Synthesis, biological evaluation, and in silico study of some unique multifunctional 1,2,4-triazole acetamides

Article abstract of DOI:10.3906/kim-1706-50

The imperative demand for antibacterial agents and enzyme inhibitors prompted us to synthesize some new compounds, 6a–6k, bearing multifunctional moieties. The target acetamides were derived from 4-phenyl-5-(1-tosylpiperidin-4-yl)-4H-1,2,4-triazole-3-thiol (3). The structural analysis was carried out using modern spectroscopic techniques including IR, NMR, and EIMS spectral analysis. The antibacterial activity was screened against five bacterial strains including three gram-negative and two gram-positive ones. Enzyme inhibition was carried out against lipoxygenase enzyme and results were supported by in silico study. The synthesized compounds were proved to be potent antibacterial agents and enzyme inhibitors.

Full text of DOI:10.3906/kim-1706-50

Turk J Chem
Turkish Journal of Chemistry
(2018) 42: 401 – 417
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1706-50
Research Article
Synthesis, biological evaluation, and in silico study of some unique
multifunctional 1,2,4-triazole acetamides
Almas SATTAR1, Aziz UR REHMAN1,∗, Muhammad Athar ABBASI1,
Sabahat Zahra SIDDIQUI1, Shahid RASOOL1, Hira KHALID2,
Muhammad Arif LODHI3, Farman Ali KHAN3
1Department of Chemistry, Government College University, Lahore, Pakistan
2Department of Chemistry, Faculty of Natural Science, Forman Christian College, Lahore, Pakistan
3Department of Biochemistry, Abdul Wali Khan University, Mardan, Pakistan
Received: 22.06.2017
•
Accepted/Published Online: 10.11.2017
•
Final Version: 27.04.2018
Abstract: The imperative demand for antibacterial agents and enzyme inhibitors prompted us to synthesize some
new compounds, 6a–6k, bearing multifunctional moieties. The target acetamides were derived from 4-phenyl-5-(1-
tosylpiperidin-4-yl)-4H -1,2,4-triazole-3-thiol (3). The structural analysis was carried out using modern spectroscopic
techniques including IR, NMR, and EIMS spectral analysis. The antibacterial activity was screened against five bacterial
strains including three gram-negative and two gram-positive ones. Enzyme inhibition was carried out against lipoxygenase
enzyme and results were supported by in silico study. The synthesized compounds were proved to be potent antibacterial
agents and enzyme inhibitors.
Key words: 1,2,4-Triazole, acetamides, antibacterial activity, lipoxygenase enzyme inhibition, in silico study
1. Introduction
The use of antibiotics is increasing to combat different infectious diseases. This excessive use of antibiotics
makes bacterial strains more resistant to presently available antibiotic drugs. These circumstances demand
an imperative supply of novel antibiotics.1,2 Enhanced expression and activity of lipoxygenase (LOX) in the
body of humans and other mammals is the cause of many diseases including asthma,3 cardiovascular diseases,4
rheumatoid arthritis,5 inflammatory bowel,6 and cancer.7
Amides, RCONHR’, are effective antibacterial8,9 and LOX inhibitors.10−12 In conjunction with an-
tibacterial and enzyme inhibition activity, amide derivatives are anti-HIV,13 antitumor,14 antipsychotic,15
analgesic,16 antiulcer,17 anticonvulsant,18 cardiotonic,19 and antiinflammatory20 agents. Keeping in view
the importance of amide functionality, it has been merged with 4-phenyl-5-(1-tosylpiperidin-4-yl)-4H -1,2,4-
triazole-3-thiol (3), which also bears other bioactive moieties including a piperidine core,21−23 sulfonamide
linkage,24,25 and 1,2,4-triazole heterocycle.26−29 The different bioactive 1,2,4-triazole derivatives include 6-aryl-
3-{(4-substitutedphenoxy)methyl}-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles26 and 3,6-disubstituted-1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazoles.27−29
Five bacterial strains were considered for antibacterial study. Bacillus subtilis is believed to cause dermal
allergy and hyper sensitivity,30 Staphylococcus aureus is a pathogenic bacterial strain,31 Salmonella typhi is a
∗Correspondence: rehman@gcu.edu.pk
401

SATTAR et al./Turk J Chem
cause of enteric fever and some other diseases,32 Escherichia coli can cause food poisoning,33 and Pseudomonas
aeruginosa is a cause of chronic infections.34 All these bacterial strains cause different diseases and so are used
as model bacterial strains for antibacterial screening.
The advancement in crystallography, nuclear magnetic resonance spectroscopy, and other structural
elucidation techniques proves very important in three-dimensional structural determinations of biomolecules
and drugs. The three-dimensional structures of proteins such as enzymes are used to study possible interactions
of drugs with enzymes using computational studies.35 The three-dimensional structure of enzyme active sites
interact with drug molecules such as ligands with the help of docking software. The software finds different
possible interactions between ligand and enzyme; such studies are regarded as virtual screening, in silico studies,
or molecular docking.36−39
The resistance to existing antibiotics1,2 and bioactive moieties21−29 prompted us to synthesize some new
bioactive compounds bearing different functionalities altogether in a single molecule. The hypothesis was that
these functionalities may boost the bioactivity potential of each other. A series of S -substituted derivatives
of 4-phenyl-5-(1-tosylpiperidin-4-yl)-4H -1,2,4-triazole-3-thiol (3) has been synthesized and most of them were
found to be potent antibacterial agents along with having notable LOX inhibition.
2. Results and discussion
The derivatives of 4-phenyl-5-(1-tosylpiperidin-4-yl)-4H−1,2,4-triazole-3-thiol (3) were synthesized according
to the Scheme and evaluated for antibacterial activity and LOX inhibition potentials. The different aryl/aralkyl
groups of the synthesized compounds are given in Table 1.
Table 1. Different N -substituted aryl/aralkyl groups.
Comp.
R
Comp.
R
Comp.
R
8''''
H3C
6''''
6''''
6''''
2''''
2''''
8''''
4''''
6a
6e
6i
2''''
4''''
4''''
7''''
H3C
H2C
7''''
CH3
7''''
CH3
9''''CH3
6''''
6''''
2''''
4''''
6''''
7''''
CH
7''''
CH3
6b
6f
6j
2''''
4''''
8''''
CH3
8''''
2CH3
2''''
4''''
7''''
O
C
H2
8''''
CH3
NO2
7''''
6''''
2''''
6''''
6''''
6c
6g
6h
6k
4''''
8''''
H3C
7''''
CH3
2''''
4''''
2''''
4''''
7''''
CH3
8''''CH3
6''''
6''''
8''''
7'''' CH3
C
H2
2''''
4''''
6d
2''''
4''''
7''''
CH3
2.1. Chemistry
Compound 6a was obtained as a white amorphous solid with percentage yield of 79%. The molecular ion peak
was observed at 561 (m/z) in EIMS and the 1 H NMR data established the formula of 6a as C29 H31 N5 O3 S2 .
402

SATTAR et al./Turk J Chem
O
O
O
O
S
S
A
N
HN
Cl
OC2H5
OC2H5
H3C
H3C
b
1
O
a
O
B
O
O
O
O
S
C
S
N
N
NH
N
H3C
NH2
SH
H3C
2
O
3
N N
D
E
HN
Br
R NH2
R
O
4a-k
5a-k
3'''
O
O
5'''
1'''
S
N
3''
O
3'
5'
1'
1''
R
N
4
5''
2'''''
S
H3C
1'''''
NH
CH2
1
2
N N
6a-k
Scheme. Outline for the synthesis of derivatives of 4-phenyl-5-(1-tosylpiperidin-4-yl)-4H -1,2,4-triazole-3-thiol (6a–6k).
Reagents and conditions: A) 5% Na2 CO3 solution, H2 O, stir for 4 h. B) N2 H4 .H2 O, EtOH, reflux for 3 h. C) i-
Phenyl isothiocyanate, EtOH, reflux for 2 h. ii- 10% NaOH, reflux for 5 h. D) BrCH2 COBr, 5% Na2 CO3 solution,
H2 O, vigorously stir 30 min. E) DMF, NaH, stir for 4–5 h.
The peaks in the IR spectrum appeared at 3447, 3071, 1738, 1629, 1536, and 1341, corresponding to N-H
stretching, C-H stretching of the aromatic ring, C=O stretching, C=N stretching, C=C aromatic stretching,
and -SO2 - stretching, respectively. The IR spectral peaks confirmed all the intended moieties and functional
groups in compound 6a. The EIMS peak at m/z420 represented the removal of p-toluene sulfonyl moiety
from compound 6a. The peak at m/z264 confirmed the removal of the phenyl group present at position 4 of
the 1,2,4-triazole ring and N -(4-ethylphenyl)-2-sulfanylacetamide moiety along with partial cleavage of triazole
ring. The peak at m/z 109 represented the presence of piperidine moiety and the peak at m/z 91 represented
the presence of toluene moiety. Other prominent fragments are given in Figure 1. In the 1 H NMR spectrum,
the p−toluenesulfonyl group was supported by two doublets and one singlet at δ (ppm) 7.59 (d,J = 8.4 Hz, 2H,
H-2′′ & H-6′′ ), 7.27 (d,J = 7.8 Hz, 2H, H-3′′ & H-5′′ ), and 2.39 (s, 3H, CH3 -7′′ ). The phenyl group directly
attached to the nitrogen of the triazole ring resonated at δ (ppm) 7.54–7.50 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ) and
7.18–7.17 (m, 2H, H-2′′′ , H-6′′′ ). The 2-methylphenyl group was confirmed by δ (ppm) 7.94 (d,J = 8.4 Hz, 1H,
403

SATTAR et al./Turk J Chem
H-6′′′′ ), 7.18–7.17 (m, 1H, H-5′′′′ ), 7.15 (d,J = 7.2 Hz, 1H, H-3′′′′ ), 7.02 (dt,J = 1.2, 7.8 Hz, 1H, H-4′′′′ ), and
2.27 (s, 3H, CH3 -7′′′′ ). The acetamoyl moiety presented two signals at δ (ppm) 9.68 (s, 1H, N-H) and 3.92 (s,
2H, H-2′′′′′ . Spectral study corroborated compound 6a as 2-(4-phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-
4H -1,2,4-triazol-3-ylthio)-N -(2-methylphenyl) acetamide. The structural elucidation of remaining compounds
under study was also performed through IR, EIMS, and 1 H NMR analysis.
O
N
S
O
N
N
S
H
CH3
N
N
H
m/z = 297 (6%)
CH3
m/ z = 206 (2%)
-C19H21N3O2S
- C13H16N2O2S
O
O
S
O
N
N
N
S
H3C
H
N N
m/z = 561 (<1%)
CH3
-C17H17N3OS
-C23H26N5OS
O
O
O O
S
S
N
H3C
m/ z = 155 (4%)
H3C
m/z = 264 (2%)
N
-SO2
-C7H7O2S
-C3H4
N
N
- HCN
H2C
m/z = 51 (2%) m/z = 91 (22%)
m/ z = 82 (31%)
m/ z = 109 (7%)
N
Figure 1. Mass fragmentation pattern of 6a.
2.2. Antibacterial activity
The antibacterial study of synthetic compounds 6a–6k was performed against five bacterial strains. The bacte-
rial strains under consideration included three gram-negative strains (S. typhi, E. coli, and P. aeruginosa) and
two gram-positive strains (B. subtilis and S. aureus). The standard antibacterial agent used was ciprofloxacin.
The results in the form of % inhibition and minimum inhibitory concentration (MIC) values are given in Tables 2
and 3. All compounds except 6g and 6h were active against all the bacterial strains under study. Compounds
404

SATTAR et al./Turk J Chem
6g and 6h were inactive only against E. coli and S. aureus, respectively, while for the remaining bacterial
strains compounds 6g and 6h showed good antibacterial activity. Compounds 6b, 6d, and 6k proved to be
proficient antibacterial agents for all microbes under study. The good activity of compounds 6b, 6d, and 6k
may be attributed to the varying moieties as 2,3-dimethylphenyl, 2,5-dimethylphenyl. and benzyl, respectively.
Compounds 6b, 6d, and 6k were proved to be the most active antibacterial agents in the following order, 6d
>6b >6k. Compounds 6d and 6k were the most active against S. aureus with MIC values of 8.15 ꢀ 0.85 and
8.21 ꢀ 0.43 µ M, respectively, with a reference of 7.57 ꢀ 0.65 µ M, the MIC value of ciprofloxacin. Compound
6b was the most active against S. typhi with MIC value of 8.11 ꢀ 0.87 with a reference of 7.47 ꢀ 0.68 µ
M. The previously synthesized acetamide analogs bearing oxadiazole as 2-(5-(1-(phenylsulfonyl)piperidin-4-yl)-
1,3,4-oxadiazol-2-ylthio)acetamide by Khalid et al.40 were moderately active against the bacterial strains under
study. The current results showed that minor changes in structure led to an enhanced antibacterial activity
potential.
Table 2. The % inhibition of bacterial activity of synthesized compounds.
% Inhibition
Compounds
S. typhi (-)
E. coli (-)
P. aeruginosa (-) B. subtilis (+) S. aureus (+)
6a
6b
6c
6d
6e
6f
76.68 ꢀ 0.51 65.25 ꢀ 0.34 74.24 ꢀ 0.17
86.17 ꢀ 0.32 73.45 ꢀ 1.13 74.38 ꢀ 0.46
77.08 ꢀ 0.46 72.32 ꢀ 0.48 71.00 ꢀ 0.81
86.81 ꢀ 0.46 88.89 ꢀ 0.76 74.11 ꢀ 0.19
70.14 ꢀ 0.87 83.23 ꢀ 0.37 63.45 ꢀ 0.70
62.56 ꢀ 0.48 52.45 ꢀ 0.97 66.18 ꢀ 0.23
70.58 ꢀ 0.91 47.18 ꢀ 0.24 76.78 ꢀ 0.67
60.41 ꢀ 0.48 79.67 ꢀ 0.26 72.21 ꢀ 0.97
63.49 ꢀ 0.53 76.67 ꢀ 0.67 57.33 ꢀ 1.13
75.00 ꢀ 0.67 72.03 ꢀ 0.37 71.97 ꢀ 1.25
86.45 ꢀ 0.67 73.53 ꢀ 0.09 72.27 ꢀ 0.97
75.24 ꢀ 0.57
75.88 ꢀ 0.23
73.36 ꢀ 0.75
79.71 ꢀ 0.67
74.13 ꢀ 0.65
62.57 ꢀ 0.28
80.78 ꢀ 0.31
73.37 ꢀ 0.52
67.35 ꢀ 0.27
77.61 ꢀ 0.56
80.65 ꢀ 0.23
91.82 ꢀ 063
59.00 ꢀ 0.53
77.91 ꢀ 0.85
60.17 ꢀ 0.56
87.54 ꢀ 0.71
70.20 ꢀ 0.18
78.07 ꢀ 0.65
81.58 ꢀ 0.67
10.31 ꢀ 0.27
62.24 ꢀ 0.43
72.18 ꢀ 0.97
90.18 ꢀ 1.56
91.89 ꢀ 0.39
6g
6h
6i
6j
6k
Ciprofloxacin 91.55 ꢀ 0.78 92.65 ꢀ 0.32 92.81 ꢀ 0.56
2.3. LOX inhibition activity
All the synthesized compounds, 6a–6k, were proved to be excellently to moderately active, except 6g. Com-
pounds 6b, 6c, 6h, 6i, and 6k were proved to be excellent LOX inhibitors. Table 4 demonstrates the %
inhibition and IC50 values. The most active compound was 6k; the enhanced activity of this compound could
be attributed to the unsubstituted benzyl moiety, which allowed compound 6k to fit into the active site of the
LOX enzyme more compatibly and inhibit the enzyme strongly. The overall inhibition order of the synthesized
compounds may be listed as 6k >6i >6h >6b >6c >6d >6e >6f >6j >6a >6g.
2.4. Molecular docking
The most active LOX inhibitors under study, 6b, 6c, 6h, 6i, and 6k, were docked to find the probable moieties
of the synthetic compounds that were the cause of LOX inhibition. The molecular docking interactions of the
most active compounds are presented in Table 5. Figure 2 shows that compound 6b inhibited LOX due to
405

SATTAR et al./Turk J Chem
Table 3. The MIC for antibacterial activity of synthesized compounds.
MIC
Compounds
S. typhi (-)
9.21 ꢀ 0.87
8.11 ꢀ 0.51
8.81 ꢀ 0.43
8.22 ꢀ 0.43
E. coli (-)
11.18 ꢀ 0.24 9.46 ꢀ 0.12
9.19 ꢀ 0.53 9.51 ꢀ 0.71
P. aeruginosa (-) B. subtilis (+) S. aureus (+)
6a
6b
6c
6d
6e
6f
9.16 ꢀ 0.96
9.14 ꢀ 0.71
9.19 ꢀ 0.93
8.73 ꢀ 0.51
9.35 ꢀ 0.77
15.32 ꢀ 0.34
8.71 ꢀ 0.27
10.47 ꢀ 0.75
12.43 ꢀ 0.76
8.61 ꢀ 0.48
8.67 ꢀ 0.54
7.43 ꢀ 0.87
15.63 ꢀ 0.48
8.37 ꢀ 0.65
15.88 ꢀ 0.39
8.15 ꢀ 0.85
10.72 ꢀ 0.45
8.63 ꢀ 0.72
8.67 ꢀ 0.54
-
10.72 ꢀ 0.20 10.71 ꢀ 0.21
8.43 ꢀ 0.65
9.67 ꢀ 0.23
14.61 ꢀ 0.65
10.14 ꢀ 0.27 8.56 ꢀ 0.97
13.83 ꢀ 0.28 19.54 ꢀ 0.56 12.76 ꢀ 0.41
6g
6h
6i
10.86 ꢀ 0.23
-
8.72 ꢀ 0.43
9.61 ꢀ 0.68
16.37 ꢀ 0.23
15.61 ꢀ 0.53 8.48 ꢀ 0.35
12.51 ꢀ 0.23 8.57 ꢀ 0.97
14.48 ꢀ 0.25
10.53 ꢀ 0.44
8.21 ꢀ 0.43
7.57 ꢀ 0.65
6j
8.46 ꢀ 0.23
8.78 ꢀ 0.35
10.09 ꢀ 0.24 9.47 ꢀ 0.36
6k
9.65 ꢀ 0.48
7.61 ꢀ 0.52
9.56 ꢀ 0.25
7.23 ꢀ 0.68
Ciprofloxacin 7.47 ꢀ 0.68
Table 4. Lipoxygenase inhibition activity of synthesized compounds.
Compounds LOX inhibition (%) at 0.5 mM IC50 (µ M)
6a
53.44 ꢀ 0.76
89.54 ꢀ 0.65
87.47 ꢀ 1.07
79.43 ꢀ 0.55
74.27 ꢀ 0.12
58.27 ꢀ 0.43
35.64 ꢀ 0.83
87.67 ꢀ 0.67
89.41 ꢀ 1.17
55.54 ꢀ 0.74
81.12 ꢀ 0.83
93.79 ꢀ 1.27
441.87 ꢀ 0.14
86.65 ꢀ 0.36
91.07 ꢀ 0.86
114.25 ꢀ 0.49
156.67 ꢀ 0.36
305.35 ꢀ 0.11
>500
6b
6c
6d
6e
6f
6g
6h
85.61 ꢀ 0.56
61.56 ꢀ 0.87
435.87 ꢀ 0.41
53.70 ꢀ 0.98
22.41 ꢀ 0.30
6i
6j
6k
Baicalein
Note: IC50 values (concentration at which there is 50% enzyme inhibition) of compounds were calculated using EZ-Fit
Enzyme kinetics software (Perella Scientific Inc., Amherst, NH, USA).
˚
an acidic interaction with His518 through the carbonyl moiety having bond length of 2.26 A. Compound 6c
made acidic and arene-cation interactions because of the sulfonyl oxygen and toluenyl ring with Thr274 and
˚
His266 with bond length of 2.29 A and on average 4.25 A, respectively (Figure 3). Compound 6h made a
˚
metallic interaction with Fe+3 by virtue of sulfonyl oxygen with bond length 2.58 A and π-π interaction with
˚
˚
His518 through the terminal phenyl ring with bond length of 4.02 A (Figure 4). Compound 6i showed three
interactions, acidic and arene-arene with His518 and strong metallic with Fe+3 , with bond lengths of 2.85 A,
˚
406

SATTAR et al./Turk J Chem
˚
3.14 A, and 2.54 A, respectively (Figure 5). The most active compound, 6k, interacted with the enzyme active
˚
site because of the sulfonyl oxygen and toluenyl ring, which made acidic and arene-cation interactions with
˚
Thr274 and His266 with bond lengths of 2.75 A and 4.25 A, respectively (Figure 6).
˚
Figure 2. 2D and 3D binding models of compound 6b.
Figure 3. 2D and 3D binding models of compound 6c.
Figure 4. 2D and 3D binding models of compound 6h.
2.5. Conclusions
The synthesized compounds were strong to moderate antibacterial agents, especially 6b, 6d, and 6k. Com-
pounds 6b, 6c, 6i, and 6k were potent LOX inhibitors. LOX inhibition was also well supported by docking
407

SATTAR et al./Turk J Chem
Figure 5. 2D and 3D binding models of compound 6i.
Figure 6. 2D and 3D binding models of compound 6k.
Table 5. Molecular docking interactions of the most active compounds.
α−Glucosidase
Compound
˚
Interactions
Acidic
Functionality
Bond distance, A
6b
6c
His518 with carbonyl oxygen
Thr274 with sulfonyl oxygen
2.26
2.29
4.25
2.58
4.02
2.85
Acidic
Arene-cation His266 with toluenyl ring
Fe+3 with sulfonyl oxygen
Metallic
π − π
Acidic
6h
6i
His518 with phenyl
His518 with carbonyl oxygen
Arene-arene
Metallic
Acidic
His518 with phenyl ethyl moiety 3.14
Fe+3 with carbonyl oxygen
2.54
2.75
4.25
Thr274 sulfonyl oxygen
6k
Arene-cation Toluenyl ring
results. The synthesized compounds were proved to be more potent antibacterial agents as compared to previ-
ously reported counterparts. The results and comparison with previous studies indicated that slight variation
408

SATTAR et al./Turk J Chem
in structure can affect biological activity tremendously. The most active synthesized compounds may be consid-
ered as new drug candidates for diseases caused by bacterial strains and LOX enzyme. Further slight structural
modifications may also result in highly bioactive compounds as suitable drug candidates for certain other dis-
eases.
3. Experimental
3.1. General
The chemicals employed for synthesis were purchased from local suppliers from Merck, Sigma-Aldrich, and Alfa
Aesar. The analytical-grade solvents were employed without further purification. An open capillary method
was used to determine the melting points of the synthesized compounds and the determined melting points were
uncorrected. TLC was employed to evaluate the purity of the synthesized compounds. TLC was performed
using precoated silica gel G-25-UV254 aluminum plates. The mobile system used for the development of TLC
was composed of different ratios of n-hexane and ethyl acetate. The components of the reaction mixture and
references were illuminated by a 254-nm UV lamp. A JASCO-320-A spectrometer was employed to obtain IR
spectra using the KBr pellet method. A Bruker spectrometer working at 400 and 600 MHz was used to obtain
1 H NMR spectra and 150 MHz for 13 C NMR spectra. A JMS-HX-110 spectrometer was used to develop EIMS
spectra.
3.2. Preparation of 4-ethoxycarbonyl-1-(4-methylphenylsulfonyl)piperidine (1)
Ethyl isonipecotate (a; 30.0 mmol) was dispersed in distilled water (40 mL), taken in a round-bottomed (RB)
flask (250 mL). p-Toluenesulfonyl chloride (b; 30.0 mmol) was added to the above reaction mixture pinch by
pinch in about 30 min. Na2 CO3 (5%) was used to maintain the pH around 9.0. The reaction mixture was
stirred at room temperature for 4 h. The reaction progress was monitored by TLC. On the completion of the
reaction, concentrated HCl (11 M, 4 mL) was added to the reaction contents and pH was adjusted to about 6.0;
the mixture was allowed to stand for 15–20 min. White solid precipitates of compound 1 were obtained after
filtration, washed with distilled water, and dried in a desiccator.
3.3. Preparation of 1-(4-methylphenylsulfonyl)-4-piperidinecarbohydrazide (2)
4-Ethoxycarbonyl-1-(4-methylphenylsulfonyl)piperidine (1; 26.0 mmol) was dissolved in ethanol (40 mL) in a
RB flask (250 mL). Hydrazine hydrate (80%, 20 mL) was added steadily to the above reaction mixture and
refluxed for 3 h. Completion of the reaction was confirmed through TLC. On completion of the reaction, excess
solvent was evaporated to yield a white crystalline product, compound 2, and the product was washed with
cold distilled water and dried.
3.4. Preparation of 4-phenyl-5-(1-tosylpiperidin-4-yl)-4H -1,2,4-triazole-3-thiol (3)
The compound, 1-(4-methylphenylsulfonyl)-4-piperidinecarbohydrazide (2; 22.0 mmol), was dissolved in ethanol
(40 mL), followed by gradual addition of phenyl isothiocyanate (22.0 mmol). The reaction was refluxed for 2
h. The completion of the reaction was confirmed via TLC. Excess solvent was evaporated to acquire the
precipitates. The dried precipitates were dispersed in 10% NaOH solution (40 mL) and refluxed for 5 h. The
completion of the reaction was also established through TLC. After it, dilute HCl was added to adjust the pH
to be slightly acidic (pH 6). White solid crystalline compound 3 was filtered, washed with distilled water, and
dried.
409

SATTAR et al./Turk J Chem
3.5. General procedure for the synthesis of compounds (5a–5k)
Aryl/aralkyl amines (4a–4k; 5.7 mmol) were added in distilled water (10 mL) in a flat-bottomed flask and 5%
Na2 CO3 solution was used to adjust the pH at 8.0 to 9.0. The reaction contents were stirred for 20 min at
room temperature. 2-Bromoacetyl bromide (0.50 mL; 5.7 mmol) was added to the reaction mixture in about 10
min along with vigorous shaking. The reaction flask was further stirred until the formation of solid precipitates.
The reaction completion was established by TLC. The solid products were filtered, washed with cold distilled
water, and dried to produce the title electrophiles, 5a–5k.
3.6. General procedure for the synthesis derivatives of 3 (6a–6k)
N, N−Dimethylformamide (DMF) (15 mL) and sodium hydride (0.1 mmol) were taken in a RB flask (50 mL)
and mixed by stirring. 4-Phenyl-5-(1-tosylpiperidin-4-yl)-4H -1,2,4-triazole-3-thiol (3; 0.1 mmol) was dissolved
in the above reaction mixture and stirred at room temperature for 30 min. After that, electrophiles 5a–5k were
added in an equimolar ratio to the reaction mixture and further stirred for 4–5 h. The progress and completion
of the reaction were assessed by TLC. Distilled water was added in excess to the reaction contents to obtain
products 6a–6k. The products thus obtained were filtered, washed, and dried.
3.7. Spectral characterization of synthesized compounds (1–3, 6a–6k)
3.7.1. 4-Ethoxycarbonyl-1-(4-methylphenylsulfonyl)piperidine (1)
White amorphous solid; yield: 89%; mp: 70–72 ◦ C; molecular formula: C15 H21 NO4 S; molecular weight: 311;
IR (KBr, cm−1) υmax : 3067 (C-H stretching of aromatic ring), 1732 (C=O stretching), 1531 (C=C aromatic
stretching), 1335 (-SO2 - stretching), 1079 (C-O bond stretching); 1 H NMR (400 MHz, CDCl3 , δ / ppm):
7.62 (d,J = 8.0 Hz, 2H, H-2′′ & H-6′′ ), 7.32 (d,J = 8.0 Hz, 2H, H-3′′ & H-5′′ ), 3.98 (q, J = 7.2 Hz, 2H,
O-CH2), 3.71–3.68 (m, 2H, He -2′ & He -6′ ), 2.73–2.62 (m, 1H, H-4′ ), 2.54–2.48 (m, 2H, Ha -2′ & Ha -6′ ), 2.42
(s, 3H, CH3 -7′′ ), 2.10–2.08 (m, 2H, He -3′ & He -5′ ), 1.60–1.86 (m, 2H, Ha -3′ & Ha -5′ ), 1.15 (t, J = 7.2
Hz, CH3); EIMS (m/z): 311 [M]+ , 266 [C13 H16 NO3 S]+ , 238 [C12 H16 NO2 S]+ , 184 [C8 H10 NO2 S]+ , 170
[C7 H8 NO2 S]+ , 155 [C7 H7 O2 S]+ , 91 [C7 H7 ]+ .
3.7.2. 1-(4-Methylphenylsulfonyl)-4-piperidinecarbohydrazide (2)
White crystalline solid; yield: 91%; mp: 128 ◦ C; molecular formula: C13 H19 N3 O3 S; molecular weight: 297; IR
(KBr, cm−1) υmax : 3348 (N-H stretching), 3063 (C-H stretching of aromatic ring), 1682 (C=O stretching), 1534
(C=C aromatic stretching), 1339 (-SO2 - stretching); 1 H NMR (400 MHz, CDCl3 , δ / ppm): 7.61 (d,J = 8.0
Hz, 2H, H-2′′ & H-6′′ ), 7.33 (d,J = 8.0 Hz, 2H, H-3′′ & H-5′′ ), 3.72–3.69 (m, 2H, He -2′ & He -6′ ), 2.73–2.62
(m, 1H, H-4′ ), 2.53–2.49 (m, 2H, Ha -2′ & Ha -6′ ), 2.42 (s, 3H, CH3 -7′′ ), 2.12–2.10 (m, 2H, He -3′ & He -5′ ),
1.58–1.84 (m, 2H, Ha -3′ & Ha -5′ ); EIMS (m/z): 297 [M]+ , 266 [C13 H16 NO3 S]+ , 238 [C12 H16 NO2 S]+ ,
184 [C8 H10 NO2 S]+ , 170 [C7 H8 NO2 S]+ , 155 [C7 H7 O2 S]+ , 91 [C7 H7 ]+ .
3.7.3. 4-Phenyl-5-(1-tosylpiperidin-4-yl)-4H-1,2,4-triazole-3-thiol (3)
White crystalline solid; yield: 95 %; mp: 155 ◦ C; molecular formula: C20 H22 N4 O2 S2 :molecular weight: 414;
IR (KBr, cm−1) υmax : 3050 (C-H stretching of aromatic ring), 2617 (S-H), 1613 (aromatic C-C stretching),
1574 (C=N stretching of triazole ring), 1469 (C=C aromatic stretching), 1347 (-SO2 - stretching), 668 (C-S
410

SATTAR et al./Turk J Chem
stretching); 1 H NMR (600 MHz, CDCl3 ,δ / ppm): 11.25 (s, 1H, S-H), 7.57 (d,J = 8.4 Hz, 2H, H-2′′
&
H-6′′ ), 7.52–7.51 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.27 (d,J = 7.8 Hz, 2H, H-3′′ & H-5′′ ), 7.27–7.23 (m, 2H,
H-2′′′ & H-6′′′ ), 3.68–3.65 (m, 2H, He -2′ & He -6′ ), 2.39 (s, 3H, CH3 -7′′ ), 2.37–2.33 (m, 1H, H-4′ ), 2.25–2.20
(m, 2H, Ha -2′ & Ha -6′ ), 1.89–1.86 (m, 2H, He -3′ & He -5′ ), 1.79–1.60 (m, 2H, Ha -3′ & Ha -5′ ); 13 C NMR
(150 MHz, CDCl3 , δ/ ppm): 156.8 (C-3), 153.3 (C-5), 143.9 (C-1′′′ ), 132.8 (C-1′′ ), 132.1 (C-4′′ ), 130.5 (C-3′′
& C-5′′ ), 129.6 (C-2′′′ & C-6′′′ ), 127.5 (C-3′′′ & C-5′′′ ), 127.3 (C-2′′ & C-6′′ ), 126.1 (C-4′′′ ), 45.3 (C-2′
& C-6′ ), 32.1 (C-4′ ), 29.2 (C-3′ & C-5′ ), 21.4 (C-7′′ ); EIMS (m/z): 414 [M]+ , 259 [C13 H15 N4 S]+ , 264
[C13 H16 N2 O2 S]+ , 150 [C7 H6 N2 S]+ , 155 [C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ , 91 [C7 H7 ]+ , 82 [C5 H8 N]+ , 59
[CHNS]+ , 51 [C4 H3 ]+ .
3.7.4. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -(2-met-
hylphenyl)acetamide (6a)
White amorphous solid; yield: 79%; mp: 181–183 ◦ C; molecular formula: C29 H31 N5 O3 S2 ; molecular weight:
561; IR (KBr, cm−1) υmax : 3447 (N-H stretching), 3071 (C-H stretching of aromatic ring), 2887 (CH2
stretching), 1738 (C=O stretching), 1629 (C=N stretching), 1536 (C=C aromatic stretching), 1341 (-SO2 -
stretching); 1 H NMR (600 MHz, CDCl3 , δ / ppm): 9.68 (s, 1H, N-H), 7.94 (d,J = 8.4 Hz, 1H, H-6′′′′ ), 7.59
(d,J = 8.4 Hz, 2H, H-2′′ & H-6′′ ), 7.54–7.50 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.27 (d, J = 7.8 Hz, 2H, H-3′′
& H-5′′ ), 7.18–7.17 (m, 3H, H-2′′′ , H-6′′′ & H-5′′′′ ), 7.15 (d,J = 7.2 Hz, 1H, H-3′′′′ ), 7.02 (dt,J = 1.2, 7.8
Hz, 1H, H-4′′′′ ), 3.92 (s, 2H, H-2′′′′′ , 3.71–3.67 (m, 2H, He -2′ & He -6′ ), 2.51–2.46 (m, 1H, H-4′ ), 2.39 (s,
3H, CH3 -7′′ ), 2.34–2.30 (m, 2H, Ha -2′ & Ha -6′ ), 2.27 (s, 3H, CH3 -7′′′′ ), 2.00–1.94 (m, 2H, He -3′ & He -5′ ),
1.82–1.80 (m, 2H, Ha -3′ & Ha -5′ ); EIMS (m/z): 561 [M]+ , 406 [C22 H24 N5 OS]+ , 297 [C16 H15 N3 OS]+ , 264
[C13 H16 N2 O2 S]+ , 206 [C10 H10 N2 OS]. + , 155 [C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ , 91 [C7 H7 ]+ , 82 [C5 H8 N]+ ,
77 [C5 H5 ]+ , 51 [C4 H3 ]+ .
3.7.5. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -(2,3-di-
methylphenyl)acetamide (6b)
White amorphous solid; yield: 85%; mp: 185–187 ◦ C; molecular formula: C30 H33 N5 O3 S2 ; molecular weight:
575; IR (KBr, cm−1) υmax : 3436 (N-H stretching), 3065 (C-H stretching of aromatic ring), 2883 (CH2
stretching), 1730 (C=O stretching), 1623 (C=N stretching), 1531 (C=C aromatic stretching), 1333 (-SO2 -
stretching); 1 H NMR (600 MHz, CDCl3 , δ / ppm): 9.60 (s, 1H, N-H), 7.60 (d,J = 8.4 Hz, 1H, H-6′′′′ ), 7.58
(d,J = 8.4 Hz, 2H, H-2′′ & H-6′′ ), 7.55–7.51 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.26 (d,J = 8.4 Hz, 2H, H-3′′
& H-5′′ ), 7.17 (dd,J = 1.2, 7.8 Hz, 2H, H-2′′′ & H-6′′′ ), 7.06 (t,J = 7.8 Hz, 1H, H-5′′′′ ), 6.96 (d,J = 7.8
Hz, 1H, H-4′′′′ ), 3.93 (s, 2H, H-2′′′′′ , 3.74–3.70 (m, 2H, He -2′ & He -6′ ), 2.48–2.45 (m, 1H, H-4′ ), 2.39 (s,
3H, CH3 -7′′ ), 2.30–2.26 (m, 2H, Ha -2′ & Ha -6′ ), 2.28 (s, 3H, CH3 -8′′′′ ), 2.16 (s, 3H, CH3 -7′′′′ ), 2.01–1.94
(m, 2H, He -3′ & He -5′ ), 1.83–1.80 (m, 2H, Ha -3′ & Ha -5′ ); EIMS (m/z): 575 [M]+ , 420 [C23 H26 N5 OS]+ ,
311 [C17 H17 N3 OS]+ , 264 [C13 H16 N2 O2 S]+ , 220 [C11 H12 N2 OS]. + , 155 [C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ ,
91 [C7 H7 ]+ , 82 [C5 H8 N]+ , 77 [C5 H5 ]+ , 51 [C4 H3 ]+ .
411

SATTAR et al./Turk J Chem
3.7.6. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -(2,4-di-
methylphenyl)acetamide (6c)
White amorphous solid; yield: 87%; mp: 181–183 ◦ C; molecular formula: C30 H33 N5 O3 S2 ; molecular weight:
575; IR (KBr, cm−1) υmax : 3439 (N-H stretching), 3067 (C-H stretching of aromatic ring), 2882 (CH2
stretching), 1731 (C=O stretching), 1628 (C=N stretching), 1533 (C=C aromatic stretching), 1337 (-SO2 -
stretching); 1 H NMR (600 MHz, CDCl3 ,δ / ppm): 9.55 (s, 1H, N-H), 7.74 (d,J = 8.4 Hz, 1H, H-6′′′′ ), 7.58
(d,J = 7.8 Hz, 2H, H-2′′ & H-6′′ ), 7.55–7.51 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.27 (d,J = 7.8 Hz, 2H, H-3′′
& H-5′′ ), 7.18–7.16 (m, 2H, H-2′′′ & H-6′′′ ), 6.97 (d,J = 7.8 Hz, 1H, H-5′′′′ ), 6.96 (s, 1H, H-3′′′′ ), 3.91 (s,
2H, H-2′′′′′ , 3.71–3.68 (m, 2H, He -2′ & He -6′ ), 2.50–2.46 (m, 1H, H-4′ ), 2.39 (s, 3H, CH3 -7′′ ), 2.33–2.30
(m, 2H, Ha -2′ & Ha -6′ ), 2.26 (s, 3H, CH3 -8′′′′ ), 2.21 (s, 3H, CH3 -7′′′′ ), 2.00–1.93 (m, 2H, He -3′ & He -5′ ),
1.83–1.80 (m, 2H, Ha -3′ & Ha -5′ ); EIMS (m/z): 575 [M]+ , 420 [C23 H26 N5 OS]+ , 311 [C17 H17 N3 OS]+ , 264
[C13 H16 N2 O2 S]+ , 220 [C11 H12 N2 OS]. + , 155 [C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ , 91 [C7 H7 ]+ , 82 [C5 H8 N]+ ,
77 [C5 H5 ]+ , 51 [C4 H3 ]+ .
3.7.7. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -(2,6-di-
methylphenyl)acetamide (6d)
White amorphous solid; yield: 81%; mp: 189–191 ◦ C; molecular formula: C30 H33 N5 O3 S2 ; molecular weight:
575; IR (KBr, cm−1) υmax : 3431 (N-H stretching), 3060 (C-H stretching of aromatic ring), 2878 (CH2
stretching), 1725 (C=O stretching), 1618 (C=N stretching), 1525 (C=C aromatic stretching), 1329 (-SO2 -
stretching); 1 H NMR (600 MHz, CDCl3 , δ / ppm): 9.27 (s, 1H, N-H), 7.58 (d,J = 8.4 Hz, 2H, H-2′′ & H-6′′ ),
7.55–7.53 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.26 (d,J = 8.4 Hz, 2H, H-3′′ & H-5′′ ), 7.17 (dd,J = 1.2, 7.8 Hz,
2H, H-2′′′ & H-6′′′ ), 7.07–704 (m, 1H, H-4′′′′ ), 7.02 (d,J = 7.2 Hz, 2H, H-3′′′′ & H-5′′′′ ), 3.94 (s, 2H, H-2′′′′′
,
3.70–3.68 (m, 2H, He -2′ & He -6′ ), 2.52–2.47 (m, 1H, H-4′ ), 2.38 (s, 3H, CH3 -7′′ ), 2.34–2.30 (m, 2H, Ha -2′
& Ha -6′ ), 2.13 (s, 6H, CH3 -7′′′′ & CH3 -8′′′′ ), 1.99–1.93 (m, 2H, He -3′ & He -5′ ), 1.83–1.80 (m, 2H, Ha -3′
& Ha -5′ ); EIMS (m/z): 575 [M]+ , 420 [C23 H26 N5 OS]+ , 311 [C17 H17 N3 OS]+ , 264 [C13 H16 N2 O2 S]+ ,
220 [C11 H12 N2 OS]. + , 155 [C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ , 91 [C7 H7 ]+ , 82 [C5 H8 N]+ , 77 [C5 H5 ]+ , 51
[C4 H3 ]+ .
3.7.8. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -(3,5-di-
methylphenyl)acetamide (6e)
White amorphous solid; yield: 84%; mp: 184–186 ◦ C; molecular formula: C30 H33 N5 O3 S2 ; molecular weight:
575; IR (KBr, cm−1) υmax : 3439 (N-H stretching), 3067 (C-H stretching of aromatic ring), 2885 (CH2
stretching), 1734 (C=O stretching), 1628 (C=N stretching), 1533 (C=C aromatic stretching), 1343 (-SO2 -
stretching); 1 H NMR (600 MHz, CDCl3 , δ / ppm): 9.97 (s, 1H, N-H), 7.59 (d,J = 8.4 Hz, 2H, H-2′′ & H-6′′ ),
7.55–7.50 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.27 (d,J = 8.4 Hz, 2H, H-3′′ & H-5′′ ), 7.21 (s, 2H, H-2′′′′ & H-6′′′′ ),
7.18–7.17 (m, 2H, H-2′′′ & H-6′′′ ), 6.72 (s, 1H, H-4′′′′ ), 3.83 (s, 2H, H-2′′′′′ , 3.76–3.73 (m, 2H, He -2′ & He -6′ ),
2.49–2.44 (m, 1H, H-4′ ), 2.39 (s, 3H, CH3 -7′′ ), 2.27 (s, 6H, CH3 -7′′′′ & CH3 -8′′′′ ), 2.26–2.24 (m, 2H, Ha -2′ &
Ha -6′ ), 2.03–1.97 (m, 2H, He -3′ & He -5′ ), 1.84–1.81 (m, 2H, Ha -3′ & Ha -5′ ); EIMS (m/z): 575 [M]+ , 420
[C23 H26 N5 OS]+ , 311 [C17 H17 N3 OS]+ , 264 [C13 H16 N2 O2 S]+ , 220 [C11 H12 N2 OS]. + , 155 [C7 H7 O2 S]+ ,
109 [C6 H9 N2 ]+ , 91 [C7 H7 ]+ , 82 [C5 H8 N]+ , 77 [C5 H5 ]+ , 51 [C4 H3 ]+ .
412

SATTAR et al./Turk J Chem
3.7.9. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -(2-ethyl-
6-methylphenyl)acetamide (6f)
Light pink amorphous solid; yield: 85%; mp: 187–189 ◦ C; molecular formula: C31 H35 N5 O3 S2 ; molecular
weight: 589; IR (KBr, cm−1) υmax : 3438 (N-H stretching), 3063 (C-H stretching of aromatic ring), 2881 (CH2
stretching), 1735 (C=O stretching), 1627 (C=N stretching), 1537 (C=C aromatic stretching), 1342 (-SO2 -
stretching); 1 H NMR (600 MHz, CDCl3 , δ / ppm): 9.28 (s, 1H, N-H), 7.58 (d,J = 8.4 Hz, 2H, H-2′′ & H-6′′ ),
7.57–7.54 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.26 (d,J = 7.8 Hz, 2H, H-3′′ & H-5′′ ), 7.18–7.16 (m, 2H, H-2′′′
&
H-6′′′ ), 7.11 (t, J = 7.8 Hz, 1H, H-4′′′′ ), 7.05–703 (m, 2H, H-3′′′′ & H-5′′′′ ), 3.95 (s, 2H, H-2′′′′′ , 3.69–3.67 (m,
2H, He -2′ & He -6′ ), 2.51–2.47 (m, 3H, H-4′ & CH2 -7′′′′′ , 2.38 (s, 3H, CH3 -7′′ ), 2.35–2.31 (m, 2H, Ha -2′ &
Ha -6′ ), 2.14 (s, 3H, CH3 -9′′′′ ), 1.99–1.92 (m, 2H, He -3′ & He -5′ ), 1.83–1.80 (m, 2H, Ha -3′ & Ha -5′ ), 1.06
(t, J = 7.8 Hz, 3H, CH3 -8′′′′ ); EIMS (m/z): 589 [M]+ , 434 [C24 H28 N5 OS]+ , 325 [C18 H19 N3 OS]+ , 264
[C13 H16 N2 O2 S]+ , 234 [C12 H14 N2 OS]. + , 155 [C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ , 91 [C7 H7 ]+ , 82 [C5 H8 N]+ ,
77 [C5 H5 ]+ , 51 [C4 H3 ]+ .
3.7.10. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -(2-
methyl-6-nitrophenyl)acetamide (6g)
Light yellow amorphous solid; yield: 88%; mp: 215–217 ◦ C; molecular formula: C29 H30 N6 O5 S2 ; molecular
weight: 606; IR (KBr, cm−1) υmax : 3435 (N-H stretching), 3064 (C-H stretching of aromatic ring), 2882 (CH2
stretching), 1731 (C=O stretching), 1624 (C=N stretching), 1556 (-NO2 stretching), 1532 (C=C aromatic
stretching), 1345 (-NO2 stretching ), 1335 (-SO2 - stretching); 1 H NMR (600 MHz, CDCl3 , δ / ppm): 9.97
(s, 1H, N-H), 7.70 (dd,J = 1.2, 8.4 Hz, 1H, H-5′′′′ ), 7.58 (d,J = 8.4 Hz, 2H, H-2′′ & H-6′′ ), 7.55–7.54 (m,
3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.45 (dd,J = 1.2, 8.4 Hz, 1H, H-3′′′′ ), 7.29–7.23 (m, 5H, H-3′′ , H-5′′ , H-2′′′
,
H-6′′′ & H-4′′′′ ), 3.97 (s, 2H, H-2′′′′′ , 3.71–3.68 (m, 2H, He -2′ & He -6′ ), 2.54–2.49 (m, 1H, H-4′ ), 2.38 (s, 3H,
CH3 -7′′ ), 2.35–2.32 (m, 2H, Ha -2′ & Ha -6′ ), 2.31 (s, 3H, CH3 -7′′′′ ), 2.02–1.96 (m, 2H, He -3′ & He -5′ ), 1.86–
1.83 (m, 2H, Ha -3′ & Ha -5′ ); EIMS (m/z): 606 [M]+ , 451 [C22 H23 N6 O3 S]+ , 342 [C16 H14 N4 O3 S]+ , 264
[C13 H16 N2 O2 S]+ , 251 [C10 H9 N3 O3 S]. + , 155 [C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ , 91 [C7 H7 ]+ , 82 [C5 H8 N]+ ,
77 [C5 H5 ]+ , 51 [C4 H3 ]+ .
3.7.11. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -(2-
ethylphenyl)acetamide (6h)
Light pink amorphous solid; yield: 89%; mp: 194–196 ◦ C; molecular formula: C30 H33 N5 O3 S2 ; molecular
weight: 575; IR (KBr, cm−1) υmax : 3446 (N-H stretching), 3071 (C-H stretching of aromatic ring), 2879 (CH2
stretching), 1728 (C=O stretching), 1626 (C=N stretching), 1537 (C=C aromatic stretching), 1345 (-SO2 -
stretching); 1 H NMR (600 MHz, CDCl3 , δ / ppm): 9.62 (s, 1H, N-H), 7.86 (d,J = 8.4 Hz, 1H, H-6′′′′ ), 7.59
(d,J = 8.4 Hz, 2H, H-2′′ & H-6′′ ), 7.55–7.51 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.28 (d,J = 8.4 Hz, 2H, H-3′′
& H-5′′ ), 7.18–7.16 (m, 4H, H-2′′′ , H-6′′′ , H-3′′′′ & H-5′′′′ ), 7.08 (dt,J = 1.2, 7.8 Hz, 1H, H-4′′′′ ), 3.94 (s,
2H, H-2′′′′′ , 3.71–3.68 (m, 2H, He -2′ & He -6′ ), 2.58 (q,J = 7.8 Hz, 2H, H-7′′′′ ), 2.52–2.48 (m, 1H, H-4′ ),
2.39 (s, 3H, CH3 -7′′ ), 2.36–2.32 (m, 2H, Ha -2′ & Ha -6′ ), 2.01–1.94 (m, 2H, He -3′ & He -5′ ), 1.83–1.80 (m,
2H, Ha -3′ & Ha -5′ ), 1.10 (t,J = 7.8 Hz, 3H, CH3 -8′′′′ ); EIMS (m/z): 575 [M]+ , 420 [C23 H26 N5 OS]+ ,
413

SATTAR et al./Turk J Chem
311 [C17 H17 N3 OS]+ , 264 [C13 H16 N2 O2 S]+ , 220 [C11 H12 N2 OS]. + , 155 [C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ ,
91 [C7 H7 ]+ , 82 [C5 H8 N]+ , 77 [C5 H5 ]+ , 51 [C4 H3 ]+ .
3.7.12. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -(4-
ethylphenyl)acetamide (6i)
White amorphous solid; yield: 86%; mp: 193–195 ◦ C; molecular formula: C30 H33 N5 O3 S2 ; molecular weight:
575; IR (KBr, cm−1) υmax : 3428 (N-H stretching), 3071 (C-H stretching of aromatic ring), 2874 (CH2 stretching
), 1746 (C=O stretching), 1632 (C=N stretching), 1526 (C=C aromatic stretching), 1345 (-SO2 - stretching);
1 H NMR (600 MHz, CDCl3 , δ / ppm): 10.09 (s, 1H, N-H), 7.59 (d,J = 8.4 Hz, 2H, H-2′′ & H-6′′ ), 7.54–7.49
(m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.49 (d,J = 8.4 Hz, 2H, H-2′′′′ & H-6′′′′ ), 7.28 (d,J = 8.4 Hz, 2H, H-3′′
&
H-5′′ ), 7.18–7.16 (m, 2H, H-2′′′ & H-6′′′ ), 7.12 (d,J = 8.4 Hz, 2H, H-3′′′′ & H-5′′′′ ), 3.83 (s, 2H, H-2′′′′′ , 3.74–
3.72 (m, 2H, He -2′ & He -6′ ), 2.57 (q,J = 7.8 Hz, 2H, H-7′′′′ ), 2.48–2.44 (m, 1H, H-4′ ), 2.39 (s, 3H, CH3 -7′′ ),
2.29–2.25 (m, 2H, Ha -2′ & Ha -6′ ), 2.02–1.95 (m, 2H, He -3′ & He -5′ ), 1.83–1.81 (m, 2H, Ha -3′ & Ha -5′ ),
1.18 (t,J = 7.8 Hz, 3H, CH3 -8′′′′ ); EIMS (m/z): 575 [M]+ , 420 [C23 H26 N5 OS]+ , 311 [C17 H17 N3 OS]+ , 264
[C13 H16 N2 O2 S]+ , 220 [C11 H12 N2 OS]. + , 155 [C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ , 91 [C7 H7 ]+ , 82 [C5 H8 N]+ ,
77 [C5 H5 ]+ , 51 [C4 H3 ]+ .
3.7.13. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -(2-
ethoxyphenyl)acetamide (6j)
Light pink solid; yield: 77%; mp: 211–213 ◦ C; molecular formula: C30 H33 N5 O4 S2 ; molecular weight: 591;
IR (KBr, cm−1) υmax : 3443 (N-H stretching), 3072 (C-H stretching of aromatic ring), 2890 (CH2 stretching),
1742 (C=O stretching), 1633 (C=N stretching), 1532 (C=C aromatic stretching), 1337 (-SO2 - stretching);
1 H NMR (600 MHz, CDCl3 , δ / ppm): 9.66 (s, 1H, N-H), 8.31 (d,J = 7.8 Hz, 1H, H-6′′′′ ), 7.58 (d,J = 7.8
Hz, 2H, H-2′′ & H-6′′ ), 7.52–7.48 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.27 (d,J = 7.8 Hz, 2H, H-3′′ & H-5′′ ),
7.17–7.16 (m, 2H, H-2′′′ & H-6′′′ ), 6.99 (dt,J = 1.2, 7.8 Hz, 1H, H-5′′′′ ), 6.89 (dt,J = 1.2, 7.2 Hz, 1H, H-4′′′′ ),
6.84 (dd,J = 1.2, 8.4 Hz, 1H, H-3′′′′ ), 4.09 (q,J = 7.2 Hz, 2H, H-7′′′′ ), 3.98 (s, 2H, H-2′′′′′ , 3.72–3.69 (m, 2H,
He -2′ & He -6′ ), 2.48–2.44 (m, 1H, H-4′ ), 2.39 (s, 3H, CH3 -7′′ ), 2.32–2.28 (m, 2H, Ha -2′ & Ha -6′ ), 2.01–1.95
(m, 2H, He -3′ & He -5′ ), 1.83–1.79 (m, 2H, Ha -3′ & Ha -5′ ), 1.49 (t,J = 7.2 Hz, 3H, CH3 -8′′′′ ); EIMS (m/z):
591 [M]+ , 436 [C23 H26 N5 O2 S]+ , 327 [C17 H17 N3 O2 S]+ , 264 [C13 H16 N2 O2 S]+ , 236 [C11 H12 N2 O2 S]. +
155 [C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ , 91 [C7 H7 ]+ , 82 [C5 H8 N]+ , 77 [C5 H5 ]+ , 51 [C4 H3 ]+ .
,
3.7.14. 2-(4-Phenyl-5-(4-methylphenylsulfonylpiperidin-4-yl)-4H -1,2,4-triazol-3-ylthio)-N -ben-
zylacetamide (6k)
White amorphous solid; yield: 76%; mp: 179–181 ◦ C; molecular formula: C29 H31 N5 O3 S2 ; molecular weight:
561; IR (KBr, cm−1) υmax : 3439 (N-H stretching), 3068 (C-H stretching of aromatic ring), 2886 (CH2
stretching), 1733 (C=O stretching), 1626 (C=N stretching), 1534 (C=C aromatic stretching), 1337 (-SO2 -
stretching); 1 H NMR (600 MHz, CDCl3 , δ / ppm): 8.06 (s, 1H, N-H), 7.58 (d,J = 8.4 Hz, 2H, H-2′′ & H-6′′ ),
7.53–7.49 (m, 3H, H-3′′′ , H-4′′′ & H-5′′′ ), 7.28–7.25 (m, 4H, H-3′′ , H-5′′ , H-2′′′ & H-6′′′ ), 7.23–7.21 (m, 3H,
H-3′′′′ , H-4′′′′ & H-5′′′′ ), 7.13–7.11 (m, 2H, H-2′′′′ & H-6′′′′ ), 4.42 (d,J = 6.0 Hz, 2H, H-7′′′′ ), 3.77 (s, 2H,
H-2′′′′′ , 3.71–3.68 (m, 2H, He -2′ & He -6′ ), 2.46–2.43 (m, 1H, H-4′ ), 2.39 (s, 3H, CH3 -7′′ ), 2.31–2.27 (m,
414

SATTAR et al./Turk J Chem
2H, Ha -2′ & Ha -6′ ), 1.97–1.91 (m, 2H, He -3′ & He -5′ ), 1.81–1.78 (m, 2H, Ha -3′ & Ha -5′ ); EIMS (m/z):
561 [M]+ , 406 [C22 H24 N5 OS]+ , 297 [C16 H15 N3 OS]+ , 264 [C13 H16 N2 O2 S]+ , 206 [C10 H10 N2 OS]. + , 155
[C7 H7 O2 S]+ , 109 [C6 H9 N2 ]+ , 91 [C7 H7 ]+ , 82 [C5 H8 N]+ , 77 [C5 H5 ]+ , 51 [C4 H3 ]+ .
3.8. Biological screening and docking studies
3.8.1. Antibacterial activity assay
Synthetic compounds 6a–6k were screened for antibacterial activity using 96-well microplates under aseptic
conditions. There is a proportional increase in the absorbance and increase in the bacterial growth. This
relationship was used as a principle in this method.41 Five bacteria were included in the study, three gram-
negative bacteria (Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa) and two gram-positive
bacteria (Staphylococcus aureus and Bacillus subtilis). An appropriate dilution of each was added to the well
(20 µ L well−1). Fresh bacterial culture, maintained overnight and diluted with nutrient broth, was added to
the wells (180 µ L well−1). The volume of each well was maintained at 200 µ L well−1 . The initial absorbance
of culture was maintained in the range of 0.12–0.19. The incubation was processed at 37 ◦ C for 24 h. The lids
of microplates were kept closed during the incubation period. A microplate reader was used to take the initial
and final absorbances. Percent inhibition was calculated using the formula given below.
Control − Test
Inhibition (%) =
× 100
Control
‘Control’ is the absorbance of only the bacterial culture and ‘Test’ is the absorbance of the bacterial culture
containing the test sample. MIC was computed with suitable dilutions (5–30 µ g/well) for each sample.
Ciprofloxacin was used as a reference standard.
3.8.2. LOX inhibition assay
Lipoxygenase inhibition activity of synthesized compounds was evaluated using a previously reported assay.42
The change in absorbance at 234 nm was taken as the index of lipoxygenase inhibition. The positive control
and reference standard used was baicalein.
3.8.3. Molecular docking protocol
For the bioactive conformations, five compounds with the highest anti-LOX potential, 6b, 6c, 6h, 6i, and 6k,
were docked into the active pocket of LOX using the default parameters of the MOE-Dock program. Before
docking of these ligands, ChemDraw Ultra 12.0 was used to draw the structures of synthesized compounds.
These were saved in .mol files and reopened in MOE 2009–2010 software. Energy minimization was preceded
up to 0.05 gradients by using the MMFF94x force field through the default parameter of the MOE energy
minimization algorithm. A database was created in which all the compounds were saved in their 3D structures
in the .mdb file format. The protein molecule of LOX (PDB code: 1IK3) was retrieved from the Protein Data
Bank. All the water molecules were released from the receptor protein and 3D protonation was carried out
using the Protonate 3D option. The energy of the protein molecule was minimized by the default parameters
of the MOE 2009–2010 energy minimization algorithm (gradient: 0.05, force field: MMFF94x). Finally, all
the compounds were docked into the binding pocket of the enzyme. A redocking procedure was also applied
to confirm the validity of the docking protocol.43 After docking with 30 conformations of each compound, the
415

SATTAR et al./Turk J Chem
best 2D images were selected for specific types of interactions and their 3D images were drawn along with their
bond lengths.
3.8.4. Statistical analysis
The results are presented as mean ꢀ SEM after three independent experiments and statistical analysis was
performed with Microsoft Excel 2010. The MIC and IC50 values were calculated using EZ-Fit software (Perrella
Scientific Inc., Amherst, NH, USA).
Acknowledgment
The authors are thankful to the Higher Education Commission of Pakistan for financial assistance regarding
spectral analysis of the synthesized compounds.
References
1. Golkar, Z.; Bagazra, O.; Pace, D. G. J. Infect. Dev. Ctries. 2014, 8, 129-136.
2. Wright, G. D. Can. J. Microbiol. 2014, 60, 147-154.
3. Lee-Do, K.; Hwang, J. U.; Baek, E. H.; Lee, K. O.; Kim, K. J.; Ha, N. J. Arch. Pharm. Res. 2008, 31, 1016-1022.
4. Yl¨a-Herttuala, S.; Rosenfeld, M. E.; Parthasarathy, S.; Sigal, E.; S¨arkioja, T.; Witztum, J. L.; Steinberg, D. J.
Clin. Investig. 1991, 87, 1146-1152.
5. Serhan, C. N. Am. J. Pathol. 2010, 177, 1576-1591.
6. Jupp, J.; Hillier, K.; Elliott, D. H.; Fine, D. R.; Bateman, A. C.; Johnson, P. A.; Cazaly, A. M.; Penrose, J. F.;
Sampson, A. P. Inflamm. Bowel Dis. 2007, 13, 537-546.
7. Wang, D.; Dubois, R. N. Nat. Rev. Cancer. 2010, 10, 181-193.
8. Priya, B. S.; Swamy, B. S. N.; Rangapa, K. S. Bioorg. Med. Chem. 2005, 13, 2623-2628.
9. Joullie, M. M.; Lassen, K. M. Arkivoc 2010, 8, 189-250.
10. Zhao, Y.; Wang, W.; Wang, Q.; Zhang, X.; Ye, L. Biochem. Biophys. Res. Commun. 2012, 418, 647-651.
11. Kerjaschki, D.; Bago-Horvath, Z.; Rudas, M.; Sexl, V.; Schneckenleithner, C.; Wolbank, S.; Bartel, G.; Krieger, S.;
Kalt, R.; Hantusch, B. et al. J. Clin. Investig. 2011, 121, 2000-2012.
12. Wu, Y.; Mao, F.; Zuo, X.; Moussalli, M. J.; Elias, E.; Xu, W.; Shureiqi, I. Cancer Med. 2014, 3, 472-484.
13. Palani, A.; Shapiro, S.; Clader, J. W.; Greenlee, W. J.; Vice, S.; McCombie, S.; Cox, K.; Strizki, J.; Baroudy, B.
M. Bioorg. Med. Chem. Lett. 2003, 13, 709-712.
14. Nezhawy, A. O. H. E.; Radwan, M. A. A.; Gaballah, S. T. Arkivoc 2009, 12, 119-130.
15. Sati, N.; Kumar, S.; Rawat, M. S. M. Ind. J. Chem. 2012, 51B, 318-322.
16. Cocco, M. T.; Congiu, C.; Onnis, V.; Morelli, M.; Cauli, O. Eur. J. Med. Chem. 2003, 38, 513-518.
17. Moffet, R. B.; Rober, A.; Skaletzky, L. L. J. Med. Chem. 1971, 14, 963-968.
18. Siddiqui, N.; Alam, M. S; Ahsan, W. Acta Pharma. 2008, 58, 445-454.
19. Hussein, A. H. M.; Gad-Elkareem, M. A. M.; El-Adasy, A. A. A. M.; Othman, I. M. Phosphorus Sulfur Silicon
Relat. Elem. 2009, 184, 2263-2280.
20. Salem, M. A.; Thabet, H. K. H.; Helal, M. H.; Abdelaal, A. S.; Ammar, Y. A. Chem. Sci. J. 2011, 32, 1-12.
21. Khalid, H.; Aziz-ur-Rehman; Abbasi, M. A.; Khan, K. M.; Ashraf, M.; Ahmad, I.; Ejaz, S. A. Pak. J. Pharm. Sci.
2013, 26, 479-485.
416

SATTAR et al./Turk J Chem
22. Prashanth, M. K.; Revanasiddappa, H. D.; Lokanatha, K. M.; Veeresh, R. B. Bioorg. Med. Chem. Lett. 2012, 22,
7065-7070.
23. Elavarasan, T.; Bhakiaraj, D. P.; Gopalakrishnan, M. ISRN Organic Chemistry 2014, 2014, 120173.
24. Hansch, C.; Sammes, P. G.; Taylor, J. B. Comprehensive Medicinal Chemistry; Pergamon Press: Oxford, UK, 1990.
25. Stokes, S. S.; Albert, R.; Buurman, E. T.; Andrews, B.; Shapiro, A. B.; Green, O. M.; McKenzie, A. R.; Otterbein,
L. R. Bioorg. Med. Chem. Lett. 2012, 22, 7019-7023.
26. Karabasanagouda, T.; Adhikari, A. V.; Shetty, N. S. Eur. J. Med. Chem. 2007, 42, 521-529.
27. Chawla, G.; Kumar, U.; Bawa, S.; Kumar, J. J. Enzy. Inhib. Med. Chem. 2012, 27, 658-665.
28. Ibrahim, D. A. Eur. J. Med. Chem. 2009, 44, 2776-2781.
29. Kritsanida, M.; Mouroutsou, A.; Marakos, P.; Pouli, N.; Papakonstantinou-Garoufalias, S.; Pannecouque, C.;
Witvrouw, M.; De-Clercq, E. Farmaco 2002, 57, 253-257.
30. Barbe, V.; Cruveiller, S.; Kunst, F.; Lenoble, P.; Meurice, G.; Sekowska, A.; Vallenet, D.; Wang, T.; Moszer, I.;
Medigue, C. et al. Microbiology 2009, 155, 1758-1775.
31. Harris, L. G.; Foster, S. J.; Richards, R. G. Eur. Cells Mater. 2002, 4, 39-60.
32. Bhattacharya, S. S.; Das, U.; Choudhury, B. K. Indian J. Med. Res. 2011, 133, 431-433.
33. Vogt, R. L.; Dippold, L. Public Health Rep. 2005, 120, 174-178.
34. Pressler, T.; Bohmova, C.; Conway, S.; Dumcius, S.; Hjelte, L.; Hoiby, N.; Kollberg, H.; Tu¨mmler, B.; Vavrova, V.
J. Cyst. Fibros. 2011, 10, 75-78.
35. Jorgensen, W. L. Science 2004, 303, 1813-1818.
36. Bajorath, J. Nat. Rev. Drug Discov. 2002, 1, 882-894.
37. Langer, T.; Hoffmann, R. D. Curr. Pharm. Des. 2001, 7, 509-527.
38. Kitchen, D. B.; Decornez, H.; Furr, J. R.; Bajorath, J. Nat. Rev. Drug Discov. 2004, 3, 935-949.
39. Gohlke, H.; Klebe, G. Angew. Chem. Int. Ed. Engl. 2002, 41, 2644-2676.
40. Khalid, H.; Aziz-ur-Rehman; Abbasi, M. A.; Malik, A.; Rasool, S.; Nafeesa, K.; Ahmad, I.; Afzal, S. J. Saudi
Chem. Soc. 2016, 20, S615-S623.
41. Kaspady, M.; Narayanaswamy, V. K.; Raju, M.; Rao, G. K. Lett. Drug Des. Discov. 2009, 6, 21-28.
42. Baylac, S.; Racine, P. Int. J. Aromather. 2003, 13, 138-142.
43. Bostro, M. J.; Greenwood, J. R.; Gottfries, J. Mol. Graph. Model. 2003, 21, 449-462.
417