Synthesis, in vitro α-glucosidase inhibitory activity and molecular docking studies of new thiazole derivatives

Article abstract of DOI:10.1016/j.bioorg.2016.08.010

Current study based on the synthesis of new thiazole derivatives via “one pot” multicomponent reaction, evaluation of their in vitro α-glucosidase inhibitory activities, and in silico studies. All synthetic compounds were fully characterized by ¹H NMR, ¹³C NMR and EIMS. CHN analysis was also performed. These newly synthesized compounds showed activities in the range of IC₅₀?=?9.06?±?0.10–82.50?±?1.70?μM as compared to standard acarbose (IC₅₀?=?38.25?±?0.12?μM). It is worth mentioning that most of the compounds such as 1 (IC₅₀?=?23.60?±?0.39?μM), 2 (IC₅₀?=?22.70?±?0.60?μM), 3 (IC₅₀?=?22.40?±?0.32?μM), 4 (IC₅₀?=?26.5?±?0.40?μM), 6 (IC₅₀?=?34.60?±?0.60?μM), 7 (IC₅₀?=?26.20?±?0.43?μM), 8 (IC₅₀?=?14.06?±?0.18?μM), 9 (IC₅₀?=?17.60?±?0.28?μM), 10 (IC₅₀?=?27.16?±?0.41?μM), 11 (IC₅₀?=?19.16?±?0.19?μM), 12 (IC₅₀?=?9.06?±?0.10?μM), 13 (IC₅₀?=?12.80?±?0.21?μM), 14 (IC₅₀?=?11.94?±?0.18?μM), 15 (IC₅₀?=?16.90?±?0.20?μM), 16 (IC₅₀?=?12.60?±?0.14?μM), 17 (IC₅₀?=?16.30?±?0.29?μM), and 18 (IC₅₀?=?32.60?±?0.61?μM) exhibited potent inhibitory potential. Molecular docking study was performed in order to understand the molecular interactions between the molecule and enzyme. Newly identified α-glucosidase inhibitors except few were found to be completely non-toxic.

Full text of DOI:10.1016/j.bioorg.2016.08.010

Accepted Manuscript
Synthesis, In Vitro α -Glucosidase Inhibitory Activity and Molecular Docking
Studies of New Thiazole Derivatives
Khalid Mohammed Khan, Saira Qurban, Uzma Salar, Muhammad Taha,
Shafqat Hussain, Shahnaz Perveen, Abdul Hameed, Nor Hadiani Ismail,
Muhammad Riaz, Abdul Wadood
PII:
S0045-2068(16)30116-X
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.08.010
YBIOO 1944
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
7 July 2016
22 August 2016
23 August 2016
Please cite this article as: K.M. Khan, S. Qurban, U. Salar, M. Taha, S. Hussain, S. Perveen, A. Hameed, N.H. Ismail,
M. Riaz, A. Wadood, Synthesis, In Vitro α -Glucosidase Inhibitory Activity and Molecular Docking Studies of New
Thiazole Derivatives, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.08.010
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Synthesis, In Vitro α-Glucosidase Inhibitory Activity and Molecular Docking Studies of
New Thiazole Derivatives
∗
a
a
a
b,c
Khalid Mohammed Khan, Saira Qurban, Uzma Salar, Muhammad Taha, Shafqat
a,d
e
a
c,d
f
Hussain, Shahnaz Perveen, Abdul Hameed, Nor Hadiani Ismail, Muhammad Riaz,
f
Abdul Wadood
a
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
b
Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA
UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E., Malaysia
(
c
Faculty of Applied Science, Universiti Teknologi MARA, Shah Alam 40450,Selangor D. E.,
Malaysia
d
Deparment of Chemistry, Karakoram International University Gilgit, Pakistan
PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-
e
7
5280, Pakistan
f
Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul
Wali Khan University Mardan, Pakistan
Abstract
Current study based on the synthesis of new thiazole derivatives via “one pot”
multicomponent reaction, evaluation of their in vitro α-glucosidase inhibitory activities, and
1
13
in silico studies. All synthetic compounds were fully characterized by H-NMR, C-NMR
and EIMS. CHN analysis was also performed. These newly synthesized compounds showed
activities in the range of IC50 = 9.06 ± 0.10-82.50 ± 1.70 µM as compared to standard
acarbose (IC50 = 38.25 ± 0.12 µM). It is worth mentioning that most of the compounds such
as 1 (IC50 = 23.60 ± 0.39 µM), 2 (IC50 = 22.70 ± 0.60 µM), 3 (IC50 = 22.40 ± 0.32 µM), 4 (IC50
=
26.5 ± 0.40 µM), 6 (IC50 = 34.60 ± 0.60 µM), 7 (IC50 = 26.20 ± 0.43 µM), 8 (IC50 = 14.06 ±
.18 µM), 9 (IC50 = 17.60 ± 0.28 µM), 10 (IC50 = 27.16 ± 0.41 µM), 11 (IC50 = 19.16 ± 0.19
µM), 12 (IC50 = 9.06 ± 0.10 µM), 13 (IC50 = 12.80 ± 0.21 µM), 14 (IC50 = 11.94 ± 0.18 µM),
0
1
1
5 (IC50 = 16.90 ± 0.20 µM), 16 (IC50 = 12.60 ± 0.14 µM), 17 (IC50 = 16.30 ± 0.29 µM), and
8 (IC50 = 32.60 ± 0.61 µM) exhibited potent inhibitory potential. Molecular docking study
was performed in order to understand the molecular interactions between the molecule and
enzyme. Newly identified α-glucosidase inhibitors except few were found to be completely
non-toxic.
*
Corresponding Author: hassaan2@super.net.pk; khalid.khan@iccs.edu; Tel. 00922134824910; Fax.
0
0922134819018

Keywords: Synthesis; thiazole; in vitro α-glucosidase; structure-activity relationship; in
silico study
Introduction
Thiazole or 1,3-thiazole is a five membered azole heterocyclic organic compound [1-2],
considered to be aromatic as it follows the Huckel rule [3]. Thiazole and its derivatives
emerged as active pharmaceutical ingredients in several drugs for their potential as
antiinflammatory [4], anticonvulsant [5], insecticidal [6], antioxidant, antitumour,
antihyperlipidemic, antihypertensive, and pesticidal [7]. Thiazole ring has privileged to have
industrial applications such as polymers, liquid crystals, photo nucleases, [8] and as
fluorescent dyes [9].Diabetes is epidemic disease worldwide and world health organization
(
WHO) has alarmed that if no proper action will be taken by 2030 there will be at least 350
million people in the world who will suffer with type-2 diabetes mellitus (T2DM) [10]. α-
Glucosidase is the key enzyme which catalyzes the final step in the digestion of
carbohydrates. The inhibitors of α-glucosidase retard the secretion of D-glucose from the
carbohydrate and slower down the absorption of glucose level. Hence, ultimately results in
the suppression of postprandial hyperglycemia [11]. Acarbose, voglibose, and miglitol are
mainly used for the treatment of type-2 diabetes mellitus. Regrettably, these drugs are 50%
less effective than other antidiabetic agents such as metformin and sulfonylurea as well as
some side effects are also associated with them such as diarrhea, flatulence and abdominal
discomfort (AG, 1994). Therefore, it is restrictive aspect to use the drug alone and often use
in combination with other antidiabetic drugs to improve the efficacy. So it is upmost task to
develop safer medication for diabetes [12].
®
Medicinally important molecule Zopolrestat having thiazole moiety in its core structure is
now on clinical use for its effectiveness in diabetic complications [13]. Similarly N-(6-
substituted-1,3-benzothiazol-2-yl) benzenesulfonamides illustrate promising antidiabetic
potential in vivo system in a NIDDM (non-insulin-dependent diabetes mellitus) rat model
[
14] (Figure-1).

Our research aim is mostly dedicated to the syntheses of diverse heterocycles and evaluation
of their biological potentials [15-21]. We have evaluated thiazole scaffold (A and B) as
potential α-glucosidase inhibitors (Figure-2) [22-24]. For current study, it was thought that
new thiazole derivatives with diverse functionalities may produce better in vitro α-
glucosidase inhibitory effects, therefore a library of new thiazole based molecules 1-24 was
synthesized via “one pot” two step reaction and screened for in vitro α-glucosidase inhibition.
To the best of our knowledge, the synthesized compounds are never reported previously.
Results and Discussion
Chemistry
New thiazole derivatives 1-24 were synthesized via “one pot” multicomponent reaction in

which a variety of aryl hydrazide/phenyl hydrazine/benzophenone hydrazone reacted with
aryl isothiocyanate in ethanol to get thiosemicarbazide intermediate within 30 minutes.
Triethylamine was used as base. After that phenacyl bromide derivative was added into the
same pot to afford new thiazole derivative. Completion of reaction was checked by periodic
TLC. Precipitates were obtained into the reaction mixture which were filtered and washed
with cold ethanol to obtained pure products in good yield (Scheme-1). The structures of all
1
13
derivatives 1-24 were confirmed by different spectroscopic techniques such as H-NMR, C-
NMR and EIMS. CHN analysis was also performed.
Scheme-1: Synthetic route for the compounds 1-24
Table-1: α-Glucosidase inhibitory activities of thiazole derivatives 1-24
a
Comp. No.
R
1
R
2
R
3
(IC50 ± SEM)
Category “A”
1
23.60 ± 0.39
2
3
4
22.70 ± 0.60
22.40 ± 0.32
26.5 ± 0.40

5
6
82.50 ± 1.70
34.60 ± 0.60
Category “B”
7
8
9
26.20 ± 0.43
14.06 ± 0.18
17.60 ± 0.28
27.16 ± 0.41
19.16 ± 0.19
9.46 ± 0.10
1
0
1
2
1
1
1
1
3
4
12.80 ± 0.21
11.94 ± 0.18
1
1
5
6
16.90 ± 0.20
12.60 ± 0.14
1
1
7
8
16.30 ± 0.29
32.60 ± 0.61
Category “C”

1
2
2
2
2
2
9
0
1
2
3
4
48.80 ± 0.60
50.10 ± 0.71
72.10 ± 1.01
72.40 ± 1.19
46.50 ± 0.50
49.50 ± 0.59
38.25 ± 0.12
b
Standard = Acarbose
a
b
SEM : standard error of mean; Acarbose : Standard Inhibitor for α-Glucosidase.
Stereochemical assignment of iminic double bond by NOESY
1
There is possibility of two isomers either (Z) or (E) (Figure-3) in the result of reaction but H-
NMR spectra of all synthetic compounds displayed the formation of single isomer. However,
nuclear Overhauser enhancement spectroscopy (NOESY) was performed on one of the
synthesized compounds (compound 2) for confirming the stereochemistry of the iminic
double bond. Out of many NOESY interactions, some confirmed the (Z) stereochemistry of
the iminic bond. Strong NOESY interactions between H-5ʹ and H-6ʹ with H-5 showed that
these rings of the molecule are close in space. Whereas, absence of any distinctive NOESY
interaction of H-2ʹʹand H-3ʹʹ with H-2ʹʹʹ and 3ʹʹʹshowed that these two rings are not close

enough to show NOESY interactions. These evidences showed the Z stereochemistry of the
iminic double bond in compounds (Figure-3a).
Furthermore, the formation of E isomer is not favorable as rings R
create steric hindrance which brings out the instability in the molecule. However, in case of
Z-isomer, rings R and R are very far apart and free from any steric hindrance. Figure-3b
1
and R
2
are much closed to
1
2
clearly showed that the formation of stable Z-isomer is more favorable as compare to unstable
E-isomer.
α-Glucosidase inhibitory activity
All synthetic compounds were evaluated for their in vitro α-glucosidase inhibitory activities.
Activity results (Table-1) showed that all derivatives found to have activity in the range of
IC50 = 9.06 ± 0.10-82.50 ± 1.70 µM as compared to standard acarbose (IC50 = 38.25 ± 0.12
µM). It is worth mentioning that except compounds 5, 19, 20, 21, 22, 23 and 24, all

derivatives were showed superior activities than standard acarbose. All structural feature such
as thiazole ring, amide linkage, amine linkage and arene moieties are seemingly played a
crucial role in exhibiting the activity. However, limited structure-activity relationship (SAR)
was established by looking at the substitution pattern at R
compounds were divided into three categories “A”, “B” and “C”. Category “A” comprised of
compounds having benzamide group as R , category “B” having aniline moiety as R and
category “C” consist of molecules having diphenyl methanimine group as R1.
1
, R
2
3
and R . For that purpose, all
1
1
Compound 12 (IC50 = 9.06 ± 0.10 µM) belongs to category “B” and has para chloro
substitutions at R and R , respectively. It was found to be the most potent analog of this
2
3
series and fourfold more active than the standard acarbose. Comparison of its activity with its
structurally similar compounds revealed that compound 17 (IC50 = 16.30 ± 0.29 µM) which
2
has meta chloro substitution at R instead of para, a slight decline in the activity observed.
Similarly, compounds having three chloro substitutions such as 14 (IC50 = 11.94 ± 0.18 µM)
and 16 (IC50 = 12.60 ± 0.14 µM) also showed excellent activity like compounds having two
chloro substituents in structure as shown in figure-4. Another compound 13 (IC50 = 12.80 ±
0
.21 µM) which has para nitro group instead of chloro at R
Similar pattern was observed in case of 15 (IC50 = 16.90 ± 0.20 µM) which also has distinctly
similar structure as 16 but only nitro group instead of chloro at R , a decline in the activity
3
, showed lesser activity than 14.
3
was noticed. Activity of compound 11 (IC50 = 19.16 ± 0.19 µM), having para methoxy group
instead of chloro, can compare with the most active compound 12 almost twice decline in the
inhibitory activity experienced (Figure-4). The above activity pattern showed that chloro
group is playing a crucial role in the activity.

Another compound 8 (IC50 = 14.06 ± 0.18 µM) from category “B” having para nitro group at
and R , also found to be potent analog. On comparison of its activity with compounds
such as 9, 7 and 10 those have methoxy, chloro and methyl groups at R , respectively, decline
R
2
3
3
in the activity from nitro to methoxy, methoxy to chloro and chloro to methyl group was
observed (Figure-5). But still these are potent analogs with much better activity than the
standard.

In case of category “A”, compounds 1 (IC50 = 23.60 ± 0.39 µM) having para chloro group, 2
IC50 = 22.70 ± 0.60 µM) having para cyano group and 3 (IC50 = 22.40 ± 0.32 µM) has no
substitution at R , although rest of the structure is same. Interestingly, all thee derivatives
(
3
showed almost similar activity (Figure-6).
Activity of other three derivatives such as 4 (IC50 = 26.5 ± 0.40µM), 5 (IC50 = 82.50 ± 1.70
µM) and 6 (IC50 = 34.60 ± 0.60 µM) was different from each other. However, the structure of
3
all three is very similar to each other but only differ at R . Compounds 4 have no substitution
at R3, while 5 and 6 have para methane sulphonyl and para chloro group, respectively.
Comparison of activity pattern of all three derivatives revealed that methane sulphonyl group
in compound 5 caused major decline in the activity (Figure-7).

1
Activity pattern of category “C” which has diphenyl methanimine group as R showed that
this category demonstrated comparatively decreased activity than category “A” and “B”.
Amongst compound 19-24, compound 20 which differ with rest of the derivatives only by the
3
substitution at R , where it has para methoxy group (Figure-8).
In order to get insights regarding the molecular interaction of these derivatives with the active
site of the enzyme, molecular docking study was performed. A plausible demonstration is
given below.
Molecular docking
Docking study was carried out by using MOE (Molecular Operating Environment) software
package. The docking results showed that all compounds were well accommodated in the
binding pocket of α-glucosidase. The docking conformation of most active compound 12
(
IC50 = 9.06 ± 0.10 µM) with chlorine group at para position of phenyl ring showed good

interaction network as well as good docking score (-11.8617). Compound 12 made two π-
interactions at the binding pocket. Phe157 made arene-arene interaction with aniline moiety
and Phe300 with chlorobenzene group (Figure-9a). When chlorine group present at meta
position of the phenyl ring in compound 17 (IC50 = 16.30 ± 0.29 µM) showed less biological
activity as well as low docking score (-9.9130). The docking conformation of compound 17
showed an arene-arene and arene-cation interaction between the chloro benzene rings of
compound with Phe157 and Arg312 of the enzyme (Figure-9b). Compound 16 (IC50 = 12.60
±
0.14 µM) having an extra chlorine group at ortho position as compared to the compound
1
2, showed two arene-arene interaction between chlorophenyl and dichlorophenyl ring of the
compound with His239 and Phe157 of the enzyme, respectively (Figure-9c). These three
chlorobenzene containing compounds with different positions and numbers showed almost
similar behavior regarding interactions but their docking scores showed their fitness in the
binding pocket of enzyme, well correlated with their biological activities. In case of
2
compound 7 (IC50 = 26.20 ± 0.43 µM), where one chlorine group is replaced with nitro (NO )
group as compared to compound 12 and 17, established an arene-cation interaction between
chlorobenzene ring of the compound with Arg312 of the enzyme (Figure-9d). The
2
substitution of nitro (NO ) group resulted in decreased docking score (-9.8135) and binding
interactions along with IC50 value as compared to compounds 16, 12, and 17. This reduced
inhibition potency might be due to the deactivating nature of nitro group by electron
withdrawing inductive effect and resonance.

In case of compounds containing methyl substituted benzamide ring 1-6, the docking
conformation of compound 1 (IC50 = 23.60 ± 0.39 µM) with a docking score (-12.5054)
established good interactions with the active side residues Asn241, His279, and Phe157.
Asn241 was observed to make hydrogen bond with the oxygen atom of 3-methyl benzamide
moiety of the compound 1, while His-279 and Phe-157 makes arene-cation and arene-arene
interactions with the methyl and chlorobenzene moieties of the compound, respectively
(
(
Figure-10a). Compounds 3 and 4 also showed almost similar mode. In case of compound 5
IC50 = 82.50 ± 1.70 µM) with a docking score of -9.9758 displayed only one arene-arene
interaction between His239 and chloro substituted phenyl ring of the compound (Figure-
0b). In this series of methyl substituted benzamide ring containing compounds which has an
electron donating inductive effect by methyl group, it was observed that compounds 1-4 and
, in which inductively electron withdrawing groups (NO , CN, Br, Cl) at the two ends of the
1
6
2
molecule were presents, showed good and almost similar docking results. Moreover, their
biological activities were also found in close range. Whereas in compound 5, addition of
electron rich species methane sulfonyl group at one end of the molecule resulted in reduced
docking score and interactions.

In case of diphenyl methanimine containing compounds 19-24, it was observed that halogen
containing compounds showed somewhat poor computational inhibition. In case of
compound 20 (IC50 = 42.90 ± 0.71 µM) with a docking score (-13.6348) showed a hydrogen
bond interaction between the oxygen atom of methoxy group with Tyr313 and arene-cation
interaction of Arg312 with the benzene moiety of the compound (Figure-11a). Whereas, in
case of compound 22 (IC50 = 72.40 ± 1.19 µM) with a docking score (-12.303) was observed
to show a single arene-cation interaction (Figure-11b)
In Vitro Cytotoxicity
Finally, thiazoles derivatives with α-glucosidase inhibitory activities were subjected for
cytotoxicity evaluation. Compound 2, 8, and 19 showed a moderate toxicity towards 3T3
mouse fibroblast cell line. All other compounds were found to be non-toxic.

Table-2: In Vitro cytotoxicity of thiazole derivatives 1-24
Comp. No. (IC50 ± SEM) Comp. No. (IC50 ± SEM) Comp. No. (IC50 ± SEM)
a
a
a
c
c
c
1
2
3
4
5
6
7
8
NT
30.55 ± 0.09
9
NTc
17
18
NTc
10
11
12
13
14
15
NTc
NTc
NT
NT
c
NT
NT
NT
NT
19
20
23.82 ± 0.43
c
c
NTc
c
c
NTc
21
22
NT
c
c
NT
>30c
NT
NTc
c
NT
23
24
NTc
NT
27.03 ± 0.14
16
c
Cycloheximide
Non Toxic (NT ); Cycloheximide (Standard inhibitor for cytotoxicity)
0.26 ± 0.1 µM
c
c
Conclusion
Newly synthesized thiazoles were showed in vitro α-glucosidase inhibitory activities in the
range of IC50 = 9.06 ± 0.10-82.50 ± 1.70 µM compared to standard acarbose (IC50 = 38.25 ±
0
.12 µM). Except compounds 5, 19, 20, 21, 22, 23 and 24, all compounds were found to be
potent. Molecular docking studies were carried out in order to understand the binding
interactions of all compounds. From this study compounds 1-4 and 6-18 were identified as
lead molecules. Further research on these non-toxic molecules may result powerful α-
glucosidase inhibitors.
Experimental
Materials and Methods
Analytical grade reagents and solvents were purchased from Sigma-Aldrich and used as
received. Thin layer chromatography was performed on pre-coated silica gel, GF-254. Spots
were visualized under ultraviolet light at 254 and 366 nm. Mass spectra were recorded under
1
13
electron impact (EI) on MAT 312 and MAT 113D mass spectrometer. The H- and C-NMR
were recorded on Bruker AM spectrometer, operating at 300, 400 and 500 MHz instruments.
The chemical shift values are presented in ppm (δ), relative to tetramethylsilane (TMS) as an
internal standard and the coupling constant (J) are in Hz. CHN analysis was performed on a
Carlo Erba Strumentazion-Mod-1106, Italy.
General Experimental Procedure for the Syntheses of Thiazole derivatives 1-24
Benzohydrazide derivative/phenyl hydrazine/benzophenone hydrazone (1 mmol), phenyl
isothiocyanate derivative (1 mmol) were taken in ethanol (10 mL) into a 100 mL round-
bottomed flask and refluxed for half an hour. Than phenacyl bromide derivatives (1 mmol)

and trimethylamine (1 mmol) were added into above mixture and further refluxed for 3 h.
TLC was taken in order to check the reaction progress. Precipitates were formed which were
filtered and washed with cold ethanol (10 mL) to afford pure products in high yields.
In Vitro α-Glucosidase Inhibition Assay
The inhibitory activities of all the thiazole derivatives were measured by using the methods
similar to those described previously [25-27]. Typically, α-glucosidase activity was assayed
in 50 mM phosphate buffer (pH 6.8) containing 5% v/v dimethyl sulfoxide and PNP
º
glycoside was used as a substrate. The inhibitors were pre-incubated with enzyme at 37 C for
º
0
.5 h. The substrate was then added and enzymatic reaction was carried out at 37 C for 60
min. The reaction was monitored spectrophotometrically by measuring the absorbance at 400
nm. All experiments were carried out in triplicates. The percent inhibition was calculated by
the following equation:
Inhibition (%) = (Abs of Control – Abs of Test / Abs of Control) × 100
Active compound solutions were suitably diluted and their inhibition studies were
determined. Data obtained was used for the determination of IC50 values (concentration at
which there is 50% enzyme inhibition) using Microsoft Excel word (2010) (Figure-12).

Representative graph for calculating IC50 values
Figure-12: α-Glucosidase inhibitory effect of compound 3, 12, and 20
Molecular Docking
Protein-ligand docking study on thiazole analogs was carried out against the active site of α-
glucosidase enzyme to understand the ligand-enzyme interactions using molecular operating
environment (MOE) software, version 2011. The X-ray crystal structures of a few bacterial α-
glucosidase have been reported. However, the 3D structure of α-glucosidase used in
biological assays from yeast has never been reported [28]. To find a proper structural
template for homology modeling, we searched for the protein data bank (PDB) from the
protein sequence data bank (http://www.ncbi.nlm.nih.gov/protein/). The crystallographic
structure of Saccharomyces cerevisiae isomaltase (PDB code 3AJ7; Resolution 1.30 Å) with
7
2.4% of sequence identity with the target was selected as a template [29]. The 3D structure
of α-glucosidase for Saccharomyces cerevisiae was predicted using MOE homology
modeling tools. The predicted model was then subjected to energy minimization up to 0.05
gradients. The 3D structures of these newly synthesized compounds were built using MOE-
builder module program implemented in MOE and save as a (mdb) file for molecular

docking. Subsequently, the energy of all compounds was minimized up to 0.05 gradient using
MMFF 94x force field. Energy minimization of all compounds was followed by the
preparation of protein for docking purposes. All the synthesized compounds was docked into
the active site of protein using the triangular matching docking method. The complex was
analyzed for interactions and their 3D images were taken by using visualizing tool PyMol.
3
T3 Cell based (mouse Fibroblast) cytotoxicity assay
Standard MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-dimethyltetrazolium bromide) colorimetric
assay was used to evaluate the cytotoxicity of thiazoles derivatives with α-glucosidase
inhibitory activities, in 96-well flat-bottomed microplates. 3T3 Cells (mouse fibroblasts) in
Dulbecco’s modified eagle’s medium was cultured for this purpose, supplemented with
streptomycin, penicillin, and 5% fetal bovine serum (FBS) by using a flask placed in a 5%
⁰
CO
2
incubator at 37 C. The growth of cells was harvested exponentially by using
hemocytometer, the harvested cells was counted and in a particular medium was diluted. Cell
cultures was prepared with a required concentration and plated onto 96-well plates. After
overnight incubation, medium was removed and fresh medium was added with different
concentrations of the compound. After 72 h, MTT was added to each well and incubation was
continued for 4 h. Subsequently, 100 µL of DMSO was added to each well to solubilize
formazan-MTT adduct, formed by the action of enzyme mitochondrial dehydrogenase. The
extent of MTT reduction to formazan within cells was calculated by measuring the
absorbance at 540 nm by using a microplate ELISA reader [30].
N-(4-(4-Chlorophenyl)-2-((4-nitrophenyl)imino)thiazol-3(2H)-yl)-3-methylbenzamide
(
1)
º
1
Yield: 89%; M.P.: 220-222 C; H NMR (300 MHz, DMSO-d
5′′′ = 9.3 Hz, 1H, H-6′′′), 8.27 (d, J3′ 6′ = 8.7 Hz, 1H, H-3′, H-5′), 7.83 (m, 3H, H-2′′′,
H-5′′′, H-6′′′), 7.57 (m, 4H, H-2′′, H-3′′, H-5′′, H-6′′), 7.18 (d, J2′ 3′ = J6′,5′ = 9.0 Hz, 2H, H-2′,
6
): δ 11.54 (s, 1H, NH), 8.31 (d,
J
6′′′
,
,2′ = J5′,
,
1
3
H-6′), 6.72 (s, 1H, H-5), 2.34 (s, 3H, CH
3
); C NMR (75 MHz, DMSO-d
6
): δ 165.7, 156.1,
1
5
38.2, 138.1, 134.0, 133.2, 131.9, 131.1, 129.4, 129.4, 128.7, 128.7, 128.6, 127.9, 127.9, 125.
+
, 125.5, 124.5, 121.5, 121.5, 116.8, 96.2, 20.8; EI MS: m/z (rel. abund. %) 464 (M ,77), 466
+
(
M +2, 32), 296 (100); Anal. Calcd for C23
H17ClN
4
O
3
S, C = 59.42, H = 3.69, N = 12.05;
-1
Found C = 59.45, H = 3.67, N = 12.08; IR (KBr, cm ): 3590 (amidic-NH), 1670 (C=O),
620, 1590, 1565 (C=C, Aromatic), 1545, 1395 (NO ).
1
2

N-(4-(4-Cyanophenyl)-2-(4-nitrophenylimino)thiazol-3(2H)-yl)-3-methylbenzamide (2)
º
1
Yield: 69%; M.P.: 230-232 C; H NMR (300 MHz, DMSO-d
6
): δ 8.18 (m, 3H, H-3′, H-5′, H-
5′′ = 8.4Hz, 1H, H-6′′), 7.94 (m, 4H, H-3′′′, H-5′′′, H-4′′), 7.57 (m, 2H, H-5, H-
′′), 7.32 (d, J2′′′ 5′′′ = 8.1Hz, 2H, H-2″′, H-6″′), 7.10 (d, J2′ 5′ = 9Hz, 2H, H-2′,
): δ 164.7, 161.8, 158.4, 155.2, 146.5,
38.7, 138.6, 134.2, 132.5, 132.0, 132.0, 131.5, 128.0, 127.2, 127.2, 125.3, 125.3, 124.7,
2
5
′′), 8.06 (d, J6′′,
,
3′′′ = J6′′′
,
,3′ = J6′,
1
3
H-6′), 2.29 (s, 3H, CH
3
); C NMR (75 MHz, DMSO-d
6
1
1
+
23.0, 123.0, 111.7, 108.4, 21.2; EI MS: m/z (rel. abund. %)) 455 (M , 2), 296 (95), 159
S, C = 63.29, H = 3.76, N = 15.38; Found C = 63.26, H =
.73, N = 15.40; IR (KBr, cm ): 3605 (amidic-NH), 2255 (CN), 1670 (C=O), 1634, 1596,
575 (C=C, Aromatic), 1557, 1418 (NO ).
(
100); Anal. Calcd for C24
H
17
N
5
O
3
-1
3
1
2
3
-Methyl-N-(2-(4-nitrophenylimino)-4-phenylthiazol-3(2H)-yl)benzamide (3)
º
1
Yield: 74%; M.P.: 285-287 C; H NMR (300 MHz, DMSO-d
3′′ = J6′′,5′′ = 9.3 Hz, 4H, H-3′, H-5′, H-2′′, H-6′′), 7.82 (d, J2′
9.3 Hz, 3H, H-2′, H-6′, H-6′′′), 7.73 (m, 3H, H-5, H-2′′′, H-4′′′), 7.49 (t, J3′′
6
): δ 11.53 (s, 1H, NH), 8.29 (d,
J
3′
,
2′ = J5′
,
6′ = J2′′
,
,
3′ = J6′,5′ = J6′′′
,
5′′′
=
,
2′′/3′′
,
4′′
=
J
5
1
1
5′′,6′′/5′′,4′′ = 7.5 Hz, 2H, H-3′′, H-5′′), 7.41 (t, J4′′
,
3′′/4′′
,5′′ = J5′′′,4′′′/5′′′,6′′′ = 7.5 Hz, 2H, H-4′′, H-
1
3
′′′), 2.40 (s, 3H, CH
3
); C NMR (75 MHz, DMSO-d ): δ 164.9, 161.7, 158.5, 155.0, 146.5,
6
38.4, 134.2, 134.0, 132.3, 131.5, 128.7, 128.7, 128.4, 128.4, 128.2, 127.7, 125.0, 125.0,
+
24.6, 123.3, 123.3, 108.4, 21.4; EI MS: m/z (rel. abund. %) 430 (M , 2), 296 (100), 159
(
92); Anal. Calcd for C23
H
18
N
4
3
O S, C = 64.17, H = 4.21, N = 13.02; Found C = 64.15, H =
4
.24, N = 13.05.
N-(2-(4-Bromophenylimino)-4-phenylthiazol-3(2H)-yl)-3-methylbenzamide (4)
º
1
Yield: 78%; M.P.: 265-267 C; H NMR (300 MHz, DMSO-d
6
): δ 10.84 (s, 1H, NH), 7.71 (s,
1
6
2
1
1
H, H-2′′′), 7.69 (d, J2′′
′, H-5″′), 7.48 (t, J3′′
H, H-4″′, H-6″′), 2.39 (s, 3H, CH
,3′′ = J6′′,5′′ = 7.5 Hz, 2H, H-2′′, H-6′′), 7.59 (m, 5H, H-2′, H-3′, H-5′, H-
,
2′′/3′′,4′′ = J5′′,4′′/5′′,6′′ = J4′′,3′′/4′′,5′′ = 7.5 Hz, 3H, H-3′′, H-4′′, H-5′′), 7.39 (m,
1
3
3
); C NMR (75 MHz, DMSO-d ): δ 164.7, 161.9, 160.6,
6
58.5, 148.1, 138.3, 135.3, 134.0, 132.7, 132.7, 132.5, 131.5, 129.7, 128.2, 124.6, 122.7,
22.7, 121.5, 120.5, 113.6, 110.6, 108.2, 55.6, 21.4; EI MS: m/z (rel. abund. %) 329 (69), 194
(
28), 159 (100); Anal. Calcd for C24
H20BrN
3
2
O S, C = 58.30, H = 4.08, N = 8.50; Found C =
5
8.33, H = 4.05, N = 8.52.

N-(2-(4-Bromophenylimino)-4-(4-(methylsulfonyl)phenyl)thiazol-3(2H)-yl)-3-
methylbenzamide (5)
º
1
Yield: 85%; M.P.: 235-237 C; H NMR (300 MHz, DMSO-d
m, 4H, H-2′′, H-3′′, H-5′′, H-6′′), 7.59 (s, 1H, H-2′′′), 7.56 (d, J6′″
.48 (d, J3′ 2′/5′,6′ = 8.7 Hz, 2H, H-3′, H-5′), 7.33 (m, 2H, H-4′′′, H-5′′′), 7.21 (s, 1H, H-5), 6.85
d, J2′ 3′ = J6′,5′ = 9.0 Hz, 2H, H-2′, H-6′), 3.21 (s, 3H, SO
75 MHz, DMSO-d ): δ 164.7, 161.8, 158.4, 148.1, 140.3, 139.3, 138.6, 134.0, 132.7, 132.7,
32.3, 131.5, 128.3, 128.3, 128.2, 127.5, 127.5, 124.6, 122.9, 122.9, 121.5, 108.4, 47.8, 21.2;
6
): δ 10.17 (s, 1H, NH), 8.00
(
,
5″′ = 7.2 Hz, 1H, H-6′′′),
7
,
13
(
(
,
2 3 3
CH ), 2.29 (s, 3H, CH ); C NMR
6
1
+
+
EI MS: m/z (rel. abund. %) 541 (M , 1.8), 543 (M +2, 2), 331 (100), 183 (100); Anal. Calcd
, C = 53.14, H = 3.72, N = 7.75; Found C = 53.16, H = 3.75, N = 7.72.
3 3 2
for C24H20BrN O S
N-(2-(4-Bromophenylimino)-4-(4-chlorophenyl)thiazol-3(2H)-yl)-3-methylbenzamide (6)
º
1
Yield: 81%; M.P.: 200-202 C; H NMR (300 MHz, DMSO-d
Hz, 2H, H-3′′, H-5′′), 7.55 (s, 1H, H-2′″), 7.52 (d, J6′′′ 5′′′ = 7.5 Hz, 1H, H-6′′′), 7.45 (m, 4H,
H-2′, H-3′, H-5′, H-6′), 7.31 (m, 2H, H-4′′′, H-5′′′), 7.00 (s, 1H, H-5), 6.82 (d, J2′′
6
): δ 7.71 (d, J3′′,2′′ = J5′′,6′′ = 8.4
,
,
5′′
3′′ = J6′′, =
1
3
8
1
1
.7 Hz, 2H, H-2′′, H-6′′), 2.27 (s, 3H, CH
3
); C NMR (75 MHz, DMSO-d ): δ 155.6, 152.5,
6
48.1, 147.1, 143.8, 143.0, 140.0, 128.7, 128.7, 127.6, 127.6, 127.0, 126.4, 126.4, 124.8,
+
24.8, 123.9, 123.9, 122.2, 122.2, 23.1; EI MS: m/z (rel. abund. %) 497 (M , 1.3), 499
+
(
M +2, 1.4), 329 (100), 331 (100); Anal. Calcd for C23
H17BrClN
3
OS, C = 55.38, H = 3.44, N
=
8.42; Found C = 55.36, H = 3.42, N = 8.44.
4
-(4-Chlorophenyl)-2-(4-nitrophenylimino)-N-phenylthiazol-3(2H)-amine (7)
º
1
Yield: 79%; M.P.: 190-192 C; H NMR (300 MHz, DMSO-d
Hz, 2H, H-3′, H-5′), 7.91 (d, J2′ 5′ = 8.7 Hz, 2H, H-2′, H-6′), 7.64 (d, J3′′
Hz, 2H, H-3′′, H-5′′), 7.57 (d, J2′′ 5′′ = 8.4 Hz, 2H, H-2′′, H-6′′), 7.48 (t, J3′′′
2′′′/3′′′
6′′′ = 7.5 Hz, 2H, H-3′″, H-5″′), 7.33 (m, 2H, H-4′′′, H-5), 7.03 (d, J2′′′ 3′′′ = J6′′′,5′′′ = 9.0
6
): δ 8.17 (d, J3′
,
2′ = J5′ 6′ = 8.7
,
,
3′ = J6′
,
,
2′′ = J5′′,6′′ = 7.5
,
3′′ = J6′′
,
,
4′′′
, =
J
5′′′
,
4′′′/5′′′
,
,
1
3
Hz, 2H, H-2′′′, H-6′′′), 4.29 (s, 1H, NH); C NMR (75 MHz, DMSO-d
6
): δ 161.7, 158.2,
1
1
1
5
1
55.0, 147.0, 146.5, 133.6, 132.4, 129.4, 129.4, 128.8, 128.8, 125.3, 125.3, 123.1, 123.1,
+
22.9, 120.4, 120.4, 113.3, 113.3, 108.2l; EI MS: m/z (rel. abund. %) 422 (M , 1), 137 (72),
05 (100); Anal. Calcd for C21 S, C = 59.65, H = 3.58, N = 13.25; Found C =
9.63, H = 3.60, N = 13.23; IR (KBr, cm ): 3586 (amidic-NH), 1675 (C=O), 1632, 1587,
570 (C=C, Aromatic), 1561, 1402 (NO ).
4 2
H15ClN O
-1
2

4
-(4-Nitrophenyl)-2-(4-nitrophenylimino)-N-phenylthiazol-3(2H)-amine (8)
º
1
Yield: 84%; M.P.: 200-202 C; H NMR (300M Hz, DMSO-d
6
): δ 8.33 (d, J3′′
,
2′′ = J5′′
,
6′′ = 8.7
Hz, 2H, H-3′′, H-5′′), 8.18 (m, 4H, H-2′, H-3′, H-5′, H-6′), 7.66 (d, J2′′′
,
3′′′ = J6′′′
,5′′′ = 7.5 Hz,
2
5
H, H-2′″, H-6′″), 7.51 (t, J3′′′
), 7.05 (d, J2′′ 5′′ = 9 Hz, 2H, H-2′′, H-6′′), 4.33 (s, 1H, NH); C NMR (75 MHz,
): δ 161.8, 158.2, 155.0, 147.2, 147.1, 146.3, 140.1, 129.3, 129.3, 126.7, 126.7,
25.1, 125.1, 123.4, 123.4, 123.3, 123.3, 122.7, 113.1, 113.1, 108.1; EI MS: m/z (rel. abund.
,2′′′ = J5′′′,6′′′ = 7.5 Hz, 2H, H-3′″, H-5′″), 7.36 (m, 2H, H-4′′′, H-
1
3
,3′′ = J6′′,
DMSO-d
6
1
+
%
15 5 4
) 433 (M , 8), 285 (24), 105 (100); Anal. Calcd for C21H N O S, C = 58.19, H = 3.49, N =
1
6.16; Found C = 58.17, H = 3.46, N = 16.18.
4
-(4-Methoxyphenyl)-2-(4-nitrophenylimino)-N-phenylthiazol-3(2H)-amine (9)
º
1
Yield: 73%; M.P.: 187-189 C; H NMR (300 MHz, DMSO-d
H, H-3′, H-5′), 7.49 (d, J2′ 3′ = J6′,5′ = 8.7 Hz, 2H, H-2′, H-6′), 7.18 (t, J3′″
8.1 Hz, 2H, H-3′″, H-5′″), 7.07 (d, J2′′ 3′′ = J6′′,5′′ = 9.0 Hz, 2H, H-2′′, H-6′′), 6.95 (d, J3′′
5′′,6′′ = 9.0 Hz, 2H, H-3′′, H-5′′), 6.77 (t, J4′′′ 3′′′ = J4′′′,5′′′ = 7.5 Hz, 1H, H-4′′′), 6.59 (m, 3H, H-
6
): δ 8.17 (d, J3′
,
2′ = J5′
,6′ = 9 Hz,
2
,
,
2′″/3′″
,
4′″ = J5′″
,
6′″/5′″
,
4′″
=
,
,
2′′
=
J
,
1
3
2
1
1
1
′′′, H-6′′′, H-5), 3.74 (s, 3H, OCH
3
); C NMR (75 MHz, DMSO-d ): δ 159.6, 156.7, 150.7,
6
47.3, 139.8, 129.3, 129.3, 129.2, 129.2, 128.8, 128.8, 122.8, 122.6, 120.7, 120.7, 119.3,
+
+
13.6, 113.6, 112.2, 112.2, 91.9, 55.1; EI MS: m/z (rel. abund. %) 416 (M , 97), 418 (M +2,
00), 386 (28), 133 (44); Anal. Calcd for C22 S, C = 63.14, H = 4.34, N = 13.39;
18 4 3
H N O
Found C = 63.12, H = 4.36, N = 13.36.
2
-(4-Nitrophenylimino)-N-phenyl-4-p-tolylthiazol-3(2H)-amine (10)
º
1
Yield: 79%; M.P.: 180-182 C; H NMR (300 MHz, DMSO-d
Hz, 2H, H-3′, H-5′), 7.79 (d, J2′ 5′ = 8.1 Hz, 2H, H-2′, H-6′), 7.65 (d, J2′′′
.5 Hz, 2H, H-2′′′, H-6′′′), 7.48 (t, J3′′′ 4′′′ = 7.5 Hz, 2H, H-3′″, H-5′″), 7.31
m, 3H, H-2′′, H-6′′, H-4′′′), 7.03 (d, J3′′ 6′′ = 9.0 Hz, 3H, H-3′′, H-5′′, H-5), 4.21 (s, 1H,
); C NMR (75 MHz, DMSO-d ): δ 161.7, 158.4, 155.2, 147.2, 146.3,
37.7, 131.0, 129.3, 129.3, 129.1, 129.1, 128.7, 128.7, 125.1, 125.1, 123.3, 123.3, 122.6,
6
): δ 8.17 (d, J3′
,
2′ = J5′
,6′ = 9.0
,
3′ = J6′
,
,
3′′′ = J6′′′
5′′′
, =
7
,
2′′′/3′′′
,
4′′′ = J5′′′
,
6′′′/5′′′
,
(
,
2′′/5′′
,
1
3
NH), 2.35 (s, 3H, CH
3
6
1
1
+
13.3, 113.3, 108.2; EI MS: m/z (rel. abund. %) 402 (M , 24), 222 (79), 117 (100); Anal.
S, C = 65.65, H = 4.51, N = 13.92; Found C = 65.63, H = 4.54, N =
Calcd for C22
18 4 2
H N O
1
3.95.

2
-(4-Chlorophenylimino)-4-(4-methoxyphenyl)-N-phenylthiazol-3(2H)-amine (11)
º
1
Yield: 75%; M.P.: 160-162 C; H NMR (300 Hz, DMSO-d ): δ 7.57 (m, 2H, H-3′, H-5′),
6
7
.30 (d, J2′′
,
3′′ = J6′′,5′′ = 8.4 Hz, 3H, H-2′′, H-6′′, H-5) ,7.06 (t, J3′′′
,
2′′′/3′′′
,4′′′ = J5′′′,3′′′/5′′′,4′′′ = 6.6
Hz, 2H, H-3″′, H-5′″), 6.86 (m, 6H, H-2′, H-6′, H-3′′, H-5′′, H-2′′′, H-6′′′), 6.64 (m, 1H, H-
1
3
4
1
1
′′′), 3.71 (s, 3H, OCH
3
); C NMR (75 MHz, DMSO-d ): δ 159.8, 146.8, 142.0, 140.1,
6
29.5, 129.5, 129.0, 129.0, 125.5, 125.5, 121.9, 121.4, 121.4, 119.8, 113.7, 113.7, 112.2,
+
+
12.2, 94.0, 55.1; EI MS: m/z (rel. abund. %) 407 (M ,29), 409 (M +2, 10), 135 (100); Anal.
OS, C = 64.78, H = 4.45, N = 10.30; Found C = 64.75, H = 4.47, N =
Calcd for C22H18ClN
3
1
0.32.
4
-(4-Chlorophenyl)-2-(4-chlorophenylimino)-N-phenylthiazol-3(2H)-amine (12)
º
1
Yield: 72%; M.P.: 175-177 C; H NMR (300 Hz, DMSO-d
6
): δ 7.58 (m, 2H, H-3′, H-5′),
2′′ = J5′′,6′′ = 8.4 Hz, 2H, H-3′′, H-5′′), 7.12 (m, 3H, H-
′″, H-5″′, H-5), 6.85 (m, 2H, H-2′, H-6′), 6.81 (d, J2′′ 3′′ = J6′′,5′′ = 8.4 Hz, 2H, H-2′′, H-6′′),
7
3
6
1
1
1
6
.38 (m, 2H, H-2′″, H-6′″), 7.31 (d, J3′′,
,
1
3
.67 (m, 1H, H-4′′′); C NMR (75 MHz, DMSO-d
6
): δ 161.8, 158.2, 147.3, 147.0, 133.6,
32.7, 132.4, 130.2, 130.2, 129.4, 129.4, 128.8, 128.8, 122.7, 122.3, 122.3, 120.3, 120.3,
+
+
+
13.1, 113.1, 108.2; EI MS: m/z (rel. abund. %) 409 (M , 8), 411 (M +2, 20), 413 (M +4,
1), 139 (100); Anal. Calcd for C21
1.15; H = 3.69, N = 10.16.
H15Cl
2
N S, C = 61.17; H = 3.67, N = 10.19; Found C =
3
2
-(2,5-Dichlorophenylimino)-4-(4-nitrophenyl)-N-phenylthiazol-3(2H)-amine (13)
º
1
Yield: 80%; M.P.: 195-197 C; H NMR (300 Hz, DMSO-d
6
): δ 10.61 (s, 1H, NH), 7.92 (m,
4
2
1
1
H, H-3″, H-5″, H-2′″, H-6″′), 7.80 (s, 1H, H-6′), 7.76 (m, 2H, H-3′, H-4′), 7.67 (m, 4H, H-
13
′′, H-6′′, H-3′′′, H-5′′′), 7.57 (m, 2H, H-4′′′, H-5); C NMR (75 MHz, DMSO-d ): δ 161.8,
6
58.2, 147.3, 147.2, 147.0, 140.2, 132.7, 130.2, 130.2, 129.3, 129.3, 126.5, 126.5, 123.2,
23.2, 122.6, 122.5, 122.5, 113.3, 113.3, 108.1; EI MS: m/z (rel. abund. %) 262 (40), 286
(
2 4 2
100); Anal. Calcd for C21H14Cl N O S, C = 55.15, H = 3.09, N = 12.25; Found C = 55.13, H
=
3.07, N = 12.28.
4
-(4-Chlorophenyl)-2-(2,5-dichlorophenylimino)-N-phenylthiazol-3(2H)-amine (14)
º
1
Yield: 76%; M.P.: 185-187 C; H NMR (300 Hz, DMSO-d
H-6′), 7.88 (d, J2′′′ 4′ = 7.8 Hz, 3H, H-2′′′, H-6′′′, H-3′), 7.83 (d, J2′′
Hz, 2H, H-2′′, H-6′′), 7.75 (dd, J3′′ 3′′ = 2.4 Hz, 2H, H-3′′, H-
6
): δ 7.92 (d, J6′
,
5′ = 2.4 Hz, 1H,
,
3′′′/6′′′
,
5′′′ = J3′
,
,3′′ = J6″,
5′ = 8.7
,
2′′ = J5′′,6′′ = 8.7 Hz, J3′′,5′′ = J5′′,

1
3
5
d
1
′′), 7.67 (m, 3H, H-3″′, H-5′′′, H-4′), 7.57 (m, 2H, H-4′′′, H-5); C NMR (75 MHz, DMSO-
): δ 161.7, 158.2, 147.2, 133.6, 132.2, 132.1, 131.4, 129.0, 129.0, 128.9, 128.9, 128.6,
25.8, 124.1, 122.7, 120.1, 120.1, 113.3, 113.3, 110.6, 108.0; EI MS: m/z (rel. abund. %) 321
S, C = 56.46, H = 3.16, N = 9.41;
6
(
14), 286 (85), 77 (100); Anal. Calcd for C21
H
14Cl
3
N
3
Found C = 56.43, H = 3.18, N = 9.39.
2
-(2,4-Dichlorophenylimino)-4-(4-nitrophenyl)-N-phenylthiazol-3(2H)-amine (15)
º
1
Yield: 58%; M.P.: 194-196 C; H NMR (300 Hz, DMSO-d
6′′ = 8.4 Hz, 2H, H-3′′, H-5′′), 7.85 (d, J2′′′
.55 (d, J3′ 5′ = 2.1, 1H, H-3′), 7.36 (dd, J5′ 6′ = 8.4 Hz, J5′
4′′′ = 7.8 Hz, 2H, H-3′″, H-5″′), 6.98 (d, J6′
5′′′ =7.5 Hz, 1H, H-4″′), 6.62 (d, J2′′
″); C NMR (75 MHz, DMSO-d ): δ 161.8, 158.2, 147.3, 147.2, 140.4, 137.3, 134.3, 131.6,
29.5, 129.5, 129.4, 128.1, 126.7, 126.5, 125.0, 123.4, 123.4, 122.7, 113.2, 113.2, 108.1; EI
6
): δ 9.14 (s, 1H, NH), 8.25 (d,
J
3′′
,
2′′ = J5′′
,
,3′′′ = J6′′′,5′′′ = 8.7 Hz, 2H, H-2′′′, H-6′′′),
7
,
,
,
3′ = 2.1 Hz, 1H, H-5′), 7.17 (t,
5′ = 8.7 Hz, 1H, H-6′), 6.87 (s,
5′′ =8.1 Hz, H-2″, H-
J
1
6
1
3′′′
,
2′′′/3′′′
,
4′′′ = J5′′′
,
6′′′/5′′′
,
,
H, H-5), 6.77 (t, J4′′′
,
3′′′ = J4′′′
,
,3′′ = J6′′,
1
3
6
+
+
MS: m/z (rel. abund. %) 454 (M , 30), 456 (M +2, 40), 330 (70); Anal. Calcd for
S, C = 55.15, H = 3.09, N = 12.25; Found C = 55.12, H = 3.07, N = 12.22.
21 2 4 2
C H14Cl N O
4
-(4-Chlorophenyl)-2-(2,4-dichlorophenylimino)-N-phenylthiazol-3(2H)-amine (16)
º
1
Yield: 59%; M.P.: 187-189 C; H NMR (300 Hz, DMSO-d
6
): δ 9.03 (s, 1H, NH), 7.58 (d,
5′ = 2.4 Hz, 1H, H-3′), 7.47 (d, J2′′
3′ = 2.1 Hz, J5′ 6′ = 8.4 Hz, 1H, H-5′), 7.17 (t,
6′″/5′″
, ,4′″ = 8.1 Hz, 2H, H-3′″, H-5′″), 6.97 (d, J6′,5′ = 8.4 Hz, 1H, H-6′), 6.77 (t,
J
J
J
J
3′′
6″
,
,
2′′ = J5′′
,
6′′ = 8.4 Hz, 2H, H-3′′, H-5′′), 7.53 (d, J3′
,
3′′
, =
5′′ = 8.7 Hz, 2H, H-2″, H-6″), 7.34 (dd, J5′
,
,
3′″
4′″
,
,
2′″/3′″
,
4″′ = J5″′
1
3
3′″ = J4′″
,
5′″ = 7.5 Hz, 1H, H-4′′′), 6.60 (m, 3H, H-2′′′, H-6′′′, H-5); C NMR (75 MHz,
): δ 161.7, 158.4, 147.3, 137.2, 134.1, 133.6, 132.4, 131.6, 129.3, 129.1, 129.1,
28.5, 128.5, 128.3, 125.2, 122.7, 120.3, 120.3, 113.3, 113.3, 108.2; EI MS: m/z (rel. abund.
DMSO-d
6
1
+
+
%
) 445 (M ,100), 447 (M +2, 97), 410 (31); Anal. Calcd for C21
3 3
H14Cl N S, C = 56.46, H =
3
.16, N = 9.41; Found C = 56.44, H = 3.18, N = 9.44.
4
-(4-Chlorophenyl)-2-(3-chlorophenylimino)-N-phenylthiazol-3(2H)-amine (17)
º
1
Yield: 73%; M.P.: 174-176 C; H NMR (300 Hz, DMSO-d
6
): δ 7.57 (d, J3′′
3′′ = J6′′,5′′ = 8.7 Hz, 2H, H-2′′, H-6′′), 7.33 (t, J5′
4′″ = 8.1 Hz, 2H, H-3″′, H-5′″), 7.05 (d, J4′
7.2 Hz, 1H, H-4′), 6.80 (m, 3H, H-2′, H-6′, H-4′′′) 6.59 (d, J2′′′,3′′′ = J6′′′,5′′′ = 8.7 Hz, 2H, H-
,
2′′ = J5′′
,6′′ = 8.4
Hz, 2H, H-3′′, H-5′′), 7.47 (d, , J2′′
,
,4′ = J5′,6′ =
8
.1 Hz, 1H, H-5′), 7.19 (t, J3′″
,
2′″/3′″
,
4′″ = J5″′
,
6″′/5′″
,
,
5′
=
1
3
2
′′′, H-6′′′), 6.56 (s, 1H, H-5); C NMR (75 MHz, DMSO-d ): δ 161.6, 158.2, 150.3, 147.2,
6

1
1
34.1, 133.4, 132.2, 131.3, 129.4, 129.4, 128.8, 128.8, 127.2, 122.7, 122.5, 120.5, 120.3,
+
+
20.3, 113.3, 113.3, 108.1; EI MS: m/z (rel. abund. %) 411 (M
,
100), 413 (M +2, 91) 320
(
25); Anal. Calcd for C21
H
15Cl
2
N
3
S, C = 61.17, H = 3.67, N = 10.19; Found C = 61.15, H =
3
.69, N = 10.16.
-(4-Methoxyphenyl)-N-phenyl-2-(phenylimino)thiazol-3(2H)-amine (18)
4
º
1
Yield: 74%; M.P.: 145-147 C; H NMR (300 Hz, DMSO-d
Hz, 2H, H-2′′, H-6′′), 7.27 (t, J3′ 4′ = 7.8 Hz, 2H, H-3′, H-5′), 7.15 (t, J3′′′
4′′′ = 8.1 Hz, 2H, H-3′″, H-5′″), 6.97 (t, J4′,3′ = J4′,5′ = 7.2 Hz, 1H, H-4′), 6.90 (d, J2′,3′
6′,5′ = 7.2 Hz, 2H, H-2′, H-6′), 6.79 (d, J2′′′ 3′′′ = J6′′′,5′′′ = 7.2 Hz, 2H, H-2′′′, H-6′′′), 6.73 (t,
6″ = 7.8 Hz, 2H, H-3′′, H-5′′), 6.27 (s,
); C NMR (75 MHz, DMSO-d ): δ 161.8, 159.6, 158.2, 149.2,
6
): δ 7.45 (d, J2′′,3′′ = J6′′,5′′ = 8.7
,
2′/3′
,
4′ = J5′
,
6′/5′
,
2′′′/3′′′ 4′′′
, ,
=
=
5′′′ 6′′′/5′′′
J , ,
J
,
J
1
1
1
4′′′,3′′′ = J4′′′,5′′′ = 7.2 Hz, 1H, H-4′′′), 6.57 (d, J3″,2″ = J5′′,
1
3
H, H-5), 3.70 (s, 3H, OCH
3
6
47.2, 130.1, 130.1, 129.6, 129.6, 129.1, 129.1, 127.3, 122.7, 126.4, 122.4, 122.4, 121.2,
+
21.2, 113.3, 113.3, 108.1, 55.7; EI MS: m/z (rel. abund. %) 373 (M , 100) , 150 (62), 132
(
86); Anal. Calcd for C22
H
19
3
N OS, C = 70.75, H = 5.13, N = 11.25; Found C = 70.73, H =
5
.16, N = 11.22.
N-(Diphenylmethylene)-4-(3-nitrophenyl)-2-(4-nitrophenylimino)thiazol-3(2H)-amine
19)
(
º
1
Yield: 89%; M.P.: 175-177 C; H NMR (300 Hz, DMSO-d ): δ 8.26 (d, J6′′,5′′ = 9.0 Hz, 1H,
6
H-6′′), 8.14 (m, 5H, H-3′, H-5′, H-2′′, H-4′′, H-5′′), 7.69 (m, 2H, H-4′′′, H-4′′′′), 7.56 (m, 4H,
H-2″′, H-2″″, H-6″′, H-6″″), 7.46 (m, 4H, H-3″′, H-3′′″, H-5″′, H-5′′′′), 7.27(m, 2H, H-2′, H-
1
3
6
1
1
′), 7.05 (s, 1H, H-5); C NMR (75 MHz, DMSO-d ): δ 161.8, 158.4, 155.5, 155.2, 147.6,
6
46.5, 135.2, 134.3, 132.8, 132.8, 131.2, 131.2, 129.6, 129.0, 129.0, 129.0, 129.0, 128.7,
28.7, 128.7, 128.7, 125.1, 125.1, 123.3, 123.3, 123.2, 120.1, 108.2; EI MS: m/z (rel. abund.
+
%
) 521 (M ,88), 180 (60), 165 (100); Anal. Calcd for C28
H
19
N
5
4
O S, C = 64.48, H = 3.67, N =
-1
1
3.43; Found C = 64.45, H = 3.69, N = 13.45; IR (KBr, cm ): 3605 (amidic-NH), 1685
(
C=O), 1607, 1586, 1570 (C=C, Aromatic), 1535, 1408 (NO ).
2
N-(Diphenylmethylene)-4-(3-methoxyphenyl)-2-(4-nitrophenylimino)thiazol-3(2H)-
amine (20)
º
1
Yield: 95%; M.P.: 165-167 C; H NMR (300 MHz, DMSO-d
6
): δ 8.26 (d, J2′′′
6′ = 9 Hz, 2H, H-3′, H-5′), 7.69 (m, 1H, H-2′′′′), 7.55 (m, 2H, H-
′′′, H-6′′′′), 7.40 (m, 8H, H-2′, H-6′, H-3′′′, H-4′′′, H-5′′′, H-3′′′′, H-4′′′′, H-5′′′′), 6.85 (dd,
,3′′′ = 9.0 Hz,
1
6
H, H-2′′′), 8.01 (d, J3′,2′ = J5′,

J
J
4′′
6′′
,
,
2′′ = 1.8 Hz, J4′′,5′′ = 6.6 Hz, 1H, H-4′′), 6.80 (s, 1H, H-5), 6.76 (bd.s, 1H, H-2′′), 6.70 (d,
1
3
5′′ = 7.5 Hz, H-6′′), 3.60 (s, 3H, OCH
3
); C NMR (75 MHz, DMSO-d ): δ 167.9, 158.9,
6
1
1
5
58.3, 145.3, 143.0, 138.2, 138.0, 135.2, 131.4, 129.8, 129.8, 129.6, 129.2, 129.0, 129.0,
28.2, 128.2, 128.1, 128.1, 127.8, 127.8, 127.3, 127.3, 123.2, 120.2, 114.4, 113.5, 103.6,
+
5.0; EI MS: m/z (rel. abund. %) 506 (M , 45), 166 (78), 165 (98), 77 (100); Anal. Calcd for
29 22 4 3
C H N O S, C = 68.76, H = 4.38, N = 11.06; Found C = 68.74, H = 4.40, N = 11.04.
4
-(4-Chlorophenyl)-N-(diphenylmethylene)-2-(4-nitrophenylimino)thiazol-3(2H)-amine
(
21)
º
1
Yield: 85%; M.P.: 177-179 C; H NMR (300 Hz, DMSO-d
6′ = 9.3 Hz, 2H, H-3′, H-5′), 7.57 (m, 4H, H-2′′′, H-2′′′′, H-6′′′, H-6′′′′), 7.43 (m, 6H,
H-3′′′, H-3′′′′, H-4′′′, H-4′′′′, H-5′′′, H-5′′′′), 7.37 (m, 4H, H-2′, H-6′, H-2′′, H-6′′), 7.27 (d,
6
): δ 7.92 (s, 1H, H-5), 7.87 (d,
3′
J ,2′ = J5′,
1
3
J
3′′
,
2′′ = J5′′
,
6′′ = 7.5 Hz, 2H, H-3′′, H-5′′); C NMR (75 MHz, DMSO-d
6
): δ 161.8, 158.2,
1
1
55.7, 155.0, 146.3, 133.4, 132.7, 132.7, 132.2, 131.2, 131.2, 129.1, 129.1, 129.1, 129.1,
28.7, 128.7, 128.6, 128.6, 128.6, 128.6, 125.3, 125.3, 123.1, 123.1, 120.3, 120.3, 108.2; EI
+
MS: m/z (rel. abund. %) 510 (M , 1), 180 (100), 139 (95); Anal. Calcd for C28
H19ClN
4
O
2
S, C
=
65.81, H = 3.75, N = 10.96; Found C = 65.84, H = 3.72, N = 10.98.
4
-(4-Bromophenyl)-N-(diphenylmethylene)-2-(4-nitrophenylimino)thiazol-3(2H)-amine
(
22)
º
1
Yield: 56%; M.P.: 179-181 C; H NMR (300 Hz, DMSO-d
6
): δ 7.92 (m, 4H, H-2′, H-3′, H-
5
′, H-6′), 7.57 (m, 2H, H-3′′, H-5′′), 7.47 (m, 10H, H-2′″, H-2′′″, H-3′″, H-3′′″, H-4″′, H-4′′″,
1
3
H-5′″, H-5′′″, H-6″′, H-6′″′), 7.26 (d, J2′′
75 MHz, DMSO-d ): δ 161.7, 158.4, 155.7, 155.2, 146.5, 133.3, 132.8, 132.8, 131.6, 131.6,
31.2, 131.2, 129.1, 129.1, 129.1, 129.1, 128.7, 128.7, 128.6, 128.6, 128.6, 128.6, 125.3,
,3′′ = J6′′,5′′ = 6.6 Hz, 3H, H-2′′, H-6′′, H-5); C NMR
(
6
1
1
+
25.3, 123.1, 123.1, 122.2, 108.2; EI MS: m/z (rel. abund. %) 554 (M , 2), 465 (59), 183
S, C = 60.55, H = 3.45, N = 10.09; Found C = 60.53, H =
(
79); Anal. Calcd for C28
H19BrN
4
O
2
3
.43, N = 10.11.
N-(Diphenylmethylene)-2-(4-nitrophenylimino)-4-p-tolylthiazol-3(2H)-amine (23)
º
1
Yield: 70%; M.P.: 160-162 C; H NMR (300 Hz, DMSO-d
6
): δ 7.85 (d, J3′
H, H-3′, H-5′), 7.73 (s, 1H, H-5), 7.58 (m, 2H, H-2′′, H-6′′), 7.45 (m, 10H, H-2′′′, H-2′′′′, H-
′′′, H-3′′′′, H-4′′′, H-4′′′′, H-5′′′, H-5′′′′, H-6′′′, H-6′′′′), 7.27 (d, J3′′ 6′′ = 6.3 Hz, 2H, H-
,2′ = J5′,6′ = 9 Hz,
2
3
,
2′′ = J5′′,

1
3
3
′′, H-5′′), 7.10 (d, J2′
MHz, DMSO-d ): δ 161.8, 158.2, 155.7, 155.2, 146.3, 137.5, 132.8, 132.8, 131.3, 131.2,
31.2, 129.3, 129.3, 129.3, 129.3, 129.1, 129.1, 128.8, 128.8, 128.7, 128.7, 128.7, 128.7,
3
,3′ = J6′,5′ = 8.1 Hz, 2H, H-2′, H-6′), 2.20 (s, 3H, CH ); C NMR (75
6
1
1
+
25.5, 125.5, 123.3, 123.3, 108.2; EI MS: m/z (rel. abund. %) 490 (M , 5), 399 (50), 180
S, C = 71.00, H = 4.52, N = 11.42; Found C =
(
100), 77 (100); Anal. Calcd for C29
H
22
N
4
O
2
7
1.03, H = 4.54, N = 11.40.
4
-(3,4-Dichlorophenyl)-N-(diphenylmethylene)-2-(4-nitrophenylimino)thiazol-3(2H)-
amine (24)
º
1
Yield: 65%; M.P.: 180-182 C; H NMR (300 Hz, DMSO-d
6′ = 9.3 Hz, 2H, H-3′, H-5′), 7.78 (d, J2′′ 6′′ = 1.5 Hz, 1H, H-2′′), 7.57 (m, 12H, H-5″,
H-6″, H-2′′′, H-2′′′′, H-3′′′, H-3′′′′, H-4′′′, H-4′′′′, H-5′′′, H-5′′′′, H-6′′′, H-6′′′′), 7.25 (d, J2′
6
): δ 8.07 (s, 1H, H-5), 7.89 (d,
J
3′
,
2′ = J5′
,
,
3′
, =
1
3
J
6′,5′ = 6.3 Hz, 2H, H-2′, H-6′); C NMR (75 MHz, DMSO-d
6
): δ 161.8, 158.2, 155.7, 155.2,
1
1
46.3, 133.6, 133.4, 132.8, 132.8, 132.5, 131.2, 131.2, 130.2, 129.3, 129.3, 129.3, 129.3,
28.7, 128.7, 128.7, 128.7, 127.9, 125.1, 125.1, 123.3, 123.3, 118.4, 108.4; EI MS: m/z (rel.
+
abund. %) 545 (M , 2), 452 (48), 180 (100); Anal. Calcd for C28
H
18Cl
2
N
4
O
2
S, C = 61.66, H =
3
.33, N = 10.27; Found C = 61.64, H = 3.31, N = 10.25.
Acknowledgements: The authors are thankful to the Higher Education Commission (HEC)
for their financial support to Project No. 20-1910 under National Research Program for
Universities.
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