Decoquinate derivatives: A new class of potent antischistosomal agents against Schistosoma japonicum

Article abstract of DOI:10.1016/j.cclet.2017.03.036

Decoquinate (1), an old and inexpensive coccidiostat, exhibited potent antimalarial activity, however, its antischistosomal activity against Schistosoma japonicum has not yet been evaluated. Based on decoquinate, a series of decoquinate derivatives was designed, synthesized, evaluated as a new class of antischistosomal agents against S. japonicum adult worms in vitro. Among them, compound 15 killed 100% of adult S. japonicum in 72?h at the concentration of 10?μmol/L in vitro, exhibited stronger worm-killing activity than PZQ in vitro and could serve as a promising lead compound to develop new antischistosomal agents.

Full text of DOI:10.1016/j.cclet.2017.03.036

Accepted Manuscript
Title: Decoquinate derivatives: A new class of potent
antischistosomal agents against Schistosoma japonicumin
Authors: Wen-Long Wang, Li-Jun Song, Bo-Chun Hu, Li
Miao, Xiao-Yu Chen, Wen-Hua Fan, Xu-Ren Yin, Shuang
Shen, Zhao-Feng Ding, Chuan-Xin Yu
PII:
S1001-8417(17)30124-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2017.03.036
CCLET 4031
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
26-12-2016
27-2-2017
26-3-2017
Please cite this article as: Wen-Long Wang, Li-Jun Song, Bo-Chun Hu,
Li Miao, Xiao-Yu Chen, Wen-Hua Fan, Xu-Ren Yin, Shuang Shen, Zhao-
Feng Ding, Chuan-Xin Yu, Decoquinate derivatives: A new class of potent
antischistosomal agents against Schistosoma japonicumin, Chinese Chemical
Lettershttp://dx.doi.org/10.1016/j.cclet.2017.03.036
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Original article
Decoquinate derivatives: A new class of potent antischistosomal agents against
Schistosoma japonicumin
Wen-Long Wang a, b, , Li-Jun Song b, #, Bo-Chun Hu a, # , Li Miao a, Xiao-Yu Chen a, Wen-Hua Fan a, Xu-
Ren Yin b, Shuang Shen b, Zhao-Feng Ding d, Chuan-Xin Yu b, c,*
a School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China
b Key Laboratory of National Health and Family Planning Commission on Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on
Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China
c Medical School, Jiangnan University, Wuxi 214122, China
d Suzhou Kemu Veterinary Pharmaceutical Co., Ltd, Meiyan High-technology Development Zone, Suzhou 215225, China
 Corresponding author.
E-mail address: wwenlong2011@163.com (W.-L. Wang), chxnyu@163.com (C.-X. Yu)
#These authors contributed equally to this work.
ARTICLE INFO
Article history:
Received 27 December 2016
Received in revised form 27 February 2017
Accepted 8 March 2017
Available online
Graphical abstract
Based on decoquinate (1), a series of decoquinate derivatives was designed, synthesized, evaluated as a new class of
antischistosomal agents. Among them, compound 15 killed 100% of adult S. japonicum in 72 hours at the concentration of 10
mol/L in vitro, exhibited stronger worm-killing activity than that of PZQ.
ABSTRACT
Decoquinate (1), an old and inexpensive coccidiostat, exhibited potent antimalarial activity, however, its antischistosomal activity against Schistosoma
japonicum has not yet been evaluated. Based on decoquinate, a series of decoquinate derivatives was designed, synthesized, evaluated as a new class of
antischistosomal agents against S. japonicum adult worms in vitro. Among them, compound 15 killed 100% of adult S. japonicum in 72 hours at the
concentration of 10 mol/L in vitro, exhibited stronger worm-killing activity than PZQ in vitro and could serve as a promising lead compound to develop new
antischistosomal agents.
Keywords:
Decoquinate
Derivatives
Worm-killing activity
Schistosoma japonicum
Structure-activity relationships (SARs)
———

1. Introduction.
Schistosomiasis is a neglected tropical disease (NTD) caused by blood-dwelling trematodes belonging to the genus Schistosoma.
Schistosoma haematobium, Schistosoma japonicum, and Schistosoma mansoni are the main species parasitizing humans [1]. It has
been estimated that 779 million people are at risk for schistosomiasis transmission, with 207 million infected in 76 countries and
territories [1, 2]. In China, the disease caused by Schistosoma japonicum remains a major public health concern with more than 280
thousand people infected [3]. In the absence of a vaccine, praziquantel (PZQ) is the only drug recommended by the World Health
Organization for the treatment and control of schistosomiasis through mass drug administration (MDA) program for more than three
decades [4]. The massive and exclusive use of PZQ has triggered concerns about the emergence of drug resistant parasite, and evidence
of drug resistance has been reported [4-6]. Therefore, it is imperative to develop new antischistosomal agents for the treatment of
schistosomiasis. Drug repositioning is attractive option in the case of tropical diseases for which the finance and resources to support
de-novo drug development are limited [7]. Artemisinin and its derivatives, which are a vital conerstone in treatment and control of
malaria, were now considered as alternative chemotherapies against S. japonicum [7]. Decoquinate (1) is an old and inexpensive
coccidiostat. Like Artemisinin, Decoquinate exhibited strong antimalarial activity. [8-10]. We hypothesized that Decoquinate, acting
similarly as Artemisinin, may also possess antischistosomal activity. Using decoquinate as a template, a series of compounds were
designed and synthesized, with the aim to improve aqueous solubility and other drug like properties, and their antischistosomal
activities were evaluated against S. japonicum adult worms in vitro. (Fig 1). Among them, compound 15 killed 100% of adult S.
japonicum in 72 hours at the concentration of 10 mol/L in vitro, while praziquantel (PZQ) (10 µmol/L) caused 37.5% of adult S.
japonicum death in 72 hours. The result indicated that compound 15 had stronger worm-killing activity than PZQ in vitro and could
serve as a promising lead compound to develop novel antischistosomal agents (Fig. 1).
2. Results and discussion
2.1 Chemistry
Based on the structure of decoquinate 1, compounds 7a-7b, 12a-12c and 13-21 were designed and synthesized (Table 1). Phenolic
compound 2 was nitrated with 65% nitric acid in the presence of catalytic amounts of iron(III) nitrate nonahydrate to give compound 3
in 52%. After alkylation of hydroxyl group on compound 3, compound 4a and 4b were obtained. Nitro compounds (4a and 4b) were
reduced by Fe/NH4Cl to produce amino compounds (5a and 5b), which reacted with ethoxymethylenemalonic diethyl ester (EMME)
to give the intermediates 6a and 6b using similar methods reported by wang group (11). The last cycle-closing reaction was carried out
by heating the intermediates 6a and 6b in diphenyl ether at 260 oC to obtain the key intermediates 7a and 7b (Scheme 1) [11]. By
removal of ethoxy group from decoquinate, intermediates 12a-12c were synthesized using similar procedures to compounds 7a-7b
(Scheme 2). Ester compounds 1, 7a-7b, 12a-12c were treated with various amine to afford the target compounds 13–21 (Scheme 3).
2.2 Worm-killing activities and structure-activity relationships
All the synthesized compounds were evaluated their worm-killing activities against adult S. japonicum in vitro, using vehicle as
negative control and PZQ as positive control [12-16], and the results were detailed in table 1. We initially prepared intermediates 7a–
7b and 12a–12c by the route outlined in Scheme 1 and Scheme 2, in which we replaced decyl group on 1 with pentyl group (compound
7a) or pent-4-en-1-yl group (compound 7b), or removed ethoxy group (compound 12a) from 1, or substituted decyl group on
compound 12a with pentyl group (compound 12b) or pent-4-en-1-yl group (compound 12c). We noticed that none of them showed
worm-killing activity against adult S. japonicum in vitro at the concentration of 100 mol/L. To explore novel antischistosomal agents
and investigate the SARs, amide compounds 13–21 were synthesized (scheme 3). As for compounds 13–16 converted from
decoquinate 1, all of them obviously exhibited worm-killing activities against adult S. japonicum in vitro. Among amide compounds
with six-membered ring at R3 position, compound 13 with morpholino group showed lower antischistosomal activities than compound
14 with 4-methylpiperazin-1-yl group, which killed 75 % of adult S. japonicum at the concentration of 10 mol/L in 72 hours. Opening
the piperazine ring on compound 14 led to compound 15 with (2-(diethylamino)ethyl)amino group, which completely killed worms at
the concentration of 10 mol/L in 72 hours and exhibited stronger worm-killing activity than PZQ in vitro. In addition, replacement of
morpholino group (compound 13) with bis(2-hydroxyethyl)amino group (compound 16) increased worm-killing activity. The results
demonstrated that ring-opening strategy at R3 position significantly improved worm-killing activity. Converting decyl group into
pentyl group, the antischistosomal activities of compound 17 was obviously decreased compared to compound 15. Introduction of C=C
bond into R1 position, the activity of compound 18 dropped significantly compared to compound 15 and 17. These results
demonstrated that long saturated alky group at R1 position could have a beneficial effect on worm-killing activity. Among the
compounds without ethoxy group (19-21), compounds 19–21 showed lower worm-killing activities than corresponding compounds
with ethoxy group (15, 17-18). These results demonstrated that ethoxy group on R2 position took positive effect on worm-killing
activity.
Furthermore, the activities against female or male adult worms were also observed. The mortality rate was 75.0% for male worms or
50.0% for female worms in 24 h at 25 μmol/L of compound 14. When the concentration was increased to 50 μmol/L, compound 14
killed 92.8% of male worms and 50.0% of female worms in 24 h. (Fig. 2A). The mortality rate was 40.0% for male worms or 27.4%
for female worms in 24 h at 10 μmol/L of compound 15 (Fig. 2B). However, the result of statistical analysis showed that there were no
differences between male and female worms killing activity of compounds 14 or 15.

The morphological alternations of Schistosoma japonicum worms incubated with compound 15 in vitro after 48 hours were observed
using an inverted microscopy (Leica, Wetzlar, Germany). The surface of worms in negative control was smooth. The head sucker and
the ovarian structure in the body were clearly visible. However, the body of worms incubated with compound 15 became dimmed. The
head and the structure in the body was blurred. The worms curled up and there were many vacuoles on the surface of worms (Fig. 3).
2.3 Cytotoxicity assay of compounds 14 and 15 on Hela cells [14]
The cytotoxic assessment of the compounds 14 and 15 on the Hela cells was observed at different concentrations (5, 10, 20, 40, 80
μmol/L) for 48 h using the MTT method [14] and the viability of the Hela cells treated with compound 14, 15 and PZQ at the
concentration of 20 mol/L was 99%, 90% and 83% respectively. The results indicated that compounds 14 showed lower cytotoxicity
than PZQ, and the difference was statistically significant (P<0.05). Meanwhile, compound 15 has the similar cytotoxicity to PZQ，and
the difference was not statistically significant (P > 0.05).
3. Conclusion
Given the emergence of PZQ resistant parasites in the near future, it is undoubtedly urgent to develop new chemotherapy for
schistosomiasis. In this paper, we synthesized a series of decoquinate derivatives as novel antischistosomal agents against adult S.
japonicum in vitro, and identified compound 15 as potent antischistosomal agent. The study of these structurally diverse decoquinate
derivatives would likely provide meaningful information for developing new potent antischistosomal agents.
4. Experimental
4.1 General procedure for the synthesis of compounds 7a-7b (exemplified by 7a)
Nitric acid (10 mL, 65%) was added dropwisely into a mixture of 2-ethoxyphenol (10.00 g, 72.50 mmol) and iron (III) nitrate
o
nonahydrate (900 mg, 2.23 mmol) in THF (250 mL) through a constant pressure dropping funnel at 0 C for 30 minutes. Then the
mixture was stirred at room temperature for 30 min and quenched by water (100 mL) and dichloromethane (150 mL). The aqueous
phase was extracted with dichloromethane (100 mL × 3), then the combined organic phases were then processed in the usual way and
chromatographed to afford compound 3 (6.90 g, 52%) as solid. The mixture of compound 3 (5.00 g, 27.30 mmol), potassium carbonate
(15.10 g，109.20 mmol) and 1-bromopentane (3.57 mL, 28.70 mmol ) in MeCN (50 mL) was refluxed overnight, then the mixture was
diluted with EtOAc (200 mL), the organic phases were then processed in the usual way and chromatographed to afford compound 4a
(5.10 g, 74%) as solid. A solution of compound 4a (5.00 g, 19.70 mmol) in ethanol/water (120 mL/120 mL) was treated with iron
powder (3.32 g, 59.30 mmol) and ammonium chloride (3.17 g, 59.30 mmol) and the mixture was heated to reflux overnight. The
mixture was then filtered and concentrated under reduced pressure. The resulting oil was partitioned between water and ethyl acetate,
and the organic phase was then washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, and concentrated
under reduced pressure to provide the desired compound 5a (2.87 g, 65%) as black oil without purification to use for next step. The
mixture of Ethoxymethylenemalonic diethyl ester (EMME, 2.42 g, 11.21 mmol) and compound 5a (2.50 g, 11.21 mmol) in EtOH (100
mL) was refluxed overnight. After removal of EtOH, the mixture was chromatographed to afford compound 6a (3.01 g, 68%) as
yellow oil. Diphenyl ether (10 mL) was heated to 260 oC and compound 6a (1.00 g, 2.54 mmol) was added. The reaction mixture was
stirred at 260 oC for15 minutes, and then cooled to room temperature. EtOAc (25 mL) was added to the mixture and the obtained solid
was filtrated, washed with EtOAc and dried to obtain 7a (590 mg, 67%) as solid.
4.2 General procedure for the synthesis of compounds 12a - 12c (exemplified by 12a)
The mixture of compound 8 (2.78 g, 20.0 mmol), potassium carbonate (11.03 g，80 mmol) and 1-bromodecane (4.55 mL, 22.0
mmol) in MeCN (50 mL) was refluxed overnight, then the mixture was diluted with EtOAc (200 mL), the organic phases were then
processed in the usual way and chromatographed to afford compound 9a (4.47 g, 80%) as solid. A solution of compound 9a (4.47 g，
16.0 mmol) in ethanol (100 mL) and water (100 mL) was treated with iron powder (2.69 g, 48.0 mmol) and ammonium chloride (2.54
g, 48.0 mmol) and the mixture was heated to reflux overnight. The mixture was then filtered and concentrated under reduced pressure.
The resulting oil was partitioned between water and ethyl acetate, and the organic phase was then washed with saturated aqueous
sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to provide the desired compound 10a
(2.59 g, 65%) as black oil without purification to use for next step. The mixture of Ethoxymethylenemalonic diethyl ester (EMME,
2.08 mL, 10.4 mmol) and compound 10a (2.59 g, 10.4 mmol) in EtOH (100 mL) was refluxed overnight. After removal of EtOH, the
mixture was chromatographed to afford compound 11a (2.97 g, 68%) as yellow oil. Diphenyl ether (10 mL) was heated to 260 oC and
compound 11a (2.97 g，7.08 mmol) was added. The reaction mixture was stirred at 260 oC for15 minutes, and then cooled to room
temperature. EtOAc (25 mL) was added to the mixture and the obtained solid was filtrated, washed with EtOAc, and dried to obtain
12a (1.45 g, 55%) as solid.
4.3 General procedure for the synthesis of compounds 13 – 21 (exemplified by 13)
The mixture of decoquinate 1 (100 mg, 0.24 mmol) and morpholine (209 L, 2.4 mmol) was stirred at 100 oC for 12 hours, cooled to
room temperature and diluted with dichloromethane (0.5 mL). Then petroleum ester was added to the mixture. The solid was
precipitated, filtrated and dried to obtain 13 (60 mg, 55%).

4.4 Worm-killing activity of compounds DQ 1, 7a-7b, 12a-12c, 13-21 on S. Japonicum adult worms in vitro
Stock solutions of compounds DQ 1, 7a-7b, 12a-12c, 13-21 and praziquantel were prepared by dissolving the drugs (1 mg) in
dimethyl sulfoxide (DMSO, 0.4 mL) and adding RPMI 1640 medium (0.6 mL). S. japonicum worms obtained from mice (C57BL/6,
female, 22–24 g, each infected with 50 cercariae) were washed in RPMI 1640 medium, kept at pH 7.5 with HEPES (20 mmol/L) and
supplemented with penicillin (100 UI/mL), streptomycin (100 mg/mL) and 10% fetal bovine serum (FBS, Gibco). After washing, 8–15
adult worms were transferred to each well of a 24-well culture plate containing 2 mL of the same medium. The worms were cultured
for 30 to 60 min at 37 °C in a humid atmosphere containing 5% CO2, and then different concentrations of compounds DQ 1, 7a-7b,
12a-12c, 13-21 (10, 25, 50, 100 µmol/L) diluted with RPMI 1640 medium were added. Control worms were treated with equal
volumes of RPMI 1640 or DMSO, and worms treated with praziquantel (10, 25, 50, 100 µmol/L) were also observed. The worm
mobility, tegumental alterations and parasite survival were monitored under an inverted microscope (Leica, Wetzlar, Germany) at 24,
48 and 72 h. Parasite death was defined as having no motor activity during 2 min of continuous observation as well as morphological
and tegumental alterations. The tests were repeated two times when compounds showed worm killing activity below the concentration
of 100 mol/L.
4.5 Cytotoxic assay of compounds 14 and 15 on Hela cells
Cytotoxicity assays were assessed using Hela cells. Cells in the exponential growth phase were collected by centrifugation and a
concentration of 4–5ꢀ×ꢀ104/mL was created. 100 μL of the cell suspension was added to each well of a 96-well plate and cultured at
37 °C in a humid atmosphere with 5 % CO2 overnight. Compounds (at 5, 10, 20, 40 and 80 μmol/L) were added. Each concentration
was assayed in triplicate, and control wells containing no drug were assayed at the same time. The cells were cultured for 48 h at 37 °C
in a humid atmosphere with 5 % CO2. Then 20 μL of MTT (5 mg/mL) was added to each well and the culture conditions were
maintained for 4 h. The medium was removed and DMSO (150 μL) was added into each well. The value of absorbance of each well
was measured (at 570 nm) with a microplate reader to assess the toxic effect of the compounds on the vertebrate cells.
4.6 Statistical analysis
Cell survival rates after exposure to compounds 14, 15 and PZQ were determined using chi-square tests. SPSS 13.0 was used for the
statistical analyses. Differences between mean values were considered to be significant at the level of 5 %.
Acknowledgment
This work was supported by Laboratory Research Fund funded by Key Laboratory of National Health and Family Planning
Commission on Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control
Technology, Parasitic Disease Prevention and Control Platform (No. WK014-002), fund from the Department of Science and
Technology of Jiangsu Province (No. BY2016022-37), fund from the National Natural Science Foundation of China (Nos. 21472069,
30972581, 8120316), fund from the Natural Science Foundation of Jiangsu Province (Nos. BK2012544, BK20151120), fund from the
scientific research projects of Jiangsu Provincial Commission of Health and Family Planning (No. H201635), fund from the scientific
research projects of Wuxi City Commission of Health and Family Planning (No. Q201656), fund from the Ministry of Education of the
People's Republic of China (No. JUSRP51516) and fund from Research project of Jiangsu Provincial Commission of Health and
Family Planning (No. H201635) and Jiangsu Science and Technology Department (No. BM2015024).
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Fig. 1. Structures of novel decoquinate derivatives
Fig. 2. Killing activity of compounds 14 (A) and 15 (B) against male and female adult S. japonicum after 24 h.
Fig. 3. Morphological investigation of Schistosoma japonicum worms after in vitro incubation with Compound 15. After 48 h or in the case of death,
schistosomes were monitored using an inverted microscopy (Leica, Wetzlar, Germany). (A) Negative control. (B) Vehicle control. (C) 10 μmol/L of Compound
15. (D) 25 μmol/L of Compound 15.

Fig. 4. Cytotoxic assessment of compounds 14, 15 and PZQ on Hela cells.
Scheme 1. Synthesis of compounds 7a and 7b. Reagents and conditions: (a) con HNO3 (65%), Fe(NO3)3.9H2O, THF, 0 oC to r. t., 52%; (b) R1Br, K2CO3,
CH3CN, reflux, 74% - 82%; (c) Fe, NH4Cl, EtOH, reflux, 65% - 70%; (d) ethoxymethylenemalonic diethyl ester (EMME), EtOH, reflux, 68% - 70%; (e)
diphenyl ether, 260 oC, 55% - 67%.
Scheme 2. Synthesis of compounds 12a-12c. Reagents and conditions: (a) R1Br, K2CO3, CH3CN, reflux, 78% - 82%; (b) Fe, NH4Cl, EtOH, reflux, 65% - 70%;
(c) ethoxymethylenemalonic diethyl ester (EMME), EtOH, reflux, 68% - 70%; (d) diphenyl ether, 260 oC, 55% - 66%.
Scheme 3. Synthesis of compounds 13-21. Reagents and conditions: (a) amine, 100 oC, 40% - 60%.

Table 1 Worm-killing activities against S. japonicum adult worms in vitro by compounds DQ 1, 7a-7b, 12a-12c, 13-21.
Comp
R1
R2
R3
Worm-killing activity a
Conc. (mol/L)b
24 h
48 h
72 h
Vehicle c
PZQ
-
-
-
-
-
-
n.e
n.e
n.e
10
n.e
12.5%
44.4%
55.6%
77.8%
n.e
37.5%
66.7%
75.0%
100%
n.e
25
37.5%
44.4%
55.6%
n.e
50
100
100
1 d
OEt
OEt
OEt
OEt
OEt
OEt
7a d
7b d
100
100
n.e
n.e
n.e
n.e
n.e
n.e
12a d
12b d
H
H
OEt
OEt
100
100
n.e
n.e
n.e
n.e
n.e
n.e
12c d
13
H
OEt
100
n.e
n.e
n.e
OEt
10
n.e
n.e
n.e
25
n.e
n.e
n.e
50
25.0%
100%
25.0%
62.5%
71.4%
100%
33.7%
100%
100%
100%
n.e
50.0%
100%
33.3%
100%
100%
100%
50.0%
100%
100%
100%
n.e
100%
100%
75%
100
10
14
15
16
17
18
19
20
OEt
OEt
OEt
OEt
OEt
H
25
100%
100%
100%
100%
100%
100%
100%
37.5%
71.4%
100%
100%
n.e
50
100
10
25
50
100
10
25
14.3%
100%
100%
n.e
57.1%
100%
100%
n.e
50
100
10
25
n.e
12.5%
100%
100%
n.e
100%
100%
100%
n.e
50
66.7%
100%
n.e
100
10
25
n.e
n.e
n.e
50
n.e
66.7%
100%
n.e
88.9%
100%
37.5%
100%
100%
100%
n.e
100
10
100%
n.e
25
62.5%
100%
100%
n.e
100%
100%
100%
n.e
50
100
10
H

25
n.e
n.e
n.e
50
n.e
66.7%
100%
n.e
88.9%
100%
n.e
100
10
100%
n.e
21
H
25
n.e
n.e
n.e
50
n.e
n.e
62.5%
100%
100
n.e
100%
a Data collected by visual examination of worm movement and shape (the values were the averages of two tests); n.e. (no effect): all worms are scored as active
in culture with typical appearance; % = number of dead worms/total number of worms observed, and dead worms judged by lack of movement within 2 minutes
in addition to morphological and tegumental alterations; b The concentration of the chemicals on S. japonicum adult worms in vitro; c RPMI 1640 medium
containing 0.5% DMSO; d Data only showed the concentration of 100 mol/L when the chemicals have no effect on the worms at 10, 25 and 50 mol/L.