Anti-leishmanial click modifiable thiosemicarbazones: Design, synthesis, biological evaluation and in silico studies

Article abstract of DOI:10.1016/j.ejmech.2018.04.003

Leishmaniasis is a devastating tropical disease with limited therapeutic options. Depending on recently reported active anti-leishmanial compounds, we designed and synthesized a series of click modifiable 1,2,3-triazole and thiosemicarbazone hybrids. Most

Full text of DOI:10.1016/j.ejmech.2018.04.003

Accepted Manuscript
Anti-leishmanial click modifiable thiosemicarbazones: Design, synthesis, biological
evaluation and in silico studies
Mohamed G. Temraz, Perihan A. Elzahhar, Alaa El-Din A. Bekhit, Adnan A. Bekhit,
Hala F. Labib, Ahmed S.F. Belal
PII:
S0223-5234(18)30330-1
10.1016/j.ejmech.2018.04.003
EJMECH 10352
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 February 2018
Revised Date: 20 March 2018
Accepted Date: 2 April 2018
Please cite this article as: M.G. Temraz, P.A. Elzahhar, A. El-Din A. Bekhit, A.A. Bekhit, H.F. Labib,
A.S.F. Belal, Anti-leishmanial click modifiable thiosemicarbazones: Design, synthesis, biological
evaluation and in silico studies, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.04.003.
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Graphical Abstract:

ACCEPTED MANUSCRIPT
Anti-Leishmanial Click Modifiable Thiosemicarbazones:
Design, Synthesis, Biological Evaluation and In Silico Studies
1
2*
3
Mohamed G. Temraz , Perihan A. Elzahhar , Alaa El-Din A. Bekhit ,
2,4
2
2*
Adnan A. Bekhit , Hala F. Labib , Ahmed S. F. Belal
1
Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt.
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria
University, Alexandria 21521, Egypt.
3
Food Sciences, University of Otago, Dunedin, New Zealand.
4
Pharmacy Program, Allied Health Department, College of Health Sciences,
University of Bahrain, P.O. Box 32038, Kingdom of Bahrain.
ABSTRACT
Leishmaniasis is a devastating tropical disease with limited therapeutic
options. Depending on recently reported active anti-leishmanial compounds,
we designed and synthesized a series of click modifiable 1,2,3-triazole and
thiosemicarbazone hybrids. Most of the synthesized compounds showed
comparable to superior activity to a well-established anti-leishmanial drug
miltefosine. Compounds 2 and 10a showed nanomolar IC s against
50
promastigotes of L. major (227.4 nM and 140.3 nM respectively, vs 7.8 µM for
miltefosine). Their antiamastigote IC s were 1.4 µM and 1 µM respectively,
50
which are 6 and 8 times the activity of miltefosine (IC50 8.09 µM). Folic and
folinic acids reversed the anti-leishmanial effects of compounds 2 and 10a
and hence we anticipate they act via an anti-folate mechanism. They
exhibited better safety profiles than that of miltefosine on VERO cell lines.
Also they were relatively safe on experimental mice when administered via
oral and parenteral routes. Docking experiments on PTR1 identified
preferential binding interactions and docking scores. Finally, drug-likeness
and ligand efficiency were assessed indicating that both 2 and 10a are
promising hits and/or leads as anti-leishmanial chemotherapeutic agents.
Keywords: Leishmania; Click reaction; Thiosemicarbazones; Anti-folate, Drug-
likeness; Ligand efficiency.
*
Corresponding authors:
Perihan A. Elzahhar, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Alexandria University, Alexandria, Egypt.

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Email: perihan.elzahhar@alexu.edu.eg
Ahmed S. F. Belal, Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Alexandria University, Alexandria, Egypt.
Email: ahmed.belal@alexu.edu.eg
1
. Introduction
Leishmaniasis is a neglected tropical disease that is caused by a protozoan
parasite belonging to the genus Leishmania. According to World Health
Organization (WHO) reports, it is the second major tropical disease after
malaria [1]. Its clinical manifestations are divided into 3 types: cutaneous,
mucocutaneous and visceral [2]. The former is the most common form and
the latter is the most severe and depleting form [2]. More than 20 species
have been reported to infect vertebrates, yet only 7 have been reported to be
associated with pathogenesis in humans [2]. Infection is transmitted through
the bite of female Phlebotomine sandfly infected with the protozoan, which
then resides in liver, spleen and bone marrow via macrophages [3]. Two
distinct morphological forms appear in their life span: promastigote in the
vector’s digestive organ and amastigote in mammalian host’s
reticuloendothelial cells [4,5]. It is endemic in over 90 countries with
approximately 2 million new cases reported each year [6]. Some research
groups developed effective vaccines against leishmanial infection, with
acceptable immunogenicity [7]. Nonetheless, no prophylactic vaccine has
entered clinical practice up till the moment [7]. Therefore, combating this
disease depends primarily on chemotherapy [7]. Pentavalent antimonials
have long been the cornerstone of first line treatment [4,8]. Several
drawbacks have been associated with antimonials including: severe side
effects, poor tolerability and critical levels of drug resistance, especially in
India [4,8]. Amphotericin B, miltefosine and pentamidine are among the drugs
that moved to the forefront of fighting visceral and cutaneous leishmaniasis,
especially in antimony resistant infections [4,8]. However, each comes along
with its own set of stumps; miltefosine has a teratogenic potential,
amphotericin B causes fever and thrombophlebitis and pentamidine promotes
induction of insulin dependent DM, not to mention costly and long duration of

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treatment [4,8]. Additionally, the impact of this disease has been found to be
extremely excruciating, owing to the lack of commercial interest to develop
new drugs against it, plus its occurrence in underdeveloped countries [9–11].
Over and above, increased rate of resistance and therapeutic failure has
reached a crucial extent, which indicates that the global health is on the losing
side of the war against this deadly disease [9–11]. Hence there’s an urgent
need of exploring new therapeutic interventions, that are readily available to
and affordable by the affected populations [5].
Click chemistry established itself as a powerful tool in medicinal chemistry to
provide for highly versatile 1.4-disubstituted 1,2,3-triazole privileged scaffolds
[
12]. Of particular interest to this study, several click modifiable triazoles have
been reported to exert an outstanding in vitro anti-leishmanial activity
Structures I-III, Figure 1) [13–15]. On the other hand, thiosemicarbazones are
(
renowned for their vast array of biological activities of which anti-leishmanial
potential stood out (Structures IV-VI, Figure 1) [16–19]. For example, Manzano
et al. identified several thiosemicarbazones with micromolar activity against
intracellular amastigotes of Leishmania donovani, the most active derivative of
which (Structure IV, Figure 1) showed an EC of 0.8 µM with very low toxicity
50
on two different mammalian cell lines [18]. Based on the above-mentioned
facts and with the objective to discover active compounds against Leishmania
that are non-toxic on mammalian cells, we envisioned that combining
thiosemicarbazone pharmacophore with click modifiable triazole on
bifunctional aromatic skeletons such as vanillin, isovanillin and 4-
hydroxyacetophenone, could provide for a good candidate for an effective and
safe anti-leishmanial agent (Inset C, Figure 1). The triazole ring was designed
to bear various aryl or phenacyl substitutions of different sizes and electronic
nature at N-1. Hence, we hereby present the design, synthesis, biological
evaluation, molecular modelling and drug-likeness assessment of some novel
triazole-thiosemicarbazone hybrids as potential anti-leishmanial agents.
2
. Results and Discussion
2
.1. Chemistry

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The synthetic strategy adopted to prepare target compounds is depicted in
Schemes 1,2 and 3.
Different propargyl derivatives 1,5 and 8 were prepared by refluxing vanillin,
isovanillin and 4-hydroxyacetophenone, respectively, in the presence of
anhydrous potassium carbonate in acetone, giving yields from 70- 80% [20–
2
2].
1
,3 dipolar cycloaddition reactions between propargyl derivatives 1,5 and 8
with appropriate azides were carried out by stirring at room temperature in
DMF in the presence of CuSO /sodium ascorbate as a catalyst; producing the
4
corresponding aldehydes and ketones equipped with 1,4-disubstituted 1,2,3
1
triazoles 3(a-f), 6(a-e) and 9(a-c) in good to excellent yields. The H-NMR of
the latter derivatives showed triazole C -H aromatic singlet around δ 8.14 -
5
13
9
.07 ppm. Their C-NMR showed characteristic peak at 142.33 – 142.78
corresponding to triazole C . Other characteristic peaks appeared at their
5
expected chemical shifts such as C=O at δ 191.79 – 192.61 ppm. Their IR
spectra showed the disappearance of ethynyl CH and C≡C stretching bands,
which evidently confirmed the formation of triazole derivatives. An alternate
route had been tried, in which propargyl 1 was reacted with thiosemicarbazide
to afford the subsequent propargyl-thiosemicarbazone 2. However, applying
click reaction on compound 2 turned out to be unsuccessful, and hence we
focused our attention on the first route.
Refluxing 3(a-f), 6(a-e) and 9(a-c) with thiosemicarbazide in ethanol and/or
acetic acid produced the corresponding thiosemicarbazones 4(a-f), 7(a-e) and
1
1
0(a-c) in 30 – 50% yields. The H-NMR of the final thiosemicarbazones 4(a-
f) and 7(a-e) showed characteristic arylidene =C-H and D O exchangeable
2
NH at δ 7.93 – 8.1 and δ 11.34 – 11.43 ppm, respectively, along with the
disappearance of the aldehydic C-H, which is in agreement with many
recently reported thiosemicarbazones [16,23].
On the other hand,
1
thiosemicarbazones 10(a-c) showed in their H-NMR a characteristic CH
3
singlet in range of δ 2.13 – 2.51 and D O exchangeable NH at δ 10.14 - 10.23
2
13
ppm. The C-NMR of all thiosemicarbazone derivatives showed characteristic
C=S peak at δ 142.61 – 179.06 ppm. Their IR spectra displayed characteristic

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-1
N-H stretching bands at ʋ 3000-3500 cm , which markedly confirmed the
formation of the target thiosemicarbazones.
2
2
.2. Biological Evaluations
.2.1. In vitro anti-leishmanial activity
Table 1
Antipromastigote and antiamastigote activities of tested compound expressed
as IC values (µM) ± SEM.
50
Antipromastigote Antiamastigote
Entry
Structure
activity (µM)
activity (µM)
2
0.2274±0.03
1.40±0.07
4
a
4.8673±0.25
4.7559±0.26
5.39±0.04
5.40±0.26
5.10±0.23
4b
O
N
N
N
S
Br
4
c
d
e
O
O
3.9687±0.03
6.9267±0.36
HN NH2
N
4
4
6.46±0.49
4.8224±0.34
4.97±0.31
5.60±0.60
4
f
5.7420±0.37
4.3969±0.32
7a
7b
7c
4.38±0.04
1.69±0.08
4.48±0.31
0.7979±0.10
5.6945±0.23
7d
2.5172±0.31
4.83±0.04

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7e
0.9903±0.36
2.18±0.09
7
f
4.3781±0.56
4.9989±0.42
4.35±0.04
4.78±0.09
7g
1
0a
0.1403±0.02
6.7940±0.08
1.00±0.08
8.26±0.31
10b
H2N
N N
N
HO
O
HN
S
1
0c
7.4104±0.14
7.8074 ± 0.34
6.04±0.53
N
O
Miltefosine
8.09 ± 0.07
All target compounds were tested against L. Major promastigotes and axenic
amastigotes, prepared in accordance to a previously reported procedure
[
24,25], to identify the most active compounds that are worthy of further
investigation. Anti-leishmanial activity was expressed in terms of IC , which is
50
the effective micromolar concentration required to achieve 50% growth
inhibition; using promastigotes and amastigotes in their exponential growth
phase. Indeed, all of the tested compounds showed substantial anti-
leishmanial activity and operated within the submicromolar to micromolar
range in terms of IC . IC s were in the range of 0.1403-7.41 µM against
50
50
promastigotes and 1-6.46 µM against amastigotes (Table 1).
Within the vanillin-derived triazole series 4, the unsubstituted phenacyl
triazole 4a showed 60% and 50 % increase in activity on both promastigotes
and amastigotes, respectively, when compared to miltefosine. Substitution
with EWG or EDG at the 4-position seemed to retain the activity against both
promastigotes and amastigotes. On the other hand, within the isovanillin-
derived triazole series 7, the unsubstituted phenacyl triazole 7a showed 78%
and 85 % increase in activity on both promastigotes and amastigotes,
respectively, when compared to miltefosine. Substitution by a small lipophilic

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EWG group such as –F (compound 7d) enhanced the anti-promastigote
activity to reach 300% that of miltefosine, while the increase in anti-
amastigote activity was still maintained at 67%. Anti-promastigote activity was
further enhanced on substitution with moderately-sized lipophilic EWG (-Cl)
(
compound 7b) and EDG (-CH ) (compound 7e) yielding one higher order of
3
magnitude of activity and IC values, for the first time in this study, to appear
50
in the nanomolar range. The anti-amastigote activities of compounds 7b and
e displayed approximately 5 and 4 times the activity of miltefosine,
7
respectively. Substitution with hydrophilic EWGs like –COOH (compound 7g)
and –SO -NH (compound 7f) exhibited lower activities compared to the
2
2
unsubstituted congeners, nonetheless they were still of higher activity
compared to the positive control miltefosine.
Intriguingly, the novel propargyl derivative 2 showed an enhanced activity on
both phenotypes, where it reached one order of magnitude higher than that of
miltefosine with IC of 0.2274 µM against promastigote. It was almost 6 times
50
more active against amastigote than miltefosine. This activity was even
superior to its triazolyl counterparts; which uplifted it to be among the most
active compounds in this study. We anticipate that the unique propargyl group
existing in compound 2 was not only acting as a physicochemical parameters
modulator, but also might be more or less an active pharmacophore that was
involved in target recognition (vide infra Molecular Docking Study).
Finally, within the 4-hydroxyacetophenone-derived triazoles 10, the 4-
bromophenacyl substituted triazole 10a exhibited the highest activity of the
entire study where it reached 140.3 nM IC against promastigote and 1 µM
50
against amastigote. Compared to miltefosine, it achieved a higher order of
magnitude and 8 times anti-leishmanial capacities against promastigotes and
amastigotes, respectively. Derivatives 10b and 10c carrying the hydrophilic
EWGs –SO NH and –COOH, respectively retained the same anti-leishmanial
2
2
magnitude as that of Miltefosine.
Owing to some unique structural features in each synthesized compound, we
could not observe clear trending structure-activity relationships. Nonetheless,
compounds 2 and 10a showed particular noteworthy nanomolar anti-

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leishmanial activity, which encouraged us to pursue further studies on them in
order to gain a better understanding of their biological activities and also to
gauge their safety as potential drug candidates. Also, it seems meaningful
that
this
inventive
1,4-disubstituted-1,2,3-triazole/thiosemicarbazone
framework serving as anti-leishmanial agent, would encourage us to
undertake further in-depth future studies, that involve wider arrays of
substituents, in order to extract sharper structure-activity relationships.
2
.2.2 Reversal of anti-leishmanial activity of most active compounds by
folic acid and folinic acid
Some reports indicated the potential of some thiosemicarbazones and 1,2,4-
triazoles as inhibitors of DHFR and PTR1, respectively [26,27]. Thus, we
anticipate that our target triazole/thiosemicarbazone hybrids would exert their
anti-leishmanial activity via inhibition of folate pathways.
In Leishmania parasites, the metabolic pathways that are associated with the
transfer of one-carbon units rely on the essential cofactors tetrahydrofolate
(H4-folate) and reduced pterin [28]. Dihydrofolate Reductase (DHFR) is a
NADPH-dependent enzyme, responsible for producing H4-folate from folate
and/or dihydrofolate [28,29]. DHFR also catalyzes the conversion of
Deoxyuridine monophosphate (dUMP) to Deoxythymidine Monophosphate
(
dTMP), and hence is recognized in some reports as DHFR-TS [28,29].
Nonetheless, most leishmania species are resistant to DHFR-TS inhibitors
28,29]. This is attributed to the presence of an alternative salvage pathway
[
regulated by pteridine reductase 1 (PTR1), which provides enough folate
supply when DHFR-TS is inhibited [30]. Interestingly, PTR1 is completely
absent in mammalian hosts [30]. Hence, the PTR1 pathway may hold promise
as an attractive molecular target for anti-leishmanial drug development.
In order to confirm the potential anti-folate mechanism of our most active
compounds (2 and 10a), we employed the approach reported by Mendoza-
Martínez et al. [8] which involved exposing the parasite to concentrations of
the tested compounds above their IC s after the addition of folic acid and
50
folinic acid. Trimethoprim was used as a positive control. The parasitic
exposure to trimethoprim after addition of folic acid led to an increase in
parasite survival up to almost 100 %. It is worth mentioning that folic acid

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competes for the active sites of DHFR and PTR1 while folinic acid contributes
to DNA synthesis without any need to undergo activation. As shown in Table
2
, reversal of anti-leishmanial effect of compounds 2 and 10a occurred upon
addition of folic acid with percentage parasite growth in the range of 89-99%
Table 2). Also, folic and folinic acids displayed more or less the same level
(
inhibition of the anti-leishmanial activity of the target compounds. From this
data, we can conclude that most, if not all, of the anti-leishmanial activities of
compounds 2 and 10a could be attributed to anti-folate mechanism, most
probably by acting on both DHFR-TS and PTR1.
On the other hand, addition of excess folic acid to parasitic cells after
exposure to the test compounds had been performed to investigate its ability
to reverse the DHFR and PTR1 inhibition (results not shown). It was found
that all test compounds showed reversibility of DHFR and PTR1 inhibition,
similar to that of trimethoprim.
Table 2
In vitro evaluation of folate pathway inhibition expressed as percentage of
survival
Entry
No competitor
added
Folic acid
20 µM 100 µM 20 µM 100 µM
Folinic acid
2
(0.25 µM)
25%
23%
69%
*
*
*
89%
91%
-
*
*
97%
99%
99%
*
*
*
90%
90%
-
*
*
98%
99%
-
*
*
10a (0.16 µM)
Trimethoprim (100 µM)
*
Percentage survival = 100 - % AP; where % AP is the percentage growth
inhibition. Retrieval of folate activity (via action on DHFR-TS and PTR1
enzymes) was obtained with both compounds 2 and 10a, along with
trimethoprim as a positive control; upon adding either folic or folinic acids
reaching nearly 100% with 100 µM of activated folate forms.
2
.2.3. In vitro cytotoxicity testing
In order to verify the safety of the most active compounds 2 and 10a, they
were tested for cytotoxicity against African green monkey kidney cells (VERO
cells) as described earlier [31]. Briefly the cells were incubated for 72h with
different dilutions of the selected compounds. 50% cytotoxic concentration
values represent the concentration of compound required to kill 50% of the
fibroblast cells were calculated (CC ). The selectivity indices were calculated
50
using the formula SI = CC /IC , against promastigotes. Interestingly, the
50
50
concentrations needed to inhibit viability of VERO cells (CC ) are 4 orders of
50

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magnitude higher than those required to inhibit the viability of promastigotes
of leishmania parasite (IC ). Furthermore, they exhibited higher selectivity
50
indices (SI) than that of miltefosine, which highlights their safety on
mammalian cells (Table 3).
Table 3
Cytotoxicity data (CC , µM) and selectivity indices (SI) of the most active
5
0
compounds
Entry
(CC )* (µM)
Selectivity
5
0
index (CC / IC )
50
50
2
827.89±2.2
508.84±2.6
99.73±1.1
3640.67
3626.80
12.77
10a
Miltefosine
*
CC50 is the concentration at which 50% cells survive and IC50 is the
concentration at which 50% leishmania death occur ± SEM.
2
.2.4. In vivo acute toxicity testing
The most active anti-leishmanial compounds, 2 and 10a were tested for their
acute toxicity in mice. The experimental mice did not have any signs of toxicity
after treatment with the test compounds. There was no significant difference
in the weight of the mice and no death cases were recorded during 3 days of
observation post administration of the test compounds (data not shown). The
test compounds were well tolerated by the experimental animals orally up to
1
25 mg/kg. Moreover, these compounds were tested for their toxicity through
the parenteral route and the results revealed that the tested compounds were
non-toxic up to 75 mg/kg.
Isolated spleen, lung, liver and kidney from mice that received 125 mg/kg
orally or 75 mg/kg parenterally, showed normal textures. Histopathological
examination of spleen, lung, liver and kidney specimens were devoid of
abnormalities.
2
2
.3. Molecular modeling and in silico predictions
.3.1. Molecular docking study
Molecular docking studies of the most active compounds (2 and 10a) into the
binding site of pteridine reductase 1 were carried out to develop a deeper
insight into the molecular mechanism of anti-leishmanial activity as well as
explore possible binding mode of interaction of these compounds, using
Molecular operating environment software (MOE 2016.0802). The crystal

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structure of Leishmania major pteridine reductase 1 enzyme (PDB ID: 2BFM)
with co-crystallized ligand trimethoprim (TOP) occupying its active site was
employed as a model for inhibitory interactions.
Selection of the docking poses was based upon the top-scored conformations
together with best binding interactions estimated by MOE scoring function and
search algorithm. Binding affinities to the active site of PTR1 were predicted in
the light of scoring functions, hydrogen bonds formed with the surrounding
amino acid residues, and the relative orientation of the docked compounds in
relation to the co-crystallized ligand TOP. Re-docking of the co-crystallized
ligand TOP into PTR1 binding site validated the docking protocol (Figure 2.)
and reproduced the initial pose generated from PDB with root mean square
deviation (RMSD) of 1.7454 Å and docking score of -4.6238 kcal/mol.
Examination of the best-docked pose of the propargyl derivative 2 revealed
that it was perfectly anchored in the active site of PTR1 enzyme with binding
energy score of -5.5028 kcal/mol. It was held in the active site through
hydrogen bonding between the acceptor sulfur atom and crucial amino acids
Leu188 and Tyr194 in addition to Thr184. The terminal amino group was
hydrogen-bonded to Gln186. As well, hydrophobic interactions with Met183,
Pro187, Leu188 and Val230 were observed (Figure 3.). Intriguingly, the
propargyl functionality shared some common hydrophobic contacts with key
residues Phe113, Leu226 and Leu229, as those found in PTR1 crystal
structure. This might spotlight the importance of this propargylic moiety as a
structural motif for target recognition.
As for the triazolyl thiosemicarbazone 10a (energy score of -6.5815 kcal/mol),
it extended smoothly in the active site of PTR1 through arene-H interaction
between p-bromophenyl ring and Ser111. It also formed two hydrogen bonds
between its bromine atom and Asn109 and the acceptor sulfur atom and
Met183. Additionally, hydrophobic contacts were observed with Leu18,
Phe113, Met179, Val180, Met183, Leu188, Leu226 and Leu229. An
interesting hallmark of 10a is the participation of 1,4-disubstituted-1,2,3-
triazole in both hydrogen bonding between its acceptor N-2 nitrogen and

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Arg17, and H-arene interaction between the exceptionally acidic C-5
hydrogen and the key amino acid Phe113 (Figure 4.). This is consistent with
the well-established attributes of the triazole ring as pharmacophoric building
block [32] actively taking part in ligand-target complex stabilization and
biomolecular target recognition rather than being just a passive linker.
Figure 5 displayed a fine overlay of docking poses of the most active
compounds; with their thiosemicarbazone groups facing each other in addition
to closely positioned propargyl (2) and triazole (10a) moieties. Moreover, both
compounds overlapped well with the co-crystallized ligand TOP and thus
adopted similar binding pattern in addition to some extra binding interactions,
which is coherent with their better energy scores. Additionally, Figure 6
showed the 3D alignment of compounds (2 and 10a) and the co-crystallized
ligand TOP in the active site of PTR1 clearly emphasizing that they occupy
the same position and spatial area of the binding pocket.
2
.3.2. In silico prediction of drug-likeness, physicochemical properties
and pharmacokinetic profile
Inappropriate ADME (absorption, distribution, metabolism, and excretion)
properties have largely been the reason behind attrition of most of new drug
candidates in clinical trials, which add up to the costs of new drug
development. Therefore, early assessment of pharmacokinetic properties of
new drug candidates is a vital step in the drug development process that can
direct lead optimization efforts into improved analogs [33].
However, it should be clear that, for a molecule that proves to be drug-like,
there is no warranty that this will eventually become a drug. Oppositely, there
are examples of molecules that do not occupy well-known oral drug-like space
that are yet clinically useful drugs [34].
The basic guidance to correlate physicochemical properties with successful
drug development has been achieved by examination of the structures of
orally administered drugs, and drug candidates, as introduced by Lipinski.
This analysis has emerged in a set of rules emphasizing the significance of
lipophilicity (octanol-water partition or LogP), number of hydrogen bond
acceptors (HBA) and donors (HBD), and molecular weight (MW). An orally

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available drug candidate is compliant with Lipinski's rule if LogP is no more
than 5, MW is less than 500, number of HBD is less than 5 and number of
HBA is less than 10 [35].
In addition, further guidelines governing oral bioavailability, that are
independent of molecular weight, have been suggested by Veber et al. [36].
They stated that molecular flexibility (represented by number of rotatable
bonds or NROTB) plays an important role in oral bioavailability; the more
flexible the molecule, the less likely it is to be orally active. Also, it has been
suggested that polar surface area (TPSA) could be used as a factor instead of
the number of hydrogen bonding groups. Compounds with 10 or fewer
2
NROTB and TPSA of less than 140 Å should present high oral bioavailability
in rats.
In the present study, we used Molinspiration [37], Molsoft [38], Pre-ADMET
[
39] and Data warrior [40] software to assess the pharmacokinetic parameters
of the most active compounds 2 and 10a.
The results presented in Table 4 showed that both compounds conform to
Lipinski's rule, with LogP values of 1.69 and 3.56 (<5), MW of 263 and 487
(<500), HBA of 5 and 8 (<10) and HBD of 3 (<5), respectively, So, they should
theoretically exhibit good passive oral absorption and differences in their
bioactivity cannot be ascribed to this property. Furthermore, the compounds
showed NROTB values of 6 and 9 (<10) and TPSA values of 68.88 and
2
2
1
07.44 Å (<140 Å ) with resultant percentage oral absorption (calculated
from %ABS = 109-0.345 TPSA) of 85.23 and 71.93%, indicating good
absorption, transport and permeability through biological membranes.
On the other hand, Molsoft software was used to estimate the solubility and
drug-likeness model score for both compounds. Aqueous solubility is known
to affect absorption and distribution characteristics considerably. In this
context, these compounds fulfilled the requirements of solubility with values of
5
7.43 and 0.73 mg/L (more than 0.0001 mg/L). Compounds showing positive
drug-likeness model scores are recognized as drug-like and can behave as
drug molecules. A positive model-score was predicted for compound 10a
(0.66) while that for compound 2 was negative (-0.11).

ACCEPTED MANUSCRIPT
Moreover, in silico estimation of the following pharmacokinetic parameters
was performed using Pre-ADMET software: Caco2 (human colon
adenocarcinoma) permeability coefficient, MDCK (Madin-Darby canine kidney
cells) permeability coefficient, Human intestinal absorption (HIA), Brain-blood
barrier partition coefficient (BBB), human plasma protein binding (PPB) and
inhibition of cytochrome P450 2D6 (CYP2D6). The results of the predicted
ADME parameters are recorded in Table 4.
Both compounds showed medium cell permeability in the Caco-2 cell model
with values of 21.21 and 20.1 nm/s. Moreover, compound 2 showed medium
cell permeability in the MDCK cell model (190.25 nm/s) while low permeability
was observed with compound 10a (0.025 nm/s). They showed high human
intestinal absorption values (95.06 and 96.43 %) indicating very well-absorbed
compounds. Furthermore, they demonstrated low BBB penetration capability
(0.07 and 0.05). Both compounds were found to be highly-bound to human
plasma proteins (100 and 95.74%). As well, they were non-inhibitors of
CYP2D6 enzyme and thus may pose no interactions with CYP2D6 inducers
and/or inhibitors.
Table 4
In silico physicochemical properties, ADME and ligand efficiency data of
compounds 2 and 10a
Compound 2
1.69
Compound 10a
3.56
a
LogPb
MW
HBA
263.32
5
487.38
8
c
d
HBD
3
3
Lipinski's violation
0
0
e
TPSA
68.88
85.23
232.45
6
57.43
-0.11
21.2054
190.251
95.063046
0.0698575
100
107.44
71.93
374.75
9
0.73
0.66
f
%
ABS
3
Volume (A)
g
NROTB
h
S (mg/L)
Drug likeness model score
i
Caco2j
20.1321
0.0254928
96.430389
0.0454235
95.740278
0.31338
4.7382
MDCK
k
HIA
l
BBB
PPB
m
n
LE (promastigote)
0.50632
5.224
o
LLE

ACCEPTED MANUSCRIPT
p
LELP
2.803
6.748
0.27438
3.8853
7
LE (amastigote)
LLE
0.44616
4.4347
3.1809
LELP
a
b
LogP: logarithm of compound partition coefficient between n-octanol and water.
c
d
MW: molecular weight. HBA: number of hydrogen bond acceptors.
HBD:
e
f
number of hydrogen bond donors. TPSA: topological polar surface area. %ABS:
g
h
percentage of absorption.
NROTB: number of rotatable bonds.
Caco2: permeability through cells derived from human colon
adenocarcinoma; Caco2 values < 4 nm/s (low permeability), values ranged from 4 to
S: aqueous
i
solubility.
j
7
0 nm/s (medium permeability) and values > 70 nm/s (high permeability). MDCK:
permeability through Madin-Darby Canine kidney cells tool for rapid permeability;
MDCK values < 25 nm/s (low permeability), values ranged from 25 to 500 nm/s
k
(
medium permeability) and values > 500 nm/s (high permeability). HIA: percentage
human intestinal absorption; HIA values ranged from 0 to 20% (poorly absorbed),
values ranged from 20 to 70% (moderately absorbed) and ranged from 70 to 100%
l
(
well absorbed). BBB: blood-brain barrier penetration; BBB values < 0.1 (low CNS
absorption), values ranged from 0.1 to 2 (medium CNS absorption) and values > 2
m
(
high CNS absorption). PPB: plasma protein binding; PPB values < 90% (poorly
n
o
bound) and values > 90% (strongly bound). LE: ligand efficiency. LLE: lipophilic
ligand efficiency. LELP: ligand-efficiency-dependent lipophilicity.
p
In recent years, the concept of Ligand Efficiency Indices (LEI) has emerged
as an advantageous tool in the optimization process of lead compounds.
Thus, these parameters were calculated for the most active compounds to
test their physicochemical and pharmacological balance [41].
Various algorithms have been proposed as regulators for hit-to-lead
optimization since the emergence of Lipinski’s rule of five, with the aim of
lowering attrition rates during clinical phase of drug development. It has been
documented that increased molecular weight and lipophilicity result in
enhanced binding potency but in the meantime, are closely correlated to
increased binding promiscuity and decreased safety thresholds. Ligand
efficiency (LE), lipophilic ligand efficiency (LLE) and ligand-efficiency-
dependent lipophilicity (LELP), are three scoring functions that penalize
molecular weight and lipophilic influence on drug potency in an attempt to
normalize for these deleterious effects [42].
Ligand Efficiency (LE = (pIC x 1.37)/non-hydrogen atoms), simply assess if
50
ligand's potency originates from optimal fit with the target protein or by making
many contacts depending on its size. Lipophilic ligand efficiency (LLE = pIC50
–
LogP), is a metric that merges both potency and lipophilicity. While, ligand-
efficiency-dependent lipophilicity (LELP = LogP/LE) is a composite descriptor
relating both molecular size and lipophilicity to potency [43].

ACCEPTED MANUSCRIPT
LE measures drug potency per heavy (non-hydrogen) atom. Regarding
antipromastigote and antiamastigote activities, compounds 2 and 10a (LE =
0
.27-0.51, Table 4) almost fulfil the suggested minimum LE of 0.3 for lead
compounds [44,45]. LLE focuses on compounds whose lipophilicity
contributes a little to their activity. LLE values presented in Table 4 for both
compounds ranged from (3.89-5.22), which closely comply with optimal value
ranges for lead compounds (LLE ≥ 3) or marketed drugs (LLE ≥ 5) [45–47].
Some studies inferred that the three ligand efficiency metrics were useful
during early stages of drug development; yet, only LELP could distinguish
between marketed drugs and unsuccessful leads [42]. As well, it strongly
correlated with safety and pharmacokinetic profile. In this context, both
compounds meet LELP requirements for leads (≤7.5) and marketed drugs
(<10) since their values ranged from 2.8-7 [45,48].
Collectively and based on the estimated ligand efficiency, drug-likeness and
pharmacokinetic predictors, these two compounds worth qualifying for further
lead optimization cycles.
3
. Conclusion
In the current study we identified new hybrid structures comprising click
modifiable 1,2,3-triazoles and thiosemicarbazones as novel anti-leishmanial
platforms. Most of the synthesized hybrids showed superior anti-leishmanial
activity to miltefosine, against both promastigotes and amastigotes of L.
Major. Compounds 2 and 10a showed nanomolar activity against
promastigotes (IC s are 227.4 nM and 140.3 nM respectively). Their IC s
50
50
against amastigotes were 1.4 µM and 1 µM respectively; reaching 6 and 8
times the activity of miltefosine, respectively. The anti-leishmanial activities of
compounds 2 and 10a were reversed by folic and folinic acids, which strongly
suggest their reliance on anti-folate mechanism, most probably via the
inhibition of both DHFR-TS and PTR1. They proved to be safe on a
representative of mammalian cell lines (VERO cell line) with selectivity indices
exceeding 3000, and also on experimental mice showing no signs of acute
toxicity when used orally up to 125 mg/kg and via parenteral route up to 75
mg/kg. Molecular docking experiments of compounds 2 and 10a on PTR1
showed perfect fitting in the binding pocket and noticeable interactions with

ACCEPTED MANUSCRIPT
key amino acids. Moreover, drug-likeness assessment via Molinspiration,
Molsoft, Pre-ADMET and Data warrior software elucidated their full
compliance with Lipinski’s rule, favorable physicochemical properties and
convenient predicted pharmacokinetic parameters. Over and above, ligand
efficiency indices for compounds 2 and 10a were calculated and they
indicated that these 2 compounds represent promising leads and/or hits that
could enrich the contemporary anti-leishmanial drug development pipeline.
4
. Experimental
4
.1. Chemistry
Melting points were recorded on electrotherm capillary tube Stuart melting
point apparatus SMP10 and are all uncorrected. Follow up of the reactions'
rates were performed by thin-layer chromatography (TLC) on silica gel-
precoated aluminum sheets (Type 60 GF254; Merck; Germany) and the spots
were visualized by exposure to iodine vapors or UV-lamp at λ 254nm for few
seconds. Infrared spectra (IR) were recorded using KBr discs on a
PerkinElmer IR spectrophotometer, Faculty of Pharmacy, Alexandria
1
13
University. Nuclear magnetic resonance ( H NMR and C NMR) spectra were
recorded on a Bruker (400 MHz) spectrophotometer, Faculty of Pharmacy;
Beni Suef University using deuterated dimethylsulfoxide (DMSO-d ) as
6
solvent, followed by recording the spectra again after addition of D O for
2
detection of D2O exchangeable peaks. The data were recorded as chemical
shifts expressed in δ (ppm) relative to tetramethylsilane (TMS) as internal
standard. Signal splitting are expressed by the following abbreviations: s =
singlet, d = doublet, t = triplet, q = quartet and m = multiplet. Microanalytical
data (C, H, N and S) were performed at the microanalytical unit, Faculty of
Science, Al-Azhar University. The propargyl derivatives 1 [20] ,5 [21] and 8
[
22] were prepared according to reported procedures.
4
.1.1. Click reaction procedure for compounds 3a-f, 6a-g and 9a-c:
To a mixture of each of propargyl derivatives 1, 5 or 8 (1 mmol) and
appropriate azide (1.5 mmol) in 10 mL of DMF, was added an aqueous
solution (5 mL) of sodium ascorbate (0.06 g, 0.34 mmol) and CuSO .5H O
4
2
(0.02 g, 0.085 mmol). The mixture was stirred at room temperature for 48h.

ACCEPTED MANUSCRIPT
After cooling, the mixture was poured on ice, filtered, washed with cold water,
dried and recrystallized from ethanol/DMF.
4
.1.1.1. 3-Methoxy-4-((1-(2-oxo-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methoxy)
benzaldehyde (3a).
-1
Orange brown powder, yield 86.4%. m.p.128~129 °C. IR (KBr, cm ):
1
068.17, 1267.51 (C-O-C), 1585.23 (C=C), 1605.25 (C=N), 1677.64 (C=O,
1
ketonic), 1710.28 (C=O, aldehydic). H NMR (400 MHz, DMSO-d ): δ 3.84 (s,
6
3
H, OCH ), 5.33 (s, 2H, CH ), 6.24 (s, 2H, OCH ), 7.42-7.45 (m, 4H,
3 2 2
benzaldehyde-C -H), 7.58 (m, 4H, benzaldehyde-C -H and phenyl-C2,4,6-H),
2,5
6
8
1
1
1
.09 (d, J = 7.6 Hz, 2H, phenyl-C -H), 8.26 (s, 1H, triazole-C -H), 9.87 (s,
3,5 5
13
H, CHO). C NMR (100 MHz, DMSO-d ): δ 192.6, 191.9, 153.4, 149.8,
6
42.4, 134.7, 134.6, 130.4, 129.7, 129.4, 129.0, 128.7, 127.2, 126.3, 113.1,
10.2, 62.2, 56.4, 56.0. Anal. Calcd (%) for C H N O (351.36): C, 64.95;
19
17
3
4
H, 4.88; N, 11.96. Found: C, 65.12; H, 5.01; N, 12.08.
4
3
.1.1.2. 4-((1-(2-(4-Chlorophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-
-methoxybenzaldehyde (3b).
-1
Yellow powder, yield 89.6%. m.p.112~113 °C. IR (KBr, cm ): 1091.86,
1
1
267.31 (C-O-C), 1587.72 (C=C), 1606.36 (C=N), 1681.86 (C=O, ketonic),
1
710.67 (C=O, aldehydic). H NMR (400 MHz, DMSO-d ): δ 3.84 (s, 3H,
6
OCH ), 5.33 (s, 2H, CH ), 6.23 (s, 2H, OCH ), 7.42-7.46 (m, 2H,
3
2
2
benzaldehyde-C -H), 7.59 (d, J = 8 Hz, 1H, benzaldehyde-C -H), 7.70 (d, J
2,5
6
=
8.4 Hz, 2H, 4-chlorophenyl-C -H), 8.09 (d, J = 8.4 Hz, 2H, 4-chlorophenyl-
2,6
13
C -H), 8.24 (s, 1H, triazole-C -H), 9.87 (s, 1H, CHO). C NMR (100 MHz,
3,5
5
DMSO-d ): δ 191.9, 191.8, 153.4, 149.8, 139.6, 133.3, 130.6, 130.4, 129.6,
6
1
27.2, 126.3, 113.1, 110.2, 62.2, 56.3, 56.0. Anal. Calcd (%) for
C H ClN O (385.80;): C, 59.15; H, 4.18; N, 10.89. Found: C, 95.43; H,
19
16
3
4
4
.33; N, 11.21.
4
3
.1.1.3. 4-((1-(2-(4-Bromophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-
-methoxybenzaldehyde (3c).
-1
Dark brown powder, yield 89.4%. m.p.130~131 °C. IR (KBr, cm ): 1044.94,
1
1
265.25 (C-O-C), 1585.68 (C=C), 1603.55 (C=N), 1697.39 (C=O, ketonic),
1
711.95 (C=O, aldehydic). H NMR (400 MHz, DMSO-d ): δ 3.83 (s, 3H,
6

ACCEPTED MANUSCRIPT
OCH ), 5.33 (s, 2H, CH ), 6.23 (s, 2H, OCH ), 7.42-7.44 (m, 2H,
3
2
2
benzaldehyde-C -H), 7.59 (d, J = 8 Hz, 1H, benzaldehyde-C -H), 7.84 (d, J
2,5
6
=
8.4 Hz, 2H, 4-bromophenyl-C -H), 8.01 (d, J = 8.4 Hz, 2H, 4-
2,6
13
bromophenyl-C -H), 8.25 (s, 1H, triazole-C -H), 9.87 (s, 1H, CHO).
C
3,5
5
NMR (100 MHz, DMSO-d ): δ 192.0, 191.9, 153.3, 149.8, 133.6, 132.5,
6
1
30.6, 130.4, 128.9, 127.2, 126.3, 113.1, 110.2, 62.2, 56.4, 56.0. Anal. Calcd
(
%) for C H BrN O (430.26): C, 53.04; H, 3.75; N, 9.77. Found: C, 53.42;
19
16
3
4
H, 3.37; N, 9.94.
4
3
.1.1.4. 4-((1-(2-(4-Fluorophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-
-methoxybenzaldehyde (3d).
-1
Beige powder, yield 87.6%. m.p.114~115 °C. IR (KBr, cm ): 1053.15,
1
1
232.35 (C-O-C), 1598.16 (C=C), 1602.33 (C=N), 1685.61 (C=O, ketonic),
1
709.02 (C=O, aldehydic). H NMR (400 MHz, DMSO-d ): δ 3.83 (s, 3H,
6
OCH ), 5.34 (s, 2H, CH ), 6.25 (s, 2H, OCH ), 7.42-7.46 (m, 4H,
3
2
2
benzaldehyde-C -H and 4-fluorophenyl-C -H), 7.58 (d, J = 8 Hz, 1H,
2,5
3,5
benzaldehyde-C -H), 8.16-8.19 (m, 2H, 4-fluorophenyl-C -H), 8.29 (s, 1H,
6
2,6
13
triazole-C -H), 9.87 (s, 1H, CHO). C NMR (100 MHz, DMSO-d ): δ 191.9,
5
6
1
1
91.3, 167.3, 164.8, 153.4, 149.8, 142.4, 131.8, 131.4, 130.4, 127.2, 126.3,
16.7, 116.5, 113.1, 110.2, 62.2, 56.3, 56.0. Anal. Calcd (%) for C H FN O
19
16
3
4
(369.35): C, 61.79; H, 4.37; N, 11.38. Found: C, 61.41; H, 4.50; N, 11.46.
4
.1.1.5. 3-Methoxy-4-((1-(2-oxo-2-(p-tolyl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy) benzaldehyde (3e).
-1
Off-white powder, yield 92.7%. m.p.170~171 °C. IR (KBr, cm ): 1026.62,
1
1
271.23 (C-O-C), 1585.27 (C=C), 1601.01 (C=N), 1674.72 (C=O, ketonic),
1
707.49 (C=O, aldehydic). H NMR (400 MHz, DMSO-d ): δ 2.43 (s, 3H,
6
CH ), 3.84 (s, 3H, OCH ), 5.33 (s, 2H, CH ), 6.19 (s, 2H, OCH ), 7.42 – 7.45
3
3
2
2
(
m, 3H, benzaldehyde-C2,5,6-H), 7.59 (d, J = 8 Hz, 2H, p-tolyl-C -H), 7.99 (d,
2,6
J = 8 Hz, 2H, p-tolyl-C -H), 8.25 (s, 1H, triazole-C -H), 9.87 (s, 1H, CHO).
3,5
5
13
C NMR (100 MHz, DMSO-d ): δ 192.1, 191.9, 153.4, 149.8, 145.3, 142.3,
6
1
32.1, 130.4, 130.0, 128.8, 127.2, 126.3, 113.1, 110.2, 62.2, 56.3, 56.0, 21.8.
Anal. Calcd (%) for C H N O (365.39): C, 65.74; H, 5.24; N, 11.50. Found:
20
19
3
4
C, 65.88; H, 5.46; N, 11.37.

ACCEPTED MANUSCRIPT
4
.1.1.6. 4-(4-((4-Formyl-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)benzoic acid (3f).
-1
Off-white powder, yield 93.8%. m.p.218~219 °C. IR (KBr, cm ): 1047.34,
1
1
264.37 (C-O-C), 1585.10 (C=C), 1606.27 (C=N), 1700.71 (C=O, aldehydic),
1
711.23 (C=O, acidic), 2600-3200 (OH, broad band). H NMR (400 MHz,
DMSO-d ): δ 3.82 (s, 3H, OCH ), 5.38 (s, 2H, CH ), 7.42 (d, J = 8 Hz, 2H,
6
3
2
benzaldehyde-C -H), 7.57 (d, J = 8 Hz, 1H, benzaldehyde-C -H), 8.06 (d, J
2,5
6
=
8.4 Hz, 2H, 4-carboxyphenyl-C -H), 8.14 (d, J = 8.4 Hz, 2H, 4-
3,5
13
carboxyphenyl-C -H), 9.09 (s, 1H, triazole-C -H), 9.85 (s, 1H, CHO).
C
2,6
5
NMR (100 MHz, DMSO-d ): δ 191.9, 153.2, 149.8, 143.9, 139.8, 131.6,
6
1
30.6, 126.3, 126.0, 124.0, 120.4, 113.2, 110.3, 62.0, 56.0. Anal. Calcd (%)
for C H N O (353.33): C, 61.19; H, 4.28; N, 11.89. Found: C, 61.42; H,
18
15
3
5
4
.35; N, 12.06.
4
.1.1.7. 4-Methoxy-3-((1-(2-oxo-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methoxy)
benzaldehyde (6a).
-1
Yellow powder, yield 90.4%. m.p.165~166 °C. IR (KBr, cm ): 1038.22,
1
1
274.36 (C-O-C), 1585.18 (C=C), 1604.22 (C=N), 1681.36 (C=O, ketonic),
1
705.44 (C=O, aldehydic). H NMR (400 MHz, DMSO-d ): δ 3.88 (s, 3H,
6
OCH ), 5.29 (s, 2H, CH ), 6.24 (s, 2H, OCH ), 7.20 (d, J = 8.4 Hz, 1H,
3
2
2
benzaldehyde-C -H), 7.60-7.64 (m, 3H, benzaldehyde-C -H and phenyl-C -
5
6
3,5
H), 7.67 (s, 1H, benzaldehyde-C -H), 7.73-7.76 (m, 1H, phenyl-C -H), 8.10
2
4
(
d, J = 7.6 Hz, 2H, phenyl-C -H), 8.25 (s, 1H, triazole-C -H), 9.88 (s, 1H,
2,6 5
13
CHO). C NMR (100 MHz, DMSO-d ): δ 192.6, 191.8, 154.9, 148.4, 142.8,
6
1
5
34.6, 134.4, 130.1, 129.5, 129.3, 128.8, 127.1, 126.7, 112.1, 64.7, 62.1,
6.3. Anal. Calcd (%) for C H N O (351.36): C, 64.95; H, 4.88; N, 11.96.
19
17
3
4
Found: C, 65.23; H, 5.05; N, 12.14.
4
4
.1.1.8. 3-((1-(2-(4-Chlorophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-
-methoxybenzaldehyde (6b).
-1
Yellow powder, yield 92.5%. m.p.168~169 °C. IR (KBr, cm ): 1050.68,
1
1
271.90 (C-O-C), 1585.26 (C=C), 1603.68 (C=N), 1682.29 (C=O, ketonic),
1
708.54 (C=O, aldehydic). H NMR (400 MHz, DMSO-d ): δ 3.88 (s, 3H,
6
OCH ), 5.28 (s, 2H, CH ), 6.22 (s, 2H, OCH ), 7.21 (d, J = 8.4 Hz, 1H,
3
2
2

ACCEPTED MANUSCRIPT
benzaldehyde-C -H), 7.61 (d, J = 8.4 Hz, 1H, benzaldehyde-C -H), 7.65 (s,
5
6
1
H, benzaldehyde-C -H), 7.7 (d, J = 8.4 Hz, 2H, 4-chlorophenyl-C -H), 8.09
2 3,5
(
d, J = 8.4 Hz, 2H, 4-chlorophenyl-C -H), 8.21 (s, 1H, triazole-C -H), 9.87 (s,
2,6 5
1
3
1
1
H, CHO). C NMR (100 MHz, DMSO-d ): δ 191.8, 154.9, 148.3, 139.6,
6
33.3, 130.6, 130.1, 129.6, 127.0, 126.7, 112.1, 112.0, 62.1, 56.4, 49.1.
Anal. Calcd (%) for C H ClN O (385.80): C, 59.15; H, 4.18; N, 10.89.
19
16
3
4
Found: C, 59.41; H, 3.89; N, 11.12.
4
4
.1.1.9. 3-((1-(2-(4-Bromophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-
-methoxybenzaldehyde (6c).
-1
Brown powder, yield 88.7%. m.p.174~175 °C. IR (KBr, cm ): 1060.50,
1
1
272.45 (C-O-C), 1585.73 (C=C), 1602.88 (C=N), 1679.25 (C=O, ketonic),
1
707.16 (C=O, aldehydic). H NMR (400 MHz, DMSO-d ): δ 3.88 (s, 3H,
6
OCH ), 5.27 (s, 2H, CH ), 6.21 (s, 2H, CH ), 7.21 (d, J = 8 Hz, 1H,
3
2
2
benzaldehyde-C -H), 7.6 (d, J = 8.4 Hz, 1H, benzaldehyde-C -H), 7.65 (s,
5
6
1
8
9
1
5
H, benzaldehyde-C -H), 7.84 (d, J = 8.4 Hz, 2H, 4-bromophenyl-C -H),
2 3,5
.01 (d, J = 8.4 Hz, 2H, 4-bromophenyl-C -H), 8.21 (s, 1H, triazole-C -H),
2,6
5
13
.87 (s, 1H, CHO). C NMR (100 MHz, DMSO-d ): δ 192, 191.8, 154.9,
6
48.3, 133.6, 132.5, 130.6, 130.1, 128.6, 127.0, 126.7, 122.1, 122.0, 62.1,
6.4. Anal. Calcd (%) for C H BrN O (430.26): C, 53.04; H, 3.75; N, 9.77.
19
16
3
4
Found: C, 52.87; H, 3.98; N, 3.98.
4
.1.1.10. 3-((1-(2-(4-Fluorophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-
yl)methoxy)-4-methoxybenzaldehyde (6d).
-1
Yellow powder, yield 96.2%. m.p.168~169 °C. IR (KBr, cm ): 1056.69,
1
1
266.60 (C-O-C), 1598.81 (C=C), 1604.74 (C=N), 1688.41 (C=O, ketonic),
1
705.24 (C=O, aldehydic). H NMR (400 MHz, DMSO-d ): δ 3.88 (s, 3H,
6
OCH ), 5.28 (s, 2H, CH ), 6.22 (s, 2H, OCH ), 7.21 (d, J = 8 Hz, 1H,
3
2
2
benzaldehyde-C -H), 7.44 – 7.48 (m, 2H, fluorophenyl-C -H), 7.61 (d, J =
5
3,5
8
8
.4 Hz, 1H, benzaldehyde-C -H), 7.65 (s, 1H, benzaldehyde-C -H), 8.16 –
6 2
.19 (m, 2H, fluorophenyl-C -H), 8.21 (s, 1H, triazole-C -H), 9.87 (s, 1H,
2,6
5
13
CHO). C NMR (100 MHz, DMSO-d ): δ 191.8, 191.3, 167.3, 164.8, 154.9,
6
1
48.3, 131.8, 131.4, 130.1, 127.0, 126.7, 116.7, 116.4, 112.1, 122.0, 62.1,

ACCEPTED MANUSCRIPT
5
1
6.4, 56.3. Anal. Calcd (%) for C H FN O (369.35): C, 61.79; H, 4.37; N,
19
16
3
4
1.38. Found: C, 62.06; H, 4.64; N, 11.60.
4
.1.1.11. 4-Methoxy-3-((1-(2-oxo-2-(p-tolyl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy) benzaldehyde (6e).
-1
Beige powder, yield 92.8%. m.p.162~163 °C. IR (KBr, cm ): 1052.80,
1
1
273.28 (C-O-C), 1584.29 (C=C), 1601.63 (C=N), 1682.35 (C=O, ketonic),
1
702.41 (C=O, aldehydic). H NMR (400 MHz, DMSO-d ): δ 2.43 (s, 3H, p-
6
tolyl-CH ) 3.88 (s, 3H, OCH ), 5.27 (s, 2H, CH ), 6.19 (s, 2H, OCH ), 7.21 (d,
3
3
2
2
J = 8 Hz, 1H, benzaldehyde-C -H), 7.43 (d, J = 8 Hz, 2H, p-tolyl-C -H), 7.61
5
3,5
(d, J = 8 Hz, 1H, benzaldehyde-C -H), 7.66 (s, 1H, benzaldehyde-C -H), 7.99
6 2
(
d, J = 7.6 Hz, 2H, p-tolyl-C -H), 8.22 (s, 1H, triazole-C -H), 9.87 (s, 1H,
2,6
5
13
CHO). C NMR (100 MHz, DMSO-d ): δ 192.1, 191.8, 154.9, 148.3, 145.3,
6
1
6
5
42.6, 132.1, 130.1, 130.0, 129.6, 129.2, 128.8, 127.0, 126.7, 112.1, 122.0,
2.1, 56.4, 56.2, 21.8. Anal. Calcd (%) for C H N O (365.39): C, 65.74; H,
20
19
3
4
.24; N, 11.50. Found: C, 56.39; H, 5.32; N, 11.82.
4
.1.1.12. 4-(4-((5-Formyl-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)benzene- sulfonamide (6f).
-1
Light brown powder, yield 93.4%. m.p.195~196 °C. IR (KBr, cm ): 1061.12,
1
270.70 (C-O-C), 1347.34 (SO2), 1596.28 (C=C), 1659.36 (C=N), 1701.05
1
(
C=O, aldehydic), 3083.08 (NH). H NMR (400 MHz, DMSO-d ): δ 3.87 (s,
6
3
7
H, OCH ), 5.33 (s, 2H, OCH ), 7.22 (d, J = 8.4 Hz, 1H, benzaldehyde-C -H),
3 2 5
.57 (s, 2H, SO NH , D O exchangeable), 7.62 (d, J = 8.4 Hz, 1H,
2
2
2
benzaldehyde-C -H), 7.67 (s, 1H, benzaldehyde-C -H), 8.05 (d, J = 8.4 Hz,
6
2
2
H, 4-benzenesulfonamide-C -H), 8.16 (d, J = 8.4 Hz, 2H, 4-
3,5
benzenesulfonamide-C -H), 9.08 (s, 1H, triazole-C -H), 9.87 (s, 1H, CHO).
2,6
5
13
C NMR (100 MHz, DMSO-d ): δ 191.9, 154.9, 148.2, 144.4, 144.3, 139,
6
1
30.1, 128.1, 128.0, 126.9, 123.9, 120.9, 112.7, 62.0, 56.4. Anal. Calcd (%)
for C H N O S (388.40): C, 52.57; H, 4.15; N, 14.43; S, 8.25. Found: C,
17
16
4
5
5
2.86; H, 4.38; N, 14.15; S, 8.41.

ACCEPTED MANUSCRIPT
4
.1.1.13. 4-(4-((5-Formyl-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)benzoic acid (6g).
-1
Light brown powder, yield 94.2%. m.p.265~266 °C. IR (KBr, cm ): 1046.69,
1
1
273.40 (C-O-C), 1585.80 (C=C), 1608.66 (C=N), 1699.38 (C=O, aldehydic),
1
714.76 (C=O, acidic), 2500-3200 (OH, broad band). H NMR (400 MHz,
DMSO-d ): δ 3.78 (s, 3H, OCH ), 5.25 (s, 2H, OCH ), 7.12 (d, J = 8.4 Hz, 1H,
6
3
2
benzaldehyde-C -H), 7.55 (d, J = 8.4 Hz, 1H, benzaldehyde-C -H), 7.58 (s,
5
6
1
8
9
1
5
H, benzaldehyde-C -H), 7.96 (d, J = 8.4 Hz, 2H, 4-carboxyphenyl-C -H),
2 3,5
.08 (d, J = 8.4 Hz, 2H, 4-carboxyphenyl-C -H), 8.92 (s, 1H, triazole-C -H),
2
,6
5
13
.77 (s, 1H, CHO). C NMR (100 MHz, DMSO-d ): δ 191.8, 166.9, 154.9,
6
48.2, 144.3, 139.8, 131.6, 130.1, 126.9, 123.8, 120.4, 112.2, 112.1, 62.0,
6.4. Anal. Calcd (%) for C H N O (353.33): C, 61.19; H, 4.28; N, 11.89.
18
15
3
5
Found: C, 61.40; H, 4.39; N, 12.23.
4
.1.1.14. 2-(4-((4-Acetylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-1-(4-
bromophenyl)ethan-1-one (9a).
-1
Light orange powder, yield 85.9%. m.p.190~191 °C. IR (KBr, cm ): 1048.04,
1
252.57 (C-O-C), 1585.73 (C=C), 1603.56 (C=N), 1692.38 (C=O, ketonic).
1
H NMR (400 MHz, DMSO-d ): δ 2.54 (s, 3H, CH ), 5.34 (s, 2H, CH ), 6.23
6
3
2
13
(
s, 2H, OCH ), 7.20-8.01 (m, 8H, Aromatic-H), 8.22 (s, 1H, triazole-C -H). C
2
5
NMR (100 MHz, DMSO-d ): δ 196.9, 192.0, 162.4, 133.6, 132.5, 130.9,
6
1
30.6, 128.9, 126.9, 115.1, 61.9, 56.4, 26.9. Anal. Calcd (%) for
C H BrN O (414.26): C, 55.09; H, 3.89; N, 10.14. Found: C, 55.37; H,
19
16
3
3
4
.11; N, 10.47.
4
.1.1.15. 4-(4-((4-Acetylphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)benzenesulfonamide (9b).
-1
Beige powder, yield 87.3%. m.p.219~220 °C. IR (KBr, cm ): 1046.95,
1
257.58 (C-O-C), 1362.63 (SO2), 1579.68 (C=C), 1601.61 (C=N), 1689.33
1
(
C=O, ketonic), 3236.15 (NH). H NMR (400 MHz, DMSO-d ): δ 2.53 (s, 3H,
6
CH ), 5.38 (s, 2H, OCH ), 7.20 (d, J = 8.4 Hz, 2H, acetophenone-C -H),
3
2
3,5
7
.56 (s, 2H, SO NH , D O exchangeable), 7.96 (d, J = 8.4 Hz, 2H, 4-
2 2 2
benzenesulfonamide-C -H), 8.05 (d, J = 8.4 Hz, 2H, 4-benzenesulfonamide-
3,5
C -H), 8.15 (d, J = 8.4 Hz, 2H, acetophenone-C -H), 9.08 (s, 1H, triazole-
2,6
2,6

ACCEPTED MANUSCRIPT
13
C -H). C NMR (100 MHz, DMSO-d ): δ 196.8, 162.2, 144.4, 144.2, 134.0,
5
6
1
31.0, 130.8, 128, 123.8, 120.9, 120, 115.1, 61.7, 26.9. Anal. Calcd (%) for
C H N O S (372.40): C, 54.83; H, 4.33; N, 15.05; S, 8.61. Found: C, 54.51;
17
16
4
4
H, 4.59; N, 15.21; S,.78.
4
.1.1.16. 4-(4-((4-Acetylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid
(9c).
-1
White powder, yield 86.1%. m.p.269~270 °C. IR (KBr, cm ): 1048.91,
1
1
275.82 (C-O-C), 1659.85 (C=C), 1605.90 (C=N), 1689.18 (C=O, ketonic),
1
714.31 (C=O, acidic), 2800-3200 (OH, broad band). H NMR (400 MHz,
DMSO-d ): δ 2.53 (s, 3H, CH ), 5.38 (s, 2H, OCH ), 7.20 (d, J = 8.4 Hz, 2H,
6
3
2
acetophenone-C -H), 7.96 (d, J = 8.4 Hz, 2H, acetophenone-C -H), 8.1 (d,
3,5
2,6
J = 8 Hz, 2H, 4-carboxyphenyl-C -H), 8.14 (d, J = 8 Hz, 2H, 4-
3,5
13
carboxyphenyl-C -H), 9.1 (s, 1H, triazole-C -H), 13.26 (br. s, 1H, OH).
C
2,6
5
NMR (100 MHz, DMSO-d ): δ 196.8, 162.2, 144.2, 139.9, 131.0, 130.8,
6
1
6
23.7, 120.5, 115.1, 61.7, 26.9. Anal. Calcd (%) for C H N O (337.34): C,
18 15 3 4
4.09; H, 4.48; N, 12.46. Found: C, 64.35; H, 4.32; N, 12.68.
4
7
.1.2. General procedure for synthesis of the thiosemicarbazones 2, 4a-f,
a-g and 10a-c:
A mixture of the appropriate aldehyde/ketone (1, 3a-f, 6a-g, or 9a-c) (1
mmole) and thiosemicarbazide (3 mmole) in absolute ethanol and/or glacial
acetic acid was refluxed for 12-16 hours. After cooling to room temperature,
the mixture was poured on ice, filtered, washed with cold water and dried. The
solid was crystallized from ethanol or ethanol/DMF to obtain the appropriate
solid products.
4
.1.2.1. 2-(3-Methoxy-4-(prop-2-yn-1-yloxy)benzylidene)hydrazine-1-
carbothioamide (2).
-1
Yellow powder, yield 43.6%. m.p.199~200 °C. IR (KBr, cm ): 1090.96,
1
261.53 (C-O-C), 1137.83 (C=S), 1530.76 (C=C), 1599.57 (C=N), 1686, 79
1
(
C=O), 2279.92 (C≡C), 3245.46 (CH, acetylenic), 3392.53 (NH). H NMR
400 MHz, DMSO-d ): δ 3.5 (s, 3H, OCH ), 3.58 (t, J= 2 Hz, 1H, CH) 4.83 (d,
(
6
3
J= 2 Hz, 2H, CH ), 7.03 (d, J = 8.4 Hz, 1H, phenyl-C -H), 7.17 (d, J = 8.4 Hz,
2
6

ACCEPTED MANUSCRIPT
1
H, phenyl-C -H), 7.54 (s, 1H, phenyl-C -H), 7.98 (s, 1H, benzylidene-
5
2
CH=N), 8.04 (s, 1H, NH, D O exchangeable), 8.18 (s, 1H, NH, D O
2
2
13
exchangeable), 11.35 (s, 1H, NH, D O exchangeable). C NMR (100 MHz,
2
DMSO-d ): δ 178.1, 149.9, 148.7, 142.7, 128.4, 122.2, 113.8, 109.4, 79.5,
6
7
4
9.0, 56.4, 56.2. Anal. Calcd (%) for C H N O S (263.32): C, 54.74; H,
12 13 3 2
.98; N, 15.96; S, 12.18. Found: C, 55.01; H, 5.13; N, 16.24; S, 12.30.
4
.1.2.2. 2-(3-Methoxy-4-((1-(2-oxo-2-phenylethyl)-1H-1,2,3-triazol-4-
yl)methoxy) benzylidene)hydrazine-1-carbothioamide (4a).
-1
Beige powder, yield 47.6%. m.p.201~202 °C. IR (KBr, cm ): 1052.78,
1
270.85 (C-O-C), 1114.84 (C=S), 1580.75 (C=C), 1612.45 (C=N), 3144.66
1
(
NH). H NMR (400 MHz, DMSO-d ): δ 3.83 (s, 3H, OCH ), 5.24 (s, 2H,
6
3
CH ), 6.23 (s, 2H, OCH ), 7.2 (m, 3H, benzylidene-C2,5,6-H), 7.56 – 7.75 (m,
2
2
3
H, phenyl-C3, 4, 5--H), 8.01 (s, 1H, benzylidene-CH=N), 8.09 (m, 2H, phenyl-
C -H), 8.18 (br. s, 2H, NH , D O exchangeable), 8.22 (s, 1H, triazole-C -H),
2,6
2
2
5
1
3
1
1
1
1.41 (s, 1H, NH, D O exchangeable). C NMR (100 MHz, DMSO-d ): δ
2 6
92.6, 149.8, 149.8, 143.4, 142.4, 134.7, 134.6, 129.5, 128.7, 127.6, 127.0,
22.5, 113.3, 109.3, 62.1, 56.4, 56.1. Anal. Calcd (%) for C H N O S
20
20
6
3
(424.48): C, 56.59; H, 4.75; N, 19.80; S, 7.55. Found: C, 56.47; H, 4.89; N,
2
0.06; S, 7.64.
4
.1.2.3. 2-(4-((1-(2-(4-Chlorophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxybenzylidene)hydrazine-1-carbothioamide (4b).
-1
Light brown powder, yield 40%. m.p.191~192 °C. IR (KBr, cm ): 1058.68,
1
268.90 (C-O-C), 1138.67 (C=S), 1558.65 (C=C), 1590.98 (C=N), 1691.80
1
(
C=O), 3272.00 (NH). H NMR (400 MHz, DMSO-d ): δ 3.83 (s, 3H, OCH ),
6
3
5
.23 (s, 2H, CH ), 6.22 (s, 2H, OCH ), 7.16–7.22 (m, 2H, benzylidene-C -
2 2 5,6
H), 7.55 (s, 1H, benzylidene-C -H), 7.69 (d, J = 8.4 Hz, 2H, 4-chlorophenyl-
2
C -H), 8.01 (s, 1H, benzylidene-CH=N), 8.09 (d, J = 8.4 Hz, 2H, 4-
3,5
chlorophenyl-C -H), 8.21 (s, 1H, triazole-C -H), 8.25 (s, 2H, NH , D O
2,6
5
2
2
13
exchangeable), 11.38 (s, 1H, NH, D O exchangeable). C NMR (100 MHz,
2
DMSO-d ): δ 191.8, 149.8, 143.4, 142.9, 139.6, 133.3, 130.6, 129.6, 127.8,
6
1
27.0, 122.5, 113.3, 109.3, 62.0, 56.4, 56.1. Anal. Calcd (%) for

ACCEPTED MANUSCRIPT
C H ClN O S (458.92): C, 52.34; H, 4.17; N, 18.31; S, 6.99. Found: C,
20
19
6
3
5
2.48; H, 4.30; N, 18.58; S, 7.13.
4
.1.2.4. 2-(4-((1-(2-(4-Bromophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxybenzylidene)hydrazine-1-carbothioamide (4c).
-1
Light brown powder, yield 45.4%. m.p.177~178 °C. IR (KBr, cm ): 1061.67,
1
266.51 (C-O-C), 1137.21 (C=S), 1585.69 (C=C), 1620.58 (C=N), 1706.10
1
(
C=O), 3152.77 (NH). H NMR (400 MHz, DMSO-d ): δ 3.83 (s, 3H, OCH ),
6
3
5
.23 (s, 2H, CH ), 6.21 (s, 2H, OCH ), 7.2 (s, 2H, benzylidene-C -H), 7.55
2 2 5,6
(
s, 1H, benzylidene-C -H), 7.84 (d, J = 8 Hz, 2H, 4-bromophenyl-C -H),
2 3,5
8
2
.00-8.02 (m, 3H, benzylidene-CH=N and 4-bromophenyl-C -H), 8.21 (s,
2,6
13
H, triazole-C -H), 11.43 (br. s, 1H, NH, D O exchangeable). C NMR (100
5
2
MHz, DMSO-d ): δ 192.0, 149.9, 149.8, 143.9, 133.6, 132.5, 131.6, 130.6,
6
1
28.9, 127.7, 122.6, 113.3, 109.3, 62.0, 56.3, 56.1. Anal. Calcd (%) for
C H BrN O S (503.38): C, 47.72; H, 3.80; N, 16.70; S, 6.37. Found: C,
20
19
6
3
4
7.91; H, 4.06; N, 16.56; S, 6.45.
4
.1.2.5. 2-(4-((1-(2-(4-Fluorophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxybenzylidene)hydrazine-1-carbothioamide (4d).
-1
Light brown powder, yield 36.9%. m.p.194~195 °C. IR (KBr, cm ): 1048.34,
1
230.63 (C-O-C), 1159.86 (C=S), 1561.42 (C=C), 1599.21 (C=N), 1698.37
1
(
C=O), 3151.75 (NH). H NMR (400 MHz, DMSO-d ): δ 3.83 (s, 3H, OCH ),
6
3
5
.23 (s, 2H, CH ), 6.22 (s, 2H, OCH ), 7.16-7.47 (m, 3H, benzylidene-C2,5,6-
2 2
H), 7.93 (s, 1H, benzylidene-CH=N), 7.99 (d, J = 8 Hz, 2H, 4-bromophenyl-
C -H), 8.80 (d, J = 8 Hz, 2H, 4-bromophenyl-C -H), 8.21 (s, 2H, triazole-
3,5
2,6
13
C -H), 11.35 (s, 1H, NH, D O exchangeable). C NMR (100 MHz, DMSO-
5
2
d6): δ 191.3, 177.9, 167.3, 164.8, 149.9, 149.7, 143.1, 142.9, 131.8, 131.7,
31.3, 127.0, 116.7, 116.5, 62.1, 56.3, 56.1. Anal. Calcd (%) for
C H FN O S (442.47): C, 54.29; H, 4.33; N, 18.99; S, 7.25. Found: C,
1
20
19
6
3
5
4.43; H, 4.41; N, 19.23; S, 7.38.
4
.1.2.6. 2-(3-Methoxy-4-((1-(2-oxo-2-(p-tolyl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy) benzylidene)hydrazine-1-carbothioamide (4e).

ACCEPTED MANUSCRIPT
-1
Yellow powder, yield 40.8%. m.p.211-212 °C. IR (KBr, cm ): 1029.77,
1
269.86 (C-O-C), 1127.61 (C=S), 1552.62 (C=C), 1601.39 (C=N), 1702.19
1
(
C=O), 3144.83 (NH). H NMR (400 MHz, DMSO-d ): δ 2.51 (s, 3H, p-tolyl-
6
CH ), 3.83 (s, 3H, OCH ), 5.23 (s, 2H, CH ), 6.18 (s, 2H, OCH ), 7.21 (m, 2H,
3
3
2
2
benzylidene-C -H), 7.43 (d, J = 7.6 Hz, 2H, p-tolyl-C -H), 7.55 (s, 1H,
5,6
3,5
benzylidene-C -H), 7.99 (d, J = 7.6 Hz, 2H, p-tolyl-C -H), 8.02 (s, 1H,
2
2,6
benzylidene-CH=N), 8.21 (s, 1H, triazole-C -H), 8.23 (s, 2H, NH , D O
5
2
2
13
exchangeable) 11.42 (s, 1H, NH, D O exchangeable). C NMR (100 MHz,
2
DMSO-d ): δ 192.1, 177.9, 149.9, 149.8, 145.3, 132.1, 130.0, 128.8, 122.6,
6
1
13.3, 109.3, 62.1, 56.2, 56.1, 21.8. Anal. Calcd (%) for C H N O S
21 22 6 3
(438.51): C, 57.52; H, 5.06; N, 19.17; S, 7.31. Found: C, 57.30; H, 5.23; N,
1
9.45; S, 7.36.
4
.1.2.7. 4-(4-((4-((2-Carbamothioylhydrazono)methyl)-2-
methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid (4f).
-1
Yellow powder, yield 48.8%. m.p.251~252 °C. IR (KBr, cm ): 1070.62,
1
270.94 (C-O-C), 1141.91 (C=S), 1543.92 (C=C), 1593.19 (C=N), 1704.99
1
(
C=O), 3315.43 (NH), 3000-3500 (OH, broad band). H NMR (400 MHz,
DMSO-d ): δ 3.83 (s, 3H, OCH ), 5.3 (s, 2H, CH ), 7.18 (d, J = 8 Hz, 1H,
6
3
2
benzylidene-C -H), 7.22 (d, J = 8.4 Hz, 1H, benzylidene-C -H), 7.55 (s, 1H,
5
6
benzylidene-C -H), 8.00 (s, 1H, benzylidene-CH=N), 8.04 (s, 2H, NH , D O
2
2
2
exchangeable), 8.08 (d, J = 8.4 Hz, 2H, 4-carboxyphenyl-C -H), 8.15 (d, J =
3,5
8
1
.4 Hz, 2H, 4-carboxyphenyl-C -H), 9.06 (s, 1H, triazole-C -H), 11.34 (s,
2,6 5
13
H, NH, D O exchangeable), 13.21 (br. s, 1H, OH). C NMR (100 MHz,
2
DMSO-d ): δ 178.0, 166.9, 149.9, 149.6, 144.4, 143.0, 139.9, 131.6, 131.2,
6
1
28.1, 123.7, 122.4, 120.4, 113.6, 109.4, 62.0, 56.2. Anal. Calcd (%) for
C H N O S (426.45): C, 53.51; H, 4.25; N, 19.71; S, 7.52. Found: C, 53.77;
19
18
6
4
H, 4.38; N, 19.89; S, 7.64.

ACCEPTED MANUSCRIPT
4
.1.2.8. 2-(4-Methoxy-3-((1-(2-oxo-2-phenylethyl)-1H-1,2,3-triazol-4-
yl)methoxy) benzylidene)hydrazine-1-carbothioamide (7a).
-1
Beige powder, yield 42.6%. m.p.202~203 °C. IR (KBr, cm ): 1014.18,
1
265.17 (C-O-C), 1133.49 (C=S), 1541.85 (C=C), 1597.68 (C=N), 1694.32
1
(
C=O), 3270.83 (NH). H NMR (400 MHz, DMSO-d ): δ 3.8 (s, 3H, OCH ),
6
3
5
.25 (s, 2H, CH ), 6.22 (s, 2H, OCH ), 7.00 (d, J = 8 Hz, 1H, benzylidene-C -
2 2 5
H), 7.19 (d, J = 7.6 Hz, 1H, benzylidene-C -H), 7.36 (d, J = 5.2 Hz, 1H,
6
benzylidene-C -H), 7.61 (d, J = 7.6 Hz, 1H, phenyl-C -H), 7.76 (d, J = 7.6
2
4-
Hz, 2H, phenyl-C -H), 8.09 (d, J = 7.6 Hz, 2H, phenyl-C -H), 8.01 (s, 1H,
3,5
2,6
benzylidene-CH=N), 8.18 (br. s, 2H, NH , D O exchangeable), 8.22 (s, 1H,
2
2
13
triazole-C -H), 11.37 (s, 1H, NH, D O exchangeable). C NMR (100 MHz,
5
2
DMSO-d ): δ 192.7, 177.9, 151.2, 148.4, 148.3, 143.0, 134.6, 129.5, 127.4,
6
1
27.0, 125.7, 123.1, 112.0, 110.3, 62.2, 56.4, 56.0. Anal. Calcd (%) for
C H N O S (424.48): C, 56.59; H, 4.75; N, 19.80; S, 7.55. Found: C, 56.80;
20
20
6
3
H, 4.89; N, 20.12; S, 7.64.
4
.1.2.9. 2-(3-((1-(2-(4-Chlorophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-
yl)methoxy)-4-methoxybenzylidene)hydrazine-1-carbothioamide (7b).
-1
Light brown powder, yield 43.7%. m.p.255~256 °C. IR (KBr, cm ): 1066.71,
1
271.90 (C-O-C), 1131.80 (C=S), 1540.53 (C=C), 1587.65 (C=N), 1685.90
1
(
C=O), 3221.06 (NH). H NMR (400 MHz, DMSO-d ): δ 3.84 (s, 3H, OCH ),
6
3
5
.25 (s, 2H, CH ), 6.23 (s, 2H, OCH ), 7.00 (d, J = 8 Hz, 1H, benzylidene-C -
2 2 5
H), 7.19 (d, J = 8 Hz, 1H, benzylidene-C -H), 7.70 (d, J = 8.4 Hz, 2H, 4-
6
chlorophenyl-C -H), 7.77 (s, 1H, benzylidene-C -H), 8.01 (s, 1H,
3,5
2
benzylidene-CH=N), 8.10 (d, J = 8.4 Hz, 2H, 4-chlorophenyl-C -H), 8.21 (s,
2,6
13
1
H, triazole-C -H), 11.38 (s, 1H, NH, D O exchangeable). C NMR (100
5
2
MHz, DMSO-d ): δ 191.8, 151.4, 148.4, 143.1, 139.6, 133.3, 133.6, 129.6,
6
1
27.3, 126.9, 123.1, 112.0, 110.3, 62.2, 56.4, 56.0. Anal. Calcd (%) for
C H ClN O S (458.92): C, 52.34; H, 4.17; N, 18.31; S, 6.99. Found: C,
20
19
6
3
5
2.08; H, 4.30; N, 18.58; S, 6.78.
4
.1.2.10. 2-(3-((1-(2-(4-Bromophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-
yl)methoxy)-4-methoxybenzylidene)hydrazine-1-carbothioamide (7c).