bands are seen in the IR
spectrum. The in-plane CH bending modes of the phenyl rings are
observed at 1282, 1070, 1017 cmꢁ1 (IR), 1283, 1018 cmꢁ1 (Raman)
for PhI, 1129 cmꢁ1 (IR), 1128 cmꢁ1 (Raman) for PhII and at
1044 cmꢁ1 (IR), 1246, 1045 cmꢁ1 (Raman) for PhIII. The corre-
sponding theoretical CH deformation modes of the phenyl rings are
in the ranges,1281e1013 cmꢁ1 for PhI,1235e1126 cmꢁ1 for PhII and
1243e1043 cmꢁ1 for PhIII [51]. The out-of-plane CH deformation
modes of the phenyl rings are assigned at 968, 910, 725 cmꢁ1 in the
IR spectrum, 971, 726 cmꢁ1in the Raman spectrum for PhI, 951, 851,
824 cmꢁ1 in the IR spectrum, 846 cmꢁ1 in the Raman spectrum for
PhII and no bands are seen experimentally for PhIII. The PED
analysis gives these modes in the ranges, 969e726 cmꢁ1 for PhI,
951e826 cmꢁ1 for PhII and 960e741 cmꢁ1 for PhIII rings,
C
26eN24 ¼ 1.3708 Å are less than the normal CeN bond (1.48 Å)
shows some resonance in this section of the molecule [46]. The
bond lengths, C6eO22 ¼ 1.3988 Å, C11eO22 ¼ 1.4273 Å and C11
]
N10 ¼ 1.3080 Å are in agreement with that of similar derivative [47].
The bond length C26]O27 ¼ 1.2509 Å is in agreement with the
reported values [48]. In the present case, the CeBr bond length is
1.9469 Å which is in agreement with literature [49]. At C13 position,
the bond angles are,C12eC13eC15 ¼ 120.7ꢀ, C12eC13eBr23 ¼ 123.7ꢀ
and C15eC13eBr23 ¼ 115.6ꢀ and this asymmetry shows the repul-
sion between bromine and benzoxazle ring. Similarly at C12 and C11
positions, the angles, C13eC12eC14 ¼ 117.4ꢀ, C13eC12eC11 ¼ 125.2ꢀ,
C
N
14eC12eC11
¼
117.4ꢀ
and
N
10eC11eC12
¼
131.5ꢀ,
10eC11eO22 ¼ 113.0ꢀ, C12eC11eO22 ¼ 115.5ꢀ which shows the
interaction between O22 and H17 atoms. The interaction between
NH and neighbouring units is revealed by the bond angles around
C26 and N24 which are respectively,
C
39eC26eN24 ¼ 116.4ꢀ,
C
39eC26eO27
¼
119.5ꢀ,
N24eC26eO27
¼
124.1ꢀ
and
C26eN24eC3 ¼ 129.3ꢀ, C26eN24eH25 ¼115.6ꢀ, C3eN24eH25 ¼115.1ꢀ.
4.3.2. IR and Raman spectra
The 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 cmꢁ1 [50], and for NBBPA the band observed at
1670 cmꢁ1 experimentally is assigned as the C]O stretching mode
which agrees with the computed value at 1673 cmꢁ1 with a PED of
73% and high IR intensity of 398.27 and Raman activity of 59.03. In
substituted benzenes, stretching vibrations of CeBr is expected in
the range 635 85 cmꢁ1 [51] and for the NBBPA, this vibration is
assigned at 687 cmꢁ1 theoretically with PED 32% and this is in
agreement with the reported values [49]. According to literature
[52] the C]N stretching modes are expected in the region
1650e1500 cmꢁ1 and for the NBBPA, this mode is assigned at
1511 cmꢁ1 theoretically with a PED of 39% while the reported

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
501
Table 3
Calculated (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
(cmꢁ1
Raman
(cmꢁ1
Assignmentsa
y
(cmꢁ1
)
IRI
RA
y
)
y
)
e
3450
3118
3094
3081
3076
3068
3066
3058
3057
3052
3049
3037
3036
2969
2934
1673
1597
85.42
18.71
1.99
4.45
3.04
11.19
11.71
6.44
21.11
1.26
3.90
2.48
5.57
2.99
11.05
398.27
32.48
322.96
82.17
94.67
190.46
115.34
282.61
226.15
48.33
43.31
58.43
114.49
76.34
14.31
3455, 3226
e
e
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
d
y
y
y
y
y
y
d
y
d
y
d
d
y
y
d
d
y
d
y
y
d
d
d
y
d
y
y
d
y
d
y
y
d
d
d
y
y
d
d
y
d
y
y
d
y
y
y
y
y
d
y
d
y
d
y
NH(99)
e
CHII(96)
CHIII(97)
CHIII(95)
CHII(96)
CHI(93)
CHIII(95)
CHII(99)
CHI(100)
CHIII(93)
CHI(99)
CHI(99)
CHI(99)
CH2(99)
CH2(99)
C]O (73)
NH(18),
C]O(12),
PhI(45),
PhII(18)
C]O(21),
PhII(46),
PhIII(51),
CHIII(23)
PhI(66),
CHI(21)
PhII(58),
CHIII(21)
NH(44),
C]N(17)
C]N(39),
NH(12)
CHI(14),
PhI(50)
CHIII(13),
PhIII(49)
PhII(46),
CH2(15),
CH2(77)
CHI(24),
PhI(42)
CHIII(23),
PhIII(40)
PhII(48),
CHII(18)
PhI(65),
CHII(10)
PhII(50),
CN(15)
CH2(56),
CHI(20)
CHI(56),
CN(17)
PhIII(77),
CHI(10)
NH(45),
CN(14)
CHIII(62),
CN(25)
PhII(18),
CHII(43),
CN(34),
CC(13),
CC(33),
CC(31),
CN(18),
CH2(62),
PhI(18)
CHI(72),
PhI(20)
CHIII(83),
PhII(11)
e
e
3082
e
e
e
e
3071
e
3065
e
e
e
e
e
e
e
3050
e
e
3033
e
3020
2975
2936
1670
1605
72.03
237.43
59.93
2936
1670
e
404.23
y
PhIII(47)
1585
1579
1566
1558
1539
1524
1511
1467
1443
1440
7.60
706.00
49.75
e
1588
e
6.91
1577
e
y
PhI(12)
7.58
1062.04
8.00
1567
e
0.97
e
35.14
3.23
450.64
2622.68
145.75
2.32
1541
1527
e
1543
1526
e
592.34
11.15
51.14
49.03
1470
1447
e
e
31.39
e
88.51
e
d
CHII(13)
1425
1409
5.96
0.45
0.85
41.06
1422
e
1423
e
1399
1396
1317
1309
1285
1281
1275
1262
1243
1235
1222
17.82
139.44
196.56
0.36
10.57
232.47
501.12
0.16
e
e
1391
1320
e
e
e
1310
e
0.01
1.91
e
5.76
314.97
24.13
694.12
5.13
1282
e
1283
e
1.60
32.38
14.04
41.57
9.38
1260
e
e
1246
e
430.58
570.34
e
yCN(15)
e
e
y
y
PhIII(22)
CN(35)
1188
1175
87.80
1.41
18.62
41.66
1190
e
1192
e
d
CH2(20)
1167
1159
1158
109.86
0.15
251.05
2.62
1165
e
1166
e
0.24
3.30
e
e
(continued on next page)

502
V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
Table 3 (continued )
B3LYP/6-311þþG (5D, 7F)
IR
Raman
(cmꢁ1
Assignmentsa
y
(cmꢁ1
)
IRI
RA
y
(cmꢁ1
)
y
)
e
1146
1137
1126
1111
1100
1060
1043
1013
1010
990
3.66
43.49
e
e
d
y
y
d
d
y
d
y
y
d
y
d
y
d
d
y
d
t
y
y
d
CHI(67),
PhI(16)
CO(35),
CHII(33)
CHII(46),
0.01
4.91
10.42
6.88
6.85
3.21
27.21
3.86
85.01
2.53
e
e
131.33
5.20
1129
e
1128
e
CN(11), yPhII(17)
CHIII(41),
PhIII(46)
CN(40),
CHII(21)
PhI(12),
CHI(45)
CC(17),
PhIII(18),
CHI(52),
PhI(16)
CHIII(39),
PhIII(18)
PhIII(46),
32.78
0.13
1098
1070
1044
1017
e
e
e
5.56
1045
1018
1010
e
dCHIII(43)
104.08
13.75
79.26
e
PhI(16),
PhI(13),
y
y
CBr(11)
PhI(40)
978
969
0.30
0.13
43.70
0.31
e
e
968
971
g
CHI(74),
PhI(14)
CHIII(94)
PhIII(22),
CO(40),
t
g
960
956
0.01
7.20
0.60
16.03
e
e
e
e
d
y
d
PhIV(27)
951
938
935
0.01
0.34
1.52
0.01
0.39
0.06
951
e
e
g
g
CHII(88)
CHI(92)
e
932
933
yCC(16),
yPhII(21), dPhII(19)
904
900
7.89
6.28
151.17
13.71
910
e
e
e
g
CHI(77)
dCH2(68),
g
g
C]O(15)
CHIII(70),
898
893
847
836
3.91
0.88
0.01
28.34
14.55
0.57
0.10
0.03
e
e
t
d
d
PhIII(10)
CN(19),
PhII(12),
e
e
y
CC(31), yPhII(10)
851
e
846
e
g
CHII(78),
PhII(10)
CHIII(68),
PhIV(26)
t
g
d
828
826
808
11.60
0.69
23.75
17.79
0.29
2.13
e
e
e
e
g
g
CHI(98)
CHII(90)
824
e
d
d
y
d
t
CH2(13),
CN(11),
CO(14),
CN(20),
PhIII(33),
CC(31),
g
CHI(11),
PhII(32)
CHIII(29)
yPhI(10)
806
789
749
741
726
710
697
687
671
668
633
27.47
2.16
0.06
10.52
2.83
0.81
0.14
1.42
5.79
0.75
0.26
3.35
12.01
804
e
807
e
d
g
g
33.83
15.02
2.46
748
e
e
y
d
PhII(50),
CN(10)
e
gCHIII(72),
t
t
PhIII(17)
PhI(22),
725
e
726
e
gCHI(47),
g
CC(10)
32.41
11.38
53.20
2.64
d
y
t
PhIII(48),
CBr(10),
PhI(56),
dCH2(17)
695
e
e
gCHI(37)
e
y
t
CBr(32),
PhII(13),
t
PhIV(18),
CC(11),
CC(18),
g
CN(11)
PhIII(10),
NH(14)
670
670
633
e
gNH(37),
t
t
t
d
d
d
d
d
t
PhIV(20),
PhIV(32),
PhIII(18),
PhIV(20),
PhI(19),
PhIII(71)
PhI(87)
PhI(28),
g
t
tCN(16)
4.26
e
g
g
5.73
640
d
PhII(14)
617
611
605
4.39
0.70
7.87
0.58
3.57
0.03
616
e
e
612
e
e
PhI(11),
CN(23),
d
CH2(23)
602
582
4.72
0.97
e
e
e
e
g
tPhII(23),
tPhIV(12), tPhI(14)
11.10
15.88
dPhIII(40),

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
503
Table 3 (continued )
B3LYP/6-311þþG (5D, 7F)
IR
Raman
(cmꢁ1
Assignmentsa
y
(cmꢁ1
)
IRI
RA
y
(cmꢁ1
)
y
)
e
d
PhIV(12)
543
536
509
500
439
429
419
21.96
2.57
37.75
0.01
6.40
1.06
3.21
0.47
3.57
3.92
0.11
0.10
1.12
e
e
e
gC]O(60),
d
d
d
t
CH2(13)
CC(37),
PhII(19)
PhIII(56),
537
512
e
e
514
e
g
t
g
CC(11),
PhI(23),
CC(17),
t
PhIV(11)
d
PhII(23)
441
429
417
e
t
g
t
g
PhIII(54),
CBr(22)
PhI(37),
CC(11),
e
d
C]O(10)
417
t
t
t
d
d
t
t
d
d
d
t
d
d
t
t
d
d
t
d
t
t
d
d
t
t
d
t
t
t
d
d
t
PhII(61),
PhIII(10)
PhI(85)
CN(26),
402
398
8.33
0.02
1.46
0.01
402
e
e
e
CC(19),
d
PhII(16)
369
360
328
314
295
294
228
217
200
185
164
118
117
72
0.01
9.14
0.24
0.61
1.97
0.15
4.10
0.11
3.38
0.14
0.39
2.15
0.47
0.01
0.18
6.59
0.36
6.42
4.28
0.23
8.07
0.87
1.40
2.20
1.72
0.69
2.85
2.00
2.42
1.32
1.31
0.04
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
PhIV(39),
PhII(13),
CBr(26),
C]O(19),
CBr(15),
t
PhIII(10),
CN(22)
CN(16),
gCN(12)
355
e
d
PhI(14),
CC(68),
CH2(10)
d
dCH2(19)
e
e
PhIII(27),
PhII(28),
CBr(30),
CN(22)
t
PhIV(13)
e
e
PhI(24),
CN(18),
d
CBr(10),
CC(10)
dPhIII(11)
e
PhII(49),
PhIV(22)
CBr(20),
e
CN(18),
d
e
PhII(14),
PhIII(13),
CBr(18),
g
CBr(12),
tPhIV(11), tCN(10), gCC(14)
162
124
e
PhI(16),
PhIII(50),
PhIV(10),
CBr(15),
dPhII(28)
gCBr(10)
CN(27),
CN(18),
g
CC(13),
d
CC(13)
CC(11)
e
g
d
g
CC(25),
CC(43),
CC(16),
g
NH(18),
t
61
e
dCH2(15)
51
e
t
t
t
t
t
d
NH(26),
CC(21),
CC(51),
CH2(36),
CC(22),
t
t
CH2(16),
NH(17)
tCN(19)
35
30
1.09
0.32
4.85
2.37
e
e
e
e
t
NH(20)
29
17
6
0.67
0.04
0.71
5.08
2.63
2.10
e
e
e
e
e
e
CH2(20),
gCC(16), dCN(37)
t
t
t
t
CH2(42),
CN(13),
CC(61),
CH2(31),
t
CC(23)
NH(10)
t
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-substituted
phenyl ring; PhIV-benzoxazole ring; potential energy distribution (%) is given in brackets in the assignment column.
respectively [51].
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 the
phenyl 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 group
is 6.2093 ppm and for the hydrogen atoms of the CH2 groups is
4.124 and 4.1239 ppm. The predicted shifts of carbon atoms are
127.7619e134.8546 ppm for PhI, 108.7313e145.5238 ppm for PhII
4.3.3. NMR spectra
The absolute isotropic chemical shielding of the NBBPA mole-
cule was calculated using B3LYP/GIAO model and the results have
been shown in Table 4. The 1H NMR experimental values are: 1H
NMR (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

504
V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
Table 4
Calculated NMR parameters (with respect to TMS).
Protons
sTMS
scalc
dcalc
¼
sTMS scalc
ꢁ
H7
H8
H9
32.7711
32.7711
32.7711
32.7711
32.7711
32.7711
32.7711
32.7711
32.7711
32.7711
32.7711
32.7711
32.7711
32.7711
32.7711
25.0983
23.3811
26.0048
23.9259
24.8866
25.0698
25.1492
26.5618
24.9907
24.85
24.9906
24.9097
24.8501
28.6471
28.6472
7.6728
9.3900
6.7663
8.8452
7.8845
7.7013
7.6219
6.2093
7.7804
7.9211
7.7805
7.8614
7.9210
4.1240
4.1239
H17
H19
H20
H21
H25
H28
H34
H36
H37
H38
H40
H41
Carbon atoms
C1
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
196.852
85.7999
79.2258
59.3433
88.1207
53.9699
51.3282
37.2263
67.762
52.7907
64.9735
60.8552
70.5372
67.3127
33.7502
66.3824
61.9974
67.7182
66.3833
69.0901
67.718
111.0521
117.6262
137.5087
108.7313
142.8821
145.5238
159.6257
129.0900
144.0613
131.8785
135.9968
126.3148
129.5393
163.1018
130.4696
134.8546
129.1338
130.4687
127.7619
129.1340
52.37120
C2
C3
C4
C5
C6
C11
C12
C13
C14
C15
C16
C18
C26
C29
C30
C31
C32
C33
C35
C39
Fig. 4. HOMO-LUMO plots of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl
acetamide.
compound, EHOMO ¼ ꢁ8.131 eV and ELUMO ¼ ꢁ5.318 eV and the
energy gap ¼ 2.813 eV. According to the literature data [67e71], the
chemical potential
m
¼ ꢁ(I þ A)/2 where I ¼ ꢁEHOMO and
A ¼ ꢁELUMO are ionization potential and electron affinity; harness,
¼ (I-A)/2 and electrophilicity index, . The different
2/2
descriptors are: ¼ 1.4065 and ¼ 16.075.
¼ 6.7245,
h
u
¼
m
u
h
m
h
144.4808
4.3.6. Molecular electrostatic potential
and 126.3148e144.0613 ppm for PhIII rings. For the carbon atoms,
C39, C26 and C11, the predicted shifts are 52.3712, 163.1018 and
159.6257 ppm respectively.
Using the molecular electrostatic potential map the charge
distribution of a molecule can be pictured which is important
because, these values determine how molecules interact with one
another as they point out the reactive sites in the molecule. In the
MEP plot (Fig. 5) blue regions are the stronger positive regions and
red regions are the lower electrostatic potential regions. From the
MEP plot it is very clear that carbonyl and NH groups are the lower
electrostatic potential sites favorable of electrophilic reactive re-
gions and other parts of the NBBPA molecule are favorable for
nucleophilic reactive regions.
4.3.4. Nonlinear optical studies
The 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 ꢂ 10ꢁ23, 6.8293 ꢂ 10ꢁ30 and ꢁ36.898 ꢂ 10ꢁ37 esu and these
values of the investigated molecule clearly reveal that they have
nonlinear optical behavior with non-zero values. The reported
values of the first hyperpolarizability of similar derivates are
2.66 ꢂ 10ꢁ30 esu [62] and 1.37 ꢂ 10ꢁ30 esu [47] and the first
hyperpolarizability of the title compound is 52.53 times that of the
standard NLO material urea [63]. The CeN bond lengths in the title
compound are intermediate between a single and double bond
which produces an extended
p-electron delocalization over the
molecular system [64]which is responsible for the nonlinearity of
the system.
4.3.5. Frontier molecular orbitals
The site selectivity and chemical reactivity such as the electronic
chemical potential, m, hardness, h, global electrophilicity, m, can be
defined successfully by DFT methods [65,66]. The HOMO-LUMO
plots are shown in Fig. 4 and HOMO is localized over the whole
molecule except the mono-substituted phenyl ring and CH2 group
while the LUMO is over the whole molecule except over the mono-
substituted phenyl ring, CH2 and acetamide group. For the NBBPA
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) 495e511
505
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.
Fig. 6. ALIE surface of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acet-
amide molecule.
ꢀ
ꢀ
ꢁ
ꢁ
d
rNþ ðrÞ ꢁ rNðrÞ
f þ
¼
¼
;
:
(2)
(3)
d
4.3.7. ALIE surface, Fukui functions and non-covalent interactions
In 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 descriptor
is based on sum of orbital energies weighted by the orbital den-
sities according to the following equation:
d
rNꢁ ðrÞ ꢁ rNðrÞ
f ꢁ
d
In equations (2) and (3), N stands for the number of electrons in
reference state of the molecule, while stands for the fraction of
d
electron which default value is set to be 0.01 [74]. Mapping of the
Fukui functions to the electron density surface enables visualiza-
tion of molecule sites where electron density increase or decrease
as a consequence of charge addition or removal, Fig. 8. In the case of
Fukui fþ function it is necessary to track the positive color (purple
color in Fig. 8a), which indicates the molecule sites where electron
density increased after the addition of charge. In this regard it can
be seen that after charge addition electron density increased in the
near vicinity of atoms of benzene ring with bromine atom and in
the near vicinity of carbon atom C11, indicating that these molecule
sites act as electrophiles. In the case of Fukui fe function it is
necessary to track the negative color (red color in Fig. 8b), which
shows the molecule sites where electron density decreased after
the removal of charge. It can be seen in Fig. 8b that electron density
decreased in the near vicinity of terminal benzene ring, showing
that this part of the molecule could act as nucleophile.
!
X
rið r Þjεij
IðrÞ ¼
:
(1)
!
r
ð r Þ
i
!
where rið r Þ denotes electronic density of the i-th molecular orbital
!
!
at the point r , εi denotes orbital energy, while
r
ð r Þ denotes total
electronic density function. ALIE indicates the energy necessary for
the removal of electron from the certain point around the mole-
cules. This quantity can be represented as maximal and minimal
value for each atom, or its values can be mapped to the electron
density 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 there
are two specific molecule sites prone to electrophilic attacks
(designated by the red color). In this regard near vicinity of bromine
atom Br23 and near vicinity of central benzene ring are charac-
terized by ALIE values which indicate that here electrons are least
tightly bound and therefore the most easily removed. These
molecule sites are characterized by the ALIE value of around
196 kcal/mol. On the other side the highest ALIE values are around
338 kcal/mol and are localized in the near vicinities of hydrogen
atoms.
Charge density analysis in the case of NBBPA molecule revealed
the existence of two intramolecular noncovalent interactions, Fig. 7.
These noncovalent interactions are located between bromine and
nitrogen atoms (Br23eN10) and between oxygen and hydrogen
atoms (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 encompassed
the calculations of Fukui functions, which are calculated in Jaguar
program by finite difference approach according to the following
equations:
4.3.8. Natural bond orbital analysis
The natural bond orbitals (NBO) calculations were performed
using NBO 3.1 program [75] as implemented in the Gaussian09
package at the DFT/B3LYP level and the results are tabulated in
Tables 5 and 6. The strong inter-molecular hyper-conjugative
interaction are: C11eC12 from N10 of n1(N10)/
from O22 of n2(O22)/ *(N10eC11), C12eC13 from Br23 of n3(Br23)/
*(C12eC13), C26eO27 from N24 of n1(N24)/ *(C26eO27), N24eC26
from O27 of n2(O27)/ *(N24eC26 having electron densities,
s*(C11eC12), N10eC11
p
p
p
s
)
0.03329, 0.30325, 0.44545, 0.29909, 0.07226e and stabilization
energies of 14.28, 28.72, 10.08, 64.93, 23.26 kJ/mol. The natural
hybrid orbital with low occupation numbers, higher energies and
considerable p-character are: n2(O22), n3(Br23), n2(O27
) with
energies, ꢁ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, lower
energies
are:
n1(O22),
n2(Br23),
n1(O27
)
with
energies, ꢁ0.60252, ꢁ0.28482, ꢁ0.67917a.u. and p-characters,
58.72, 99.34, 38.97% and occupation numbers, 1.97241, 1.97414,

506
V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
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 5
Second-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
C3eC4
C5eC6
C5eC6
C5eC6
N10eC11
LP N10
LP O22
LP O22
LP Br23
LP N24
LP N24
LP O27
LP O27
p
p
p
p
p
p
s
p
p
n
1.68597
1.68597
1.60101
1.60101
1.60101
1.86715
1.90927
1.74929
1.74929
1.92825
1.65272
1.65272
1.86624
1.86624
C1eC2
C5eC6
C1eC2
C3eC4
p
p
p
p
p
p
*
*
*
*
*
*
0.33296
0.46084
0.33296
0.38314
0.30325
0.46084
0.03329
0.46084
0.30325
0.44545
0.38314
0.29909
0.07226
0.06509
17.93
20.70
19.33
18.67
10.97
14.53
14.28
21.58
28.72
10.08
31.93
64.93
23.26
18.66
0.29
0.28
0.29
0.28
0.26
0.34
0.64
0.35
0.33
0.29
0.29
0.27
0.70
0.60
0.065
0.071
0.068
0.065
0.049
0.069
0.086
0.082
0.089
0.053
0.086
0.119
0.116
0.096
N10eC11
C5eC6
C11eC12
C5eC6
N10eC11
C12eC13
C3eC4
C26eO27
N24eC26
C26eC39
s*
p*
p*
p*
p*
p*
s
s
p
p
s
s
*
*
a
E(2) means energy of hyper-conjugative interactions (stabilization energy kJ/mol).
Energy difference between donor and acceptor i and j NBO orbitals.
F(i,j) is the Fock matrix element between i and j NBO orbitals.
b
c
1.97473.
4.3.9. Reactive and degradation properties based on autoxidation
and hydrolysis
Table 6
NBO results showing the formation of Lewis and non Lewis orbitals.
Computational analysis of molecules based on the combination
of DFT calculations and MD simulations allows inexpensive initial
prediction of degradation properties. In this regard it is useful to
note that bond dissociation energy (BDE) for hydrogen abstraction
indicate to what extent is some molecule prone to autoxidation
mechanism, while BDE values for the rest of the single acyclic
bonds can serve for the classification of bonds according to their
strengths. These results are important for the rationalization and
optimization of procedures related to forced degradation studies
[76e79]. Analysis of BDEs for hydrogen abstraction can indicate
whether some molecule is prone to autoxidation mechanism or
not. According to the Wright et al. [80], for investigated pharma-
ceutical molecule can be concluded that it is vulnerable towards
autoxidation mechanism if the BDE ranges from 70 to 85 kcal/mol.
Gryn'ova et al. [81] agrees with this statement, but also states that
in this regard CeH dissociation is questionable for the BDE values in
the range between 85 and 90 kcal/mol, meaning that BDE values for
hydrogen abstraction in this range could also be important for the
autoxidation mechanism. When BDE values for hydrogen abstrac-
tion are lower than 70 kcal/mol, formed radicals are resistant for
the O2insertion [25,80e82] and these values are not suitable for
autoxidation mechanism.
Bond (A-B) ED/ea
EDA% EDB% NBO
s%
p%
p
C3-C4
1.68597
49.21 50.79 0.7015 (sp1.00)C
0.00
100.00
100.00
ꢁ0.25489
1.60101
e
e
þ0.7126 (sp1.00)C 0.00
e
p
C5-C6
49.78 50.22 0.7055 (sp1.00)C
e
58.40 41.60 0.7642 (sp1.00)N
e
0.00
100.00
100.00
ꢁ0.25756
1.86715
e
þ0.7087 (sp1.00)C 0.00
e
p
N10-C11
0.00
100.00
100.00
ꢁ0.31882
1.90927
e
þ0.6450 (sp1.00)C 0.00
e
n1 N10
e
e
e
e
sp2.02
33.07 66.93
e
ꢁ0.36852
1.97241
e
e
e
n1 O22
e
e
e
e
sp1.43
e
41.18 58.72
e
ꢁ0.60252
1.74929
e
e
n2 O22
e
e
e
e
sp1.00
e
0.00
e
100.00
e
ꢁ0.32784
1.99512
e
n1 Br23
e
e
e
e
sp0.15
e
86.85 13.15
e
ꢁ0.93118
1.97414
e
e
n2 Br23
e
e
e
e
sp99.99
e
0.66
e
99.34
e
ꢁ0.28482
1.92825
e
n3 Br23
e
e
e
e
sp1.00
e
0.00
e
100.00
e
ꢁ0.28331
1.65272
e
n1 N24
e
e
e
e
sp1.00
e
0.00
e
100.00
e
ꢁ0.26022
1.97473
e
n1 O27
e
e
e
e
sp0.64
e
61.03 38.97
e
ꢁ0.67917
1.86624
e
e
n2 O27
e
e
e
e
sp99.99
e
0.05
e
99.95
e
In Fig. 9 all BDE values have been provided. Values provided in
red color corresponds to the BDE values for hydrogen abstraction,
while values provided in blue corresponds to the BDE values for the
ꢁ0.23752
e
a
ED/e is expressed in a.u.

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
507
rest of the single acyclic bonds. Results presented in Fig. 9 indicate
that NBBPAmolecule is very stable in open air and in the presence of
oxygen since there are no BDE values for hydrogen abstraction in
the range from 70 to 85 kcal/mol. The lowest BDE value for
hydrogen abstraction is 91.53 kcal/mol, which is higher even than
upper limit defined by Wright and Gryn'ova. On the other side the
lowest BDE values for the rest of the single acyclic bonds have been
calculated for bonds denoted with numbers 13 and 15, indicating
that degradation could start by detaching of bromine atom or ter-
minal benzene ring.
Comparison of atoms of NBBPAmolecule with regard to their
interactions 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 most
important interactions with water molecules are presented in
Fig. 10. According to the results presented in Fig. 10, six atoms of
NBBPAmolecule have pronounced interactions with water. g(r)
curves of carbon atoms C1, C4 and C26 are very similar in terms of
profile and peak distance (around 3.5 Å), with atom C4 having
somewhat lower maximal g(r) value comparing with C1 and C26.
g(r) curve of bromine atom Br23 is also similar with carbons, but it's
maximal g(r) value is the highest of all atoms with significant in-
teractions with water molecules. However, the most important
interactions with water molecules according to RDFs can be seen
for 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 the
case of H25 maximal g(r) value is located at around 1.8 Å, indicating
strong interactions with water in both cases.
Fig. 10. RDFs of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acetamide's
atoms with significant interactions with water molecules.
4.3.10. Molecular docking studies
DNA 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 in
subunit B of DNA gyrase, and defined by N45, E49, D72, R75, I77,
K102, V117, and T164 residues in T. thermophilus and E. coli DNA
gyrase subunit B (GyrB). This region is thought to be a drug target
for antibacterial agents [84e86]. High resolution crystal structure
of DNA gyrase B form complex was downloaded from the protein
data 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. 11. Receptor ligand interactions which were determined by Autodock. The weak
noncovalent interactions with N45, E49, I77, F103, E104, K109, V117 amino acid
residues.
Fig. 9. BDEs of all single acyclic bonds of N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-phenyl acetamidemolecule

508
V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
binded well the active site was analyzed for detailed interactions
with ADT and Discover Studio Visualizer 3.5 software [90]. The
ligand binds at the active site of the substrate (Figs. 11e13) by weak
noncovalent interactions with N45, E49, I77, F103, E104, K109, V117
amino acid residues. Discover Studio Visualizer was also predicted
electrostatic interactions with N45, D48, E104, K109, V117 and van
der Waals interactions with E49, I77, F103, Q105, T166. E104 forms
pe2
bond with benzene ring which attaches to bromine and K109
also forms -cation bond with the other benzene rings of com-
pound. The docked ligand title compound forms a stable complex
with GyrB complex and gives a binding affinity ( G in kcal/mol)
p
D
value of ꢁ9.1 (Table 7). These preliminary results suggest that the
compound 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 showed
that
phenylacetamide compound exhibit higher activity against E. coli
and S. aureus, with MIC values of 128 g/ml and 64 g/ml respec-
N-[2-(2-bromophenyl)-1,3-benzoxazol-5-yl]-2-
Fig. 12. Molecular surface model of Autodock analyzing.
m
m
tively, than the standard drug Gentamycin. Additionally, it was
found that the activity against E. faecalis was more potent than to
removed and then the receptor was prepared by the cleaning of all
heteroatom's (i.e., nonreceptor atoms such as water, ions, co-
crystallized ligand, etc.). The Auto Dock Tools (ADT) graphical
user interface was used to calculate Gasteiger charges and polar
hydrogen's. Ligand and receptor input files were arranged in PDBQT
format. The docking area was defined around the ATP binding site,
which was determined in crystal structure, by a grid box of
40 Å ꢂ 40 Å ꢂ 40 Å using 0.375 Å grid point spacing in AutoGrid.
The docking conformations of ligands in the binding sites of the
receptor were searched with Lamarkian genetic Algorithm (LGA) in
Autodock. The cocrystallized inhibitor was extracted from receptor
and docked again for the affordance of the docking protocol. The
docking protocol predicted the same conformation as was present
in the crystal structure with RMSD value well within the reliable
range of 2 Å [89]. Amongst the docked conformations, one which
Table 7
The binding affinity values of different poses of the title compound predicted by
AutodockVina
Mode
Affinity (kcal/mol)
Distance from best mode (Å)
e
RMSD l.b.
RMSD u.b.
e
1
2
3
4
5
6
7
ꢁ9.1
ꢁ8.4
ꢁ7.9
ꢁ7.8
ꢁ7.3
ꢁ6.9
ꢁ6.8
0.000
1.661
2.733
6.935
5.849
5.934
6.121
0.000
2.464
2.931
10.325
12.456
9.303
12.734
Fig. 13. Receptor ligand interactions which were determined by Discovery Studio Client 3.5. pe2 bond between benzene ring which bounds bromine and e104. K109 also forms p-
cation bond with the other benzene rings of compound.

V.V. Aswathy et al. / Journal of Molecular Structure 1141 (2017) 495e511
(9) (1997) 788e790.
509
other tested microorganisms. The red shift and splitting of NH
stretching mode shows the weakening of the NH bond, while the
first hyperpolarizability of the title compound is 52.53 times that of
the standard NLO material e urea. It was also concluded that CeN
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bond lengths an extended
p-electron delocalization over the mo-
lecular system, which is responsible for the nonlinearity of the
molecule. The carbonyl and NH groups are favorable sites for
electrophilic attacks, while ALIE surface indicated the significance
of bromine atom in terms of electrophilic attacks. HOMO is spread
over the whole molecule, except the mono-substituted phenyl ring
and CH2 group, while LUMO is spread over the whole molecule,
except over the mono-substituted phenyl ring, CH2 and acetamide
group. HOMO and LUMO visualization indicates charge transfer
within the molecule. Fukui functions show that benzene ring with
bromine atom and terminal benzenering could have significant
importance with the addition and removal of charge. Analysis of
electron density determined that there are two intramolecular
noncovalent interactions. BDE values for hydrogen abstraction
indicate high stability of the title molecule in the open air or in the
presence of oxygen, while BDE values of the rest of the single
acyclic bonds indicate that degradation could start with the
detaching bromine atom. RDFs obtained after MD simulations
indicate that there are six atoms of NBBPA molecule with signifi-
cant interactions with water molecules among which the most
important are hydrogen atom H25 and oxygen atom O27. The
docked title compound forms a stable complex with GyrB complex
and gives a binding affinity value of ꢁ9.1 kcal/mol and might
exhibit inhibitory activity against GyrB complex. Obtained results
concerning antimicrobial activity emphasize the potential of NBBPA
molecule for further studies, while other results encompassed by
this study provide important insights into the reactivity of the title
compound.
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Acknowledgments
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[22] D.V. Sojic, D.Z. Orcic, D.D. Cetojevic-Simin, N.D. Banic, B.F. Abramovic, Efficient
removal of sulcotrione and its formulated compound Tangenta® in aqueous
TiO 2 suspension: stability, photoproducts assessment and toxicity, Chemo-
sphere 138 (2015) 988e994.
Part of this work has been performed thanks to the support
€
received from Schrodinger Inc. Part of this study was conducted
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ꢀ
[23] D.V. Sojic, D.Z. Orcic, D.D. Cetojevic-Simin, V.N. Despotovic, B.F. Abramovic,
Kinetics and the mechanism of the photocatalytic degradation of mesotrione
in aqueous suspension and toxicity of its degradation mixtures, J. Mol. Catal. A
Chem. 392 (2014) 67e75.
within the projects supported by the Ministry of Education, Science
and Technological Development of Serbia, grant numbers OI 171039
and TR 34019. Additionally, this study is supported by a grant
(Project Number:16H0237002) from Scientific Research Projects
Committee of Ankara University.
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[24] D.D. Cetojevic-Simin, S.J. Armakovic, D.V. Sojic, B.F. Abramovic, Toxicity
assessment of metoprolol and its photodegradation mixtures obtained by
using different type of TiO 2 catalysts in the mammalian cell lines, Sci. Total
Environ. 463 (2013) 968e974.
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