Synthesis, characterization, in vitro tissue-nonspecific alkaline phosphatase (TNAP) and intestinal alkaline phosphatase (IAP) inhibition studies and computational evaluation of novel thiazole derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.104088

Alkaline phosphatases (APs) are a class of homodimeric enzymes which physiologically possess the dephosphorylation ability. APs catalyzes the hydrolysis of monoesters into phosphoric acid which in turn catalyze a transphosphorylation reaction. Thiazoles are nitrogen and sulfur containing aromatic heterocycles considered as effective APs inhibitors. In this context, the current research paper presents the successful synthesis, spectroscopic characterization and in vitro alkaline phosphatase inhibitory potential of new thiazole derivatives. The structure activity relationship and molecular docking studies were performed to find out the binding modes of the screened compounds with the target site of tissue non-specific alkaline phosphatase (h-TNAP) as well as intestinal alkaline phosphatase (h-IAP). Compound 5e was found to be potent inhibitor of h-TNAP with IC₅₀ value of 0.17 ± 0.01 μM. Additionally, compounds 5a and 5i were found to be highly selective toward h-TNAP with IC₅₀ values of 0.25 ± 0.01 μM and 0.21 ± 0.02 μM, respectively. In case of h-IAP compound 5f was the most potent inhibitor with IC₅₀ value of 1.33 ± 0.10 μM. The most active compounds were resort to molecular docking studies on h-TNAP and h-IAP to explore the possible binding interactions of enzyme-ligand complexes. Molecular dynamic simulations were carried out to investigate the overall stability of protein in apo and holo state.

Full text of DOI:10.1016/j.bioorg.2020.104088

Journal Pre-proofs
Synthesis, characterization, in vitro tissue-nonspecific alkaline phosphatase
(TNAP) and intestinal alkaline phosphatase (IAP) inhibition studies and com-
putational evaluation of novel thiazole derivatives
Hamid Aziz, Abid Mahmood, Sumera Zaib, Aamer Saeed, Zahid Shafiq, Julie
Pelletier, Jean Sévigny, Jamshed Iqbal
PII:
S0045-2068(20)31385-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.104088
YBIOO 104088
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
8 April 2020
3 July 2020
7 July 2020
Please cite this article as: H. Aziz, A. Mahmood, S. Zaib, A. Saeed, Z. Shafiq, J. Pelletier, J. Sévigny, J. Iqbal,
Synthesis, characterization, in vitro tissue-nonspecific alkaline phosphatase (TNAP) and intestinal alkaline
phosphatase (IAP) inhibition studies and computational evaluation of novel thiazole derivatives, Bioorganic
Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104088
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Synthesis, characterization, in vitro tissue-nonspecific alkaline phosphatase (TNAP) and
intestinal alkaline phosphatase (IAP) inhibition studies and computational evaluation of
novel thiazole derivatives
Hamid Aziza, Abid Mahmoodb, Sumera Zaibb, Aamer Saeeda* Zahid Shafiqc Julie Pelletierc,
Jean Sévignyc,d, Jamshed Iqbalb*
aDepartment of Chemistry Quaid-I-Azam University Islamabad, 45320 Pakistan
bCentre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad-22060, Pakistan.
cInstitute of Chemical Sciences, Bahauddin Zakariya University, Multan-60800
Pakistan
dCentre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2,
Canada.
eDépartement de microbiologie-infectiologie et d'immunologie, Faculté de Médecine,
Université Laval, Québec, QC, G1V 0A6, Canada.
*Corresponding authors: e-mail address: aamersaeed@yahoo.com;
drjamshed@cuiatd.edu.pk;
Address: Department of Chemistry Quaid-I-Azam University Islamabad, 45320 Pakistan [Prof.
Dr. Aamer Saeed]; Centre for Advanced Drug Research, COMSATS University Islamabad,
Abbottabad Campus, Abbottabad-22060, Pakistan [Prof. Dr. Jamshed Iqbal]

Highlights
 New thiosemicarbazides (3a-3e) were heterocyclized with 1,3-dicarbonyls to afford
thiazoles (5a-f) and with phenacyl halides to furnish the thiazoles (5g-l).
 The functionalized thiazoles (5a-l), were subjected to h-TNAP and h-IAP assay and 5e
and 5f were identified as most active molecules.
 Molecular docking of 5e and 5f on h-TNAP and h-IAP to explore the binding
interactions was carried out
 Molecular dynamic simulations were carried out to investigate the overall stability of
protein in apo and holo state.

Graphical abstract:
Binding interactions of compound 5e in h-IAP (left) and compound 5e h-TNAP (right)
showing competitive mode of inhibition in both the enzymes

Abstract
Alkaline phosphatases (APs) are a class of homodimeric enzymes which
physiologically possess the dephosphorylation ability. APs catalyzes the hydrolysis of
monoesters into phosphoric acid which in turn catalyze a transphosphorylation reaction.
Thiazoles are nitrogen and sulfur containing aromatic heterocycles considered as
effective APs inhibitors. In this context, the current research paper presents the
successful synthesis, spectroscopic characterization and in vitro alkaline phosphatase
inhibitory potential of new thiazole derivatives. The structure activity relationship and
molecular docking studies were performed to find out the binding modes of the
screened compounds with the target site of tissue non-specific alkaline phosphatase (h-
TNAP) as well as intestinal alkaline phosphatase (h-IAP). Compound 5e was found to
be potent inhibitor of h-TNAP with IC50 value of 0.17±0.01 µM. Additionally,
compounds 5a and 5i were found to be highly selective toward h-TNAP with IC50
values of 0.25±0.01 µM and 0.21±0.02 µM, respectively. In case of h-IAP compound
5f was the most potent inhibitor with IC50 value of 1.33±0.10 µM. The most active
compounds were resort to molecular docking studies on h-TNAP and h-IAP to explore
the possible binding interactions of enzyme-ligand complexes. Molecular dynamic
simulations were carried out to investigate the overall stability of protein in apo and
holo state.
Key words: Thiazole; Structure Activity Relationship; Tissue Specific Alkaline Phosphatase;
Hydrogen Bonding. Xx

1. Introduction
Enzymes are macromolecular proteins used as biological catalysts in fundamental as well as
clinical research. Alkaline phosphatases (APs, EC 3.1.3.1) from the family of ectonucleotidases
are dimeric enzymes having two Zn-atoms, one Mg-atom and five cysteine residues [1-3]. APs
are found in blood serum from bone (50-60%), intestinal (30%) and hepatic (10-20%) fractions
[4]. APs hydrolyze O/S-phosphorothioates, phosphoramidates, thiophosphates, phosphate and
oxyphosphate monoesters into phosphate at alkaline pH. They are categorized as tissue specific
(placental, germ cells, intestinal) and tissue non-specific isozymes found in kidneys, bones and
central nervous system [5]. Intestinal alkaline phosphatases (h-IAPs) are found on the border
of intestine and regulated lipid absorption, bicarbonate secretion, maintain pH at duodenum
surface and controls bacterial endotoxins inflammation. The h-IAPs perform the detoxification
of intestinal lipopolysaccharide and maintain homeostasis. Human tissue non-specific alkaline
phosphatases (h-TNAP) hydrolyze inorganic pyrophosphate to phosphate and regulate mineral
deposition in bones. APs facilitate the absorption of nutrients across cell membranes and serve
as a mediator in physiological processes to regulate cellular functions including metabolism,
gene expression, molecular transportation and signal transduction. The isozymes increase the
concentration of inorganic phosphate and decreases the extracellular pyrophosphate [6,7]. The
deficiency of h-IAPs cause inflammation owing to the insufficient detoxification of commensal
bacteria. Similarly, the increase in APs cause disproportionate intracellular fat depots and
mineralization disorders. This in turn damage musculoskeletal system and induces metabolism
related disorders which ultimately damages liver and kidney cells. Thus, the selective inhibition
and effective regulation of APs isozymes are required to develop useful therapies against
osteoarthritis, rickets, arterial calcification and myocardial infection. In this regard, literature
survey reveals heterocyclic analogues particularly 1,3-thiazoles as potent inhibitors of APs
isozyme [8-9].

Thiazoles are five-member aromatic heterocycles containing sulfur and nitrogen atoms at 1,3-
positions. Thiazoles are isomeric with 1, 2-azoles, containing sulfur and nitrogen atoms at 1,2-
positions termed as isothiazole [10]. Thiazoles are easily metabolized by biological reactions
and are non-carcinogenic in nature. Thiazole, a prime component of vitamin B1, are essential
scaffolds in pharmaceuticals, functional organic materials, asymmetric ligands and
agrochemicals [11]. Among the biologically active natural products, thiazoles have been found
to display diverse biological potential including antimicrobial [12], anticancer [13],
anticonvulsant [14], neuroprotective analgesic [15], antioxidant and anti-inflammatory [16].
Thiazole core skeleton is present in many drugs including Tiazofurin (antineoplastic),
Nitazoxanide (antiparasitic), Nizatidine (antiulcer), Thiamethoxam (insecticide), Fentiazac and
Meloxicam (anti-inflammatory) as shown in Figure 1. Triazolothiadiazoles possess significant
potential as APs inhibitors and some of the investigated thiazole analogues are proven to be
more potent inhibitors than the reference drugs [17].
Thus, keeping in view the physiological profile of AP isozymes, and the diverse
pharmacological potential of thiazole core and their ability to effectively inhibit the APs
isozymes, the synthesis of functionalized thiazoles is required. In this regard, the current
research reports the facile synthesis, spectroscopic characterization, APs inhibition assay and
molecular modelling studies of thiazolyl ethanone derivatives the thiazolyl hydrazine [18]. The
synthesis is performed in an efficient way using dry ethanol as a solvent and a catalytic amount
of concentrated sulphuric acid.

O
O
N
N
O
CH3
NH
NO2
N
S
O2N
S
N
H
N
N
O2N
S
S
Cl
N
N
H
O
CH3
N
Nizatidine
Nitazoxanide
Thiamethoxam
S
HO
OH
O
N
O
N
O
HO
HO
O
N
N
H
S
NH2
N
S
O
CH3
S
O
OH
Cl
Meloxicam
Fentiazac
Tiazofurin
Figure 1. Some literature reported potent thiazole based drugs
2. Results and discussion
2.1. Synthesis of thiazole derivatives (5a-l)
The synthetic pathyway employed for the synthesis of the target thiazolyl ethanone (5a-f) and
thiazolyl hydrazine derivatives (5g-l) is displayed in Scheme 1. Thus, the common
semicarbazones as key intermediates (3a-e), were obtained by the acid catalysed condensation
of suitably substituted benzaldehydes with thiosemicarbazide in dry ethanol. The thiazolyl
ethanone derivatives (5a-f) were obtained by reaction of (3a-e), with 1,3-dicarbonyl
compounds (4a) in dry ethanol in the absence of any catalyst under reflux conditions. A similar
reaction of substituted phenacyl halides (4b) with intermediates (3a-e) afforded the thiazolyl
hydrazines (5g-l). All compounds were recrystallized from ethanol at room temperature to
afford the pure products in high purity and good to excellent yields (scheme 1).
2.2. Characterization

The structures of the intermediates (3a-e), thiazolyl ethanone derivatives (5a-f) and as well as
the thiazolyl hydrazines (5g-l) were established through elemental analysis (CHNS) and
1
13
spectroscopic techniques (FT-IR, H and C NMR). The C, H, N and S analyzer provided
percent data highly aligned with the elements present in the synthesized compounds.
The FT-IR spectrum of a typical thiosemicarbazone (3e) showed the stretchings for NH2 at
3238, for NH 3178, 1620 for HC=N and 1159.67 cm-1 for C=S, while rest of signals appeared
at their respective regions. Compound (3e) exhibited the NH protons as characteristic broad
singlets at 10.20 and 7.90 and at 8.45 for OH, whilst the presence of HC=N at 8.43 ppm in the
1H NMR spectrum was noted. The thiocarbonyl C=S appeared at 181.43 and C=N carbons at
143.30 C=N ppm in the 13C NMR spectrum.
The stretchings for NH at 320, 3098, 2958 for (C=C-H), 2914.23, 2840.29 for CH3, 1625.73
for HC=N were observed in In the FT-IR spectrum of a typical thiazolyl ethanone (5a).
Similarly, the 1H NMR displayed broad singlet for 11.08 for NH, singlet at 8.10 for H-C=N,
and 3.88 singlet for methyl of thiazole ring and 3.74 for methyl of carboxylate moiety. In 13C
NMR absence of thiocarbonyl and appearance of signals at 170.52 for C=N, 166.34 (C=O),
143.43 (C=N ring) and at 10.26, 28.49 for methyl of thiazole ring and carboxylate
respectively was noted.
The thiazolyl hydrazine (5h) showed stretchings for 3276 (NH), 2987 (C=C-H), 1623 (HC=N),
cm-1 FT-IR spectrum and broad singlets at 11.20 for NH, characteristic 1H singlet at 8.20 for
HC=N, another at 6.64 for C=C-H, besides those for aromatic protons were noted in 1H NMR.
13
The important signals of C NMR include those at δ 171.80 (C=N), 146.50 (C=C), 143.22
(HC=N) ppm.

S
R2
N
N
N
H
R3
R1
(5g-5l)
O
Dry EtOH / Reflux
X
R3
(4b)
CH3
S
O
X
S
R2
O
R2
N
R2
N
O
O
N
H
NH2
N
N
S
H
Dry EtOH
+
H3C
X
NH2
R1
R1
H2N
N
R1
H
H+ / Reflux
Dry EtOH/ Reflux
(5a-5f)
(1a-1e)
(3a-3e)
(2)
(4a)
Scheme 1. Synthetic route employed for the thiazole derivatives (5a-l)
The different substituents attached to the synthesized thiazole derivatives (5a-l) are listed in
Table-1.
Table 1. Structures of the synthesized thiazole derivatives (5a-l)
Compound
R1
R2
H
X
Compound
R1
R2
R3
4-Br
5a
5b
5c
5d
5e
5f
3-OCH3,4-OH
OCH3
CH3
5g
5h
5i
3-OCH3,4-OH
H
4-Cl
H
H
3-OCH3,4-OH
H
H
4-Cl
4-Br
CH3 OCH3
4-Cl
H
H
CH3
H
CH3
CH3
CH3
5j
CH3 4-Br
CH3 4-Cl
3-NO2
4-OH
5k
5l
H
H
3-NO2
H
4-Br

2.3. Biological activities
2.3.1. Structure activity relationship (SAR) of thiazole derivatives
The inhibitory potential of the synthesized thiazolyl ethanone derivatives (5a-f) and as well as
thiazolyl hydrazines (5g-h) for h-TNAP as well as h-IAP was analyzed using human
recombinant enzymes in both the cases (Table 2). Different substitutions and unsubstitution
patterns were correlated with their ability to inhibit alkaline phosphatases. Such types of
correlations help to develop SAR among the synthesized screened compounds. Levamisole is
a thaizole derivative and has been reported as a known inhibitor of h-TNAP. Thiazole ring is
the structural part of thiamine (Vitamin B1), carboxylase, epothilones and macrocyclic
thiopeptide antibiotics. This structural unit has broad range of biological activities including
antimicrobial, anticonvulsant, antiviral, antibacterial, antituberculosis, antimalarial, anticancer,
anti-inflammation, hypertension, schizophrenia and hypnotic. In the current study, thiazole
derivatives were synthesized in order to find out more potent and selective antagonists of
alkaline phosphatase. Among the synthesized derivatives, compounds 5a, 5e and 5i were found
to be more potent against h-TNAP. The presence of nitro group at 3rd position of benzene ring
in 5e, made it highly potent against h-TNAP with IC50 of 0.17±0.01 µM which is 100 times
more potent as compared to Levamisole with IC50 value of 25.2±1.90 µM. Structural
comparison of compounds 5a and 5f, exhibited that methoxy group in 3-methoxybenzylidene
ring, gave selectivity toward h-TNAP to 5a with the IC50 value of 0.25±0.01 µM. 5b exhibited
inhibitory activity against both isoenzymes h-TNAP and h-IAP with IC50 3.43±0.01 µM and
4.71± 0.03 µM, respectively, as compared to 5d having no inhibitory activity. This activity is
due to the presence of electron withdrawing group (chlorine) at para-position of the benzene
ring. Compound 5f was found to be more potent for h-IAP with IC50 value of 1.33±0.10 µM
which is 75 times more potent than L-phenylalanine (IC50: 100±3.00 µM). Mono-substitutions
on benzene ring of the synthesized derivatives exhibited non-selectivity for both isozymes of

alkaline phosphatase. Such behavior of mono-substitution on benzene ring can be observed by
comparing the structures of 5b, 5e, 5f and 5l. Removal of substitutions on benzene ring
eliminated the inhibitory potential of molecules as can be noted from inhibitory activities of
compounds 5c, 5d, 5j and 5k. Chlorine and bromine at para-position of the phenyl ring
attached to thiazole the ring conferred 5h and 5i molecules selectivity towards h-TNAP with
an IC50 values of 3.02±0.02 µM and 0.21±0.01 µM. 5j and 5k were found to be relatively less
potent for both isozymes due to removal of substitution on phenyl ring, although their phenyl
ring has halogens substitution. Incorporation of nitro group at meta-position of benzene ring
and bromophenyl with thiazole ring in 5l showed selective inhibition against h-TNAP with
IC50 1.38 ± 0.02 µM. Presence of chlorophenyl at position 4 of thiazole ring have no inhibitory
activity against both isoenzymes h-TNAP and h-IAP as in 5k molecule but incorporation of
methoxy group at position 3 and OH group at position 4 of benzene ring show selective
inhibition against h-TNAP with IC50 value of 3.02 ± 0.02 µM (5h).
Table 2. APs inhibition of the screened thiazolyl ethanone (5a-f) and thiazolyl hydrazines
derivatives (5g-l)
Compound
h-TNAP
h-IAP
IC50±SEM (µM) / % inhibition
5a
5b
5c
5d
5e
5f
0.25±0.01
3.43±0.01
14.96%
12.34%
4.71±0.03
3.15%
11.22%
17.21%
0.17±0.01
4.56±0.03
3.77±0.02
1.33±0.10

5g
1.45±0.02
3.02±0.02
0.21±0.01
11.10%
45.14%
1.38±0.02
25.2±1.90
-
5.14±0.06
10.46%
11.23%
10.43%
11.67%
12.16%
-
5h
5i
5j
5k
5l
Levamisole
L-phenylalanine
100±3.00
2.3.2. Enzyme kinetics study for h-TNAP and h-IAP inhibition
Enzyme kinetics studies were performed to determine the mode of inhibition involves for h-
TNAP and h-IAP. Most potent compounds were selected for such type of studies. 5e molecule
exhibited competitive mode of inhibition for both h-TNAP and h-IAP. 0, 0.1, 0.5 and 1.0 µM
concentrations were used for inhibitor 5e and substrate concentration were 0, 12.5, 25, 50 and
100 µM. In addition, enzyme kinetic studies were also performed for 5i, and compound
exhibited mixed type of enzyme inhibition for h-TNAP (Figure 2).
0 M
0.00025
0.00020
0.00015
0.00010
0 M
0.1 M
0.5 M
1.0M
0.0006
0.0004
0.0002
0.1 M
0.5 M
1.0M
0 M
0.00020
0.00015
0.00010
2.0 M
4.0 M
6.0M
-0.04
-0.02
0.0000
0.02
0.04
0.06
0.08
0.10
-0.04 -0.02
0.00
0.02
0.04
0.06
0.08
0.10
-0.04 -0.02
0.00
0.02
0.04
0.06
0.08
1/[S](M)
1/[S](M)
1/[S](M)
Figure 2. Double-reciprocal plots of the inhibition kinetics of h-TNAP by compounds 5e
(left) and of h-IAP by compound 5e (middle) indicating competitive mode of inhibition. 5i
exhibited mixed type of inhibition (right) for h-TNAP.

2.3.4. Molecular modeling investigation
2.3.4.1.
Molecular docking studies against human tissue non-specific alkaline
phosphatase (h-TNAP)
The binding mechanism for the screened compounds against h-TNAP was validated by the
binding mechanism of its positive control Levamisole (Figure 3). In vitro analysis found 5a,
5e, 5g, 5i, and 5l as promising inhibitors of the h-TNAP protein, imitating the interactions of
Levamisole. Zn2+483 ion and His154 were involved in interaction with all the selected screened
compounds. Arg167, Asp320, and His321 were the other prominent residues to interact with
the screened compounds. Major interactions depicted were hydrogen bonding, π-π interactions,
π-alkyl interactions and π-cation/anionic interactions between the residues and compounds.
The compound 5a showed additional interactions with Glu108 and Arg119 (Figure 4).
Compound 5e exhibited chemical interaction with the two Zn2+ ions, hydrogen bonding with
His154, His321 and His437 residues, and π-π interaction with His324 residue (Figure 5).
Compound 5g interacted with a Zn2+ ion along with the His154, Arg167, Asp320, His324 and
Thr436 residues (Figure 6). Compound 5i was least reactive with the protein among the
selected compounds exhibiting interactions with Zn2+ ion and Arg151, His154, His321 and
His437 residues of the protein (Figure 7). Compound 5l exhibited the usual interactions as
displayed by other compounds of the group, involving interactions with both of Zn2+ ions,
hydrogen bonding with His154 and His321 residues, π-anion interaction with arg167 and
Asp320, π-π interactions with His324 and π-σ interactions with His434 residue in the active
site of protein (Figure 8).

Figure 3: 3D & 2D interactions of Levamisole with h-TNAP
Figure 4: 3D & 2D interactions of 5a with h-TNAP
Figure 5: 3D & 2D interactions of 5e with h-TNAP

Figure 6: 3D & 2D interactions of 5g with h-TNAP
Figure 7: 3D & 2D interactions of 5i with h-TNAP

Figure 8: 3D & 2D interactions of 5l with h-TNAP
Molecular docking studies against h-IAP
2.3.4.2.
L-phenylalanine was initially docked into the h-IAP protein to find the binding site residues to
validate the bindings for the test compounds. L-phenylalanine exhibited interactions with the
two Zn2+ ions along with hydrogen bonding with Ser92, His153, His317, His358 and His432,
while π- cationic interactions Asp42, Arg166, and Asp316 residues (Figure 9). In vitro analysis
found 5e and 5f as potent inhibitors of the h-IAP protein, and the molecular docking provided
the validation for it by imitating the interactions of L-phenylalanine. Both compounds, 5e and
5f, interacted with Zn2+ ion as illustrated in Figure 10 and 11, respectively. Ser92 and His153
was involved in the formation of hydrogen bonding with all the compounds. Asp42, Arg166,
Asp316, His317, His320 were among the common involved in the interactions with the test
compounds as indicated by 2D and 3D interaction figures.

Figure 9: 3D & 2D interactions of L-phenylalanine with h-IAP
Figure 10: 3D & 2D interactions of 5e with h-IAP

Figure 11: 3D & 2D interactions of 5f with h-IAP
2.3.5. HYDE assessment of selective and potent compounds against h-IAP and h-TNAP
Pose selection criteria for selected potent compounds was further narrowed down by running
HYDE affinity assessment tool of LeadIT and it served in the selection of appropriate binding
mode. The FlexX score of the selective compounds and their binding free energy ΔG are given
in table 3.
Table 3. FlexX score and binding free energy of ΔG of selected compounds
FlexX score of the top-
ranking pose
Binding free energy ΔG
(kJꢀmol−1)
Compound
5e
5f
-30.4873
-16.2262
-16.5975
-29.6870
-0
-11
-16
-5
5a
5e

5g
5i
5l
-11.9777
-9.0874
-11
-10
-2
-24.6684
Molecular dynamic simulations
RMSD analysis
The RMSD values provide the information of overall stability of the protein and its complex
with selected inhibitor when deviations need to calculate in the initial structure. As shown in
Figure 12, three selected compounds (5a, 5e and 5i) are significantly stable and depicted less
deviation within the range of 0.2 to 0.35 nm. The slight variation was consistent in the structure
of h-TNAP alone, whereas, all the complex structures were found as significantly stable. The
structure of 5e+protein showed slight deviation around 8-10 ns while, 5a+protein was found to
show variability around 14 ns and remaining simulations were found stable. The structure of
5i+protein was quite stable during the simulation time period. When the structure of IAP was
monitored in apo and holo form, Cα backbone was prompted with an average of 0.3 nm for
IAP and 0.28 nm for 5f+protein. The stability was suggested with the overall RMSD of around
0.29 nm. A continuous negligible fluctuations were found throughout the simulation time. The
results of both the protein structures showed the stability in terms of internal motion in systems.

Figure 12. Root Mean Square Deviation (RMSD) of amino acid residues of h-TNAP and h-IAP
protein during 20 ns MD-simulation run in the presence and absence of selected compounds
RMSF analysis
The knowledge of RMSF values also provide the information regarding the calculation that
were carried out to investigate the flexibility of the protein structure in the presence and absence
of selected compounds. As represented in Figure 13, both the systems presented significant
pattern of fluctuations. However, the overall values of RMSF for TNAP and its complexes
were around 0.25 nm, with slight increase in complex of 5i, which is around 0.3 nm. However,
the IAP and its complex delineated the fluctuations around 0.3 nm as depicted in Figure 13.
The region having loop was found to show slight fluctuations while active site region was
significantly stable during the whole simulations time course. The results indicated that all the
complexes result in stability of the system.

Figure 13. Root Mean Square Fluctuation (RMSF) of amino acid residues of h-TNAP and h-IAP
protein during 20 ns MD-simulation run in the presence and absence of selected compound
Radius of gyration
The results of calculations carried out for the investigation of radius of gyration (Rg) presented
the information about compactness of the system with time frame. Radius of gyration described
the folding and unfolding of structure of protein in the presence and absence of selected
inhibitors. The results were illustrated in Figure 14, suggesting that in comparison to TNAP
alone, the complexes of 5a and 5e were more compact, while less compactness was shown by
5i+TNAP complex. Figure 6 showed that an average score of Rg for TNAP and its complexes
was 2.25 nm while for IAP and its complex, it was 2.2 nm. Our results depicted the
compactness of systems throughout the simulations.

Figure 4. Radius of gyration (Rg) of amino acid residues of h-TsNAP and h-IAP protein structures
during 20 ns MD-simulation run in the presence and absence of selected compounds
3. Conclusions
The current research study reports the successful synthesis and characterization of thiazolyl
ethanone derivatives (5a-f) and thiazolyl hydrazines (5g-l). The compounds were evaluated to
determine their inhibitory potential against APs (h-TNAP and h-IAP). The compounds
exhibiting most prominent inhibitory activity during in vitro analysis were subjected to
molecular docking studies on h-TNAP and h-IAP, revealing the significant binding modes
between the selected compounds and target proteins evincing inhibition of the respective
enzymes.
4. Experimental
4.1. Chemistry
All the reagents involved were purchased from Sigma Aldrich and were used without any
further purification. The glass ware was dried in an oven at 70 ℃ for one 60 minutes. Bruker
1
13
Avance AVIII spectrometer was used to record the H and C nuclear magnetic resonance
(NMR) spectra at 400 and 100 MHz respectively in deuterated DMSO-d6 as a solvent and
TMS as an internal reference. 1H-NMR data is reported as δ values in ppm relative to residual

signals from deuterated solvents, while coupling constants are indicated by J in Hz. 13C-NMR
data were reported as position (δ) and assignment of the atom. The infrared spectra measured
in wave numbers (cm-1) was recorded using PerkinElmer Fourier transform infrared ATR
spectrometer. Elemental analysis (C, H, N, S) was performed to determine the % presence of
each element present. The progress of the reaction monitored using Thin-layer chromatography
on silica gel 60 F254 precoated plates (0.25 mm) in a single spot/two solvent systems. Stuart
MP-D melting point apparatus was used to determine the melting points of the synthesized
compounds.
4.2. Synthesis of 2-benzylidenehydrazine-1-carbothioamide (3a-e)
For the synthesis of 2-benzylidenehydrazine-1-carbothioamide derivatives (3a-e) a reported
methodology from literature with slight modifications was employed. For this purpose,
appropriate benzaldehyde (1.20 mmol) was completely dissolved in distilled ethanol (05 mL)
at room temperature and a drop of concentrated sulphuric acid was added as a catalyst. After
30 minutes of continuous stirring, a solution of thiosemicarbazide (1.20 mmol) prepared in
distilled ethanol (10 mL) was dropwise added to the reaction mixture and the mixture was
refluxed for next 120 minutes at 100 ℃. TLC in a solvent system of n-hexane: ethyl acetate
(4:1) was constantly employed to check the extent of reaction. After 120 minutes, the reaction
mixture was cooled to room temperature which resulted in the formation of solid precipitates.
The solid precipitates obtained were filtered, dried in an oven at 70 ℃ and recrystallized from
ethanol to purify the corresponding 2-benzylidenehydrazine-1-carbothioamide derivatives 3(a-
e) [18-19].
4.2.1. 1-(4-Hydroxy-3-methoxybenzylidene)thiosemicarbazide (3a)
Rf= 0.28 (n-hexane: ethyl acetate, 4:1); m.p 222 ℃; Mol. wt: 225.27; Orange powder; (Yield
73%): IR (Pure, cm-1) ν: 3283.06 (NH2), 3122.79 (NH), 3050.84 (sp2 C-H), 2965.80 (sp3 C-H),
1620.20 (HC=N), 1562.57, 1515.64, 1495.91 (Ar-C=C), 1154.32 (C=S); 1H NMR (400 MHz,

DMSO-d6, δ): 11.42 (b, s, 1H, NH), 9.19 (s, 1H, HC=N), 8.41 (b, s, 1H, OH), 8.12 (b, s, 1H,
NH), 7.90 (b, s, 1H, NH), 7.53 (d, 1H, J = 7.80 Hz, Ar-H), 6.937 (d, 1H, J = 7.80 Hz, Ar-H),
6.75 (s, 1H, Ar-H), 3.79 (s, 3H, O-C-H); 13C NMR (100 MHz, DMSO-d6, δ): 178.08 (C=S),
148.32 (Ar), 146.40 (Ar), 139.91 (HC=N), 121.19 (Ar), 119.47 (Ar), 118.55 (Ar), 113.22 (Ar),
56.31 (O-C); Anal. Cald. For C9H11N3O2S: C, 47.99; H, 4.92; N, 18.65; S, 14.30; Found: C,
47.90; H, 4.95; N, 18.72; S, 14.10.
4.2.2. 1-(4-Chlorobenzylidene) thiosemicarbazide (3b)
Rf= 0.19 (n-hexane: ethyl acetate, 4:1); m.p 210 ℃; Mol. wt: 213.69; Light yellow powder;
(Yield 76%): IR (Pure, cm-1) ν: 3180.38 (NH2), 3106.65 (NH), 29621.65 (sp2 C-H), 1596.64
(HC=N), 1575.19, 1519.73, 1489.17 (Ar-C=C), 1199.89 (C=S); 1H NMR (`400 MHz, DMSO-
d6, δ): 10.26 (b, s, 1H, NH), 8.38 (b, s, 1H, NH), 7.89 (b, s, 1H, NH), 8.13 (s, 1H, HC=N), 7.69
(d, 2H, J = 8.10 Hz, Ar-H), 7.39 (d, 2H, J= 8.10 Hz, Ar-H); 13C NMR (100 MHz, DMSO-d6,
δ): 181.43 (C=S), 143.30 (HC=N), 136.63 (Ar), 131.93 (Ar), 130.63 (Ar), 129.13 (Ar); Anal.
Cald. For C8H8 ClN3S: C, 44.97; H, 3.77; N, 19.66; S, 15.01; Found: C, 44.87; H, 3.81; N,
19.69; S, 15.1.
4.2.3. 1-(1-Phenylethylidene) thiosemicarbazide (3c)
Rf= 0.47 (n-hexane: ethyl acetate, 4:1); m.p 132-134 ℃; Mol. wt: 193.27; Light yellow powder;
(Yield 81%): IR (Pure, cm-1) ν: 3368 (NH2), 3229.10 (NH), 3174.54 (sp2 C-H), 2969.93 (sp3
C-H), 1639.57 (HC=N), 1578.56, 1548.86, 1499.54 (Ar-C=C), 1159.48 (C=S); 1H NMR (400
MHz, DMSO-d6, δ): 10.21 (b, s, 1H, NH), 8.28 (b, s, 1H, NH), 7.94 ( b, s, 1H, NH), 7.92 (m,
2H, Ar-H), 7.389 (m, 3H, Ar-H), 2.30 (3H, C-H); 13C NMR (100 MHz, DMSO-d6, δ): 179.36
(C=S), 148.28 (C=N), 138.11 (Ar), 129.66 (Ar), 128.69 (Ar), 127.06 (Ar), 14.46 (C-H); Anal.
Cald. For C9H11N3S: C, 55.93; H, 5.74; N, 21.74; S, 16.59; Found: C, 55.80; H, 5.82; N, 21.80;
S, 16.62.
4.2.4.1-(3-Nitrobenzylidene) thiosemicarbazide (3d)

Rf= 0.27 (n-hexane: ethyl acetate, 4:1); m.p 218 ℃; Mol. wt: 224.24; Yellow powder; (Yield
81%): IR (Pure) ν: 3370.55 (NH2), 3253.32 (NH), 3185.34, 2956.08 (sp2 C-H), 1644.34
(HC=N), 1579.56, 1569.48, 1451.69 (Ar-C=C), 1120.34 (C=S); 1H NMR (400 MHz, DMSO-
d6) δ: 11.08 (b, s, 1H, NH), 8.21 (b, s, 1H, NH), 7.98 (b, s, 1H, NH), 8.63 (s, 1H, Ar-H), 8.23
(d, 1H, J= 8.2 Hz, Ar-H), 8.19 (s, 1H, HC=N), 8.13 (d, 1H, J = 8.2 Hz, Ar-H), 7.70 (t, 1H, J
= 8.0 Hz, Ar-H); 13C NMR (100 MHz, DMSO-d6) δ: 182.39 (C=S), 148.53 (Ar), 143.30
(HC=N), 135.33 (Ar), 134.73 (Ar), 129.83 (Ar), 124.13 (Ar), 123.43 (Ar); Anal. Cald. For
C8H8N4O2S: C, 42.85; H, 3.60; N, 24.99; S, 14.30; Found: C, 42.80; H, 3.55; N, 24.95; S,
14.34.
4.2.5. 1-(4-Hydroxybenzylidene) thiosemicarbazide (3e)
Rf= 0.37 (n-hexane: ethyl acetate, 4:1); m.p 212-216 ℃; Mol. wt: 195.24; Red powder; (Yield
82%): IR (Pure, cm-1) ν: 3238.10 (NH2), 3178.63 (NH), 1620.64 (HC=N), 1582.98, 1561.47,
1490.70 (Ar-C=C), 1159.67 (C=S); 1H NMR (400 MHz, DMSO-d6, δ): 10.20 (b, s, 1H, NH),
8.45 (b, s, 1H, OH), 8.24 (b, s, 1H, NH), 7.90 (b, s, 1H, NH), 8.43 (s, 1H, HC=N), 7.43 (d, 2H,
13
J = 7.52 Hz, Ar-H), 6.83 (d, 2H, J = 7.52 Hz, Ar-H); C NMR (100 MHz, DMSO-d6, δ):
181.43 (C=S), 161.10 (Ar), 143.30 (HC=N), 130.90 (Ar), 126.43 (Ar), 116.30 (Ar); Anal. Cald.
For C8H9N3O3S: C, 49.21; H, 4.65; N, 21.52; S, 16.42; Found: C, 49.12; H, 4.74; N, 21.42; S,
16.46.
4.3. Procedure for the synthesis of thiazolyl ethanones (5a-f) and thiazolyl hydrazines (5g-
l)
The synthesis of 2-(2-benzylidenehydrazineyl) thiazole derivatives (5a-l) was achieved by
slight modification of literature method. Briefly, an equimolar ratio of 2-benzylidenehydrazine-
1-carbothioamide derivatives (3a-e) and phenacyl halides/1,3-dicarbonyl compounds were
refluxed in distilled ethanol at 100 ℃ for 1-2 h. TLC was constantly employed to check the
extent of reaction in a solvent system of n-hexane: ethyl acetate (4:1). Once the reaction was

completed, ethanol was removed under reduced pressure and the crude products obtained were
recrystallized from ethanol at room temperature to purify the synthesized thiazole derivatives
(5a-l) [18-19].
4.3.1. (E)-Methyl 2-(2-(4-hydroxy-3-methoxybenzylidene)hydrazinyl)-4-methylthiazole-
5-carboxylate (5a)
Rf=0.36 (n-hexane: ethyl acetate, 4:1); m.p 244 ℃; Mol. wt: 321.35; Light yellow powder;
(Yield 76%): IR (Pure) ν: 3201.32 (NH), 3098.65, 2958.67 (C=C-H), 2914.23, 2840.29 (-CH3),
1625.73 (HC=N), 1592.34, 1514.56, 1498.52, 1446.15 (Ar-C=C), cm-1; 1H NMR (400 MHz,
DMSO-d6, δ): 11.08 (b, s, 1H, NH), 8.10 (s, 1H, HC=N), 7.10 (d, 1H, J = 8.02 Hz, Ar-H), 7.0
(s, 1H, Ar-H), 6.79 (d, 1H, J = 8.02 Hz, Ar-H); 3.88 (s, 3H, C-H), 3.74 (s, 3H, C-H), 2.48 (s,
13
3H, C-H); C NMR (100 MHz, DMSO-d6, δ): 170.52 (C=N), 166.34 (C=O), 156.78 (C=C),
151.56 (Ar), 148.66 (Ar), 143.43 (HC=N), 127.46 (Ar), 122.93 (Ar), 117.59 (Ar), 115.45
(C=C), 114.86 (Ar), 56.49 (O-C), 51.30 (O-C), 10.26 (C-H); Anal. Cald. For C14H15N3O4S: C,
52.33; H, 4.70; N, 13.08; S, 9.98; Found: C, 52.30; H, 4.68; N, 13.05; S, 9.96.
4.3.2. (E)-1-(2-(2-(4-chlorobenzylidene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (5b)
Rf= 0.34 (n-hexane: ethyl acetate, 4:1); m.p 230 ℃; Mol. wt: 293.77; Yellow powder; (Yield
79%): IR (Pure) ν: 3212.46 (NH), 3052.86, 2921.73 (C=C-H), 2901.38, 2813.54 (-CH3),
1613.24 (HC=N), 1595.66, 1548.84, 1552.58, 1477.16 (Ar-C=C), cm-1; 1H NMR (400 MHz,
DMSO-d6, δ): 11.19 (b, s, 1H, NH), 8.16 (s, 1H, HC=N), 7.64 (d, 2H, J = 8.10 Hz, Ar-H), 7.39
(d, 2H, J = 8.10 Hz, Ar-H), 2.56 (s, 3H, C-H); 2.49 (s, 3H, C-H); 13C NMR (100 MHz, DMSO-
d6, δ): 196.89 (C=O), 170.56 (C=N), 156.78 (C=C), 144.30 (HC=N), 136.66 (Ar), 131.42 (Ar),
130.59 (Ar), 129.16 (Ar), 103.45 (C=C), 28.49 (C-H), 10.26 (C-H); Anal. Cald. For
C13H12ClN3OS: C, 53.15; H, 4.12; N, 14.30; S, 10.91; Found: C, 53.09; H, 4.15; N, 14.35; S,
10.89.

4.3.3. (E)-methyl 4-methyl-2-(2-(1-phenylethylidene)hydrazinyl)thiazole-5-carboxylate
(5c)
Rf=0.52 (n-hexane: ethyl acetate, 4:1); m.p 123 ℃; Mol. wt: 289.35; Light Yellow powder;
(Yield 80%): IR (Pure) ν: 3192.50 (NH), 3051.73, 2961.57 (C=C-H), 2920.13, 2890.74 (-CH3),
1
1629.38 (C=N), 1591.20, 1540.75, 1504.62, 1476.56 (Ar-C=C), cm-1; H NMR (400 MHz,
DMSO-d6, δ): 11.0 (b, s, 1H, NH), 7.67 (d, 2H, J = 8.2 Hz, Ar-H ), 7.38 (t, 2H, J = 8.2 Hz, Ar-
H), 7.10 (t, 1H, J = 8.2 Hz, Ar-H), 3.89 (s, 3H, C-H); 2.56 (s, 3H, C-H); 0.90 (s, 3H, C-H); 13C
NMR (100 MHz, DMSO-d6, δ): 170.52 (C=N), 168.43 (C=N), 166.82 (C=O), 156.78 (C=C),
140.76 (Ar), 130.82 (Ar), 129.25 (Ar), 129.16 (Ar), 115.45 (C=C), 51.49 (O-C), 19.49 (C-H),
10.26 (C-H); Anal. Cald. For C14H15N3O2S: C, 58.11; H, 5.23; N, 14.52; S, 11.08; Found: C,
58.19; H, 5.20; N, 14.57; S, 11.08.
4.3.4. (E)-1-(4-methyl-2-(2-(1-phenylethylidene)hydrazinyl)thiazol-5-yl)ethanone (5d)
Rf=0.54 (n-hexane: ethyl acetate, 4:1); m.p℃; Mol. wt: 273.95; Yellow powder; (Yield 90%):
IR (Pure) ν: 3352.56 (NH), 3092.48, 2912.67 (C=C-H), 2910.34, 2861.34 (-CH3), 1632.12
(C=N), 1590.63, 1584.54, 1545.25, 1472.16 (Ar-C=C), cm-1; 1H NMR (400 MHz, DMSO-d6,
δ): 11.08 (b, s, 1H, NH), 7.56 (d, 2H, J = 8.2 Hz, A r-H), 7.19 (t, 2H, J = 8.2 Hz, Ar-H), 7.10
(t, 1H, , J = 8.2 Hz, Ar-H), 2.56 (s, 3H, C-H); 2.49 (s, 3H, C-H), 0.97 (s, 3H, C-H); 13C NMR
(100 MHz, DMSO-d6, δ): 196.82 (C=O), 170.59 (C=N), 168.78 (C=N), 156.43 (C=C), 140.66
(Ar), 130.42 (Ar), 129.59 (Ar), 129.36 (Ar), 103.45 (C=C), 28.49 (C-H), 14.49 (C-H), 10.26
(C-H); Anal. Cald. For C14H15N3OS: C, 61.51; H, 5.53; N, 15.37; S, 11.73; Found: C, 61.56;
H, 5.55; N, 15.35; S, 11.74.
4.3.5 (E)-(1-(4-methyl-2-(2-(3-nitrobenzylidene)hydrazineyl)thiazol-5-yl)ethan-1-one (5e)
Rf=0.31(n-hexane: ethyl acetate, 4:1); m.p 255 ℃; Mol. wt: 304.32; Yellow powder; (Yield
90%): IR (Pure) ν: 3262.27 (NH), 3125.48, 2954.63 (C=C-H), 2934.68, 2835.84 (-CH3),
1632.74 (HC=N), 1593.64, 1543.84, 1525.28, 1470.72 (Ar-C=C), cm-1; 1H NMR (400 MHz,

DMSO-d6, δ): 11.20 (b, s, 1H, NH), 8.64 (s, 1H, Ar-H), 8.26 (d, 1H, J = 8.2 Hz, Ar-H), 8.15
(d, 1H, J = 8.2 Hz, Ar-H), 8.14 (s, 1H, HC=N), 8.05 (t, 1H, J = 8.0 Hz, Ar-H), 2.56 (s, 3H,
13
C-H); 2.49 (s, 3H, C-H); C NMR (100 MHz, DMSO-d6, δ): 196.90 (C=O), 170.59 (C=N),
156.78 (C=C), 148.56 (Ar), 143.46 (HC=N), 139.26 (Ar), 135.22 (Ar), 134.52 (Ar), 129.59
(Ar), 124.36 (Ar), 103.45 (C=C), 28.48 (C-H), 10.16 (C-H); Anal. Cald. For C13H12N4O3S: C,
51.31; H, 3.97; N, 18.41; S, 10.54; Found: C, 51.23; H, 3.95; N, 18.46; S, 10.59.
4.3.6. (E)-1-(2-(2-(4-hydroxybenzylidene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (5f)
Rf=0.40 (n-hexane: ethyl acetate, 4:1); m.p 185 ℃; Mol. wt: 275.33; Yellow powder; (Yield
70%): IR (Pure) ν: 3222.60 (NH), 3012.64, 2910.17 (C=C-H), 2902.13, 2806.14 (-CH3),
1630.22 (HC=N), 1591.03, 1541.35, 1500.25, 1470.26 (Ar-C=C), cm-1; 1H NMR (400 MHz,
DMSO-d6, δ): 11.04 (b, s, 1H, NH), 8.12 (s, 1H, HC=N), 7.45 (d, 2H, J = 7.52 Hz, Ar-H), 6.86
(d, 2H, J = 7.52 Hz, Ar-H), 2.56 (s, 3H, C-H); 2.49 (s, 3H, C-H); 13C NMR (100 MHz, DMSO-
d6, δ): 196.83 (C=O), 170.53 (C=N), 160.65 (Ar), 156.78 (C=C), 144.48 (HC=N), 130.42 (Ar),
126.59 (Ar), 116.36 (Ar), 103.45 (C=C), 28.49 (C-H), 10.26 (C-H); Anal. Cald. For
C13H13N3O2S: C, 56.71; H, 4.76; N, 15.26; S, 11.65; Found: C, 56.62; H, 4.79; N, 15.32; S,
11.69.
4.3.7.
(5g)
(E)-4-((2-(4-(4-bromophenyl)thiazol-2-yl)hydrazono)methyl)-2-methoxyphenol
Rf=0.27 (n-hexane: ethyl acetate, 4:1); m.p 165 ℃; Mol. wt: 404.28; Yellow powder; (Yield
73%): IR (Pure) ν: 3328.93 (OH), 3246.90 (NH), 3087.46, 2974.59 (C=C-H), 2924.28 (-CH3),
1620.54 (HC=N), 1593.68, 1567.26, 1536.76, 1470.78 (Ar-C=C), cm-1; 1H NMR (400 MHz,
DMSO-d6, δ): 11.0 (b, s, 1H, NH), 8.09 (s, 1H, HC=N), 7.52 (d, 2H, J = 8.20 Hz, Ar-H), 7.48
(d, 2H, J = 8.20 Hz, Ar-H), 7.10 (s, 1H, Ar-H), 7.04 (d, 1H, J = 8.02 Hz, Ar-H), 6.90 (d, 1H, J
13
= 8.02 Hz, Ar-H), 6.65 (s, 1H, C=C-H), 4.50 (b, s, 1H, OH), 3.75 (s, C-H); C NMR (100

MHz, DMSO-d6, δ): 171.80 (C=N), 151.50 (Ar), 148.19 (C=C), 148.10 (Ar), 143.42 (HC=N),
132.85 (Ar), 132.42 (Ar), 129.74 (Ar), 127.42 (Ar), 123.29 (Ar), 123.15 (Ar), 117.43 (Ar),
114.43 (Ar), 100.43 (C=C), 56.50 (O-C); Anal. Cald. For C17H14BrN3O2S: C, 50.50; H, 3.49;
N, 10.39; S, 7.93; Found: C, 50.45; H, 3.54; N, 10.43; S, 7.93.
4.3.8. 1-(4-Hydroxy-3-methoxybenzylidene)-2-(4-(4-chlorophenyl)thiazol-2-yl) hydrazine
(5h)
Rf=0.30 (n-hexane: ethyl acetate, 4:1); m.p 130 ℃; Mol. wt: 359.83; Yellow powder; (Yield
70%): IR (Pure) ν: 3317.85 (OH), 3219.57 (NH), 3052.56, 2989.34 (C=C-H), 2943.19 (-CH3),
1632.48 (HC=N), 1567.26, 1520.63, 1479.20 (Ar-C=C), cm-1; 1H NMR (400 MHz, DMSO-d6,
δ): 11.04 (b, s, 1H, NH), 8.10 (s, 1H, HC=N), 7.54 (d, 2H, J = 8.20 Hz, Ar-H), 7.48 (d, 2H, J
= 8.20 Hz, Ar-H), 7.32 (s, 1H, Ar-H); 7.10 (d, 1H, J = 8.02 Hz, Ar-H), 6.70 (d, 1H, J = 8.02
Hz, Ar-H), 6.60 (s, 1H, C=C-H), 4.99 (b, s, 1H, OH), 3.76 (s, 3H, C-H); 13C NMR (100 MHz,
DMSO-d6, δ): 171.86 (C=N), 151.56 (Ar), 148.51 (C=C), 147.99 (Ar), 143.18 (HC=N), 134.48
(Ar), 131.86 (Ar), 129.40 (Ar), 128.93 (Ar), 127.40 (Ar), 122.90 (Ar), 117.56 (Ar), 114.89
(Ar), 100.48 (C=C), 56.50 (O-C); Anal. Cald. For C17H14ClN3O2S: C, 56.74; H, 3.92; N, 11.68;
S, 8.91; Found: C, 56.69; H, 3.96; N, 11.74; S, 8.95.
4.3.9. (E)-4-(4-bromophenyl)-2-(2-(4-chlorobenzylidene)hydrazinyl)thiazole (5i)
Rf= 0.39 (n-hexane: ethyl acetate, 4:1); m.p 135 ℃; Mol. wt: 392.70; Yellow powder; (Yield
74%): IR (Pure) ν: 3276.10 (NH), 2987.59 (C=C-H), 1623.45 (HC=N), 1597.56, 1568.26,
1
1537.62, 1479.08 (Ar-C=C), cm-1; H NMR (400 MHz, DMSO-d6, δ): 11.20 (b, s, 1H, NH),
8.20 (s, 1H, HC=N), 7.65 (d, 2H, J = 8.10 Hz, Ar-H), 7.50 (d, 2H, J = 8.2 Hz, Ar-H), 7.38 (d,
2H, J = 8.20 Hz, Ar-H); 7.30 (d, 2H, J = 8.10 Hz, Ar-H), 6.64 (s, 1H, C=C-H); 13C NMR (100
MHz, DMSO-d6, δ): 171.80 (C=N), 146.50 (C=C), 143.22 (HC=N), 134.44 (Ar), 131.76 (Ar),
130.85 (Ar), 129.42 (Ar), 129.24 (Ar), 129.12 (Ar), 128.95 (Ar), 123.05 (Ar), 100.49 (C=C);