Click one pot synthesis, spectral analyses, crystal structures, DFT studies and brine shrimp cytotoxicity assay of two newly synthesized 1,4,5-trisubstituted 1,2,3-triazoles

Article abstract of DOI:10.1016/j.molstruc.2015.11.010

Methyl-2-(1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)-2-oxoacetate (1) and ethyl-2-(1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)-2-oxoacetate (2) were synthesized by one pot three component strategy, and characterized by FT-IR, NMR (¹H and ¹³

Full text of DOI:10.1016/j.molstruc.2015.11.010

Journal of Molecular Structure 1106 (2016) 430e439
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Click one pot synthesis, spectral analyses, crystal structures, DFT
studies and brine shrimp cytotoxicity assay of two newly synthesized
1,4,5-trisubstituted 1,2,3-triazoles
,
Muhammad Naeem Ahmed a **, Khawaja Ansar Yasin a, Khurshid Ayub b,
,
Tariq Mahmood b *, M. Nawaz Tahir c, Bilal Ahmad Khan a, Muhammad Hafeez a,
Madiha Ahmed d, Ihsan ul-Haq d
a Department of Chemistry, The University of Azad Jammu and Kashmir, Muzaffarabad, 13100, Pakistan
b Department of Chemistry, COMSATS Institute of Information Technology, University Road, Tobe Camp, 22060, Abbottabad, Pakistan
c Department of Physics, University of Sargodha, Sargodha, Pakistan
d Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 54320, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Methyl-2-(1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)-2-oxoacetate (1) and ethyl-2-(1-benzyl-4-phenyl-
1H-1,2,3-triazol-5-yl)-2-oxoacetate (2) were synthesized by one pot three component strategy, and
characterized by FT-IR, NMR (1H and 13C) spectroscopy and TOF-MS spectrometry. Finally, the structures
were unequivocally confirmed by single crystal X-ray diffraction analyses. Both compounds, 1 and 2 exist
in monoclinic crystal packing having space group P21/n and P21/c, respectively. Crystal structures in-
vestigations revealed that the molecular structures of the title compounds are stabilized by weak
intermolecular hydrogen bonding interactions to form dimers. Density functional theory (DFT) calcula-
tions were performed not only to compare with the experimental spectroscopic results but also to probe
structural properties. The molecular electrostatic potential (MEP) mapped over the entire stabilized
geometries of the molecules delivered information about the electrophilic and nucleophilic sites.
Furthermore, frontier molecular orbital analysis gave the idea about stability and reactivity of com-
pounds. Both compounds were also screened for brine shrimp cytotoxicity assay.
Received 25 June 2015
Received in revised form
18 September 2015
Accepted 9 November 2015
Available online 14 November 2015
Keywords:
Click chemistry
Triazole
X-ray diffraction
DFT
Brine shrimp assay
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
can still promptly create molecular diversity through the use of
reactive modular building blocks. In search for new compounds
Click chemistry has recently emerged as an important tool in
synthetic chemistry [1]. In recent years, the design and synthesis of
pharmacologically relevant heterocyclic molecules by combinato-
rial techniques is proven as a promising approach in the search for
new pharmacological lead structures [2]. Click chemistry is one of
the leading reactions to make carbon-heteroatom-carbon (C-X-C)
bonds in aqueous environment. Structures bearing C-X-C moiety
possess a wide variety of chemical and biological applications in
various fields [3e6]. Click reactions require only benign reaction
conditions, simple workup including purification procedures, and
through these reliable and efficient reactions, click chemistry may
accelerate the process of discovery and optimization [3,7].
Importance and applications of triazole chemistry regarding the
click reactions is not much hidden, and has been explored by the
scientific community extensively [8]. Click chemistry has been
successfully applied to synthesize compounds for drug discovery,
enzyme inhibition, receptor-ligand binding studies, for DNA label-
ing and for studying the biological systems [1].
In continuation of our ongoing research regarding the synthesis
of 1,4,5-trisubstituted 1,2,3-triazole [9] derivatives via click reaction
and density functional theory studies of different classes [10e12],
here we are reporting the synthesis, structural investigations and
brine shrimp cytotoxic assay of two new 1,4,5-trisubstituted 1,2,3-
triazoles. Both compounds were synthesized in good yields, char-
acterized by spectroscopic analysis and finally, the structures were
* Corresponding author.
** Corresponding author.
E-mail addresses: aromatics790@gmail.com (M.N. Ahmed), mahmood@ciit.net.
pk (T. Mahmood).
http://dx.doi.org/10.1016/j.molstruc.2015.11.010
0022-2860/© 2015 Elsevier B.V. All rights reserved.

M.N. Ahmed et al. / Journal of Molecular Structure 1106 (2016) 430e439
431
confirmed unambiguously by X-ray diffraction studies. The DFT
C
18H15N3O3, [MþH]þ 322.1186; observed 322.1187.
simulations were performed not only to validate the spectroscopic
results, but also to investigate other structural properties like
frontier molecular orbital (FMOs) analysis, molecular electrostatic
potential (MEP). Both compounds were also screened for their
brine Shrimp cytotoxicity assay.
2.2.1.2. Ethyl 2-(1-benzyl-4-phenyl-1H-1, 2, 3-triazol-5-yl)-2-
oxoacetate (2). White crystalline solid, m.p. 74e76 ꢀC,
Yield ¼ 89%, IR (ATR, cmꢁ1): nmax 3057 (CHarom.), 2981 (CH), 1741
(COOCH3), 1683 (C]O), 1537 (C]C), 1477 (C]C), 1224 (N]N); 1H-
NMR
d
ppm 7.52e7.28 (m, 10H), 5.88 (s, 2H), 3.71 (q, 2H, J ¼ 7.2 Hz),
2. Materials and methods
0.92 (t, 3H, J ¼ 7.2 Hz); 13C-NMR
d ppm 177.3, 160.8, 152.9, 134.1,
129.6, 128.9, 128.7, 128.6, 128.5, 128.1, 127.2, 62.8, 54.1, 13.2. HRMS
(ESI-TOF) (m/z): calculated for C19H17N3O3, [MþH]þ 336.1343;
observed 336.1345.
2.1. Experimental
Different alkyl and aryl azides were purchased from J and K
chemicals China, and were used without further purification.
Phenylacetylides were prepared according to the procedures re-
ported in the literature [13]. Solvents of analytical reagent (AR)
grades were purchased from Sigma Aldrich, and used without pu-
rification. Melting points were determined on a Yanaco melting
point apparatus, and are reported as uncorrected. Thin layer chro-
matography (TLC) was carried out using pre coated silica gel 60
HF254 aluminum sheets (Merck). IR spectra were recorded on a
Nicolet FT-IR 5DX spectrometer, using ATR method. The 1H NMR
(300 MHz) and 13C NMR (75 MHz) spectra were recorded in CDCl3
on a JEOL JNM-ECA 300 spectrometer. TMS was used as an internal
reference and J values were calculated in Hz. HR-MS were obtained
on a Bruker microTOF-QII spectrometer.
2.3. Crystallography
Suitable crystals of both compounds 1 and 2, having proper size
and shape were selected and analyzed by single crystal X-ray
diffraction technique. Selected crystal of each compound was
coated with paratone 8772 oil and mounted on a glass fiber. All
measurements were made on Bruker Kappa APEX-IICCD diffrac-
tometer with graphite monochromatic Mo-Ka radiation. The
structures were solved by direct method and refined by using
SHELXL 2013 (Sheldrick, 2013) [15]. The figures were plotted with
the aid of ORTEP II.
The cif files of both compounds have been assigned CCDC
numbers 983913 and 994384 and can be obtained free of charge on
application to CCDC 12 Union Road, Cambridge CB21 EZ, UK. (Fax:
(þ44) 1223 336-033; e-mail: data_request@ccdc.cam.ac.uk).
2.2. Synthesis
The synthesis of triazole derivatives (1 and 2) was carried out by
adopting click one pot three component synthetic methodology
(for synthetic scheme see Fig. 1).
2.4. Computational details
Computational investigations were performed at density func-
tional theory level by using Gaussian 09 software [16]. The visual-
ization of the results/optimized geometries was achieved through
GuassView 5.0 [17]. Optimization of both triazole derivatives 1 and
2 was carried out at B3LYP/6-31G (d, p) level of theory. Frequency
simulations were performed at the same level, to confirm the
optimized geometries as true minima (no imaginary frequency).
Furthermore, frequency output files were used for simulated
vibrational analysis. Theoretical nuclear magnetic resonance (1H
and 13C NMR) studies were performed at B3LYP/6-311Gþ(2d,p)
level, by adopting GIAO formalism, and the chemical shift were
referred with reference to tetramethylsilane. Molecular electro-
static potential (MEP) and frontier molecular orbital (FMOs) were
simulated at B3LYP/6-31G (d, p) level of DFT.
2.2.1. General procedure for the synthesis of triazoles 1 and 2
Synthesis of both compounds was accomplished by slight
modification of the procedure already described in the literature
[9,14].
Methoxalyl chloride for compound 1 and ethoxalyl chloride for
compound 2 (0.07 g, 0.5 mmol) was added to a suspension of
benzylazide (0.08 g, 0.6 mmol), copper (1) phenylacetylide (0.09 g,
0.5 mmol) and chlorobenzene (2 ml). The resultant mixture was
stirred at room temperature for 4 h, and finally subjected to flash
column chromatography eluting with 10% ethylacetate in petro-
leum ether to obtain 1 and 2 as white solids.
2.2.1.1. Methyl 2-(1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)-2-
oxoacetate (1). White crystalline solid, m. p. 75e77 ꢀC,
Yield ¼ 91%, IR (ATR, cmꢁ1): nmax 3031 (CHarom.), 2954 (CH), 1739
(COOCH3), 1687 (C]O), 1484 (C]C), 1455 (C]C), 1220 (N]N); 1H-
2.5. Brine shrimp cytotoxic lethality assay
A 24 h LC50 lethality test was performed in a 96 well plate using
Brine shrimp (Artemia salina) larvae using literature method with
some modifications [18]. Six graded concentrations (300, 100, 33.3,
NMR
ppm 176.9, 161.2, 153.2, 134.2, 129.7, 129.5, 129.0, 128.8, 128.6,
128.5, 128.1, 127.2, 54.2, 52.7. HRMS (ESI-TOF) (m/z): calculated for
d
ppm 7.50e7.30 (m, 10H), 5.90 (s, 2H), 3.29 (s, 3H); 13C-NMR
d
11.1, 3.7, 1.3
mg/ml) in triplicate for test extracts were used.
Fig. 1. Synthetic scheme for triazole derivatives 1 and 2.

432
M.N. Ahmed et al. / Journal of Molecular Structure 1106 (2016) 430e439
Doxorubicin was employed as reference standard and DMSO as
negative control. Eggs of test organism A. salina Leach (Ocean 90,
USA) were kept for hatching (48 h) in simulated sterile sea water
3.2. Geometry optimization
DFT is a valuable tool not only to compare and validate the
experimental data, but also to look inside the structural properties
of compounds. Both compounds (1 and 2) were optimized at
B3LYP/6-31G (d, p) level to compare with the X-ray diffraction data
(Fig. 4). Comparison of some important bond lengths and bond
angles is given in Table 1 (bond lengths) and Table S2 (bond angles).
Some important X-ray diffraction bond lengths in 1 such as
O1eC16, O2eC17, O2eC18A, O3eC17, N1eN2, N1eC8, N1eC7,
N2eN3 and N3eC9 found at 1.207 Å, 1.240 Å, 1.480 Å, 1.246 Å,
1.331 Å, 1.361 Å, 1.472 Å, 1.323 Å and 1.352 Å respectively, whereas
the computed values of these bond lengths were depicted at
1.223 Å, 1.339 Å, 1.444 Å, 1.207 Å, 1.333 Å, 1.376 Å, 1.475 Å, 1.310 Å
and 1.361 Å. Similarly, the X-ray diffraction values of some impor-
tant bond lengths such as O1eC16, O2eC17, O3eC17, O3eC18,
N1eN2, N1eC7, N2eN3, N3eC8 and N3eC9 in 2 are observed at
1.208 Å, 1.195 Å, 1.317 Å, 1.465 Å, 1.315 Å, 1.361 Å, 1.333 Å, 1.369 Å
and 1.453 Å respectively. Simulated values of these bond lengths
are 1.222 Å, 1.208 Å, 1.337 Å, 1.455 Å, 1.310 Å, 1.361 Å, 1.333 Å,
1.375 Å and 1.474 Å, respectively. Maximum deviation in computed
and X-ray bond lengths of both triazole derivatives 1 and 2
with constant oxygen supply in
a specially designed two-
compartment plastic tray under a 60 W lamp, providing direct
light and warmth (30e32 ꢀC). The mature nauplii were then used
for the cytotoxicity test and the number of survivors was counted
after 24 h. Larvae were considered dead if they did not exhibit any
internal or external movement during several seconds of observa-
tion. The median lethal concentration (LC50) of the test samples was
calculated using table curve 2D version 5.01 software.
3. Results and discussion
Both triazole based derivatives, methyl 2-(1-benzyl-4-phenyl-
1H-1,2,3-triazol-5-yl)-2-oxoacetate (1) and ethyl 2-(1-benzyl-4-
phenyl-1H-1,2,3-triazol-5-yl)-2-oxoacetate (2) were synthesized
in good yields from commercially available starting materials by
adopting one pot three component strategy (details have been
naratted in the experimental section). After accomplishing the
successful synthesis, the final structures were characterized by
spectroscopic techniques like FT-IR, NMR (1H and 13C), and finally
the structures were confirmed by single crystal X-ray diffraction
studies.
observed in the range 0.002e0.099
respectively.
Å and 0.00e0.034 Å
In 1, X-ray values of some important bond angles such as
C17eO2eC18A, N2eN1eC8, N2eN1eC7, C8eN1eC7, N3eN2eN1,
N2eN3eC9, N1eC7eC1, N1eC8eC9, O1eC16eC8, O1eC16eC17,
O2eC17eO3 and O2eC17eC16 are depicted at 116.0ꢀ,110.3ꢀ, 118.5ꢀ,
131.1ꢀ, 108.2ꢀ, 108.7ꢀ, 112.3ꢀ, 104.5ꢀ, 123.4ꢀ, 118.6ꢀ, 126.2ꢀ and 117.6ꢀ
respectively. These experimental values correlated nicely with
theoretical ones appearing at 115.4ꢀ, 110.6ꢀ, 118.3ꢀ, 131.0ꢀ, 108.5ꢀ,
109.2ꢀ, 112.7ꢀ, 103.5ꢀ, 123.9ꢀ, 116.8ꢀ, 125.9ꢀ and 123.2ꢀ respectively.
Similarly, the experimental and computed bond angles in 2 showed
excellent correlation to each other (for individual values see
Table S2). Maximum deviation in X-ray and computed bond angles
of both compounds 1 and 2 observed in the range 0.1e5.6ꢀ and
0.0e3.3ꢀ respectively. After analyzing carefully the data, it is
concluded that very good correlation exists between the X-ray and
computed bond lengths and bond angle values.
3.1. X-ray diffraction analysis
Compounds 1 and 2 crystallized as white solid, and X-ray
diffraction analysis was performed to ensure the final structures,
and to study their three dimensional patterns. The complete crystal
data parameters are narrated in (Table S1; supplementary
information's) and ORTEP views of both 1 and 2 are shown in Fig. 2.
Both compounds are closely related to (E)-1-(benzyl-5-methyl-
1H-1, 2, 3-triazol-4-yl)-3-phenylprop-2-en-1-one with different
substitution on position 4 and 5 of the triazole ring [19,20]. The
compound (1) consisting of benzyl, phenyl and methoxalyl moiety
attached to the central 1, 2, 3-triazole ring crystallized in the
monoclinic system having space group P21/n. Compound 2 which
consists of benzyl, phenyl and ethoxalyl substituents attached to
the 1,2,3-triazole ring also crystallized in same crystal system but
with different space group P21/c. Packing diagrams of the title
compounds showed that the molecules exist as dimers via several
non-bonding interactions (Fig. 3). Packing patterns of compound 1
revealed that dimerization stabilized via H7eO1 hydrogen bonding
whereas in 2 dimers linked via H19eN1 and O3eH9 hydrogen
bonding interactions.
3.3. Vibrational analysis
Experimental FT-IR spectra of triazole derivatives 1 and 2 were
recorded by using ATR method, whereas simulated vibrational
spectra were extracted from frequency calculation. Both experi-
mental as well as simulated spectra are shown in Fig. S1
Fig. 2. ORTEP plot of compound (1) and (2) at 50% probability level. H-atoms are omitted for clarity purpose.

M.N. Ahmed et al. / Journal of Molecular Structure 1106 (2016) 430e439
433
Fig. 3. Packing patterns of compound (1) showing dimers linked via H7eO1 and compound (2) showing dimers linked via H19eN1 and O3eH9.
(Supplementary information) and Fig. 5, respectively. Detailed
comparison of experimental and calculated vibrational frequencies
is narrated in Table 2. In order to minimize the theoretical error,
simulated vibrations above 1700 cmꢁ1 were scaled by using a
scaling factor of 0.958 and for less than 1700 cmꢁ1 scaling factor
was 0.9627 [21]. Both compounds have mainly aromatic, carbonyl,
ester, CH2, and CH3 functional groups. From Table 4, it is clear that
there exists an outstanding agreement between the experimental
and theoretical vibrations.
3.3.3. CH2 and Me group vibrations
The simulated stretching (symmetric/asymmetric) aliphatic CH
vibrational frequency lie in the range of 2900e3100 cmꢁ1 and these
vibrations are usually weak due to less change in dipole moment.
Both 1 and 2 showed simulated CH stretching vibrations in the
range 3024e2943 cmꢁ1 and 3015-2930 cmꢁ1, respectively. These
simulated vibrations showed nice agreement with their experi-
mental counterparts, at 2954 cmꢁ1 for compound 1 and 3036 cmꢁ1
,
2981 cmꢁ1, 2937 cmꢁ1 for 2. Other than stretching vibrations,
several scissoring, in-plane and out of plane bending vibrations
were observed for CH2 and CH3 groups of both 1 and 2, in the
simulated as well as experimental spectra and found in good
agreement (for individual values see Table 4).
3.3.1. C]O vibrations
Among all functional groups, carbonyl is the one that can be
easily identified by using vibrational spectroscopy, and experi-
mentally a strong stretching vibration appears in the range
1650e1800 cmꢁ1 [22]. The title compounds 1 and 2 have two
different carbonyl functional groups, and their very strong
stretching frequencies in simulated spectra appeared at 1755 cmꢁ1
(COOCH3), 1670 cmꢁ1 (C]O) for 1, and 1751 cmꢁ1 (COOEt),
1671 cmꢁ1 (C]O) for 2. Experimental values of both carbonyl
groups found at 1738 cmꢁ1, 1682 cmꢁ1 for 1, 1741 cmꢁ1, 1682 cmꢁ1
for 2, and coincide excellently with the computed values.
3.3.4. Triazole ring vibrations
Triazole ring is combination of C]N, N]N and CeN functional
groups, and stretching vibrations of these groups lies in the range of
1200e1500 cmꢁ1 [25]. Computed symmetric and asymmetric C]N
stretching vibrations appeared at 1242 cmꢁ1, 1198 cmꢁ1 for 1 and
1240 cmꢁ1, 1196 cmꢁ1 for compound 2. The experimental C]N
stretching vibrations appeared at 1271 cmꢁ1,1193 cmꢁ1 (compound
1) and 1262 cmꢁ1, 1201 cmꢁ1 (compound 2), respectively. Similarly,
simulated as well as experimental N]N and CeN vibrations in both
compounds showed an excellent correlation to each other. Very
strong N]N symmetric stretching peak in the computed spectra of
both compounds appeared at 1224 cmꢁ1, whereas respective
3.3.2. Aromatic vibrations
Low intensity aromatic (CH) stretching vibrations generally
appear in the region 2800e3100 cmꢁ1 [23]. In the simulated
spectra, the prominent aromatic CH stretching (symmetric/asym-
experimental value was observed at 1220 cmꢁ1 for
1 and
1224 cmꢁ1 for 2. Theoretical CeN stretching vibration of 1 was
observed at 1312 cmꢁ1 and for 2 at 1311 cmꢁ1, whereas experi-
mental CeN vibration of both appeared at 1311 cmꢁ1 (1) and
1333 cmꢁ1 (2) simultaneously, and showed very good correlation.
metric) vibrations of both
1 and 2 appeared in the range
3071e3062 cmꢁ1. These simulated aromatic CH stretching vibra-
tions correlate nicely with the experimental values; 3031 cmꢁ1 and
3057 cmꢁ1 for 1 and 2, respectively. The symmetric and asymmetric
stretching vibrational region of aromatic ring (C]C) of medium
intensity normally lies in the range of 1600e1200 cmꢁ1 [24]. The
simulated IR spectrum of 1 showed the aromatic C]C symmetric
and asymmetric stretching vibrations at 1505 cmꢁ1, 1483 cmꢁ1
1467 cmꢁ1 and 1434 cmꢁ1. Similarly the computed aromatic
3.4. Nuclear magnetic resonance (NMR) studies (1H and 13C)
Since last two to three decades, nuclear magnetic resonance
spectroscopy has been used extensively for the structural elucida-
tion of compounds. Besides the single crystal X-ray data, the 1H and
13C chemical shifts contain very important information about the
structure of compounds. Nowadays, the DFT simulations are play-
ing very active role to predict theoretical NMR chemical shifts and
to compare with experimental results. Experimental NMR spectra
stretching C]C peaks of 2 appeared at 1513 cmꢁ1, 1483 cmꢁ1
,
1467 cmꢁ1 and 1434 cmꢁ1. The simulated aromatic C]C stretching
vibrations showed strong agreement with their experimental
counter parts depicted at 1484 cmꢁ1, 1455 cmꢁ1 and 1431 cmꢁ1 for
compound 1, and 1537 cmꢁ1 and 1477 cmꢁ1 for compound 2.

434
M.N. Ahmed et al. / Journal of Molecular Structure 1106 (2016) 430e439
Fig. 4. Optimized geometries of 1 and 2 at 6-31G (d, p) level of DFT.
(both 1H and 13C) were recorded in CDCl3, and are being shown in
electronic supplementary information Fig. S2eS5. Simulated NMR
spectra of both compounds 1 and 2 were computed by adopting
GIAO method at B3LYP/6-311þG(2d,p) level of DFT. It is very well
documented in the literature, that higher basis set works very well
for accurate measurements of chemical shift values as compare to
lower basis set [26]. The detailed comparison of simulated and
experimental 1H NMR chemical shifts of both compounds is
narrated in Table 3.
Both compounds mainly have aromatic, CH2 and CH3 protons, in
the experimental 1H NMR spectrum, the aromatic protons appeared
experimentally in the range 7.47e7.34 ppm (compound 1) and
7.45e7.33 ppm (compound 2), whereas computed aromatic CeH
chemical shifts (with respect to TMS) appeared at 8.48e7.39 ppm
(1)/8.59e7.40 (2) ppm. The methylene protons attached directly to
the aromatic ring experimentally are depicted at 5.90 ppm and
5.88 ppm for 1 and 2, respectively. The same protons in the
simulated spectra appeared at 5.90e5.83 ppm (1)/5.88 ppm (2), and
showed an excellent correlation with the experimental values.
Similarly, the experimental and simulated chemical shifts of methyl
protons in both compounds correlated excellently.
A comparison of experimental and computed 13C NMR chemical
shift is narrated in Table S3, and an excellent correlation is observed
themselves. In the experimental scan of 1, chemical shifts at
176.8 ppm, 161.2 ppm and 153.1 ppm were assigned to the qua-
ternary carbons C7, C8 and C2 (atomic labeling is in accordance
with Fig. 4) respectively, whereas the computed values for these
carbons appeared at 186.3 ppm, 172.2 ppm and 162.2 ppm. The
experimental aromatic CH signals in
1
appeared at
128.9e128.1 ppm, which agree nicely with the computed values
found in 136.5e131.9 ppm range. The chemical shift of CH2 in 1
(directly attached to aromatic ring) observed at 54.2 ppm correlates
nicely with simulated value appeared at 56.7 ppm. The experi-
mental and computed CH3 value in 1 showed an excellent

M.N. Ahmed et al. / Journal of Molecular Structure 1106 (2016) 430e439
435
agreement to each other as well. Similarly, the 13C chemical shifts of
all carbons in 2 showed excellent agreement to each other. The
quaternary carbons C7, C8 and C2 of 2, appeared experimentally at
177.3 ppm, 160.7 ppm and 152.8 ppm, whereas the simulated sig-
nals of these carbons were found at 186.7 ppm, 171.6 ppm and
Table 1
Some selected X-ray and simulated bond lengths (Å) of 1 and 2, (Atomic labels are
with reference to Fig. 2).
(1)
X-ray
Calc. (B3LYP) (2)
X-ray
Calc. (B3LYP)
1.222
O1eC16
O2eC17
O2eC18A 1.480 (6) 1.444
O3eC17
N1eN2
N1eC8
N1eC7
N2eN3
N3eC9
C1eC6
C1eC2
C1eC7
C2eC3
C4eC5
C5eC6
C10eC11
C10eC15
C11eC12
C12eC13
C13eC14
C14eC15
C16eC17
1.207 (2) 1.223
1.240 (3) 1.339
O1eC16
O2eC17
O3eC17
O3eC18
N1eN2
N1eC7
N2eN3
N3eC8
N3eC9
C1eC2
C1eC6
C1eC7
C2eC3
C3eC4
C4eC5
C5eC6
C7eC8
C8eC16
C9eC10
C10eC11 1.367 (3)
C10eC15 1.370 (3)
C11eC12 1.362 (5)
C12eC13 1.364 (5)
C13eC14 1.357 (4)
C14eC15 1.379 (3)
C16eC17 1.527 (2)
C18eC19 1.463 (3)
1.208 (2)
1.195 (18) 1.208
162.1 ppm. Experimental aromatic (CH) signal of
2
at
1.317 (2)
1.465 (2)
1.315 (2)
1.361 (2)
1.333 (2)
1.369 (2)
1.453 (2)
1.387 (3)
1.391 (3)
1.467 (3)
1.383 (3)
1.362 (4)
1.372 (4)
1.386 (4)
1.384 (2)
1.467 (2)
1.500 (3)
1.337
1.455
1.310
1.361
1.333
1.375
1.474
1.403
1.405
1.470
1.395
1.395
1.397
1.392
1.403
1.464
1.517
1.398
1.401
1.396
1.394
1.397
1.393
1.540
1.514
134.0e128.0 ppm, showed an agreement with simulated one's at
136.2e131.8 ppm. Similarly, the CH2 and CH3 computed and
experimental signals showed an excellent correlation.
1.246 (3) 1.207
1.331 (2) 1.333
1.361 (3) 1.376
1.472 (2) 1.475
1.323 (2) 1.310
1.352 (3) 1.361
1.372 (3) 1.401
1.379 (3) 1.398
1.509 (3) 1.516
1.373 (3) 1.396
1.363 (5) 1.397
1.389 (4) 1.393
1.387 (3) 1.405
1.387 (3) 1.403
1.374 (3) 1.392
1.381 (4) 1.397
1.370 (4) 1.395
1.372 (3) 1.395
1.530 (3) 1.539
3.5. Molecular electrostatic potential (MEP)
Molecular electrostatic potential (MEP) mapping in quantum
mechanical chemistry is a valuable tool not only to identify the
reactive sites in a compound but also helpful to understand the
molecular recognition process [27]. It explains the reactivity of
chemical system by predicting electrophilic as well as nucleophilic
sites inside any molecule [28]. MEP mapping provides the visual
understanding of relative polarity [29], and can be defined math-
ematically by the following expression.
Z
ZA
jRA ꢁ rj
rðr0Þ
dr0
X
VðrÞ ¼
ꢁ
jr0 ꢁ rj
P
Summation ( ) runs over all nuclei, Z is charge of nucleus
located at distance RA and
mapping, electrophilic and nucleophilic regions are explained by
the appearance of different colors, the preferred nucleophilic site is
represented by red color, electrophilic site is represented by blue
A
r
(r0) is electron density. During MEP
4000
3000
2000
Wavenumber cm-1
1000
4000
3000
2000
Wavenumber (cm-1)
1000
Fig. 5. Simulated vibrational spectra of compound 1 (above) and 2 (below).

436
M.N. Ahmed et al. / Journal of Molecular Structure 1106 (2016) 430e439
Table 2
Experimental and simulated vibrational (cmꢁ1) frequencies of 1 and 2, (only those simulated values are narrated, those have intensity above 10).
1 Calc. (Intensity)
1 (Exp.)
Assignment
ys yasCHarom.
yas, ysCHarom.
yas, ysCHarom.
yasMe
2 Calc. (Intensity)
2 (Exp.)
Assignment
3071(20.8)
3069(24.9)
3062(19.3)
3024(11.0)
2974(11.8)
e
,
3071(21.4)
3069(23.4)
3062(19.4)
3015(25.4)
3002(17.7)
2973(11.7)
2945(14.2)
2930(13.7)
1751(182.9)
1671(237.6)
1513(23.3)
1483(11.6)
1467(58.7)
1434(13.5)
1427(28.1)
1411(43.8)
1385(12.4)
1329(18.9)
1315(14.2)
1311(93.3)
1240(96.7)
1224(125.8)
1196(99.6)
1123(37.2)
1117(11.5)
1097(12.0)
1005(103.3)
987(32.1)
e
ys
ys
,
,
yasCHarom.
yasCHarom.
3031
e
3057
e
yasCHarom.
yasMe,ysCH2
yasMe
ysCH2
ysCH2
ysMe
ysCOOCH3
ysCO
e
3036
e
e
ysCH2
2981
e
2943(23.1)
1755(170.5)
1670(246.9)
1505(24.1)
1483(11.6)
1467(62.2)
1434(12.9)
1426(30.6)
1409(48.7)
1328(17.5)
1315(14.8)
1312(87.4)
1242(82.6)
1224(109.6)
1198(76.7)
1122(32.1)
1114(16.1)
993(118.0)
975(15.0)
921(21.6)
783(18.4)
764(43.5)
759(17.6)
724(39.9)
711(40.7)
687(20.8)
687(30.0)
2954
1739
1687
e
ysMe
ysCOOCH3
ysC ¼ O
2937
1741
1683
1537
e
ysC ¼ C
1484
1455
1431
e
yasC ¼ Carom.
yasC ¼ Carom.
yasC ¼ Carom.
ysC ¼ C
bCHarom.
1477
e
ysC ¼ Carom.
r
CH2
bCHarom.
e
ysC-N
e
rCH2
1331
e
r
N-C-C
1453
e
ysC ¼ N
b
CHarom.
ysCH2-CH3
1311
1271
1220
1193
e
ysC-N
e
rN-C-C
yasC ¼ N
ysN ¼ N
ysC ¼ N
e
bCHarom
1333
1262
1224
1201
1142
e
ysC-N
ysC ¼ N
ysN ¼ N
ysC ¼ N
ysN-N
t
t
CH2
CH2
e
1015
978
927
782
e
ysO-Me
b
g
Ph
Ph
t
CH2
Me
ysO-Et
Ph
e
u
ysCH2-Ph
1013
971
e
gCHarom.
gCHarom.
gCHarom.
gCHarom.
gCHarom.
gCHarom.
b
e
952(44.8)
810(14.7)
787(34.5)
774(22.4)
e
731
704
684
664
796
777
e
gCHarom.
ysCH2-Ph
gCH
724(45.8)
730
gCHarom.
ys, Symmetric treching; yas, Asymmetric streching; b, In plane bending; g, Out of plane bending; t, twisting; r, Scissoring; u wagging.
Table 3
Comparison of experimental and simulated 1H NMR of 1 and 2 (ppm), (Atomic labels are with reference to Fig. 4).
Proton (1)
Exp.
Calc. (B3LYP)
Proton (2)
Exp.
Calc. (B3LYP)
H19 (aromatic)
7.47-
7.34
(aromatic
protons)
8.48
8.43
8.21
7.81
7.70
7.69
7.61
7.55
7.49
7.39
5.90
5.83
3.74
3.62
3.52
H18 (aromatic)
H27 (aliphatic)
H16 (aromatic)
H31 (aromatic)
H33 (aromatic)
H21 (aromatic)
H20 (aromatic)
H22 (aromatic)
H32 (aromatic)
H29 (aromatic)
H6 (CH2)
H5 (CH2)
H10 (CH2)
H11 (CH2)
H40 (CH3)
H42 (CH3)
H41 (CH3)
7.45-
7.33
(aromatic
protons)
8.59
8.51
8.24
7.77
7.67
7.66
7.58
7.55
7.54
7.40
5.88
5.88
4.11
3.87
1.56
1.47
1.18
H
H
H
H
H
H
H
H
H
H
H
H
H
H
28 (aliphatic)
17 (aromatic)
32 (aromatic)
34 (aromatic)
22 (aromatic)
23 (aromatic)
21 (aromatic)
33 (aromatic)
30 (aromatic)
5 (CH2)
5.90
5.90
3.28
3.28
3.28
5.88
5.88
3.71
3.71
0.91
0.91
0.91
6 (CH2)
11 (CH3)
10 (CH3)
12 (CH3)
color and green region represents close to zero potential. The
electrostatic potential increases in the order
3.6. Frontier molecular orbitals (FMOs) analysis
red < orange < yellow < green < blue. Molecular electrostatic po-
tential mapping of 1 and 2 was simulated at the same level of
theory as used to obtain energy minima structures and surfaces are
shown in the (Fig. 6).
It is clear from MEP surfaces that both compounds 1 and 2, are
nucleophilic in nature and negative region is concentrated on tri-
azole and oxalyl moieties, and these are preferred sites for elec-
trophiles or positive charge containing species. MEP value was
ranged from ꢁ0.0496 a. u. to 0.0496 a. u. for 1 and ꢁ0.0498 a. u. to
0.0498 a. u. for compound 2.
The FMOs analysis play has a vital role to understand the ab-
sorptions, electronic as well as optical properties of chemical
compounds [30]. The energy gap between the highest occupied
orbital (HOMO) and lowest unoccupied orbital (LUMO) is very
important in term of explaining the chemical behavior of any
compound. Small HOMOeLUMO energy gap means high chemical
reactivity, low kinetic stability, and vice versa [31]. The surfaces of
the HOMO and LUMO orbitals were simulated at the B3LYP/6-31G
(d, p) level of theory in gas phase and are shown in Fig. 7.
As it is reflected from Fig. 7, that the electronic cloud in HUMO of

M.N. Ahmed et al. / Journal of Molecular Structure 1106 (2016) 430e439
437
Fig. 6. MEP surfaces of 1 and 2 at B3LYP/6-31G (d, p) level of DFT.
both 1 and 2 is mainly localized on aromatic and triazole moieties,
whereas LUMO electrons are mainly located on the aromatic ring
directly attached to triazole and oxalyl moiety. The energy of HOMO
orbital corresponds to the ionization potential (I. P) and energy of
LUMO orbital corresponds to the electron affinity (E. A.). FMOs
analysis of 1 revealed that there are total 84 filled orbitals and
energy difference between HOMO-LUMO is 4.216 eV. Whereas
compounds 2 have 88 filled orbitals and the energy difference
between HOMO-LUMO is equal to 4.24 eV. HOMO-LUMO energy
difference revealed that both compounds are highly reactive and
kinetically less stable, furthermore both compounds have almost
employed successfully for the exploration of commercially impor-
tant bioactive compounds. In the present study, compound 2 was
more potent (LC50 12.58
mg/ml) as compared to compound 1 (LC50
13.3 g/ml) (Table 4). The degree of lethality was found to be
m
directly proportional to descending concentration of extract as
highest mortality (100%) was found at maximum concentration
(300 mg/ml), minimum mortality was found at the lowest concen-
tration (Fig. 8). These results can be associated with the previous
findings in which bis-triazole derivatives depicted high mortality
percentage (50%) at concentrations ranging from 50 to 150 mg/ml
[32]. However, on the contrary findings of another report suggest
the less cytotoxic behavior of various thiazolo- and 1, 2, 3-
thiadiazolo-4-H-1,2,4-triazoles derivatives against brine shrimps
same energy difference (DE) therefore have same sort of reactivity.
at concentrations of 100 and 10 mg/ml [33]. Previous documents
3.7. Brine shrimp cytotoxic lethality assay
propose that due to commercial availability and bearing most
sensitive biological system, newly hatched larvae of A. salina L., is
one of the most suitable living models to evaluate the bioactivity of
wide variety of samples. It is apparent from the present study that
Brine shrimp cytotoxicity assay is a simple and inexpensive
methodology employed for the detection and isolation of bioactive
compounds with significant cytotoxic potential. This assay has been
Fig. 7. HOMO-LUMO surfaces of both compounds 1 and 2.

438
M.N. Ahmed et al. / Journal of Molecular Structure 1106 (2016) 430e439
Table 4
Brine shrimp lethality assay of both compounds 1 and 2.
Compound
Brine shrimp lethality assay (concentration
m
g/mL)
33.3
% Mortality
300
LC50
100
90 0.98
11.1
3.7
1.3
1
2
100 1.21
100 1.18
70 0.72
90 0.88
45 0.62
40 0.59
15 0.56
15 0.60
0
5
13.3 0.45
12.58 0.39
100 1.02
0.43
experimental values. MEP mapping revealed that both compounds
are nucleophilic in nature and negative region is concentrated on
triazole and oxalyl moieties, and these are preferred site for elec-
trophiles. Charge separation ranged from ꢁ0.0496 a. u. to 0.0496 a.
u. for 1 and ꢁ0.0498 a. u. to 0.0498 a. u. for compound 2. FMOs
analysis showed that both compounds are kinetically less stable
having low HOMO-LUMO energy gap i. e. equal to 4.24 eV. Brine
shrimp cytotoxicity assay proved that 2 was more potent (LC50
12.58 mg/ml) as compared to 1 (LC50 13.3 mg/ml) at nontoxic level of
concentration.
Acknowledgments
The authors are highly thankful to Higher Education Commis-
sion Pakistan (HEC) for financial support (Grant no. 20-3013/NRPU/
R&D/HEC/14/525).
Appendix A. Supplementary data
Fig. 8. LC50 values of both compounds 1 and 2 at different concentrations.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.molstruc.2015.11.010.
cytotoxicity profile of the tested compounds is higher even at lower
concentrations. This shows that tested compounds have very
strong capability of interaction with the model biological system.
Most of the drugs exert their pharmacological effects by interaction
with the biological system through receptors, subcellular compo-
nents and enzyme. So, this study hypothesize that further detailed
investigation of these compounds can explore the potential useful
pharmacological effects of these compounds. This is strongly sup-
ported by Silva et al. (2009) who described that brine shrimp assay
had served the purpose of exploration of numerous pharmacolog-
ical properties of natural products as well as synthesized com-
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