N-(5-Methyl-1,3-Thiazol-2-yl)-2-{[5-((Un)Substituted- Phenyl)1,3,4-Oxadiazol-2-yl]Sulfanyl}acetamides. Unique Biheterocycles as Promising Therapeutic Agents

Article abstract of DOI:10.1134/S106816201806002X

An electrophile, 2-bromo-N-(5-methyl-1,3-thiazol-2-yl)acetamide, was synthesized by the reaction of 5-methyl-1,3-thiazol-2-amine and bromoacetyl bromide in an aqueous medium. In a parallel scheme, a series of (un)substituted benzoic acids was converted sequentially into respective esters, acid hydrazides, and then into 1,3,4-oxadiazole heterocyclic cores. The electrophile was coupled with the aforementioned 1,3,4-oxadiazoles to obtain the targeted bi-heterocyles. Structural analysis of the synthesized compounds was performed by IR, EI-MS, ¹H NMR, and ¹³C NMR. The enzyme inhibition study of these molecules was carried out against four enzymes, namely, acetylcholinesterase, butyrylcholinesterase, α-glucosidase, and urease. The interactions of these compounds with respective enzymes were recognized by their in silico study. Moreover, their cytotoxicity was also determined to find out their utility as possible therapeutic agents.

Full text of DOI:10.1134/S106816201806002X

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2018, Vol. 44, No. 6, pp. 801–811. © Pleiades Publishing, Ltd., 2018.
N-(5-Methyl-1,3-Thiazol-2-yl)-2-{[5-((Un)Substituted-
Phenyl)1,3,4-Oxadiazol-2-yl]Sulfanyl}acetamides. Unique
Biheterocycles as Promising Therapeutic Agents1
M. A. Abbasia, b, 2, M. S. Ramzanb, Aziz-ur-Rehmanb, S. Z. Siddiquib, S. A. A. Shahc, d,
M. Hassana, S.-Y. Seoa, M. Ashrafe, B. Mirzaf, and H. Ismailf
aCollege of Natural Science, Department of Biological Sciences, Kongju National University, Gongju, 32588 South Korea
bDepartment of Chemistry, Government College University, Lahore, 54000 Pakistan
cFaculty of Pharmacy, University Technology MARA, Puncak Alam Campus,
Bandar Puncak Alam, Selangor Darul Ehsan, 42300 Malaysia
dAtta-ur-Rahman Institute for Natural Products Discovery (AuRIns), University Technology MARA,
Puncak Alam Campus, Bandar Puncak Alam, Selangor Darul Ehsan, 42300 Malaysia
eDepartment of Chemistry, Islamia University of Bahawalpur, Bahawalpur, 63100 Pakistan
fDepartment of Biochemistry, Quaid-i-Azam University, Islamabad, 45320 Pakistan
Received June 7, 2018; in final form, July 10, 2018
Abstract⎯An electrophile, 2-bromo-N-(5-methyl-1,3-thiazol-2-yl)acetamide, was synthesized by the reac-
tion of 5-methyl-1,3-thiazol-2-amine and bromoacetyl bromide in an aqueous medium. In a parallel scheme,
a series of (un)substituted benzoic acids was converted sequentially into respective esters, acid hydrazides,
and then into 1,3,4-oxadiazole heterocyclic cores. The electrophile was coupled with the aforementioned
1,3,4-oxadiazoles to obtain the targeted bi-heterocyles. Structural analysis of the synthesized compounds was
performed by IR, EI-MS, 1H NMR, and 13C NMR. The enzyme inhibition study of these molecules was car-
ried out against four enzymes, namely, acetylcholinesterase, butyrylcholinesterase, α-glucosidase, and ure-
ase. The interactions of these compounds with respective enzymes were recognized by their in silico study.
Moreover, their cytotoxicity was also determined to find out their utility as possible therapeutic agents.
Keywords: 5-methyl-1,3-thiazol-2-amine, 1,3,4-oxadiazole, acetamide, cholinesterases, glucosidase, urease,
brine shrimps
DOI: 10.1134/S106816201806002X
INTRODUCTION
diazoles belong to a group of heterocyclic compounds
that have enjoyed attention of researchers for the last
two decades; numerous derivatives have been synthe-
sized and evaluated for an extensive range of biological
properties. Molecules with 1,3,4-oxadiazole nucleus
are known to exhibit unique anti-edema [3] and anti-
inflammatory activities [4, 5]. Literature survey
revealed that minor modification in the structure of
1,3,4-oxadiazole can lead to quantitative, as well as
qualitative, changes in biological activity. These het-
erocyclic derivatives have inhibitory effect against HIV
replication, leukemia, colon, and breast cancer [6, 7].
1,3,4-Oxadiazole has two extendable positions at sec-
ond and fifth carbons; therefore a large number of
2,5-disubstituted derivatives have been synthesized and
attracted attention due to their broad range of biological
activity, such as anti-inflammatory, antifungal [8], anti-
parasitic [9], and antimicrobial effects [10].
Heterocyclic compounds containing five-mem-
bered heterocyclic units have a diversity of valuable
biological effects. In medicinal chemistry, heterocy-
clic nucleus imparts an important function and serves
as a key template for the development of various ther-
apeutic agents. Different classes of heterocyclic com-
pounds possess a broad range of pharmacological
activities. Compounds bearing thiazole ring are used
as drugs in treatment of cancer, lowering blood pres-
sure, and treatment of infection [1]. Researchers have
proven the in vitro potency of some thiazole deriva-
tives to inhibit the bacterial pathogens [2]. 1,3,4-Oxa-
Abbreviations: AChE, acetylcholinesterase; AGIs, α-glucosi-
dase inhibitors ; BChE, butyrylcholinesterase.
1
2
The article is submitted in the original.
Corresponding author: phone: (+82)-416-8508503; e-mail:
abbasi@gcu.edu.pk.
801

802
ABBASI et al.
O
C
N
O
C
N
(a)
+
Br
Br
NH2
O
N
H
H3C
H3C
R1
S
Br
S
(I)
(II)
(III)
3'''
(e)
N
2
4
5
3
C
S
2'
2''
O
1'''
1
S
R1
1'
N
5''
1''
5'''
H3C
H
(VIIIa−n)
3''N
R2
HS
N 4''
O
2-{[5-(Un/substitued-phenyl)-1,3,4-oxadiazol-2-yl]
sulfanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide
R2
(VIIa−n)
N N
(d)
O
O
C
O
C
R1
NH2
R1
C
N
H
R1
OH
OEt
(b)
(c)
(IVa−n)
(Va−n)
(VIa−n)
R2
R2
R2
Scheme 1. Protocol for the synthesis of 2-{[5-((un)substituted-phenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-N-(5-methyl-
1,3-thiazol-2-yl)acetamide (VIIIa–n). Reagents and conditions: (a) H2O, 20% Na2CO3 solution, stirring for 2 h.
(b) H2SO4, EtOH, refluxing for 4–5 h; (c) N2H4, EtOH, refluxing for 3–4 h; (d) CS2, KOH, EtOH, refluxing for 5–6 h;
(e) DMF, LiH, stirring for 4–6 h.
Acetylcholinesterase (AChE, or acetylhydrolase; and controlling urease activity is a promising
EC 3.1.1.7) is the primary cholinesterase in the body, approach.
which catalyzes breakdown of acetylcholine and some
Diabetes mellitus is an endocrine disorder charac-
terized by hyperglycemia and associated with distur-
bances of carbohydrate, fat, and protein metabolism
resulting from defects in insulin secretion, insulin
action, or both. α-Glucosidase inhibitors (AGIs) are
drugs that inhibit the absorption of carbohydrates
from the gut and may be used in treatment of patients
with diabetes type 2 or impaired glucose tolerance [14,
15]. α-Glucosidase inhibitors, such as acarbose and
miglitol, have been approved for clinical use in man-
agement of diabetes type 2, as well as the treatment of
diabetic complications. A main drawback of the cur-
rently used α-glucosidases, such as acarbose, are the
side effects such as abdominal distention, flatulence,
meteorism, and possible diarrhea [16].
other choline esters functioning as neurotransmitters.
Its activity serves to terminate synaptic transmission.
It belongs to carboxylesterase family of enzymes.
Butyrylcholinesterase (BChE) is of pharmacological
and toxicological importance, because it hydrolyzes
ester-containing drugs and scavenges cholinesterase
inhibitors, including potent organophosphorus nerve
agents, before they reach their synaptic targets [10, 11].
It has been found that BChE inhibition is an effective
tool to cure Alzheimer’s disease and dementias. For
the treatment of Alzheimer’s and related diseases, it is
of great importance to search for new cholinesterase
inhibitors as possible drug candidates [12].
Urease (urea amidohydrolase; EC 3.5.1.5) occurs
throughout the animal and plant kingdom. Many
microorganisms use this enzyme to provide a source of
nitrogen for growth; it plays an important role in plant
nitrogen metabolism during the germination process.
Of medical and veterinary interest, urease is a viru-
lence factor in certain human and animal pathogens;
it participates in the development of kidney stones,
pyelonephritis, peptic ulcers, and other disease states.
Meanwhile, the obvious remedy for treating bacterial
infection by Helicobacter pylori with antimicrobials has
Molecular docking analysis approximates the syn-
thesized ligands regarding their orientation and con-
formation at binding site of target protein. Precise
forecast of activity and structural modeling can be
achieved by docking studies. Furthermore, it eluci-
dates interactions between the target protein active site
and an inhibitor [17].
Based upon the aforementioned biological activi-
often been proven futile and only a few combination ties of various heterocyclic molecules, in the present
regimens have reached clinical practice. Thus, the research, amalgamation of two heterocyclic moieties,
need for alternative or novel treatment is evident [13] i.e. 1,3-thiazole and 1,3,4-oxadiazole, was carried out
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N-(5-METHYL-1,3-THIAZOL-2-YL)-2-…
803
Table 1. Different substituents (R1 and R2) in Scheme 1
to impart the designed molecules with possible thera-
peutic properties.
–R1
–R2
Compds.
(IV–VIIIa)
(IV–VIIIb)
(IV–VIIIc)
(IV–VIIId)
(IV–VIIIe)
(IV–VIIIf)
(IV–VIIIg)
(IV–VIIIh)
(IV–VIIIi)
(IV–VIIIj)
(IV–VIIIk)
(IV–VIIIl)
(IV–VIIIm)
(IV–VIIIn)
–H
4-CH3
4-OH
2-OCH3
2-Cl
3-Cl
4-Cl
2-Cl
3-NH2
4-NH2
2-NO2
3-NO2
4-NO2
3-NO2
–H
–H
RESULTS AND DISCUSSION
Chemistry
–H
–H
Various biheterocycles were obtained by starting
the synthesis from 5-methyl-1,3-thiazol-2-amine (I),
which was reacted with 2-bromoethanoyl bromide (II)
in a basic aqueous medium to obtain an electrophile,
2-bromo-N-(5-methyl-1,3-thiazol-2-yl)acetamide (III).
In a parallel series of reactions, different nucleophiles
were synthesized starting from (un)substituted-ben-
zoic acids (IVa–n) and converting them sequentially
into respective esters (Va–n), acid hydrazides (VIa–n),
and 5-(un/substituted-phenyl)-1,3,4-oxadiazole-2-
thiols (VIIa–n). The thiols, serving as nucleophiles,
were finally coupled one by one with compound (III),
in an aprotic polar solvent, i.e. DMF, using LiH as
a base, to obtain the targeted biheterocyclic mole-
cules (VIIIa–n). This synthesis is outlined in Scheme
and the substituents are listed in Table 1. Structures
of synthesized derivatives are corroborated by their
–H
–H
–H
4-Cl
–H
–H
–H
–H
–H
5-NO2
coupling in them. However, in its 13C NMR spectrum,
the appearance of four carbon resonances for this
methylated heterocyclic moiety at δ 155.82 (C-2),
134.90 (C-4), 126.67 (C-5), and 11.07 (C-6) was very
rational. Similarly, the other heterocyclic core of the
molecule, i.e. 1,3,4-oxadiazol-2-yl, was also affirmed
by two quaternary carbon resonances at δ 163.99 (C-5'')
and 162.73 (C-2''). Central C- and N-substituted acet-
amido group, which connects the two heterocycles in
the molecule, was inferred by two characteristic peaks
in both its 1H NMR and 13C NMR spectra. In the for-
mer spectrum, the peaks appeared at δ 12.36 (s, 1H,
CONH) and 4.38 (br.s, 2H, CH2-2') while in the latter
spectrum, the carbon resonances appeared at δ 164.97
(C-1') and 35.29 (C-2'). The mass fragmentation data
of this molecule, as described in experimental section,
was also in complete agreement with the above-dis-
cussed structural assignments. So, on the basis of col-
lective spectral evidences, the structure of (VIIId) was
confirmed and it was named 2-{[5-(2-methoxyphe-
nyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-N-(5-methyl-1,3-
thiazol-2-yl)acetamide (VIIId). A similar pattern was
adopted for the structural characterization of other
derivatives in the synthesized series.
1
IR, EI-MS, H NMR, and 13C NMR spectral data.
Structural assignment of one of the compounds is dis-
cussed hereby in detail for the convenience of the
readers. Compound (VIIId) was obtained as dull white
solid. The reaction yield was found to be 77% and the
melting point of the compound was 205–206°C.
Molecular formula of this compound, C15H14N4O3S2,
was predicted by the molecular ion peak at m/z 362 in
its EI-MS spectrum and by counting the number of
protons in its 1H NMR spectrum. The number of car-
bon resonances in its 13C NMR spectrum also sup-
ported the assignment. Various functional groups in
this molecule were identified by the IR data. Therein,
different absorption bands at ν 3358 (NH stretching),
2977 (CH stretching of aromatic ring), 2823 (OCH3
stretching, 1674 (C=N stretching), 1644 (C=O
stretching), 1575 (C=C stretching of aromatic ring),
1163 (C–O–C stretching) were observed for the spec-
ulated functionalities. The 2-methoxyphenyl group in
this molecule was ascribed by a set of discrete signals
1
in the aromatic region of its H NMR spectrum at
δ 7.77 (dd, J = 1.5, 7.7 Hz, 1H, H-6'''), 7.59 (br.t, J =
8.6 Hz, 1H, H-5'''), 7.24 (br.d, J = 8.5 Hz, 1H, H-3'''),
and 7.09 (br.t, J = 7.6 Hz, 1H, H-4''') along with a
methoxy group signal at 3.85 (br.s, 3H, CH3-7''').
Structural assignment of this unit was also fully sup-
ported by seven typical carbon resonances in its
13C NMR spectrum at δ 157.26 (C-2'''), 133.60 (C-5'''),
130.0 (C-6'''), 120.69 (C-4'''), 112.62 (C-3'''), 111.78
(C-1'''), and 55.92 (C-7'''). N-(5-Methyl-1,3-thiazol-
2-yl) moiety of the molecule was characterized by two
Enzyme Inhibition, Molecular Docking,
and Structure-Activity Relationship
All the synthesized compounds (VIIIa–n) were
assessed for their enzyme inhibition activity against
four enzymes, namely acetylcholinesterase, butyryl-
cholinesterase, urease, and α-glucosidase. All com-
pounds exhibited enzyme inhibition activity but dif-
fered in their potency compared to the standard refer-
1
distorted doublets in its H NMR spectrum at δ 7.16
(distorted d, J = 1.1 Hz, 1H, H-4) and 2.34 (distorted d,
J = 0.6 Hz, 3H, CH3-6). The unique multiplicity of
both these signals was an attribute of the mutual allylic ence used. The outcomes are recorded as percent
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 6 2018

804
Table 2. Percent inhibition at 0.5 mM and IC50 values for cholinesterases, urease, and α-glucosidase
AChE BChE Urease α-Glucosidase
inhibition, inhibition,
ABBASI et al.
Compd.
IC50, µM
IC50, µM
IC50, µM
IC50, µM
inhibition, %
inhibition, %
%
%
(VIIIa)
(VIIIb)
(VIIIc)
(VIIId)
(VIIIe)
(VIIIf)
(VIIIg)
(VIIIh)
(VIIIi)
(VIIIj)
(VIIIk)
(VIIIl)
49.19 0.15 43.15 0.13 55.15 0.15 157.35 0.30 63.27 0.25 117.22 0.45 71.15 0.45 297.29 0.26
89.29 0.21 57.51 0.17 49.27 0.26 121.17 0.55 71.55 0.15 220.31 0.22 73.55 0.15 198.12 0.21
87.29 0.35 55.17 0.07 77.14 0.25 141.15 0.19 81.35 0.15 245.15 0.19 89.17 0.25 52.11 1.2
79.37 0.30 50.41 0.19 89.62 0.34 74.52 0.25 91.15 0.45 84.17 0.23 75.17 0.35 235.33 0.22
63.43 0.29 51.15 0.19 77.23 0.35 115.05 0.21 87.17 0.25 222.30 0.35 83.35 0.15 24.23 0.29
75.55 0.21 59.25 0.21 49.27 0.35 105.29 0.15 79.43 0.35 245.13 0.23 64.17 0.71 291.15 0.17
89.29 0.19 83.17 0.09 53.14 0.35 89.17 0.35 43.19 0.30 149.45 0.17 70.49 0.19 232.55 0.19
93.27 0.44 11.49 0.13 73.42 0.45 215.64 0.29 49.29 0.25 199.35 0.55 50.35 0.17 272.44 0.21
89.49 0.37 81.55 0.19 89.74 0.63 69.27 0.35 67.15 0.45 145.21 0.17 50.29 0.25 289.27 0.24
88.77 0.39 19.35 0.10 61.26 0.15 113.85 0.45 90.23 0.17 76.26 0.25 81.43 0.35 270.33 0.25
83.76 0.30 35.34 0.12 77.47 0.16 163.23 0.17 77.17 0.35 105.25 0.15 75.15 0.45 140.25 0.26
81.41 0.19 29.19 0.04 55.47 0.27 83.35 0.41 51.29 0.22 97.15 0.29 73.29 0.22 242.11 0.29
(VIIIm) 70.87 0.21 43.35 0.01 71.35 0.36 171.25 0.17 61.17 0.71 249.11 0.17 69.23 0.17 310.17 0.11
(VIIIn)
Eserine
Thiourea
Acarbose
59.19 0.35 77.60 0.50 73.27 0.29 113.21 0.49 69.35 0.17 217.29 0.45 73.25 0.29 326.21 0.35
91.27 1.17 0.04 0.01 82.82 1.09 0.85 0.01
98.12 0.18 21.11 0.12
92.23 0.16 37.38 0.12
inhibition and IC50 (50% inhibitory concentration) in ing compounds (VIIIi) and (VIIIj), the observed order
was para > meta. For the mono-nitro compounds
(VIIIk–m), the order of activity was found to be meta >
ortho > para. Derivatives, (VIIIa–n), were docked into
the active pocket of this enzyme. From the docking
results, we have identified that compound (VIIIh) cre-
ated a couple of π–π and arene–cation interactions
with Trp82, Phe329, and His438 residues, with bond
Table 2. Cytotoxicity of these molecules was also eval-
uated and LD50 values for brine shrimps are reported
in Table 3.
All the molecules are noticeable inhibitors of
AChE. The most promising results against this enzyme
were shown by compounds (VIIIh) and (VIIIj) with
IC50 values of 11.49
0.13 and 19.35
0.10 µM, lengths of 3.39, 3.28, and 4.73 Å, respectively (Fig. 1).
respectively. This good inhibitory potential of (VIIIh)
might be attributed to the presence of 2,4-dichloro
groups, while that of (VIIIj) might be related to the
presence of 4-amino group in this molecule. Eserine
with IC50 value of 0.04 0.01 µM was used as standard
inhibitor against this enzyme. Among mono-chloro
compounds (VIIIe–g), the order of activity was found
to be as ortho > meta > para. However, for amino bear-
Against BChE,
a noticeable inhibition was
observed for compounds (VIIId) and (VIIIi) with IC50
values of 74.52 0.25 and 69.27 0.35 µM, respec-
tively. This inhibition might be due to the incorpora-
tion of 2-methoxy and 2-amino groups, respectively.
The reference used for BChE was also eserine with
IC50 value of 0.85 0.01 µM. Synthesized compounds
can be arranged in the following row according to their
inhibitory activity: (VIIIi) > (VIIId) > (VIIIl) >
(VIIIg) > (VIIIf) > (VIIIn) > (VIIIj) > (VIIIe) >
(VIIIb) > (VIIIc) > (VIIIm) > (VIIIk) > (VIIIh) (see
Table 2 for IC50 values). As follows from Fig. 2, arene–
arene interactions have been established for com-
pound (VIIId) with Tyr121 and Trp279, with bond
lengths of 2.14 and 3.54 Å, respectively.
Table 3. Brine shrimp activity assay
LD50,
mM
LD50,
mM
LD50,
mM
Compd.
Compd.
Compd.
(VIIIa)
(VIIIb)
(VIIIc) 275.5 (VIIIh)
(VIIId)
(VIIIe)
1.9
(VIIIf)
1.8
(VIIIk)
(VIIIl)
(VIIIm)
(VIIIn)
–
4.7 (VIIIg) 255.8
1.3
(VIIIi) 184.5
(VIIIj)
5.7
9.1
4.3
Against urease, all the active compounds displayed
moderate inhibitory potential when contrasted with
the reference, thiourea, with IC50 value of 21.11 0.12 μM.
The most active urease inhibitors were compounds
(VIIIj) and (VIIId) with IC50 values of 76.26 0.25
2.1
1.9
1.4 Doxorubicin 5.21
Doxorubicin was used as reference standard.
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N-(5-METHYL-1,3-THIAZOL-2-YL)-2-…
805
(a)
(b)
His
A438
Gly
A439
His438
Phe
A329
Glu
A197
Tyr
3.28
A332
4.73
Gly
A115
Ala
A328
Trp82
Trp
A82
Phe329
3.39
Pro
A285
Gly
A116
Thr
A120
2D
3D
Fig. 1. The 2D (a) and 3D (b) interaction analysis of compound (VIIIh) against AChE.
(a)
(b)
Asp
72
Tyr121
Tyr
121
Trp
279
Trp279
Asn
85
3.54
(64%)
2.14
Phe
330
Tyr
334
Trp
84
Phe
331
Gly
335
2D
3D
Fig. 2. The 2D (a) and 3D (b) interaction analysis of compound (VIIId) against BChE.
and 84.17 0.23 μM, respectively. Synthesized com- amidic nitrogen and 4-aminophenyl group with
Asp224 and Glu-166 at distances of 1.52 and 1.85 Å,
respectively. It also exhibited strong acidic interaction
through its acetamidic carbonyl oxygen with His323
showing a bond length of 1.58 Å.
pounds can be arranged in the following row according
to their inhibitory activity: (VIIIj) > (VIIId) > (VIIIl) >
(VIIIk) > (VIIIa) > (VIIIi) > (VIIIg) > (VIIIh) >
(VIIIn) > (VIIIb) > (VIIIe) > (VIIIf) > (VIIIc) >
(VIIIm) (see Table 2 for IC50 values). Conclusions
drawn from the inhibitory activity experiments were
supported by molecular docking studies. Figure 3
depicts docking results for the most active compound
(VIIIj). It demonstrates two very strong polar interac-
Against α-glucosidase, the reference used was acar-
bose with IC50 value of 37.38 0.12 µM. Compound
(VIIIe) with IC50 value of 24.11 0.29 μM was the
most active one against α-glucosidase. Synthesized
compounds can be arranged in the following row
tions through 5-methyl-1,3-thiazol-2-yl bearing acet- according to their inhibitory activity: (VIIIe) > (VIIIc) >
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 6 2018

806
ABBASI et al.
(a)
(b)
Cys
322
Ala
170
His
323
Glu
223
Lys
169
Asp
224
Glu
166
2D
3D
Fig. 3. The 2D (a) and 3D (b) interaction analysis of compound (VIIIj) against urease.
(a)
(b)
Asn
347
Asp
408
Arg
439
Phe
157
Asp
349
Gln
350
Arg
312
Phe
177
Asp
68
Phe
300
Glu
304
Val
303
2D
3D
Fig. 4. The 2D (a) and 3D (b) interaction analysis of compound (VIIIe) against α-glucosidase.
(VIIIn) > (VIIIk) > (VIIIb) > (VIIIg) > (VIIId) > tions with 2-chlorophenyl, 1,3,4-oxadiazole, and 5-
methyl-1,3-thiazol-2-yl units of the ligand, respectively.
(VIIIl) > (VIIIj) > (VIIIh) > (VIIIi) > (VIIIf) >
(VIIIa) > (VIIIm) > (VIIIn) (see Table 2 for IC50 val-
ues). The inhibitory activity experiment results were
likewise helped by molecular docking. Figure 4 shows
the docking results for the most active compound
(VIIIe), which was strongly bound with the active
pocket of α-glucosidase through five interactions.
Phe177 and Phe300 were found to be involved in a sep-
arate π–π interactions with 5-methyl-1,3-thiazol-2-yl
and 2-chlorophenyl moieties. Similarly, Arg312 and
Inhibitory activity of the newly synthesized com-
pounds against the four enzymes showed that mole-
cules bearing chloro, amino, hydroxy, and methoxy
groups were found to be better inhibitors, probably
because they fit well into the active sites of the
enzymes. Also, these functional groups are able to
generate better π–π interactions with the active site.
Thus, compounds (VIIId), (VIIIe), (VIIIh), (VIIIc),
and (VIIIj) were significantly more efficient among
Arg439 have also given three arene–cation interac- the whole series of synthesized molecules.
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N-(5-METHYL-1,3-THIAZOL-2-YL)-2-…
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Brine Shrimp Activity
General procedure for synthesis of (un)substituted-
benzohydrazides (VIa–n). Ethyl ester (Va–n)
(0.04 mol) was refluxed with 80% N2H4 · H2O
(7.2 mL) for 3–4 h in 20 mL EtOH in a round-bottom
flask (100 mL). The reaction was monitoredby TLC.
At completion, excess ice-cold distilled water (60 mL)
was added to get the precipitate, which was filtered,
washed with distilled water, and dried to acquire title
compounds (VIa–n) [18, 19].
Cytotoxicity of the compounds was analyzed by
brine shrimp activity assay. The high LD50 values of
compounds proved their low toxicity. Therefore, these
compounds might be suitable for use as drugs. Doxo-
rubicin was used as reference with LD50 value of
5.21 mM. Compounds (VIIIc), (VIIIg), (VIIIi), and
(VIIIp) with LD50 values of 275.5, 255.8, 184.5, and
186.5 mM were the most suitable ones.
General procedure for synthesis of 5-((un)substi-
tuted-phenyl)-1,3,4-oxadiazol-2-thiols
(VIIa–n).
Solid KOH (0.03 mol) was dissolved in 25 mL EtOH
on reflux in a 100 mL round-bottom flask. (Un)Sub-
stituted benzohydrazide (VIa–n) (0.03 mol) was
refluxed with CS2 (0.06 mol) in this basified EtOH for
5–6 h. The reaction was monitored by TLC. At com-
pletion, excess ice-cold distilled water (60 mL) was
added to form homogeneous solution. pH was
adjusted to 5–6 by pouring dilute HCl; precipitate
thus formed was filtered, washed with distilled water
and dried. The formed products (VIIa–n) were also
re-crystallized from EtOH [18, 19].
EXPERIMENTAL
General
All the chemicals, along with analytical grade sol-
vents, were purchased from Sigma Aldrich, Alfa Aesar
(Germany), or Merck through local suppliers. Pre-
coated silica gel Al-plates were used for TLC with ethyl
acetate and n-hexane as solvent system. Spots were
detected by UV254. Gallonkamp apparatus was used to
detect melting points in capillary tubes. IR spectra
(νmax, cm–1) were recorded by KBr pellet method in
the Jasco-320-A spectrophotometer. 1H NMR spectra
(δ, ppm) were recorded at 600 MHz and 13C NMR
spectra, at 150 MHz, in DMSO-d6 using the Bruker
Advance III 600 Ascend spectrometer using the BBO
probe. EI-MS spectra were measured on a JEOL
JMS-600H instrument with native data processing
system.
General procedure for synthesis of 2-{[5-((un)sub-
stituted-phenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-N-(5-
methyl-1,3-thiazol-2-yl)acetamides
(VIIIa–n).
5-((Un)substituted-phenyl)-1,3,4-oxadiazol-2-thiol
(VIIa–n) (0.004 mol) was dissolved in DMF (11 mL)
in
a 50-mL round-bottom flask. Then, LiH
(0.004 mol) was added and the mixture was stirred for
0.5 h. N-(5-Methyl-1,3-thiazol-2-yl)-2-bromoacet-
amide (III) (0.004 mol) was poured in and the mixture
was further stirred for 4–6 h. Completion of the reac-
tion was confirmed by TLC. Then, excess ice-cold dis-
tilled water (25 mL) was poured in small portions
along with continuous stirring. Aqueous NaOH was
added drop-wise upon gentle shaking to adjust the pH
to 8–10. The reaction mixture was aged for 1 h and the
precipitate of (VIIIa–n) was filtered, washed with dis-
tilled water, and dried.
Synthesis
N-(5-Methyl-1,3-thiazol-2-yl)-2-bromoacetamide
(III). 2-Amino-5-methyl-1,3-thiazole (I) (0.038 mol)
was dispersed in 25 mL distilled water in iodine flask
(100 mL) and 20% aq. Na2CO3 solution was poured to
adjust pH to 9–10. 2-Bromoethanoyl bromide (II)
(0.038 mol) was poured in small patches upon vigor-
ous shaking and then set to stir for further 2 h. Reac-
tion completion was monitored by TLC. Excess ice-
cold distilled water (40 mL) was added and the formed
precipitate was collected by filtration. The precipitate
of compound (III) was washed with distilled water and
dried.
General procedure for synthesis of ethyl (un)substi-
tuted benzoates (Va–n). (Un)Substituted benzoic acid
(IVa–n) (0.02 mol) was refluxed with 60 mL EtOH for
4–5 h in the presence of conc. H2SO4 (1.25 mL) in a
round-bottom flask (250 mL). TLC plates were used to
monitor the reaction. Excess distilled water (150 mL) was
added after reaction completion and pH was adjusted
to 8–10 by 20% aq. Na2CO3 solution. The product was
collected through sequential extraction with CHCl3
(20 mL × 3). Chloroform was distilled off to collect
the product. In some cases, the product was collected
through filtration. Esters (Va–n) were used in further
synthesis [18, 19].
N-(5-Methyl-1,3-thiazol-2-yl)-2-[(5-phenyl-1,3,4-
oxadiazol-2-yl)sulfanyl]acetamide (VIIIa). Yellowish
amorphous solid; yield 71%; mp 187–188°C. Mol.
formula C14H12N4O2S2; mol. mass 332 g mol–1; IR:
3347 (N–H stretching), 2976 (C–H stretching of aro-
matic ring), 1678 (C=N stretching), 1644 (C=O
stretching), 1570 (C=C stretching of aromatic ring),
1
1154 (C–O–C stretching); H NMR: 12.38 (s, 1H,
CONH), 7.95 (dd, J = 1.6, 8.6 Hz, 2H, H-2''' and H-
6'''), 7.63 (br.t, J = 7.4 Hz, 1H, H-4'''), 7.59 (br.t, J =
7.8 Hz, 2H, H-3''' and H-5'''), 7.17 (distorted d, J = 1.3
Hz, due to allylic coupling, 1H, H-4), 4.43 (br.s, 2H,
CH2-2'), 2.34 (distorted d, J = 1.3 Hz, due to allylic
coupling, 3H, CH3-6); 13C NMR: 165.26 (C-1'),
165.01 (C-5''), 162.98 (C-2''), 156.86 (C-2), 136.88
(C-4), 132.06 (C-4'''), 129.38 (C-3''' and C-5'''),
126.36 (C-2''' and C-6'''), 122.89 (C-1'''), 113.91 (C-5),
35.37 (C-2'), 11.07 (C-6). EI-MS: m/z 332 [M]+, 219
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 6 2018

808
ABBASI et al.
[C10H7N2O2S]+, 192 [C9H7N2OS]+, 114 [C3H2N2OS]+, Hz, due to allylic coupling, 3H, CH3-6); 13C NMR:
77 [C6H5]+.
2-{[5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl]sul-
fanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIb).
Light brown solid; yield 73%; mp 229–230°C. Mol.
formula C15H14N4O2S2; mol. mass 346 g mol–1; IR:
3358 (N–H stretching), 2977 (C–H stretching of aro-
matic ring), 1675 (C=N stretching), 1645 (C=O
164.97 (C-1'), 163.99 (C-5''), 162.73 (C-2''), 157.26
(C-2'''), 155.82 (C-2), 134.90 (C-4), 133.60 (C-5'''), 130.0
(C-6'''), 126.67 (C-5), 120.69 (C-4'''), 112.62 (C-3'''),
111.78 (C-1'''), 55.92 (CH3O-2'''), 35.29 (C-2'), 11.07
(C-6). EI-MS: m/z 362 [M]+, 249 [C11H9N2O3S]+,
222 [C10H9N2O2S]+, 189 [C6H9N2OS2]+, 175
[C9H7N2O2]+, 141 [C5H5N2OS]+, 135 [C8H7O]+, 114
stretching), 1576 (C=C stretching of aromatic ring), [C3H2N2OS]+, 92 [C7H8]+, 77 [C6H5]+.
1
1169 (C–O–C stretching); H NMR: 12.38 (s, 1H,
2-{[5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-yl]sul-
CONH), 7.82 (br.d, J = 7.9 Hz, 2H, H-2''' and H-6'''),
7.38 (br.d, J = 7.8 Hz, 2H, H-3''' and H-5'''), 7.17 (br.s,
1H, H-4), 4.37 (br.s, 2H, CH2-2'), 2.38 (s, 3H, CH3-4'''),
fanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIe).
Light brown amorphous solid; yield 89%; mp 236–
237°C. Mol. formula C13H11ClN4O2S2; mol. mass
2.34 (s, 3H, CH3-6); 13C NMR: 165.34 (C-1'), 165.04
(C-5''), 162.61 (C-2''), 156.01 (C-2), 142.26 (C-4'''),
134.94 (C-4), 129.94 (C-2''' and C-6'''), 126.71 (C-5),
126.32 (C-3''' and C-5'''), 120.15 (C-1'''), 35.30 (C-2'),
21.07 (CH3-4'''), 11.07 (C-6). EI-MS: m/z 346 [M]+,
366 g mol–1; IR: 3350 (N–H stretching), 2973 (C–H
stretching of aromatic ring), 1670 (C=N stretching),
1640 (C=O stretching), 1572 (C=C stretching of aro-
matic ring), 1159 (C–O–C stretching), 684 (C–Cl
1
stretching); H NMR: 12.37 (s, 1H, CONH), 7.95
233 [C11H9N2O2S]+, 206 [C10H9N2OS]+, 192
(br.d, J = 7.5 Hz, 1H, H-6'''), 7.71 (br.d, J = 7.9 Hz,
[C8H5N3OS]+, 159 [C9H7N2O]+, 141 [C5H5N2OS]+, 1H, H-3'''), 7.64 (br.t, J = 7.3 Hz, 1H, H-5'''), 7.52
119 [C8H7O]+, 104 [C7H4O]+, 91 [C7H7]+, 65 [C5H5]+.
(br.t, J = 7.5 Hz, 1H, H-4'''), 7.17 (br.s, 1H, H-4), 4.41
(br.s, 2H, CH2-2'), 2.34 (s, 3H, CH3-6); 13C NMR:
164.86 (C-1'), 163.71 (C-5''), 163.30 (C-2''), 155.80
(C-2), 149.15 (C-4'''), 134.91 (C-4), 133.28 (C-4'''),
131.65 (C-2'''), 131.13 (C-3'''), 131.10 (C-6'''), 127.82
(C-5'''), 126.67 (C-5), 122.05 (C-1'''), 35.36 (C-2'),
11.07 (C-6); EI-MS: m/z 366 [M]+, 257
2-{[5-(4-Hydroxyphenyl)-1,3,4-oxadiazol-2-yl]-
sulfanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide
(VIIIc). Brown solid; yield 85%; mp 231–232°C. Mol.
formula C14H12N4O3S2; mol. mass 348 g mol–1; IR:
3356 (N–H stretching), 2979 (C–H stretching of aro-
matic ring), 1667 (C=N stretching), 1646 (C=O
stretching), 1571 (C=C stretching of aromatic ring),
[C8H9N4O2S2]+,
226
[C8H4ClN2OS]+, 141 [C5H5N2OS]+, 75 [C6H3]+.
[C9H6ClN2OS]+,
179
1
1148 (C–O–C stretching); H NMR: 12.38 (s, 1H,
CONH), 7.76 (d, J = 8.7 Hz, 2H, H-3''' and H-5'''),
7.17 (distorted d, J = 1.4 Hz, due to allylic coupling,
1H, H-4), 6.91 (d, J = 8.7 Hz, 2H, H-2''' and H-6'''),
4.34 (br.s, 2H, CH2-2'), 2.34 (distorted d, J = 1.3 Hz,
2-{[5-(3-Chlorophenyl)-1,3,4-oxadiazol-2-yl]sul-
fanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIf).
Off-white amorphous powder; yield 83%; mp 213–
214°C. Mol. formula C13H11ClN4O2S2; mol. mass
due to allylic coupling, 3H, CH3-6); 13C NMR: 165.47
(C-1'), 161.70 (C-5''), 160.81 (C-2''), 156.01 (C-2),
134.92 (C-4), 133.28 (C-4'''), 128.35 (C-2''' and C-6'''),
113.62 (C-1'''), 116.12 (C-3''' and C-5'''), 113.62 (C-5),
35.32 (C-2'), 11.06 (C-6). EI-MS: m/z 348 [M]+, 228
366 g mol–1; IR: 3368 (N–H stretching), 2979 (C–H
stretching of aromatic ring), 1674 (C=N stretching),
1646 (C=O stretching), 1571 (C=C stretching of aro-
matic ring), 1155 (C–O–C stretching), 689 (C–Cl
1
stretching); H NMR: 12.37 (s, 1H, CONH), 7.99
[C7H7N3O2S2]+, 208 [C9H8N2O2S]+, 175 [C9H7N2O2]+, (br.d, J = 8.5 Hz, 1H, H-6'''), 7.96 (br.t, J = 8.5 Hz,
161 [C9H9N2O]+, 141 [C5H5N2OS]+, 121 [C7H5O2]+,
93 [C6H5O]+.
1H, H-5'''), 7.52 (d, J = 2.1 Hz, 1H, H-2'''), 7.49 (dis-
torted dd, J = 2.1, 8.5 Hz, 1H, H-4'''), 7.17 (br.s, 1H,
H-4), 4.41 (br.s, 2H, CH2-2'), 2.34 (s, 3H, CH3-6);
2-{[5-(2-Methoxyphenyl)-1,3,4-oxadiazol-2-yl]-
sulfanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide
(VIIId). Dull white solid; yield 77%; mp 205–206°C.
Mol. formula C15H14N4O3S2; mol. mass 362 g mol–1;
IR: 3358 (N–H stretching), 2977 (C–H stretching of
aromatic ring), 2823 (O–CH3 stretching), 1674 (C=N
stretching), 1644 (C=O stretching), 1575 (C=C
stretching of aromatic ring), 1163 (C–O–C stretch-
13C NMR: 165.15 (C-1'), 163.70 (C-5''), 163.32 (C-2''),
157.65 (C-2), 137.81 (C-3'''), 134.81 (C-4), 133.28 (C-4'''),
131.14 (C-2'''), 131.09 (C-6'''), 127.82 (C-5'''), 126.91
(C-5), 122.07 (C-1'''), 35.38 (C-2'), 11.07 (C-6); EI-MS:
m/z 366 [M]+, 226 [C9H6ClN2OS]+, 193 [C8H6N3OS]+,
179 [C8H4ClN2OS]+, 139 [C5H3N2OS]+, 125 [C7H5Cl]+,
114 [C3H2N2OS]+, 75 [C6H3]+.
1
ing); H NMR: 12.36 (s, 1H, CONH), 7.77 (dd, J =
2-{[5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl]sul-
1.5, 7.7 Hz, 1H, H-6'''), 7.59 (br.t, J = 8.6 Hz, 1H, H- fanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIg).
5'''), 7.24 (br.d, J = 8.5 Hz, 1H, H-3'''), 7.16 (distorted Brown solid; yield 88%; mp 207–208°C; Mol. for-
mula C14H11ClN4O2S2; mol. mass 366 g mol–1; IR:
3347 (N–H stretching), 2979 (C–H stretching of aro-
matic ring), 1681 (C=N stretching), 1636 (C=O
d, J = 1.1 Hz, due to allylic coupling, 1H, H-4), 7.09
(br.t, J = 7.6 Hz, 1H, H-4'''), 4.38 (br.s, 2H, CH2-2'),
3.85 (br.s, 3H, CH3O-2'''), 2.34 (distorted d, J = 0.6
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Vol. 44
No. 6
2018

N-(5-METHYL-1,3-THIAZOL-2-YL)-2-…
809
stretching), 1584 (C=C stretching of aromatic ring), IR: 3376 (N–H stretching), 2966 (C–H stretching of
1165 (C–O–C stretching), 691 (C–Cl stretching); aromatic ring), 1683 (C=N stretching), 1657 (C=O
1H NMR: 12.38 (s, 1H, CONH), 7.91 (br.d, J = 8.5 Hz,
stretching), 1563 (C=C stretching of aromatic ring),
1
2H, H-3''' and H-5'''), 7.49 (br.d, J = 8.5 Hz, 2H, H-2''' 1168 (C–O–C stretching); H NMR: 12.70 (s, 1H,
and H-6'''), 7.17 (br.s, 1H, H-4), 4.43 (br.s, 2H, CH2-2'), CONH), 7.56 (br.d, J = 8.6 Hz, 2H, H-2''' and H-6'''),
2.34 (s, 3H, CH3-6); 13C NMR: 165.15 (C-1'), 163.70
(C-5''), 163.32 (C-2''), 157.65 (C-2), 137.81 (C-3''' and
C-5'''), 134.81 (C-4), 133.28 (C-4'''), 131.14 (C-2''' and
C-6'''), 126.91 (C-5), 122.07 (C-1'''), 35.38 (C-2'), 11.07
(C-6); EI-MS: m/z 366 [M]+, 226 [C9H6ClN2OS]+,
7.16 (d, J = 1.1 Hz, due to allylic coupling, 1H, H-4), 6.62
(br.d, J = 8.6 Hz, 1H, H-3''' and H-5'''); 4.30 (br.s, 2H,
CH2-2'), 2.34 (d, J = 0.7 Hz, due to allylic coupling,
3H, CH3-6); 13C-NMR: 166.09 (C-1'), 165.22 (C-5''),
160.57 (C-2''), 156.01 (C-2), 152.40 (C-4'''), 134.92
(C-4), 127.89 (C-2''' and C-6'''), 126.68 (C-5), 113.47
(C-3''' and C-5'''), 109.13 (C-1'''), 35.28 (C-2'), 11.07
(C-6). EI-MS: m/z 347 [M]+, 207 [C9H8N3OS], 193
179 [C8H4ClN2OS]+, 139 [C5H3N2OS]+, 125
[C7H5Cl]+, 114 [C3H2N2OS]+, 75 [C6H3]+.
[C8H6N3OS], 160 [C8H6N3O]+, 141 [C5H5N2OS], 133
2-{[5-(2,4-Dichlorophenyl)-1,3,4-oxadiazol-2-yl]-
sulfanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIh).
Brown greasy liquid; yield 79%; Mol. formula
C13H9Cl2N4O2S2; mol. mass 400 g mol–1; IR: 3379
(N–H stretching), 2982 (C–H stretching of aromatic
ring), 1677 (C=N stretching), 1647 (C=O stretching),
1575 (C=C stretching of aromatic ring), 1164 (C–O–
C stretching), 694 (C–Cl stretching); 1H NMR: 12.35
(s, 1H, CONH), 7.98 (d, J = 8.5 Hz, 1H, H-6'''), 7.91
(d, J = 2.1 Hz, 1H H-3'''), 7.65 (dd, J = 1.2, 8.5 Hz,
1H, H-5'''), 7.17 (distorted d, J = 1.2 Hz, due to allylic
coupling, 1H, H-4), 4.41 (br.s, 2H, CH2-2'), 2.34
(distorted d, J = 1.3 Hz, due to allylic coupling, 3H,
CH3-6); 13C NMR: 165.01 (C-1'), 163.88 (C-5''),
162.64 (C-2''), 156.01 (C-2), 137.18 (C-4'''), 134.90
(C-4), 132.72 (C-1'''), 132.23 (C-6'''), 130.71 (C-3'''),
128.13 (C-5'''), 126.65 (C-5), 121.0 (C-2'''), 35.40 (C-2'),
11.0 (C-6). EI-MS: m/z 400 [M]+, 260 [C9H5ClN2O2S]+,
[C8H7NO], 118 [C3HNOS], 92 [C6H6N], 65 [C5H5]+.
2-{[5-(2-Nitrophenyl)-1,3,4-oxadiazol-2-yl]sulfa-
nyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIk).
Light yellow solid; yield 79%; mp 217–218°C; Mol.
formula C14H11N5O4S2; mol. mass 377 g mol–1; IR:
3364 (N–H stretching), 2982 (C–H stretching of aro-
matic ring), 1673 (C=N stretching), 1649 (C=O
stretching), 1578 (C=C stretching of aromatic ring),
1157 (C–O–C stretching of ether); 1H NMR: 12.36 (s,
1H, CONH), 8.19-8.17 (m, 1H, H-3'''), 8.01 (m, 1H,
H-6'''), 7.94-7.90 (m, 2H, H-4''' and H-5'''), 7.16 (dis-
torted d, J = 1.2 Hz, due to allylic coupling, 1H, H-4),
4.40 (br.s, 2H, CH2-2'), 2.34 (d, J = 0.9 Hz, due to
allylic coupling, 3H, CH3-6); 13C NMR: 165.0 (C-1'),
164.3 (C-5''), 161.61 (C-2''), 156.0 (C-2), 147.5 (C-2'''),
134.85 (C-4), 133.61 (C-4'''), 133.43 (C-5'''), 131.09
(C-3'''), 124.70 (C-6'''), 116.43 (C-1'''), 113.91 (C-5),
35.33 (C-2'), 11.07 (C-6). EI-MS: m/z 377 [M]+, 227
173
[C3H2N2OS]+.
[C7H3Cl2N]+,
141
[C5H5N2OS]+,
114
[C8H7N2O2S2]+,
[C3H2N2OS]+, 76 [C6H4]+.
141
[C5H5N2OS]+,
114
2-{[5-(3-Aminophenyl)-1,3,4-oxadiazol-2-yl]sul-
fanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIi).
White solid; yield: 77%; mp 206–207°C; Mol. for-
mula C14H13N5O2S2; Mol. mass g mol–1; IR: 3378 (N–
H stretching), 2962 (C–H stretching of aromatic
ring), 1680 (C=N stretching), 1653 (C=O stretching),
1566 (C=C stretching of aromatic ring), 1164 (C–O–
C stretching); 1H NMR: 12.38 (s, 1H, CON-H), 7.93
(dd, J = 2.1, 8.5 Hz, 1H, H-6'''), 7.88 (br.t, J = 8.5 Hz,
1H, H-5'''), 7.48 (d, J = 2.1 Hz, 1H, H-2'''), 7.47 (dd,
J = 2.1, 8.5 Hz, 1H, H-4'''), 7.17 (br.s, 1H, H-4), 4.43
(br.s, 2H, CH2-2'), 2.34 (s, 3H, CH3-6); 13C NMR:
165.26 (C-1'), 162.99 (C-5''), 160.46 (C-2''), 157.71
(C-2), 134.80 (C-4), 132.21 (C-3'''), 132.04 (C-1'''),
129.41 (C-5'''), 129.38 (C-6'''), 126.35 (C-4'''), 126.02
(C-2'''), 126.01 (C-5), 35.36 (C-2'), 11.07(C-6). EI-
MS: m/z 347 [M]+, 207 [C9H8N3OS]+, 193
2-{[5-(3-Nitrophenyl)-1,3,4-oxadiazol-2-yl]sulfa-
nyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIl).
Light yellow solid; yield 84%; mp 223–224°C; Mol.
formula C14H11N5O4S2; mol. mass 377 g mol–1; IR:
3360 (N–H stretching), 2963 (C–H stretching of aro-
matic ring), 1660 (C=N stretching), 1650 (C=O
stretching), 1562 (C=C stretching of aromatic ring),
1
1169 (C–O–C stretching); H NMR: 12.37 (s, 1H,
CONH), 8.62 (distorted d, J = 1.8 Hz, 1H, H-2'''),
8.45 (dd, J = 1.3, 8.2 Hz, 1H, H-6'''), 8.38 (br.d, J =
7.8 Hz, 1H, H-4'''), 7.89 (br.t, J = 7.3 Hz, 1H, H-5'''),
7.16 (distorted d, J = 1.1 Hz, due to allylic coupling,
1H, H-4), 4.43 (br.s, 2H, CH2-2'), 2.34 (distorted d,
J = 0.84 Hz, due to allylic coupling, 3H, CH3-6);
13C NMR: 164.93 (C-1'), 164.06 (C-5''), 163.77 (C-2''),
155.0 (C-2), 148.21 (C-3'''), 134.90 (C-4), 132.41
(C-6'''), 131.31 (C-5'''), 126.37 (C-4'''), 124.40 (C-1'''),
120.88 (C-2'''), 113.91 (C-5), 35.41 (C-2'), 11.07
(C-6). EI-MS: m/z 377 [M]+, 150[C7H4NO3]+, 141
[C8H6N3OS]+, 141 [C5H5N2OS]+, 133 [C8H7NO]+,
118 [C3HNOS]+, 92 [C6H6N]+, 65 [C5H5]+.
2-{[5-(4-Aminophenyl)-1,3,4-oxadiazol-2-yl]sul-
fanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIj).
Brown amorphous powder; yield 81%; mp 225–226°C;
Mol. formula C14H13N5O2S2; mol. mass 347 g mol–1;
[C5H5N2OS]+, 114 [C3H2N2OS]+, 76 [C6H4]+.
2-{[5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-yl]sulfa-
nyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIm).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 6 2018

810
ABBASI et al.
Light yellow solid; yield 81%; mp 216–217°C; Mol. well) was used as a positive control. The percent inhi-
formula C14H11N5O4S2; mol. mass 377 g mol–1; IR:
3364 (N–H stretching), 2987 (C–H stretching of aro-
matic ring), 1684 (C=N stretching), 1654 (C=O
stretching), 1586 (C=C stretching of aromatic ring),
bition was calculated using the following formula:
Control − Test
Ingibiton (%) =
×100,
Control
1
where Control is the total enzyme activity without
inhibitor and Test, the activity in the presence of test
compound.
IC50 values were calculated using the EZ–Fit
Enzyme kinetics software (Perrella Scientific Inc.
Amherst, US).
1173 (C–O–C stretching of ether); H NMR: 12.38
(s, 1H, CONH), 8.42 (br.d, J = 7.0 Hz, 2H, H-3'''
and H-5'''), 8.21 (br.d, J = 7.0 Hz, 2H, H-2''' and H-6'''),
7.17 (distorted d, J = 1.2 Hz, due to allylic coupling,
1H, H-4), 4.43 (br.s, 2H, CH2-2'), 2.34 (d, J = 0.9 Hz,
due to allylic coupling, 3H, CH3-6); 13C NMR: 165.00
(C-1'), 164.51 (C-5''), 163.93 (C-2''), 156.01 (C-2),
149.15 (C-4'''), 134.95 (C-4), 127.74 (C-2''' and C-6'''),
124.58 (C-3''' and C-5'''), 122.01 (C-1'''), 113.91 (C-5),
35.38 (C-2'), 11.07(C-6); EI-MS: m/z 377 [M]+, 227
[C8H7N2O2S2]+, 150[C7H4NO3]+, 141 [C5H5N2OS]+,
Urease. This enzyme assay is the customized form
of the commonly known Berthelot assay [22]. The
assay mixture (85 µL per well of a 96-well plate) con-
tains 10 µL of phosphate buffer, pH 7.0, 10µL of sample
solution and 25 µL of enzyme solution (0.135 units). The
contents were pre-incubated at 37°C for 5 min; 40 µL of
urea stock solution (20 mM) was added to each well
with incubation for 10 min at 37°C. Then, 115 µL phe-
nol hypochlorite reagents (freshly prepared by mixing
45 µL phenol with 70 µL of alkali) were added into
each well. For color development, incubation was car-
ried out for further 10 min at 37°C. Absorbance was
measured at 625 nm. Percentage enzyme inhibition
and IC50 values were calculated by the same procedure
as mentioned above.
α-Glucosidase. α-Glucosidase inhibition activity
was performed according to the slightly modified
method [23]. Total volume of the reaction mixture was
100 µL; it contained 70 µL phosphate buffer saline
(50 mM) with pH of 6.8, 10 µL of test compound
(0.5 mM), and 10 µL enzyme (0.057 units). The con-
tents were mixed, pre-incubated for 10 min at 37°C,
and pre-read at 400 nm. The reaction was initiated by
the addition of 10 µL of 0.5 mM substrate (p-nitro-
phenylglucopyranoside). Acarbose was used as posi-
tive control. After 30 min of incubation at 37°C, absor-
bance was measured at 400 nm using the Synergy HT
microplate reader. All experiments were carried out in
duplicates. The percent inhibition and IC50 values
were calculated by the same equation as described for
cholinesterase enzymes above.
114 [C3H2N2OS]+, 76 [C6H4]+.
2-{[5-(3,5-Dinitrophenyl)-1,3,4-oxadiazol-2-yl]-
sulfanyl}-N-(5-methyl-1,3-thiazol-2-yl)acetamide (VIIIn).
Light yellow amorphous solid; yield 83%; mp 273–
274°C; Mol. formula C14H10N6O6S2; mol. mass
422 g mol–1; IR: 3366 (N–H stretching), 2985 (C–H
stretching of aromatic ring), 1685 (C=N stretching),
1658 (C=O stretching), 1589 (C=C stretching of aro-
1
matic ring), 1192 (C–O–C stretching); H NMR:
12.38 (s, 1H, CONH), 8.92 (d, J = 2.4 Hz, 2H, H-2'''
and H-6'''), 8.66 (d, J = 2.4 Hz, 1H, H-4'''), 7.17 (br.s,
1H, H-4), 4.43 (br.s, 2H, CH2-2'), 2.34 (s, 3H, CH3-6);
13C NMR: 165.12 (C-1'), 163.88 (C-5''), 162.67 (C-
2''), 157.71 (C-2), 144.97 (C-3''' and C-5'''), 134.88
(C-4), 132.60 (C-1'''), 128.14 (C-2''' and C-6'''), 118.19
(C-4'''), 113.91 (C-5), 35.40 (C-2'), 11.07 (C-6); EI-MS:
m/z 422 [M]+, 303 [C12H8N4O2S2]+, 193 [C8H6N3OS]+,
140 [C5H5N2OS]+, 114 [C3H2N2OS]+, 104 [C7H4O]+,
76 [C6H4]+.
Enzyme Inhibition Assays
Cholinesterases. AChE and BChE inhibition activ-
ities were evaluated according to Ellman method with
slight modifications [20, 21]. Total volume of the reac-
tion mixture was 100 µL. It contained 60 µL Na2HPO4
buffer with concentration of 50 mM and pH 7.7; 10 µL
test compound (0.5 mM per well) was added, followed
Molecular Docking
To prove the bioactive conformations, the synthe-
by the addition of 10 µL (0.005 unit per well AChE or sized compounds were docked into the binding pock-
0.5 unit per well BChE) enzyme. The contents were ets of the proteins under study using the default
mixed and pre-read at 405 nm. Then contents were parameters of the MOE-Dock method [24]. Before
pre-incubated for 10 min at 37°C. The reaction was docking, the molecules of acetylcholinesterase, butyr-
initiated by the addition of 10 µL of 0.5 mM substrate ylcholinesterase, urease, and α-glucosidase were
(acetylthiocholine iodide for AChE or butyrylthio- retrieved from the Protein Data Bank (PDB ID codes
choline chloride for BChE) per well, followed by the 1GQR, 1POP, 4UBP, and 3NO4, respectively). MOE
addition of 10 µL DTNB (0.5 mM per well). After applications were used for performing 3D protonation
15 min of incubation at 37°C absorbance was measured of the protein molecules. Energy minimization algo-
at 405 nm using a Synergy HT (Biotek, US) 96-well plate rithm of MOE tool was used to minimize the protein
reader. All experiments were carried out with their molecule energy in the gradient of 0.05 Force Field of
respective controls in triplicate. Eserine (0.5 mM per MMFF94X with the default parameters of MOE
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 44
No. 6
2018

N-(5-METHYL-1,3-THIAZOL-2-YL)-2-…
811
2. Knadler, M.P., Bergstrom, R.F., Callaghan, J.T., and
2009-10. Then all the ligands (3D protonated and
energy minimized) were docked into the binding
pockets (selective residues/amino acids are shown in
the docking images in Figs. 1–4) of the above-men-
tioned proteins using the Triangular Matching dock-
ing method. Re-docking procedure was also applied in
order to validate the docking protocol. Each complex
was analyzed for the type of interactions; bond dis-
tances and their 3D images were taken.
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Cytotoxicity assay. Cytotoxicity was studied by the
brine shrimp cytotoxic assay [25]. Artificial sea water
was prepared using sea salt, 34 g/L. Brine shrimp
(Artemia salina) eggs (Sera, Heidelberg, Germany)
were hatched in shallow rectangular dish (22 × 32 cm)
under constant aeration for 48 h at room temperature.
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and used for the assay. Ten shrimps were transferred to
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artificial sea water with 200, 20, 2 and 0.2 µg/mL final
concentration of test compound from their stock solu-
tion. The vials were maintained under illumination at
room temperature 25 to 28°C. After 24 h, the number
of surviving shrimps was counted. Experiment was
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Statistical Analysis
Statistical analysis was performed by Microsoft
Excel 2010 for all the thrice measured values and the
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pp. 1189–1197.
17. Dong, L. and Ni J.M., Red. Asian J. Trad. Med., 2008,
We can conclude that the incorporation of electron
donating groups (OCH3, NH2, OH) in the thiazole–
oxadiazole biheterocyclic core renders good inhibitory
potential against AChE, BChE, urease, and α-gluco-
sidase. Similarly, from the cytotoxicity study, it was
inferred that most of the compounds possess low tox-
icity and hence, these molecules can find their utility
as promising therapeutic agents.
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ACKNOWLEDGMENTS
21. Ellman, G.L., Courtney, K.D., Andres, V., and Feath-
erstone, R.M., Bio. Pharm., 1961, vol. 7, pp. 88–95.
The present study was supported by the Basic Sci-
ence Research Program through the National
Research Foundation of Korea (NRF) funded by the
Ministry of Education (2017R1D1A1B03034948).
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