Synthesis, characterization, computational study, and screening of novel 1-phenyl-4-(2-phenylacetyl)-thiosemicarbazide derivatives for their antioxidant and antimicrobial activities

Article abstract of DOI:10.1016/j.molliq.2021.115977

Condensation reaction of phenyl acetyl isothiocyanate (1) and phenyl hydrazine was performed to obtain 1-phenyl-4-(2-phenylacetyl) thiosemicarbazide (2). Compound (2) was utilized as starting material for synthesis of different fused 1,2,4-triazole compou

Full text of DOI:10.1016/j.molliq.2021.115977

Journal of Molecular Liquids 333 (2021) 115977
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Synthesis, characterization, computational study, and screening of novel
1-phenyl-4-(2-phenylacetyl)-thiosemicarbazide derivatives for their
antioxidant and antimicrobial activities
c,
b
Amal A. Altalhi a, Heba E. Hashem b, Nabel A. Negm c, , Eslam A. Mohamed , Eman M. Azmy
⇑
⇑
a Department of Chemistry, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
b Chemistry Department, College for Girls, Ain Shams University, Heliopolis, Cairo, Egypt
c Egyptian Petroleum Research Institute (EPRI), Nasr City, Cairo 11727, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Condensation reaction of phenyl acetyl isothiocyanate (1) and phenyl hydrazine was performed to obtain
1-phenyl-4-(2-phenylacetyl) thiosemicarbazide (2). Compound (2) was utilized as starting material for
synthesis of different fused 1,2,4-triazole compounds. Reaction of (2) with hydrazine hydrate, anthranilic
acid and ethyl glycinate under microwave irradiation and traditional thermal conditions afforded 1,2,4-
traizol derivatives (4, 5 and 6) respectively. On the other hand, cyclization reaction of compound (2)
under microwave irradiation gave 1,2,4-triazole-3-thione derivative (3). The microwave assessed reac-
tion produced higher yield reaction and purity than the traditional thermal reaction. The chemical struc-
tures of the synthesized compounds were elucidated using elemental analysis, FTIR, 1H NMR, and GC/MS
spectroscopy. The synthesized compounds (2–7) were evaluated for their antimicrobial and antioxidant
potentials. Density functional theory (DFT) was evaluated at B3LYP/3-21G level to study the electronic
and geometric characteristics of (4–6) compounds including the optimized structures, relative binding
energies, atomic charges, position of HOMO and LUMO of the molecules.
Received 7 February 2021
Revised 15 March 2021
Accepted 23 March 2021
Available online 27 March 2021
Keywords:
Microwave assessed cyclization
Density functional theory
Antimicrobial activity
Antioxidant efficiency
Ó 2021 Elsevier B.V. All rights reserved.
1. Introduction
responses in this manner harming the cell biomolecules, for exam-
ple, proteins, lipids, and nucleic acids. Along these lines, a few
Acyl isothiocyanate derivatives have wide applications in the
synthesis of various heterocyclic compounds [1–3], such as dia-
zoles and triazoles which have numerous biological activities [4]
such as: antibacterial and antifungal [5], anti-inflammatory [6],
anticonvulsant [7], anti-HIV [8], antineoplastic, and anti-
proliferative [9–14]. Several methods were described for the
preparation of acyl isothiocyanates which were mainly based on
the reaction of acyl halides and thiocyanate ions [15].
Receptive oxygen species (ROS) have been examined for their
association in balancing an assortment of cell flagging occasions
that influence significant physiological capacities. Among ROS,
hydroxyl radicals (OH) are created much of the time under physi-
ological conditions and are firmly connected with different models
of pathologies putatively inferring oxidative pressure. Additionally,
when ROS is created more than the cell cancer prevention agent
resistance is deficient; it can invigorate free extreme chain
investigations have recommended that harm to cell biomolecules
reflects deliberate oxidative worry in cell frameworks. Specifically,
the action of oxidative distress because of expanded creation of
ROS or irregular characteristics in the body’s redox potential is a
sign of ceaseless inflammatory infections including disease [16].
It has been reported that structural properties of triazoles, like
moderate dipole character, hydrogen bonding capability, rigidity
and stability under in-vivo conditions are the main reasons for
their superior pharmacological activities [17]. The measurement
of antioxidant activities of traditional and synthetic antioxidants
has received considerable attention. Several methods are used,
either in vitro or in vivo, to determine the antioxidant activities
of natural antioxidants. Although there is a great multiplicity of
the methods used to determine antioxidant activity, there is no
approved standardized in vitro method to evaluate the antioxi-
dants of interest. The most known and applicable assays are 1,1-
diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2),
nitric oxide, peroxynitrite radical, total radical-trapping antioxi-
dant parameter (TRAP), ferric reducing-antioxidant power (FRAP),
superoxide radical (SOD), hydroxyl radical activity, hydroxyl
⇑
Corresponding authors.
E-mail addresses: nabelnegm@hotmail.com (N.A. Negm), Eslamazmy60@yahoo.
com (E.A. Mohamed).
https://doi.org/10.1016/j.molliq.2021.115977
0167-7322/Ó 2021 Elsevier B.V. All rights reserved.

A.A. Altalhi, H.E. Hashem, N.A. Negm et al.
Journal of Molecular Liquids 333 (2021) 115977
radical averting capacity (HORAC), reducing power (RP), ferric thio-
cyanate (FTC), cupric ion reducing antioxidant capacity (CUPRAC),
and metal chelating activity [18]. 2,2-azino-bis(3-ethylbenzthiazo
line-6-sulfonic acid) (ABTSꢀ+) method uses a diode-array spec-
trophotometer to measure the color loss of ABTSꢀ+. The antioxidant
reduces ABTSꢀ+ to ABTS and decolorize it. ABTSꢀ+ is a stable radical
not found in the human body. Antioxidant activity can be mea-
sured [19]. Accordingly, the presented work aimed to use micro-
wave assessed irradiation for synthesis of new substituted
triazole, fused triazole-quinazolinone and imidazolo-triazole
derivatives with anticipated antioxidant and antimicrobial activi-
ties. The incorporated mixes have been concentrated hypotheti-
cally by semi-observational sub-atomic orbital hypothesis at the
degree of PM3 of hypothesis and Density Functional Theory
(DFT) at the B3LYP/3-21G degrees of hypothesis.
was separated-off and recrystallized from ethanol to give: 5-ben
zyl-1-phenyl-1,2-dihydro-3H-1,2,4-triazole-3-thione (3).
2.2.2.1. Compound (3) 5-benzyl-1-phenyl-1,2-dihydro-3H-1,2,4-tria-
zole-3-thione. Yield: 89%; yellow crystals m.p. 218–220 °C. IR (KBr)
(t
, cmꢁ1): 3200 (NH), 3069 (CH arom), 2997, 2915, 2849 (CH alipha),
1594 (C@N), 1240 (C@S). 1H NMR (DMSO d6) d(ppm): 3.97 (s, 2H,
CH2Ar), 7.73–7.24 (m, 10H, Ar-H), 13.73 (br. s, 1H, NH, exchange-
able). Anal (%): Calc. MWt. 267.35 g/mol, C: 67.39, H: 4.90, N:
15.72; Found: C: 67.29, H: 4.85, N: 15.65; MS (70 eV) m/z (%):
267(Mꢀ+, 100), 266(74), 188(2), 91(33), 77(13).
2.2.3. Reaction of 1-phenyl-4-(2-phenylacetyl)-thiosemicarbazide (2)
with hydrazine hydrate, anthranilic acid, methyl glycinate
hydrochloride
Microwave assessed synthetic route: Equimolar amount of
1-phenyl-4-(2-phenylacetyl)-thiosemicarbazide (2) and three
amines namely: hydrazine hydrate, anthranilic acid, and methyl
glycinate hydrochloride individually were reacted under dry
condition in an open vessel irradiated in microwave oven for
2–4 min. The adducted solids were cooled, washed with distilled
water, filtered off and were recrystallized from ethanol to yield
compounds (4, 5, 6) respectively.
Thermal synthetic route: 1-phenyl-4-(2-phenylacetyl)-thiose
micarbazide (2)(10 mmol) and hydrazine hydrate, anthranilic acid
and methyl glycinate hydrochloride (0.01 mol), individually, were
dissolved in ethanol (30 mL), and refluxed for 3–6 h to obtain the
solid product after cooling to room temperature, then the different
products were filtered-off, and recrystallized from ethanol as
recrystallizing solvent.
2. Experimental section
2.1. Instrumental
Melting points were measured using Gallenkamp electric melt-
ing point apparatus. FTIR spectra were carried out using KBr disks
on FTIR Thermo-Electron Nicolet 7600 (USA). 1H NMR spectra run
at 300 MHz on GEMINI-300BB NMR spectrometer using tetram-
ethyl silane as internal standard in deuterated dimethyl sulfoxide
(DMSO d6). The mass spectra were recorded using Shimadzu GC/
MSQP-1000EX mass spectrometer operating at 70 eV. TLC was car-
ried on Merck Kiesel gel 60-F254 aluminum backed plates and
detected by UV spectra at 254–365 nm. Microwave irradiation
was carried out using Galanz microwave oven, WP1000AP30-2.
Biological activity was evaluated using disk-diffusion method in
Pharmacology Department, Faculty of Pharmacy, Mansoura
University, Egypt.
2.2.3.1. Compound (4) 3-Benzyl-5-(2-phenylhydrazinyl)-4H-1,2,4-tri-
azole. Yield 85%; white crystals m.p. 208–210 °C; IR (KBr) (t,
cmꢁ1): 3285, 3202, 3153 (NH), 3063 (CHarom), 2912 (CHaliph),
1598 (C@N); 1H NMR (DMSO d6) d(ppm): 3.97 (s, 2H, CH2Ar),
7.07–7.34 (m, 10H, Ar-H), 7.71 (br. s, 1H, NHNHPh, exchangeable),
9.85 (br. s, 1H, NHtriazole, exchangeable). Anal (%): Calc. MWt.
265.31 g/mol, C: 67.90, H: 5.70, N: 26.40. Found: C: 67.88, H:
5.67, N: 26.38. MS (70 eV) m/z (%): 265 (Mꢀ+, 43), 264 (2), 116
(6), 105 (2), 91 (1 0 0), 77 (47), 64 (24).
2.2. Synthesis
2.2.1. Synthesis of 1-phenyl-4-(2-phenylacetyl)-thiosemicarbazide (2)
Phenyl hydrazine (0.01 mol) was added drop wise to a stirred
solution of phenyl acetyl isothiocyanat (1) (0.01 mol) in dry ace-
tonitrile (20 mL) at room temperature for 3 h. The solid product
obtained was filtered off and recrystallized from ethanol to yield
product (2).
2.2.3.2. Compound (5) 2-Benzyl-1-phenyl-[1,2,4]triazolo[5,1-b]
quinazolin-9(1H)-one. Yield: 68%; pale brown crystals m.p. 178–
180 °C; IR (KBr) (t
, cmꢁ1): 3071, 3000 (CHarom), 2916, 2915, 2849
(CHaliph), 1660 (C@O), 1619 (C@N), 1595 (C@C); 1H NMR (DMSO d6)
d(ppm): 3.96 (s, 2H, CH2Ar), 7.26–7.92 (m, 14H, Ar-H). Anal (%):
Calc. MWt. 352.39 g/mol, C: 74.98, H: 4.58, N: 15.90. Found: C:
74.91, H: 4.53, N: 15.89. MS (70 eV) m/z (%): 352 (Mꢀ+, 13), 340
(16), 338 (34), 276 (9), 264 (25), 244 (14), 173 (5), 91 (1 0 0), 77
(28).
2.2.1.1 Compound (2) 1-phenyl-4-(2-phenylacetyl)-thiosemicar-
bazide. Yield: 90%; white crystals, m.p. 158–160 °C; IR (KBr) (t,
cmꢁ1): 3284 (NH), 3084, 3026 (CHarom), 2918, 2850, 2712 (CHaliph),
1664 (C@O), 1242 (C@S); 1H NMR (DMSO d6) d(ppm): 3.47 (s, 2H,
CH2Ph), 7.05–7.50 (m, 10H, Ar-H), 7.71 (br. s, 1H, HN-Ar, exchange-
able), 9.04 (br. s, 1H, NHNHAr, exchangeable), 9.84 (br. s, 1H,
CSNHCO, exchangeable). Anal (%): Calc. (MWt. 285.36 g/mol), C:
63.13, H: 5.30, N: 14.73; Found: C: 63.10, H: 5.27, N: 14.68. MS
(70 eV) m/z (%): 285 (Mꢀ+, 3), 225 (6), 194 (3), 167 (9), 108 (27),
106 (10), 91 (1 0 0), 77 (40).
2.2.3.3. Compound (6) 2-Benzyl-1-phenyl-1,5-dihydro-6H-imidazo
[1,2-b][1,2,4]triazol-6-one. Yield: 74%; white needles m.p. 198–
200 °C. IR (KBr) (t
, cmꢁ1): 3067 (CHarom), 2901 (CHaliph), 1745
(C@O), 1664 (C@C), 1598 (C@N). 1H NMR (DMSO d6) d(ppm):
3.00 (s, 2H, CH2CO); 3.97 (s, 2H, CH2Ar), 7.24–7.73 (m, 10H, Ar-
H). Anal (%): Calc. MWt. 290.32 g/mol; C: 70.33, H: 4.86, N:
19.30. Found: C: 70.3, H: 4.84, N: 19.28. MS (70 eV) m/z (%): 290
(Mꢀ+, 18), 276 (1), 268 (22), 267 (41), 266 (30), 134 (2), 116 (13),
165 (3), 91 (1 0 0), 77 (52).
2.2.2. Synthesis of 5-benzyl-1-phenyl-1,2-dihydro-3H-1,2,4-triazole-
3-thione (3)
Microwave assessed synthetic route: Grinded mixture of 1-p
henyl-4-(2-phenylacetyl)-thiosemicarbazide (2) (0.5 g) in ethanol
and three drops of HCl in an open vessel was irradiated in micro-
wave oven for 2 min. The obtained product was washed by distilled
water, filtered off, dried and recrystallized from ethanol to yield: 5-
benzyl-1-phenyl-1,2-dihydro-3H-1,2,4-triazole-3-thione (3).
Thermal synthetic route: A solution of 1-phenyl-4-(2-phenyla
cetyl)-thiosemicarbazide (2) (0.01 mol) in absolute ethanol
(25 mL) and HCl (5 mL) was refluxed for 3 h until a yellow product
2.2.4. Reaction of 5-benzyl-1-phenyl-1,2-dihydro-3H-1,2,4-triazole-3-
thione (3) with electrophiles
Microwave assessed synthetic route: Equimolar amount of 5-
benzyl-1-phenyl-1,2-dihydro-3H-1,2,4-triazole-3-thione (3) and
anthranilic acid or ethyl chloroacetate, individually, were reacted
2

A.A. Altalhi, H.E. Hashem, N.A. Negm et al.
Journal of Molecular Liquids 333 (2021) 115977
Scheme 1. Synthesis of compound (2).
Fig. 2. Analysis of 5-benzyl-1-phenyl-1,2-dihydro-3H-1,2,4-triazole-3-thione (3).
Fig. 1. Analysis of 1-phenyl-4-(2-phenylacetyl)-thiosemicarbazide (2).
(20 mL) as a solvent for 6 h. The solvent was evaporated under
reduced pressure and treated with ice/HCl. The formed solid pro-
duct was filtered off, and recrystallized from ethanol to give
ethyl-2-(3-benzyl-2-phenyl-5-thioxo-2,5-dihydro-1H-1,2,4-triazol-1-
yl) acetate (7).
in an open vessel in microwave for 2–4 min. The obtained solid
product after cooling to room temperature was washed by distilled
water, filtered off, and finally recrystallized from ethyl alcohol giv-
ing products (5, 7) respectively.
Thermal synthetic route: A mixture of 5-benzyl-1-phenyl-1,2-
dihydro-3H-1,2,4-triazole-3-thione (3) (0.01 mol) and ethyl
chloroacetate (0.01 mol) was heated under reflux in pyridine
2.2.4.1. Compound (7) ethyl-2-(3-benzyl-2-phenyl-5-thioxo-2,5-dihy-
dro-1H-1,2,4-triazol-1-yl) acetate. Yield: 60%; white crystals m.p.
3

A.A. Altalhi, H.E. Hashem, N.A. Negm et al.
Journal of Molecular Liquids 333 (2021) 115977
Scheme 2. Reaction of compound (2) to yield compounds (4, 5, 6).
Scheme 3. Synthesis of compound (3).
200–202 °C. IR (KBr) (
t
, cmꢁ1): 3068 (CHarom), 2997, 2901, 2813
2.3.1.2. Growing of microorganisms. The bacterial strains were
refined on supplement medium, while the bacterial strains were
refined on malt medium. For microscopic organisms, the stock
media were hatched for 24 h. The stock media were hatched for
roughly 48 h, with resulting separating of the way of life through
a meager layer of clean Sintered Glass G2 to expel mycelia parts
before the arrangement containing the spores was utilized for
inoculation.
(CHaliph), 1736 (C@O), 1635 (C@N), 1241 (C@S). 1H NMR (DMSO d6)
d(ppm): 1.09 (t, 3H, CH2CH3), 4.03 (q, 2H, CH2CH3), 4.10 (s, 2H, CH2-
CO), 3.97 (s, 2H, CH2Ar), 7.93–7.26 (m, 10H, Ar-H). Anal (%): Calc.
MWt. 353.44 g/mol, C: 64.57, H: 5.42, N: 11.89. Found: C: 64.52,
H: 5.40, N: 11.85. MS (70 eV) m/z (%): 353 (Mꢀ+, 10), 352 (1), 268
(19), 267 (1 0 0), 265 (8), 91 (88), 77 (39).
2.3. Evaluation
2.3.1.3. Measurements of resistance and susceptibility. For discs
preparation, 1 mL of inocula were added to 50 mL of agar media
(40 °C) and blended. The agar was filled 120 mm Petri dishes and
permitted to cool to room temperature. Wells (6 mm in width)
were cut in the agar plates utilizing appropriate sterile cylinders.
At that point, openings topped off to the outside of agar with
0.1 mL of the blended tested compounds broke up in DMF
2.3.1. Antimicrobial activity
2.3.1.1. Microorganisms. The antimicrobial assay of compounds (2–
7) was performed against various bacterial strains of S. aureus,
E. coli, P. aeruginosa, B. subtilis, Candida Albicans, and Aspergillus
flavus.
4

A.A. Altalhi, H.E. Hashem, N.A. Negm et al.
Journal of Molecular Liquids 333 (2021) 115977
Fig. 4. Analysis of 2-Benzyl-1-phenyl-[1,2,4]triazolo[5,1-b]quinazolin-9(1H)-one
(5).
Fig. 3. Analysis of 3-Benzyl-5-(2-phenylhydrazinyl)-4H-1,2,4-triazole (4).
Na/K cushion of pH 7). Ascorbic acid was utilized as a standard
for the cell reinforcement estimations. A standard alignment bend
is built for ascorbic acid at 0–350 mM. Tests were weakened prop-
erly as antioxidant agent in Na/K at pH 7. Antioxidant compounds
were blended with 200 mL of ABTSꢀ+ radical cation solution and
absorbance was perused at 750 nm after 5 min.
(1 mg/mL DMF). The plates were left on a leveled surface, hatched
for 24 h at 37 °C for microscopic organisms and 48 h for growth,
and afterward the distances across of the hindrance zones were
estimated. The restraint zone shaped by these mixes against the
specific test bacterial strain decided the antibacterial exercises of
the manufactured mixes [20]. The mean of three individual dupli-
cates was utilized to figure the zone of development restraint of
each compound. The outcomes were contrasted and compared by
Ciprofloxacin [21] as an antibacterial reference and Colitrimazole
as an antifungal reference. Both antimicrobial results were deter-
mined as a mean of three replicates.
3. Results and discussion
3.1. Chemistry of 1-phenyl-4-(2-phenylacetyl)-thiosemicarbazide (2)
1-phenyl-4-(2-phenylacetyl)-thiosemicarbazide (2) was syn-
thesized by the reaction of phenyl acetyl isothiocyanate (1) with
phenyl hydrazine in acetonitrile at room temperature according
to Scheme 1.
The spectral information of 2 was in agreement with their pro-
posed structure, where the infrared spectrum showed absorption
characteristic for C@O, NH, and C@S groups. 1H NMR spectrum
exhibited three broad singlet signals for NH proton exchangeable
with D2O. On the other hand, the inspection of mass spectrum gave
2.3.2. Antioxidant activity
This method uses UV–Vis spectra to measure the loss in color
when an antioxidant is added to blue-green chromophore ABTSꢀ+
(2,2-azino-bis(3-ethyl benzthiazoline-6-sulfonic acid)). The
antioxidant reduces the stable ABTSꢀ+ radicals to uncolored ABTS.
Antioxidant efficacy can be measured as described [22–23]. ABTS
radical particles are set up by including manganese dioxide
(80 mg) to a 5 mM fluid stock of ABTS (20 mL utilizing a 75 mM
5

A.A. Altalhi, H.E. Hashem, N.A. Negm et al.
Journal of Molecular Liquids 333 (2021) 115977
Fig. 5. Analysis of 2-Benzyl-1-phenyl-1,5-dihydro-6H-imidazo[1,2-b][1,2,4]triazol-6-one (6).
the correct molecular ion peaks and some other important peaks
appearance of a characteristic band at 1594 cmꢁ1 corresponded
(Fig. 1a-c).
to C@N (Fig. 2a-c).
3.3. Reaction of compound (2)
3.2. Chemistry of 5-benzyl-1,2-dihydro-1-phenyl-1,2,4-triazole-3-
thione (3)
Solvent-free microwave assessed reactions of compounds 2
with hydrazine hydrate yielded 3-benzyl-5-(2-phenylhydrazinyl)-
4H-1,2,4-triazole (4) (Scheme 2).
Spectral data of compound 4 confirmed the presented structure.
IR spectrum did not show any bands for C@O and C@S. 1H NMR dis-
played three wide-ranging singlet signal for NH protons in addition
to multiple signals for aromatic protons. The GC–MS spectrum of
compound 4 gives the accurate molecular ion signal and several
plentiful peaks (Fig. 3a-c).
Compound 5-benzyl-1,2-dihydro-1-phenyl-1,2,4-triazole-3-thi
one (3) was synthesized via cyclization reaction by solvent-free
microwave irradiation of 1-phenyl-4-(2-phenylacetyl)-thiosemicar
bazide (2) for 2 min (Scheme 3), and also chemically in the pres-
ence of ethanol and HCl for 3 h. It was found that the yields of
the two reaction pathways (thermal and microwave assessed)
are identical, but microwave route gave higher yield and purity.
The chemical structure of compound (3) was confirmed by the dis-
appearance of the absorption band of C@O at 1664 cmꢁ1 and the
On the other hand, reaction of compound 2 with anthranilic
acid and methyl glycinate hydrochloride, individually, under
6

A.A. Altalhi, H.E. Hashem, N.A. Negm et al.
Journal of Molecular Liquids 333 (2021) 115977
Scheme 4. Reaction of compound (3) to yield compounds (5, 7).
Zoneof inhibitionbytestcompoundðdiametreÞ
Zoneof inhibitionbystandardðdiametreÞ
microwave irradiation gave fused triazolo-quinazolinone (5) or
imidazole-triazole (6), respectively (Scheme 2).
%Acti
v
ityIndex ¼
X100
The structures of compounds 5 and 6 were elucidated from
their spectral data. The infrared spectra of compound 5 and 6
showed the corresponding characteristic absorption bands at
1660 and 1745 cmꢁ1 for C@O group of quinazoline ring or imida-
zolinone ring, respectively. The absence of any signals in the down
field region of 1H NMR spectrum as well as the presence of signals
corresponding to aromatic and aliphatic protons are in accordance
with the suggested structure of compounds 5 and 6 (Fig. 4a–c;
Fig. 5a,b). More improved evidences were obtained for the struc-
tures of compounds 5 and 6 gained from mass spectra (Fig. 4c,
Fig. 5c).
The results of the antimicrobial study summarized in Table 1
showed that the formed heterocyclic compounds 4, 5 and 7 have
highest efficacy towards bacteria and fungi. While the started com-
pounds 2 and 3 have moderate activity (Table 1).
It is clear that the prepared compounds exhibited high inhibi-
tion zone diameters comparable to the controls used for both bac-
teria and fungi. Compounds 5–7 exhibited the highest activities
against both of bacteria and fungi.
3.5.2. Antioxidant activity
The new synthesized compounds were screened for their
antioxidant activities by using 2,2-azino-bis(3-ethylbenzthiazo
line-6-sulfonic acid (ABTS) method [24–25]. The antioxidant activ-
ity of the prepared triazole derivatives have indicated that the
presence of active scavenging centers such as amino groups and
the nitrogen atoms within the conjugated systems of the different
molecules could undergo a role in OH scavenging efficiency. The
content of the active scavenging centers may affect the antioxidant
activity for triazole derivatives [26,27].
The obtained results of the prepared triazole derivatives as
antioxidant showed that compounds 2, 4, 5 and 7 have high
antioxidant activities at (81.7%, 83.8%, 84.5%, 84.9%) compared to
ascorbic acid (90.2%) (Table 2). The data showed that, compound
7 exhibited high antioxidant activity at 84.9% compared to ABTS
as a standard, and that may be due to the presence of the electron
donating group (CH2COOC2H5). On the other hand, compound 5
also showed high activity with inhibition of 84.5%, that can be
rationalized due to the presence of C@O in cyclic resonance sys-
tem. In a similar way, compound 4 has three NH groups exhibit
high antioxidant inhibition activity at 83.8%. Compound 3 has
moderate antioxidant activity, which can be ascribed to the pres-
ence of C@S and Ar groups surrounding the NH group. That
decreases the activity of the NH group towards scavenging the free
radicals in the medium.
3.4. Reaction of compound 3
Compound 3 reacted with ethyl chloroacetate and anthranilic
acid, individually under solvent-free microwave irradiation or reflux-
ing in pyridine gave ethyl 2-(3-benzyl-2-phenyl-5-thioxo-2,5-dihy
dro-1H-1,2,4-triazol-1-yl)acetate (7) and 2-Benzyl-1-phenyl-[1,2,
4]-triazolo [5,1-b]quinazolin-9(1H)-one (5), respectively (Scheme 4).
Structure of compound 7 was elucidated using its spectral data.
Infrared spectrum showed absorption bands correlated to C@S,
C@O and C@N groups. 1H NMR spectrum was devoid of down field
signals instead of quartet signal for CH2, triplet signal for CH3, and
singlet signal for CH2CO protons. Further proof was achieved from
the mass spectrum (Fig. 6a-c).
Reaction of compound (7) with hydrazine hydrate under micro-
wave irradiation or reflux in ethanol gave a product which was
identified as compound (3) using melting point and TLC identifica-
tions (Scheme 4).
3.5. Pharmacology
3.5.1. Antimicrobial activity
The synthesized heterocyclic compounds (2–7) were examined
for their antimicrobial activities against Gram-negative bacteria
(E. coli and P. aeruginosa), Gram-positive bacteria (S. aureus and B.
subtilis), and fungi (C. albicans and A. Flavus). Ciprofloxacin and
clotrimazole were used as standards against bacteria and fungi,
respectively. The activity index (%) of the complex was calculated
by the formula:
The gained outcomes of antimicrobial and antioxidant studies
revealed that the thermal and microwave assessed modifications
of 5-benzyl-1-phenyl-1,2-dihydro-3H-1,2,4-triazole-3-thione (3)
and 1-phenyl-4-(2-phenylacetyl)thiosemicarbazide (2) consider-
ably were improved the antioxidant and antimicrobial properties
of their corresponding derivatives (4–7).
7

A.A. Altalhi, H.E. Hashem, N.A. Negm et al.
Journal of Molecular Liquids 333 (2021) 115977
Table 2
Antioxidant activity results for the prepared triazole derivatives using ABTS method.
ABTS Absstandard-Abstest/Absstandard ꢂ 100
Compound
Compounds
Inhibition, %
Absorbance ofsamples
0.0%
0.518
0.058
0.209
0.095
0.084
0.080
0.085
0.078
Control of ABTS
Ascorbic acid
90.2%
59.6%
81.7%
83.8%
84.5%
83.2%
84.9%
3
2
4
5
6
7
and HOMO-LUMO energies of the studied compounds were calcu-
lated by the 6–311 + G (d,p) higher basis set level. The optimized
molecular structures with minimum energies obtained from the cal-
culations of the investigated compounds are shown in Fig. 7.
The experimental data showed that the reaction of hydrazine
hydrate and anthranilic acid with compound 2 occurs on C13
and C14 and give the cyclic triazole and fused triazolo-
quinazolinone products 4 and 5 which could be explained from
the calculated quantum chemical parameters where it elucidated
that the positive regions are mainly concentrated on C13 and
C14, which have Mulliken charges +0.603924 and +0.389610,
respectively (Table 3) which represent the highest electrophilicity
index where anthranilic acid and hydrazine hydrate react through
nucleophilic attack.
The bond angle and bond length of compound 2 and the formed
cyclic triazole derivatives 4 and 5 differ from each other. Calcula-
tion of C@O and C@S groups substituted to triazole moiety in com-
pound 4 are 1.3765 and 1.41679 Ao, respectively, and C-N-Ar bond
distance in compound 5 is 1.31151Ao. While C@O and C@S bond
distance of molecule 2 are 1.2391 and 1.71539 Ao, respectively.
On the other hand, the bond angle of C-NH-C of triazole moiety
in compound 4 and 5 is smaller than that in the linear molecule
2 (Table 4). The two phenyl rings of the two molecules gave nearly
similar bond lengths.
The frontier orbital energy difference (HOMO-LUMO) reflects
the molecules chemical reactivity and kinetic stability; smaller
frontier orbital, the higher chemical reactivity and lower kinetic
stability [28].
The 3D plots of the frontier orbitals HOMO, LUMO and the
Molecular electrostatic potential (MESP), Fig. 8. Compound 2
HOMO is spread over the terminal (ArANHNHAC@S). The LUMO
is also found to be uniformly distributed over the entire molecule
(COANHAC@S) and reflects a lot of anti-bonding character. While,
compounds 4 and 5, HOMO orbital spread over triazole, N-phenyl
and quinazoline ring in compound 5 and localized on benzyl and
triazole rings in compound 4. On the other hand, LUMO orbital
localized on the same rings of compound 4 and mainly on triazole
and benzyl ring in compound 5. LUMO–HOMO energy difference
Fig. 6. Analysis of ethyl-2-(3-benzyl-2-phenyl-5-thioxo-2,5-dihydro-1H-1,2,4-tria-
zol-1-yl)acetate (7).
3.6. Computational study (DFT)
The computational studies were performed by operating the
Gaussian09–20softwarepackage.Fullgeometryoptimizationswere
carried out using Density Function Theory (DFT) at B3LYP level for
compounds 2, 4, and 5. Properties, Mulliken charge distributions
Table 1
Diameter of inhibition zone (mm) and activity index % for all synthesized compounds (A%: activity index %, D (mm): diameter of inhibition zone).
Compound
E. coli
P. aeuroginosa
S. aureus
B. subtilis
C. Albicans
A. flavus
D (mm)
A, %
D (mm)
A, %
D (mm)
A, %
D (mm)
A, %
D (mm)
A, %
D (mm)
A, %
2
3
4
5
6
7
7
26.9
38.5
38.5
50.0
51.5
53.8
100
–
10
13
13
15
15
16
23
NA
43.5
56.5
56.5
65.2
66
69.6
100
–
12
15
16
17
18
19
24
NA
50.0
62.5
66.7
70.8
73.9
79.2
100
–
11
15
17
18
18
20
23
NA
47.8
65.2
73.9
78.3
81.2
86.9
100
–
7
9
9
10
11
12
NA
27
25.9
33.3
33.3
37.0
42.5
44.4
–
9
36
44
48
56
58
60
–
10
10
13
13
14
26
NA
11
12
14
14
15
NA
25
Ciprofloxacin
Colitrimazole
100
100
8

A.A. Altalhi, H.E. Hashem, N.A. Negm et al.
Journal of Molecular Liquids 333 (2021) 115977
Fig. 7. Optimized structures of compounds 2, 4, and 5 using DFT/6–31+G(d).
9

Table 3
Mulliken atomic charge distribution of compounds 2, 4, 5.
Compound 2
Compound 4
Atom
Compound 5
Atom
Atom
Mulliken charge
Mulliken charge
Mulliken charge
1C
2C
3C
4C
5C
6C
7H
8H
9H
10H
11H
12C
ꢁ0.145414
ꢁ0.075868
ꢁ0.090906
ꢁ0.173163
ꢁ0.144644
ꢁ0.164567
0.180487
0.179506
0.173065
0.173985
0.173049
1C
2C
0.765435
0.185700
1C
2C
3C
4C
5C
6C
7C
8C
0.463909
0.071392
ꢁ0.115151
0.610750
0.321998
ꢁ0.147829
ꢁ0.112977
ꢁ0.167569
ꢁ0.130756
0.174138
0.175057
0.175433
0.180360
3H
4N
5N
6N
7N
8N
9H
10H
11C
12C
13C
0.391128
ꢁ0.709836
ꢁ0.274997
ꢁ0.323843
ꢁ0.544010
ꢁ0.591056
0.377870
0.360690
9C
10H
11H
12H
13H
0.272486
ꢁ0.197521
ꢁ0.181316
ꢁ0.585021
0.603924
13C
14C
ꢁ0.156856
14N
ꢁ0.379253
14C
15N
16N
17N
18O
19S
0.389610
ꢁ0.712679
ꢁ0.497056
ꢁ0.571500
ꢁ0.411165
ꢁ0.132434
0.384800
0.361319
0.284910
0.241036
0.253057
ꢁ0.114810
ꢁ0.182246
ꢁ0.160834
ꢁ0.180955
ꢁ0.202302
0.287580
0.182852
0.164438
0.164788
0.165724
0.181431
15C
16C
17H
18H
19H
20H
21H
22C
23C
24C
25C
26C
27C
28C
29H
30H
31H
32H
33H
ꢁ0.183886
ꢁ0.114888
0.183652
0.167562
0.167052
0.167041
0.186449
0.076713
ꢁ0.181000
ꢁ0.060209
ꢁ0.225070
ꢁ0.118400
ꢁ0.161964
ꢁ0.137243
0.173675
0.178722
0.156670
0.174215
0.177033
15N
16N
17N
18C
19C
20C
21C
22H
23C
24H
25C
26H
27H
28H
29C
30O
31C
32C
33C
34C
35C
36C
37H
38H
39H
40H
41H
ꢁ0.534214
ꢁ0.570099
ꢁ0.402874
0.299976
ꢁ0.051895
ꢁ0.162603
ꢁ0.189771
0.197427
ꢁ0.182618
0.197866
ꢁ0.132775
0.177709
0.177782
0.177672
0.086618
ꢁ0.445326
ꢁ0.152381
ꢁ0.095474
ꢁ0.171429
ꢁ0.000725
ꢁ0.282435
ꢁ0.042048
0.195943
0.19858
20H
21H
22H
23H
24H
25C
26C
27C
28C
29C
30C
31H
32H
33H
34H
35H
0.196282
0.201918
0.188115

Table 4
Bond length and bond angle of compound 3, 4 and 5.
Compound 2
Bond
Angle
Bond lengths (Ao)
Bond angles (o)
C14-N15
C13-N15
N16-N17
N16-H20
N17-H21
C13-O18
C14-S19
C13-C12
C14-N16
C13-C14
C14-N15-C13
N15-C14-N16
C14-N16-N17
N17-C30-C25
N15-C13-C12
C13-C12-C2
C13- O18-S19
C14- S19-O18
C14-N15-H22
H20-N16-N17
H21-N17-N16
1.39006
1.39664
1.38607
1.01090
1.00919
1.23917
1.71539
1.52971
1.36640
2.52475
129.91599
111.25909
124.01403
121.95303
114.69209
116.92404
87.12393
67.97133
116.11477
115.47282
114.50633
Compound 4
Bond
C1-N4
C2-N4
N7-N8
Angle
Bond lengths (Ao)
1.34116
1.41679
1.39215
1.01055
1.01053
1.41739
1.35167
1.39985
Bond angles (o)
107.66493
126.48099
120.29512
121.75718
122.27928
120.38830
109.66851
106.85752
126.94066
120.70665
114.08509
C1-N4-C2
N4-C1-N7
C1-N7-N8
N8-C11-C16
N4-C2-C22
C2-C22-C25
C2-N5-N6
C1-N6-N5
H3-N4-C1
H9-N7-N8
H18-N8-N7
N7-H9
N8-H18
N8-C11
C1-N7
C2-C22
C1-N6
1.37653
2.22693
1.36506
C1-C2
N5-N6
Compound 5
Bond
Angle
Bond lengths (Ao)
1.31151
1.41746
1.37654
1.40528
1.39721
1.35547
1.25388
Bond angles (o)
106.60772
109.09743
111.79254
105.41503
127.72122
122.02758
C1-N14
C1-N15
C1-N17
N15-N16
C5-N16
C5-N17
C4-O30
C1-N17-C5
N15-C1-N17
N16-C5-N17
C5-N16-N15
N17-C1-C14
N15-C4-O30

A.A. Altalhi, H.E. Hashem, N.A. Negm et al.
Journal of Molecular Liquids 333 (2021) 115977
Fig. 8. 3D HOMO-LUMO plots of compounds 2, 4 and 5.
Table 5
Calculated energies, dipole moment and frontier molecular orbital energies of compounds 2, 4, 6 using B3LYP/6–311+G (d,p) basis set calculations.
Compound
Ea
HOMOb
LUMOc
D
Ed
Dipole moment
2
4
5
ꢁ1218.28145453
ꢁ853.00233903
ꢁ1142.04767505
ꢁ0.22196
ꢁ0.19954
ꢁ0.16785
ꢁ0.06751
ꢁ0.11519
ꢁ0.05070
0.15445
0.08435
0.11715
9.9776
6.3960
11.1303
a
b
c
E: the total energy (a.u.).
HOMO: highest occupied molecular orbital (eV).
LUMO: lowest unoccupied molecular orbital (eV).
d
D
E: ELUMO-EHOMO (eV).
(D
E) and dipole moment (
l, Debye) for the studied molecules were
4. Conclusion
listed in Table 5. This study gave important results on the reactivity
of such compounds and the most active site towards nucleophilic
and electrophilic substitution.
1-phenyl-4-(2-phenylacetyl) thiosemicarbazide was prepared
successfully using thermal and microwave assessed reaction. Sev-
12

A.A. Altalhi, H.E. Hashem, N.A. Negm et al.
Journal of Molecular Liquids 333 (2021) 115977
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CRediT authorship contribution statement
Amal A. Altalhi: Conceptualization, Methodology, Writing -
review & editing, Project administration. Heba E. Hashem: Investi-
gation. Nabel A. Negm: Conceptualization, Methodology, Writing -
review & editing, Project administration. Eslam A. Mohamed: Con-
ceptualization, Methodology. Eman M. Azmy: Conceptualization,
Methodology.
Declaration of Competing Interest
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cationic surfactants: Surface and thermodynamic properties and applicability
in bacterial growth and metal corrosion prevention, J. Surfac. Deterg. 14 (2011)
505–514.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
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Acknowledgement
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I would like to thank Taif University Researcher supporting pro-
ject number (TURP-2020/243), Taif University, Taif, Saudi Arabia.
Also, I would like to thank Prof. Dr. Nabel A. Negm, thanks to the
Egyptian Petroleum Research Institute (EPRI).
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13