Reactive, spectroscopic and antimicrobial assessments of 5-[(4-methylphenyl) acetamido]-2-(4-tert-butylphenyl)benzoxazole: Combined experimental and computational study

Article abstract of DOI:10.1016/j.molstruc.2016.09.024

The synthesis, FT-IR, FT-Raman and NMR spectral analysis of an antimicrobial active benzoxazole derivative, 5-[(4-methylphenyl)acetamido]-2-(4-tert-butylphenyl) benzoxazole (MPATB) is reported. The localization of HOMO, LUMO plots in the title compound ov

Full text of DOI:10.1016/j.molstruc.2016.09.024

Accepted Manuscript
Reactive, spectroscopic and antimicrobial assessments of 5-[(4-methylphenyl)
acetamido]-2-(4-tert-butylphenyl)benzoxazole: Combined experimental and
computational study
Y. Sheena Mary, Nourah Z. Alzoman, Vidya V. Menon, Ebtehal S. Al-Abdullah, Ali A.
El-Emam, C. Yohannan Panicker, Ozlem Temiz-Arpaci, Stevan Armaković, Sanja J.
Armaković, C. Van Alsenoy
PII:
S0022-2860(16)30951-6
10.1016/j.molstruc.2016.09.024
MOLSTR 22936
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 5 August 2016
Revised Date: 8 September 2016
Accepted Date: 8 September 2016
Please cite this article as: Y.S. Mary, N.Z. Alzoman, V.V. Menon, E.S. Al-Abdullah, A.A. El-Emam, C.Y.
Panicker, O. Temiz-Arpaci, S. Armaković, S.J. Armaković, C. Van Alsenoy, Reactive, spectroscopic
and antimicrobial assessments of 5-[(4-methylphenyl) acetamido]-2-(4-tert-butylphenyl)benzoxazole:
Combined experimental and computational study, Journal of Molecular Structure (2016), doi: 10.1016/
j.molstruc.2016.09.024.
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ACCEPTED MANUSCRIPT
Reactive, spectroscopic and antimicrobial assessments of 5-[(4-methylphenyl)
acetamido]-2-(4-tert-butylphenyl)benzoxazole: Combined experimental and
computational study
Sheena Mary Ya*, Nourah Z. Alzomanb, Vidya V.Menonc,d, Ebtehal S. Al-Abdullahb, Ali
A. El-Emamb, C.Yohannan Panickera, Ozlem Temiz-Arpacie , Stevan Armakovićf, Sanja
J. Armakovićg, C.Van Alsenoyh
aDepartment of Physics, Fatima Mata National College, Kollam, Kerala, India
bDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia
cDepartment of Physics, IES College of Engineering, Chittilappily, Thrissur, Kerala,
India
dRD, Bharathiar University, Coimbatore, Tamilnadu, India
eAnkara University Faculty of Pharmacy, Department of Pharmaceutical Chemistry, TR-
06100, Tandogan, Ankara, Turkey
f University of Novi Sad, Faculty of Sciences, Department of Physics,
Trg D. Obradovića 4, 21000 Novi Sad, Serbia
gUniversity of Novi Sad, Faculty of Sciences, Department of Chemistry, Biochemistry
and Environmental Protection, Trg D. Obradovića 3, 21000 Novi Sad, Serbia
hDepartment of Chemistry, University of Antwerp, Groenenborgerlaan 171, B-2020,
Antwerp, Belgium
*author for corresponence email: sypanicker@rediffmail.com
Abstract
The synthesis, FT-IR, FT-Raman and NMR spectral analysis of an antimicrobial active
benzoxazole
derivative,
5-[(4-methylphenyl)acetamido]-2-(4-tert-butylphenyl)
benzoxazole (MPATB) is reported. The localization of HOMO, LUMO plots in the title
compound over the title molecule shows the charge transfer in the molecular system
through the conjugated paths.The electrophilic and nucleophilic sites are revealed from
the molecular electrostatic potential map. The first hyperpolarizability of the title
compound is greater than that of the standard nonlinear optical material urea and the title
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compound and its derivatives are good objects for further research in nonlinear optical
analysis. Molecule sites prone to electrophilic attacks have been detected by calculation
of average local ionization energies, while calculations of Fukui functions have provided
additional information about the local reactivity properties. Bond dissociation energies
have been calculated in order to investigate autoxidation possibilities of the title
molecule, as well as to determine the weakest bonds and therefore the sites where process
of degradation could start. Reactive properties with water have been investigated by
molecular dynamics simulations and calculations of radial distribution functions. The
compound possessed broad spectrum activity against all of the tested Gram-positive and
Gram-negative bacteria and yeasts, their minimum inhibitory concentrations ranging
between 8-128 ꢀg/ml. The compound exhibited significant antifungal activity (64ꢀg/ml)
against Candida krusei, at same potency with the compared standard drugs fluconazole.
The docked title compound forms a stable complex with thymidylate synthase and got a
binding affinity value of -8.5kcal/mol and the title compound can be a lead compound for
developing new anti-cancerous drug.
Keywords: Benzoxazole; DFT; ALIE; BDE; Molecular docking.
1.
Introduction
Heterocyclic compounds such as benzoxazoles have attracted attention due to
their diverse pharmacological and biological properties like antibacterial, antifungal, anti-
tubercular, anti-tumor and antiviral [1-6]. Benzoxazoles are the structural bio-isoesters of
nucleotides such as guanine and adenine and they interact easily with biopolymers of
living system [7, 8] and they inhibit essential bacterial enzymes, such as hyaluronanlyase
[9] and isocitratelyase [10] as well as bacterial two component systems [11, 12]. Mabied
et al. [13] reported the crystal structure of stereochemistry study of 2-substituted
benzoxazole derivatives and Jayana et al. [14] reported the synthesis, antibacterial and
antioxidant evaluation of novel 1-(5,7-dichloro-1,3-benzoxazol-2-yl)-1H-pyrazolo[3,4-b]
quinoline derivatives.
Studies of reactive properties of newly synthetized organic molecules with
potential important biological activities are very important for the development and
improvement of methods for water purification. Namely, molecules that are active
components of pharmaceutical products are synthetized to be very stable, thus natural
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conditions and conventional purification methods are not enough effective for their
degradation [15-17.]
Unfortunately, due to various reasons drugs are entering the environment and are
accumulating especially in the water resources, where they are toxic to aquatic
organisms. So far these types of compounds have been detected in all types of waters
[18]. Fortunately, advanced oxidation processes are seen as the alternative when it comes
to the degradation of these compounds [19-21]. Forced degradation experiments are very
important in the process of making of new pharmacological formulations [22]. These
experiments serve as tools by which it is possible to evaluate degradation mechanisms
and toxicity. These experiments are expensive and tedious, but principles of molecular
modeling are very useful for their rationalization. In this regard it is important to note that
DFT calculations and MD simulations are able to provide important reactive properties
thanks to which autoxidation and hydrolysis properties can be effectively initially
assessed. Taking this into account in this work we have also been devoted to the
investigation of specific local reactivity properties based on DFT calculations and MD
simulations.
2.
Experimental
2.1
Materials and methods
The chemicals and solvents were purchased from Sigma-Aldrich (Munich,
Germany) and Fisher Scientific (Pittsburgh, PA, USA); they were used without
purification. Silica gel HF254 chromatoplates (0.3 mm) were used for thin layer
chromatography, and the mobile phase was chloroform/methanol (10:0.5). Melting point
was recorded on a Stuart Scientific SMP1 instrument (Bibby Scientific Limited,
Staffordshire, UK) and is uncorrected. NMR spectra were recorded on a Varian Mercury
400 MHz NMR spectrometer (Palo Alto, CA, USA); trimethylsilane (TMS) was used as
an internal standard. The mass spectra was recorded on a Waters ZQ Micromass LC-MS
spectrometer (Milford, MA, USA) using the ESI(+) method.
The FT-IR spectrum (Fig. S1-supporting material) was recorded using KBr pellets
on a DR/Jasco FT-IR 6300 spectrometer. The FT-Raman spectrum (Fig. S2-supporting
material) 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
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150 mW, measurement on solid sample. The spectral resolution after apodization was 2
cm-1.
Materials used in the microbiology study were; Mueller Hinton agar (MHA)
(Merck, Darmstadt, Germany), Mueller Hinton broth (MHB) (Merck), Sabouraud
dextrose agar (SDA) (Merck), RPMI-1640 medium with L-glutamine (Sigma-Aldrich), 3-
(N-morpholino)-propane-sulfonic acid (MOPS) (Sigma-Aldrich), 96-well micro plates
(BD, Franklin Lakes, NJ, USA), ampicillin (Mustafa Nevzat Pharmaceuticals, Istanbul,
Turkey), gentamycin sulfate (Paninkret Chem.-Pharm., Pinneberg, Germany), ofloxacin
(Zhejiang Huangyan East Asia Chemical Co. Ltd., Huangyan, Zhejiang, China),
vancomycin (Mayne Pharma, Salisbury South, SA, Australia), meropenem (Astra
Zeneca, Istanbul, Turkey), fluconazole (Sigma-Aldrich), amphotericin B (Riedel de
Haen, Seelze, Germany), isoniazid (Sigma-Aldrich), ethambutol (Sigma-Aldrich), DMSO
(Riedel de Haen).
Microorganisms used in the assay were; Klebsiella pneumoniae clinical isolate
(extended beta lactamase spectrum (ESBL)), Escherichia coli isolate (ESBL),
Enterococcus faecalisisolate (resistant to vancomycin (VRE)), and Staphylococcus
aureus isolate (resistant to methicilline (MRSA)), K. pneumoniae RSKK 574, E. coli
ATCC 25922, E. faecalis ATCC 29212, S. aureus ATCC 29213, Candida albicans
ATCC 10231, Candida krusei ATCC 6258, Mycobacterium tuberculosis H37RV ATCC
27294 and a clinical isolate of M. tuberculosis. Reference strains and clinical isolates
were provided by Gazi University, Faculty of Pharmacy, Department of Pharmaceutical
Microbiology, Culture Collection, Ankara, Turkey, and Gazi University Hospital
Microbiology Laboratory, Ankara, Turkey, respectively.
2.2
Chemistry
General procedure for the preparation of 5-[(4-methylphenyl)acetamido]-2-(4-tert-
butylphenyl)benzoxazole
Firstly, 5-amino-2-(4-tert-butylphenyl)-benzoxazole was synthesized by heating
0.02 mol 2,4-diaminophenol·2.HCl with 0.02mol 4-tert-butylbenzoic acid in 25 g
polyphosphoric acid (PPA) and stirring for 3-4 h. At the end of the reaction period, the
residue was poured into an ice/water mixture and the solution was neutralized with 10%
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NaOH. The resulting precipitate was filtered, washed with distilled water, dissolved in
boiling ethanol with 0.2 g charcoal, and filtered off. Then distilled water was added to the
filtrate slowly in order to stimulate crystallization. The crude compound 3 was obtained
by filtering and drying the crystalline material [2]. Then, 4-methylphenyl acetic acid (0.5
mmol) and thionylchloride (1.5 ml) were refluxed in benzene (5 ml) at 80°C for 3h.
excess thionylchloride was removed in vacuo. The 4-methylphenyl acetic acid chloride
(5) was dissolved in ether (10 ml) and this solution added during 1 h to a stirred, ice-cold
mixture
of
5-amino-2-[4-tert-butylphenyl]benzoxazole
(3)
(0.5
mmol),
sodiumbicarbonate (0.5 mmol), diethylether (10 ml) and water (10 ml). The mixture was
kept stirred overnight at room temperature and filtered. The precipitate was washed with
water, 2M HCl and water, respectively and finally with ether to give compound. The
product was re-crystallized from ethanol-water as needles which are dried in vacuo.
(scheme).
2.3
Microbiological assays
For microbiological assays, standard powders of ampicillin, meropenem,
gentamycin sulfate, ofloxacin, vancomycin, fluconazole, amphotericin B, isoniazid, and
ethambutol were dissolved in appropriate solvents recommended by Clinical and
Laboratory Standards Institute (CLSI) guidelines [23, 24]. Stock solutions of the test
compounds were prepared in DMSO. Bacterial susceptibility testing was performed
according to the guidelines of CLSI M100-S18 [24]. MHB was added to each well of the
micro plates. The bacterial suspensions used for inoculation were prepared at 106 CFU/ml
by diluting fresh cultures at McFarland 0.5 density. Suspensions of the bacteria at 106
CFU/ml were inoculated to the two-fold diluted solution of the compounds. A 10-µl
bacterium inoculum was added to each well of the micro plates. There were 105 CFU/ml
bacteria in the wells after inoculations. Micro plates were incubated at 37°C over night.
Fungal susceptibility testing was performed according to the guidelines of CLSI M27-A3
[23]. RPMI-1640 medium with L-glutamine buffered to pH 7 with MOPS was added to
each well of the micro plates. The colonies were suspended in sterile saline, and the
resulting suspension was adjusted to McFarland 0.5 density. A working suspension was
prepared by appropriate dilution of the stock suspension. A 10-µl bacterium inoculum
was added to each well of the micro plates resulting in 103 CFU/ml yeasts in the wells.
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Micro plates were incubated at 35°C for 24-48 hours. After incubation, the lowest
concentration of the compounds that completely inhibited macroscopic growth was
determined and reported as minimum inhibitory concentration (MIC). All solvents and
diluents, pure microorganisms, and pure media were used in control wells. All
experiments were done in three parallel series. The data on the antimicrobial activity of
the compounds and the control drugs as MIC (µg/ml) values are given in table 1. The
synthesized compound was found to possess MIC values between 8- 128 ꢀg/ml for
Mycobacterium tuberculosis, some Gram-negative, Gram-positive bacteria and their
isolates and some fungi. The synthesized compound exhibited the same antifungal
activity against C. krusei with MIC values of 64ꢀg/ml with the tested reference drug
fluconazole. However, all compounds showed lower antibacterial and anti-mycobacterial
activity against the microorganisms than the control drugs.
3.
Computational details
The molecular geometry optimization, polarizabilities and natural bond orbital
analysis for the title compound are calculated by density functional using B3LYP/6-
311++G(d,p) [25, 26] level of theory using Gaussian09 software [27]. The calculated
scaled wave numbers are scaled by using scaling factor as reported in literature [28].
With the help of potential energy distribution analysis [29] and Gaussview program [30]
the vibrational assignments were carried out. The optimized geometrical (Fig.1)
parameters are given in table S1 (supporting material).
Jaguar 9.0 program [31], as implemented in Schrödinger Materials Science Suite
2015-4, was also used for the DFT calculations of ALIE, Fukui functions and BDEs with
B3LYP exchange-correlation functional [32], together with 6-311++G(d,p), 6-
31++G(d,p) and 6-311G(d,p) basis sets, respectively. For MD simulations Desmond
program [33-36], also as implemented in Schrödinger Materials Science Suite 2015-4,
was used with OPLS 2005 force field [37] within NPT ensemble class. Simulation time
was set to 10 ns, while the whole system was modeled by placing one MPATB molecule
in the cubic box with ~3000 water molecule. Other parameters include cut off radius set
to 12 Å, temperature to 300 K and pressure to 1.0325 bar. Solvent was treated within
simple point charge (SPC) model [38]. Noncovalent interactions were determined by
using the method of Johnson [39, 40], as implemented in Jaguar 9.0 program. In all cases
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when Jaguar and Desmond were used, input and output files were manipulated by
Maestro graphical user interface application for Schrödinger Materials Science Suite
2015-4.
4.
Results and discussion
In the following discussions, the rings, C41-C43-C47-C45-C42-C40, C29-C27-C25-C26-
C28-C31, N35-C24-O34-C25-C26 and C1-C2-C3-C4-C5-C6 are designated as PhI, PhII, PhIII
and PhIV, respectively.
4.1
Geometrical parameters
For the title compound, the bond lengths in the phenyl rings are in the range
1.4002-1.3929 Å for PhI, 1.4140-1.3829 Å for PhII and 1.4062-1.3929 Å for PhIV which
are in agreement with that of literature [41]. The C38=O39 bond length of the title
compound is 1.2188 Å which is in agreement with literature [42]. The bond lengths, C25-
O34 = 1.3711 Å, C24-O34 =1.3807 Å, C24=N35 = 1.2975 Å, C38-N36 = 1.3695 Å, C31-N36
=1.4138 Å and C26-N35 = 1.3912 Å are different which is due to different environment in
the molecular system and C24-N35 assumes a double bond character as reported in
literature [41].
The bond angles around, C47 is C45-C47-C43 = 117.8°, C45-C47-C53 = 121.1°, C43-
C47-C53 = 121.1° and this asymmetry in angles is due to interaction between phenyl ring
PhI and H48, 49. Similarly at C40 position, the angles are, C41-C40-C42 = 117.8°, C42-C40-C50
= 121.1° and C41-C40-C50 = 121.1° which is due to the repulsion between the CH2 and
H44,46 of PhI. At C25 and C26 positions, C26-C25-C27 = 123.1°, C26-C25-O34 = 107.7°, C27-
C25-O34 = 129.1Å and C25-C26-C28 = 121.1°, C25-C26-N35 = 108.4°, C28-C26-N35 = 130.5°
and this is due to the interaction between rings PhII and PhIII. The bond angles, C50-C38-
N36 = 115.5°, C50-C38-O39 = 119.9°, N36-C38-O39 = 124.6° around C38 position gives the
interaction is due to the neighboring units CH2, NH. Similarly, at C24, the angles are O34-
C24-N35 = 114.7°, O34-C24-C6 = 117.5° and N35-C24-C6 = 127.9° and this asymmetry is
due to interaction between PhIII and neighboring units. The interaction between NH and
PhII is given by the bond angles around N36 which are C31-N36-C38 = 129.2°, C31-N36-H37
= 115.4°, C38-N36 – H37 = 115.4°. The carbonyl group is tilted from the phenyl ring PhI as
is evident from the torsion angles, C42-C40-C50-C38 = 89.5°, C45-C42-C40-C50 = -178.8°,
C41-C40-C50-C38 = -89.6°, C43-C41-C40-C50 = 178.8°.
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4.2
IR and Raman spectra
The observed IR, Raman bands, calculated scaled wave numbers and assignments
are given in Table 2.
According to literature [43, 44], the NH vibrations are expected in the following
regions: stretching mode: 3500-3300 cm-1; deformation modes: around 1500, 1250 and
750-600 cm-1. For the title compound, the NH stretching modes are assigned at 3440 cm-1
in the IR spectrum, 3449 cm-1 in the Raman spectrum and at 3453 cm-1 theoretically and
the NH deformations are assigned at 1507, 1263, 646 cm-1 theoretically and
experimentally bands are observed at 1498, 644 cm-1 in the Raman spectrum and 644 cm-
1
in the IR spectrum. The NH stretching mode has a PED of 100% with IR intensity
108.48 and Raman activity 440.07. The PED of the NH deformation modes are in
between 40 and 50%. For the mode at 646 cm-1, the IR intensity and Raman activity are
very low, less than 10. The reported values of NH modes are at 3462 cm-1 in the IR
spectrum, 3450 cm-1 in the Raman spectrum, 3400 cm-1 theoretically (stretching modes),
1508, 1219, 655 cm-1 (DFT) (deformation modes) [42] and 1587, 1250, 650 cm-1 (IR),
1580, 1227, 652 cm-1 (DFT) (deformation modes) [45].
The C=N stretching mode of the title compound is assigned at 1521 cm-1 in the IR
spectrum, 1519 cm-1 in the Raman spectrum and at 1522 cm-1 theoretically as expected
[46, 47]. The C-N stretching modes of the title compound are observed at 1192, 1115 cm-
in the IR spectrum, 1242, 1189 cm-1 in the Raman spectrum and at 1244, 1189, 1118
1
cm-1 theoretically which are expected in the range 1300-1100 cm-1 [47]. All the CN
stretching modes have PEDs from 36 to 42% and for the modes, 1522, 1244 cm-1 the
Raman activity is very high and bands are seen the Raman spectrum.
In the present case, the C=O stretching mode is observed at 1677 cm-1 in the IR
spectrum, 1670 cm-1 in the Raman spectrum, 1674 cm-1 theoretically while the C-O
stretching modes are assigned at 905 cm-1 in the Raman spectrum and at 1174, 903 cm-1
theoretically. For the modes, 1674 and 903 cm-1 the Raman activities are high, 64.43 and
181.17 and experimentally bands are observed in the Raman spectrum and the PEDs are
74 and 39%. Also the mode at 1674 cm-1 has a high IR intensity of 334.39 and a PED of
74%. According to literature, the C=O stretching modes are expected in the region 1850-
1550 cm-1 [48] and the C-O-C stretching modes are in the region 1200-900 cm-1 [47, 49].
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Sebastian et al reported the carbonyl stretching mode at 1694 cm-1 in the IR spectrum,
1696 cm-1 in the Raman spectrum and at 1699 cm-1 theoretically [42]. For a benzoxazole
derivative, the C-O-C stretching modes are reported at 1173, 890 cm-1 in the IR spectrum,
1174, 888 cm-1 in the Raman spectrum, 1172, 893 cm-1 theoretically by Parveen et al.
[41].
The methyl stretching modes of the title compound are observed at 2960, 2903
cm-1 in the IR spectrum and at 2983, 2955, 2914 cm-1 in the Raman spectrum as expected
[43, 46]. The bending modes of the methyl groups are observed at 1427, 1361, 1342,
1018, 984 cm-1 in the IR spectrum and at 1461, 1433, 1359, 1338, 1002, 969, 905 cm-1 in
the Raman spectrum. The DFT calculations give these modes in the ranges 2984- 2904
cm-1 (stretching) and 1463-901 cm-1 (deformation modes) [43, 46]. The CH2 modes of the
title compound are observed at 1410, 1149 cm-1 in the IR spectrum, 1276, 1156 cm-1 in
the Raman spectrum experimentally and the PED analysis gives these modes at 2968,
2933 cm-1 (stretching) and 1408, 1276, 1159, 893 cm-1 (deformation modes) which are in
agreement with the literature [43, 468].
The CC3 stretching modes are expected in the ranges 1295-1175 cm-1 and 890-710
cm-1 [46] and in the present case, these modes are assigned at 1235, 809 cm-1 in the IR
spectrum, 812 cm-1 in the Raman spectrum and at 1234, 1176, 811 cm-1 theoretically with
PEDs 47, 48 and 52%. For the mode 1234 cm-1 the IR intensity is 40.31 and for the other
two modes the IR intensity is less than 10.00. Joseph et al. reported the CC3 stretching
modes at 1244, 907 cm-1 in the IR spectrum, 1247 cm-1 in Raman spectrum and at 1286,
1249, 906 cm-1 theoretically [50]. The deformation modes of the CC3 group are expected
in the regions, 435± 85, 335 ± 80 and 300 ± 80 cm-1 (total five modes, two asymmetric,
one symmetric and two rocking) and for the title compound, these deformation modes
are assigned at 446, 384, 374, 342, 284 cm-1 theoretically with PEDs, 62, 35, 38, 43, 33%
and the IR intensity and Raman activity values are less than 10% and the reported values
are 496, 326, 313, 290, 219 cm-1 theoretically [50].
The phenyl CH stretching modes are observed at 3020 cm-1 in the Raman
spectrum for PhI, 3078 cm-1 in the IR spectrum, 3121 cm-1 in the Raman spectrum for
PhII and 3052 cm-1 in the IR spectrum, 3085, 3053 cm-1 in the Raman spectrum for PhIV
[46]. Theoretically these CH stretching modes are assigned in the ranges, 3049-3031 cm-1
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for PhI, 3124-3041 cm-1 for PhII and 3083-3052 cm-1 for PhIV as expected [46]. The
phenyl ring stretching modes are assigned in the ranges 1589-1296 cm-1 for PhI, 1595-
1320 cm-1 for PhII and 1586-1280 cm-1 for PhIV while experimentally bands are
observed at 1578, 1545, 1521, 1279 cm-1 in the IR spectrum and at 1610, 1580, 1551,
1519, 1397, 1322 cm-1 in the Raman spectrum [46].
Trisubstituted phenyl rings have three frequency intervals for the ring breathing
mode: 500-660, 1050-1100 and 600-750 cm-1 for light substituent, heavy substituent and
mixed substituent according to literature [51]. For the title compound, the ring breathing
mode of tri-substituted benzene ring is assigned at 1100 cm-1 theoretically. with a PED 04
44% and the IR intensity and Raman activity values are low. The ring breathing mode of
tri-substituted phenyl rings are reported at 1110, 1129 cm-1 [52] and at 1109 cm-1 in the
IR spectrum, 1100 cm-1 theoretically 1109 cm-1 (IR), 1100 cm-1 (DFT) [53]. The ring
breathing mode of para-substituted phenyl rings with entirely different substituent are
expected in the range 780-880 cm-1 according to literature [51] and in the present case,
this is confirmed by the bands at 824 cm-1 and 783 cm-1 by PED analysis with PEDs 42
and 48%. For the mode at 783 cm-1 the IR intensity is less than 10%. The phenyl ring
breathing mode of para substituted phenyl rings was reported at 795 cm-1 [54], at 873 cm-
1
in the IR spectrum and at 861 cm-1 theoretically [55] and at 753 cm-1 in IR spectrum,
793, 759 cm-1 theoretically [56].
The in-plane CH bending modes of the phenyl rings are assigned as 1018 cm-1
(IR), 1277, 1188, 1162, 1020 cm-1 (DFT) for PhI, 1106 cm-1 (Raman), 1236, 1103, 1094
cm-1 (DFT) for PhII and 1002 cm-1 (Raman), 1293, 1174, 1161, 1000 cm-1 (DFT) for
PhIV as expected [46]. The out-of-plane CH bending modes of the phenyl rings are
assigned at 928 cm-1 (IR), 928 cm-1 (Raman), 947, 930, 822, 797 cm-1 (DFT) for PhI, 875
cm-1 (IR), 880 cm-1 (Raman), 895, 880, 773 cm-1 (DFT) for PhII and at 956 cm-1 (IR),
959, 841, 829, 818 cm-1 (DFT) for PhIV [46].
4.3
NMR spectra
1
GIAO H and 13C NMR chemical shifts with respect to trimethylsilane were
calculated (Table S2-supporting material) using the B3LYP/6-311++G(d,p). The
predicted chemical shifts of aromatic protons of the title compound are in the range
7.7001-7.7117 for PhI, 6.2699-9.5267 for PhII and 7.7705-8.9744 for PhIV while the
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experimental protons shifts are 7.60-8.11 for PhI, 7.52-8.09 for PhII and 7.11-7.24 for
PhIV. For the tert-butyl group, the proton shifts are in the range, 1.6036-2.1832
(calculated), 1.31 (experimental) while in the methyl group, the chemical shift is in the
range 2.6774-3.0564 (calculated), 2.26 (experimental) and in methylene group the shifts
are 4.0823, 4.0833 (calculated), 7.61 (experimental). The hydrogen atom of amide group
in this compound appears at a higher chemical shift of 10.30 experimentally and 6.0895
ppm theoretically. We noted that, δcalc of the NH protons strongly deviate from the
experimental data due to the high polarity of these bonds. The range for 13C NMR
chemical shift of the typical organic molecule usually is greater than 100 ppm [57] the
accuracy ensures reliable interpretation of spectroscopic parameters. The carbon atom of
the carbonyl group has a low lying excited state involving the movement of electrons
from the oxygen lone pair to anti-bonding π orbital which generates a paramagnetic
current [58]. This n→π* transition produces a large shift to a higher wave number, and
hence the carbonyl carbon signals have a weaker intensity and appear in the range, 150-
220 ppm [59, 60]. Thus it is easy to recognize the carbonyl absorptions from the other
resonances and for the title compound carbonyl shift is 162.9137. The aromatic carbons
give resonance in overlapped areas of NMR spectrum with chemical shift values from
100 to 150 ppm [61] and in the present case, the chemical shifts of the aromatic carbon
atoms are, 129.9396-132.4818 for PhI, 109.535-146.2932 for PhII and 124.8705-
152.1844 for PhIV. The chemical shifts of the other carbon atoms are 28.4974 (C53),
43.6937 (C11), 38.7895 (C13), 35.071 (C14), 38.785 (C12) and 51.8989 (C50).
4.4
Frontier Molecular analysis
Using frontier molecular orbital analysis characterization of intra molecular
charge transfer through conjugated paths can be explained through the donor – acceptor
groups [62]. In the present case the HOMO and LUMO energies are -8.136 and -5.272
eV, respectively. The ionization energy I = -EHOMO = 8.136eV and electron affinity A = -
ELUMO = 5.272eV and energy gap = 2.864 eV. The global chemical descriptors are given
as hardness η = (I-A)/2, chemical potential ꢀ = -(I+A)/2 and electronegativity index ω
=ꢀ2/2η [63, 64]. In the present case η, ꢀ and ω are 1.432, -6.704 and 15.693 respectively.
In the HOMO-LUMO plot (Fig. S3-supporting material), the HOMO is localized over the
acetamido group, benzoxazole, trisubstituted phenyl ring and the phenyl ring attached
11

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with the tert-butyl group, while the LUMO is over the tertbutyl phenyl ring, tert-butyl
group, benzoxazole ring and trisubstituted phenyl ring. This shows the charge transfer in
the molecular system through the conjugated paths.
4.5
Molecular Electrostatic Potential Map
MEP is used for predicting sites in studies of biological recognition and hydrogen
bonding interactions and relative reactivity’s towards electrophilic attack [65]. Using the
B3LYP/6-311++G(d,p) method the reactive sites for electrophilic and nucleophilic attack
for the title compound is calculated and shown in Fig. S4 (supporting material).The
different values of the electrostatic potential are represented by different colors and
increases in the order red < orange < yellow < green < blue. Red indicates the strongest
repulsion while blue represents the strongest attraction. From the MEP plot it is clear that
the carbonyl group and nitrogen atom in the benzoxazole moiety are the strongest
repulsion regions and NH group is the strongest attraction centers.
4.6
ALIE surfaces, non-covalent interactions and Fukui functions
Sites of molecule that are possibly prone to electrophilic attacks are characterized
by the lowest values of energy necessary for the removal of electrons. In this work these
sites were effectively determined by calculations of average local ionization energy
(ALIE), as introduced by Sjoberg et al. [66, 67]. This important quantum-molecular
descriptor is defined as a sum of orbital energies weighted by the orbital densities:
r
ρi
(
r
)
εi
)
I
(
r =
)
,
(1)
r
∑
ρ
(
r
i
r
r
ρ
( )
r
where
r
represents the electronic density of the i-th molecular orbital at the point ,
i
r
ρ
(
r
)
εi represents the orbital energy and
is the total electronic density function.
Visualization of ALIE results is the most effective if the corresponding ALIE values are
mapped to the electron density surface, which is the case in this work, Fig.2.
ALIE surface of MPATB molecule indicate that areas with the lowest ALIE values
are located within the all three benzene rings. Beside benzene rings there is one more
location where ALIE value is the lowest and that is the near vicinity of nitrogen atom
N35, where ALIE has the value of ~199 kcal/mol. On the other side the highest ALIE
value for the MPATB molecule was calculated to be ~333 kcal/mol and it is mostly
12

ACCEPTED MANUSCRIPT
located in the near vicinity of hydrogen atoms. Analysis of intra-molecular non-covalent
interactions revealed only one such interaction, involving oxygen atom O39 and
hydrogen atom H32.
Other possibly important molecule locations of the MPATB molecule in this work
have been detected employing the concept of Fukui functions. These functions show how
electron density changes with the addition or removal of charge. In this way one
practically obtains response properties in terms of electron density when molecule acts as
electrophile or nucleophile. In Jaguar program the values of Fukui f + and f – functions are
calculated in finite difference approximation according to the following equations:
(
ρ N +δ
(
r
)
−
ρ N
(
r
)
)
f + =
f − =
,
,
(2)
(3)
δ
(
ρ N −δ
(r
)
δ
−
ρ N
(r
))
where
N represents the number of electrons in reference state of the molecule, while δ is
fraction of electron which default value is set to be 0.01 [68]. Same as in the case of
ALIE values, values of Fukui functions have been mapped to the electron density surface,
Fig.3.
In Fig.3 purple colour has been used as positive one, while the red colour is used
+
as negative one. In the case of Fukui
f
function purple colour indicates molecule areas
where electron density increase with the addition of charge while in the case of Fukui f –
function red colour indicates molecule areas where electron density decreases with the
charge removal. Both Fukui functions confirm the importance of benzene rings in terms
of reactivity. Namely, purple colour in case of Fukui f + function is located in the benzene
–
ring in the vicinity of three methyl groups, while red colour in the case of Fukui
f
function is located again within the benzene ring, this time the one with only one methyl
group.
4.7
Nonlinear Optical properties
The polarizability, first and second hyperpolarzabilities of the title compound are
respectively, 5.4051×10-23, 1.328×10-30 and -603.64×0-37 esu. The reported value of a
similar derivative is 1.37×10-30 esu [69] and in the present case the first
hyperpolarizability of the title compound is 10.22 times that of the standard NLO
13

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material urea [70]. The hyperpolarizability values can give information about the physical
properties of materials to gain knowledge about the third harmonic signals in molecular
systems [71-73]. From the values of hyperpolarizabilities of the title compound, we can
conclude that the title compound and its derivatives are good objects for further research
in nonlinear optical analysis.
4.8
Natural Bond Orbital Analysis
NBO 3.1 program [74] as implemented in the Gaussian09 package is used for the
NBO calculations of the title compound in order to understand various second-order
interactions and the important interactions are given in tables S3 and S4 (supporting
materials). The strong inter molecular hyper-conjugative interactions are: C24-N35 from
O34 of n2(O34)→π*(C24-N35) with electron density, 0.30911e and stabilization energy
34.08 kJ/mol; C24-O34 from N35 of n1(N35)→σ*(C24-O34) with electron density 0.06285e
and stabilization energy 14.40 kJ/mol; C38-O39 from N36 of n1(N36)→π*(C38-O39) with
electron density 0.28747e and stabilization energy 63.34 kJ/mol; N36-C38 from O39 of
n2(O39)→σ* (N36-C38) with electron density 0.0768e and stabilization energy 26.20
kJ/mol. The different bonding in the molecular system are: n2(O34) and n2 (O39) with
higher orbital energies, -0.3291 and -0.23508a.u), considerable p-characters, 100 and
99.97%, low occupation number 1.973332 and 1.85687. While the orbital, n1(O34) and
n1(O39) occupy lower energies, -0.58285 and -0.66097a.u with p-characters, 62.30 and
42.05% and high occupation numbers, 1.96958 and 1.97442.Thus, a very close to pure p-
type lone pair orbital participates in the electron donation to the π*(C24-N35) orbital for n2
(O34) →π*(C24-N35), σ* (C24-O34) orbital for n1 (N35) →σ* (C24-O34), π*(C38-O39)orbital
for n1 (N36) →π*(C38-O39) andσ*(N36-C38) orbital for n2 (O39) →σ* (N36-C38)interaction
in the compound.
4.9
Reactive and degradation properties based on autoxidation and hydrolysis
Molecular modeling provides important results thanks to which forced
degradation experiments can be significantly rationalized and optimized [75]. Namely,
there is clear correlation between the mechanism of autoxidation and BDE for hydrogen
abstraction. Concretely, if the BDE for hydrogen abstraction is in the proper interval then
particular molecule location can be considered as possible starting point for the
mechanism of autoxidation. Concerning the proper interval of BDE values it is important
14

ACCEPTED MANUSCRIPT
to know that all peroxy radicals have similar BDE values (87-92 kcal/mol) which can be
considered as independent of the chemical surrounding [76, 77]. This implies that if the
BDE for hydrogen abstraction at some location is in this interval it can be considered that
autoxidation mechanism is possible. However, the study of Wright et al. [78] have shown
that autoxidation mechanism is the most probable for molecule where BDE for hydrogen
atom is in the interval between 75 and 85 kcal/mol. Beside calculations of BDE for
hydrogen abstraction it is also useful to calculate BDE values for the remaining single
acyclic bonds since these indicate the weakest bonds, and thus the locations where
degradation could start. BDE values for all single acyclic bonds are presented in Fig.4.
Results presented in Fig.4 indicate that it is not likely for MPATB molecule to be
prone to autoxidation mechanism since all calculated BDE values for hydrogen
abstraction are higher than 92 kcal/mol, although there are two bonds (denoted with 10
and 11) with BDE values close to 92 kca/mol. This further indicates that MPATB
molecule is stable in the open air and in the presence of oxygen. Of the remaining single
acyclic bonds there are two with the lowest BDE values, around 70 kcal/mol each. Those
bonds are denoted with numbers 14 and 17 and they could be the locations where
degradation could start.
In order to investigate which atoms of MPATB molecule have pronounced
interactions with water molecules, we have calculated RDF as obtained after MD
simulations. RDF, g(r), gives the probability of finding a particle in the distance
r from
another particle [79]. In Fig.5 RDFs of atoms with significant interactions with water
molecules have been presented. According to the calculated RDFs there are five carbon
atoms and four non-carbon atoms with significant interactions with water molecules.
Carbon atoms with significant interactions with water molecules are C1, C5, C11, C47
and C53. Of these five, three of them, C1, C5 and C53 have lower peak distances
(between 3.5 Å and 4 Å), than atoms C11 and C47 (that have peak distances between 4.5
Å and 5.0 Å). On the other side two carbon atoms with the highest g(r) values are carbon
atoms C11 and C53. The fact that carbon atoms C47 and C53 have pronounced
interactions with water molecules is very important because the BDE value for the
abstraction of nearby hydrogen atom is close to 92 kcal/mol. This further indicates that
autoxidation mechanism for MPATB molecule is hard to be expected since oxidation and
15

ACCEPTED MANUSCRIPT
hydrolysis could compete at the mentioned molecule location. Concerning the non-carbon
atoms the most important RDF is calculated for hydrogen atom H37, for which the
highest g(r) value is somewhat higher than 0.9, while the peak distance is located at
below 2 Å. Other atoms with significant interactions with water include hydrogen atom
H7, nitrogen atom N35 and oxygen atom O39. Oxygen atom O39 has the highest g(r)
value of almost 1.0, while its peak distance is located at around 2.7 Å. The importance of
nitrogen atom N35 lies in the fact that this atom is also recognized as important reactive
center according to the ALIE results.
4.10 Molecular docking studies
Based on the structure of a compound, PASS (Prediction of Activity Spectra) [80]
is an online tool which predicts different types of activities. PASS analysis of the title
compound predicts activities given in the Table S5 (supporting material), thymidylate
synthase activity with probability to be active (Pa) value of 0.754. Thymidylate synthase
(TS) is a key enzyme in the synthesis of 2'-deoxythymidine-5'-monophosphate, an
essential precursor for DNA biosynthesis. For this reason, this enzyme is a critical target
in cancer chemotherapy [81, 82]. Benzoxazole derivatives are used as antitumor activities
against human breast cancer cell lines [83, 84]. Thus we choose thymidylate synthase and
used as target for docking study. High resolution crystal structure of thymidylate synthase
was downloaded from the RSCB protein data bank website with PDB ID: 3TMS. All
molecular docking calculations were performed on Auto Dock-Vina software [85] and as
in literature [41, 42]. With the help of Discovery Studio Visualizer 4.0 software the active
site which binds well are analysed [86] and the weak non-covalent interactions are given
in Fig. 6. Amino acid Trp101 forms two π-sigma, π-π T-shaped, π-alkyl interaction with
CH3 group, phenyl rings respectively. Phe149 forms π-π T-shaped and Tyr164 forms π-
stacked interactions with phenyl ring. Ser131 shows H-bond with benzoxazole ring. The
docked ligand forms a stable complex with thymidylate synthase (Fig. S5-supporting
material) and got a binding affinity value of -8.5kcal/mol (Table 3). These studies show
that the title compound can be used for developing new anti-cancerous drug.
5.
Conclusion
5-[(4-methylphenyl)acetamido]-2-(4-tertbutylphenyl)benzoxazole was synthesized and
characterised by experimental and theoretical methods. The structure, vibrational wave
16

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numbers, NMR, frontier molecular orbital, MEP, NLO and NBO analysis of the title
compound is carried out by DFT level using the B3LYP/6-311++G(d,p) basis set. The
calculated geometrical parameters were found to be in good agreement with that of
similar derivatives. The stability and intermolecular interaction have been interpreted by
NBO analysis. ALIE surface recognized benzene rings and nitrogen atom N35 as
important reactive centres possibly prone to electrophilic attacks. Fukui functions also
indicated the importance of benzene rings from the aspect of reactivity. BDE values for
hydrogen abstraction indicate that MPATB molecule is highly stable in the open air and
in the presence of oxygen. RDF indicate that five carbon atoms and four non-carbon
atoms have significant interactions with water molecule, among which the most
important is the hydrogen atom H37. The title compound binds at the active site of the
substrate by weak non-covalent interactions and the amino acid Trp101 forms two π-
sigma, π-π T-shaped, π-alkyl interaction with CH3 group, phenyl rings respectively;
Phe149 forms π-π T-shaped and Tyr164 forms π-stacked interactions with phenyl ring.
Ser131 shows H-bond with benzoxazole ring.
Acknowledgments
The authors would like to extend their appreciation to the Deanship of Scientific
Research at KingSaud University for funding this study through the research group
project No. RGP-1435-062. The authors are thankful to University of Antwerp for access
to the University’s CalcUA supercomputer cluster and the support received from
Schrödinger Inc.
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Figure captions
Fig.1 Optimized geometry of 5-[(4-methylphenyl)acetamido]-2-(4-tert-butylphenyl)
benzoxazole
Fig.2 ALIE surface of 5-[(4-methylphenyl)acetamido]-2-(4-tert-butylphenyl)
benzoxazole molecule
Fig.3 Fukui functions a) f + and b) f – of 5-[(4-methylphenyl)acetamido]-2-(4-tert-
butylphenyl) benzoxazole molecule
Fig.4 BDEs of all single acyclic bonds of 5-[(4-methylphenyl)acetamido]-2-(4-tert-
butylphenyl) benzoxazole molecule
Fig.5 RDFs of atoms of 5-[(4-methylphenyl)acetamido]-2-(4-tert-butylphenyl)
benzoxazole with pronounced interactions with water: a) carbon atoms and b) non-carbon
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Fig.6 Amino acids of thymidylate synthase receptor interaction with the docked ligand
and receptor surface as background
27

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Table 1
Antimicrobial and antitubercular activity (MIC µg/ml) of synthesized compound 6 and the standard drugs.
Microorganismsa
Compound
-
E.c.
32
n.d.b
E.c*
64
K.p.
32
K.p*
128
n.d.
256
S.a.
128
1
S.a*
128
1
E.f.
64
1
E.f*
128
32
C.a
64
C.k.
64
M.t.
8
M.t.*
8
Vancomycin
Gentamycin
sulfate
n.d.
1024
n.d.
0.5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1
0.125 32
4
32
Meropenem
Ofloxacin
Amipicillin
0.0625 <0.0625 0.03
<0.0625 64 0.125 0.5
>2048
<0.0625 0.125
0.125 0.25
>2048 0.5 >2048
n.d.
-
2
0.5
4
n.d.
n.d.
n.d.
0.25
1
n.d.
n.d.
n.d.
2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1
2
2
1
0.5
n.d.
n.d.
n.d.
n.d.
AmphotericinB n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Fluconzaloe
Isoniazid
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
64
n.d.
n.d.
n.d.
n.d.
<0.25 <0.25
Ethambutol
2
2
aE.c., Escherichia coli ATCC 25922; E.c.*,Escherichia coli isolate (ESBL); K.p., Klebsiella pneumoniae RSKK 574; K.p.*, Klebsiella pneumoniae isolate
(ESBL); S.a.,Staphylococcus aureus ATCC 29213; S.a.*, Staphylococcus aureus isolate (MRSA); E.f., Enterococcus faecalis ATCC 29212; E.f.*: Enterococcus
faecalis isolate (VRE); C.a.,Candida albicans ATCC 10231;C.k., Candida krusei ATCC 6258; M.t., Mycobacterium tuberculosis H37RV ATCC 27294; M.t.*,
Mycobacterium tuberculosis isolate.
bn.d., not determined (microbiological assays were not performed due to following reasons: antibacterial drugs were not assayed against fungi and M.
tuberculosis; antifungal drugs were not assayed against bacteria and M. tuberculosis; antitubercular drugs were not assayed against bacteria and fungi; Gram-
negative bacteria employed in the study are naturally resistant to vancomycin).
1