Probing phenylcarbamoylazinane-1,2,4-triazole amides derivatives as lipoxygenase inhibitors along with cytotoxic, ADME and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2020.104525

Hunting small molecules as anti-inflammatory agents/drugs is an expanding and successful approach to treat several inflammatory diseases such as cancer, asthma, arthritis, and psoriasis. Besides other methods, inflammatory diseases can be treated by lipoxygenase inhibitors, which have a profound influence on the development and progression of inflammation. In the present study, a series of new N-alkyl/aralky/aryl derivatives (7a-o) of 2-(4-phenyl-5-(1-phenylcarbamoyl)piperidine-4H-1,2,4-triazol-3-ylthio)acetamide was synthesized and screened for their inhibitory potential against the enzyme 15-lipoxygenase. The simple precursor ethyl piperidine-4-carboxylate (a) was successively converted into phenylcarbamoyl derivative (1), hydrazide (2), semicarbazide (3) and N-phenylated 5-(1-phenylcarbamoyl)piperidine-1,2,4-triazole (4), then in combination with electrophiles (6a-o) through further multistep synthesis, final products (7a-o) were generated. All the synthesized compounds were characterized by FTIR, ¹H, ¹³C NMR spectroscopy, EIMS, and HREIMS spectrometry. Almost all the synthesized compounds showed excellent inhibitory potential against the tested enzyme. Compounds 7c, 7f, 7d, and 7g displayed potent inhibitory potential (IC₅₀ 9.25 ± 0.26 to 21.82 ± 0.35 μM), followed by the compounds 7n, 7h, 7e, 7a, 7b, 7l, and 7o with IC₅₀ values in the range of 24.56 ± 0.45 to 46.91 ± 0.57 μM. Compounds 7c, 7f, 7d exhibited 71.5 to 83.5% cellular viability by MTT assay compared with standard curcumin (76.9%) when assayed at 0.125 mM concentration. In silico ADME studies supported the drug-likeness of most of the molecules. In vitro inhibition studies were substantiated by molecular docking wherein the phenyl group attached to the triazole ring was making a π-δ interaction with Leu607. This work reveals the possibility of a synthetic approach of compounds in relation to lipoxygenase inhibition as potential lead compounds in drug discovery.

Full text of DOI:10.1016/j.bioorg.2020.104525

Bioorganic Chemistry 107 (2021) 104525
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Probing phenylcarbamoylazinane-1,2,4-triazole amides derivatives as
lipoxygenase inhibitors along with cytotoxic, ADME and molecular
docking studies
,
Saima Muzaffara, Wardah Shahida, Naheed Riaza *, Muhammad Saleema,
,
Muhammad Ashrafa *, Aziz-ur-Rehmanb, Bushra Bashira, Ayesha Kaleema, Mariya al-Rashidac,
Bikash Barald, Keshab Bhattaraie, Harald Grosse
a Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
b Department of Chemistry, Government College University Lahore, Lahore 54000, Pakistan
c Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur Road Lahore, Lahore 54600, Pakistan
d
¨
Department of Biochemistry, University of Turku, Tykistokatu 6, Finland
e Department of Pharmaceutical Biology, Auf der Morgenstelle 8, 72076, University of Tuebingen, Tuebingen, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hunting small molecules as anti-inflammatory agents/drugs is an expanding and successful approach to treat
several inflammatory diseases such as cancer, asthma, arthritis, and psoriasis. Besides other methods, inflam-
matory diseases can be treated by lipoxygenase inhibitors, which have a profound influence on the development
and progression of inflammation. In the present study, a series of new N-alkyl/aralky/aryl derivatives (7a-o) of
2-(4-phenyl-5-(1-phenylcarbamoyl)piperidine-4H-1,2,4-triazol-3-ylthio)acetamide was synthesized and screened
for their inhibitory potential against the enzyme 15-lipoxygenase. The simple precursor ethyl piperidine-4-
carboxylate (a) was successively converted into phenylcarbamoyl derivative (1), hydrazide (2), semicarbazide
(3) and N-phenylated 5-(1-phenylcarbamoyl)piperidine-1,2,4-triazole (4), then in combination with electro-
philes (6a-o) through further multistep synthesis, final products (7a-o) were generated. All the synthesized
compounds were characterized by FTIR, 1H, 13C NMR spectroscopy, EIMS, and HREIMS spectrometry. Almost all
the synthesized compounds showed excellent inhibitory potential against the tested enzyme. Compounds 7c, 7f,
7d, and 7g displayed potent inhibitory potential (IC50 9.25 ± 0.26 to 21.82 ± 0.35 µM), followed by the
compounds 7n, 7h, 7e, 7a, 7b, 7l, and 7o with IC50 values in the range of 24.56 ± 0.45 to 46.91 ± 0.57 µM.
Compounds 7c, 7f, 7d exhibited 71.5 to 83.5% cellular viability by MTT assay compared with standard curcumin
(76.9%) when assayed at 0.125 mM concentration. In silico ADME studies supported the drug-likeness of most of
the molecules. In vitro inhibition studies were substantiated by molecular docking wherein the phenyl group
Lipoxygenase inhibition
Cytotoxicity
ADME & Molecular docking studies
Azinanetriazoles
Synthesis
attached to the triazole ring was making a π-δ interaction with Leu607. This work reveals the possibility of a
synthetic approach of compounds in relation to lipoxygenase inhibition as potential lead compounds in drug
discovery.
1. Introduction
pathway. Both these pathways result in the formation of functionally
active metabolites which act in autocrine, paracrine and endocrine
Inflammation is a complex biological defense mechanism elicited in
response to allergens, pathogens and chemicals. When tissue cells are
damaged by these effectors, the cell membranes which is enriched in
phospholipids are hydrolysed by activated phospholipase A2 to yield
arachidonic acid which in turn enters two separate oxidative pathways
called cyclooxygenase (COX) pathway and lipoxygenase (LOX)
manner [1]. COX-I (EC 1.14.99.1) is constitutive enzyme and is involved
in renal homeostasis and platelet aggregation. COX-II (EC 1.14.99.2) is
inducible enzyme that converts arachidonic acid into prostaglandin G2
(PGG2) which further yields thromboxanes, prostacyclin, prostaglandin
E2 and their increased levels mediate the pain, heat, redness, swelling
and the fever [2]. On the other hand, LOX-pathway is characterized by a
* Corresponding authors.
E-mail addresses: nrch322@yaoo.com (N. Riaz), dr.m.ashraf@gmail.com (M. Ashraf).
https://doi.org/10.1016/j.bioorg.2020.104525
Received 2 September 2020; Received in revised form 24 October 2020; Accepted 30 November 2020
Available online 3 December 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
family of enzymes involved to convert arachidonic acid into a variety of
hydroperoxy acids and ultimately end up in another class of functionally
active molecules called leukotrienes (LT) like LTA4, LTB4, LTC4, LTD4,
LTE4 etc. and these leukotrienes are mediators of allergic response and
inflammation [3]. The symptoms include fatigue, sinusitis, cataracts,
pain, heart disease, stroke, asthma, arthritis, colitis etc. Therefore, the
inhibition of COX and LOX pathways by drugs can give some relief to
patients with asthma, pain, allergy and other inflammatory diseases like
cancer. In LOX pathway, lipoxygenases (LOXs, EC 1.13.11.12) are family
of enzymes present across multiple species from plants to mammals [3].
In humans, six family members have been sequenced and characterized.
Seven members have been described in mice. Classification is based on
the product produced due to the specificity of the substrate, linoleic acid
or arachidonic acid. Stereospecific incorporation of molecular oxygen
occurs at carbons 5, 8, 9, 11, 12, and 15 leading to the corresponding
regioisomeric hydroperoxyeicosatetraenoic acids (HPETEs) [4]. This
diversity in the function across this family of enzymes has resulted in
confounding results, which are recently beginning to be clarified. All
these enzymes trigger the synthesis of important biological secondary
metabolites and have been involved in inflammation [3]. For example,
12-LOX is involved in the pathogenesis and complications of diabetes
mellitus [5]. 15-LOX is involved in the promotion of cancer by ampli-
fying peroxisome proliferator-activated receptor-gamma (PPARγ) [6].
Recently, it has been reviewed that 15-LOX inhibitors might be suitable
as chemotherapy agents in the near future especially for the prostrate
cancer [7]. The metal cofactor of these enzymes alters its oxidation state
Fe+2, Fe+3 in their catalytic redox reaction, while inserting oxygen into
the polyunsaturated fatty acid substrate (arachidonic acid or linoleic
acid), synthesizing a large variety of biologically active intermediates
called leukotrienes. Leukotrienes are inflammatory mediators playing a
pathophysiological role in different diseases like asthma, allergic
rhinitis, cardiovascular diseases, Alzheimer’s disease, osteoporosis,
atherosclerosis, Crohn’s disease, multiple sclerosis, osteoarthritis, pso-
riasis, rheumatoid arthritis, diabetes mellitus, carcinoma, bacterial or
viral infections which leads to severe inflammation and certain types of
cancers [8]. Therefore, the regulation of polyunsaturated fatty acid
metabolizing enzymes is a great challenge and many inflammatory and
allergic disorders could be cured by inhibition of family of LOX enzymes.
However, the development of specific inhibitors has been hampered by
homology of LOX family members. Todate no pharmaceutical product
from 15-LOX inhibitors has been approved for therapeutic usage except
Zileuton (N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea) that is mar-
keted for the treatment of asthma [9]. Its side effects include sinusitis,
nausea and pharyngolaryngeal pain. Thus, the search for LOX inhibitors
with minimal side effects is crucial. Among others, the triazoles were
identified as good inhibitors of human 15-LOX and 5-LOX [10].
carbonic anhydrase [16], xanthine oxidoreductase [17], methionine
aminopeptidase-2 [18], lipase, β-hydroxysteroid dehydrogenase type-1,
CYP26A1, adenosine deaminase, thymidine phosphorylase [19], and
LOX [20].
The purpose of the present research work was to explore a new series
of biologically active mercapto N-alkyl/aralkyl/aryl acetamide triazoles
which may assist in designing new synthetic drug leads. In the present
studies, N-substituted derivatives (7a-o) of 1,2,4-triazol-3-ylthio)acet-
amide were synthesized in a series of steps and were subsequently
screened against 15-LOX.
2. Results and discussion
2.1. Chemistry
The present study aimed to synthesize new phenyl carbamoyl
piperidine-1,2,4-triazole amides (7a-o) (Fig. 1) containing a variety of
substituents and to investigate them for their enzyme inhibitory po-
tential against enzyme 15-LOX. All the synthetic protocols are outlined
in Scheme 1. The simplest starting material isonepacotate (a), was
rubbed with phenylisocyanate and then treated with hydrazine hydrate
to get hydrazide 2 (98% yield). The 1H NMR spectrum of 2 showed two
singlets at δ 4.02 (1H, –NH) and 8.90 (2H, –NH2) attributed to –NHNH2
moiety. The reaction of the hydrazide (2) with phenyl isothiocyanate
yielded N-phenyl-4-(2-(phenylcarbamothioyl)hydrazinecarbonyl)piper-
idine-1-carboxamide (3) which was refluxed under alkaline conditions
(10% NaOH), to get an intramolecular cyclized product, 4-(5-mercapto-
4-phenyl-4H-1,2,4-triazol-3-yl)-N-phenylpiperdine-1-carboxamide (4).
These intermediates were identified through 1H NMR analysis, since N-
phenyl nuclei in the spectrum of 3 resonated at δ 7.25 (5H, br s, N-Ph) in
addition to signals of phenyl carbamoyl moiety at δ 7.19 (1H, t, J = 7.5
Hz, H-4ʹ), 7.43 (2H, t, J = 7.5 Hz, H-3ʹ,5ʹ), 7.61 (2H, d, J = 7.5 Hz, H-
2ʹ,6ʹ). Further, the C S carbon nucleus resonated at δ 181.1 in the 13C
–
–
NMR spectrum of 3, whereas, the -SH proton displayed its position at δ
11.2 in the 1H NMR spectrum of compound 4. The absence of –NH signal
and carbonyl carbon resonance in their respective NMR spectra sub-
stantiated the successful conversion of 3 into phenyl substituted 1,2,4-
triazol-3-thiol (4). The N-alkyl/aralky/aryl-2-bromoacetamides (6a-o),
the second precursors of target compounds were synthesized by stirring
alkyl/aralkyl/aryl amines (5a-o) (Table 1) with 2-bromoacetyl bromide
in a basic medium (pH 9–10). The final step was the coupling of com-
pound 4 separately with the precursors 6a-o to get target compounds
7a-o, respectively. This task was accomplished according to the pro-
cedures given in the experimental section.
The compound 7a was synthesized as a colorless amorphous powder
(93% yield; M.P. 98-100˚C). Its IR spectrum displayed the absorption
bands at 3365, 3045, 2941, 1681, 1662, 1619–1544, 1260 cmꢀ 1 rep-
1,2,4-Triazoles, a group of heterocyclic compounds, and their fused
heterocyclic derivatives have acquired much attention in the last few
decades due to their diverse chemistry [11]. They interact at the active
site of a receptor as hydrogen bond acceptor and as a donor, triazole core
acts as an important pharmacophore [12]. Moreover, good stability to
acid-base hydrolysis and oxidative-reductive conditions and resistance
to metabolic degradation are such desirable properties that make it a
medicinal backbone and important area of research in a wide range of
applications in organic and bioorganic synthesis [13], like plant growth
regulators, drug designing, surfactants, agrochemicals and complexa-
tion agents [11]. Literature quest revealed that 1,2,4-triazoles de-
rivatives possess significant antibacterial, antiviral, antifungal,
antituberculosis, antidepressant, antiinflammatory, anticancer, antiox-
idant, antitumor, anticonvulsant [14], molluscicidal, analgesic, antihy-
pertensive, anti-HIV-1, diuretic, anthelmintic, bactericidal, herbicidal,
insecticidal, acaricidal, fungicidal, antileishmanial, anxiolytic, hypo-
lipidemic, antimycotic, antimigraine, antinociceptive, CNS stimulants,
antianxiety and antiplatelet activities [15]. Furthermore, 1,2,4-triazole
derivatives have been reported to exhibit good inhibitory potential
against many enzymes like aromatase, cholinesterase, tumor-associated
– – –
–
–
–
resented the presence of N H, Ar-H, C H, C O, C C, C N, C N
– – –
functional groups, respectively. The 1H NMR spectrum exhibited two
sets of N-ethyl groups at δ 1.10 (3H, t, J = 7.1 Hz, H-2′′′′), 1.22 (3H, t, J
= 7.1 Hz, H-2′′′′′), and 3.35 (2H, q, J = 7.1 Hz, H-1ʹʹʹʹ), 3.45 (2H, q, J =
7.1 Hz, H-1′′′′′), respectively. The signals at δ 1.81 (4H, m, H-3ʹ,5ʹ), 1.86
(1H, m, H-4ʹ) and 2.84 (4H, m, H-2ʹ,6ʹ) supported the existence of azi-
nane ring coupled to 1,2,4 triazole. Methylene of acetamide was deter-
mined by singlet appeared at δ 4.14 (2H, s, H-2′′′). The signal appeared
at δ 7.48 (2H, m), 7.64 (3H, m) was assigned to the phenyl group bonded
to a nitrogen atom of triazole while the presence of two triplets at δ 7.00
(1H, J = 7.5 Hz, H-4ʹʹ), 7.24 (2H, J = 7.5 Hz, H-3ʹʹ,5ʹʹ) together with a
doublet at δ 7.32 (2H, J = 7.5 Hz, H-2ʹʹ,6ʹʹ) in aromatic zone confirmed
the presence of second phenyl group in the molecule. The BB & DEPT
spectra of 13C NMR showed altogether twenty carbon signals for 26
carbons which supported the presence of two methyl, five methylene,
seven methine, and six quaternary carbon atoms. The most downfield
peaks at δ 166.8, 158.7, 156.4, and 151.4 were allotted to two amide
carbonyls and two quaternary carbons of triazole ring, respectively. The
signal at δ 35.4 was attested for methylene carbon atom bonded to S-
2

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
1
1
CH3
N
N
2'''''
N
N
1'''''
H
2'''
2'''
2'''
3
2''''
3''''
2'''
1'''
N
3
5
3'
1'''
N
CH3
5
N
N
N
S
S
S
CH3
3'
1''''
N
N
S
S
1''''
H
N
5'
N1'
O
H
N
5'
N1'
O
5''
5''
5''
1''
1''
1''
5''
1''
7b
O
O
O
O
O
3''
3''
3''
7a
3''
1
1
2''''
4''''
N
N
3
N
N
3
H
N
H
N
2'''
2''''
1'''
1'''
5
5
3'
3'
7''''
6''''
H
N
H
N
5'
5'
N1'
O
N1'
O
4''''
6''''
5''
1''
7d
3''
7c
1
1
N
N
3
N
N
3
CH3
2''''
H
N
H
N
2'''
2''''
1'''
CH3
1'''
5
5
3'
3'
N
S
H
N
H
N
5'
5'
N1'
7f
O
4''''
6''''
4''''
N1'
O
6''''
5''
1''
O
7e
3''
1
1
CH3
N
N
3
N
N
3
H
N
H
N
2'''
2'''
2''''
2''''
1'''
1'''
5
3'
5
3'
N
N
S
S
N
N
S
S
H
N
H
N
5'
4''''
5'
4''''
N1'
O
6''''
N1'
6''''
O
CH3
5''
5''
1''
1''
O
O
O
O
7g
3''
3''
7h
1
1
N
N
3
CH3
2''''
N
N
3
CH3
H
N
H
N
2'''
2'''
2''''
1'''
1'''
CH3
5
5
3'
3'
H
N
H
N
4''''
5'
5'
N1'
O
6''''
4''''
N1'
O
6''''
5''
CH3
5''
1''
1''
7j
7i
3''
3''
1
1
N
N
3
CH3
2''''
N
N
3
CH3
H
N
H
N
2'''
2'''
2''''
1'''
1'''
5
5
3'
3'
N
S
N
S
H
N
H
N
4''''
5'
5'
N1'
6''''
4''''
N1'
O
6''''
O
5''
H3C
5''
1''
1''
O
O
CH3
7l
7k
3''
3''
1
1
N
N
3
N
N
3
H
N
H
2'''
2'''
2''''
2''''
1'''
CH3
CH3
1'''
N
CH3
5
5
3'
3'
N
S
N
S
H
N
H
4''''
5'
5'
4''''
N1'
O
N
1''
N1'
6''''
6''''
O
5''
5''
1''
O
O
CH3
7n
7m
3''
3''
1
N
N
3
H
2'''
2''''
1'''
N
5
3'
N
S
H
N
5'
4''''
N1'
O
6''''
5''
1''
O
7o
3''
Fig. 1. Chemical scaffolds of the synthesized compounds.
atom. The presence of azinane ring was confirmed due to the carbon
signals at δ 43.5, 32.8, and 29.9. The signals for N-ethyl groups reso-
nated at δ 42.5, 40.7, 13.0, and 11.7. The C-peaks at δ 139.6, 128.2,
122.7, and 120.8 were assigned to the phenyl ring of phenylcarbamoyl
moiety, whereas, the signals at δ 132.8, 130.4, 130.0, 127.5 were
attributed to the phenyl moiety attached to triazole nucleus. The mo-
lecular formula C26H32N6O2S of 7a was deduced through HR-EI-MS
which showed the molecular ion [M]+ peak at m/z 442.2330. The
structure elucidation of the remaining compounds 7b-o of the series was
done in the similar fashion and their spectral data is presented in the
3

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
Scheme 1. Protocol for the synthesis of triazole amides (7a-o) from isonepacotate.
Table 1
. Alkyl/aralkyl/aryl-substituted amines.
Compd
R
Compd
f
R
Compd
k
R
a
b
g
l
c
h
i
m
n
d
e
j
o
4

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
experimental section.
and 24.56 ± 0.45 µM, respectively. When comparing the activities of 7g
(21.82 ± 0.35 µM) and 7h (32.54 ± 0.63 µM) bearing the same sub-
stituent, the ethyl group at ortho and para positions, respectively, it may
be deduced that the electron-donating effect of ethyl group attached to
phenyl group is lessened in para isomer as compared to ortho so as
showing somewhat lower inhibitory activity. Meanwhile, comparing the
activity of 7g (21.82 ± 0.35 µM) and 7e (32.61 ± 0.46 µM); unexpect-
edly the activity is decreased, though the methyl group is more electron-
donating than the ethyl group, and indicates that the decrease in size of
the substituent subsides activity.
2.2. Lipoxygenase inhibitory activities and SAR studies
In vitro 15-LOX inhibition of compounds 7a-o was ascertained using
quercetin (IC50 4.86 ± 0.14 µM) as standard; the results are given in
Table 2. All the compounds exhibited significant inhibitory activity
(9.25 ± 0.26 to 56.35 ± 0.84 µM) against the tested enzyme. 7c and 7f
showed the most potent 15-LOX inhibition with IC50 values of 9.25 ±
0.26 and 9.54 ± 0.17 µM, respectively. Even though the activity is cu-
mulative of the entire molecule, so for a minimal SAR was conceded by
analyzing the consequence of different alkyl/aralkyl/aryl groups on the
inhibitory capacity, the way it was the one-off divergent part and all
other parts were same in all molecules. The general structural bits of the
studied acetamides are sported in Fig. 2.
Equipotent activities were manifested by 7a, 7b, 7l and 7o, that are
42.53 ± 0.75, 45.47 ± 0.82, 46.38 ± 0.57 and 46.91 ± 0.57 µM,
respectively. These comparatively lower activities of 7a and 7b
demonstrate that alkyl-substituted compounds have lesser inhibitory
potential as compared to the aryl-substituted but conversely, 7l and 7o
demonstrated the same activities. This may be due to the less electronic
charge or resonance in phenyl ring. Inspection of the dimethyl
substituted derivatives reveales that their inhibitory potential may be on
account of stearic effects. From the discussion of the SAR within the
synthesized derivatives, it can be conferred that both the positions and
type of substituents at the aryl part are playing vital roles in 15-LOX
inhibitory activities.
Comparison of the activities of 7c (9.25 ± 0.26 µM) and 7d (13.64 ±
0.24) evinced that unsaturation resulted in a lesser activity that may be
caused by the waning of electronic charge, while the comparison of the
activity of 7d (13.64 ± 0.24 µM) with 7f (9.54 ± 0.17 µM) revealed that
substitution of methyl group at meta-position on phenyl ring caused the
higher inhibitory potential that may be attributed to mesomaric effect of
the methyl group. It is also noticed that the activity is vaster in the ni-
trogen atom having benzyl structures bound by a methyl linker instead
of the compounds directly attached by the phenyl ring. This philoso-
phises that the unpaired electrons of the N-atom bonded to the phenyl
ring are involved in resonance and that the nitrogen offers little inter-
action to the enzyme by decreasing the potential for protonation. This
methylene bridge effect is supported by the value of diminished inhib-
iting activity displayed by 7o (46.91 ± 0.57 µM). Likewise, 7g and 7n
exhibited potent inhibitory activities with IC50 values as 21.82 ± 0.35
2.3. Cell viability studies
Compounds 7a-o were screened for their cytotoxicity against
mononuclear cells purified from fresh blood and used in MTT assay at
0.125 mM concentration [21] (Table 2). Compound 7c was found to be
the least toxic and showed 83.5 ± 1.3% cell viability followed by 7d that
exhibited 78.4 ± 1.2% viability. The highly toxic compound 7m showed
28.5 ± 1.3% cell viability and was also inactive against the enzyme 15-
LOX. The order of increments in cell toxicity was found as 7c < 7d < 7f
< 7j < 7a < 7b < 7o < 7i. This data reveals that potent inhibitors show
greater cell viability and offer their potential candidature as ‘lead’
compounds against 15-LOX. Compound 7m was inactive as a 15-LOX
inhibitor and was also highly cytotoxic that killed about 73% cells at
0.125 mM concentration in the assayed conditions, even worse than the
standard drugs cyclophosphamide, cisplatin, and curcumin (Table 2).
Table 2
15-LOX inhibitory and cytotoxicity profiles of compounds 7a-o (Mean ± SEM, n
= 3).
Compound
Soybean 15-LOX assay
Cell viability assay
Inhibition (%) at
0.125 mM
IC50 (µM)
Cell viability (%) at
0.125 mM
7a
7b
7c
7d
7e
7f
59.81 ± 1.32
53.46 ± 1.24
76.54 ± 1.13
74.21 ± 1.29
83.25 ± 1.43
85.58 ± 1.21
67.61 ± 1.25
68.48 ± 1.32
58.19 ± 1.46
56.42 ± 1.47
84.51 ± 1.53
56.47 ± 1.36
42.53 ±
0.75
63.2 ± 1.7
62.3 ± 1.6
83.5 ± 1.3
78.4 ± 1.2
49.3 ± 1.4
71.5 ± 1.3
45.7 ± 1.5
52.3 ± 1.7
57.4 ± 1.5
65.5 ± 1.7
38.3 ± 1.3
32.7 ± 1.5
2.4. ADME studies
45.47 ±
0.82
9.25 ±
0.26
The computational methods are routinely used for the prediction of
molecular properties of the drug molecues which are necessary for their
pharmacokinetics studies. These ADME (absorption, distribution,
metabolism, and excretion) properties are predicted by Lipinski’s rule of
five [22]. MedChem Designer software ver 3.0 has been used for these
studies (Table 3). The rule states that poor absorption or permeation of
drug occurs when it violates this rule, that is, it has >5 hydrogen bond
donors, molecular weight is >500, log P is >5 and the sum of N and O
atoms is >10.
13.64 ±
0.24
32.61 ±
0.46
9.54 ±
0.17
7g
7h
7i
21.82 ±
0.35
32.54 ±
0.63
The lipophilicity is determined by logP, its value < 5.0 predicts a
good partition coefficient. S + logP and MlogP are octanol–water dis-
tribution coefficients and based on in silico analysis their values < 5
indicate that these molecules have drug-like properties. Higher logD,
logP values, and lower number of hydrogen bonds predict higher
bioavailability of drugs. TPSA is the topological polar surface area and
should be >140 Å2 which is linked with low blood–brain barrier pene-
tration and poor membrane permeability. Further, the sum of hydrogen
bond donor and acceptor (M_NO + HBDH) should be ≤12. The data
indicates that all these molecules meet the Lipinski’s and Veber’s rules of
good drug-likeness property [22,23].
56.35 ±
0.84
7j
52.62 ±
0.83
7k
7l
51.72 ±
0.72
46.38 ±
0.57
7m
7n
45.42 ± 1.35
64.13 ± 1.43
Inactive
24.56 ±
0.45
28.5 ± 1.3
45.6 ± 1.2
7o
75.56 ± 1.56
92.63 ± 1.24
46.91 ±
0.57
58.4 ± 1.5
Quercetin
4.86 ±
0.14
–
2.5. Molecular docking studies with soybean 15-LOX enzyme
Cyclophosphamide
Cisplatin
–
–
–
–
58.5 ± 1.6
54.7 ± 1.7
76.9 ± 1.5
–
Molecular docking studies were carried out both for soybean 15-LOX
and human 5-LOX. A logical similarity exists amongst the soybean 15-
Curcumin
–
5

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
Fig. 2. General structural features of compounds 7a-o.
binding free energy using HYDE utility of LeadIT software, the confor-
mation with most favorable binding free energy was selected for
detailed binding site interactions (Table 4).
Table 3
ADME properties of compounds 7a-o.
Comp
MlogP
S þ
S þ
M. Wt.
M_NO
T_PSA
HBDH
The docked conformation of compound 7c is shown in Fig. 3. Lys278
was making hydrogen bonds with triazole nitrogen and carbonyl oxygen
logP
logD
7a
7b
7c
7d
7e
7f
3.614
3.410
4.209
3.995
4.263
4.263
4.456
4.456
4.456
4.456
4.456
4.456
4.456
4.456
4.068
2.871
2.648
3.987
3.168
3.704
3.787
4.096
4.188
4.021
4.061
4.087
3.988
4.105
4.162
3.465
2.871
2.648
3.987
3.168
3.704
3.787
4.096
4.188
4.021
4.061
4.087
3.988
4.105
4.162
3.465
492.647
478.620
532.712
526.664
526.664
526.664
540.691
540.691
540.691
540.691
540.691
540.691
540.691
540.691
512.637
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
83.36
92.15
92.15
92.15
92.15
92.15
92.15
92.15
92.15
92.15
92.15
92.15
92.15
92.15
92.15
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
atom. A
π-anion interaction was observed between Asp553 and the
phenyl ring connected to piperidinyl amide moiety. A number of hy-
drophobic interactions were also observed. Tyr275 was making a
π
-sigma interaction with the cyclohexyl group. An alkyl interaction was
observed between Lys278 and the cyclohexyl group. Phe272 and Ala263
7g
7h
7i
were making -alkyl interactions with piperaine moiety and N-
π
substituted phenyl ring. Leu273, Ala777 and Ile857 made π-alkyl in-
teractions with the phenyl ring connected to piperidinyl amide moiety.
The docked conformation of compound 7d is shown in Fig. 4. Lys278
was making a hydrogen bond with the oxygen atom of the carbonyl
7j
7k
7l
7m
7n
7o
group. A π-anion interaction was observed between Asp553 and the
phenyl ring connected to piperidinyl amide moiety. A
π
-sigma interac-
tion was observed between Ala263 and N-substituted phenyl ring. A
number of
π-alkyl interactions were observed. Phe272 was making
LOX and human 5-LOX that become indistinguishable for more than
50% within 10 Å in the active site pocket [24]. Soybean 15-LOX is the
most easily accessible than the human LOX so that its results could be
generalized and extendable to human 5-LOX. That is why the docking of
compounds has been carried out in the active sites of the enzyme only,
15 Å area around the active site.
π
-alkyl interaction with the piperidine moiety. Val256 and Leu563 were
making
π
-alkyl interactions with the phenyl ring attached to the amide
group. Leu273, Ala777 and Ile857 made π-alkyl interactions with the
phenyl ring connected to piperidinyl amide moiety.
The docked conformation of compound 7f is shown in Fig. 5. Lys278
was making hydrogen bonds with the nitrogen atom of the triazole ring.
The crystal structure of soybean 15-LOX enzyme (PDB id: 1rrh, 2.0 Å)
was downloaded from the Protein Data Bank (PDB). All steps of receptor
preparation, and subsequesnt docking studies were carried out using
BioSolveIT’s LeadIT software (LeadIT version 2.3.2; BioSolveIT GmbH,
Sankt Augustin, Germany, 2017, www.biosolveit.de/LeadIT). Out of top
10 docked conformations, each conformation was analyzed for its
Phe272 was making a
π-sulfur interaction with the sulfur atom. A
π
-anion interaction was observed between Asp553 and the phenyl ring
connected to piperidinyl amide moiety. A -sigma interaction was
observed between Ala263 and N-substituted phenyl ring. A number of
-alkyl interactions were observed. His266 was making a -akyl inter-
action with the methyl group of the phenyl ring. Tyr275 was making a
π
π
π
Table 4
Binding free energies and non-bonded interactions of soybean 15-LOX inhibitors.
Ligand
Binding Free
Energy (kJ/
mol)
Hydrophobic interactions
Hydrogen bond interactions
Hydrogen bond
distance (Å)
Other interactions and
distance (Å)
7c
7d
7f
ꢀ 20
ꢀ 18
ꢀ 21
ꢀ 15
Tyr275 (
π
-δ), Lys278 (alkyl), Phe272 (
π
-alkyl),
Lys278 (H-bond donor)
Lys278 (H-bond donor)
2.61 and 2.01
2.51
Asp553 (electrostatic,
Ala263 (
π
-alkyl), Leu273 (
π
-alkyl), Ala777
π-anion)
(
π
-alkyl), Ile857 (
π
-alkyl)
Ala263 (
Leu563 (
π
-δ), Phe272 (
π
-alkyl), Val256 (
π
-alkyl),
Asp553 (electrostatic,
-anion)
π
-alkyl), Leu 273 (
π
-alkyl), Ala777
π
(
π
-alkyl), Ile857 (
Val26 (alkyl), His266 (
Leu273 ( -alkyl), Ala263 (
π
-alkyl)
-alkyl), Tyr275 (
-alkyl)
π
π
-alkyl),
Thr274 (H-bond donor), Tyr275 (H-bond
donor), Lys278 (H-bond donor), Asn556 (H-
bond donor), His266 (H-bond donor)
Lys278 (H-bond donor)
2.1, 1.80 and 2.4,
2.71 and 2.4, 2.26,
2.8
Asp553 (electrostatic,
π
π
π
-anion), Phe272
(
π-Sulfur)
7 g
Tyr275 (
-alkyl), Tyr275 (
Leu273 ( -alkyl), Ala777 (
π
-
π
stacked), Lys278 (alkyl), Phe272
-alkyl), Ala263 ( -alkyl),
-alkyl), Ile857 ( -alkyl)
2.53 and 2.20
Asp553 (electrostatic,
(
π
π
π
π-anion)
π
π
π
6

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
Fig. 3. Docked conformation of 15-LOX inhibitor 7c.
Fig. 4. Docked conformation of 15-LOX inhibitor 7d.
Fig. 5. Docked conformation of 15-LOX inhibitor 7f.
7

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
π
π
-alkyl interaction with the piperidine moiety. Leu273 was making a
-alkyl interaction with methyl substituted phenyl ring. Ala263 was
the carbonyl group next to it is making a hydrogen bond with Asn554.
The nitrogen atoms of the triazole ring made hydrogen bonds with
Gln557. The sulfur atom was in direct contact with the active site’s iron
atom. The other NH group in compound 7d was making a hydrogen
bond with Gln363, while this interaction was absent for compound 7g,
this may in part, explain the difference in activity between the two.
One of the most striking feature observed, while comparing binding
site interactions of soybean 15-LOX and human 5-LOX inhibitors, was
that in case of human 5-LOX inhibition three out of five inhibitors
indicated direct binding with the Fe2+ ion of the enzyme’s active site.
This metal–ligand interaction was not observed in case of soybean 15-
LOX inhibitor. This leads us to speculate that the compounds
described herein can be better inhibitors of 5-LOX over 15-LOX, and thus
may have the potential to be developed further as potent anti-
inflammatory agents.
making a π-alkyl interaction with the N-substituted phenyl ring.
The docked conformation of compound 7g is shown in Fig. 6. Lys278
made hydrogen bonds with the nitrogen atom of the triazole ring.
Asp553 was making a π-anion interaction with the phenyl ring con-
nected to piperidinyl amide moiety. A
observed between Tyr275 and ethyl substituted benzyl ring. Lys278 and
Tyr 275 were making alkyl and -alkyl interactions with the ethyl group
of benzyl ring respectively. A number of hydrophobic -alkyl in-
teractions were observed. Phe272 was making a -alkyl interaction with
the piperidinyl ring. Ala263 was making a -alkyl interaction with the N-
-alkyl
π-π stacked interaction was
π
π
π
π
substituted phenyl ring. Leu273, Ala777 and Ile857 were making
π
interactions with the phenyl ring connected to piperidinyl amide
moiety.
3. Conclusion
2.6. Molecular docking studies with human 5-LOX enzyme
In our endeavor to accomplish the design, synthesis, and character-
ization of new derivatives of 2-(4-phenyl-5-(1-phenylcarbamoyl)piper-
idine-4H-1,2,4-triazol-3-ylthio)acetamide (7a-o) as potential 15-LOX
inhibitors, compounds 7c, 7f, 7d, and 7g are potent inhibitors against
the enzyme and are less toxic with potential drug-likeness properties as
determined by ADME studies. SAR reveals that both the position and
nature of the substituent on the phenyl of acetamides are important for
15-LOX inhibition. In silico studies of revealed that the active molecules
(7c, 7f, 7d, and 7g) can be better inhibitors of 5-LOX over 15-LOX.
Further investigations regarding stability under biological conditions
and pharmacokinetic parameters are required which could be helpful to
design more potent anti-inflammatory agents for therapeutic use.
Arachidonic acid is the substrate of 5-LOX, and is converted to
leukotriene A4 (LTA4) which promotes inflammation. 5-LOX is also
overexpressed in several types of cancers. Hence 5-LOX is an important
drug target for anti-inflammatory and anti-cancer drugs. Therefore, the
most active 15-LOX inhibitors were also docked against human 5-LOX.
The crystal structure of the human 5-LOX enzyme in complex with its
substrate, arachidonic acid, was downloaded from the PDB (PDB id:
3V99, 2.2 Å). Docking of the most active inhibitors was carried out
(Table 5). All compounds were found to bind in the same area as that of
arachidonic acid (Fig. 7).
The docked conformation of the most active inhibitor 7c is shown in
Fig. 8. One of the NH groups was making hydrogen bonds with Ala672,
the carbonyl group adjacent to it was making a hydrogen bond with
Asn554. The other NH group made a hydrogen bond with Gln363. The
nitrogen atom of the triazole ring was making a hydrogen bond with
4. Experimental
4.1. Materials and methods
Gln557. The phenyl group attached to the triazole ring made a
interaction with Leu607.
π-δ
All the chemicals and solvents of analytical grade were procured
from Sigma, Aldrich, and Alfa Aesar, until unless specified. The 1H and
13C NMR spectra were recorded on Bruker instrument operating at 400
and 100 MHz, respectively, using tetramethylsilane as an internal
standard. IR spectra were obtained on Shimadzu 460 FTIR spectrometer
as KBr pellets. The mass spectra were measured on JMSA 500 mass
spectrometer and JMS H × 110 spectrometer with a data system.
Melting points were determined by using Gallen Kemp electrothermal
apparatus.
Similar interactions were observed for 7f, which had comparable
inhibition to 7c. Two different NH groups bind with Asn554 and Gln363,
respectively, by separate hydrogen bonds. The nitrogen atoms of the
triazole ring were making a hydrogen bond with Gln557. Furthermore,
the sulfur atom was in direct contact with the iron atom of the enzyme’s
active site. The triazole ring was also making a
π
-δ interaction with
His367, the toluene ring made a
π-alkyl interaction with Leu607 (Fig. 9).
The docked conformations of the next most active inhibitors 7d and
7g are shown in Figs. 10, 11. They show mostly similar interactions, that
is, one of the NH groups is making a hydrogen bond with Ala672, while
Fig. 6. Docked conformation of 15-LOX inhibitor 7g.
8

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
Table 5
Binding free energies and non-bonded interactions of human 5-LOX inhibitors.
Ligand
Binding Free
Hydrophobic interactions
Hydrogen bond interactions
Hydrogen bond
Other interactions and
distance (Å)
Energy (kJ/mol)
distance (Å)
7c
7d
7f
ꢀ 20
ꢀ 30
ꢀ 31
–23
Leu607 ( -δ), Ala672 (alkyl)
π
Ala672 (H-bond donor), Gln363 (H-bond donor),
Asn554 (H-bond donor), Gln557 (H-bond donor)
Ala672 (H-bond donor), Gln363 (H-bond donor),
Asn554 (H-bond donor)
1.8, 2.09, 2.09, 2.14
–
His367 (
-alkyl)
His367 (
-alkyl)
His367 (
Ala410 (
π
-δ), Ala672
-δ), Leu607
1.72, 2.09, 2.06
2.09, 2.1, 2.05
2.01, 2.09, 2.09
Fe-ligand (2.39)
Fe-ligand (2.37)
Fe-ligand (2.37)
(π
π
Asn554 (H-bond donor), Gln363 (H-bond donor),
Asn557 (H-bond donor)
(π
7g
π
-δ), Ala672 (
π
-δ),
Ala672 (H-bond donor), Asn554 (H-bond donor),
Asn557 (H-bond donor)
π
-alkyl)
Fig. 7. Overlap of arachidonic acid (black) with 5-LOX inhibitors 7c (light pink), 7d (grey), 7f (dark pink), and 7g (orange). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. Docked conformations of 5-LOX inhibitor 7c.
–
–
–
2950 (C H), 1735, 1660 (C O),
–
4.2. Synthesis of ethyl 1-(phenylcarbamoyl)piperidine-4-carboxylate (1)
3396 (N H), 3041
(Ar-H), – –
1
–
–
1615–1560 (Ar-C C), 1260 (C O, C N); H NMR (400 MHz, CDCl3,
Ethyl piperidine-4-carboxylate (a; 0.025 mol, 3.8 mL) was taken in a
watch glass and phenyl isocyanate (0.025 mol, 2.98 mL) was added drop
wise with constant rubbing, which continued (15–20 min) till the
appearance of a single spot on TLC. The physical and spectroscopic data
of compound 1 is given below:
ppm): δ 1.29 (3H, t, J = 7.0 Hz, CH3-CH2-O), 1.89 (4H, m, H-3,5), 3.34
(1H, m, H-4), 3.39 (2H, m, H-2α,6α), 4.13 (2H, q, J = 7.0 Hz, CH3-CH2-
O), 4.34 (2H, br d, J = 13.2 Hz, H-2β,6β), 7.19 (1H, t, J = 7.5 Hz, H-4ʹ),
7.43 (2H, t, J = 7.5 Hz, H-3ʹ,5ʹ), 7.61 (2H, d, J = 7.5 Hz, H-2ʹ,6ʹ); 13
C
NMR (100 MHz, CD3OD): δ 14.1 (CH3), 28.5 (C-3,5), 40.4 (C-4), 46.5 (C-
2,6), 62.1 (CH2), 121.6 (C-2ʹ,6ʹ), 128.0 (C-4ʹ), 128.9 (C-3ʹ,5ʹ), 139.4 (C-
White solid; Yield: 98%; m.p. (105–108 ◦C); IR (KBr disk, cmꢀ 1):
9

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
Fig. 9. Docked conformations of 5-LOX inhibitor 7f.
Fig. 10. Docked conformations of 5-LOX inhibitor 7d.
Fig. 11. Docked conformations of 5-LOX inhibitor 7g.
+
–
1ʹ), 158.1 (C O), 175.8 (COOEt); HR-EI-MS (m/z): 276.1491 [M]
4.3. Synthesis of 4-(hydrazinecarbonyl)-N-phenylpiperidine-1-
carboxamide (2)
–
calculated for C15H20N2O3; 276.1473.
In 100 mL round bottom flask containing ethyl 1-
a
10

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
(phenylcarbamoyl)piperidine-4-carboxylate (1; 0.024 mol, 6.7 g), 30
mL of hydrazine hydrate (80%) was added slowly with constant stirring
for 4–5 h. White fluffy precipitates were formed on cooling which were
filtered, washed with cold distilled water and dried. The physical and
spectroscopic data of 2 is as follows:
1–2 h. The solid product was filtered, washed with cold water, and dried
to get the respective electrophile(s), N-alkyl/aralkyl/aryl substituted-2-
bromoacetamides (6a-o) and their names and physic-chemical constants
are as follows:
◦
White shiny powder; Yield: 98%; m.p. (160–168 C); IR (KBr disk,
4.6.1. 2-Bromo-N,N-diethylacetamide (6a)
Colorless liquid; Pungent smell; Yield: 98%; B.P.: 217-219˚C; Solu-
bility (CH3OH).
cmꢀ 1): 3396, 3364 (N H), 3040 (Ar-H), 2955 (C H), 1680, 1660
–
–
1
–
–
–
–
–
(C O), 1614–1561 (Ar-C C), 1240 (C N); H NMR (400 MHz, CDCl3,
ppm): δ 1.87 (4H, m, H-3,5), 3.33 (1H, m, H-4), 3.39 (2H, m, H-2α,6α),
4.35 (2H, br d, J = 13.2 Hz, H-2β,6β), 7.19 (1H, t, J = 7.5 Hz, H-4ʹ), 7.43
(2H, t, J = 7.5 Hz, H-3ʹ,5ʹ), 7.61 (2H, d, J = 7.5 Hz, H-2ʹ,6ʹ); 13C NMR
(100 MHz, CD3OD): δ 29.4 (C-3,5), 40.1 (C-4), 47.0 (C-2,6), 121.6 (C-
4.6.2. 2-Bromo-N-propylacetamide (6b)
Colorless liquid; Pungent smell; Yield: 99%; B.P.: 201-206˚C; Solu-
bility (CH3OH).
–
–
2ʹ,6ʹ), 128.0 (C-4ʹ), 128.9 (C-3ʹ,5ʹ), 139.4 (C-1ʹ), 158.1 (C O), 178.2
(CONH); HR-EI-MS (m/z): 262.1449 [M]+ calculated for C13H18N4O2;
4.6.3. 2-Bromo-N-(cyclohexylmethyl)acetamide (6c)
White amorphous powder; Odorless Yield: 99%; M.P.: 98-100˚C;
Solubility (CH3OH).
262.1429.
4.4. Synthesis of N-phenyl-4-(2-(phenylcarbamothioyl)
4.6.4. 2-Bromo N-benzylacetamide (6d)
White amorphous powder; Odorless; Yield: 97%; M.P.: 122-124˚C;
Solubility (CH3OH).
hydrazinecarbonyl)piperidine-1-carboxamide (3)
Phenyl isothiocyanate (2.63 mL, 0.022 mol) was added drop wise
with stirring to a 100 mL round bottom flask already furnished with
compound 2 (5.9 g, 0.022 mol) dissolved in 30 mL of methanol. The
mixture was refluxed with stirring for 3–4 h and the precipitates were
formed on cooling which were filtered, washed and dried. Below is the
physical and spectroscopic data of 3:
4.6.5. 2-Bromo-N-(o-tolyl)acetamide (6e)
White amorphous powder; Odorless; Yield: 99%; M.P.: 107-110˚C;
Solubility (CH3OH).
White powder; Yield: 98%; m.p. (180–183 ◦C); IR (KBr disk, cmꢀ 1):
4.6.6. 2-Bromo-N-(m-tolyl)acetamide (6f)
Pink amorphous powder; Odorless; Yield: 99%; M.P.: 104-106˚C;
Solubility (CH3OH).
–
–
–
3364 (N H), 3033 (Ar-H), 2939 (C H), 1678, 1660 (C O),
–
1
–
–
–
1617–1550 (Ar-C C), 1215 (C N); H NMR (400 MHz, CDCl3, ppm): δ
1.87 (4H, m, H-3,5), 3.34 (1H, m, H-4), 3.39 (2H, m, H-2α,6α), 4.35 (2H,
4.6.7. 2-Bromo-N-(2-ethylphenyl)acetamide (6g)
White amorphous powder; Odorless; Yield: 99%; M.P.: 90-91˚C; Sol-
ubility (CH3OH).
br d, J = 13.2 Hz, H-2β,6β), 7.19 (1H, t, J = 7.5 Hz, H-4ʹ), 7.25 (5H, br s,
N-Ph), 7.43 (2H, t, J = 7.5 Hz, H-3ʹ,5ʹ), 7.61 (2H, d, J = 7.5 Hz, H-2ʹ,6ʹ);
13C NMR (100 MHz, CD3OD): δ 29.4 (C-3,5), 40.7 (C-4), 47.0 (C-2,6),
121.6 (C-2ʹ,6ʹ), 128.0 (C-4ʹ), 128.9 (C-3ʹ,5ʹ), 139.4 (C-1ʹ), N-Ph [126.6
–
–
4.6.8. 2-Bromo-N-(4-ethylphenyl)acetamide (6h)
Yellow amorphous powder; Odorless; Yield: 92%; M.P.: 118-119˚C;
Solubility (CH3OH).
(C-2,6), 128.4 (C-4), 129.0 (C-3,5), 138.5 (C-1)], 158.1 (C O), 173.2
+
–
–
(CO), 181.1 (C S); HR-EI-MS (m/z): 397.1593 [M] calculated for
C
20H23N5O2S; 397.1572.
4.6.9. 2-Bromo-N-(2,3-dimethylphenyl)acetamide (6i)
White amorphous powder; Odorless; Yield: 99%; M.P.: 123-125 ˚C;
Solubility (CH3OH).
4.5. Synthesis of 4-(5-mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)-N-
phenylpiperdine-1-carboxamide (4)
Compound 3 (0.021 mol, 8.5 g) was dissolved in 10% aqueous NaOH
(30 mL) in a 100 mL round bottom flask and was refluxed for 4 h. The
reaction mixture was transferred into ice-cold water, acidified to pH 5–6
by using dilute HCl, precipitates were formed which were filtered,
washed, and dried. The physical and spectroscopic data of 4 is as
follows:
4.6.10. 2-Bromo-N-(2,4-dimethylphenyl)acetamide (6j)
Off white amorphous powder; Odorless; 99%; M.P.: 150-153˚C;
Solubility (CH3OH).
4.6.11. 2-Bromo-N-(2,5-dimethylphenyl)acetamide (6k)
White amorphous powder; Odorless; 99%; M.P.: 138-140˚C; Solubi-
lity (CH3OH).
White powder; yield: 95%; m.p. (262–266 ◦C); IR (KBr disk, cmꢀ 1):
–
–
–
H), 1661 (C O), 1619–1560 (Ar-
3369 (N
– –H), 3031 (Ar-H), 2930 (C
–
1
–
C
C, C N), 1244 (C N); H NMR (400 MHz, CDCl3, ppm): δ 1.92 (4H,
m–, H-3ʹ,–5ʹ), 3.35 (1H, m, H-4ʹ), 3.39 (4H, m, H-2aʹ,6aʹ), 4.33 (2H, br d, J
4.6.12. 2-Bromo-N-(2,6-dimethylphenyl)acetamide (6l)
White amorphous powder; Odorless; Yield: 99%; M.P.: 140-143 ˚C;
Solubility (CH3OH).
= 13.2 Hz, H-2bʹ,6bʹ), 7.19 (1H, t, J = 7.5 Hz, H-4ʹʹ), 7.43 (2H, t, J = 7.5
Hz, H-3ʹʹ,5ʹʹ), 7.61 (2H, d, J = 7.5 Hz, H-2ʹʹ,6ʹʹ), N-Ph [7.41 (2H, m, H-
2,6), 7.60 (3H, m, H-3–5)]; 13C NMR (100 MHz, CD3OD): δ 29.4 (C-
3ʹ,5ʹ), 40.7 (C-4ʹ), 47.0 (C-2ʹ,6ʹ), 121.6 (C-2ʹʹ,6ʹʹ), 128.0 (C-4ʹʹ), 128.9 (C-
3ʹʹ,5ʹʹ), 139.4 (C-1ʹʹ), N-Ph [126.6 (C-2,6), 128.4 (C-4), 129.0 (C-2,5),
4.6.13. 2-Bromo-N-(3,4-dimethylphenyl)acetamide (6m)
Yellow amorphous powder; Odorless; 93%; M.P.: 127-129 ˚C; Solu-
bility (CH3OH).
–
–
–
–
138.5 (C-1)], 158.1 (C O), 173.2 (C O), 181.1 (C S). HR-EI-MS (m/
–
–
z): 379.1451 [M]+ calculated for C20H21N5OS; 379.1466.
4.6.14. 2-Bromo-N-(3,5-dimethylphenyl)acetamide (6n)
Yellow amorphous powder; Odorless; 90%; M.P.: 124-126 ˚C; Solu-
bility (CH3OH).
4.6. General procedure for the synthesis of compounds 6a-o
Computed amounts of alkyl/aralkyl/aryl amines (5a-o, 0.01 mol)
were added separately in a quick fit Erlenmeyer flask already having
20% aqueous Na2CO3 solution to set the pH 9–10. The bromoacetyl
bromide (0.01 mol) was added drop-wise into the flask over 2–5 min
with continuous vigorous shaking till the formation of precipitates fol-
lowed by further stirring to get homogenous precipitates over a period of
4.6.15. 2-Bromo-N-phenylacetamide (6o)
White amorphous powder; Odorless; Yield: 99%; M.P.: 112-115 ˚C;
Solubility (CH3OH).
11

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
–
– –
νmax: 3355 (N H), 3039 (Ar-H), 2957 (C H), 1682, 1661 (C O),
–
4.7. General method for the synthesis of compounds 7a-o
1
– –
– –
–
1622–1540 (Ar-C C, C N), 1261 (C N); H NMR (400 MHz, CD3OD):
In a 50 mL round bottom flask, 0.2 g (0.5 mmol) of compound 4 was
dissolved in methanol (5 mL) and 30 mL ethanolic KOH (0.5 mmol) was
added and the mixture was stirred for 30 min at room temperature. The
electrophiles (6a-o) separately were added slowly to the mixture and set
to reflux for 4–5 h. An excess amount of cold distilled water was added
and the precipitates of the final products, 7a-o, were formed separately,
which were filtered, washed, and dried.
δ 1.82 (4H, m, H-3ʹ,5ʹ), 2.75 (1H, m, H-4ʹ), 2.79 (2H, m, H-2αʹ,6αʹ), 3.87
(2H, s, H-2′′′), 4.13 (2H, br d, J = 13.2 Hz, H-2βʹ,6βʹ), 4.36 (2H, s, H-
7′′′′), 6.98 (1H, t, J = 8.0 Hz, H-4ʹʹ), 7.22 (5H, m, H-2ʹʹʹʹ-6ʹʹʹʹ), 7.24 (2H, t,
J = 8.0 Hz, H-3ʹʹ,5ʹʹ), 7.32 (2H, d, J = 8.0 Hz, H-2ʹʹ,6ʹʹ), N-Ph [7.34 (2H,
m, H-2,6), 7.60 (3H, m, H-3–5)]; 13C NMR (100 MHz, CDCl3 + CD3OD):
δ 30.6 (C-3ʹ,5ʹ), 33.6 (C-4ʹ), 36.1 (C-2ʹʹʹ), 44.3 (C-7ʹʹʹʹ), 44.4 (C-2ʹ,6ʹ),
121.7 (C-2ʹʹ,6ʹʹ), 123.7 (C-4ʹʹ), 127.9 (C-4ʹʹʹʹ), 128.0 (C-3ʹʹ,5ʹʹ), 129.1 (C-
2ʹʹʹʹ,3ʹʹʹʹ,5ʹʹʹʹ,6ʹʹʹʹ), N-Ph [127.9 (C-2,6), 131.0 (C-3,5), 131.5 (C-4), 133.2
–
–
4.7.1. 4-(5-(2-(Diethylamino)-2-oxoethylthio)-4-phenyl-4H-1,2,4-triazol-
(C-1)], 138.6 (C-2ʹʹʹʹ), 140.1 (C-1ʹʹ), 152.0 (C-3), 157.1 (C O), 159.0 (C-
3-yl)-N-phenylpiperidine-1-carboxamide (7a)
5), 169.1 (C-1ʹʹʹ); HR-EI-MS (m/z): 526.2169 [M]+ calculated for
White amorphous powder; Yield: 93%; M.P.: 98-100˚C; IR (KBr) νmax
:
C29H30N6O2S; 526.2150.
–
–
–
3365 (N H), 3045 (Ar-H), 2941 (C H), 1681, 1662 (C O),
–
1
– –
–
1619–1544 (Ar-C C, C N), 1260 (C N); H NMR (400 MHz, CD3OD):
4.7.5. 4-(5-(2-Oxo-2-(2-methylphenylamino)ethylthio)-4-phenyl-4H-
1,2,4-triazol-3-yl)-N-phenylpiperidine-1-carboxamide (7e)
δ 1.10 (2H, t, J =–7.1 H–z, H-2ʹʹʹʹ), 1.22 (2H, t, J = 7.1 Hz, H-2ʹʹʹʹʹ), 1.81
(4H, m, H-3ʹ,5ʹ), 2.80 (1H, m, H-4ʹ), 2.84 (2H, m, H-2
α
ʹ,6
α
ʹ), 3.35 (2H, q,
White amorphous powder; Yield: 88%; M.P.: 176-180˚C; IR (KBr)
–
– –
νmax: 3354 (N H), 3038 (Ar-H), 2951 (C H), 1680, 1664 (C O),
–
J = 7.1 Hz, H-1ʹʹʹʹ), 3.45 (2H, q, J = 7.1 Hz, H-1ʹʹʹʹʹ), 4.15 (2H, br d, J =
13.2 Hz, H-2βʹ,6βʹ), 4.17 (2H, s, H-2′′′), 7.00 (1H, t, J = 7.5 Hz, H-4ʹʹ),
7.24 (2H, t, J = 8.0 Hz, H-3ʹʹ,5ʹʹ), 7.32 (2H, d, J = 7.5 Hz, H-2ʹʹ,6ʹʹ), N-Ph
[7.48 (2H, m, H-2,6), 7.64 (3H, m, H-3–5)]; 13C NMR (100 MHz,
CD3OD): δ 11.7 (C-2′′′′), 13.0 (C-2′′′′′), 29.9 (C-3ʹ,5ʹ), 32.8 (C-4ʹ), 35.4
(C-2ʹʹʹ), 40.7 (C-1′′′′), 42.5 (C-1′′′′′), 43.5 (C-2ʹ,6ʹ), 120.8 (C-2ʹʹ,6ʹʹ), 122.7
1
– –
– –
–
1621–1530 (Ar-C C, C N), 1260 (C N); H NMR (400 MHz, CD3OD):
δ 1.82 (4H, m, H-3ʹ,5ʹ), 2.76 (1H, m, H-4ʹ), 2.22 (3H, s, CH3), 2.81 (2H,
m, H-2
α
ʹ,6
α
ʹ), 4.02 (2H, s, H-2′′′), 4.15 (2H, br d, J = 13.2 Hz, H-2βʹ,6βʹ),
6.98 (1H, t, J = 8.5 Hz, H-4ʹʹ), 7.06 (1H, d, J = 8.5 Hz, H-6ʹʹʹʹ), 7.16 (1H,
t, J = 8.5 Hz, H-5ʹʹʹʹ), 7.19 (1H, t, J = 8.5 Hz, H-4ʹʹʹʹ), 7.22 (2H, t, J = 8.5
Hz, H-3ʹʹ,5ʹʹ), 7.30 (2H, d, J = 8.5 Hz, H-2ʹʹ,6ʹʹ), 7.49 (1H, t, J = 8.5 Hz,
H-3ʹʹʹʹ), N-Ph [7.37 (2H, m, H-2,6), 7.61 (3H, m, H-3–5)]; 13C NMR (100
MHz, CDCl3 + CD3OD): δ 18.2 (CH3), 30.7 (C-3ʹ,5ʹ), 33.7 (C-4ʹ), 36.1 (C-
2ʹʹʹ), 44.5 (C-2ʹ,6ʹ), 121.8 (C-2ʹʹ,6ʹʹ), 123.8 (C-4ʹʹ), 125.2 (C-6ʹʹʹʹ), 126.8
(C-3′′′′), 127.0 (C-4ʹʹʹʹ), 129.2 (C-3ʹʹ,5ʹʹ), 131.2 (C-5ʹʹʹʹ), 132.4 (C-2ʹʹʹʹ), N-
Ph [128.1 (C-2,6), 131.1 (C-3,5), 131.6 (C-4), 133.3 (C-1)], 136.1 (C-
(C-4ʹʹ), 128.2 (C-3ʹʹ,5ʹʹ), N-Ph [127.5 (C-2,6), 130.0 (C-3,5), 130.4 (C-4),
–
132.8 (C-1)], 139.6 (C-1ʹʹ), 151.4 (C-3), 156.4 (C O), 158.7 (C-5),
–
166.8 (C-1ʹʹʹ); HR-EI-MS (m/z): 442.2330 [M]+ calculated for
C
26H32N6O2S; 442.2307.
4.7.2. 4-(5-(2-Oxo-2-(propylamino)ethylthio)-4-phenyl-4H-1,2,4-triazol-
3-yl)-N-phenylpiperidine-1-carboxamide (7b)
–
–
1ʹʹʹʹ), 140.2 (C-1ʹʹ), 152.6 (C-3), 157.2 (C O), 159.7 (C-5), 168.2 (C-1ʹʹʹ);
White amorphous powder; Yield: 95%; M.P.: 115-120˚C; IR (KBr)
HR-EI-MS (m/z): 526.2170 [M]+ calculated for C29H30N6O2S; 526.2150.
–
–
–
–
νmax: 3360 (N H), 3035 (Ar-H), 2951 (C H), 1680, 1660 (C O),
1
– –
–
1620–1540 (Ar-C C, C N), 1261 (C N); H NMR (400 MHz, CD3OD):
– –
4.7.6. 4-(5-(2-Oxo-2-(3-methylphenylamino)ethylthio)-4-phenyl-4H-
1,2,4-triazol-3-yl)-N-phenylpiperidine-1-carboxamide (7f)
δ 0.89 (3H, t, J = 7.0 Hz, H-3ʹʹʹʹ), 1.50 (2H, sexet, J = 7.0 Hz, H-2ʹʹʹʹ), 1.80
(4H, m, H-3ʹ,5ʹ), 2.81 (1H, m, H-4ʹ), 2.84 (2H, m, H-2
αʹ,6
α
ʹ), 3.13 (2H, t,
White amorphous powder; Yield: 89%; M.P.: 127-130˚C; IR (KBr)
J = 7.0 Hz, H-1ʹʹʹʹ), 3.83 (2H, s, H-2′′′), 4.15 (2H, br d, J = 13.2 Hz, H-
2βʹ,6βʹ), 7.00 (1H, t, J = 8.0 Hz, H-4ʹʹ), 7.24 (2H, t, J = 8.0 Hz, H-3ʹʹ,5ʹʹ),
7.32 (2H, d, J = 8.0 Hz, H-2ʹʹ,6ʹʹ), N-Ph [7.46 (2H, m, H-2,6), 7.64 (3H,
m, H-3–5)]; 13C NMR (100 MHz, CD3OD): δ 11.7 (C-3ʹʹʹʹ), 23.5 (C-2ʹʹʹʹ),
31.2 (C-3ʹ,5ʹ), 34.2 (C-4ʹ), 36.7 (C-2ʹʹʹ), 42.7 (C-1ʹʹʹʹ), 45.0 (C-2ʹ,6ʹ), 122.2
νmax: 3350 (N H), 3035 (Ar-H), 2950 (C H), 1681, 1661 (C O),
–
–
–
–
1
– –
–
1619–1525 (Ar-C C, C N), 1262 (C N); H NMR (400 MHz, CD3OD):
– –
δ 1.85 (4H, m, H-3ʹ,5ʹ), 2.31 (3H, s, CH3), 2.79 (1H, m, H-4ʹ), 2.83 (2H,
m, H-2
α
ʹ,6
α
ʹ), 3.73 (2H, s, H-2′′′), 4.16 (2H, br d, J = 13.2 Hz, H-2βʹ,6βʹ),
6.92 (1H, d, J = 7.5 Hz, H-6ʹʹʹʹ), 6.99 (1H, t, J = 7.5 Hz, H-4ʹʹ), 7.17 (1H,
t, J = 7.5 Hz, H-5ʹʹʹʹ), 7.23 (2H, t, J = 8.0 Hz, H-3ʹʹ,5ʹʹ), 7.31 (2H, d, J =
7.5 Hz, H-2ʹʹ,6ʹʹ), 7.33 (1H, d, J = 7.5 Hz, H-4ʹʹʹʹ), 7.35 (1H, s, H-2ʹʹʹʹ), N-
Ph [7.43 (2H, m, H-2,6), 7.60 (3H, m, H-3–5)]; 13C NMR (100 MHz,
CDCl3 + CD3OD): δ 21.6 (CH3), 31.0 (C-3ʹ,5ʹ), 33.2 (C-4ʹ), 34.1 (C-2ʹʹʹ),
44.8 (C-2ʹ,6ʹ), 118.1 (C-2ʹʹʹʹ), 121.5 (C-6ʹʹʹʹ), 122.1 (C-2ʹʹ,6ʹʹ), 124.0 (C-
4ʹʹ), 126.1 (C-4ʹʹʹʹ), 129.4 (C-3ʹʹ,5ʹʹ), 129.6 (C-5ʹʹʹʹ), N-Ph [128.6 (C-
2,6),131.3 (C3,5), 131.7 (C-4), 133.9 (C-1)], 139.2 (C-3ʹʹʹʹ), 139.7 (C-
(C-2ʹʹ,6ʹʹ), 124.1 (C-4ʹʹ), 129.5 (C-3ʹʹ,5ʹʹ), N-Ph [128.8 (C-2,6), 131.4 (C-
–
–
3,5), 131.8 (C-4), 134.1 (C-1)], 140.9 (C-1ʹʹ), 152.6 (C-3), 157.8 (C O),
160.2 (C-5), 169.8 (C-1ʹʹʹ); HR-EI-MS (m/z): 478.2169 [M]+ calculated
for C25H30N6O2S; 478.2150.
4.7.3. 4-(5-(2-(Cyclohexylamino)-2-oxoethylthio)-4-phenyl-4H-1,2,4-
triazol-3-yl)-N-phenylpiperidine-1-carboxamide (7c)
–
–
White amorphous powder; Yield: 93%; M.P.: 210-220˚C; IR (KBr)
1ʹʹʹʹ), 140.7 (C-1ʹʹ), 152.5 (C-3), 157.6 (C O), 160.1 (C-5), 167.8 (C-1ʹʹʹ);
HR-EI-MS (m/z): 526.2170 [M]+ calculated for C29H30N6O2S; 526.2150.
–
–
–
–
νmax: 3351 (N H), 3033 (Ar-H), 2955 (C H), 1681, 1665 (C O),
1
– –
–
1625–1540 (Ar-C C, C N), 1260 (C N); H NMR (400 MHz, CD3OD):
– –
δ 1.19 (2H, m, H-3ʹʹʹʹ,5ʹʹʹʹ), 1.31 (4H, m, H-2ʹʹʹʹ,6ʹʹʹʹ), 1.34 (2H, m, H-4ʹʹʹʹ),
4.7.7. 4-(5-(2-(2-Ethylphenylamino)-2-oxoethylthio)-4-phenyl-4H-1,2,4-
1.59 (1H, m, H-1ʹʹʹʹ), 1.70 (2H, m, H-3ʹʹʹʹ,5ʹʹʹʹ), 1.79 (4H, m, H-3ʹ,5ʹ), 2.76
triazol-3-yl)-N-phenylpiperidine-1-carboxamide (7g)
(1H, m, H-4ʹ), 2.80 (2H, m, H-2
αʹ,6
α
ʹ), 3.61 (2H, t, J = 7.0 Hz, H-1ʹʹʹʹ),
White amorphous powder; Yield: 91%; M.P.: 95-105˚C; IR (KBr) νmax:
3.77 (2H, s, H-2′′′), 4.15 (2H, br d, J = 13.2 Hz, H-2βʹ,6βʹ), 6.98 (1H, t, J
= 8.0 Hz, H-4ʹʹ), 7.22 (2H, t, J = 8.0 Hz, H-3ʹʹ,5ʹʹ), 7.29 (2H, d, J = 8.0
Hz, H-2ʹʹ,6ʹʹ), N-Ph [7.33 (2H, m, H-2,6), 7.61 (3H, m, H-3–5)]; 13C NMR
(100 MHz, CDCl3 + CD3OD): δ 25.3 (C-2ʹʹʹʹ,6ʹʹʹʹ), 26.1 (C-4ʹʹʹʹ), 30.1 (C-
3ʹʹʹʹ,5ʹʹʹʹ), 33.0 (C-1ʹʹʹʹ), 33.0 (C-3ʹ,5ʹ), 33.6 (C-4ʹ), 36.4 (C-2ʹʹʹ), 44.4 (C-
2ʹ,6ʹ), 121.7 (C-2ʹʹ,6ʹʹ), 123.7 (C-4ʹʹ), 129.2 (C-3ʹʹ,5ʹʹ), N-Ph [128.1 (C-
3352 (N H), 3031 (Ar-H), 2951 (C H), 1680, 1663 (C O),
–
–
–
–
1
– –
–
1618–1519 (Ar-C C, C N), 1260 (C N); H NMR (400 MHz, CD3OD):
– –
δ 1.13 (3H, t, J = 7.2 Hz, CH3-CH2), 1.84 (4H, m, H-3ʹ,5ʹ), 2.59 (2H, q, J
= 7.2 Hz, CH3-CH2), 2.79 (1H, m, H-4ʹ), 2.84 (2H, m, H-2αʹ,6αʹ), 4.06
(2H, s, H-2′′′), 4.15 (2H, br d, J = 13.2 Hz, H-2βʹ,6βʹ), 6.99 (1H, t, J = 7.5
Hz, H-4ʹʹ), 7.17 (2H, m, H-4ʹʹʹʹ,6ʹʹʹʹ), 7.23 (2H, t, J = 8.0 Hz, H-3ʹʹ,5ʹʹ),
7.25 (1H, t, J = 8.0 Hz, H-5ʹʹʹʹ), 7.32 (2H, d, J = 8.0 Hz, H-2ʹʹ,6ʹʹ), 7.38
(1H, d, J = 7.5 Hz, H-3ʹʹʹʹ), N-Ph [7.46 (2H, m, H-2,6), 7.63 (3H, m, H-
3–5)]; 13C NMR (100 MHz, CD3OD): δ 14.9 (CH3), 26.2 (CH2), 31.2 (C-
3ʹ,5ʹ), 34.2 (C-4ʹ), 37.0 (C-2ʹʹʹ), 44.9 (C-2ʹ,6ʹ), 122.2 (C-2ʹʹ,6ʹʹ), 124.1 (C-
4ʹʹ), 127.0 (C-6ʹʹʹʹ), 127.3 (C-4ʹʹʹʹ), 127.8 (C-3ʹʹʹʹ), 129.9 (C-5ʹʹʹʹ), 129.5 (C-
3ʹʹ,5ʹʹ), N-Ph [128.8 (C-2,6), 131.4 (C-3,5), 131.9 (C-4), 134.0 (C-1)],
2,6), 131.0 (C-3,5), 131.5 (C-4), 133.3 (C-1)], 140.2 (C-1ʹʹ), 152.4 (C-3),
–
–
157.2 (C O), 159.4 (C-5), 168.1 (C-1ʹʹʹ); HR-EI-MS (m/z): 518.2483
[M]+ calculated for C29H36N6O2S; 518.2463.
4.7.4. 4-(5-(2-(Benzylamino)-2-oxoethylthio)-4-phenyl-4H-1,2,4-triazol-
3-yl)-N-phenylpiperidine-1-carboxamide (7d)
–
White amorphous powder; Yield: 93%; M.P.: 195-200˚C; IR (KBr)
135.9 (C-2ʹʹʹʹ), 139.8 (C-1ʹʹʹʹ), 140.9 (C-1ʹʹ), 152.8 (C-3), 157.7 (C O),
–
12

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
160.4 (C-5), 169.2 (C-1ʹʹʹ); HR-EI-MS (m/z): 540. 2325 [M]+ calculated
2βʹ,6βʹ), 6.90 (1H, d, J = 7.6 Hz, H-4ʹʹʹʹ), 6.98 (1H, t, J = 8.0 Hz, H-4ʹʹ),
7.05 (1H, d, J = 7.6 Hz, H-3ʹʹʹʹ), 7.22 (2H, t, J = 8.0 Hz, H-3ʹʹ,5ʹʹ), 7.30
(2H, d, J = 8.0 Hz, H-2ʹʹ,6ʹʹ), 7.32 (1H, s, H-6ʹʹʹʹ), N-Ph [7.37 (2H, m, H-
2,6), 7.61 (3H, m, H-3,5)]; 13C NMR (100 MHz, CDCl3 + CD3OD): δ 17.7
(CH3), 21.1 (CH3), 30.7 (C-3ʹ,5ʹ), 33.7 (C-4ʹ), 36.7 (C-1ʹʹʹ), 44.5 (C-2ʹ,6ʹ),
121.7 (C-2ʹʹ,6ʹʹ), 123.7 (C-4ʹʹ), 125.7 (C-6ʹʹʹʹ), 127.5 (C-4ʹʹʹʹ), 129.2 (C-
3ʹʹ,5ʹʹ), 131.0 (C-3ʹʹʹʹ), 133.2 (C-2ʹʹʹʹ), N-Ph [128.1 (C2,6), 131.1 (C-3,5),
131.3 (C-4), 133.3 (C-1)], 135.8 (C-5ʹʹʹʹ), 136.7 (C-1ʹʹʹʹ), 140.2 (C-1ʹʹ),
for C30H32N6O2S; 540.2307.
4.7.8. 4-(5-(2-(4-Ethylphenylamino)-2-oxoethylthio)-4-phenyl-4H-1,2,4-
triazol-3-yl)-N-phenylpiperidine-1-carboxamide (7h)
White amorphous powder; Yield: 96%; M.P.: 100-104˚C; IR (KBr)
–
–
–
νmax: 3351 (N H), 3036 (Ar-H), 2961 (C H), 1683, 1661 (C O),
–
1
– –
–
1617–1517 (Ar-C C, C N), 1261 (C N); H NMR (400 MHz, CD3OD):
– –
′′′
–
δ 1.18 (3H. t, J = 7.5 Hz, CH3-CH2), 1.80 (4H, m, H-3ʹ,5ʹ), 2.58 (2H, q, J
152.6 (C-3), 157.2 (C O), 159.7 (C-5), 168.1 (C-1 ); HR-EI-MS (m/z):
–
= 7.5 Hz, CH3-CH2), 2.74 (1H, m, H-4ʹ), 2.79 (2H, m, H-2
αʹ,6
α
ʹ), 3.96
540.2325 [M]+ calculated for C30H32N6O2S; 540.2307.
(2H, s, H-2′′′), 4.13 (2H, br d, J = 13.2 Hz, H-2βʹ,6βʹ), 6.98 (1H, t, J = 8.0
Hz, H-4ʹʹ), 7.11 (2H, d, J = 8.0 Hz, H-2ʹʹʹʹ,6ʹʹʹʹ), 7.22 (2H, t, J = 8.0 Hz, H-
3ʹʹ,5ʹʹ), 7.32 (2H, d, J = 8.0 Hz, H-2ʹʹ,6ʹʹ), 7.42 (2H, t, J = 8.0 Hz, H-
3ʹʹʹʹ,5ʹʹʹʹ), N-Ph [7.35 (2H, m, H-2,6), 7.59 (3H, m, H-3–5)]; 13C NMR
(100 MHz, CDCl3 + CD3OD): δ 16.0 (CH3), 28.8 (CH2), 30.5 (C-3ʹ,5ʹ),
34.0 (C-2ʹʹʹ), 33.5 (C-4ʹ), 37.1 (C-2ʹʹʹ), 44.3 (C-2ʹ,6ʹ), 120.7 (C-2ʹʹʹʹ,6ʹʹʹʹ),
121.5 (C-2ʹʹ,6ʹʹ), 123.6 (C-4ʹʹ), 128.7 (C-3ʹʹʹʹ,5ʹʹʹʹ), 129.1 (C-3ʹʹ,5ʹʹ), N-Ph
[(127.9 (C2,6), 130.9 (C-3,5), 131.5 (C-4), 133.0 (C-1)], 136.2 (C-4ʹʹʹʹ),
4.7.8.4. 4-(5-(2-(2,6-Dimethylphenylamino)-2-oxoethylthio)-4-phenyl-
4H-1,2,4-triazol-3-yl)-N-phenylpiperidine-1-carboxamide
(7l). White
amorphous powder; Yield: 91%; M.P.: 190-148˚C; IR (KBr) νmax: 3359
–
–
– –
–
H), 1681, 1665 (C O), 1617–1517 (Ar-
(N
– H),–3042 (Ar-H), 2969 (C
–
1
–
C
C, C N), 1261 (C N); H NMR (400 MHz, CD3OD): δ 1.84 (4H, m,
H-3ʹ,5ʹ), 2.14 (6H, s, CH3), 2.76 (1H, m, H-4ʹ), 2.80 (2H, m, H-2αʹ,6αʹ),
4.05 (2H, s, H-2′′′), 4.14 (2H, br d, J = 13.2 Hz, H-2βʹ,6βʹ), 6.96 (1H, t, J
= 8.5 Hz, H-4ʹʹ), 7.05 (3H, m, H-3′′′-5ʹʹʹʹ), 7.22 (2H, t, J = 8.5 Hz, H-
3ʹʹ,5ʹʹ), 7.29 (2H, d, J = 8.5 Hz, H-2ʹʹ,6ʹʹ), N-Ph [7.36 (2H, m, H-2,6), 7.61
(3H, m, H-3–5)]; 13C NMR (100 MHz, CDCl3 + CD3OD): δ 18.4 (2 ×
CH3), 30.6 (C-3ʹ,5ʹ), 33.6 (C-4ʹ), 36.0 (C-1ʹʹʹ), 44.4 (C-2ʹ,6ʹ), 121.6 (C-
2ʹʹ,6ʹʹ), 123.7 (C-4ʹʹ), 128.1 (C-4ʹʹʹʹ), 128.7 (C-3ʹʹʹʹ,5ʹʹʹʹ), 129.1 (C-3ʹʹ,5ʹʹ),
N-Ph [128.0 (C-2,6), 131.1 (C-3,5), 131.5 (C-4), 133.1 (C-1)], 134.3 (C-
–
–
140.0 (C-1ʹʹʹʹ), 141.3 (C-1ʹʹ), 152.6 (C-3), 157.0 (C O), 159.4 (C-5),
167.0 (C-1ʹʹʹ); HR-EI-MS (m/z): 540.2325 [M]+ calculated for
C
30H32N6O2S; 540.2307.
4.7.8.1. 4-(5-(2-(2,3-Dimethylphenylamino)-2-oxoethylthio)-4-phenyl-
4H-1,2,4-triazol-3-yl)-N-phenylpiperidine-1-carboxamide
(7i). White
–
–
amorphous powder; Yield: 93%; M.P.: 160-167˚C; IR (KBr) νmax: 3355
1ʹʹʹʹ), 136.1 (C-2ʹʹʹʹ,6ʹʹʹʹ), 140.1 (C-1ʹʹ), 152.5 (C-3), 157.1 (C O), 159.5
(N
(C-5), 168.1 (C-1′′′); HR-EI-MS (m/z): 540.2325 [M]+ calculated for
–
–
– –
–
H), 1682, 1664 (C O), 1615–1515 (Ar-
– H),–3039 (Ar-H), 2965 (C
–
1
–
C
C, C N), 1260 (C N); H NMR (400 MHz, CD3OD): δ 1.83 (4H, m,
C30H32N6O2S; 540.2307.
H-3ʹ,5ʹ), 2.11 (3H, s, CH3), 2.29 (3H, s, CH3), 2.80 (1H, m, H-4ʹ), 2.84
(4H, m, H-2
α
ʹ,6
α
ʹ), 4.05 (2H, s, H-2′′′), 4.17 (2H, br d, J = 13.2 Hz, H-
4.7.8.5. 4-(5-(2-(3,4-Dimethylphenylamino)-2-oxoethylthio)-4-phenyl-
2βʹ,6βʹ), 7.00 (1H, t, J = 8.0 Hz, H-4ʹʹ), 7.05 (2H, d, J = 8.0 Hz, H-
4ʹʹʹʹ,6ʹʹʹʹ), 7.15 (1H, t, J = 8.0 Hz, H-5ʹʹʹʹ), 7.24 (2H, t, J = 8.0 Hz, H-
3ʹʹ,5ʹʹ), 7.32 (1H, d, J = 8.0 Hz, H-2ʹʹ,6ʹʹ), N-Ph [7.47 (2H, m, H-2,6), 7.64
(3H, m, H-3–5)]; 13C NMR (100 MHz, CD3OD): δ 14.3 (CH3), 20.5 (CH3),
31.2 (C-3ʹ,5ʹ), 34.2 (C-4ʹ), 37.1 (C-1ʹʹʹ), 44.9 (C-2ʹ,6ʹ), 122.2 (C-2ʹʹ,6ʹʹ),
124.1 (C-4ʹʹ), 124.6 (C-6ʹʹʹʹ), 126.6 (C-4ʹʹʹʹ), 129.1 (C-5ʹʹʹʹ), 129.5 (C-
3ʹʹ,5ʹʹ), 133.0 (C-3ʹʹʹʹ), 134.1 (C-2ʹʹʹʹ), N-Ph [128.9 (C-2,6), 131.4 (C-3,5),
4H-1,2,4-triazol-3-yl)-N-phenylpiperidine-1-carboxamide
(7m). White
amorphous powder; Yield: 90%; M.P.: 116-118˚C; IR (KBr) νmax: 3356
–
–
– –
–
H), 1686, 1669 (C O), 1619–1519 (Ar-
(N
– H),–3049 (Ar-H), 2965 (C
–
1
–
C
C, C N), 1260 (C N); H NMR (400 MHz, CD3OD): δ 1.83 (4H, m,
H-3ʹ,5ʹ), 2.18 (3H, s, CH3), 2.21 (3H, s, CH3), 2.74 (1H, m, H-4ʹ), 2.78
(2H, m, H-2
α
ʹ,6α
ʹ), 3.94 (2H, s, H-2′′′), 4.13 (2H, br d, J = 13.2 Hz, H-
2βʹ,6βʹ), 6.97 (1H, t, J = 8.0 Hz, H-4ʹʹ), 7.02 (1H, d, J = 8.0 Hz, H-6ʹʹʹʹ),
7.22 (2H, t, J = 8.0 Hz, H-3ʹʹ,5ʹʹ), 7.27 (2H, m, H-2ʹʹʹʹ,5ʹʹʹʹ), 7.30 (2H, d, J
131.8 (C-4), 136.4 (C-1)], 138.8 (C-1ʹʹʹʹ), 140.9 (C-1ʹʹ), 152.7 (C-3),
′′′
= 8.0 Hz, H-2ʹʹ,6ʹʹ), N-Ph [7.33 (2H, m, H-2,6), 7.58 (3H, m, H-3–5)]; 13
C
–
157.8 (C O), 160.4 (C-5), 168.9 (C-1 ); HR-EI-MS (m/z): 540.2325
–
[M]+ calculated for C30H32N6O2S; 540.2307.
NMR (100 MHz, CDCl3 + CD3OD): δ 19.3 (CH3), 20.0 (CH3), 30.5 (C-
3ʹ,5ʹ), 33.5 (C-4ʹ), 37.1 (C-1ʹʹʹ), 44.3 (C-2ʹ,6ʹ), 121.5 (C-2ʹʹ,6ʹʹ), 118.1 (C-
2ʹʹʹʹ), 121.8 (C-6ʹʹʹʹ), 123.6 (C-4ʹʹ), 129.1 (C-3ʹʹ,5ʹʹ), 130.3 (C-5ʹʹʹʹ), 133.0
(C-4ʹʹʹʹ), N-Ph [127.8 (C-2,6), 130.9 (C-3,5), 131.4 (C-4), 133.4 (C-1)],
4.7.8.2. 4-(5-(2-(2,4-Dimethylphenylamino)-2-oxoethylthio)-4-phenyl-
4H-1,2,4-triazol-3-yl)-N-phenylpiperidine-1-carboxamide
(7j). White
–
–
amorphous powder; Yield: 92%; M.P.: 90-100˚C; IR (KBr) νmax: 3351
136.2 (C-3ʹʹʹʹ), 137.6 (C-1ʹʹʹʹ), 139.9 (C-1ʹʹ), 152.5 (C-3), 156.9 (C O),
(N
159.3 (C-5), 167.0 (C-1′′′); HR-EI-MS (m/z): 540.2325 [M]+ calculated
–
–
– –
–
H), 1681, 1661 (C O), 1611–1513 (Ar-
– H),–3041 (Ar-H), 2969 (C
–
1
–
C
C, C N), 1261 (C N); H NMR (400 MHz, CD3OD): δ 1.82 (4H, m,
for C30H32N6O2S; 540.2307.
H-3ʹ,5ʹ), 2.17 (3H, s, CH3), 2.26 (3H, s, CH3), 2.78 (1H, m, H-4ʹ), 2.81
(2H, m, H-2
α
ʹ,6
α
ʹ), 4.00 (2H, s, H-2′′′), 4.15 (2H, br d, J = 13.2 Hz, H-
4.7.8.6. 4-(5-(2-(Phenylamino)-2-oxoethylthio)-4-phenyl-4H-1,2,4-tri-
2βʹ,6βʹ), 6.97 (1H, t, J = 8.0 Hz, H-4ʹʹ), 7.00 (1H, d, J = 8.0 Hz, H-6ʹʹʹʹ),
7.03 (1H, s, H-3ʹʹʹʹ), 7.23 (2H, t, J = 8.0 Hz, H-3ʹʹ,5ʹʹ), 7.30 (3H, d, J = 8.0
Hz, H-2ʹʹ,6ʹʹ), 7.33 (1H, d, J = 8.0 Hz, H-5ʹʹʹʹ), N-Ph [7.38 (2H, m, H-2,6),
7.61 (3H, m, H-3–5)]; 13C NMR (100 MHz, CDCl3 + CD3OD): δ 18.1
(CH3), 21.0 (CH3), 30.8 (C-3ʹ,5ʹ), 33.8 (C-4ʹ), 36.7 (C-1ʹʹʹ), 44.5 (C-2ʹ,6ʹ),
121.8 (C-2ʹʹ,6ʹʹ), 123.8 (C-4ʹʹ), 125.4 (C-6ʹʹʹʹ), 127.6 (C-3ʹʹʹʹ), 129.2 (C-
3ʹʹ,5ʹʹ), 131.6 (C-5ʹʹʹʹ), 133.4 (C-4ʹʹʹʹ), N-Ph [128.2 (C-2,6), 131.2 (C-3,5),
azol-3-yl)-N-phenylpiperidine-1-carboxamide (7n). White amorphous
–
powder; Yield: 88%; M.P.: 145-155˚C; IR (KBr) νmax: 3359 (N
–
–
– H), –3045
–
(Ar-H), 2966 (C H), 1682, 1665 (C O), 1617–1513 (Ar-C C, C N),
– –
1
–
1261 (C N); H NMR (400 MHz, CD3OD): δ 1.85 (4H, m, H-3ʹ,5ʹ), 2.17
(3H, s, CH3), 2.27 (3H, s, CH3), 2.80 (1H, m, H-4ʹ), 2.84 (2H, m, H-
2α
ʹ,6
α
ʹ), 4.03 (2H, s, H-2′′′), 4.15 (2H, br d, J = 13.2 Hz, H-2βʹ,6βʹ), 6.97
(1H, t, J = 8.5 Hz, H-4ʹʹ), 7.03 (2H, s, H-2ʹʹʹʹ,6ʹʹʹʹ), 7.24 (2H, t, J = 8.5 Hz,
H-3ʹʹ,5ʹʹ), 7.26 (1H, s, H-4ʹʹʹʹ), 7.32 (2H, d, J = 8.0 Hz, H-2ʹʹ,6ʹʹ), N-Ph
[7.46 (2H, m, H-2,6), 7.62 (3H, m, H-3–5)]; 13C NMR (100 MHz,
CD3OD): δ 18.1 (CH3), 21.0 (CH3), 31.2 (C-3ʹ,5ʹ), 34.2 (C-4ʹ), 37.1 (C-
1ʹʹʹ), 44.9 (C-2ʹ,6ʹ), 122.2 (C-2ʹʹ,6ʹʹ), 124.1 (C-4ʹʹ), 126.2 (C-2ʹʹʹʹ), 127.8
(C-6ʹʹʹʹ), 129.5 (C-3ʹʹ,5ʹʹ), 132.1 (C-4ʹʹʹʹ), 133.5 (C-5ʹʹʹʹ), N-Ph [128.8 (C-
131.9 (C-4), 133.5 (C-1)], 133.4 (C-4′′′′), 133.6 (C-2ʹʹʹʹ), 136.7 (C-1ʹʹʹʹ),
′′′
–
140.3 (C-1ʹʹ), 152.6 (C-3), 157.3 (C O), 159.8 (C-5), 168.3 (C-1 ); HR-
–
EI-MS (m/z): 540.2325 [M]+ calculated for C30H32N6O2S; 540.2307.
4.7.8.3. 4-(5-(2-(2,5-Dimethylphenylamino)-2-oxoethylthio)-4-phenyl-
4H-1,2,4-triazol-3-yl)-N-phenylpiperidine-1-carboxamide
(7k). White
2,6), 131.4 (C-3,5), 131.8 (C-4), 134.0 (C-1)], 134.1 (C-3ʹʹʹʹ), 137.2 (C-
′′′
–
amorphous powder; Yield: 92%; M.P.: 175-180˚C; IR (KBr) νmax: 3353
1ʹʹʹʹ), 140.9 (C-1ʹʹ), 152.7 (C-3), 157.8 (C O), 160.4 (C-5), 168.8 (C-1 );
–
(N
HR-EI-MS (m/z): 540.2325 [M]+ calculated for C30H32N6O2S; 540.2307.
–
–
– –
–
H), 1682, 1660 (C O), 1612–1514 (Ar-
– H),–3044 (Ar-H), 2967 (C
–
1
–
C
C, C N), 1262 (C N); H NMR (400 MHz, CD3OD): δ 1.84 (4H, m,
H-3ʹ,5ʹ), 2.17 (3H, s, CH3), 2.26 (3H, s, CH3), 2.77 (1H, m, H-4ʹ), 2.81
4.7.8.7. 4-(5-(2-(3,6-Dimethylphenylamino)-2-oxoethylthio)-4-phenyl-
(2H, m, H-2
α
ʹ,6
α
ʹ), 4.01 (2H, s, H-2′′′), 4.14 (2H, br d, J = 13.2 Hz, H-
4H-1,2,4-triazol-3-yl)-N-phenylpiperidine-1-carboxamide
(7o). White
13

S. Muzaffar et al.
Bioorganic Chemistry 107 (2021) 104525
amorphous powder; Yield: 91%; M.P.: 208-210˚C; IR (KBr) νmax: 3353
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added per
well. The plate was pre-read at 490 nm and incubated overnight for
–
–
–
H), 1680, 1666 (C O), 1619–1519 (Ar-
(N
– H),–3041 (Ar-H), 2961 (C
–
1
–
C
C, C N), 1262 (C N); H NMR (400 MHz, CD3OD): δ 1.83 (4H, m,
18–20 h in an incubator at 37˚C. After a given time, DMSO (100 μL) was
H–-3ʹ,5ʹ),–2.76 (1H, m, H-4ʹ), 2.79 (2H, m, H-2
αʹ,6
α
ʹ), 3.96 (2H, s, H-2′′′),
added to dissolve the formazon crystals to make a total volume of 200
4.12 (2H, br d, J = 13.2 Hz, H-2βʹ,6βʹ), 6.98 (1H, t, J = 7.5 Hz, H-4ʹʹ),
7.07 (1H, t, J = 7.5 Hz, H-4ʹʹʹʹ), 7.22 (2H, t, J = 7.5 Hz, H-3ʹʹ,5ʹʹ), 7.29
(2H, t, J = 7.5 Hz, H-3ʹʹʹʹ,5ʹʹʹʹ), 7.33 (2H, d, J = 7.5 Hz, H-2ʹʹ,6ʹʹ), 7.36
(2H, d, J = 7.5 Hz, H-2ʹʹʹʹ,6ʹʹʹʹ), N-Ph [7.52 (2H, m, H-2,6), 7.58 (3H, m,
H-3–5)]; 13C NMR (100 MHz, CDCl3 + CD3OD): δ 30.5 (C-3ʹ,5ʹ), 33.4 (C-
4ʹ), 37.1 (C-2ʹʹʹ), 44.3 (C-2ʹ,6ʹ), 120.5 (C-2ʹʹʹʹ,6ʹʹʹʹ), 121.4 (C-2ʹʹ,6ʹʹ), 123.6
(C-4ʹʹ), 125.0 (C-4ʹʹʹʹ), 127.8 (C-3ʹʹ,5ʹʹ), 129.1 (C-3ʹʹʹʹ,5ʹʹʹʹ), N-Ph [129.4
μL. Absorbance was measured after two hours. Experiments were carried
out in triplicates along with the positive and negative controls. Cell
viability was measured by the following equation.
Cell viability (%) = (Abs. of sam ple – Abs. of blank / Abs. of control – Abs. of
blank) × 100.
(C-2,6), 130.9 (C-3,5), 131.4 (C-4), 132.9 (C-1)], 138.5 (C-1ʹʹʹʹ), 139.9
′′′
–
(C-1ʹʹ), 152.5 (C-3), 156.9 (C O), 159.3 (C-5), 167.2 (C-1 ); HR-EI-MS
–
(m/z): 512.2014 [M]+ calculated for C28H28N6O2S; 512.1994.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.8. 15-LOX inhibition assay
The assay was performed according to the chemiluminescence
method of Kondo and coworkers [25] with some minor modifications. A
total volume of 100 µL of reaction mixture contained 60 µL of borate
buffer (200 mM, pH 9.0), 10 µL of test compound or solvent, and 10 µL
soybean 15-LOX enzyme. This mixture was incubated at 25˚C in dark for
5 min. Then 10 µL solution of luminol (3 nM) containing cytochrome-C
(1 nM) was added to each well and luminescence was measured by
BioTek HTX 96-well plate reader in luminescence mode. The reaction
was initiated by the addition of 10 µL substrate solution (the substrate
linoleic acid (155 µL) and Tween-20 (257 µL) were dissolved in 5 mL of
deionized water followed by the addition of 0.6 mL 1 N NaOH and the
mixture was emulsified). After dilution to 20 mL, this substrate solution
was flushed with nitrogen gas to avoid the substrate from oxidation and
used). Chemiluminescence of the reaction contents was measured for
100 to 300 s. All experiments were performed in triplicates. Quercetin
was used as a positive control. The percentage of inhibition was calcu-
lated using the following formula:
Acknowledgments
Thanks are due to the Higher Education Commission of Pakistan for
funding the NRPU Project No. 4950 to M.A. and research assistantship to
W.S. as well as the Alexander von Humboldt (AvH) Foundation, Ger-
many for their financial support in the form of equipment grant.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104525.
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