Synthesis of oxadiazole-2-oxide derivatives as potential drug candidates for schistosomiasis targeting SjTGR

Article abstract of DOI:10.1186/s13071-021-04634-4

Background: Schistosomiasis is a chronic parasitic disease that affects millions of people’s health worldwide. Because of the increasing drug resistance to praziquantel (PZQ), which is the primary drug for schistosomiasis, developing new drugs to treat schistosomiasis is crucial. Oxadiazole-2-oxides have been identified as potential anti-schistosomiasis reagents targeting thioredoxin glutathione reductase (TGR). Methods: In this work, one of the oxadiazole-2-oxides derivatives furoxan was used as the lead compound to exploit a series of novel furoxan derivatives for studying inhibitory activity against both recombinant Schistosoma japonicum TGR containing selenium (rSjTGR-Sec) and soluble worm antigen protein (SWAP) containing wild-type Schistosoma japonicum TGR (wtSjTGR), in order to develop a new leading compound for schistosomiasis. Thirty-nine novel derivatives were prepared to test their activity toward both enzymes. The docking method was used to detect the binding site between the active molecule and SjTGR. The structure–activity relationship (SAR) of these novel furoxan derivatives was preliminarily analyzed. Results: It was found that several new derivatives, including compounds 6a–6d, 9ab, 9bd and 9be, demonstrated greater activity toward rSjTGR-Sec or SWAP containing wtSjTGR than did furoxan. Interestingly, all intermediates bearing hydroxy (6a–6d) showed excellent inhibitory activity against both enzymes. In particular, compound 6d with trifluoromethyl on a pyridine ring was found to have much higher inhibition toward both rSjTGR-Sec (half-maximal inhibitory concentration, IC₅₀,7.5nM) and SWAP containing wtSjTGR (IC₅₀ 55.8nM) than furoxan. Additionally, the docking method identified the possible matching sites between 6d and Schistosoma japonicum TGR (SjTGR), which theoretically lends support to the inhibitory activity of 6d. Conclusion: The data obtained herein showed that 6d with trifluoromethyl on a pyridine ring could be a valuable leading compound for further study.

Full text of DOI:10.1186/s13071-021-04634-4

Li et al. Parasites Vectors
https://doi.org/10.1186/s13071-021-04634-4
(2021) 14:225
Parasites & Vectors
Open Access
RESEARCH
Synthesis of oxadiazole-2-oxide derivatives
as potential drug candidates for schistosomiasis
targeting SjTGR
1,3
2,3†
1,3
1
1,3
3
2*
Gongming Li , Qingqing Guo , Chao Feng , Huan Chen , Wenjiao Zhao , Shu Li , Yang Hong and
Dequn Sun1*
Abstract
Background: Schistosomiasis is a chronic parasitic disease that afects millions oꢀ people’s health worldwide. Because
oꢀ the increasing drug resistance to praziquantel (PZQ), which is the primary drug ꢀor schistosomiasis, developing new
drugs to treat schistosomiasis is crucial. Oxadiazole-2-oxides have been identiꢁed as potential anti-schistosomiasis
reagents targeting thioredoxin glutathione reductase (TGR).
Methods: In this work, one oꢀ the oxadiazole-2-oxides derivatives ꢀuroxan was used as the lead compound to exploit
a series oꢀ novel ꢀuroxan derivatives ꢀor studying inhibitory activity against both recombinant Schistosoma japonicum
TGR containing selenium (rSjTGR-Sec) and soluble worm antigen protein (SWAP) containing wild-type Schistosoma
japonicum TGR (wtSjTGR), in order to develop a new leading compound ꢀor schistosomiasis. Thirty-nine novel deriva-
tives were prepared to test their activity toward both enzymes. The docking method was used to detect the binding
site between the active molecule and SjTGR. The structure–activity relationship (SAR) oꢀ these novel ꢀuroxan deriva-
tives was preliminarily analyzed.
Results: It was ꢀound that several new derivatives, including compounds 6a–6d, 9ab, 9bd and 9be, demonstrated
greater activity toward rSjTGR-Sec or SWAP containing wtSjTGR than did ꢀuroxan. Interestingly, all intermediates bear-
ing hydroxy (6a–6d) showed excellent inhibitory activity against both enzymes. In particular, compound 6d with
triꢂuoromethyl on a pyridine ring was ꢀound to have much higher inhibition toward both rSjTGR-Sec (halꢀ-maximal
inhibitory concentration, IC ,7.5nM) and SWAP containing wtSjTGR (IC 55.8nM) than ꢀuroxan. Additionally, the
5
0
50
docking method identiꢁed the possible matching sites between 6d and Schistosoma japonicum TGR (SjTGR), which
theoretically lends support to the inhibitory activity oꢀ 6d.
Conclusion: The data obtained herein showed that 6d with triꢂuoromethyl on a pyridine ring could be a valuable
leading compound ꢀor ꢀurther study.
Keywords: Anti-schistosomiasis, Schistosoma japonicum, SjTGR, Oxadiazole-2-oxides, Furoxan
*
Correspondence: hongyang@shvri.ac.cn; dqsun@swust.edu.cn
Background
†
Gongming Li and Qingqing Guo contributed equally to this work
1
Schistosomiasis is one of the most widespread parasitic
diseases in the world, and is one of the main tropical
endemic diseases in Asia, Africa and South America [1].
It is estimated that approximately 779 million individuals
around the world are at risk of schistosomes infection [2].
Schistosomiasis is caused by ꢀve species of the trematode
School oꢀ Liꢀe Science and Engineering, Southwest University oꢀ Science
and Technology, Mianyang 621010, China
National Reꢀerence Laboratory oꢀ Animal Schistosomiasis, Key
Laboratory oꢀ Animal Parasitology oꢀ Ministry oꢀ Agriculture, Shanghai
Veterinary Research Institute, Chinese Academy oꢀ Agricultural Sciences,
Shanghai 200241, China
2
Full list oꢀ author inꢀormation is available at the end oꢀ the article
©
The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or ꢀormat, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate iꢀ changes were made. The images or other third party material
in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. Iꢀ material
is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the
permitted use, you will need to obtain permission directly ꢀrom the copyright holder. To view a copy oꢀ this licence, visit http://creativeco
mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/
zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Li et al. Parasites Vectors
(2021) 14:225
Page 2 of 11
ꢁ
atworm: Schistosoma japonicum (S. japonicum), Schisto- applying bioisosteres was exploited to design novel oxa-
soma mansoni (S. mansoni), Schistosoma haematobium, diazole-2-oxide derivatives.
Schistosoma mekongi and Schistosoma intercalatum.
On the other hand, the prokaryotic expression system
When the human body is infected, the worms will per- has been used extensively for protein expression due to
sist in the liver, hepatic portal system or the urinary tract its rapid growth rate, capacity for continuous fermenta-
system, leading to inꢁammation and obstructive disease tion, and relatively low cost. Due to the lack of the post-
[
3]. In China, schistosomiasis is caused mainly by Schis- translational modiꢀcations of this expression system,
tosoma japonicum, and more than 77 thousand people the bioactivity, function, structure, solubility and so on
are infected [4]. will be aꢂected to the expressed functional products.
Te most eꢂective drug for the treatment of schisto- Although eukaryotic expression systems can resolve this
somiasis is praziquantel (PZQ) (1), which has been used problem, just like a two-edged sword, some disadvan-
extensively since the 1980s [5] in the clinic without any tages such as low yield, demanding culture conditions
backup drugs. Te overuse of praziquantel has caused and higher cost cannot be totally avoided [14]. Tus, the
and accelerated the emergence of drug resistance. While special soluble worm antigen protein (SWAP) containing
PZQ is very eꢂective against the adult forms of all schis- wtSjꢃGR extracted from worms might be a better alter-
tosome species, it is weak for juvenile forms of all schis- native target for studying the bioactivity. It would reꢁect
tosomes, which might be the reason that PZQ cannot the actual situation in advance when Schistosoma japoni-
cure schistosomiasis completely. Although R-PZQ was cum is treated with chemical compounds. Terefore, the
approved by the Food and Drug Administration (FDA) target compounds herein were also applied to test the
as an orphan drug in 2018, it is also at risk of resist- inhibition activity on SWAP containing wtSjꢃGR in addi-
ance, and there is still no alternative drug against the tion to rSjꢃGR-Sec.
migratory juvenile and subadult worms [6–9]. Tus,
In this work, two series of novel furoxan derivatives (7
there is an urgent need to look for potential reagents to and 9) (Fig. 2) were obtained through two methods: either
discover novel alternatives to PZQ for the treatment of by replacing the phenyl moiety of furoxan with its bioisos-
schistosomiasis.
teres pyridine or substituted pyridine, or by modifying
It has been reported [1, 10–13] that instead of separate the cyano moiety into an ester or amine group. Because
thioredoxin reductase (ꢃrxR) and glutathione reductase the ꢁuorine atom possesses a strong electron-withdraw-
(
GR) enzymes of mammalian hosts, a single multifunc- ing eꢂect and high lipophilicity, appropriate introduc-
tional selenocysteine-containing ꢁavoenzyme, thiore- tion of ꢁuorine atoms into molecules can improve their
doxin glutathione reductase (ꢃGR), plays a critical role biofunction [15]. Terefore, several ꢁuorine-containing
in maintaining proper redox balance for parasite survival derivatives were also intentionally synthesized. Te target
in both S. mansoni and S. japonicum (Fig. 1). Te cellu- compounds (7 and 9) and their corresponding intermedi-
lar redox system is important in the physiological func- ates (6) (Fig. 2) were tested for their inhibitory activity on
tion of an organism. Given the biochemical diꢂerences ꢃrxR from rSjꢃGR-Sec and SWAP containing wtSjꢃGR..
between the redox metabolism of schistosomes and host Based on the result obtained, the SAR of these novel furo-
(
mammalian), ꢃGR is inferred to be a potential target for xan derivatives was preliminarily analyzed.
new drug design.
In previous studies [1, 2, 10–13], some oxadiazole- Methods
2
-oxide analogues have been shown to eꢂectively inhibit Reagent
SmꢃGR and SjꢃGR, even exhibiting prominent worm All chemical reagents involved in synthetic procedures
killing activity in vivo. Importantly, some oxadiazole- were purchased commercially from J&K Chemicals.
2
-oxides showed good juvenile and adult S. japonicum Te chemical reagents used in activity measurement,
killing activity in vitro [2]. Modiꢀcations around an including dimethyl sulfoxide (DMSO), ethylene diamine
oxadiazole-2-oxide skeleton have been demonstrated as tetraacetic acid (EDꢃA), dithionitrobenzoic acid (DꢃNB),
a valuable strategy for discovering potential antischisto- and Mꢃꢃ, were purchased from Sigma-Aldrich Inc.
somal agents. Te application of bioisosteres is an eꢂec-
tive strategy in drug design. In the design of compounds, Synthesis of oxadiazole‑2‑oxide derivatives
previous studies [1, 2, 10–13] have mainly substituted on Te synthesis of compounds 6a–d commenced with the
the benzene ring and changed the CN moiety, but did synthesis of α,β-unsaturated esters (4a–d) from substi-
not pay attention to modiꢀcation of the benzene ring and tuted nicotinaldehydes (3a–d) by Wittig reaction [16].
other moieties in the furoxan structure with correspond- Compounds 4a–d were then reduced using diisobuty-
ing bioisosteres. Terefore, in this work, the strategy of laluminum hydride (DIBAL-H) [17] to produce alcohols

Li et al. Parasites Vectors
(2021) 14:225
Page 3 of 11
Fig. 1 Redox pathways in mammals and S. mansoni.
5
a–d. Subsequent sodium nitrate-mediated cyclization
[
17] gave oxadiazole-2-oxides (6a–d) (Scheme 1).
In Scheme 2, compound 7 was readily prepared by
esteriꢀcation of alcohol 6a–d with various acyl chlorides
using triethylamine and dimethylaminopyridine (DMAP)
as base and catalyst, respectively. Te synthesis of target
compounds 9 began with the treatment of compounds
6
a–d with benzenesulfonyl chloride to generate unsta-
ble intermediates 8a–d, which were aminated in situ
to aꢂord various target amines 9. As a result, a total of
3
9 novel oxadiazole-2-oxide derivatives were synthe-
sized and their structures were determined by nuclear
magnetic resonance (NMR) and high-resolution mass
spectrometry (HRMS). Te preparation process and
spectrum of the compounds are detailed in Additional
Fig. 2 The structure oꢀ praziquantel (PZQ) and ꢀuroxan and the
general structure oꢀ compounds 6, 7 and 9..
ꢀ
le 1.
Crystallography
crystal was kept at 150 K during data collection. Using
Olex2 [18], the structure was solved with the Superꢁip
structure solution program using charge-ꢁipping and
reꢀned with the ShelXL reꢀnement package using least
squares minimization.
Normally, two isomers 6 and 6’ were obtained (Scheme 1)
and one of them was the main product after cyclization.
In order to conꢀrm the conformation of main product,
its crystal was selected and was measured on a Super-
Nova diꢂractometer (dual-source, Cu at zero, EosS2). Te

Li et al. Parasites Vectors
(2021) 14:225
Page 4 of 11
Preparation of the rSjTGR‑Sec and the SWAP containing
wtSjTGR
the inhibitory activity of the compounds against the ꢃrxR
activity of SWAP containing wtSjꢃGR and rSjꢃGR-Sec
Te rSjꢃGR-Sec was expressed and harvested as described in this paper was also based on a previously
described previously [13, 19]. Most of rSjꢃGR-Sec was described method [2]. Te reaction was performed in 0.1
expressed in E. coli as a soluble His-tagged fusion protein M potassium phosphate buꢂer (pH 7.0) containing 10
when bacterial growth occurred at 24 °C. Te rSjꢃGR- mM EDꢃA, 100 µM nicotinamide adenine dinucleotide
™
Sec was puriꢀed by His Mag Agarose beads accord- phosphate (NADPH), 0.2 µg supernatant of SWAP con-
ing to manufacturer’s instructions. Te concentration of taining wtSjꢃGR or 0.6 µg rSjꢃGR-Sec, and the studied
puriꢀed rSjꢃGR-Sec was determined using a Bradford compounds with diꢂerent concentrations at 25 °C in a
assay kit (Sangon, China), and the protein was identiꢀed ꢀnal volume of 200 μL, and was initiated by adding 3 mM
by sodium dodecyl sulfate polyacrylamide gel electro- DꢃNB. Te increase of the absorbance at 412 nm was
phoresis (SDS-PAGE) and stored at 4 °C [20].
monitored, and the IC values of the compounds were
50
Te SWAP containing wtSjꢃGR was prepared from calculated by GraphPad Prism v6.0c software; Te results
adult schistosomes. Rabbits were infected with cer- are reported in ꢃable 1. All assays were done in triplicate.
cariae of S. japonicum by exposing their abdominal skin
and were sacriꢀced at 42 days after infection to collect Docking study
the adult schistosomes by perfusion. Te worms were Te crystal structure of SjꢃGR is a homodimer, and we
washed three times with phosphate-buꢂered saline retained chain B for docking studies [22]. Te dock-
(
PBS). After suꢄcient grinding of the adult schistosomes ing was conducted by AutoDock 4, and the results are
in PBS, a suitable amount of benzoyl sulfonyl ꢁuoride displayed in Figs. 4 and 5. Te results were further pro-
(
PMSF) solution was added to obtain a ꢀnal concentra- cessed and displayed by PyMOL2.3.2. Te hydrophobic
tion of 1 mM. Te homogenate was centrifuged at 4 °C, eꢂects between inhibitors and protein were analyzed by
+
1
2,000×g for 20 min [1, 10]. Te concentration of super- LigPlot v.2.1, and the result is shown in Fig. 5.
natant containing wtSjꢃGR was determined by Bradford
assay kit (Sangon, China).
Results and discussion
Synthesis and crystallography
Compound inhibition of rSjTGR‑Sec and SWAP containing
wtSjTGR
In total, 39 novel oxadiazole-2-oxide derivatives were
synthesized following Schemes 1 and 2. Te structures
Te novel furoxan derivatives in this work were dissolved of these compounds are detailed in ꢃable 1. During syn-
in DMSO with a storage solution concentration of 64 mil- thesis of the important intermediate 6, two isomers were
limoles per liter. Te analysis of ꢃrxR activity of SWAP inevitably formed when the oxadiazole-N-oxide ring was
containing wtSjꢃGR and rSjꢃGR-Sec was performed constructed by sodium nitrate-mediated cyclization.
based on methods described previously [21]. Analysis of Gasco and co-workers reported [23] that intramolecular
Scheme 1 Synthesis oꢀ compounds 6a–6d. Reagents and conditions: (1) NaH, THF, triethyl-phosphonoacetate, r.t; (2) DIBAL-H, DCM, −78 °C; (3)
NaNO , AcOH, 60 °C.THF, tetrahydroꢀuran; DIBAL-H, diisobutylaluminum hydride; DCM, dichloromethane; r.t., room temperature.
2

Li et al. Parasites Vectors
(2021) 14:225
Page 5 of 11
Scheme 2 Synthesis oꢀ compound 7 and 9. Reagents and conditions: (1) Acyl chloride, Et N, DCM, DMAP, r.t; (2) benzene sulꢀonyl chloride, Et N,
3
3
DCM, DMAP, r.t; (3) amine, K CO , NaI, MeCN, r.t. DCM, dichloromethane; DMAP, dimethylaminopyridine; r.t., room temperature.
2
3
hydrogen bond played a crucial role on the formation of in ꢃable 1, all intermediates bearing hydroxy (6a–6d)
the main isomer during cyclization and could stabilize showed better inhibitory activity against both enzymes
the structure of the isomer a (Scheme 3).
than furoxan. Te pyridine ring in the structure of com-
For our case, two isomers, 6 and 6’, were obtained pound 6a had a stronger electron-withdrawing eꢂect on
Scheme 1), and one of them was the main product after the oxadiazole-2-oxide ring than compound 6b, and 6a
(
cyclization. ꢃo conꢀrm the position of the N-oxide moi- (IC 70nM of SWAP containing wtSjꢃGR, IC 36nM
5
0
50
ety, and whether the main product was 6 or 6 ’, the crystal of rSjꢃGR-Sec) had better inhibitory activity on both
of the main product was obtained. It was interesting that enzymes than 6b (IC 0.14µM of SWAP containing
5
0
the X-ray conꢀrmed that the main product had a similar wtSjꢃGR, IC 0.39µM of rSjꢃGR-Sec). Compared with
5
0
conformation with isomer a in Scheme 3, and speciꢀcally, 6b, the electron-donating methoxy group at the ortho
the main product was 6a (Scheme 1). Te crystal struc- position of pyridine (like compound 6c) decreased the
ture showed that there were two independent molecules inhibitory activity (6c, IC 1.1µM of SWAP containing
5
0
in an asymmetric unit (Fig. 3a). One molecule’s nitro- wtSjꢃGR, IC 2.4µM of rSjꢃGR-Sec). Notably, electron-
5
0
gen atoms were labeled N1, N2 and N3, and the other withdrawing group triꢁuoromethyl made 6d exhibited
molecule’s nitrogen atoms were labeled N4, N5 and N6. excellent inhibitory eꢂect (IC 7.5nM of SWAP contain-
5
0
However, intermolecular hydrogen bonds, instead of ing wtSjꢃGR, IC 55.8nM of rSjꢃGR-Sec). Tese data
5
0
intramolecular hydrogen bonds, were found in the crys- indicated that the electron-withdrawing substituents
tal of 6a. Te molecule’s nitrogen atoms labeled N1, N2 very possibly provided enhanced potency.
and N3 formed a hydrogen bond helix chain (Fig. 3b),
ꢃo further investigate the relationship between the
while the nitrogen atoms labeled N4, N5 and N6 formed structure and bioactivity, modiꢀcation of hydroxyl group
a hydrogen bond zigzag chain (Fig. 3c). Tis result also into the ester group and amine, respectively, aꢂorded
indicated that after the pyridine replaced the benzene, compounds 7 and 9. It was found that the ester group
the intramolecular hydrogen bond shown in Scheme 3 could not eꢂectively improve the inhibitory eꢂect,
might not be necessary for the formation of main isomer although the ethyl ester (7ca), chloracetyl ester (7ae,
6
a–d.
7be), benzoyl ester (7af, 7cf) and acryloyl ester (7bj)
exhibited inhibitory eꢂects similar to furoxan. Only 7ca
(IC 34.77µM of SWAP containing wtSjꢃGR) showed
TGR activity assessment
5
0
With furoxan as the positive control, oxadiazole-2-ox- inhibitory eꢂects against SWAP containing wtSjꢃGR,
ides (6, 7, 9) were evaluated for their inhibitory activity but not as good as furoxan. Among chloracetyl esters,
against both rSjꢃGR-Sec and SWAP containing wtSjꢃGR perhaps because the pyridine ring of 7ae had a stronger
under diꢂerent concentrations (ꢃable 1). As shown electron-withdrawing eꢂect, 7ae (IC 4.23µM of SWAP
5
0

Li et al. Parasites Vectors
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Table 1 Structures oꢀ oxadiazole-2-oxide derivatives and their inhibitory activity against the TrxR activity oꢀ SWAP containing wtSjTGR
and rSjTGR-Sec
Compounds
IC50 of SWAP containing
R’
IC50 of rSjTGR-Sec (µM)
5.87±±.ꢁ7
wtSjTGR (µM)
Furoxan
/
/
/
/
/
4.00±0.4ꢀ
0.07±0.0±
6
a
0.0ꢁ6±0.00ꢀ
6
b
0.±4±0.0ꢁ
0.ꢁ9±0.0ꢀ
6
c
±.±ꢀ±0.ꢀ7
ꢀ.ꢁ8±0.4ꢀ
6
d
0.00747±0.00095
0.05580±0.00ꢁ±8
7
aa
ab
>50
>50
>50
>50
>50
>50
7
7
ac
ad
ae
af
7
>50
>50
>50
7
4.ꢀꢁ±0.54
8.87±0.46
7
ꢁ.ꢀꢁ±0.05
7
ag
ah
>50
>50
>50
>50
>50
>50
7
7
ai
9
aa
ab
ꢀ±.57±±.44
0.6ꢀ±0.0ꢁ
>50
>50
±.5ꢀ±0.ꢀ0
>50
9
9
ac
9
ad
ba
>50
>50
7
>50
>50
>50
>50
>50
>50
7
bb
bc
7
7
bd
>50
>50

Li et al. Parasites Vectors
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Page 7 of 11
Table 1 (continued)
Compounds
IC50 of SWAP containing
R’
IC50 of rSjTGR-Sec (µM)
wtSjTGR (µM)
7
be
bf
bg
bh
±6.40±0.67
±4.8ꢀ±ꢁ.67
>50
7
>50
>50
>50
7
>50
>50
7
7
bi
>50
5.80±±.55
>50
>50
>50
>50
7
bj
7
bk
9
ba
bb
bc
>50
>50
>50
>50
>50
>50
9
9
9
bd
be
ca
cf
ca
da
df
da
0.04ꢀ±0.0±
NA
9
0.6ꢁ±0.±±
ꢁ4.77±±.ꢁꢁ
±ꢀ.5±±0.68
0.ꢀꢀ±0.0±
>50
7
7
>50
9
>50
>50
>50
>50
>50
>50
7
7
9
±±.6ꢀ±±.9ꢁ
ꢁ.7ꢁ±±.67
IC50 values > 50 μM signify lack of ꢀtted curve through the dose-response data—that is, ꢁat response within the range tested
NA not applicable
containing wtSjꢃGR) showed a slight advantage over but 7cf did not show an inhibitory eꢂect on rSjꢃGR-Sec.
be (IC 16.40µM of SWAP containing wtSjꢃGR, IC Among acrylate compounds, only 7bj showed inhibi-
4.82µM of rSjꢃGR-Sec). However, 7ae did not show tory activity against SWAP containing wtSjꢃGR (IC
7
50
50
1
5
0
an eꢂective inhibitory eꢂect on rSjꢃGR-Sec. As for 5.80µM).
phenyl derivatives, 7af (IC 8.87µM of SWAP contain-
Some amine derivatives (9aa, 9ab, 9bd, 9be, 9da)
showed inhibitory activity, among which 9bd had an
5
0
ing wtSjꢃGR, IC 3.23µM of rSjꢃGR-Sec) and 7cf (IC
50
50
1
2.51µM of SWAP containing wtSjꢃGR) did not diꢂer inhibitory eꢂect against SWAP containing wtSjꢃGR at
signiꢀcantly in inhibiting SWAP containing wtSjꢃGR, the nanomolar level (IC = 42nM). Compared with
5
0

Li et al. Parasites Vectors
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Scheme 3 The mechanism oꢀ ꢀorming an oxadiazole-N-oxide ring reported by Gasco and co-workers.
Fig. 3 a Crystal structure oꢀ 6a; b hydrogen bond helix chain ꢀormed by molecule’s nitrogen atoms labeled N1, N2 and N3; c hydrogen bond zigzag
chain ꢀormed by the molecule’s nitrogen atoms labeled N4, N5 and N6.
phenyl amino derivative 9aa, 9ba, 9ca and 9da, the pyri- good inhibitory activity against SWAP containing
dine with strong electron-withdrawing ability improved wtSjꢃGR (IC50 0.042µM). Interestingly, compound 9be
the activity, such as compounds 9aa (IC 21.57µM to is an intermediate of a synthetic target molecule, but it
5
0
SWAP containing wtSjꢃGR) and 9da (IC 11.62µM to displayed much better inhibitory eꢂects (IC50 0.63µM to
5
0
SWAP containing wtSjꢃGR, IC 3.73µM to rSjꢃGR- SWAP containing wtSjꢃGR, IC50 0.22µM to rSjꢃGR-Sec)
5
0
Sec) showed better inhibition eꢂects than 9ba (IC
>
0
than furoxan. Perhaps the sulfamine or Si-O-N moieties
5
5
0µM) and 9ca (IC > 50µM). Similar to phenyl amino in molecule 9be has some inꢁuence on enzyme inhibi-
5
0
derivatives, 4-triꢁuoromethoxyphenyl amino deriva- tion ability; this result might provide a new direction for
tive 9ab (IC 0.62µM to SWAP containing wtSjꢃGR, the design of additional compounds.
50
IC 1.52µM to rSjꢃGR-Sec) showed better inhibitory
50
eꢂects than 9bb (IC > 50µM both to SWAP containing Docking studies
5
0
wtSjꢃGR and rSjꢃGR-Sec). However, among piperazine ꢃo rationalize the obtained bioactivity data and to
substituted derivatives (9ac, 9ad and 9bd), 9bd exhibited understand how the synthesized inhibitors interact with

Li et al. Parasites Vectors
(2021) 14:225
Page 9 of 11
Fig. 4 In a–c, the protein (SjTGR) is shown as ribbons, the synthesized inhibitors are shown as orange sticks, and the residues that can interact
with inhibitors are shown as green sticks. a Compound 6d ꢀorms a π-cation interaction (red dashed line) with Arg393 and hydrogen bonds (yellow
dashed lines) with Ser117, Thr153 and Asp433. b Compound 7af ꢀorms a π-cation interaction with Arg393 and hydrogen bonds with Cys154, and
Asp433. c Compound 9ab ꢀorms a π-cation interaction with Arg393, a π–π stacking interaction (blue dashed line) with Tyr296, and a hydrogen
bond with Ser276. d Superposition oꢀ docking poses oꢀ compounds 6d (blue), 7af (purple), 9ab (green) in the binding pocket oꢀ SjTGR. SjTGR,
Schistosoma japonicum thioredoxin glutathione reductase.
+
Fig. 5 LigPlot generated two-dimensional schematic overview oꢀ molecular interactions between SjTGR and compounds 6d, 7af and 9ab.
Hydrogen bonds are indicated by green dashed lines with corresponding distances between the atoms given in Å. Hydrophobic contacts are
shown by red arcs with spokes radiating toward the atoms involved. SjTGR, Schistosoma japonicum thioredoxin glutathione reductase.
schistosomal proteins, the selected compounds with (Fig. 4b), while the pyridine ring and oxadiazole ring did
high activity 6d, 7af and 9ab were docked to the avail- not form any type of interactions with the residues of
able crystal structure of SjꢃGR (thioredoxin glutathione Arg393. Compound 9ab formed one hydrogen bond with
reductase from Schistosoma japonicum, PDB ID:4LA1). Ser276, a π-cation interaction with Arg393 and a π-π
Te crystal structure of SjꢃGR is a homodimer, and we stacking interaction with ꢃyr296. It was observed that the
retained chain B for docking studies. Te results are dis- oxadiazole ring participated in the formation of chemi-
played in Figs. 4 and 5.
cal bonds, to constitute more interaction bonds. Te dis-
Compound 6d formed four hydrogen bonds with tance between the oxadiazole ring and other ring-shaped
Asp433, Ser117 and Tr153, and a π-cation interaction conjugated structures should be taken into consideration.
with Arg393 (Fig. 4a), and two strong hydrogen bonds After superimposing three compounds (6d, 7af and 9ab)
existed between the hydroxyl moiety of 6d and SjꢃGR. together (Fig. 4d), each of them formed one π-cation
Tese results could explain the highest activity of 6d. interaction with Arg393; therefore, it was speculated that
Compound 7af formed two hydrogen bonds with Cys154 Arg393 played an anchor role during the binding process.
and Asp433, and a π-cation interaction with Arg393

Li et al. Parasites Vectors
(2021) 14:225
Page 10 of 11
Authors’ contributions
On the other hand, a hydrophobic eꢂect was shown
by red arcs with spokes radiating toward the atoms
involved (Fig. 5). Te numbers of the residues that had
hydrophobic contacts with the inhibitors were nega-
tively associated with enzyme inhibitory activity. Among
three compounds (6d, 7af and 9ab), compound 7af had
the most hydrophobic contacting residues and it had the
lowest activity, while compound 6d had the least hydro-
phobic contacting residues and it exhibited the highest
activity.
DS, YH and GL designed the experiments and draꢀted the manuscript. GL, HC
and WZ prepared and identiꢁed the compound. QG and SL perꢀormed the
biological experiments and conducted the data analysis. CF perꢀormed the
docking study. GL, QG and CF draꢀted the initial version oꢀ the manuscript. DS,
YH and GL draꢀted the ꢁnal version oꢀ the manuscript. All authors read and
approved the ꢁnal manuscript.
Funding
This work was supported by the National Natural Science Foundation oꢀ China
(No. 81773560 and No. 31402192).
Availability of data and materials
Data supporting the conclusions oꢀ this article are included within the article
and its additional ꢁle.
Conclusions
Ethics approval and consent to participate
Not applicable.
Using furoxan as the leading compound, a series of novel
esters and amine derivatives have been synthesized by
modifying the cyano moiety into an ester or amine group
and replacing phenyl moiety with its bioisosteres pyridine
or substituted pyridine. Te structure of the key interme-
diate for target compounds was conꢀrmed by crystallog-
raphy. Te inhibitory activity of all title compounds and
key intermediates was evaluated against the ꢃrxR activ-
ity of both rSjꢃGR-Sec and SWAP containing wtSjꢃGR
for the ꢀrst time. Several new derivatives, 6a–d, 9ab,
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
School oꢀ Liꢀe Science and Engineering, Southwest University oꢀ Science
2
and Technology, Mianyang 621010, China. National Reꢀerence Laboratory
oꢀ Animal Schistosomiasis, Key Laboratory oꢀ Animal Parasitology oꢀ Ministry
oꢀ Agriculture, Shanghai Veterinary Research Institute, Chinese Academy
oꢀ Agricultural Sciences, Shanghai 200241, China. Marine College, Shandong
University (Weihai), Weihai 264209, China.
9bd and 9be, showed better activity toward rSjꢃGR-Sec
3
or SWAP containing wtSjꢃGR than furoxan. Te SAR of
these novel furoxan derivatives was preliminarily ana-
lyzed. A pyridine ring with strong electron-withdrawing
ability might be beneꢀcial for enhancing the inhibitory
eꢂect. Docking studies showed that hydroxyl moiety can
form two strong hydrogen bonds with SjꢃGR, which is
of great help in enhancing the inhibitory activity of the
compounds. Compound 6d with triꢁuoromethyl on the
pyridine ring has the highest activity and is a good SjꢃGR
inhibitor and could be a potent antischistosomal agent.
Received: 27 July 2020 Accepted: 9 February 2021
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