Synthesis and pharmacological screening: Sulfa derivatives of 2-pipecoline-bearing 1,3,4-oxadiazole core

Article abstract of DOI:10.1134/S1068162017030025

An electrophile, 1-(4-(bromomethylbenzenesulfonyl)-2-methylpiperidine, was synthesized by the reaction of 2-methylpiperidine (2-pipecoline) and 4-bromomethylbenzenesulfonyl chloride in a weak basic medium under pH control. A series of nucleophiles, 5-aryl/aralkyl-1,3,4-oxadiazol-2-thiols, were synthesized from corresponding carboxylic acids in three steps. The title molecules were synthesized by coupling the electrophile to nucleophiles in an aprotic medium using LiH as an activator. The structures of all synthesized compounds were corroborated through IR, ¹H NMR, and EI-MS techniques. All the compounds were screened for their pharmacological behavior, particularly, antibacterial and enzyme inhibitory activities. Notably efficient results were obtained against both gram-positive and gram-negative bacterial strains. Regarding enzyme inhibition, compounds were efficient against acetylcholinesterase and butyrylcholinesterase.

Full text of DOI:10.1134/S1068162017030025

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 3, pp. 328–339. © Pleiades Publishing, Ltd., 2017.
Synthesis and Pharmacological Screening: Sulfa Derivatives
of 2-Pipecoline-Bearing 1,3,4-Oxadiazole Core1
Aziz-ur-Rehmana, 2, A. Arifa, M. A. Abbasia, S. Z. Siddiquia, S. Rasoola, and S. A. A. Shahb, c
aDepartment of Chemistry, Government College University, Lahore, 54000 Pakistan
bFaculty of Pharmacy, University of Technology MARA, Puncak Alam Campus, Bandar Puncak Alam,
Selangor Darul Ehsan, 42300 Malaysia
cAtta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Level 9, FF3, University of Technology MARA,
Puncak Alam Campus, Bandar Puncak Alam, Selangor Darul Ehsan, 42300 Malaysia
Received May 23, 2016; in final form, October 4, 2016
Abstract⎯An electrophile, 1-(4-(bromomethylbenzenesulfonyl)-2-methylpiperidine, was synthesized by the
reaction of 2-methylpiperidine (2-pipecoline) and 4-bromomethylbenzenesulfonyl chloride in a weak basic
medium under pH control. A series of nucleophiles, 5-aryl/aralkyl-1,3,4-oxadiazol-2-thiols, were synthe-
sized from corresponding carboxylic acids in three steps. The title molecules were synthesized by coupling the
electrophile to nucleophiles in an aprotic medium using LiH as an activator. The structures of all synthesized
1
compounds were corroborated through IR, H NMR, and EI-MS techniques. All the compounds were
screened for their pharmacological behavior, particularly, antibacterial and enzyme inhibitory activities. Notably
efficient results were obtained against both gram-positive and gram-negative bacterial strains. Regarding enzyme
inhibition, compounds were efficient against acetylcholinesterase and butyrylcholinesterase.
Keywords: 1,3,4-oxadiazole, 2-methylpiperidine, antimicrobial activity, anti-enzymatic activity, sulfonamide
DOI: 10.1134/S1068162017030025
INTRODUCTION
electron deficient ring system, such as pyridine, furan,
thiophene, and naphthalene [5].
Substituted 1,3,4-oxadiazoles are efficacious phar-
macological intermediates in drug development.
Many non-natural antitumor compounds possess
oxadiazole ring [1]. Drug molecules containing such
moiety were also found to have various other biologi-
cal activities including analgesic, anti-inflammatory,
anti-proliferative [2], antibacterial, herbicidal, and
antifungal [3]. Epilepsy, a neurological disorder, is the
third wide spread disorder all over the world. Epileptic
patients suffer from severe fits. The anticonvulsants
are to stop the firing of neurons that starts seizures and
also function as mood stabilizers. Semicarbazones
based on 2,5-disubstituted oxadiazole moiety are
extraordinary anticonvulsant drug for epileptic
patients [4]. Owing to their biological activities, 1,3,4-
oxadiazoles are also utilized in agricultural field as
insecticides, fungicides, and herbicides. In material
sciences, electronic properties of oxadiazoles make
them potentially advantageous target molecules.
Many are used as organic light emitting diodes
(OLEDs) and laser dyes. 1,3,4-Oxadiazole also exhib-
its electroluminescent properties when it is fused with
Sulfonamides are synthetic antibiotics that are
clinically used to control bacterial infection in humans
and animals. In animal husbandry, sulfonamides are
fed to poultry animals to fight against gastrointestinal
and respiratory disease. These have some adverse
effects in humans like accumulation in tissues, hyper-
tension, and allergy. Prolonged exposure of these
medicines may be carcinogenic; also, the organism
becomes bacteria-resistant [6]. Cyclic sulfonamides,
like sultams, are the structural part of drugs such as
HIV integrase inhibitors, MMP-2 inhibitors, and car-
bonic anhydrase inhibitors and anti-AIDS agents [7].
Piperidine is an abundant structural moiety, which
is present in several biologically active natural and syn-
thetic compounds [8]. Piperidine has various com-
mercial, pharmacological, and curative applications.
It is used as solvent, curing agent for rubber, food
additive, and intermediate for inorganic synthesis [9].
Enzymes belonging to cholinesterase family con-
stitute operational enzyme system in our body. Acetyl-
choline (ACh), a serine protease neurotransmitter, is
responsible for serine hydrolysis. AChE stops trans-
mission of signal through hydrolysis of acetylcholine
[10–12]. BChE is known as “pseudo” or “non-neuro-
1
The article is published in the original.
2
Corresponding author: phone: (+92)-42-111000010, ext. 450;
e-mail: rehman@gcu.edu.pk; azizryk@yahoo.com
328

SYNTHESIS AND PHARMACOLOGICAL SCREENING
Table 1. Different aralkyl/aryl groups
329
Compd.
R
Compd.
R
Compd.
R
Cl
2'
Cl
2'
6'
2'
(VIa)
(VIf)
(VIk)
4'
6'
4'
HO
6'
4'
O2N
2'
H2C 11'
O2N
2'
6'
4'
(VIb)
(VIc)
(VIg)
(VIh)
(VIl)
8'
6'
4'
2'
4'
6'
NO2
Cl
7'
H2N
CH2
2'
2'
2'
(VIm)
6'
4'
6'
4'
6'
4'
Cl
Cl
O2N
2'
2'
2'
6'
(VId)
(VIe)
(VIi)
(VIj)
(VIn)
4'
6'
H37C'
6'
4'
4'
NO2
OCH3
2'
7'
6'
4'
2'
O2N
6'
4'
nal cholinesterase”. BChE has been reported a valu- groups of different nature, like electron donating or
able agent as an investigative marker for Alzheimer
disease [13, 14]. Urease catalyzes the hydrolysis of urea
and it protects the bacteria in the acidic environment
of the stomach [15]. α-Glucosidase causes type-2 dia-
betes mellitus [16].
withdrawing, along with variation in their position,
were employed in the synthesis to evaluate their anti-
bacterial and enzyme-inhibitory activities.
RESULTS AND DISCUSSION
In search for new drug candidates, a large number
of organic compounds are being synthesized and eval-
uated for pharmacological behavior. In continuation
of our previous work [12–14], this research project was
a successful effort to fuse three bioactive moieties
within one core. The synthesized molecules were
explored for their pharmacological behavior regarding
antibacterial effects against gram-negative and gram-
positive bacteria and enzyme inhibitory effects against
cholinesterase, urease, and α-glucosidase enzymes.
The size, nature, and position of substituents in a mol-
A series of compounds bearing different function-
alities was developed in order to evaluate the role of the
functional groups in biological activity and to get more
bioactive compounds. The general protocol of synthe-
sis is sketched in Scheme and the varying aralkyl/aryl
groups are listed in Table 1. The synthesized molecules
were subjected to structural and pharmacological
analysis. The results of structural analysis are given
under spectral characterization and that of pharma-
ecule greatly affect the biological activities [17]. So cology are given in Tables 2 and 3.
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330
AZIZ-UR-REHMAN et al.
Table 2. MIC values for antibacterial inhibition study of synthesized compounds
MIC, mg/mL
Compounds
S. typhi (–)
E. coli (–)
P. aeruginosa (–)
B. subtilis (+)
S. aureus (+)
(VIa)
(VIb)
(VIc)
(VId)
(VIe)
(VIf)
(VIg)
(VIh)
(VIi)
(VIj)
(VIk)
(VIl)
(VIm)
(VIn)
–
10.92 0.18
15.86 0.52
–
10.76 0.61
12.78 0.90
–
9.64 0.12
12.53 0.55
11.32 0.88
–
9.85 0.90
10.53 0.49
8.83 0.57
9.42 0.90
10.64 0.90
–
9.79 0.18
9.80 0.70
–
14.69 0.77
18.79 0.10
–
10.87 0.64
–
10.24 0.65
9.76 0.65
12.71 0.65
10.54 0.32
–
18.78 0.46
–
11.76 0.54
–
–
–
–
–
16.75 0.59
9.36 0.64
9.58 0.41
–
10.87 0.87
15.24 0.73
12.54 0.37
9.75 0.50
14.25 0.85
9.72 0.51
9.93 0.38
–
14.58 0.56
9.11 0.12
9.17 0.42
12.87 0.53
12.65 0.58
11.43 0.64
13.87 0.59
–
19.79 0.41
12.33 0.62
14.90 0.46
17.86 0.20
16.98 0.10
14.28 0.90
10.27 0.78
–
10.59 0.41
16.90 0.52
9.65 0.15
12.83 0.26
Ciprofloxacin
7.83 0.78
8.01 0.12
7.98 0.89
7.22 0.67
7.00 1.54
C2H5OH/H+
O
NH2NH2
O
O
Reflux, 3–5 h
Reflux, 4–6 h
NH2
NH
R
OC2H5
IIa−n
R
OH
Ia−n
R
IIIa−n
N4 3N
Reflux, 5–7 h
CS2, KOH
7''
5
2
1
R
O
S
1''
3''
5'''
1'''
N
N
R
N
3'''
5''
VIa−n
S
O
SH
O
CH3
O
IVa−n
Stir for 4 h
LiH, HCON(CH3)2
HN
H2O, Na2CO3, Stir for 4 h
O
O
S
a
O
O
N
H3C
S
Br
Cl
H3C
Br
V
b
Scheme. Outline for the synthesis
of 5-substituted-2-(4-(2-methylpiperidin-1-yl-sulfonyl)benzylthio)-1,3,4-oxadiazole (VIa–n).
Chemistry
compound (V) in dimethyl formamide (DMF) using
LiH as activator to get final compounds. The com-
pound (VIk) was separated as sticky solid. The IR
spectrum of 5-(3-chlorophenyl)-2-(4-(2-meth-
ylpiperidin-1-yl-sulfonyl)benzylthio)-1,3,4-oxadiazole
in KBr solid showed absorption bands at frequen-
cies3035 (Ar C–H), 2534 (S–H), 1659 (C=N), 1526
(Ar C=C), 1432 (SO2), 1227, 1059 (C–O–C), and 618
Compound (V), an electrophile, was synthesized
from 2-methylpiperidine and 4-bromomethylben-
zenesulfonyl chloride in the presence of aqueous weak
basic medium. The product was filtered out at pH 5–6.
Dilute HCl was employed for this purpose but too low
pH has negative effect. A number of carboxylic acids
are converted into heterocyclic 1,3,4-oxadiazoles, the
nucleophiles. These nucleophiles were mixed with (C–S). The molecular formula was affirmed by EI-
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SYNTHESIS AND PHARMACOLOGICAL SCREENING
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AZIZ-UR-REHMAN et al.
MS indicating molecular ion peak at m/z 464 and ues, which were 11.32 0.88, 12.53 0.55, 12.78
characteristic peaks at (m/z) 98 and 111 showing the 0.90, and 15.86 0.52 mg/mL, respectively. Against
presence of 2-methylpiperidinyl and 3-chlorophenyl
group in the molecule, respectively. The EI-MS data
of (VIk) is elucidated in Figure as mass fragmentation
pattern, which is helpful to study mass spectrum of all
targeted molecules. In the 1H NMR spectrum, signals
of aromatic protons appearing at δ (ppm) 7.76 (d, J =
8.0 Hz, 2H, H-3", H-5") and 7.54 (d, J = 7.6 Hz, 2H,
H-2", H-6") justified the benzene sulfonyl ring. The
signals of aromatic protons appearing at δ (ppm) 7.98
(s, 1H, H-2') and 7.44–7.39 (m, 3H, H-4', H-5', H-6')
confirmed the presence of 3-chlorophenyl ring. Also,
aliphatic protons resonated at δ (ppm) 4.23–4.12 (m,
1H, He-6'''), 3.67 (br.s, 1H, Ha-6'"), 3.23 (br.s, 1H, H-
2'"), 3.02–2.86 (m, 2H, He-4', He-5'"), and 1.54–1.23
(m, 4H, He-3'", Ha-3"', Ha-4'", Ha-5"') for piperidine
ring. The aliphatic protons for methylene and methyl
carbons resonated at δ (ppm) 4.77 (s, 2H, H-7") and
1.02 (d, J = 7.2 Hz, 3H, CH3-7"'). On the basis of
described affirmations, the compound (VIk) was named as
2-(3-chlorophenyl)-5-(4-(2-methylpiperidin-1-ylsul-
phonyl)benzylthio)-1,3,4-oxadiazole. On the basis of
IR, 1H NMR, and EI-MS data, the synthesized com-
pounds (VIa–n) were elucidated as described in the
Experimental section.
P. aeruginosa, (VIl) showed best inhibition with MIC
value of 9.11
0.12 mg/mL. Compounds (VIm),
(VIh), (VIc), (VId), and (VIi) also proved as promising
inhibitors with MIC values of 9.17 0.42 < 9.72 0.51 <
9.79
0.18 < 9.80
0.70 < 9.93
0.38 mg/mL,
respectively. Compounds (VIa), (VIn), and (VIk)
showed good inhibition potential depicted by their
MIC values of 10.64 0.90 < 12.87 0.53 < 14.58
0.56 mg/mL.
Against S. aureus, compounds (VIb) and (VIm)
were the best inhibitors having MIC values of 9.65
0.15 and 9.76
0.65 mg/mL, respectively. Com-
pounds (VIa) and (VIi) showed good inhibition poten-
tial against this bacterium compared to reference, cip-
rofloxacin with MIC of 7.00
1.54 mg/mL. Their
MICs were rather close, that is, 10.24 0.65 and 10.54
0.32 mg/mL. The varying groups in these two com-
pounds were p-hydroxy and p-methyl having electron
donating nature. Compounds (VId) and (VIk) had
MIC values of 10.54 0.32 and 10.59 0.41 mg/mL,
respectively. Compounds (VIc) and (VIh) had very
close inhibition potential against S. aureus. Com-
pounds (VIg), (VIl), and (VIn) also showed good inhi-
bition potential. Compounds (VIe), (VIf), and (VIj)
were inactive against this bacterial strain. Against
B. subtilus, (VIc), (VIe), (VIf), and (VIj) were inactive.
Compound (VId) showed good inhibition against this
bacterium. Compounds (VIh), (VIl), (VIg), (VIi),
(VIa), (VIb), (VIn), and (VIk), all exhibited moderate
activity as shown by their MIC values given in Table 2.
Antibacterial Activity
All the target compounds were evaluated against
gram-positive (B. subtilis and S. aureus) and gram-
negative bacterial strains (S. typhi, E. coli, and P. aeru-
ginosa) using ciprofloxacin as a reference drug. Per-
centage inhibition and minimum inhibitory concen-
tration (MIC) values of all synthesized compounds are
given in Table 2.
Enzyme Inhibition Studies
All the synthesized compounds were screened
against AChE and BChE against eserine used as a
standard. Their inhibitory potential values are
expressed as IC50 values in Table 3. Amongst all deriv-
atives, 2-(4-(2-methylpiperidin-1-yl-sulfonyl)benz-
ylthio)-5-(3-aminophenyl)-1,3,4-oxadiazole (VIc)
showed very good inhibition with IC50 value of 8.51
0.004 μM against BChE. The enhanced potential of
this derivative may be because of the 3-aminophenyl
ring as substituent, which has electron donating char-
acter. On the other hand, it was inactive against AChE.
Target molecule 2-(4-(2-methylpiperidin-1-ylsul-
phonyl)benzylthio)-5-(3-nitrophenyl)-1,3,4-oxadi-
azole (VIg) possessing IC50 of 9.72 0.01 μM also
showed good inhibitory potential against BChE.
Compounds (VIj) < (VIl) < (VIi) with IC50 values
(μM) 24.1 0.05 < 31.2 0.02 < 33.2 0.03 were also
found moderate inhibitors against BChE when com-
pared with reference eserine with IC50 value of 0.04
Against S. typhi, (VIh), (VIi), and (VIn) showed
very good inhibition potential as indicated by their
MIC values of 9.36 0.64, 9.58 0.41, and 9.75
0.50 mg/mL, respectively. The other more efficient
compounds against this strain were (VIk), (VId), and
(VIm). Compounds (VIb), (VIg), and (VIl) showed
moderate activity against this bacterial strain. The
compounds (VIa), (VIc), (VIe), (VIf), and (VIj) were
potentially inactive. Against E. coli, the most active
compound with excellent inhibitory potential was
(VIm) with MIC of 8.83 0.57 mg/mL as compared
to that of ciprofloxacin, 8.01 0.12 mg/mL. Com-
pounds (VIc), (VIf), and (VIj) were inactive. Com-
pounds (VIg), (VIk), (VIn), and (VIl) possessed best
inhibitory potential against this gram-negative bacte-
ria, probably because of 3-nitrophenyl, 3-clorophenyl,
2-cloro-3,5-dinitrophenyl, and 3,5-dinitrophenyl
substituents attached within the core of these target
compounds, respectively. The compounds (VIa) and
(VId) also showed good inhibitory potential. The com- 0.0001 μM. All other compounds showed moderate
pounds (VIi), (VIh), (VIe), and (VIb) showed moder- inhibition activity against this class of enzymes while
ate inhibition potential as depicted by their MIC val- (VIh), (VIk), and (VIn) remained inactive.
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SYNTHESIS AND PHARMACOLOGICAL SCREENING
Cl
333
H3C
Cl
O
+
N
+
+
N
N
N
CH2
S
S
O
O
O
m/z = 211
m/z = 153
m/z = 252
-C14H18N2O2S2
-C13H18NO2S
-C9H7N2OS
H3C
O
Cl
N
N
+
S
N
CH2
S
O
O
-C15H10ClN2OS
-C14H18N3O2S2
[M]+ m/z = 464
H3C
O
Cl
-C7H15NO2S
+
O
+
S
N
O
Cl
m/z = 139
m/z = 162
N
N
+
S
CH2
O
-SO2
-CO
m/z = 301
H3C
N
Cl
+
+
m/z = 98
m/z = 111
Mass fragmentation pattern of 2-(3-chlorophenyl)-5-(4-(2-methylpiperidin-1-ylsulfonyl)benzylthio)-1,3,4-oxadiazole (VIk).
Сompound (VIg) showed very good inhibition uated for anti-urease enzyme inhibition activity. Stan-
against AChE with IC50 of 17.2 0.01 μM, which is dard used for this was thiourea. All derivatives showed
weak inhibition potential with reference of thiourea.
Compounds (VIc), (VIg), and (VIj) remained inactive
against the urease enzyme.
best among the whole series against AChE. Com-
pounds (VIa) and (VIl) showed moderate potential
against AChE as indicated by their IC50 values of
27.4 0.03 < 39.2 0.07 μM, respectively with refer-
ence to standard eserine, 0.85
0.0001 μM. The
EXPERIMENTAL
somewhat higher activity of these compounds was
probably because of p-hydroxy, 3-nitrophenyl, and
3,5-dinitrophenyl moieties present within the cores of
synthesized molecules. Compounds (VIi), (VId), and
(VIj) exhibited weak inhibitory potential against AChE
All the chemicals were purchased from Alfa Aesar,
Sigma Aldrich, and Merck through local suppliers.
The solvents used were of analytical grade and were
processed without further purification. Reactions
were monitored by TLC (thin layer chromatography)
on pre-coated silica gel G-25-UV254 plates using ethyl
acetate and n-hexane as solvent system. Melting
points were checked on Gallonkamp melting point
apparatus by open capillary tube and were uncor-
rected. IR spectra (KBr, υmax, cm–1) were recorded on
with IC50 378.7
0.09 < 443.2
0.06 < 443.7
0.09 μM; while (VIb), (VIc), (VIe), (VIf), (VIh),
(VIk), (VIm), and (VIn) remained inactive.
All target compounds were evaluated against yeast
α-glucosidase. Reference used was acarbose. The
inhibition potential values are given in Table 3. Only
compounds (VIl), (VIg), and (VIa) showed inhibition
having IC50 values of 148.52 0.19, 157.45 0.18, and
229.34 0.25, respectively. All other derivatives were
a MIDAC M 2000 spectrometer. 1H NMR spectra (δ,
ppm; J, Hz) were recorded in CDCl3 on a Bruker
spectrometer operating at 400 (1H NMR) or 100 MHz
inactive. Synthesized target molecules were also eval- (13C NMR) at 25°C. Mass spectra (EI-MS) were mea-
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334
AZIZ-UR-REHMAN et al.
sured on a JEOL JMS-600H instrument along with tion completion. At the end of reaction, the mixture
the data system.
was acidified with dilute hydrochloric acid and kept at
pH 5–7 to remove excessive base. Precipitates were
filtered, washed with cold distilled water, and dried
[20, 21]. 1H NMR: 7.73 (d, J = 8.0 Hz, 2H, H-3", H-5"),
7.59 (d, J = 7.6 Hz, 2H, H-2", H-6"), 4.23 (s, 2H, H-7"),
General Procedure for Synthesis of Ethyl Esters (IIa–n)
Aryl/aralkyl carboxylic acids (Ia–n) (0.032 mol)
were homogenized in ethanol (99%, 30 mL) in a 250 4.46 (br.s, 1H, He-6"'), 3.69 (br.s, 1H, Ha-6"'), 3.24
mL round bottom flask. Concentrated sulfuric acid
(2.5 mL) was added in the mixture and set to reflux for
3–5 h. TLC was developed for monitoring reaction
completion. At maximum completion, the 10% aque-
ous sodium carbonate solution was poured to neutral-
ize the mixture up to pH of 9–10 after addition of 150
mL distilled water. This step converted untreated
organic acid and sulfuric acid into salts washed away
by aqueous layer. The esters were filtered or extracted
by solvent extraction technique using 50 mL chloroform
or diethyl ether from a separating funnel. Chloroform was
distilled off to collect esters (IIa–n) [18, 19].
(br.s, 1H, H-2'"), 3.10–2.94 (m, 2H, He-4'", He-5"'),
1.59–1.26 (m, 4H, He-3'", Ha-3"', Ha-4'", Ha-5"'),
1.05 (d, J = 8.0 Hz, 3H, CH3-7'"); 13C NMR: 146.3
(C-4"), 143.6 (C-1"), 132.7 (C-2", C-6"), 127.1 (C-3",
C-5"), 56.4 (C-2"'), 43.6 (C-6'"), 33.8 (C-3"'), 31.2
(C-7'), 24.9 (C-5'"), 19.3 (C-4"'), 18.1 (C-7'").
General Procedure for Synthesis of S-Substituted
Derivatives (VIa–n)
The synthesized nuclephiles (IVa–n) (0.005 mol)
were homogenized in N,N-dimethylformamide
(10 mL) in a round bottom flask (50 mL) and then
solid lithium hydride (0.005 mol) was added along
with continuous stirring. The 0.005 mole of electro-
phile (V) was poured into homogeneous solution after
0.25 h and the solution was further stirred for 4–6 h.
After a single-spot TLC, ice cold distilled water was
introduced into the reaction contents along with aque-
ous sodium hydroxide to adjust pH to 10–12. The pre-
cipitates were filtered, washed with distilled water, and
dried [13].
General Procedures for Synthesis of Aryl/Aralkyl
Carbohydrazides (IIIa–n)
The synthesized aryl/aralkyl esters (IIa–n)
(3.5 mL) were diluted in 250 mL ethanol followed by
addition of 4.8 mL 80% hydrazine. Refluxing was
continued for 4–6 h. After monitoring by TLC, ice
cold distilled H2O was poured to acquire the precipi-
tates of (IIIa–n), which were filtered and washed off
with cold distilled H2O [18, 19].
5-(4-Hydroxyphenyl)-2-(4-(2-methylpiperidin-1-
yl-sulfonyl)benzylthio)-1,3,4-oxadiazole (VIa). Light
brown sticky solid; yield 78%; molecular formula:
C21H23N3O4S2; molecular mass 445.5 g mol–1; IR:
3031 (Ar C-H), 2450 (S–H), 1675 (C=N), 1523 (Ar
C=C), 1398 (SO2), 1231, 1059 (C–O–C), 615 (C-S);
General Procedures for Synthesis of 5-Substituted-
1,3,4-Oxadiazol-2-Thiols (IVa–n)
Aryl/aralkyl carbohydrazides (IIIa–n) (0.012 mol)
were shaken well with absolute ethanol (20 mL) and
set to reflux after the addition of carbon disulfide
(0.024 mol) and solid potassium hydroxide
(0.012 mol) for 5–7 h. After the final TLC, distilled
water would be added along with diluted hydrochloric
acid to make pH of 3 to acquire the precipitates. The
addition of acid is crucial to transform the salt form of
5-substituted-1,3,4-oxadiazol-2-thiol into acidic one,
but limited amount, because the excess reduces the
amount of product. The precipitates were further fil-
tered, washed with distilled water, and re-crystallized
from methanol [18, 19].
1H NMR: 7.91 (d, J = 7.6 Hz, 2H, H-2', H-6'), 7.84
(d, J = 7.2 Hz, 2H, H-3', H-5'), 7.75 (d, J = 8.4 Hz,
2H, H-3", H-5"), 7.56 (d, J = 7.6 Hz, 2H, H-2", H-
6"), 4.48 (br.s, 1H, He-6"'), 4.21 (s, 2H, H-7"), 3.68
(br.s, 1H, Ha-6"'), 3.22 (br.s, 1H, H-2"'), 3.05–2.87
(m, 2H, He-4"', He-5"'), 1.56–1.23 (m, 4H, He-3"',
Ha-3"', Ha-4"', Ha-5"'), 1.09 (d, J = 8.0 Hz, 3H, CH3-
7"'); 13C NMR: 168.4 (C-5), 164.3 (C-2), 159.4 (C-4'),
145.2 (C-4"), 141.8 (C-1"), 131.3 (C-2", C-6"), 125.3
(C-3", C-5"), 123.9 (C-1'), 123.3 (C-2', C-6'), 117.7
(C-3', C-5'), 56.7 (C-2"'), 43.2 (C-6"'), 33.5 (C-3"'),
31.4 (C-7"), 24.2 (C-5"'), 19.4 (C-4"'), 18.3 (C-7"');
EI-MS (m/z): 445 [M]+, 283 [C15H11N2O2S]+, 252
Procedure for Synthesis of 1-(4-Bromomethyl-
benzenesulfonyl)-2-Methylpiperidine (V)
[C13H18NO2S]+, 193 [C8H5N2O2S]+, 162 [C6H12-
NO2S]+, 135 [C7H5NO2]+, 121 [C7H5O2]+, 98
[C6H12N]+, 93 [C6H5O]+.
2-Methylpiperidine (a) (0.0127 mol) was taken in a
round bottom flask and diluted with 4–5 mL of dis-
tilled water. 4-Bromomethylbenzenesulfonyl chloride
(b) (0.0127 mol) was added pinch by pinch to the mix-
ture during vigorous stirring at room temperature. The
10% aqueous sodium carbonate solution was gradually
added to maintain pH around 9. The reaction was
5-(Naphthalen-1-ylmethyl)-2-(4-(2-methylpipridin-
1-ylsulfonyl)benzylthio)-1,3,4-oxadiazole
(VIb).
Creamy amorphous solid; yield 79%; mp 82–84°C;
molecular formula: C26H27N3O3S2; molecular mass
stirred for 4 h and monitored with TLC to check reac- 493.6 g mol–1; IR: 3029 (Ar C-H), 2560 (S–H), 1662
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SYNTHESIS AND PHARMACOLOGICAL SCREENING
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Ha-5"'), 1.02 (d, J = 6.4 Hz, 3H, CH3-7"'); 13C NMR:
(C=N), 1520 (Ar C=C), 1390 (–SO2), 1233, 1055 (C–
O–C), 616 (C–S); 1H NMR: 7.91 (d, J = 8.0 Hz, 1H, 167.7 (C-5), 164.6 (C-2), 145.7 (C-4"), 142.5 (C-1"),
H-8'), 7.84 (d, J = 8.0 Hz, 2H, H-3", H-5"), 7.62 (d, 133.2 (C-3', C-5'), 131.5 (C-2', C-6'), 129.7 (C-4'),
J = 8.4 Hz, 1H, H-5'), 7.53 (d, J = 6.4 Hz, 1H, H-4'),
7.50–7.43 (m, H-3', H-6', H-7'), 7.42 (d, J = 7.2 Hz,
H-2", H-6"), 7.34 (d, J = 8.0 Hz, 1H, H-2'), 4.73 (s,
2H, H-11'), 4.57 (s, 2H, H-7"), 4.22 (br.s, 1H, He-6"'),
3.69–3.60 (m, 1H, Ha-6"'), 3.24 (br.s, 1H, H-2"'),
3.05-2.89 (m, 2H, He-4"', He-5"'), 1.54-1.23 (m, 4H,
He-3"', Ha-3"' Ha-4"', Ha-5"'), 1.03 (d, J = 7.0 Hz, 3H,
129.0 (C-1'), 127.6 (C-2", C-6"), 125.2 (C-3", C-5"),
56.4 (C-2"'), 43.3 (C-6"'), 33.4 (C-3"'), 31.9 (C-7"),
23.7 (C-5"'), 18.9 (C-4"'), 18.1 (C-7"'); EI-MS (m/z):
429 [M]+, 251 [C13H17O2NS]+, 238 [C12H16NO2S]+,
163 [C6H12NO2S]+, 119 [C7H5NO]+, 105 [C7H5O]+,
77 [C6H5]+, 98 [C6H12N]+.
13
5-(4-Nitrophenyl)-2-(4-(2-methylpiperidin-1-yl-
sulfonyl)benzylthio)-1,3,4-oxadiazole (VIe). Red
amorphous solid; Yield 76%; mp 132–134°C; molec-
ular formula: C21H22N4O5S2; molecular mass 474.5 g
CH3-7"'); C NMR: 165.2 (C-5), 164.6 (C-2), 148.6
(C-1'), 144.4 (C-4"), 140.0 (C-1"), 130.9 (C-5'), 130.4
(C-4'), 129.6 (C-10'), 128.8 (C-2", C-6"), 127.8 (C-
7'), 126.7 (C-3", C-5"), 125.8 (C-6'), 125.3 (C-3'),
124.8 (C-2'), 122.9 (C-8'), 120.4 (C-9'), 55.4 (C-2"'), mol–1; IR: 3037 (Ar C–H), 2535 (S–H), 1668 (C=N),
43.2 (C-6"'), 33.6 (C-3"'), 32.5 (C-11'), 31.2 (C-7"), 1529 (Ar C=C), 1430 (SO2), 1232, 1064 (C–O–C),
613 (C–S); 1H NMR: 8.34 (d, J = 8.4 Hz, 2H, H-3',
H-5'), 8.15 (d, J = 8.4 Hz, 2H, H-2', H-6'), 7.92 (d, J
= 7.2 Hz, 2H, H-3", H-5"), 7.77 (d, J = 8.0 Hz, 2H,
H-2", H-6"), 4.52 (s, 2H, H-7"), 4.24 (br.s, 1H, He-
6"'), 3.69 (br.s, 1H, Ha-6"'), 3.22 (m, 1H, H-2"'),
3.02–2.96 (m, 2H, He-4"', He-5"'), 1.70–1.23 (m, 4H,
He-3"', Ha-3"', Ha-4"', Ha-5"'), 1.05 (d, J = 7.2 Hz,
25.7 (C-5"'), 18.9 (C-4"'), 17.8 (C-7"'); EI-MS (m/z):
493 [M]+, 332 [C20H16N2OS]+, 251 [C13H17O2NS]+,
241 [C13H9N2OS]+, 238 [C12H16NO2S]+, 213
[C13H9N2O]+, 163 [C6H12NO2S]+, 183 [C12H9NO]+,
169 [C12H9O]+, 141 [C11H9]+, 98 [C6H12N]+.
5-(3-Aminophenyl)-2-(4-(2-methylpiperidin-1-yl-
sulfonyl)benzylthio)-1,3,4-oxadiazole (VIc). Light
brown sticky solid; yield 80%; molecular formula:
C21H24N4O3S2; molecular mass 444.5 g mol–1; IR:
3031 (Ar C–H), 2432 (S–H), 1688 (C=N), 1522 (Ar
C=C), 1420 (–SO2), 1222, 1056 (C–O–C), 615 (C–
3H, CH3-7"'); 13C NMR: 165.3 (C-5), 164.2 (C-2),
149.1 (C-4'), 144.6 (C-4"), 141.8 (C-1"), 138.5 (C-1'),
128.6 (C-2", C-6"), 125.8 (C-3", C-5"), 124.0 (C-2',
C-6'), 123.5 (C-3', C-5'), 55.8 (C-2"'), 43.9 (C-6"'),
34.7 (C-3"'), 32.3 (C-7"), 24.2 (C-5"'), 19.1 (C-4"'),
18.3 (C-7"'); EI-MS (m/z): 474 [M]+, 312
[C15H10N3O3S]+, 251 [C13H17O2NS]+, 238 [C12H16-
S); 1H NMR: 7.95 (d, J = 8.0 Hz, 1H, H-6'), 7.81 (s,
1H, H-2'), 7.75 (d, J = 7.6 Hz, 2H, H-3", H-5"), 7.57
(d, J = 8.0 Hz, 2H, H-2"-H-6"), 7.50–7.46 (m, 2H,
H-4', H-5'), 4.52 (s, 2H, H-7"), 4.21 (br.s, 1H, He-6"'),
3.66 (br.s, 1H, Ha-6"'), 3.23–3.16 (m, 1H, H-2"'),
3.01–2.91 (m, 2H, He-4"', He-5"'), 1.54–1.23 (m, 4H,
He-3"', Ha-3"', Ha-4"', Ha-5"'), 1.02 (d, J = 6.4 Hz,
NO2S]+, 222 [C8H4N3O3S]+, 163 [C6H12NO2S], 164
[C7H4N2O3]+, 122 [C6H4NO2]+ ,150 [C7H4NO3]+, 98
[C6H12N]+
5-(2-Chlorophenyl)-2-(4-(2-methylpiperidin-1-yl-
sulfonyl)benzylthio)-1,3,4-oxadiazole (VIf). Light yel-
low amorphous solid; yield 73%; molecular formula:
C21H22ClN3O3S2; molecular mass 464.0 g mol–1; IR:
3031 (Ar C–H), 2530 (S–H), 1665 (C=N), 1523 (Ar
C=C), 1399 (SO2), 1224, 1059 (C–O–C), 615 (C–S);
3H, CH3-7"'); 13C NMR: 171.1 (C-5), 164.7 (C-2),
147.5 (C-3'), 144.7 (C-4"), 140.6 (C-1"), 132.5 (C-5'),
130.2 (C-1'), 128.7 (C-2", C-6"), 126.4 (C-3", C-5"),
122.9 (C-6'), 118.3 (C-4'), 116.3 (C-2'), 55.6 (C-2"'),
43.7 (C-6"'), 33.9 (C-3"'), 31.3 (C-7"), 25.2 (C-5"'),
18.7 (C-4"'), 17.5 (C-7"'); EI-MS (m/z): 444 [M]+,
282 [C15H12N3OS]+, 252 [C13H18NO2S]+, 192
1H NMR: 7.89 (d, J = 8.0 Hz, 1H, H-6'), 7.76 (d, J =
8.4 Hz, 2H, H-3", H-5"), 7.57 (d, J = 8.0 Hz, 2H, H-
2", H-6"), 7.52 (d, J = 8.0 Hz, 1H, H-3'), 7.44 (t, J =
7.6 Hz, 1H, H-5'), 7.37 (t, J = 7.6 Hz, 1H, H-4'), 4.53
(s, 2H, H-7"), 4.21 (br.s, 1H, He-6"'), 3.72–3.65 (m,
1H, Ha-6"'), 3.24–3.13 (m, 1H, H-2"'), 3.02–2.99 (m,
2H, He-4"', He-5"'), 1.55–1.23 (m, 4H, He-3"', Ha-3"'
[C8H6N3OS]+, 162 [C6H12NO2S]+, 134 [C7H6N2O]+,
120 [C7H6NO]+, 98 [C6H12N]+, 92 [C6H6N]+.
5-Phenyl-2-(4-(2-methylpiperidin-1-yl-sulfonyl)benz-
ylthio)-1,3,4-oxadiazole (VId). Light brown sticky
solid; yield 77%; molecular formula: C21H23N3O3S2;
Ha-4"', Ha-5"'), 1.03 (d, J = 6.4 Hz, 3H, CH3-7"'); 13C
NMR: 165.2 (C-5), 161.9 (C-2), 144.3 (C-4"), 140.5
(C-1"), 138.3 (C-4'), 136.3 (C-2'), 135.1 (C-3'), 128.8
(C-2", C-6"), 127.2 (C-6'), 126.4 (C-5'), 126.0 (C-3",
C-5"), 124.1 (C-1'), 55.1 (C-2"'), 43.9 (C-6"'), 33.4
(C-3"'), 31.7 (C-7"), 25.6 (C-5"'), 18.8 (C-4"'), 17.9
(C-7"'); EI- MS (m/z): 466 [M+2]+, 464 [M]+, 301
[C15H10ClN2O3S2]+, 251 [C13H17O2NS]+, 238
molecular mass 429.5 g mol–1; IR: 3027 (Ar C-H),
2589 (S-H), 1671 (C=N), 1521 (Ar C=C), 1431 (SO2),
1231, 1059 (C–O–C), 610 (C–S bond str.); 1H NMR:
7.95 (d, J = 7.2 Hz, 1H, H-2', H-6'), 7.75 (d, J = 7.6
Hz, 2H, H-3", H-5"), 7.57 (d, J = 8.0 Hz, 2H, H-2",
H-6"), 7.52–7.45 (m, 3H, H-3', H-4', H-5'), 4.52 (s,
2H, H-7"), 4.20 (br.s, 1H, He-6"'), 3.64 (br.s, 1H, Ha-
6"'), 3.23–3.16 (m, 1H, H-2"'), 3.02–2.91 (m, 2H, He-
4"', He-5"'), 1.54–1.22 (m, 4H, He-3"', Ha-3"', Ha-4"', [C12H16NO2S]+, 211 [C8H4ClN2OS]+, 162 [C6H12-
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336
AZIZ-UR-REHMAN et al.
NO2S]+, 153 [C7H4ClNO]+, 139 [C7H4ClN]+, 111
[C6H4Cl]+, 98[C6H12N]+ .
(d, J = 8.0 Hz, 2H, H-3", H-5"), 7.56 (d, J = 7.6, 2H,
H-2", H-6"), 7.27 (d, J = 8.0 Hz, 2H, H-3', H-5'),
4.52 (s, 2H, H-7"), 4.21 (br.s, 1H, He-6"'), 3.66-3.58
(m, 1H, Ha-6"'), 3.24 (br.s, 1H, H-2"'), 3.02–2.83 (m,
2H, He-4"', He-5"'), 1.54–1.23 (m, 4H, He-3"', Ha-3"',
Ha-4"', Ha-5"'), 2.39 (s, 3H, H-7'), 1.03 (d, J = 6.4 Hz,
5-(3-Nitrophenyl)-2-(4-(2-methylpiperidin-1-yl-
sulfonyl)benzylthio)-1,3,4-oxadiazole (VIg). Light yel-
low amorphous solid; yield 83%; mp 110–112°C;
molecular formula: C21H22N4O5S2; molecular mass
3H, CH3-7"'); 13C NMR: 164.9 (C-5), 164.1 (C-2),
144.9 (C-4"), 142.7 (C-4'), 141.3 (C-1"), 130.5 (C-2',
C-6'), 129.4 (C-1'), 128.4 (C-2", C-6"), 127.6 (C-3',
C-5'), 125.4 (C-3", C-5"), 56.2 (C-2"'), 43.3 (C-6"'),
34.5 (C-3"'), 32.1 (C-7"), 24.7 (C-5"'), 20.4 (C-7'),
19.3 (C-4"'), 18.5 (C-7"'); EI-MS (m/z): 443 [M]+,
281 [C16H13N2OS]+, 251 [C13H17O2NS]+, 238 [C12H16-
474.5 g mol–1; IR: 3032 (Ar C–H), 2532 (S–H), 1651
(C=N), 1520 (Ar C=C), 1427 (SO2), 1230, 1057 (C–
O–C), 613 (C–S); 1H NMR: 7.89 (s, 1H, H-2'), 7.76
(d, J = 8.4 Hz, 2H, H-3", H-5"), 7.65 (d, J = 8.0 Hz,
1H, H-6'), 7.57 (d, J = 8.0, 2H, H-2", H-6"), 7.47 (t,
J = 7.6 Hz, 1H, H-4'), 7.35 (t, J = 7.6 Hz, 1H, H-5'),
4.53 (s, 2H, H-7"), 4.21 (br.s, 1H, He-6"'), 3.72–3.65
(m, 1H, Ha-6"'), 3.24-3.13 (m, H-2"'), 3.02–2.99 (m,
2H, He-4"', He-5"'), 1.55–1.23 (m, 4H, He-3"', Ha-3"'
NO2S]+, 191 [C9H8N2OS]+, 163 [C6H12NO2S]+, 133
[C8H7NO]+, 119 [C8H7O]+, 98 [C6H12N]+, 91
[C6H4CH3]+.
5-(2-Methoxyphenyl)-2-(4-(2-methylpiperidin-1-
yl-sulfonyl)benzylthio)-1,3,4-oxadiazole (VIj). Brown
sticky solid; yield 75%; molecular formula:
C22H25N3O4S2; molecular mass: 459.5 g mol–1; IR:
3041 (Ar C–H), 2540 (S–H), 1645 (C=N), 1533 (Ar
C=C), 1439 (SO2), 1225, 1069 (C–O–C), 625 (C–S);
Ha-4"', Ha-5"'), 1.03 (d, J = 6.4 Hz, 3H, CH3-7"'); 13C
NMR: 165.9 (C-5), 163.7 (C-2), 145.9 (C-3'), 144.1
(C-4"), 141.4 (C-1"), 134.4 (C-6'), 132.3 (C-1'), 130.5
(C-5'), 128.6 (C-2", C-6"), 127.1 (C-3", C-5"), 125.3
(C-4'), 120.7 (C-2'), 57.4 (C-2"'), 44.5 (C-6"'), 34.1
(C-3"'), 32.3 (C-7"), 25.8 (C-5"'), 19.2 (C-4"'), 18.0
(C-7"'); EI-MS (m/z): 474 [M]+, 312 [C15H10N3O3S]+,
251 [C13H17O2NS]+, 238 [C12H16NO2S]+, 222
[C8H4N3O3S]+, 162 [C6H12NO2S]+, 164 [C7H4N2O3]+,
122 [C6H4NO2]+, 150 [C7H4NO3]+, 98 [C6H12N]+ .
5-Benzyl-2-(4-(2-methylpiperidin-1-yl-sulfonyl)-
benzylthio)-1,3,4-oxadiazole (VIh). Light green amor-
phous solid; yield 76%; molecular formula:
C22H25N3O3S2; molecular mass: 443.5 g mol–1; IR:
3035 (Ar C–H), 2536 (S–H), 1659 (C=N), 1526 (Ar
1H NMR: 7.84 (d, J = 7.6 Hz, 1H, H-6'), 7.79 (t, J =
8.8 Hz, 1H, H-4'), 7.75 (d, J = 8.0 Hz, 2H, H-3", H-
5"), 7.57 (d, J = 7.6, 2H, H-2", H-6"), 7.47 (t, J = 8.0
Hz, 1H, H-5'), 4.50 (s, 2H, H-7"), 4.21 (br.s, 1H, He-
6"'), 3.96 (s, 3H, H-7'), 3.68 (br.s, 1H, Ha-6"'), 3.23
(br.s, 1H, H-2"'), 3.02–2.92 (m, 2H, He-4"', He-5"'),
1.53–1.23 (m, 4H, He-3"', Ha-3"', Ha-4"', Ha-5"'),
1.03 (d, J = 6.4 Hz, 3H, CH3-7"'); 13C NMR: 169.9
C=C), 1432 (SO2), 1229, 1059 (C–O–C), 618 (C–S); (C-5), 164.5 (C-2), 154.6 (C-2'), 143.8 (C-4"), 141.8
1H NMR: 7.75 (d, J = 8.0 Hz, 2H, H-3", H-5"), 7.57
(d, J = 7.6 Hz, 2H, H-2", H-6"), 7.33 (d, J = 7.6 Hz,
2H, H-3', H-5'), 7.26 (t, J = 8.0 Hz, 1H, H-4'), 7.23
(d, J = 7.6 Hz, 2H, H-2', H-6'), 4.77 (s, 2H, H-7'),
4.53 (s, 2H, H-7"), 4.23-4.12 (m, 1H, He-6"'), 3.67
(C-1"), 130.7 (C-4'), 128.2 (C-2", C-6"), 127.6 (C-3",
C-5"), 124.4 (C-6'), 122.3 (C-5'), 113.5 (C-3'), 108.7
(C-1'), 57.1 (C-2"'), 56.4 (C-7'), 44.8 (C-6"'), 34.2 (C-
3"'), 31.9 (C-7"), 25.4 (C-5"'), 19.4 (C-4"'), 18.6 (C-
7"'); EI-MS (m/z): 495 [M]+, 207 [C9H7N2O2S]+, 251
(br.s, 1H, Ha-6"'), 3.23 (br.s, 1H, H-2"'), 3.02–2.86 [C13H17O2NS]+,
238
[C12H16NO2S]+,
297
[C16H13N2O2S]+, 163 [C6H12NO2S]+, 149 [C8H7-
NO2]+, 135 [C8H7O2]+, 107 [C7H7O]+, 98 [C6H12N]+.
(m, 2H, He-4"'-He-5"'), 1.54–1.23 (m, 4H, He-3"',
Ha-3"', Ha-4"', Ha-5"'), 1.02 (d, J = 7.2 Hz, 3H, CH3-
7"'); 13C NMR: 165.6 (C-5), 164.7 (C-2), 144.3 (C-
4"), 141.0 (C-1"), 136.2 (C-1'), 128.8 (C-2", C-6"),
128.5 (C-3', C-5'), 128.3 (C-2', C-6'), 127.2 (C-3", C-
5"), 125.9 (C-4'), 55.2 (C-2"'), 43.4 (C-6"'), 34.2 (C-
3"'), 33.1 (C-7'), 32.7 (C-7"), 24.5 (C-5"'), 19.4 (C-
4"'), 18.6 (C-7"'); EI-MS (m/z): 443 [M]+, 281
[C16H13N2OS2]+, 251 [C13H17O2NS]+, 238 [C12H16-
5-(3-Chlorophenyl)-2-(4-(2-methylpiperidin-1-yl-
sulfonyl)benzylthio)-1,3,4-oxadiazole (VIk). Light
brown sticky solid; yield 79%; molecular formula:
C21H22ClN3O3S2; molecular mass: 464.0 g mol–1; IR:
3035 (Ar C–H), 2534 (S–H), 1659 (C=N), 1526 (Ar
C=C), 1432 (SO2), 1227, 1059 (C–O–C), 618 (C–S);
1H NMR: 7.98 (s, 1H, H-2'), 7.76 (d, J = 8.0 Hz, 2H,
H-3", H-5"), 7.54 (d, J = 7.6 Hz, 2H, H-2", H-6"),
7.44–7.39 (m, 3H, H-4', H-5', H-6'), 4.77 (s, 2H, H-
7"), 4.23–4.12 (m, 1H, He-6"'), 3.67 (br.s, 1H, Ha-
NO2S]+, 193 [C9H9N2OS]+, 162 [C6H12NO2S]+, 133
[C8H7NO]+, 91 [C7H7]+, 119 [C8H7O]+, 98 [C6H12N]+ .
5-(4-Methylphenyl)-2-(4-(2-methylpiperidin-1-yl-
sulfonyl)benzylthio)-1,3,4-oxadiazole (VIi). Light yel- 6"'), 3.23 (br.s, 1H, H-2"'), 3.02–2.86 (m, 2H, He-4"',
low amorphous solid; yield 81%; molecular formula:
C22H25N3O3S2; molecular mass 443.5 g mol–1; IR:
3039 (Ar C–H), 2537 (S-H), 1641 (C=N), 1533 (Ar
C=C), 1439 (SO2), 1223, 1079 (C–O–C), 620 (C–S);
He-5"'), 1.54–1.23 (m, 4H, He-3"', Ha-3"', Ha-4"', Ha-
5"'), 1.02 (d, J = 7.2 Hz, 3H, CH3-7"'); 13C NMR:
165.7 (C-5), 164.1 (C-2), 143.5 (C-4"), 141.3 (C-1"),
133.1 (C-3'), 130.9 (C-2'), 130.1 (C-1'), 128.9 (C-2",
1H NMR: 7.85 (d, J = 7.2 Hz, 2H, H-2', H-6'), 7.75 C-6"), 128.3 (C-4'), 128.0 (C-6'), 127.7 (C-5'), 127.1
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SYNTHESIS AND PHARMACOLOGICAL SCREENING
337
(C-3", C-5"), 57.3 (C-2"'), 44.5 (C-6"'), 34.6 (C-3"'), 32.4 (VIn). Light green amorphous solid; yield 80%; mp
(C-7"), 25.7 (C-5"'), 19.2 (C-4"'), 18.4 (C-7"'); EI-MS 150–152°C; molecular formula: C21H20ClN5O7S2;
(m/z): 466 [M+2]+, 464 [M]+, 301 [C15H10ClN2OS]+,
252 [C13H18NO2S]+, 211 [C8H4ClN2OS]+, 162
[C6H12NO2S]+, 153 [C7H4ClNO]+, 139 [C7H4ClO]+,
111 [C6H4Cl]+, 98 [C6H12N]+.
molecular mass 554 g mol–1; IR: 3035 (Ar C–H),
2534 (S–H), 1679 (C=N), 1526 (Ar C=C), 1432
1
(SO2), 1224, 1059 (C–O–C), 618 (C–S); H NMR:
7.98 (s, 1H, H-6'), 7.83 (s, 1H, H-4'), 7.75 (d, J = 8.0
Hz, 2H, H-3", H-5"), 7.57 (d, J = 7.6 Hz, 2H, H-2",
H-6"), 4.77 (s, 2H, H-7"), 4.23–4.12 (m, 1H, He-6"'),
3.67 (br.s, 1H, Ha-6"'), 3.23 (br.s, 1H, H-2"'), 3.02–
2.86 (m, 2H, He-4"', He-5"'), 1.54–1.23 (m, 4H, He-
3"', Ha-3"', Ha-4"', Ha-5"'), 1.02 (d, J = 7.2 Hz, 3H,
5-(3,5-Dinitrophenyl)-2-(4-(2-methylpiperidin-1-
yl-sulfonyl)benzylthio)-1,3,4-oxadiazole (VIl). Green
amorphous solid; Yield 80%; mp 112–114°C; molecu-
lar formula: C21H21N5O7S2; molecular mass 519.5 g
mol–1; IR: 3035 (Ar C–H), 2534 (S–H), 1669
(C=N), 1526 (Ar C=C), 1432 (SO2), 1234, 1059 (C–
CH3-7"'); 13C NMR: 170.6 (C-5), 164.2 (C-2), 153.6
(C-3'), 147.4 (C-5'), 143.9 (C-4"), 139.1 (C-1"), 135.8
(C-1'), 133.2 (C-2'), 128.8 (C-2", C-6"), 127.3 (C-3",
C-5"), 126.3 (C-6'), 123.4 (C-4'), 57.1 (C-2"'), 44.3
(C-6"'), 34.7 (C-3"'), 32.2 (C-7"), 25.4 (C-5"'), 19.2
(C-4"'), 18.6 (C-7"'); EI-MS (m/z): 555 [M+2]+, 553
[M]+, 392 [C15H8ClN4O5S]+, 301 [C8H2ClN4O5S]+,
1
O–C), 618" (C–S); H NMR: 8.85 (s, 2H, H-2', H-
6'), 8.52 (s, 1H, H-4'), 7.75 (d, J = 8.0 Hz, 2H, H-3",
H-5"), 7.57 (d, J = 7.6 Hz, 2H, H-2", H-6"), 4.77 (s,
2H, H-7"), 4.23–4.12 (m, 1H, He-6"'), 3.67 (br.s, 1H,
Ha-6"'), 3.23 (br.s, 1H, H-2"'), 3.02–2.86 (m, 2H, He-
4"', He-5"'), 1.54–1.23 (m, 4H, He-3"', Ha-3"', Ha-4"',
251 [C13H17O2NS]+, 238 [C12H16NO2S]+, 162 [C6H12-
NO2S]+, 243 [C7H2ClN3O5]+, 229 [C7H2ClN2O5]+,
201 [C6H2ClN2O4]+, 98 [C6H12N]+.
Ha-5"'), 1.02 (d, J = 7.2 Hz, 3H, CH3-7"'); 13C NMR:
169.7 (C-5), 164.5 (C-2), 147.3 (C-3', C-5'), 144.6 (C-
4"), 140.1 (C-1"), 135.4 (C-1'), 128.7 (C-2", C-6"),
128.1 (C-2', C-6'), 126.8 (C-3", C-5"), 114.7 (C-4'),
56.6 (C-2"'), 43.8 (C-6"'"'), 34.3 (C-3"'), 32.6 (C-7"),
24.2 (C-5"'), 19.5 (C-4"'), 18.8 (C-7"'); EI-MS (m/z):
519 [M]+, 357 [C15H9N4O5S]+, 251 [C13H17O2NS]+,
Antibacterial Assay
The activity was measured in sterile 96-well micro
plates under aseptic conditions. The method is based
on the principle that microbial cell number increases
as the microbial growth proceeds in a log phase of
growth, which results in increased absorbance of broth
medium [22]. Three gram-negative (Escherichia coli,
Pseudomonas aeruginosa, and Salmonella typhi) and
two gram-positive bacteria (Bacillus subtilis, Staphylo-
coccus aureus) were included in the study. The organ-
isms were maintained on stock culture agar medium.
The test samples with suitable solvents and dilutions
were pipetted into wells (20 μg/well) and maintained
overnight. After suitable dilution with fresh nutrient
broth, thefresh overnight bacterial culture was poured
into wells (180 μL). The initial absorbance of the cul-
ture was strictly maintained between 0.12–0.19 at
540 nm. The total volume in each well was kept to
238 [C12H16NO2S]+, 267 [C8H4N4O5S]+, 163 [C6H12-
NO2S]+, 209 [C7H3N3O5]+, 195 [C7H3N2O5]+, 167
[C6H3N2O4]+, 98 [C6H12N]+ .
5-(2,4-Dichlorophenyl)-2-(4-(2-methylpiperidin-
1-yl-sulfonyl)benzylthio)-1,3,4-oxadiazole (VIm). Off
white amorphous solid; yield 80%; mp 86–88°C;
molecular formula: C21H21Cl2N3O3S2; molecular
mass 498.4 g mol–1; IR: 3035 (Ar C–H), 2534 (S–H),
1689 (C=N), 1526 (Ar C=C), 1432 (SO2), 1227, 1060
(C–O–C), 618 (C–S); 1H NMR: 7.93 (s, 1H, H-3'),
7.85 (d, J = 7.6 Hz, 1H, H-6'), 7.75 (d, J = 8.0 Hz, 2H,
H-3", H-5"), 7.48 (d, J = 7.6 Hz, 2H, H-2", H-6"),
7.44 (d, J = 8.0 Hz, 1H, H-5'), 4.77 (s, 2H, H-7"),
4.23-4.12 (m, 1H, He-6"'), 3.67 (br.s, 1H, Ha-6"'), 3.23
(br.s, 1H, H-2"'), 3.02–2.86 (m, 2H, He-4"', He-5"'), 200 μL. The incubation was done at 37°C for 16–24 h
with lid on the micro plate. The absorbance was mea-
sured at 540 nm using micro plate reader, before and
after incubation, and difference was noted as an index
of bacterial growth. The percent inhibition was calcu-
lated using the formula:
1.54–1.23 (m, 4H, He-3"', Ha-3"', Ha-4"', Ha-5"'), 1.02
13
(d, J = 7.2 Hz, 3H, CH3-7"'); C NMR: 167.2 (C-5),
164.6 (C-2), 143.7 (C-4"), 139.3 (C-1"), 135.3 (C-4'),
133.8 (C-2'), 131.4 (C-6'), 130.7 (C-3'), 128.6 (C-2",
C-6"), 127.4 (C-5'), 127.0 (C-3", C-5"), 125.8 (C-1'),
57.6 (C-2"'), 44.2 (C-6"'), 34.5 (C-3"'), 32.7 (C-7"),
25.1 (C-5"'), 19.4 (C-4"'), 18.8 (C-7"'); EI-MS (m/z):
501 [M+4]+, 499 [M+2]+, 497 [M]+, 336 [C15H9-
Inhibition (%) = [(X – Y)/X] × 100,
where X is absorbance in control with bacterial culture
and Y is absorbance in test sample. Results are mean
of triplicate (n = 3) SEM. Ciprofloxacin was taken
as standard.
Cl2N2OS]+, 251 [C13H17O2NS]+, 238 [C12H16NO2S]+,
246 [C8H3Cl2N2OS]+, 162 [C6H12NO2S]+, 186
[C7H3Cl2NO]+, 145 [C6H3Cl2]+, 172 [C7H3Cl2O]+, 98
[C6H12N]+.
Cholinesterase Enzyme Inhibition Assay
5-(2-Chloro-3,5-dinitrophenyl)-2-(4-(2-meth-
ylpiperidin-1-yl-sulfonyl)benzylthio)-1,3,4-oxadiazole
The acetylcholinesterase and butyrylcholinesterase
activity was measured according to the method
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 3 2017

338
AZIZ-UR-REHMAN et al.
described [23] with minor modification in ration of mixing 45 μL phenol reagent with 70 μL of alkali
quantities taken. Total volume of reaction mixture was reagent). For color development, incubation was per-
taken as 100 μL; it contained 60 μL of Na2HPO4 as formed at 37°C for another 10 min. Absorbance was
measured at 625 nm using the 96-well plate reader
Synergy HT BioTek, USA. The percentage inhibition
and IC50 values were calculated similarly to cholines-
buffer (50 mM) and pH was maintained at 7.7. Test
sample (10 μL/well, 0.5 mM) was added followed by
addition of AChE or BChE (10 μL, 0.5 units/well;
Sigma Inc.). The contents were homogenized, pre- terase assay.
read at 405 nm, pre-incubated at 37°C for 10 min. The
reaction was initiated by the addition of 10 μL of
0.5 mM acetylthiocholine chloride (for AChE) or
CONCLUSION
butyrylthiocholine chloride (for BChE) as substrate.
Then, 10 μL of DTNB was added, which was 0.5 mM
per well. After 15 min of incubation at 37◦C, absor-
bance was measured at 405 nm (using 96-well plate
reader Synergy HT, Biotek, USA). All experiments
were performed as triplicates with reference to their
suitable standard. Eserine 0.5 mM per well was taken
as control. The percent inhibition was calculated by
using the formula:
All the molecules were synthesized in excellent
yields given in experimental section. The structures of
all target molecules were well corroborated by their
spectral data of IR, 1H and 13C NMR, and EI-MS. All
the compounds were subjected to antibacterial and
enzyme inhibition analysis. The different bacterial
strains taken into account included gram-positive
(B. subtilis and S. aureus) and gram-negative bacterial
strains (S. typhi, E. coli, and P. aeruginosa). The
enzyme inhibition study involved cholinesterases
(AChE, BChE), α-glucosidase, and urease enzyme.
The compounds remained more efficient against
gram-negative bacterial strains as compared to gram-
positive ones. The BChE enzyme is more efficiently
inhibited by these compounds as compared to others.
In this way, the most active compounds could be
potential target in the drug invention and drug devel-
opment program.
Inhibition (%) = [(Control – test)/control] × 100.
IC50 values were calculated by using EZ Fit
“enzyme kinetics software”, Parrella Scientific Inc.,
Amherst, USA.
α-Glucosidase Enzyme Inhibition Assay
The enzyme inhibition activity against α-glucosi-
dase was measured with small modification in the
reported procedure [24]. α-Glucosidase assay was
made with addition of 70 μL phosphate buffer at pH
6.8 with 50 mM concentration to 10μL (0.5 mM) of
test compound, which was further mixed with 10 μL
(0.057 units) enzyme to make the total volume of the
reaction mixture 100 μL. This blank sample was mixed
and the absorbance was measured at 400 nm, which
was followed by incubation at 37°C for 10 min. Subse-
quently, the enzymatic activity was preceded by the
addition of 10 μL of p-nitrophenylglucopyranoside
(0.5 mM) substrate. Acarbose was utilized as positive
control for this enzymatic activity. Again, absorbance
was taken at 400 nm followed by incubation for 30 min
at 37°C. All tests were performed in twice. The per-
centage inhibition and IC50 were calculated similarly
to cholinesterase assay.
ACKNOWLEDGMENTS
The authors are thankful to the Higher Education
Commission (HEC) of Pakistan for the financial
assistance.
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