Synthesis, molecular docking, acetylcholinesterase and butyrylcholinesterase inhibitory potential of thiazole analogs as new inhibitors for Alzheimer disease

Article abstract of DOI:10.1016/j.bioorg.2015.08.002

A series of thirty (30) thiazole analogs were prepared, characterized by ¹H NMR, ¹³C NMR and EI-MS and evaluated for Acetylcholinesterase and butyrylcholinesterase inhibitory potential. All analogs exhibited varied butyrylcholinester

Full text of DOI:10.1016/j.bioorg.2015.08.002

Accepted Manuscript
Synthesis, Molecular Docking, Acetylcholinesterase and Butyrylcholinesterase
Inhibitory Potential of Thiazole Analogs as New Inhibitors for Alzheimer Dis-
ease
Fazal Rahim, Muhammad Tariq Javed, Hayat Ullah, Abdul Wadood,
Muhammad Taha, Muhammad Ashraf, Qurat-ul-Aine, Muhammad Anas Khan,
Fahad Khan, Salma Mirza, Khalid M. Khan
PII:
S0045-2068(15)30011-0
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.08.002
YBIOO 1834
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 July 2015
12 August 2015
16 August 2015
Please cite this article as: F. Rahim, M.T. Javed, H. Ullah, A. Wadood, M. Taha, M. Ashraf, Qurat-ul-Aine, M.A.
Khan, F. Khan, S. Mirza, K.M. Khan, Synthesis, Molecular Docking, Acetylcholinesterase and
Butyrylcholinesterase Inhibitory Potential of Thiazole Analogs as New Inhibitors for Alzheimer Disease, Bioorganic
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.08.002
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Synthesis, Molecular Docking, Acetylcholinesterase and Butyrylcholinesterase Inhibitory
Potential of Thiazole Analogs as New Inhibitors for Alzheimer Disease
Fazal Rahim a*, Muhammad Tariq Javed a, Hayat Ullah a, Abdul Wadood b, Muhammad Taha c,d,
Muhammad Ashraf e, Qurat-ul-Ainee, Muhammad Anas Khan f, Fahad Khan a, Salma Mirzag,
Khalid M Khang
b
aDepartment of Chemistry, Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan. Department
of Biochemistry, Abdul Wali Khan University Mardan, Mardan-23200, Pakistan. cAtta-ur-Rahman
Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus,
d
42300 Bandar Puncak Alam, Selangor, Malaysia. Faculty of Applied Science UiTM, 40450 Shah Alam,
Selangor, Malaysia. eDepartment of Biochemistry and Biotechnology, The Islamia University of
Bahawalpur, Bahawalpur 63100. Pakistan. fNational Centre of Excellence in Physical Chemistry,
g
University of Peshawar, KPK, Pakistan. H. E. J. Research Institute of Chemistry, International Center
for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
ABSTRACT
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A series of thirty (30) thiazole analogs were prepared, characterized by HNMR, C NMR and
EI-MS and evaluated for Acetylcholinesterase and butyrylcholinesterase inhibitory potential. All
analogs exhibited varied butyrylcholinesterase inhibitory activity with IC50 value ranging
between 1.59 ± 0.01-389.25 ± 1.75 μM when compared with the standard eserine (IC50,
0.85±0.0001μM). Analogs 15, 7, 12, 9, 14, 1, 30 with IC50 values 1.59 ± 0.01, 1.77 ± 0.01, 6.21
± 0.01, 7.56 ± 0.01, 8.46 ± 0.01, 14.81 ± 0.32 and 16.54 ± 0.21 μM respectively showed
excellent inhibitory potential. Seven analogs 15, 20, 19, 24, 28, 30 and 25 exhibited good
acetylcholinesterase inhibitory potential with IC50 values 21.3 ± 0.50, 35.3 ± 0.64, 36.6 ± 0.70,
44.81 ± 0.81, 46.36 ± 0.84, 48.2 ± 0.06 and 48.72 ± 0.91 μM respectively. All other analogs also
exhibited well to moderate enzyme inhibition. The binding mode of these compounds was
confirmed through molecular docking.
Keywords: Alzheimer's disease, Acetylcholinesterase inhibition, Butyrylcholinesterase
Inhibition, Thiazole, Synthesis, Molecular docking
*Corresponding Author: fazalstar@gmail.com, Tel.: 0092-335-9528343;
1

1.
Introduction
Alzheimer’s disease is an irretrievable, complex neurodegenerative disorder characterized by
progressive cognitive impairment, various neuropsychiatric and behavioral disturbances, and
restrictions in activities of daily life. It is a pathologically complex disease implicating
interactions between environmental and genetic risk factors [1]. Alzheimer’s is an age related
disease and is the most common reason of dementia in old people, being diagnosed after the age
of 56, and affecting up to 10 % of the population over the age of 65. The disease affects 30% or
more of the population over the age of 80. In the developed world, AD is the fourth major cause
of death after cardiovascular disease, cancer, and cerebral accidents. Worldwide there are
approximately 35 million peoples with AD, and that number is expected to grow to 107 million
by 2050 [2]. Alzheimer disease was reported to be associated with cardiovascular risk factors
such as hypertension and increased serum cholesterol [3]. It involves the degeneration of
cholinergic neurons and loss of cholinergic transmission. Since AD is a multi-pathogenic illness,
a current drug-discovery strategy is to develop novel anti-Alzheimer agents with multiple
potencies such as inhibition of both acetylcholinesterase and butyrylcholinesterase [4].
Acetylcholine plays an important role in cognitive functions particularly in memory. It reduced
cholinergic neurotransmission in the brain which have an important role in cognitive impairment
associated with Alzheimer's disease [5, 6]. BChE (EC 3.1.1.8) is a sister enzyme of AChE [7].
Interest in BChE has been growing because of its possible role in Alzheimer disease and the
introduction of anticholinesterase treatments for this disorder [8]. BChE enzymatic activity was
higher in patients who had hypertension, hyperlipidemia, and high body weight and lower in
patients who had suffered acute myocardial infarction or undergone treatment with beta blockers
[9–11]. BChE inhibit the activity of the enzyme which affects the transmission of the
neurotransmitter and makes patient discomfort including dizziness, blurred vision, vomiting,
fever and even death [12, 13]. BChE specific inhibition is unlikely to be associated with adverse
events and may show efficacy without remarkable side effects [14]. Therefore BChE may be
considered as an important target for novel drug development to treat Alzheimer disease. In the
future, the development of specific BChE inhibitors and the continued use of cholinesterase
inhibitors may lead to improved clinical outcomes [15].
The heterocyclic compounds are reported to posses various biological activity [16]. Thiazole
moiety is the key pharmacophore and intermediate for synthesizing pharmaceuticals and in the
2

field of agro-chemicals [17–19]. Thiazole are important class of heterocyclic compounds found
in many potent biologically active molecules such as thiobendazol (anthelmintic drug) [20],
riluzolr (anticonvulsant drug) [21] and talipexzol (antiparkinsonian drug) [22]. Its application
also found in drug development for the treatment of inflammation [23]. Recently thiazole
analogs have been also reported as antiglycating agent, anti-diabetic and as a potent inhibitor for
cholinesterase [24-26].
Our research group is continuously doing an effort in search of lead molecules [27]. Herein we
are going to report thiazole derivatives as new class of Acetylcholinesterase and
butyrylcholinesterase inhibitors.
2. Results and discussion
2.1 Chemistry
Different acetophenone/benzaldehyde (1mmol) were reacted and refluxed with
thiosemicarbazide (1mmol) in methanol in the presence of catalytic glacial acetic acid for 3-5
hours. After completion of reaction the mixture were filtered and washed with hexane to yield
pure product. The obtained products (1mmol) were then reacted and refluxed with (1mmol) of
methoxy or chloro substituted phenacyl bromide for 3-5 hours. After reaction completion the
mixture was filter and wash with hexane to yield pure products (1-30) [23, 28, 29]. Different
1
13
spectroscopic techniques, such as EI-MS, H NMR and C NMR were used to determine the
structure of all analogs.
Insert Scheme-1 here
Insert Table-1 here
2.2. Acetyl cholinesterase and Butyrylcholinesterase inhibition
Analogs 1-30 showed acetylcholinesterase inhibition with IC50 values ranging between
21.3±0.08 to 452.1±0.27 μM as compared with standard eserine with IC50, 0.04 ± 0.0001 μM.
Seven analogs 15, 20, 19, 24, 28, 30 and 25 exhibited good acetylcholinesterase inhibitory
potential with IC50 values 21.3 ± 0.50, 35.3 ± 0.64, 36.6 ± 0.70, 44.81 ± 0.81, 46.36 ± 0.84, 48.2
± 0.06 and 48.72 ± 0.91 μM respectively. Analog 15 was found the most potent among the series.
This compound has two hydroxyl groups on one phenyl ring and one chloro group on other
3

phenyl ring. The presence of these two hydroxyl group seems to be play an important role in this
inhibition. The hydroxyl might be involved in hydrogen bonding. Analog 20 was found to be
second active among the series. This compound illustrate activity have one methoxy group on
one phenyl ring and also methoxy group on other phenyl part. Compound 19 have two chloro
groups on one phenyl ring and one methoxy group on other phenyl ring. Analog 30 have two
hydroxyl groups on one phenyl ring. Similarly other active analogs have either EWG or EDG on
phenyl ring, whose position, nature and arrangement on phenyl ring greatly affect the inhibition.
Analogs 1-30 also showed a variable degree of butyrylcholinesterase inhibition with IC50values
ranging between 1.59 ± 0.01-389.25 ± 1.75 μM as compared with standard eserine (IC50,0.85 ±
0.0001 μM). Seven analogs 15, 7, 12, 9, 14, 1 and 30 exhibited potent butyrylcholinesterase
inhibitory potential with IC50 values 1.59 ± 0.01, 1.77 ± 0.01, 6.21 ± 0.01, 7.56 ± 0.01, 8.46 ±
0.01, 14.81 ± 0.32 and 16.54 ± 0.21 μM respectively. Compound 15 was also found to be the
most potent among the series. This compound has two hydroxyl groups on one phenyl ring and
one chloro group on other phenyl ring. The presence of these two hydroxyl group seems to be
play an important role in this inhibition. The hydroxyl group is might be involved in hydrogen
bonding. Analog 7 was found to be second most active among the series. The compound have
anthracene moiety and chlorinated phenyl ring. The activity is may be due to arene-interaction.
Analog 12 have one hydroxyl group on one phenyl ring and one chloro group on other phenyl
ring. Its potential is less than compound 15; this might be due less number of hydroxyl groups.
Analog 9 having secondary amine group on one phenyl part and chloro group on the other
phenyl part. Analog 14 illustrate activity have one methoxy and one hydroxyl group on one
phenyl ring and also chloro group on other phenyl part. Analogs 1 and 30 also showed good
potential, but surprisingly in analog 1 on both phenyl parts electron withdrawing groups are
present and in analog 30 on both phenyl parts electron donating groups are present. It means that
those analogs which have electron donating or withdrawing groups on both the phenyl part show
less potency as compared to those analogs which have electron donating group on one phenyl
part and electron withdrawing group on the other phenyl part. All other analogs also showed well
to moderate activities. All analogs except 3, 5 and 29 showed both acetylcholinesterase and
butyrylcholinesterase inhibitory potential, which might be due to position of substituent on
phenyl ring that not suitably interact with the active site of enzyme. The binding interaction of
these analogs for butyrylcholinesterase inhibition was confirmed through molecular docking.
4

2.3. Molecular Docking
From the docking simulation and in vitro studies, it was observed that compound 15 (IC50 value
1.59 µM) is the most active analog among all the 30 compounds which included in this study.
Compound 07 (IC50 value 1.77 µM) also the second ranked most active compound. These
compounds were bound extremely into the binding cavity of butyrylcholinesterase enzyme making
interactions with the active site residues. In active site, two most important residues of BChE
(TYR128 and TYR332) are frequently involved in hydrogen bonding and play an important
inhibitory role [30].
The docking results showed that all the compounds fit well in active site of
butyrylcholinesterase. The top-ranked docking conformation of the most active compound
(compound 15) showed that the compound established five hydrogen bonds and two arene-arene
interations with the active site residues (Figure-1).
The hydroxyl groups attached to the phenyl ring of the compound formed hydrogen bonds with
Trp82, Gly115 Thr120 and Tyr128. One hydrogen bond between the amine moiety of the compound and
His 438 was also observed. Furthermore, two arene-arene interactions between the compound and the
active site residues (Trp82 and Tyr332) were also observed (Figure-1). The highest activity of
compound 15 might be due to the presence of two hydroxyl groups attached to the same benzene
ring. Both the hydroxyl groups are involved in bonding (backbone acceptor and side chain
acceptor). The hydroxyl groups are well-built activating groups which polarize the molecule and
facilitate it to make several interactions with other residues.
Insert Figure-1 here
The second most active compound (Compound 07) in the series was observed to establish four
interactions with important active site residues (Figure-2). As shown in Figure-2, Thr284
interact with the NH group of the compound, which is directly attached to the Azomethine
group, Ser287 make interaction with the nitrogen atom of thiazole group and with the NH attach
next to azomethine group and Tyr332 establish π-interaction with the chloro-substituted benzene
ring.
5

Insert Figure-2 here
The third most active compound (Compound 12) in the series was observed in establishing two hydrogen
bonds with the important active site residues, as shown in (Figure-3). Glu197 establish interaction with
the NH next attach to the azomethine group and Leu286with para-hydroxyl moiety of the
compound 12.
The fourth, fifth and sixth most active compound (Compound 09, Compound 14 and Compound
01) in the series of the synthesized compounds was observed in making interactions with the
active site residues of the BChE enzyme, Trp82 and His438 was found in phi-interaction with
benzene ring of the compound (compound 09) through arene-arene bond and His438 interact
with thiazole ring through arene-cation bond. Trp82& Leu286 making interactions with thiazole
moiety of the compound and Leu286 interact with meta-hydroxyl moiety attaches to the benzene
ring of the compound (compound 14).
Glu197 was found in interaction with compound 01 through hydrogen bonding as shown in
(Figure-3).
Insert Figure-3 here
Compound 02, 08, 10 and compound 11 also exhibited weak potency against BChE enzyme.
Compound 3, 4, 5, 6, 13 and 16-30 reflects no interaction between compounds and active site
residues of BChE, which might be due to the inverted orientation, and these compounds also
show least inhibitory activity (IC50 value) against the human BChE enzyme which contain much
differences, if compared with the standard substrate (Eserine with IC50 value 0.85 ±
0.0001).Therefore, these compounds were not further evaluated for further study.
The structural difference between the most active compound 15 and the least active compounds are
the Meta and Para-hydroxyl moiety of the compound 15. This OH moiety in compound 15 may
provide enzymatic potency against butyrylcholinesterase enzyme.
6

Docking results of compound 15 and 07 with BChE provided valuable information about the
nature of the binding interactions that were delightfully associated with the experimental studies.
This information could be utilized to design new leads against the BChE.
2.4. Computational Methods
The study was designed to dock thiazole derivatives against butyrylcholinesterase enzyme with
the following communications; Intel(R) xenon(R) CPU E5620@2.40GHz system having 3.8GB
RAM with the open 11.4 (X 86_64) operating platform. Protein-Ligand docking was carried out
using the Molecular Operating Environment (MOE 2010.11) software package. Among thirty-
one X-ray crystal structures of human BChE in the protein data bank [31, 32] (Pdb code 1P0P
with 2.30Å resolution) was selected as the target protein based on suitable resolution and co-
crystallized ligand, BChE. The entire system (target protein) was energy minimized by
MMFF94x force field [33], after adding the missing hydrogen atoms, 3D structures of all 30
synthesized compounds were drawn by molecule builder which is incorporated in MOE modeling
package and the structures (Ligands) were subjected to MMFF94x for energy minimization.
Subsequently for the evaluation of potential energy, partial charges were calculated by
MMFF94x force field [34]. Both prepared systems (protein and Ligand) were introduced for
molecular docking simulation. Docking simulations were performed by using Triangle matcher
placement method. A total of 30 conformations were generated for each Ligand protein complex
with docking score. Each complex was analyzed for interactions and their 3D images were taken.
2.5. Butyrylcholinesterase Assay
Ellman et al spectrophotometric method was to measure the ChEs inhibitory assay [33]. For
(AChE) and (BChE) inhibition the substrate (AChE) and (BChE) substrate were used
respectively. 140µl sodium phosphate buffer (pH 8.0), 20 µl of either AChE/BChE solution and
test sample of 20µl were incubated for 15 min at room temperature. The reaction was initiated
with AChE/BChE 10µl with addition of DTNB respectively. For 15 min,AChE or BChE was
hydrolyzing the reaction of DTNB with thiocoline, unconfined by the enzymatic hydrolysis of
AChE and BChE. The 10% analytical grade ethanol were dissolve test samples and positive
control of serine, tecrine and berberine. The percentage (%) inhibition was calculated from E-S/E
˟100, where E&S are enzyme activities with and without test sample. Each sample was expressed
7

ChEs inhibitory activity in term of IC50 value (µg/ml) or µm essential to inhibit the hydrolysis of
substrate.
2.6. Acetylcholinesterase Assay
The AChE inhibition activity was performed according to the method [33] with slight
modifications. Total volume of the reaction mixture was 100 µL. It contained 60 µL Na2H PO4
buffer with concentration of 50 mM and pH 7.7. Ten µL test compound (0.5 mM well-1) was
added, followed by the addition of 10 µL (0.005 unit well-1) enzyme. The contents were mixed
and pre-read at 405 nm. Then contents were pre-incubated for 10 min at 37ºC. The reaction was
initiated by the addition of 10 µL of 0.5 mM well-1 substrate (acetylthiocholine iodide), followed
by the addition of 10 µL DTNB (0.5 mM well-1). After 15 min of incubation at 37ºC absorbance
was measured at 405 nm using 96-well plate reader Synergy HT, Biotek, USA. All experiments
were carried out with their respective controls in triplicate. Eserine (0.5 mM well-1) was used as a
positive control. The percent inhibition was calculated by the help of following equation
Inhibition (%) = Control – Test x 100
Control
IC50 values were calculated using EZ–Fit Enzyme kinetics software (Perrella Scientific Inc.
Amherst, USA).
3. Conclusion
Thirty analogs were prepared and evaluate for acetylcholinesterase and butyrylcholinesterase
inhibition. Seven analogs 15, 7, 12, 9, 14, 1, 30 with IC50 values 1.59±0.003 μM, 1.77
±0.002μM, 6.21 ±0.005μM, 7.56 ±0.003μM, 8.46 ±0.003μM, 14.81 ±0.02μMand 16.54 ±0.11μM
respectively showed excellent butyrylcholinesterase inhibitory potential comparable with the
standard eserine (IC50, 0.85±0.0001μM). Seven analogs 15, 20, 19, 24, 28, 30 and 25 also
exhibited good acetylcholinesterase inhibitory potential with IC50 values 21.3±0.08, 35.3±0.04,
36.6±0.06, 44.81±0.05, 46.36±0.02, 48.2±0.06 and 48.72±0.09 μM respectively. All other
analogs also exhibited well to moderate enzyme inhibition. The binding affinity was confirmed
1
through molecular docking studies. All compounds were characterized through HNMR, 13C
NMR and EI-MS.
8

4. Experimental Section
4.1. General methods
1H NMR spectra were run on an Avance AV-300-400 MHz in d6-DMSO. Chemical shifts (δ) are
reported as values in ppm in DMSO. TMS was used as an internal standard. Electron ionization
(EI) mass spectra (MS) were recorded on Jeol JMS-600H. Reactions were checked by thin TLC
on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany); spots were
visualized by UV at 254 and 365nm.
4.2. General procedure for the synthesis of compounds (1–30)
Different acetophenone/benzaldehyde (1mmol) were reacted and refluxed with
thiosemicarbazide (1mmol) in methanol in the presence of catalytic glacial acetic acid for 3-5
hours. After completion of reaction the mixture were filtered and washed with hexane to yield
pure product. The obtained products (1mmol) was then reacted and refluxed with (1mmol) of
methoxy or chloro substituted phenacyl bromide for 3-5 hours. After reaction completion the
mixture was filter and wash with hexane to yield pure products (1-30) [26, 27]. Different
1
13
spectroscopic techniques, such as EI-MS, H NMR and C NMR were used to determine the
structure of all analogs.
4.3. Characterization of compounds
4.3.1. (E)-4-(4-chlorophenyl)-2-(2-(-(3-nitrophenyl)ethylidene)hydraiznyl)thiazole. (1)
Yield: 33%; 1HNMR: (DMSO-d6, 300 MHz): δ 11.5 (s, 1H, NH), 8.5 (Hr.s, 1H, H-2), 8.2 (t,J4/5,6
= 8.4 Hz , 2H, H-4/6), 7.9 (d, J3″, 2″/5″,6″ = 8.4 Hz , 2H, H-3″/5″), 7.7 (t, J5/4,6 = 7.6 Hz, 1H,
6/4,5
H-5),7.4 (m, 3H, H-5′/2″/6″); IR (ATR): 3012, 1657, 1522, , 720, 695 cm−1; 13C NMR (75 MHz,
DMSO-d6): 171.4, 154.4, 150.1, 147.6, 138.2, 135.0, 134.2, 131.0,129.5, 129.2, 129.2, 128.4,
128.4, 123.0, 120.6, 105.1, 31.1,; EI-MS: m/z (rel. int. %): 372 (M+, 100), 223 (37), 209 (67),
174 (95), 168 (73).
4.3.2. (E)-4-(4-chlorophenyl)-2-(2-(2-(4-nitrophenyl)ethylidene)hydrazinyl)thiazole.(2)
Yield: 50%; 1H-NMR: (DMSO-d6, 300 MHz): δ 12.6 (s, 1H, NH), 8.2 (d, J3,2/5,6 = 8.7Hz, 2H, H-
3/5), 8.1(s, 1H, HC=N), 8.4 (m, 4H,H-2,6/2″,6″), 8.1 (m, 3H, H-5ꞌ/2″/6″); IR (ATR): 3018, 1660,
1528, , 740, 691 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.5, 154.4, 150.1, 144.6, 143.4, 134.1,
9

131.0, 129.1, 129.1, 128.8, 128.8, 128.7, 128.5, 123.6, 123.6, 105.0, 32.1; EI-MS: m/z (rel. int.
%): 210 (M+, 100), 168 (50), 358 (45), 372 (3), 236 (5).
4.3.3. (E)-2-(2-(1-(2-bromo-4-nitrophenyl)ethylidene)hydrazinyl)-4-(4-chlorophenyl) thiazole.
(3)
1
yield: 50%; Solid m.p. 262 °C; H-NMR: (DMSO-d6, 300 MHz): δ 12.8 (s, 1H, NH), 8.2 (m,
2H, H-3/5), 8.1 (d, J3″,2″/5″,6″ = 6.3Hz , 2H, H-3″/5″), 8.0 (m, 1H, H-6), 8.1 (d, J2″,3″/6″,5″ =
8.7Hz , 2H, H-2″/6″), 7.5 (s, 1H, H-5′), 3.3 (s, 3H, Me); IR (ATR): 3030, 1654, 1540, , 680, 555
13
cm−1; C NMR (75 MHz, DMSO-d6): 171.4, 168.4, 152.2, 150.0, 141.5, 134.1, 132.2, 131.0,
129.1, 129.1, 128.6, 128.6, 126.5, 123.1, 122.4, 105.1, 16.2; EI-MS: m/z (rel. int. %): 449 (M+,
18), 221 (17), 149 (15), 137 (31), 83 (100); MS: as calculated 449.9553 and found 449.9545.
4.3.4. (E)-4-(4-chlorophenyl)-2-(2-(2,4-dichlorohenzylidene)hydrazinyl)thiazole. (4)
Yield: 50%; 1H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 1H, NH), 8.3 (Hs, 1H, HC=N), 7.9 (m,
3H, H-3/3″/5″), 7.6 (s, 1H, H-5ꞌ), 7.5 (m, 3H, H-6/2"/6"); IR (ATR): 3030, 1654, 690, 560 cm−1;
13C NMR (75 MHz, DMSO-d6): 171.6, 150.0, 143.3, 134.2, 132.6, 131.0, 131.0, 129.1,
129.1129.0, 129.0, 128.7, 128.6, 128.2, 127.0, 105.1; EI-MS: m/z (rel. int. %): 381 (M+, 15), 210
(100), 168 (36).
4.3.5. (E)-4-(4-chlorophenyl)-2-(2-(2-nitrobenzylidene)hydrazinyl)thiazole.(5)
Yield: 50%; Solid m.p. 260 °C; 1H-NMR: (DMSO-d6, 300 MHz): δ 12.5 (s, 1H, NH), 8.4 (s, 1H,
HC=N), 8.0 (d, J3″, 2″/5″,6″ = 8.1 Hz , 2H, H-3″/5″), 8.0 (d, J3,4/6,5 = 8.4 Hz , 2H, H-3/6), 7.7
(t, J4/3,5 = 7.5 Hz, 1H, H-4), 7.6 (t, J5/4,6 = 7.2 Hz, 1H, H-5), 7.4 (m, 3H, H-5′/2″/6″); IR
(ATR): 3020, 1674, 1560, , 695, 540 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.4, 150.1, 147.6,
143.1, 134.6, 134.1, 131.7, 131.0, 130.2, 129.1, 129.1, 128.6, 128.6, 128.2, 124.1, 105.2; EI-MS:
m/z (rel. int. %): 358 (M+, 54), 210 (100), 168 (91), 83 (68), 44 (56); MS: as calculated 358.0291
and found 358.0295.
4.3.6. (E)-4-(4-chlorophenyl)-2-(2-(1-(4-methoxyphenyl)ethylidene)hydrazinyl)thiazole
.(6)
Yield: 81%; 1H-NMR: (DMSO-d6, 400 MHz): δ7.9 (m, 4H,H-2",3"/5″,6″), 7.5 (d,J2,3/6,5 = 8.8 Hz
, 2H, H-2/6), 7.4(s, 1H, H-5ꞌ)7.0 (d,J3,2/5,6 = 8.8 Hz , 2H, H-3/5); IR (ATR): 3035, 1658, 1240,
10

680, 555 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.4, 168.6, 162.4, 150.1, 134.1, 131.0, 129.6,
129.1, 129.1, 128.8, 128.8, 128.6, 128.6, 114.2, 114.3, 105.1, 55.6, 17.0; EI-MS: m/z (rel. int.
%): 357 (M+,33 ), 342 (3), 171 (1), 148 (100), 134 (50).
4.3.7. (E)-2-(2-(anthracen-9-ylmethylene)hydrazinyl)-4-(4-clorophenyl)thiazole.(7)
1
Yield: 81%; H-NMR: (DMSO-d6, 400 MHz): δ11.2 (s, 1H, NH), 9.4 (s, 1H, HC=N) 8.8
(d,J3",2"/5",6" = 8.8 Hz , 2H, H-3"/5"), 8.6 (s, 1H, H-5ꞌ) 8.1 (d,J2″,3″/6″,5″ = 8.4 Hz , 2H, H-2″/6″), 7.9
(d,J2,3/10,9 = 8.8 Hz , 2H, H-2/10), 7.6 (m, 4H, H-3,4/9,8), 7.4 (d,J5,4/7,8 = 8.4 Hz , 2H, H-5/7),
7.2 (s, 1H, H-6); IR (ATR): 3105, 1680, 690, 564 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.6,
105.0, 128.7, 129.1, 134.2, 129.1, 150.1, 128.7, 131.0, 143.2, 128.0, 128.7, 128.0, 125.4, 125.4,
128.7, 131.6, 128.0, 123.7, 125.5, 128.7, 131.6, 126.0, 125.5; EI-MS: m/z (rel. int. %): 411 (M+,
33), 237 (8), 210 (20), 202 (100), 176 (36).
4.3.8. (E)-4-(4-chlorophenyl)-2-(2-(1-(4-(piperidin-1-yl)phenyl)ethylidene)hydrazinyl)
thiazole.(8)
1
Yield: 43%; Solid m.p. 280 °C; H-NMR: (DMSO-d6, 300 MHz): δ 7.9 (m, 4H, H-3″/5″/2/6),
7.4 (m, 4H, H-2″/6″/3/5), 7.2 (s, 1H, H-5′), 3.3 (s, 3H, Me), 3.0 (s, 10H, H-2‴/3‴/4‴/5‴/6‴); IR
(ATR): 3095, 1680, 1630, 695, 560 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.6, 168.7, 151.8,
150.1, 134.2, 131.0, 130.0, 130.0, 129.1, 129.1, 128.7, 128.7, 127.0, 111.8, 111.8, 105.1, 54.7,
54.7, 25.4, 25.4, 24.3, 17.1; EI-MS: m/z (rel. int. %): 410 (M+, 54), 375 (20), 299 (29), 210
(100), 168 (91), 83 (68), 44 (56); MS: as calculated 410.1332 and found 410.1327.
4.3.9. (E)-4-((2-(4-(4-chlorophenyl)thiazol-2-yl)hydrazono)methyl)-N,N-dimethyl aniline.(9)
1
Yield: 38%; Solid m.p. 270 °C; H-NMR: (DMSO-d6, 300 MHz): δ 8.0 (s, 1H, HC=N), 7.8 (d,
J3″,2″/5″,6″ = 6.3 Hz , 2H, H-3″/5″), 7.6 (m, 2H, H-2/6), 7.4 (d, J2″,3″/6″,5″ = 6.6 Hz, 2H, H-
2″/6″), 7.3 (d, J3,2 = 6.3 Hz , 1H, H-3), 7.2 (d, J5,6 = 6.3 Hz , 1H, H-5), 7.2 (s, 1H, H-5′), 3.0 (s,
6H, 2Me); IR (ATR): 3045, 2960, 1680, 1060, 685, 548 cm−1; 13C NMR (75 MHz, DMSO-d6):
171.5, 153.2, 150.1, 143.2, 134.2, 131.0, 129.1, 129.1, 128.8, 128.8, 128.1, 128.1, 123.1, 111.8,
111.8, 105.0, 41.2, 41.2; EI-MS: m/z (rel. int. %): 356 (M+, 3), 210 (100), 168 (39), 147 (46), 43
(87); MS: as calculated 356.0862 and found 356.0854.
11

4.3.10. (E)-4(4-chlorophenyl)-2-(2-(naphthalen-2-yl-methylene)hydrazinyl)thiazole.(10)
1
Yield: 32%; Solid m.p. 276 °C; H-NMR: (DMSO-d6, 300 MHz): δ 8.3 (s, 1H, HC=N, 8.0 (d,
J3″, 2″/5″,6″ = 8.4 Hz , 2H, H-3″/5″), 7.9 (m, 5H, H-2/3/4/5/8), 7.5 (m, 2H, H-7/6), 7.4 (d,
J2″,3″/6″,5″ = 8.4 Hz , 2H, H-2″/6″), 7.2 (s, 1H, H-5′); IR (ATR): 3115, 1680, 1640, 700, 568
13
cm−1; C NMR (75 MHz, DMSOd6): 171.6, 150.1, 143.2, 134.2, 136.0, 133.8, 131.0, 129.1,
128.6, 128.6, 128.4, 128.0, 127.9, 127.9, 127.1, 126.8, 126.1, 126.1, 105.1; EI-MS: m/z (rel. int.
%): 363 (M+, 18), 210 (87), 168 (35), 153 (100), 127 (33); MS: as calculated 363.0597 and
found 363.0590.
4.3.11. (E)-2-(2-(4-(benzyloxy)benzylidene)hydrazinyl)-4-(4-chlorophenyl)thiazole.(11)
1
Yield: 81%; Solid m.p. 278 °C; H-NMR: (DMSO-d6, 300 MHz): δ 8.0 (s, 1H, HC=N), 7.8 (d,
J3″,2″/5″,6″ = 6.3 Hz , 2H, H-3″/5″), 7.6 (d, J2″,3″/6″,5″ = 6.3 Hz , 2H, H-2″/6″), 7.4 (d,
J2,3/6,5 = 5.4 Hz , 2H, H-2/6), 7.3 (m, 5H, H-2‴/3‴/4″′/5″′/6″′), 7.2 (s, 1H, H-5′), 7.0 (d, J3,2/5,6
= 6.6 Hz , 2H, H-3/5), 5.1 (s, 2H, OCH2); IR (ATR): 3025, 1665, 690, 570 cm−1; 13C NMR (75
MHz, DMSO-d6): 171.6, 161.2, 150.1, 143.2, 136.5, 134.2, 131.0, 130.1, 130.1, 129.1, 129.1,
128.7, 128.7, 128.7, 128.7, 127.5, 127.0, 127.0, 126.1, 114.2, 114.2, 105.1, 70.7; EI-MS: m/z
(rel. int. %): 429 (M+, 17), 214 (53), 91 (100), 65 (22); MS: as calculated 419.0859 and found
419.0848.
4.3.12. (E)-2,6-di-tert-butyl-4-((2-(4-(4-chlorophenyl)thiazol-2-yl)hydrazono)methyl)
phenol.(12)
Yield: 100%; Solid m.p. 287 °C; 1H-NMR: (DMSO-d6, 300 MHz): δ 7.9 (s, 1H, HC=N), 7.7 (d,
J3″,2″/5″,6″ = 6.3 Hz , 2H, H-3″/5″), 7.4 (s, 2H, H-2/6), 7.0 (s, 1H, H-5′), 6.9 (d, J2″,3″/6″,5″ =
6.6 Hz , 2H, H-2″/6″), 3.7 (s, 3H, OMe), 1.3 (s, 18H, 6Me); IR (ATR): 3025, 2972 1655, 1060,
680, 560 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.6, 150.1, 156.8, 143.2, 136.1, 136.1, 134.2,
131.0, 129.1, 129.1, 128.7, 128.7, 125.4, 123.6, 123.6, 105.1, 36.4, 36.1, 36.1, 36.1, 34.2, 34.2,
31.4, 31.4; EIMS: m/z (rel. int. %): 441 (M+, 18), 216 (35), 210 (100), 174 (37), 168 (59); MS:
as calculated 441.1642 and found 441.1650.
4.3.13. 1-((E(2-(4-(4-chlorophenyl)thiazol-2-yl)hydrazono)methyl)-3-((Z)-2-(4-(4-chloro
phenyl)thiazol-2-yl)hydrazono)methyl)benzene.(13)
1
Yield: 85%; Solid m.p. 278 °C; H-NMR: (DMSO-d6, 300 MHz): δ 12.2 (s, 2H, 2xNH), 8.0 (s,
2H, 2xHC=N), 7.8 (d, J3″,2″/5″,6″ = 8.4 Hz , 4H, 2xH-3″/5″), 7.6 (s, 2H, 2xH -5′), 7.4 (m, 8H,
12

13
H-2/3/5/6, 2xH-2″/6″); IR (ATR): 3030, 1650, 695, 580 cm−1; C NMR (75 MHz, DMSO-d6):
171.6, 171.6, 150.1, 150.1, 143.1, 143.1, 136.1, 136.1, 134.2, 134.2, 133.7, 133.7, 131.4, 131.3,
129.2, 129.2, 129.2, 129.2, 128.7, 128.7, 128.7, 128.7, 128.6, 128.2, 105.1, 105.1; EI-MS: m/z
(rel. int. %): 549 (M+, 18), 338 (22), 210 (100), 168 (59), 82 (13); MS: as calculated 549.4973
and found 549.4964.
4.3.14. (E)-5-((2-(4-(4-chlorophenyl)thiazol-2-yl)hydrazono)methyl)-2-methoxyphenol. (14)
Yield: 52%; Solid m.p. 281 °C; 1H-NMR: (DMSO-d6, 400 MHz): δ 8.1 (s, 1H, HC=N), 7.8 (d,
J3″,2″/5″,6″ = 6.3 Hz , 2H, H-3″/5″), 7.4 (d, J2″,3″/6″,5″ = 6.3 Hz , 2H, H-2″/6″), 7.3 (m, 2H, H-
2/5), 7.1 (dd, J 6,2 = 1.8, J6,5 = 6.3 Hz , 1H, H-6), 7.0 (s, 1H, H-5′); IR (ATR): 3040, 1670,
1290, 690, 575 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.6, 152.1, 150.1, 147.1, 143.1, 134.2,
131.0, 130.1, 129.1, 129.1, 128.7, 128.7, 122.7, 115.8, 112.2, 105.1, 56.0; EI-MS: m/z (rel. int.
%): 359 (M+, 18), 210 (60), 101 (32), 59 (81), 43 (100); MS: as calculated 359.0495 and found
359.0486.
4.3.15. (E)-4-((2-(4-(4-chlorophenyl)thiazol-2-yl)hydrazono)methyl)benzene-1,2-diol.(15)
Yield: 32%; 1H-NMR: (DMSO-d6, 400 MHz): δ 11.8 (s, 1H, NH), 7.8 (m, 3H, H-3"/5"/HC=N),
7.4 (d,J2″,3″/6″,5″ = 8.4 Hz , 2H, H-2″/6″), 7.3 (s, 1H, H-5ꞌ), 7.1 (s 1HH-2) 6.8 (m, 1H, H-6), 6.7 (d,
J2 = 8 Hz , 1H, H-5); IR (ATR): 3460, 3030, 1650, 1270, 695, 570 cm−1; 13C NMR (75 MHz,
DMSO-d6): 171.6, 150.1, 149.5, 146.0, 143.2, 134.2, 131.2, 131.0, 129.1, 129.1, 128.7, 128.7,
123.1, 116.1, 117.2, 105.0; EI-MS: m/z (rel. int. %): 355 (M+, 30), 206 (100), 191 (27), 164 (54),
149 (43).
4.3.16. (E)-4-(4-Methoxyphenyl)-2-(2-(1-(3-nitro phenyl) ethylidene) hydrazinyl)thaizole (16)
Yield: 74%; Solid m.p. 290 °C; 1H-NMR: (DMSO-d6, 300 MHz): δ 8.5 (s, 1H, H-2), 8.2 (t, J4/5,6
6/5,4 = 7.8 Hz, 2H, H-4/6), 7.8 (d, J2″,3″/6″,5″ = 8.7 Hz , 2H, H-2″/6″), 7.7 (t, J5/6,4 = 8.1 Hz, 1H, H-
5), 7.1 (s, 1H, H-5′), 6.9 (d, J3″,2″/5″,6″ = 8.7 Hz , 2H, H-3″/5″), 3.7 (s, 3H, OMe), 2.4 (s, 3H, Me);
IR (ATR): 3020, 1660, 1360, 1270, 690, 575 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.6,
168.6, 160.4, 150.1, 134.8, 134.2, 131.7, 131.7, 128.4, 128.4, 126.0, 126.4, 125.2, 114.7, 114.7,
13

105.1, 55.7, 17.0; EI-MS: m/z (rel. int. %): 368 (M+, 100), 205 (77), 164 (93), 149 (26), 82 (46);
MS: as calculated 368.4096 and found 368.4096.
4.3.17. (E)-4-(4-methoxyphenyl)-2-(2-(4-nitrobenzylidene)hydrazinyl)thiazole (17)
1
Yield: 51%; H-NMR: (DMSO-d6, 300 MHz): δ 11.3 (s, 1H, NH), 8.3 (d, J3,2 /5,6 = 8.7 Hz, 2H,
H-3/5), 8.2 (s, 1H, CH=N), 7.9 (d, J2,3/6,5 = 8.7 Hz, 2H, H-2/6), 7.8 (d, J2’’,3’’/6’’,5’’ = 9 Hz, 2H, H-
2″/6″), 7.0 (s, 1H, H-5′), 6.9 (d, J3″,2″/5″,6″ = 9 Hz , 2H, H-3″/5″), 3.8 (s, 3H, OMe); IR (ATR):
3025, 1670, 1340, 1280, 695, 575 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.6, 160.5, 150.1,
150.1, 143.2, 139.7, 125.1, 128.4, 128.4, 124.1, 124.1, 124.0, 124.0, 114.6, 114.6, 105.0, 55.6;
EI-MS: m/z (rel. int. %): 354 (M+, 100), 206 (93), 191 (24), 164 (75), 148 (34).
4.3.18. (E)-4-(4-methoxyphenyl)-2-(2-(3-nitrobenzylidene)hydrazinyl)thiazole (18)
Yield: 28 %; 1H-NMR: (DMSO-d6, 300MHz): δ 11 (s, 1H, NH), 8.5 (d, J2,6 = 1.8 Hz, 1H, H-2),
8.2 (s, 1H, CH=N), 8.2 (m, 1H, H-4), 8.1 (d, J6 ,5 = 7.8 Hz , 1H, H-6), 7.8 (d, J2″,3″/6″,5″ = 6.9 Hz,
2H, H-2″/6″), 7.7 (t, J5/4,6 = 8.1 Hz, 1H, H-5), 7.0 (s, 1H, H-5′), 6.9 (d, J3″,2″/5″,6″ = 9 Hz, 2H, H-
3″/5″), 3.8 (s, 3H, OMe); IR (ATR): 3035, 1655, 1370, 1290, 690, 570 cm−1; 13C NMR (75
MHz, DMSO-d6): 171.6, 160.5, 150.1, 148.0, 143.1, 134.3, 132.4, 129.6, 128.3, 128.3, 126.1,
121.5, 125.1, 114.7, 114.7, 105.0, 55.6; EI-MS: m/z (rel. int. %): 354 (M+, 71), 206 (93), 191
(22), 164 (100), 149 (55).
4.3.19. (E)-2-(2-(2,4-dichlorobenzylidene)hydrazinyl)-4-(4-methoxyphenyl)thiazole (19)
Yield: 34 %; 1H-NMR: (DMSO-d6, 300MHz): δ 8.3 (s, 1H, H-3), 7.9 (d, J5,6 = 8.4Hz, 1H, H-5),
7.7 (d, J2″,3″/6″,5″ = 8.7Hz, 2H, H-2″/6″), 7.6 (bs, 1H, CH=N), 7.5 (d, J6,5 = 8.7Hz, 1H, H-6), 7.1 (s,
1H, H-5′), 6.9 (d, J3″,2″/5″,6″ = 8.7Hz, 2H, H-3″/5″), 3.7 (s, 3H, OMe); IR (ATR): 3030, 1650,
1280, 710, 590 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.6, 160.5, 150.1, 143.2, 132.6, 131.0,
129.0, 129.0, 128.1, 128.3, 128.3, 127.0, 125.1, 114.6, 114.6, 105.0, 55.6; EI-MS: m/z (rel.int.
%): 376 (M+ 45), 341 (11), 205 (100), 163 (48), 148 (23).
4.3.20. (E)-4-(4-methoxyphenyl)-2-(2-(1-(4-methoxyphenyl)ethylidene)hydrazinyl)thiazole. (20)
Yield: 65 %; 1H-NMR: (DMSO-d6, 300MHz): δ 7.8 (m, 4H, H-2/6/2″/6″), 7.1 (s, 1H, H-5′), 7.0
(m, 4H, H-3/5/3″/5″), 3.8 (s, 6H, OMe), 2.4 (s, 3H, Me); IR (ATR): 3040, 1680, 1265, 690, 580
cm−1; 13C NMR (75 MHz, DMSO-d6): 171.6, 168.6, 162.7, 160.4, 150.1, 129.7, 128.6, 128.6,
14

128.3, 128.3, 125.1, 114.6, 114.6, 114.2, 114.2, 105.0, 55.6, 55.6, 17.0; EI-MS: m/z (rel.int. %):
211 (M+, 29), 196 (42), 153 (73), 137 (100), 122 (67).
4.3.21. (E)-2-(2-(Anthracen-9-yl-methylene)hydrazinyl)-4-(4-methoxyphenyl) thiazole (21)
Yield: 73%; Solid m.p. 277 °C; 1H-NMR: (DMSO-d6, 300 MHz): δ 12.3 (s, 1H, NH), 9.2 (s, 1H,
HC=N), 8.6 (m, 3H, H-2/6/10), 8.1 (d, J5,4/7,8 = 8.1 Hz , 2H, H-5/7), 7.8 (d, J2″,3″/6″,5″ = 8.4
Hz , 2H, H-2″/6″), 7.6 (m, 4H, H-3/4/8/9), 7.1 (s, 1H, H-5′), 6.9 (d, J3″,2″/5″,6″ = 8.7 Hz , 2H,
H-3″/5″), 3.7 (s, 3H, OMe); IR (ATR): 3120, 1670, 1630, 1280, 695, 575 cm−1; 13C NMR (75
MHz, DMSO-d6): 171.6, 160.5, 150.1, 143.2, 131.7, 131.7, 128.7, 128.7, 128.7, 128.4, 128.4,
128.0, 128.0, 128.0, 128.0, 125.4, 125.4, 125.4, 125.4, 125.1, 123.8, 114.7, 114.7, 105.1, 55.7;
EI-MS: m/z (rel. int. %): 409 (M+, 45), 203 (100), 191(21), 176 (26), 101 (15), 88 (25); MS: as
calculated 409.5029 and found 409.5020.
4.3.22. (E)-4-((2-(4-(4-Methoxyphenyl)thiazol-2-yl)hydrazono)methyl)-N,N-dimethylaniline (22)
1
Yield: 38%; Solid m.p. 264 °C; H-NMR: (DMSO-d6, 300 MHz): δ 7.9 (s, 1H, HC=N), 7.7 (d,
J2,3/6,5 = 8.7 Hz , 2H, H-2/6), 7.4 (d, J2″,3″/6″,5″ = 8.7 Hz , 2H, H-2″/6″), 7.0 (s, 1H, H-5′), 6.9
(d, J3,2/5,6 = 8.7 Hz , 2H, H-3/5), 6.7 (d, J3″,2″/5″,6″ = 9.6 Hz , 2H, H-3″/5″), 3.7 (s, 3H, OMe),
2.9 (s, 6H, 2Me); IR (ATR): 3020, 1680, 1320, 705, 585 cm−1; 13C NMR (75 MHz, DMSO-d6):
171.6, 160.5, 153.2, 150.1, 143.2, 128.4, 128.4, 128.1, 128.1, 125.2, 123.1, 114.7, 114.7, 111.7,
111.7, 105.1, 55.7, 41.0, 41.0; EI-MS: m/z (rel. int. %): 352 (M+, 100), 206(100), 191 (91), 164
(65), 147 (67); MS: as calculated 352.1358 and found 352.1350.
4.3.23. (E)-4-(4-Methoxyphenyl)-2-(2-(naphthalen-2-yl-methylene)hydrazinyl)thiazole (23)
Yield: 32%; Solid m.p. 291 °C; 1H-NMR: (DMSO-d6, 300 MHz): δ 12.2 (s, 1H, NH), 8.1 (s, 1H,
HC=N), 8.0 (s, 1H, H-8), 7.9 (m, 4H, H-/3/4/5/8), 7.8 (d, J2″,3″/6″,5″ = 6.6 Hz , 2H, H-2″/6″),
7.5 (m, 2H, H-6/7), 7.1 (s, 1H, H-5′), 6.9 (d, J3″,2″/5″,6″ = 6.6 Hz , 2H, H-3″/5″), 3.7 (s, 3H,
OMe); IR (ATR): 3105, 1670, 1270, 695, 570 cm−1; EI-MS: m/z (rel. int. %): 359 (M+, 48), 206
(100), 191 (22), 164 (33), 153 (79); MS: as calculated 359.1092 and found 359.1099.
4.3.24. (E)-2(2-(4-(Benzyloxy)benzylidene)hydrainyl)-4-(4-methoxyphenyl)thiazole (24)
15

1
Yield: 81%; Solid m.p. 310 °C; H-NMR: (DMSO-d6, 300 MHz): δ 7.9 (s, 1H, HC=N), 8.0 (d,
J2,3/6,5 = 6.6 Hz , 2H, H-2/6), 7.5 (d, J2″′,3″′/6″′,5″′ = 6.3 Hz , 2H, H-2″′/6″′), 7.4 (d,
J2″,3″/6″,5″ = 5.4 Hz , 2H, H-2″/6″), 7.3 (m, 2H, H-3″′/5″′), 7.2 (t, J4″′/3″′,5″′ = 6 Hz, 1H, H-
4″′), 7.1 (s, 1H, H-5′), 7.0 (d, J3,2/5,6 = 6.6 Hz , 2H, H-3/5), 6.9 (d, J3″,2″/5″,6″ = 6.3 Hz , 2H,
H-3″/5″), 5.1 (s, 2H, OCH2), 3.7 (s, 3H, OMe); IR (ATR): 3040, 1660, 1270, 570, 520 cm−1; 13C
NMR (75 MHz, DMSO-d6): 128.7, 127.5, 128.7, 127.0, 127.0, 136.5, 114.3, 161.2, 144.2, 70.5,
130.1, 126.0, 130.3, 143.1, 171.6, 128.4, 114.7, 105.1, 128.4, 114.7, 125.2, 150.1, 160.4, 55.6;
EI-MS: m/z (rel. int. %): 415 (M+, 10), 206 (88), 191 (17), 149 (18), 91 (100); MS: as calculated
415.1354 and found 415.1349.
4.3.25.(E)-2,6-di-tert-butyl-4-((2-(4-(4-Methoxyphenyl)thiazol-2-yl)hdrazono)-methyl)phenol
(25)
Yield: 99%; Solid m.p. 282 °C; 1H-NMR: (DMSO-d6, 300 MHz): δ 7.9 (s, 1H, HC=N), 7.7 (d,
J2″,3″/6″,5″ = 6.3 Hz , 2H, H-2″/6″), 7.4 (s, 2H, H-2/6), 7.0 (s, 1H, H-5′), 6.9 (d, J3″,2″/5″,6″ =
6.6 Hz , 2H, H-3″/5″), 3.7 (s, 3H, OMe), 1.3 (s, 18H, 6Me); IR (ATR): 3020, 2965, 1670, 1260,
670, 520 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.6, 160.5, 156.8, 150.1, 143.2, 136.1, 136.1,
128.4, 128.4, 125.2, 125.4, 123.6, 123.6, 114.7, 114.7, 105.1, 55.8, 34.3, 34.3, 31.4, 31.4, 31.4,
31.4, 31.4, 31.4; EI-MS: m/z (rel. int. %): 437 (M+, 22), 261 (32), 206 (100), 191 (30), 164 (61);
MS: as calculated 437.2137 and found 437.2130.
4.3.26.1-((E)-(2-(4-(4-Methoxyphenyl)thiazol-2-yl)hydrazno)methyl)-3-((Z)-(2-(4-(4-methoxy
phenyl)thiazol-2-yl)hydrazono)methyl)benze (26)
Yield: 86%; Solid m.p. 279 °C; 1H-NMR: (DMSO-d6, 300 MHz): δ 11.5 (s, 2H, NH), 8.2 (s,
1H, H-2), 8.0 (s, 2H, 2xHC=N), 7.9 (s, 2H, 2xH-5′), 7.7 (d, J2″,3″/6″,5″ = 8.7 Hz, 4H, 2xH-
2″/6″), 7.6 (d, J4,5/6,5 = 7.5 Hz , 2H, H-4/6), 7.5 (m, 1H, H-5), 6.9 (d, J3″,2″/5″,6″ = 8.7 Hz ,
4H, 2xH-3″/5″), 3.7 (s, 6H, 2-OMe); IR (ATR): 3010, 2965, 1680, 1290, 675, 550 cm−1; 13C
NMR (75 MHz, DMSO-d6): 171.6, 171.6, 160.4, 160.4, 150.1, 150.1, 143.2, 143.2, 133.7, 133.7,
131.4, 131.1, 128.4, 128.4, 128.4, 128.4, 128.3, 128.3, 125.2, 125.2, 124.8, 124.6, 114.6, 114.6,
105.1, 105.1, 55.7, 55.7; ESI/MS; calculated for and found EI-MS: m/z (rel. int. %): 540 (M+,
59), 309 (66), 206 (100), 191 (42), 149 (46), 128 (37); MS: as calculated 540.1402 and found
540.1410.
16

4.3.27.
5-(4-methoxyphenyl)-2-((E)-2-(4-((E)-(2-(4-(4-methoxyphenyl)thiazol-2-
yl)hydrazono)methyl)benzylidene)hydrazinyl)thiazole (27)
Yield: 85%; Solid m.p. 284 °C; 1H-NMR: (DMSO-d6, 300 MHz): δ 11.5 (s, 1H, NH), 8.0 (s, 2H,
2xHC=N), 7.9 (s, 2H, 2xH-5′), 7.7 (d, J2″,3″/6″,5″ = 8.7 Hz , 4H, 2xH-2″/6″), 7.6 (d, J 2,3/6,5 =
7.5 Hz , 2H, H-2/6), 7.6 (d, J3,2/5,6 = 7.5 Hz , 2H, H-3/5), 6.9 (d, J3″,2″/5″,6″ = 8.7 Hz , 4H,
2xH-3″/5″), 3.7 (s, 6H, 2OMe); IR (ATR): 3010, 2970, 1654, 1260, 690, 570 cm−1; 13C NMR
(75 MHz, DMSO-d6): 171.6, 160.4, 160.4, 150.1, 150.1, 143.2, 143.2, 133.7, 133.7, 131.2,
131.2, 128.4, 128.4, 128.4, 128.4, 128.3, 128.3, 125.2, 125.2, 125.1, 125.1, 114.6, 114.6, 105.1,
105.1, 55.7, 55.7; EI-MS: m/z (rel. int. %): 540 (M+, 29), 390 (54), 206 (100), 191 (37), 149
(35); MS: as calculated 540.1402 and found 540.1415.
4.3.28. (E)-2-(2-(3,4-dimethoxybenzylidene)hydraziny)-4-(4-methoxyphenyl)thiazole (28)
1
Yield: 76 %; H-NMR: (DMSO-d6, 400MHz): δ 12.0 (s, 1H, NH), 7.9 (s, 1H, CH=N), 7.7 (d,
J2″,3″/6″,5″ = 8.8 Hz, 2H, H-2″/6″), 7.2 (d, J2,6 = 1.2Hz, 1H, H-2), 7.1 (d, J6,2 = 8.4Hz, 1H, H-6), 7.1
(s, 1H, H-5′), 6.9 (m, 3H, H-3″/5″/3), 3.7 (s, 9H, OMe); IR (ATR): 3030, 2980, 1690, 1280, 695,
570 cm−1; 13C NMR (75 MHz, DMSO-d6): 171.5, 160.4, 152.0, 150.0, 149.6, 143.2, 130.4,
128.4, 128.4, 125.2, 122.6, 114.6, 114.6, 111.6, 109.1, 105.0, 55.7, 56.0, 56.0; EI-MS: m/z
(rel.int. %): 206 (M+, 59), 191 (68), 164 (43), 149 (60), 82 (100).
4.3.29. (E)-2-methoxy-5-((2-(4-(4-methoyphenyl)thiazol-2-yl)hydrazono)methyl)-phenol (29)
1
Yield: 52%; Solid m.p. 270 °C; H-NMR: (DMSO-d6, 300 MHz): δ 7.8 (s, 1H, HC=N), 7.7(d,
J2″,3″/6″,5″ = 6.6 Hz , 2H, H-2″/6″), 7.2 (d, J2,6 = 0.9 Hz , 1H, H-2), 7.1 (s, 1H, H-5′), 6.9 (m,
4H, H-3″/5″/5/6), 3.7 (s, 6H, 2OMe); IR (ATR): 3450, 3020, 2970, 1670, 1290, 690, 575 cm−1;
13C NMR (75 MHz, DMSO-d6): 171.6, 160.4, 152.1, 150.0, 147.0, 143.1, 131.2, 128.2, 128.2,
125.1, 122.6, 115.8, 114.7, 114.7, 112.2, 105.1, 56.2, 55.7; EI-MS: m/z (rel. int. %): 355 (M+,
30), 206 (100), 191 (27), 164 (54), 149 (43); MS: as calculated 355.0991and found 355.0999.
4.3.30. (E)-4-((2-(4-(4-Methoxyphenyl)thiazol-2-yl)hydrazon)methy)benzone-1,2-diol (30)
Yield: 86%; Solid m.p. 287 °C; 1H-NMR: (DMSO-d6, 300 MHz): δ 7.8 (s, 1H, HC=N), 7.7 (d,
J5,6 = 6.6 Hz , 1H, H-5), 7.2 (d, J6,5 = 6.6 Hz , 1H, H-6), 7.1(d, J2,6 = 1.5 Hz , 1H, H-2), 7.0 (s,
1H, H-5′), 6.9 (d, J2″,3″/6″,5″ = 6.6 Hz , 2H, H-2″/6″), 6.7 (d, J3″,2″/5″,6″ = 6 Hz, 2H, H-3″/5″),
3.7 (s, 3H, OMe); IR (ATR): 3430, 3010, 2975, 1680, 1275, 670, 595 cm−1; 13C NMR (75
MHz, DMSO-d6): 171.5, 150.1, 149.4, 146.0, 143.2, 133.1, 131.1, 129.1, 129.1, 128.6, 127.4,
17

127.4, 123.1, 117.2, 116.2, 105.1, 46.5; EI-MS: m/z (rel. int. %): 341 (M+, 30), 217 (100), 191
(27), 164 (54), 149 (43); MS: as calculated 341.0834 and found 341.0823.
Acknowledgment
The authors are thankful to Higher Education Commission of Pakistan for financial support.
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20

Captions
Scheme-1: Synthesis of thiazoleanalogs (1-30)
Table-1: Butyrylcholinesterase activity of thiazole derivatives
Table-2: Acetyl cholinesterase activity of thiazole derivatives
Figure-1: Putative binding mode of compound 15
Figure-2: Putative binding mode of compound 07
Figure-3: Putative binding mode of compound 12, 09, 14 and 01
Scheme-1;
O
S
R2
H
H2N
Methanol
+
N
NH2
R1
R2
N
NH2
R1
N
Reflux, 3-5
hrs, Acetic
acid
H
S
O
(glacial)
Reflux
3-5 hrs
Methanol
Br
R3
R1
N
S
N
R2 HN
R3
(1-30)
21

Figure-1:
Figure -2:
22

Figure -3:
23

Table-1:
S.No
IC50 ± SEMa (µM)
IC50 ± SEMa (µM)
R1
R2
R3
Buterycholinesterase Acetylcholinesterase
CH3
H
4-Cl
4-Cl
14.81 ± 0.32
344.8 ± 1.21
1
2
29.62 ± 0.40
306.5 ± 1.35
CH3
4-Cl
35.56 ± 0.46
-
3
H
H
4-Cl
4-Cl
4-Cl
45.43 ± 0.45
452.1 ± 1 .27
4
5
6
Cl
Cl
37.18 ± 0.42
-
CH3
112.73 ± 1.09
348.4 ± 1.36
H
CH3
H
4-Cl
4-Cl
4-Cl
1.77 ± 0.01
7
8
9
412.2 ± 1.22
42.38 ± 0.47
159.3 ± 1.09
7.56 ± 0.01
355.2 ± 1.25
24

H
H
4-Cl
4-Cl
23.61 ± 0.24
30.28 ± 0.35
10
11
321.4 ±1.23
211.6 ± 1.12
H
4-Cl
6.21 ± 0.01
12
93.12 ± 1.05
H
H
4-Cl
4-Cl
113.18 ± 1.08
8.46 ± 0.01
13
14
357.7 ± 1.63
85.22 ± 1.21
H
4-Cl
1.59 ± 0.01
216.14 ± 1.53
389.25 ± 1.75
318.92 ± 1.95
263.97 ± 1 .84
389.24 ± 1.51
15
16
17
18
19
20
21.3 ± 0.50
379.9 ± 1.17
376.8±0.31
363.3 ± 1.16
36.6 ± 0.70
35.3 ± 0.64
CH3 4-OMe
H
H
H
4-OMe
4-OMe
4-OMe
Cl
Cl
CH3 4-OMe
25

H
87.25 ± 1.17
21
4-OMe
305.1 ± 1.25
H
H
151.59 ± 1.54
49.17 ± 0.65
22
23
4-OMe
4-OMe
91.21 ± 1.23
368.41 ± 0.81
H
H
161.85 ± 1.73
96.57 ± 1.08
24
25
4-OMe
4-OMe
44.81 ± 0.84
48.72 ± 1.09
H
H
99.24 ± 1.21
219.93 ± 1.85
26
27
4-OMe
4-OMe
211.25 ± 1.25
94.31 ± 1.17
H
H
144.37 ± 1.53
99.85 ± 1.31
28
29
4-OMe
4-OMe
46.36 ± 1.02
-
H
4-OMe
48.2 ± 1.06
16.54 ± 0.21
30
26

Eserine
-
-
0.85 ± 0.0001
0.04 ± 0.0001
-
27

Graphical
HO
HO
H
N
NH
N
S
Cl
(Compound 15)
IC50 = 1.59 ± 0.003
Potent butyrylcholinesterase Inhibitor
28