Design, synthesis and antitrypanosomal activity of heteroaryl-based 1,2,4-triazole and 1,3,4-oxadiazole derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.103933

Two series of novel 1,2,4-triazol-3-yl-thioacetamide 3a-b and 4a-b and 5-pyrazin-2-yl-3H-[1,3,4]oxadiazole-2-thiones 9a-h were designed and synthesized. The compounds prepared have been identified using ¹H NMR, ¹³C NMR and elemental analyses. The synthesized compounds 3a, 3b, 4a, 4b, 9a, 9b, 9d-e and 9f have been evaluated with α-difluoromethylornithine (DFMO) as a control drug for their in vitro antitrypanosomal activity against Trypanosoma brucei. Results showed that 3b was the most active compound in general and also more potent than control DFMO. 3b was 8 folds more potent than the reference with IC₅₀ of 0.79 μM and IC₉₀ of 1.35 μM, respectively compared to DFMO (IC₅₀ = 6.10 μM and IC₉₀ of 8.66 μM). The tested compounds showed moderate cytotoxicity with selectivity indices ranging from 12 (9d) to 102 (3b) against L6 cells. Docking study was performed into ten of T. brucei enzymes which have been identified as potential/valid targets for most of the antitrypanosomal agents. The results of the docking study revealed high binding scores toward many of the selected enzymes. A good correlation was observed only between log (IC₅₀) of antitrypanosomal activity of the new compounds and their calculated Ki values against TryR enzyme (R² = 0.726). Compound 3b, the most active as antitrypanosomal agents exhibited similar binding orientation and interaction to those of WP6 against TryR enzyme. However, in a next round of work, a complementary studies will be carried out to clarify the mechanism of action of these compounds.

Full text of DOI:10.1016/j.bioorg.2020.103933

Journal Pre-proofs
Design, synthesis and antitrypanosomal activity of heteroaryl-based 1,2,4-tria-
zole and 1,3,4-oxadiazole derivatives
Montaser Sh. Shaykoon, Adel A. Marzouk, Osama M. Soltan, Amira S.
Wanas, Mohamed M. Radwan, Ahmed M. Gouda, Bahaa G. M. Youssif,
Mohamed Abdel-Aziz
PII:
S0045-2068(20)30376-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.103933
YBIOO 103933
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
16 February 2020
10 April 2020
11 May 2020
Please cite this article as: M. Sh. Shaykoon, A.A. Marzouk, O.M. Soltan, A.S. Wanas, M.M. Radwan, A.M.
Gouda, B. G. M. Youssif, M. Abdel-Aziz, Design, synthesis and antitrypanosomal activity of heteroaryl-based
1
,2,4-triazole and 1,3,4-oxadiazole derivatives, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/
j.bioorg.2020.103933
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier Inc.

Design, synthesis and antitrypanosomal activity of heteroaryl-based 1,2,4-
triazole and 1,3,4-oxadiazole derivatives
Montaser Sh. Shaykoon , Adel A. Marzouk , Osama M. Soltan , Amira S. Wanas b,c,
a
a
a
b
d
e
f
Mohamed M. Radwan , Ahmed M. Gouda , Bahaa G. M. Youssif , Mohamed Abdel-Aziz
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Assiut
b
Branch, Assiut-71524, Egypt; National Center for Natural Products Research, University of
Mississippi, MS 38677, USA; cPharmacognosy Department, Faculty of Pharmacy, Minia
d
University, 61519-Minia, Egypt; Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-
e
Suef University, Beni-Suef 62514, Egypt; Pharmaceutical Organic Chemistry Department, Faculty
f
of Pharmacy, Assiut University, Assiut 71526, Egypt. Department of Medicinal Chemistry, Faculty
of Pharmacy, Minia University, 61519-Minia, Egypt.
Short running title: Antitrypanosomal activity of new 1,2,4-triazole and 1,3,4-oxadiazole
derivatives
*
To whom correspondence should be addressed:
Mohamed Abdel-Aziz, Ph.D. Department of Medicinal Chemistry, Faculty of Pharmacy,
Minia University, 61519-Minia, Egypt.
Tel.:(002)-01003311327
E-mail address: Abulnil@hotmail.com
Bahaa G. M. Youssif. Ph.D. Pharmaceutical Organic Chemistry Department, Faculty of
Pharmacy, Assiut University, Assiut 71526, Egypt.
Tel.: (002)-01098294419
E-mail address: bgyoussif@ju.edu.sa, bahaa.youssif@pharm.aun.edu.eg

Abstract
Two series of novel 1,2,4-triazol-3-yl-thioacetamide 3a-b and 4a-b and 5-pyrazin-2-yl-3H-
[1,3,4]oxadiazole-2-thiones 9a-h were designed and synthesized. The compounds prepared
have been identified using H NMR, 13C NMR and elemental analyses. The synthesized
compounds 3a, 3b, 4a, 4b, 9a, 9b, 9d-e and 9f have been evaluated with α-
difluoromethylornithine (DFMO) as a control drug for their in vitro antitrypanosomal activity
against Trypanosoma brucei. Results showed that 3b was the most active compound in general
and also more potent than control DFMO. 3b was 8 folds more potent than the reference with
IC of 0.79 μM and IC of 1.35 μM, respectively compared to DFMO (IC = 6.10 μM and
1
5
0
90
50
IC of 8.66 μM). The tested compounds showed moderate cytotoxicity with selectivity indices
9
0
ranging from 12 (9d) to 102 (3b) against L6 cells. Docking study was performed into ten of T.
brucei enzymes which have been identified as potential/valid targets for most of the
antitrypanosomal agents. The results of the docking study revealed high binding scores toward
many of the selected enzymes. A good correlation was observed only between log (IC ) of
5
0
antitrypanosomal activity of the new compounds and their calculated Ki values against TryR
2
enzyme (R = 0.726). Compound 3b, the most active as antitrypanosomal agents exhibited
similar binding orientation and interaction to those of WP6 against TryR enzyme. However, in
a next round of work, a complementary studies will be carried out to clarify the mechanism of
action of these compounds.
Keywords: 1,2,4-triazole, 1,3,4-oxadiazole, Pyrazine, Antitrypanosomal, Docking.

Graphical Abstract

Highlights

Two series of heteroaryl-based 1,2,4-triazole and 1,3,4-oxadiazole derivatives were designed
and synthesized.


Compounds 3a-b and 4a-b showed higher antitrypanosomal activity than 9a-h.
Compound 3b was the most active compound and even more potent than control DFMO.

Docking study into ten of the target enzymes in T. brucei revealed a good correlation
between antitrypanosomal activity and Ki values against TryR.

The triaryl bearing triazoles 3 and 4 showed higher affinities toward TryR than the diaryl
bearing oxadiazole derivatives 9a-h.

1
. INTRODUCTION
Human African trypanosomiasis (HAT), also known as sleeping sickness, caused by
Trypanosoma brucei rhodesiense or T. b. gambiense is a major threat to communities in
Africa's sub-Saharan region [1,2]. The parasites attack the central nervous system after
infection and if left untreated, causing behavioral changes, coma (sleeping sickness) and
ultimately death. All the medications commonly used to treat HAT are ineffective with their
low efficacy and adverse side effects [3]. Therefore, there is an urgent need for better drugs to
treat HAT more effectively, which are in safe, low-cost and easy to use. 1,3,4,-oxadiazole has
become an important building motif for the development of new drugs among heterocyclic
compounds. Compounds with 1,3,4,-oxadiazole core have a wide range of biological activity
including antibacterial, antifungal, anti-inflammatory [4], analgesic, antiviral and anticancer
effects. The ability to undergo various chemical reactions of 1,3,4,-oxadiazole has made them
an essential center of medicinal chemistry [5]. Some derivatives of 1,3,4-oxadiazole-2-thione
and their Mannich bases have been reported to be anti-inflammatory [6], anti-tubercular [7],
antifungal [8] and anticancer [9,10]. Pyrazine and pyridine derivatives are well known
biologically active compounds, they have antimycobacterial [11], antimalarial [12, 13],
antileishmanial [14], antitrypanosomal [15, 16]. In addition, 1,2,4-triazole derivatives are an
important class of heterocyclic compounds and have various biological activities such as
antimicrobial [17, 18], anti-tubercular [19], anti-cancer [20-22] and antitrypanosomal [23]
activities. On the other hand, Nitric Oxide (NO) plays an important role in the treatment of
parasite infection, many theories have been made to explain the antiparasitic effect of NO,
including inhibition of the in vitro growth of many bacteria and parasites [24]. Other studies
have shown that murine macrophages can produce sufficient NO to kill leishmania parasite in
vitro [24]. In addition, a report stated that the endogenous and exogenous NO has anti-parasitic
effects on Protozoa (including Leishmania, Trypanosoma, Giardia, Trichomonas, Naegleria,

Entamoeba, Plasmodium, Toxoplasma, and Babesia) [25]. NO has been found to block the
Trypanosoma cruzi life cycle both in vitro and in vivo [25].
Rational Design of the new compounds
Large numbers of compounds have been synthesized and tested over the last two decades for
their effectiveness against different species of trypanosomes [26, 27]. However, few of these
agents are nitrogen/oxygen-based heterocyclic compounds, Figure 1. Among the heterocyclic-
base antitrypanosomal agents, the mesoionic 1,3,4-thiadiazolium-2-aminide I exhibited potent
inhibitory activity against trypanothione reductase (TryR) activity in Trypanosoma cruzi and
Leishmania infantum [28]. Compound II also displayed antitrypanosomal activity mediated by
inhibition of TryR (IC = 0.93 μM) [29].
5
0
N
H N
2
O
b
N
N
Cl
a
H
a
c
N
N
N
N
N
Cl
S
N
a
c
O
S
N
H
S
H
S
b
II
b
NO2
I
III
R2
c
N
N
H
X
N
NH
N
S
a
X
c
N
N
X
Y
O
N
S
b
O
CH3
a
Scaffold A
Scaffold B
N
H
O
N
OCH3
c
N
N
O
H
N
N
c
O N
N
2
O
N
N
O
O
H
c
N
H
O
O
OCH3
Nifuroxazide
OH
N
NH2
VI
IV
V
0
007
T. cruzi TR
Inhibition of TS2 reduction [85%]
Figure 1. Heterocycle-based antitrypanosomal agents and rational design of scaffolds A and B
Moreover, nifuroxazide VI reduced TryR activity by 85% (at 100 μM) and exhibited complete
inhibition in the growth of T. cruzi and T. brucei at 10 μM concentration [30]. On the other

hand, the antitrypanosomal activity of compounds III-V was mediated by inhibition of T. cruzi
cysteine protease cruzain enzyme [31-33].
The chemical structure of compounds I-VI have similar pharmacophoric groups which include
a heterocyclic nucleus attached directly or through a spacer to two or three
aromatic/heteroaromatic rings, Figure 1. In the current study, we have designed two new
scaffolds A and B based on the chemical structure of compounds I-VI to investigate their
antitrypanosomal activity. The study of structure activity relationship of scaffold A was
achieved by variation in the heterocyclic ring (a) (pyrazine and pyridine) and replacement of
the acetyl oxygen (Y) by hydroxyimino group. Moreover, the phenyl ring (c) in scaffold B was
substituted with both electron withdrawing (F) and donating (CH /OCH ) groups at ortho/para
3
3
positions to evaluate the impact of their electronic effects on the antitrypanosomal potency,
Figure 1.
2
2
. RESULTS AND DISCUSSION
.1. Chemistry
The target ketone intermediates 3a-b were prepared with N-(4-acetylphenyl)-2-
bromoacetamide 1 in absolute ethanol in the presence of K CO by refluxing 1,2,4-triazole-3-
2
3
thiol derivatives 2a-b. Reflux of compounds 3a-b with hydroxylamine hydrochloride in
ethanol gave the corresponding oximes 4a-b, Scheme 1. The IR spectra of compounds 3a-b
-1
-1
showed significant stretching bands at 3269-3246 cm related to (NH) and at 1596 cm related
-1
to (C=N). In addition, the (C=O) stretching band appears at 1674-1631 cm related to (CO-
1
NH and CO-CH ). H NMR spectra for 3a-b showed three singlet peaks at δ 10.70-10.75 ppm
3
related to (NH) proton, δ 4.30-4.36 ppm related to (S-CH -CO) and δ 2.11-2.50 ppm related to
2
(
CO-CH ). The 13C NMR spectra for compounds 3a-b showed characteristic ketonic
3

resonances at δ 196.58, 196.96 ppm and at δ 166.07, 166.48 ppm which related to carbonyl of
the amide linker.
IR spectra of 4a-b are characterized by the appearance of intense broad bands at 3500-3100
-1
-1
cm , related to NH and OH groups, stretching vibration at 1682-1657 cm , related to (CO-
NH). In addition, the disappearance of the ketonic (C=O) stretching vibrations. A characteristic
1
feature of the H NMR spectra for oximes 4a-b is the appearance of downfield singlets in the
range of δ11.10-11.11 ppm, related to the hydroxyl group. The resonances of (NH), (S-CH2)
and CH protons were observed in the expected regions at δ10.50-10.51 ppm, 4.26-4.28 ppm,
3
1
3
and 2.11-2.12 ppm, respectively. The C NMR spectra of compounds 4a-b showed one amide
carbonyl group at δ165-166 ppm and the disappearance of the ketonic carbonyl due to its
conversion to ketoxime group (C=N-OH). Moreover, the methyl group adjacent to ketoxime
appears at δ11.42 and 11.43 ppm, respectively which more upfield shifted by 15 ppm than the
corresponding ketones . The methylene group (SCH ) appears at δ 36.84 ppm.
2

O
O
O
CH3
CH3
(a)
Br
N
H N
2
H
p-aminoacetophenone
1
N
N
SH
(b)
N
Ar
2a, b
O
N
N
N
O
N
N
S
CH3
(c)
NH
COCH3
S
NH
Ar
N
Ar
NOH
3a, b
4a, b
N
N
1
-4 a, Ar =
; 1-4 b,Ar = N
Reagents and conditions: (a) bromoacetyl bromide, K CO , CH Cl ; (b) K CO , absolute
2
3
2
2
2
3
ethanol; (c) NH OH.HCl, EtOH, reflux.
2
Scheme 1: Synthesis of compounds 3a-b and 4a-b
A mixture of hydrazide 7, potassium hydroxide, carbon disulfide and ethanol was heated under
reflux to afford 8. Formalin 40% was added to a stirred solution of 5-(pyrazin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione 8 in absolute ethanol followed by addition of an ethanolic solution of the
appropriate amine. The formed precipitate was filtered off to give the newly synthesized Mannich
1
13
bases 9a-h. IR, H NMR, C NMR, electro spray ionization mass spectrometry (ESI-MS) and
elemental analysis have confirmed the structures of the prepared compounds. IR spectra of
-1
compounds 9a-h showed significant broad bands at 3500-3200 cm related to (NH) group, at 1661-
-1
-1
1
602 cm related to (C=N) and at 1441-1418 cm related to (C=S). In addition, compounds 9e-h
-1
1
showed significant stretching band at 1286- 1263 cm related to (O-C-H ). H NMR spectra of
3
compounds 9a-h showed two common peaks appeared at δ 6.05-5.58 ppm related to the protons of
the (CH ) bridge (duplet) and at δ 5.33-5.58 ppm related to (NH) proton (triplet). Moreover, the
2
two Mannich bases 9c and 9d showed a characteristic single peak at δ 2.31 ppm which related to

(
ph-CH ) protons. Furthermore, p-methoxy derivative 9f showed a singlet signal at δ 3.77 ppm
3
related to the methoxy group in addition to the doublet of doublet pattern in the aromatic proton’s
region at 6.99 ppm and at 7.51 ppm. Also, the dimethoxy derivative 9g showed two singlets related
to the two methoxy groups at δ 3.74-3.77 ppm. Moreover, the trimethoxy derivative 9h spectrum
1
3
displays two singlets for the three methoxy groups at δ 3.76-3.85 ppm. The C NMR spectra of
compounds 9a-h showed characteristic absorption at δ 58.35-58.90 ppm due to the CH carbon. In
2
addition, 13C NMR spectra of 9a-h showed characteristic aromatic carbons at their expected
absorption at δ 110.79-158.00 ppm. Furthermore, compounds 9e and 9f were characterized by
signals at δ 56.95 and 55.48 ppm related to O-CH On the other hand, compounds 9a-h showed a
3
.
characteristic signal at δ 175.00-176.44 ppm related to C=S. Finally, data from the elemental
analyses further confirmed the assigned structures.
O
O
N
N
COOH
N
N
N
(a)
O
(b)
NHNH2
N
6
5
7
X
NH
S
NH
N
8
N N
O
S
N
N
(c)
N
O
(d)
N
9a-h
Reagents and conditions: (a) Ethanol, H SO , reflux; (b) NH NH , absolute ethanol, reflux;
2
4
2
2
(
c) CS ,KOH, absolute ethanol, reflux, 2) HCl; (d) HCHO 40%, different amines, EtOH, rt.
2
Scheme 2: Synthesis of compounds 9a-h
2
.2. Antitrypanosomal activity

The synthesized compounds 3a, 3b, 4a, 4b, 9a, 9b, 9d-e and 9f have been evaluated for their
in vitro antitrypanosomal activity against Trypanosoma brucei. α-difluoromethylornithine
(
DFMO) was used as a control. The average values of antitrypanosomal activity (IC μM and
50
IC μM) and cytotoxicity on Rat myoblast cell line L6 were reported in Table 1 along with
9
0
the selectivity index (cytotoxicity IC /antitrypanosomal activityIC ). With respect to 1,2,4-
5
0
50
triazole-based compounds, the effect of 5-position substituents on the 1,2,4-triazole moiety and
its oxime derivatives has been investigated. Among the 1,2,4-triazoles, the non-oxime
derivative 3b with 5-position pyridin-4-yl had greater potency than its counterpart 3a pyrazin-
2
-yl-substituted. Compound 3b was the most active compound in general and also more potent
than control DFMO. 3b was 8 folds more potent than the reference with IC value of 0.79 μM
5
0
and IC of 1.35 μM, respectively compared to DFMO (IC = 6.10 μM and IC of 8.66 μM).
9
0
50
90
Replacement of the pyridin-4-yl moiety in compound 3b by pyrazin-2-yl in compound 3a
resulted in at least 6 folds reduction of the IC value. Similarly, oxime-substituted 1,2,4-
5
0
triazoles 4a and 4b exhibited significant in vitro antitrypanosomal activity against
Trypanosoma brucei with IC of 4.87 μM, 4.18 μM, respectively and with IC of 7.65 μM, >
5
0
90
1
0 μM, respectively compared to DFMO. About1,3,4-oxadiazole-2-thiones, results showed
that the antitrypanosomal activity decreased in the following order: 4-O-CH > 4-CH > 4-F. 2-
3
3
substitution on the pendant phenyl moiety of 1,3,4-oxadiazole-2-thiones (9a and 9e) had no
significant potency against T. brucei (IC > 10 μM), revealing that 2-substitutions in the
5
0
pendant phenyl moiety of 1,3,4-oxadiazole-2-thiones were unprofitable. Compounds 9f and 9d
X = 4-OCH and 4-CH , respectively) exhibited remarkable in vitro antitrypanosomal activity
(
3
3
against Trypanosoma brucei with IC of 3.94 μM and 6.33 μM, respectively and with IC of
5
0
90
>
10 μM and 4.32 μM and > 10 μM, respectively compared to DFMO. Other tested compounds
exhibited weak antitrypanosomal activity with IC and IC > 10 μM. The results showed that
5
0
90
the ketone of 1,2,4-triazole pyrazine or pyridine derivatives with N-(4-acetylphenyl)-2-

bromoacetamide (3a and 3b) and their oximes (4a and 4b) showed significant in vitro
antitrypanosomal activity against Trypanosoma brucei. In addition, Mannich bases of 1,3,4-
oxadiazole-2-thiones 9d and 9f with electron donation groups showed remarkable in vitro
antitrypanosomal activity against Trypanosoma brucei compared to other Mannich bases with
electron withdrawing function groups.
Table 1: Antitrypanosomal activity of compounds 3a-b, 4a, 4b, 9a, 9b, 9d-e and 9f
R1
N
N
H
N
NH
N
S
Het
N
N
O
Y
N
N
S
O
CH3
Scaffold A
Scaffold B
T. brucei
Comp. No Scaffold
Het
Y
R1
IC50
IC90
3
a
A
A
A
A
B
B
B
B
B
-
Pyrazin-2-yl
Pyridine-4-yl
Pyrazin-2-yl
O
O
-
5.12
0.79
4.87
4.18
> 10
> 10
6.33
> 10
3.94
6.1
8.4
3
b
-
1.35
7.65
> 10
> 10
> 10
> 10
> 10
4.32
8.66
4
a
NOH
-
-
4
b
Pyridine-4-yl NOH
9
a
-
-
-
-
-
-
-
-
-
-
-
-
2-F
9
9
b
d
4-F
4-CH3
2-OCH3
4-OCH3
-
9
e
9
f
DFMO

Further, the myoblast L6 rat was tested for the cytotoxic effect of these analogs. The
cytotoxicity and antitrypanosomal activity IC were used to classify the selectivity index (SI).
5
0
The result is shown in Table 2. The compounds showed moderate cytotoxicity with selectivity
indices ranging from 12 (9d) to 102 (3b) when tested against L6 cells, Table 2 and Figure 2.
However, the SI of 3b (102.46) was higher than the reference DFMO (12.17), which led to the
conclusion that this compound was of interest and would be subject to additional structural
modifications to improve its potency, affinity and/or selectivity.
Table 2: cytotoxicity activity of compounds 3a-b, 4a, 4b, 9a, 9b, 9d-e and 9f against L-6, IC .
50
Selectivity Index
(SI)
Comp.
No
a,b
IC (µM)
5
0
1
3.86
3
a
70.97±3.3
80.95±2.8
88.78±2.6
77.22±2.2
ND
1
02.46
3
b
1
8.22
8.47
4
a
1
4
b
-
-
-
9
a
-
9
9
b
d
ND
1
2.03
76.19±2.8
ND
--
9
e
1
1
8.39
2.17
9
f
72.47±3.1
74.29±2.9
DFMO
a
b
IC is theconcentration of test compound which reduce cellular growth to 50% after treatment for72 h.
5
0
Results represent mean IC value ± S.D. (n = 3).
5
0

1
1
20
00
8
6
4
2
0
0
0
0
0
3
a
3b
4a
4b
9d
9f
DFMO
Compound
Figure 2: Selectively indices of compounds 3a,b, 4a,b, 9d and 9f for T. brucei.
.3. Molecular Docking study
2
In this work, a comparative molecular docking study was performed to investigate the potential
molecular target(s) which could mediate the antitrypanosomal activity of the new compounds.
the study was performed using ten of T. brucei enzymes which represent validated/potential
targets for most of the antitrypanosomal agents. Of these enzymes, T. brucei rhodesain (pdb
code 2P7U) [34] which have about 80% similarity with T. cruzi cruzain and TryR (pdb code
2
WP6) [29] were selected as potential targets for the new compounds based on their rational
design, Figure 1. In addition, the sterol 14α-demethylase enzyme (CYP51, pdb code: 3GW9)
[
35], pteridine reductase (PTR1, pdb code 3JQ7) [36], purine nucleosidase (pdb code 4I72)
37], adenosine kinase (TbrAK, pdb code: 3OTX) [38], ornithine decarboxylase (ODC, pdb
[
code 1NJJ) [39], UDP-galactose 4'-epimerase (TbGalE, pdb code: 1GY8) [40], dihydrofolate
reductase (tb DHFR, pdb code 3QFX) [41] and triphosphate isomerase (pdb code: 1IIH) [42]
were also used in this study. The crystal structure of the ten enzymes were downloaded from
protein data bank (https://www.rcsb.org/).
The study was performed using AutoDock 4.2 [43]. The docking procedures were essentially
the same for each of the ten proteins. Preparation of ligand and protein files and calculation of

atomic charges was done according to the previous report [44,45]. Grid parameter files were
prepared following the previous report [46] and docking parameters were set to the default
values following the previous report [47]. Visualization of binding interactions and orientations
of the new compounds was done using discovery studio visualizer [48]. Validation of the
docking results into each of the ten targets was performed by re-docking of the native ligand
into the corresponding protein and comparing the binding mode, orientation and interactions
with those of the co-crystallized one. The plots showing the correlation between
antitrypanosomal activity log (IC ) of the new compounds and their calculated inhibition
5
0
constant (Ki) values obtained from docking results are provided in supplementary data
Figures S1-S10). The results of the docking study revealed high binding scores toward many
(
of the selected enzymes. However, a good correlation was observed only between the
2
biological results and inhibition constant of the new compounds against TryR enzyme (R =
0
.726).
The results of the docking study into sterol 14α-demethylase enzyme (CYP51, pdb code: 3GW9)
[
35] revealed low binding affinity (G = -7.98 to -10.95 kcal/mol) compared to -12.1 kcal/mol for
b
the native ligand (VNI). Investigation of the binding orientation of the co-crystallized ligand VNI
revealed that the imidazole ring of VNI adopted an orientation in the front of the heme molecule
of CYP51, Figure 3(A). On the other hand, the triazole ring of compound 3b adopted an opposite
orientation to VNI where the triazole ring is away from iron atom.
The docking study of the new compounds into the active site pteridine reductase (PTR1, pdb
code 3JQ7) [36] revealed low binding affinities (G = -7.78 to -7.90 kcal/mol) compared to -
b
9
.65 kcal/mol for the native ligand (DX2). Investigation of the binding mode of DX2 revealed
that the pteridine ring is sandwiched between the phenyl ring of Phe97 and nicotinamide ring
of cofactor NADP. Similar binding orientations was also observed for several PTR1 inhibitors

(pdb codes: 2YHI, 2YHU and 4WCF) [49]. On the other hand, the triazole ring in the best fit
conformation of compound 3b adopted a perpendicular orientation to DX2, Figure 3(B).
Figure 3: 3D Binding modes of compound 3b (shown as sticks colored by element) into: A) CYP51
pdb code: 3GW9), VNI (colored in yellow), imidazole ring (encircled in pink color) of VNI adopted
(
an orientation in the front of the iron in heme molecule, triazole ring (encircled in blue color) of
compound 3b adopted a different orientation from VNI; B) PTR1 (pdb code 3JQ7), DX2 (colored in
deep green) adopted an orientation sandwiched between the phenyl ring of Phe97 (encircled by blue
circle) and nicotinamide ring (encircled by pink circle) of NADP. The triazole ring in compound 3b
adopted a different orientation from DX2; C) 6-oxopurine nucleosidase (pdb code 4I72) overlaid with
immucillin A (UA2, galidesivir, colored in yellow); D) rhodesain (pdb code 2P7U), D1R (shown as
yellow sticks) adopted an orientation which occupy the S1-S3 subsites in the active site of rhodesain by
the four rings, receptor shown as hydrophobicity surface, hydrogen are hidden for clarity.
The results of the docking study into purine nucleosidase (pdb code 4I72) [37] revealed very
bad binding score suggesting that this enzyme may not be a potential target for the new
compounds. On the other hand, docking results of the new compounds into rhodesain (pdb
code 2P7U) [34], the major cysteine protease of T. brucei rhodesiense revealed comparable
binding affinities to that of the co-crystallized ligand D1R (G = -6.97). Investigation of the
b
3
D binding mode of compound 3b, into the active site of rhodesain revealed that it can occupy

two subsites (S2 and S3) and the pyridinyl ring span partially to the S1 subsite of rhodesain
while D1R span the four subsites (S1-S3) showing an important interaction with thiol group of
cysteine (Cys25), Figure 3(D).
Figure 4: 3D binding modes of compound 3b (shown as sticks colored by element) into: A) TbrAK (pdb
code: 3OTX) showing the binding site of compound 3a away from the adenosine binding site, PPM
(colored in blue) and AP5A (shown as yellow sticks); B) into ODC (pdb code 1NJJ), GET (shown as
yellow sticks);C) TbGalE, pdb code: 1GY8), chlorobiocin (shown as blue sticks), and UDP (shown as
yellow sticks); D) tbDHFR (pdb code 3QFX), CP6 (shown as yellow sticks).
Molecular docking analyses of the new compounds into T. brucei rhodesiense adenosine kinase
(TbrAK, pdb code: 3OTX) [38] revealed comparable binding free energy scores compared to
that of 4-(3-(4-phenoxyphenyl)-1H-pyrazol-5-yl)morpholine (PPM) which can target
adenosine kinase (TbrAK) of the parasite. However, the best fit conformation of compound 3b
exhibited completely different binding modes, orientations and interactions from those of PPM.
Compound 3b binds in a different position away from the binding sites of AP5A or PPM,

Figure 4(A). On the other hand, much lower binding affinities were observed for the new
compounds into ornithine decarboxylase (ODC, pdb code 1NJJ) [39] compared to geneticin
(GET) which already has weak inhibitory activity against ODC. Moreover, the results of the
docking studies of the new compounds into T. brucei UDP-galactose 4'-epimerase (TbGalE,
pdb code: 1GY8) [40] revealed also lower binding affinities than both the native ligand uridine-
5
-diphosphate (UDP) and the previously reported inhibitor, chlorobiocin [50]. The results of
the docking study of the new compounds into T. brucei dihydrofolate reductase (tbDHFR, pdb
code 3QFX) [41] showed high binding free energy in the range of -7.16 to -9.93 kcal/mol
compared to -8.08 kcal/mol for the native ligand, 5-(4-chlorophenyl)-6-ethyl-pyrimidine-2,4-
diamine (CP6). The best fit conformation of compound 3a adopted an orientation in which the
acetyl phenyl moeity overlay with the pyrimidine ring of CP6. Hovever, several important H-
bonding interactions with Asp54, Val33, Tyr166, and Thr184 are missing for compound 3b,
Figure 4. On the other hand, docking study of the new compounds into T. brucei triphosphate
isomerase (pdb code: 1IIH) [42] revealed binding free energy comparable to that of the native
ligand, 3-phosphoglyceric acid (3PG). However, no correlation observed between
antitrypanosomal activity and K values of the tested compounds, Figure 5.
i
Docking of the new compounds into TryR (pdb: 2WP6) [29] revealed high binding affinities
comparable to that of the native ligand II (Supplementary data, Table S1). A good correlation was
observed between the antitrypanosomal activity (Log IC ) results and K values of the new
5
0
i
2
compounds (R = 0.726). Moreover, among these compounds, the triazole derivative (3a,b and
4
a,b) exhibited higher binding affinities to TryR than the oxadiazole derivatives 9a-h. These
results were in concordance with the biological results of the new compounds, Table 1.
Investigation of the binding modes of the new compounds into TryR revealed different types
of hydrogen bonding, hydrophobic and pi-sulfur interactions (Supplementary data, Table S1).

Compound 3b exhibited a similar binding mode and interaction to those of compound II. The
triazole ring in the best fit conformation of compound 3b overlaid partially on the pyrimidine
ring of compound II, Figure 5. In addition, the three aromatic rings in compound 3b overlaid
partially on the three rings in compound II and occupy their binding sites. Like compound II,
the triazole 3b exhibited a hydrogen bonding interactions with Glu18. Compound 3b also
displayed multiple hydrophobic interactionsof pi-sigma, pi-pi, and pi-alkyl with Leu17, Trp21,
Tyr110, and Met113 residues.
Figure 5: 3D binding modes of compound 3b (shown as sticks colored by element) into: A) trypanosomal
triosephosphate isomerase (TIM, pdb code: 1IIH), 3PG shown as yellow sticks; B) TryR (pdb: 2WP6),
3
,4- dihydroquinazoline inhibitor WP6 shown as yellow sticks, receptor shown as H-bond surface,
hydrogen atoms were omitted for clarity.
3
. Conclusion
This research has developed two series of novels 1,2,4-triazol-3-yl-thioacetamide 3a-b, 4a-b,
and 5-pyrazine-2-yl-3H-[1,3,4]oxadiazol-2-thiones 9a-h. Spectral and elemental analyses have

confirmed the new compounds' chemical structure. In vitro antitrypanosomal activity on
trypanosoma brucei was evaluated against the synthesized compounds 3a, 3b, 4a, 4b, 9a, 9b,
9
d-e and 9f. Such compounds were also tested for cytotoxicity of L6 cells. Results showed the
ability of most new compounds to substantially stop trypanosoma brucei replication.
Compound 3b was generally the highest active compound and also stronger (8 folds) than
DFMO. The tested compounds demonstrated moderate cytotoxicity with selectivity indices
ranging from 12 (9d) to 102 (3b) when tested with L6 cells. However, the SI of 3b (102.46)
was higher than the reference DFMO (12.17), which led to the conclusion that this compound
was of interest and would be subject to additional structural modifications to improve its
potency, affinity and/or selectivity. Docking study was performed into ten of T. brucei enzymes
which have been identified as potential/valid targets for most of the antitrypanosoomal agents.
The results of the docking study revealed high binding scores toward many of the selected
enzymes. A good correlation was observed only between log (IC ) of antitrypanosomal
5
0
2
activity of the new compounds and their calculated Ki values against TryR enzyme (R =
.726). Compound 3b, the most active as antitrypanosomal agents exhibited similar binding
0
orintation and interaction to those of WP6 against TryR enzyme. However, a complementary
studies will be carried out to clarify the mechanism of action of these compunds.
4
4
. Experimental
.1. Chemistry
General details [See Appendix A]

Compounds 1[51], 2a-b [51] and 8 [52] were prepared according to previously reported
procedures.
4
1
.1.1. General procedure for the synthesis of N-(4-acetylphenyl)-2-(4-phenyl-5-(aryl)-4H-
,2,4-triazol-3-ylthio) acetamide derivatives (3a-b)
A mixture of 2a-b (1 mmol), N-(4-acetylphenyl)-2-bromoacetamide 1 (1 mmol) and K CO
2
3
(0.165 g, 1.2 mmol) in absolute ethanol was heated at reflux for 4-8 h. The reaction mixture
was evaporated under reduced pressure; the obtained residue was crystallized from aqueous
ethanol affording the pure products 3a-b in a good yield.
N-(4-Acetylphenyl)-2-(4-phenyl-5-(pyrazin-2-yl)-4H-1,2,4-triazol-3-ylthio)acetamide
(3a)
°
-1
Yellowish white powder (0.17 g, 81% yield); mp: 192-193 C; IR V /cm 3246 (NH), 1662
max
1
(
CO-CH ), 1596 (CO-NH); H NMR (DMSO-d ) δ (ppm): 2.50 (s, 3H, CH ), 4.30 (s, 2H, CH ),
3
6
3
2
7
2
.41-7.44 (m, 2H, Ar-H), 7.49-7.51 (m, 3H, Ar-H), 7.70 (d, 2H, J = 8.00 Hz, Ar-H), 7.92 (d,
H, J = 8.00 Hz, Ar-H), 8.37 (d,1H, J = 2.40 Hz, H5 of pyrazine), 8.59 (d, 1H, J = 2.40 Hz,
1
3
H6 of pyrazine), 9.17 (d,1H, J = 1.20 Hz, H3 of pyrazine), 10.75 (s, 1H, NH); C NMR
DMSO-d ) δ (ppm): 26.51, 39.31, 118.43, 127.29, 129.59, 129.63, 129.84, 131.97, 142.22,
(
6
1
43.13, 143.72, 144.24, 145.08, 151.52, 153.57, 166.07, 196.58; ESI-MS: m/z [M+H]:
4
31.141; Anal. Calcd. for C H N O S (430.48): C, 61.38; H, 4.21; N, 19.52. Found: C, 61.62;
2
2
18
6
2
H, 4.30; N, 19.75.
N-(4-Acetylphenyl)-2-(4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-ylthio)
acetamide (3b)
°
-1
Yellowish white powder (0.16 g, 79% yield); mp: 183-185 C; IR V /cm 3269 (br, NH), 1674
max
1
(
CO-CH ), 1631 (CO-NH), 1596 (C=N); H NMR (DMSO-d ) δ (ppm): 2.49 (s, 3H, CH ), 4.26
3
6
3
(
s, 2H, CH ), 7.26 (d, 2H, J = 8.00 Hz, Ar-H), 7.47-7.49 (m, 2H, Ar-H), 7.57-7.59 (m, 3H, Ar-
2

H), 7.69 (d, 2H, J = 8.00 Hz, Ar-H),7.92 (d, 2H, J = 8.00 Hz, pyridine-H), 8.53 (d, 2H, J = 8.00
1
3
pyridine-H), 10.70 (s, 1H, NH); C NMR (DMSO-d ) δ (ppm): 26.90, 39.36, 118.86, 121.96,
6
1
27.97, 127.99, 130.09, 130.75, 134.00, 143.51, 150.48, 150.57, 152.67, 152.77, 153.41,
1
57.95, 166.48, 196.96; ESI-MS: m/z [M+H]: 430.146; Anal. Calcd. for C H N O S (429.49):
2
3
19
5
2
C, 64.32; H, 4.46; N, 16.31. Found: C, 64.49; H, 4.43; N, 16.49.
General procedure for the synthesis of N-(4-(1-(hydroxyimino)ethyl)phenyl)-2-(4-phenyl-
-(aryl)-4H-1,2,4-triazol-3-ylthio)acetamide derivatives (4a-b)
5
Amixture of the appropriate ketone derivatives 3a-b (1 mmol) and hydroxyl amine
hydrochloride (0.083 g, 1.2 mmol) in 30 mL absolute ethanol was heated under reflux for 8-12
h and then left to cool. The separated solid was filtered off, washed with diluted ammonia
followed by distilled water, dried and recrystallized from aqueous ethanol affording oxime
derivatives 4a-b in a good yield.
N-(4-(1-(Hydroxyimino)ethyl)phenyl)-2-(4-phenyl-5-(pyrazin-2-yl)-4H-1,2,4-triazol-3-
ylthio)acetamide (4a)
°
-1
White powder (0.29 g, 75% yield); mp: 148-149 C; IR V / cm 3350-3100 (br, OH), 1657
max
1
(
CO-NH), 1631 (C=N); H NMR (DMSO-d ) δ (ppm): 2.11 (s, 3H, CH ), 4.28 (s, 2H, CH ),
6
3
2
7
8
.43 (d, 2H, J = 8.00 Hz, Ar-H), 7.54 (d, 2H, J = 8.00 Hz, Ar-H), 7.60-7.64 (m, 5H, Ar-H),
.40 (d,1H, J = 2.40 Hz, H5 of pyrazine), 8.62 (d, 1H, J = 2.40 Hz, H6 of pyrazine), 9.19
1
3
(
s,1H, H3 of pyrazine), 10.51 (s, 1H, NH), 11.10 (s, 1H, OH); C NMR (DMSO-d ) δ (ppm):
6
1
1
1.42, 36.84, 118.79, 126.14, 127.30, 129.60, 129.84, 132.08, 134.03, 139.17, 142.24, 143.73,
44.25, 145.09, 151.49, 152.39, 153.59, 165.47; ESI-MS: m/z [M+H]: 446.152; Anal. Calcd.
for C H N O S (445.49): C, 59.31; H, 4.30; N, 22.01. Found: C, 59.57; H, 4.37; N, 22.34.
2
2
19
7
2
N-(4-(1-(Hydroxyimino)ethyl)phenyl)-2-(4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-
ylthio)acetamide (4b)

°
-1
White powder (0.29 g, 77 % yield); mp: 251-252 C; IR V /cm 3450-3100 (br, OH), 3051
max
1
(
C-H, aromatic), 2930 (C-H, aliphatic), 1682 (CO-NH), 1603 (C=N); H NMR (DMSO-d ) δ
6
(
ppm): 2.12 (s, 3H, CH ), 4.26 (s, 2H, CH ), 7.29 (d, 2H, J = 8.00 Hz, Ar-H), 7.50 (d, 2H, J =
3
2
8
.00 Hz, Ar-H), 7.56-7.61(m, 7H, 5 Ar-H + 2H, pyridine-H), 8.56 (d, 2H, J = 8.00 Hz,
1
3
pyridine-H), 10.50 (s, 1H, NH), 11.11 (s, 1H, OH); C NMR (DMSO-d ) δ (ppm): 11.43,
6
3
1
6.89, 118.79, 121.55, 126.13, 127.58, 130.29, 130.62, 132.08, 133.32, 133.82, 139.15, 150.15,
52.31, 153.40, 165.13; ESI-MS: m/z [M+H]: 445.156; Anal. Calcd. for C H N O S
2
3
20
6
2
(
444.50): C, 62.15; H, 4.54; N, 18.91. Found: C, 62.37; H, 4.61; N, 19.23.
General procedure for the synthesis of 3-[(Substituted phenyl amino)methyl] 5-pyrazin-
-yl-3H-[1,3,4]oxadiazole-2-thiones (9a-h)
2
Formalin 40% (1.5 mL, 0.02 mol) was added to a stirred solution of 5-(Pyrazin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione 8 (3.6 g, 0.02 mol) in 40 mL absolute ethanol. An ethanolic solution
(10 mL) of the appropriate amine (0.02 mol) was added in a drop wise manner to the reaction
mixture, stirred for 3 h at room temperature, and left overnight in the refrigerator, the precipitate
formed was filtered, washed with cold ethanol, dried and crystallized from ethanol
3
-((2-Flourophenylamino)methyl)-5-(pyrazin-2-yl)-1,3,4-oxadiazole-2(3H)-thione (9a)
°
-1
Yellowish white powder (2.45 g, 88% yield); mp: 187-189 C; IR V / cm : 3350-3500 (br,
max
1
NH), 1660 (C=N), 1428 (C=S), 1215 and 1166 (C-O-C); H NMR (CDCl ) δ (ppm): 5.35 (bs,
3
1
H, NH), 5.57 (d, 2H, J = 7.4 Hz, CH ), 6.91-7.20 (m, 4H, Ar-H), 8.67 (d, 1H, J = 2.30 Hz, H5
2
1
3
of pyrazine), 8.69 (d, 1H, J = 2.30 Hz, H6 of pyrazine), 9.12 (s, 1H, H3 of pyrazine). C NMR
(
CDCl ) δ (ppm): 58.35, 113.75, 115.34, 115.40, 118.05, 125.28, 138.30, 143.65, 145.41,
3
1
2
45.54, 147.77, 157.20, 176.44; Anal. Calcd. for C H FN OS (303.31): C, 51.48; H, 3.32; N,
13 10 5
3.09. Found: C, 51.62; H, 3.30; N, 23.26.
-((4-Flourophenylamino)methyl)-5-(pyrazin-2-yl)-1,3,4-oxadiazole-2-(3H)-thione (9b)
3

°
-1
Yellowish white powder (2.31 g, 83 % yield); mp: 185-186 C; IR V /cm : 3300-3500 (br,
max
1
NH), 1661 (C=N), 1436 (C=S), 1222 and 1169 (C-O-C); H NMR (DMSO-d ) δ (ppm): 5.53
6
(
t, 1H, J = 7.20 Hz, NH), 6.05 (d, 2H, J = 7.40 Hz, CH ), 7.29 (d, 2H, J = 8.00 Hz, Ar-H), 7.66
2
(d, 2H, J = 8.00 Hz, Ar-H), 8.74 (d, 1H, J = 2.40 Hz, H5 of pyrazine), 8.86 (d, 1H, J = 2.40
1
3
Hz, H6 of pyrazine), 9.16 (s, 1H, H3 of pyrazine), C NMR (DMSO-d ) δ (ppm): 58.90,
6
1
14.42, 116.86, 127.80, 129.47, 133.29, 144.25, 145.24, 146.26, 147.56, 175.01; EI-MS (70
+
eV) m/z (%) 303.05 [M ]; Anal. Calcd. for C H FN OS (303.31): C, 51.48; H, 3.32; N, 23.09.
1
3
10
5
Found: C, 51.62; H, 3.29; N, 23.41.
-(Pyrazin-2-yl)-3-((o-tolylamino)methyl)-1,3,4-oxadiazole-2-(3H)-thione (9c)
5
°
-1
Brownish yellow powder (2.10 g, 78% yield); mp: 158-160 C; IR V /cm : 3200-3400 (br,
max
1
NH), 1633 (C=N), 1418 (C=S), 1247 and 1165 (C-O-C); H NMR (DMSO-d ) δ (ppm): 2.31
6
(
s, 3H, CH ), 5.47 (t, 1H, J = 7.20 Hz, NH), 5.99 (d, 2H, J = 7.20 Hz, CH ), 7.05-7.67 (m, 4H,
3
2
Ar-H), 8.70 (d, 1H, J = 2.40 Hz, H5 of pyrazine), 8.84 (d, 1H, J = 2.40 Hz, H6 of pyrazine),
1
3
9
1
.23 (s,1H, H3 of pyrazine); C NMR (DMSO-d ) δ (ppm): 18.15, 70.64, 124.67, 125.84,
6
28.79, 129.21, 129.49, 135.49, 138.22, 140.22, 142.42, 145.89, 147.26, 176.40;Anal. Calcd.
for C H N OS (299.35): C, 56.17; H, 4.38; N, 23.40. Found: C, 56.43; H, 4.43; N, 23.58.
1
4
13
5
5
-(Pyrazin-2-yl)-3-((p-tolylamino)methyl)-1,3,4-oxadiazole-2-(3H)-thione (9d)
°
-1
Brownish yellow powder (2.13 g, 79% yield); mp: 179-180 C; IR V /cm : 3300-3450 (br,
max
1
NH), 1654 (C=N), 1429 (C=S), 1226 and 1169 (C-O-C); H NMR (DMSO-d ) δ (ppm): 2.31
6
(
s, 3H, CH ), 5.51 (t, 1H, J = 7.20 Hz, NH), 6.04 (d, 2H, J = 7.90 Hz, CH ), 7.23 (d, 2H, J =
3
2
8
8
.00 Hz, Ar-H), 7.51 (d, 2H, J = 8.00 Hz, Ar-H), 8.74 (d,1H, J = 2.40 Hz, H5 of pyrazine),
1
3
.85 (d, 1H, J = 2.40 Hz, H6 of pyrazine), 9.20 (s, 1H, H3 of pyrazine); C NMR (DMSO-d )
6
δ (ppm): 21.04, 70.20, 113.67, 120.98, 125.11, 126.10, 129.96, 134.95, 136.69, 142.55, 144.40,
1
45.92, 147.29, 176.51;ESI-MS: m/z [M+H]: 300.10; Anal. Calcd. for C H N OS (299.35):
14 13 5
C, 56.17; H, 4.38; N, 23.40. Found: C, 56.39; H, 4.44; N, 23.57.

3
-((2-Methoxyphenylamino)methyl)-5-(pyrazin-2-yl)-1,3,4-oxadiazole-2(3H)-thione (9e)
°
-1
Yellowish white powder (1.80 g, 64% yield); mp: 176-178 C; IR V /cm : 3300-3500 (br,
max
NH), 1652 (C=N), 1577 (C=C, aromatic), 1423 (C=S), 1286 ( C-O), 1240 and 1167 (C-O-C);
1
H NMR (DMSO-d ) δ (ppm): 3.81 (s, 3H, O-CH ), 5.33 (t, 1H, J = 7.20 Hz, NH), 5.88 (d, 2H,
6
3
J = 7.40 Hz, CH ), 6.99-7.40 (m, 4H, Ar-H), 8.65 (d, 1H, J = 2.40 Hz, H5 of pyrazine), 8.86
2
1
3
(
d, 1H, J = 2.40 Hz, H6 of pyrazine), 9.21 (s,1H, H3 of pyrazine), C NMR (DMSO-d ) δ
6
(ppm): 55.95, 58.80, 110.99, 112.62, 120.72, 125.18, 130.93, 131.21, 143.48, 144.88, 147.01,
1
48.38, 156.40, 176.43; Anal. Calcd. for C H N O S (315.350): C, 53.32; H, 4.16; N, 22.21.
14 13 5 2
Found: C, 53.45; H, 4.21; N, 22.47.
3
-((4-Methoxyphenylamino)methyl)-5-(pyrazin-2-yl)-1,3,4-oxadiazole-2(3H)-thione (9f)
°
-1
Yellowish white powder (1.93 g, 69% yield); mp: 195-197 C; IR V /cm : 3300-3450 (br,
max
1
NH), 1632 (C=N), 1436 (C=S), 1246 and 1170 (C-O-C); H NMR (DMSO-d ) δ (ppm): 3.77
6
(
s, 3H, O-CH ), 5.52 (t, 1H, J = 7.20 Hz, NH), 6.02 (d, 2H, J = 7.90 Hz,-CH ), 6.99 (d, 2H, J
3
2
=
8.00 Hz, Ar-H), 7.51(d, 2H, J = 8.00 Hz, ArH), 8.73 (d, 1H, J = 2.40 Hz, H5 of pyrazine),
1
3
8
.85 (d, 1H, J = 2.40 Hz, H6 of pyrazine), 9.20 (s,1H, H3 of pyrazine), C NMR (DMSO-d )
6
δ (ppm): 55.48, 58.70, 114.49, 114.79, 116.48, 127.45, 130.12, 143.44, 145.70, 145.97, 147.32,
+
1
58.46, 175.04; EI-MS (70 eV) m/z (%) 315.48 [M ]; Anal. Calcd. for C H N O S (315.350):
1
4
13
5
2
C, 53.32; H, 4.16; N, 22.21. Found: C, 53.47; H, 4.23; N, 22.37.
3
-((3,4-Dimethoxyphenylamino)methyl)-5-(pyrazin-2-yl)-1,3,4-oxadiazole-2(3H)-thione
(9g)
°
-1
Red powder (2.03 g, 71% yield); mp: 160-161 C; IR V /cm : 3400 (NH), 1659 (C=N), 1441
max
1
(
C=S), 1263 (C-O), 1215 and 1172 (C-O-C); H NMR (DMSO-d ) δ (ppm): 3.74 (s, 3H, O-
6
CH ), 3.77 (s, 3H, O-CH ), 5.57 (t, 1H, J = 7.20 Hz, NH), 6.02 (d, 2H, J = 7.40 Hz, CH ),
3
3
2
7
.00-7.15 (m, 2H, Ar-H), 7.33 (s, 1H, Ar-H), 8.74 (d, 1H, J = 2.40 Hz, H5 of pyrazine), 8.84

1
3
(
d, 1H, J = 2.40 Hz, H6 of pyrazine), 9.20 (s,1H, H3 of pyrazine), C NMR (DMSO-d ) δ
6
(ppm): 56.01, 56.47, 57.82,112.48, 114.57, 119.60, 128.28, 135.04, 141.17, 145.21, 146.92,
1
2
5
48.46, 150.97, 158.47, 175.20,Anal.Calcd. for C H N O S (345.376): C, 52.16; H, 4.38 N,
15 15 5 3
0.28. Found: C, 52.38; H, 4.45; N, 20.43.
-(Pyrazin-2-yl)-3-((3,4,5-trimethoxyphenylamino)methyl)-1,3,4-oxadiazole-2(3H)-thione
(9h)
°
-1
Yellowish powder (1.98 g, 68% yield); mp: 157-158 C; IR V /cm : 3300-3450 (br, NH),
max
1
1
3
602 (C=N), 1423 (C=S), 1275 (C-O), 1205 and 1168 (C-O-C); H NMR (DMSO-d ) δ (ppm):
6
.76 (s, 3H, O-CH ), 3.85 (s, 6H, 2-O-CH ), 5.57 (t, 1H, J = 7.2 Hz, NH), 6.04 (d, 2H, J = 7.20
3
3
Hz, CH ), 6.97(s, 2H, Ar-H), 8.85 (d,1H, J = 2.40 Hz, H5 of pyrazine), 8.87 (d, 1H, J = 2.40
2
1
3
Hz, H6 of pyrazine), 9.21 (s,1H, H3 of pyrazine); C NMR (DMSO-d ) δ (ppm): 54.41, 56.52,
6
6
0.81, 71.08, 91.41, 104.22, 112.56, 128.34, 142.12, 145.08, 147.10, 149.92, 152.80, 156.68,
58.67, 176.82, Anal. Calcd. for C H N O S (375.402): C, 51.19; H, 4.56 N, 18.66. Found:
1
1
6
17
5
4
C, 51.42; H, 4.62; N, 18.91.
4
.2. Antitrypanosomal and Cytotoxicity Assays
Antitrypanosomal and cytotoxicity Assayswere performed at National Center for Natural
Products Research, School of Pharmacy, University of Mississippi, USA as previously
described [53, 54].
4
.3. Molecular docking study
The pdb file of TryR (pdb code 2WP6) [29], T. brucei rhodesain (pdb code 2P7U) [34],
sterol14α-demethylase enzyme (CYP51, pdb code: 3GW9) [35], pteridine reductase (PTR1,
pdb code 3JQ7) [36], purine nucleosidase (pdb code 4I72) [37], adenosine kinase (pdb code:
3
OTX) [38], ornithine decarboxylase (pdb code 1NJJ) [39], UDP-galactose 4'-epimerase (pdb
code: 1GY8) [40], dihydrofolate reductase (pdb code 3QFX) [41] and triphosphate isomerase

(pdb code: 1IIH) [42] were also used in this study. The crystal structure of the ten enzymes
were downloaded from protein data bank (https://www.rcsb.org/). The docking study was
carried out using AutoDock 4.2 [43]. The docking procedures were essentially the same for
each of the ten proteins. Preparation of ligand and protein files and calculation of atomic
charges was done according to the previous report [44, 45]. Grid parameter files were prepared
following the previous report [46] and docking parameters were set to the default values
following the previous report [47]. Discovery studio visualizer was used to visualize binding
interactions and orientations of the new compounds [48]. The results were presented in Figures
3
-5 and in supplementary data (Tables S1-11and Figures S1-10). List of abbreviations and
chemical structure of the native ligand of the enzymes (supplementary data).
Conflicts of interest
The authors declare no conflict of interest

References
1
. http://www.dndi.org/
2
3
4
. Steverding, D. Steverding, Parasites & Vectors 3 (2010) 15.
. A. H. Fairamb, Trends Parasitol. 19 (2003) 488.
. S. M. El-Moghazy, F. F. Barsoum, H. M. Abdel-Rahman, A. A. Marzouk, Synthesis and
anti-inflammatory activity of some pyrazole derivatives. Med. Chem. Res. 21 (2012) 1722-
1
733.

5
. C. S. De Oliveira, B. F. Lira, J. M. Barbosa-Filho, J. G. F. Lorenzo, P. F. de Athayde-Filho,
Synthetic approaches and pharmacological activity of 1, 3, 4-oxadiazoles: a review of the
literature from 2000–2012. Molecules 17 (2012), 10192-10231.
6
7
. F. Omar, N. Mahfouz, M. Rahman, Design, synthesis and anti-inflammatory activity of
some 1, 3, 4-oxadiazole derivatives. Eur. J. Med. Chem 31 (1996) 819-825.
. M. G. Mamolo, D. Zampieri, L. Vio, M. Fermeglia, M. Ferrone, S. Pricl, G. Scialino, E.
Banfi, Antimycobacterial activity of new 3-substituted 5-(pyridin-4-yl)-3H-1, 3, 4-
oxadiazol-2-one and 2-thione derivatives. Preliminary molecular modeling investigations.
Bioorg. Med. Chem. 13 (2005) 3797-3809.
8
9
1
1
1
. S. G. Küçükgüzel, S. Rollas, H. Erdeniz, M. Kiraz, Synthesis, characterization and
antimicrobial evaluation of ethyl 2-arylhydrazono-3-oxobutyrates. Eur. J. Med. Chem 34
(1999) 153-160.
. C. Loetchutinat, F. Chau, S. Mankhetkorn, Synthesis and Evaluation of 5-Aryl-3-(4-
hydroxyphenyl)-1,3,4-oxadiazole-2-(3H)-thiones as P-Glycoprotein Inhibitors. Chem.
Pharm. Bull. 51 (2003) 728-730.
0. A. H. Abadi, A. A. H. Eissa, G. S. Hassan, Synthesis of novel 1,3,4-trisubstituted pyrazole
derivatives and their evaluation as antitumor and antiangiogenic agents. Chem. Pharm.
Bull. 51 (2003) 838-844.
1. C. Pierre-Audigier, C. Surcouf, V. Cadet-Daniel, A. Namouchi, S. Heng, A. Murray, B.
Guillard, B. Gicquel, Fluoroquinolone and pyrazinamide resistance in multidrug-resistant
tuberculosis [Short communication]. Int. J Tuberc. Lung Dis. 16 (2012) 221-223.
2. Y. Younis, F. Douelle, D. González Cabrera, C. Le Manach, A. T. Nchinda, T. Paquet, L.
J. Street, K. L. White, K. M. Zabiulla, J. T. Joseph, Structure–Activity-Relationship Studies
around the 2-Amino Group and Pyridine Core of Antimalarial 3,5-Diarylaminopyridines