Praziquantel derivatives with antischistosomal activity: Aromatic ring modification

Article abstract of DOI:10.1111/cbdd.12153

A series of aromatic ring-modified praziquantel derivatives were prepared and evaluated against juvenile and adult stage of Schistosoma japonicumin. Several analogs comparable in activity to the drug praziquantel have been identified based on in vitro and in vivo japonuicum schistosomes worm viability assay. Structure and activity relationship of these praziquantel aromatic ring-modified compounds was revealed. Specifically, a compound in which a bromine has been introduced in the aromatic ring of praziquantel demonstrated close antischistosomal activity to praziquantel in vivo.

Full text of DOI:10.1111/cbdd.12153

Chem Biol Drug Des 2013; 82: 216–225
Research Article
Praziquantel Derivatives with Antischistosomal
Activity: Aromatic Ring Modification
to decrease worm infection rate in vivo (6). Inspired by this
Zhi-xia Wang, Jing-lei Chen and Chunhua Qiao*
discovery, we believe that extensive investigation in the
aromatic ring might provide analogs with improved worm
killing ability. We are particularly interested in those
analogs displaying juvenile worm killing capability in vivo. In
the course of our investigation, another research group
reported several more aromatic ring-modified PZQ analogs
(7) (Figure 1). The in vitro efficacy study demonstrated that
variation of the aromatic ring led to slightly decreased
activity, and a smaller thiophene replacement of phenyl
ring displayed almost comparable activity with the parent
PZQ. Nonetheless, no in vivo efficacy was disclosed for
these compounds. Herein, we report our continued effort
for the SAR study in the aromatic ring of PZQ.
College of Pharmaceutical Science, Soochow University,
199 RenAi Road, Suzhou 215123, Jiangsu, China
*Corresponding author: Chunhua Qiao,
qiaochunhua@suda.edu.cn
A series of aromatic ring-modified praziquantel deriva-
tives were prepared and evaluated against juvenile and
adult stage of Schistosoma japonicumin. Several ana-
logs comparable in activity to the drug praziquantel
have been identified based on in vitro and in vivo
japonuicum schistosomes worm viability assay. Struc-
ture and activity relationship of these praziquantel aro-
matic ring-modified compounds was revealed.
Specifically, a compound in which a bromine has been
introduced in the aromatic ring of praziquantel demon-
strated close antischistosomal activity to praziquantel
in vivo.
Experimental Section
General chemistry
All chemicals (reagent grade) used were purchased from
Sigma-Aldrich (St. Louis, MO, USA) and Sinopharm Chem-
ical Reagent Co. Ltd. (Shanghai, China). 1H NMR spectra
were measured on Varian Unity Inova 300/400 MHz NMR
Spectrometer at 25 °C and referenced to TMS. Chemical
shifts are reported in ppm using the residual solvent line
as internal standard. Splitting patterns are designed as s,
singlet; d, doublet; t, triplet; m, multiplet. HRMS spectra
were acquired on Bruker Esquire Liquid Chromatography-
Ion Trap Mass Spectrometer. Analytical thin-layer chroma-
tography (TLC) was performed on the glass-backed silica
Key words: antischistosomal, aromatic ring modification,
praziquantel derivatives
Received 15 December 2012, revised 19 February 2013 and
accepted for publication 15 April 2013
Schistosomiasis is one of the most burdensome
nevertheless neglected tropical diseases. The World Health
Organization (WHO) estimates that the number of people
that treated for schistosomiasis has risen from 12.4 million
in 2006 to 33.5 million in 2010.a Over the past 40 years,
praziquantel (PZQ, Figure 1) has been used as the only
effective drug for schistosomal disease (1,2). There are no
backup drugs for the treatment of schistosomiasis should
PZQ become less effective. Thus, there is an urgent need
to develop replacements of PZQ.
ꢀ
gel sheets (silica gel 60 A GF254). All compounds were
detected using UV light (254 or 365 nm). Analytical HPLC
was conducted on SHIMADZU LC-20AD. Prior to biologi-
cal evaluation, all compounds were determined to be
>95% pure using appropriate analytical methods (MeOH/
H2O 80% v/v, 5 min; MeOH/H2O 75% v/v, 15 min;
MeOH/H2O 60% v/v, 10 min; MeOH/H2O 80% v/v, 5 min)
based on the peak area percentage.
Ever since PZQ was first introduced by Bayer and E.
Merck in the 1970s,b much interest has been attracted in
the elucidation of the structure–activity relationship (SAR)
of PZQ. There are five positions amenable to modification
in this molecule (Figure 1). Among these, only the
exocyclic amide position R1 was heavily investigated in the
original patent and literatures (3,4). In 2007, Mathew
Group first reported the modification in the aromatic ring,
10-NH2 and 10-NO2 PZQ derivatives. Unfortunately, these
two variants displayed significantly decreased worm killing
ability compared with the parent compound (5). We
recently reported 10-hydroxy PZQ, with modest capability
General procedure for synthesis of compounds
2a–h
To 2-phenylethylamine derivatives 1a–h (1.0 mmol) was
added ethylformate (820 mg, 12 mmol, 0.9 mL) at 0 °C
and the resulting solution was stirred for 0.5 h. The mix-
ture was then heated at reflux for 12 h. The reaction was
quenched by addition of water and extracted with ethyl
acetate (3 9 15 mL). The organic layer was washed with
brine (2 9 10 mL), dried over MgSO4, and concentrated in
216
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12153

Praziquantel Derivatives with Anti-Schistosomal Activity
6.5 Hz, 1H), 7.07–6.92 (m, 3H), 5.80 (s, 1H), 3.64–3.50
(m, 2H), 2.85–2.71 (m, 2H), 2.39–2.27 (m, 3H).
2-(2-Methylphenyl) ethylformamide 2h. 95% yield. 1H
NMR (400 MHz, CDCl3) d 8.16 (s, 1H), 7.24–7.08 (m, 4H),
5.60 (s, 1H), 3.54 (dd, J = 13.5, 6.7 Hz, 2H), 2.85 (dd,
J = 15.1, 7.9 Hz, 2H), 2.33 (d, J = 8.2 Hz, 3H).
General procedure for synthesis of
2-phenylethylisocyanide 3a–h
To a solution of 2-phenylethylformamide 2a–h (1.0 mmol)
in 15 mL CH2Cl2 at À10 °C was added TEA (10 mmol)
and a solution of POCl3 (1.5 mmol) in CH2Cl2 (5 mL) drop-
wise under nitrogen atmosphere. The resulting suspension
was stirred for 3 h at À10 °C and quenched by addition
of 10 mL saturated NaHCO3 to adjust the pH to 8. The
organic layer was separated, washed with water
(2 9 20 mL) and brine (2 9 20 mL), dried over sodium
sulfate, and evaporated. The crude material was purified
by column chromatography (petroleum ether/ethyl ace-
tate = 50:1) to give the isocyanide 3a–h as a yellow oil.
Figure 1: Structure of praziquantel and reported derivatives.
vacuum to give the crude product that was purified by
flash column chromatography (petroleum ether: ethyl ace-
tate = 2:1) to afford compounds 2a–h as colorless oil.
2-(2-Methoxyphenyl) ethylformamide 2a. 93% yield.
1H NMR (400 MHz, CDCl3) d 8.06 (s, 1H), 7.22 (dd,
J = 17.7, 10.0 Hz, 1H), 7.10 (dd, J = 15.9, 7.3 Hz, 1H),
6.87 (dd, J = 16.2, 7.8 Hz, 2H), 6.00 (s, 1H), 3.81 (s, 3H),
3.46 (dq, J = 19.5, 6.3 Hz, 2H), 2.87–2.77 (m, 2H).
2-(2-Methoxyphenyl) ethylisocyanide 3a. 90% yield.
1H NMR (400 MHz, CDCl3) d 7.25 (t, J = 7.7 Hz, 1H),
7.16 (d, J = 7.1 Hz, 1H), 6.91 (t, J = 7.0 Hz, 1H), 6.86 (d,
J = 8.0 Hz, 1H), 3.81 (s, 3H), 3.58 (s, 2H), 2.98 (s, 2H).
2-(4-Bromophenyl) ethylisocyanide 3b. 80% yield. 1H
NMR (400 MHz, CDCl3) d 7.47 (d, J = 8.0 Hz, 2H), 7.11
(d, J = 7.9 Hz, 2H), 3.60 (t, J = 6.3 Hz, 2H), 2.93 (s, 2H).
2-(4-Bromophenyl) ethylformamide 2b. 97% yield. 1H
NMR (400 MHz, CDCl3)
d 8.11 (s, 1H), 7.43 (d,
J = 8.0 Hz, 2H), 7.06 (t, J = 9.5 Hz, 2H), 5.67 (s, 1H),
3.56–3.45 (m, 2H), 2.80 (t, J = 6.9 Hz, 2H).
2-(2-Bromophenyl) ethylisocyanide 3c. 83% yield. 1H
NMR (300 MHz, CDCl3) d 7.57 (d, J = 7.7 Hz, 1H), 7.30
(s, 2H), 7.17 (s, 1H), 3.67 (s, 2H), 3.13 (s, 2H).
2-(2-Bromophenyl) ethylformamide 2c. 92% yield. 1H
NMR (300 MHz, CDCl3)
d 8.14 (s, 1H), 7.55 (d,
J = 7.5 Hz, 1H), 7.25 (s, 2H), 7.12 (d, J = 5.4 Hz, 1H),
5.94 (s, 1H), 3.54 (dd, J = 24.5, 6.5 Hz, 2H), 2.99 (d,
J = 6.8 Hz, 2H), 1.99 (s, 1H).
2-(3-Methoxyphenyl) ethylisocyanide 3d. 94% yield.
1H NMR (400 MHz, CDCl3) d 7.24 (d, J = 7.8 Hz, 1H),
6.81 (d, J = 7.8 Hz, 2H), 6.76 (s, 1H), 3.80 (s, 3H), 3.60 (t,
J = 6.6 Hz, 2H), 2.96 (d, J = 6.3 Hz, 2H).
2-(3-Methoxyphenyl) ethylformamide 2d. 93% yield.
1H NMR (400 MHz, CDCl3) d 8.14 (s, 1H), 7.23 (d,
J = 7.8 Hz, 1H), 6.79 (d, J = 7.4 Hz, 2H), 6.73 (d,
J = 15.1 Hz, 1H), 5.54 (s, 1H), 3.80 (s, 3H), 3.58 (dd,
J = 12.8, 6.4 Hz, 2H), 2.81 (dd, J = 16.0, 9.4 Hz, 2H).
2-(3-Bromophenyl) ethylisocyanide 3e. 81% yield. 1H
NMR (400 MHz, CDCl3) d 7.40 (d, J = 13.0 Hz, 2H), 7.19
(s, 2H), 3.62 (s, 2H), 2.96 (s, 2H).
2-(4-Methylphenyl) ethylisocyanide 3f. 89% yield. 1H
NMR (400 MHz, CDCl3) d 7.14 (q, J = 7.8 Hz, 4H), 3.58
(t, J = 6.1 Hz, 2H), 2.95 (s, 2H), 2.35 (s, 3H).
2-(3-Bromophenyl) ethylformamide 2e. 97% yield. 1H
NMR (400 MHz, CDCl3)
d 8.13 (s, 1H), 7.37 (d,
J = 8.2 Hz, 2H), 7.25–7.08 (m, 2H), 5.78 (s, 1H), 3.53 (dt,
J = 23.4, 6.1 Hz, 2H), 2.82 (t, J = 6.8 Hz, 2H).
2-(3-Methylphenyl) ethylisocyanide 3g. 76% yield. 1H
NMR (400 MHz, CDCl3) d 8.41 (d, J = 8.5 Hz, 1H), 6.38
(d, J = 8.5 Hz, 1H), 4.47–4.24 (m, 2H), 1.58 (d,
J = 0.9 Hz, 4H).
2-(4-Methylphenyl) ethylformamide 2f. 84% yield. 1H
NMR (400 MHz, CDCl3) d 8.12 (s, 1H), 7.09 (dt, J = 13.8,
7.7 Hz, 4H), 5.57 (s, 1H), 3.59–3.42 (m, 2H), 2.80 (dd,
J = 12.5, 5.8 Hz, 2H), 2.33 (s, 3H).
2-(2-Methylphenyl) ethylisocyanide 3h. 81% yield. 1H
NMR (400 MHz, CDCl3) d 7.04 (d, J = 3.5 Hz, 4H), 3.42
(d, J = 7.2 Hz, 2H), 2.87 (d, J = 7.0 Hz, 2H), 2.19 (s, 3H).
13C NMR (75 MHz, CDCl3) d 136.11 (s), 134.79 (s),
2-(3-Methylphenyl) ethylformamide 2g. 98% yield. 1H
NMR (400 MHz, CDCl3) d 8.10 (s, 1H), 7.19 (dd, J = 13.8,
Chem Biol Drug Des 2013; 82: 216–225
217

Wang et al.
130.67 (s), 129.37 (s), 127.46 (s), 126.43 (s), 41.77 (s),
33.06 (s), 19.33 (s).
J = 3.4 Hz, 2H), 3.37 (s, 3H), 3.33 (s, 3H), 2.81–2.72 (m,
2H), 2.57 (s, 1H), 2.23 (s, 1H), 2.05 (d, J = 8.6 Hz, 1H),
1.75 (s, 3H), 1.67 (s, 3H), 1.44 (d, J = 9.5 Hz, 3H), 1.24
(d, J = 5.2 Hz, 3H).
General procedure for synthesis of compounds
4a–h
N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(2-(3-bromophenyl)
ethylamino) ethylcyclohexanecarboxamide 4e. 92%
yield. 1H NMR (400 MHz, CDCl3) d 7.35 (d, J = 9.3 Hz,
2H), 7.14 (dd, J = 14.5, 7.3 Hz, 3H), 6.55 (s, 1H), 4.61 (s,
1H), 4.40 (s, 1H), 3.99 (d, J = 7.3 Hz, 2H), 3.54 (dd,
J = 12.8, 6.4 Hz, 1H), 3.48 (s, 1H), 3.44 (d, J = 4.4 Hz,
2H), 3.39 (s, 3H), 3.35 (s, 3H), 2.85–2.72 (m, 2H), 2.58 (s,
1H), 2.23 (s, 1H), 1.77 (s, 2H), 1.68 (s, 1H), 1.61 (s, 2H),
1.49–1.40 (m, 2H), 1.23 (s, 1H).
To a mixture of paraformaldehyde (1.0 mmol), aminoacet-
aldehyde dimethyl acetal (1.0 mmol), and cyclohexane-
carboxylic acid (1.0 mmol, 1.0 equiv) in 10 mL methanol
was added (2-isocyanoethyl)benzene derivatives 3a–h
(1.0 mmol) dropwise at 0 °C. The solution was stirred for
48 h at room temperature and concentrated in vacuo. The
residue was redissolved in 10 mL CH2Cl2 and washed
with water (2 9 10 mL) and brine (1 9 10 mL) and dried
over anhydrous magnesium sulfate. The drying agent was
removed by filtration. After concentration, the crude mate-
rial was purified by column chromatography (petroleum
ether/ethyl acetate = 1:1) to give the colorless oil com-
pounds 4a–h.
N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(2-(4-methylphenyl)
ethylamino) ethylcyclohexanecarboxamide 4f. 74%
yield. 1H NMR (400 MHz, CDCl3) d 7.08 (d, J = 6.6 Hz,
4H), 6.93 (s, 1H), 6.43 (s, 1H), 4.58 (s, 1H), 4.39 (s, 1H),
3.98 (d, J = 3.9 Hz, 2H), 3.53 (d, J = 6.3 Hz, 1H), 3.46 (d,
J = 6.2 Hz, 1H), 3.41 (s, 2H), 3.38 (s, 3H), 3.34 (s, 3H),
2.81–2.70 (m, 2H), 2.57 (s, 1H), 2.31 (s, 3H), 2.22 (t,
J = 11.3 Hz, 1H), 1.75 (s, 2H), 1.67 (s, 1H), 1.61–1.54 (m,
2H), 1.51–1.36 (m, 3H), 1.22 (s, 2H), 0.86 (d, J = 6.7 Hz,
3H).
N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(2-(2-methoxyphenyl)
ethyl amino)ethylcyclohexanecarboxamide 4a. 96%
yield. 1H NMR (400 MHz, CDCl3) d 7.22 (d, J = 7.5 Hz,
1H), 7.11 (d, J = 7.1 Hz, 1H), 6.94–6.80 (m, 2H), 6.41 (s,
0.5 H), 4.55 (t, J = 4.9 Hz, 0.5H), 4.40 (t, J = 4.9 Hz, 1H),
3.99 (d, J = 8.6 Hz, 2H), 3.82 (d, J = 3.1 Hz, 3H), 3.51
(dd, J = 12.5, 6.4 Hz, 1H), 3.45 (d, J = 8.8 Hz, 1H), 3.40
(d, J = 9.8 Hz, 3H), 3.35 (s, 3H), 2.88–2.75 (m, 2H), 2.59
(s, 0H), 2.24 (d, J = 11.5 Hz, 1H), 1.76 (s, 2H), 1.66 (s,
2H), 1.61 (s, 3H), 1.47 (s, 2H), 1.22 (s, 2H).
N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(2-(3-methylphenyl)
ethylamino) ethylcyclohexanecarboxamide 4g. 87%
yield. 1H NMR (300 MHz, CDCl3) d 7.15 (d, J = 6.7 Hz,
1H), 7.00 (d, J = 10.7 Hz, 3H), 6.45 (s, 1H), 4.55 (s, 1H),
4.37 (s, 1H), 3.97 (s, 2H), 3.57–3.44 (m, 2H), 3.40 (s, 2H),
3.36 (s, 3H), 3.32 (s, 3H), 2.74 (d, J = 9.6 Hz, 2H), 2.54
(d, J = 11.5 Hz, 1H), 2.30 (s, 3H), 2.23 (s, 1H), 2.06 (d,
J = 21.2 Hz, 1H), 1.82–1.53 (m, 6H), 1.42 (s, 2H), 1.20 (s,
1H).
N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(2-(4-bromophenyl)
ethylamino) ethylcyclohexanecarboxamide 4b. 92%
yield. 1H NMR (300 MHz, CDCl3) d 7.38 (s, 2H), 7.06 (s,
3H), 6.54 (s, 1H), 4.58 (s, 1H), 4.38 (s, 1H), 3.95 (s, 2H),
3.50 (s, 1H), 3.41 (s, 2H), 3.37 (s, 3H), 3.33 (s, 3H), 2.72
(s, 2H), 2.56 (s, 1H), 2.18 (d, J = 11.8 Hz, 1H), 1.75 (s,
2H), 1.62 (d, J = 24.3 Hz, 3H), 1.40 (d, J = 11.8 Hz, 2H),
1.20 (s, 2H), 0.86 (s, 1H).
N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(2-(2-methylphenyl)
ethylamino) ethylcyclohexanecarboxamide 4h. 79%
yield. 1H NMR (400 MHz, CDCl3) d 7.12 (d, J = 4.1 Hz,
4H), 7.09–7.03 (m, 1H), 6.53 (s, 1H), 4.60 (s, 1H), 4.40 (s,
1H), 3.99 (d, J = 8.3 Hz, 2H), 3.45 (s, 3H), 3.38 (s, 3H),
3.35 (s, 3H), 2.85–2.74 (m, 2H), 2.59 (s, 1H), 2.32 (d,
J = 5.4 Hz, 3H), 2.27 (s, 1H), 1.76 (s, 2H), 1.64 (d,
J = 14.9 Hz, 3H), 1.53–1.37 (m, 2H), 1.23 (s, 2H).
N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(2-(2-bromophenyl)
ethylamino) ethylcyclohexanecarboxamide 4c. 77%
yield. 1H NMR (400 MHz, CDCl3) d 7.53 (t, J = 7.1 Hz,
1H), 7.23 (s, 2H), 7.14 (s, 1H), 7.08 (d, J = 5.9 Hz, 1H),
6.53 (s, 1H), 4.61 (t, J = 4.7 Hz, 1H), 4.40 (t, J = 4.9 Hz,
1H), 3.99 (d, J = 9.4 Hz, 2H), 3.55 (dd, J = 13.4, 6.7 Hz,
2H), 3.44 (dd, J = 10.0, 5.1 Hz, 3H), 3.38 (s, 3H), 3.35
(s, 3H), 2.94 (dt, J = 14.4, 7.1 Hz, 2H), 2.59 (t,
J = 11.3 Hz, 1H), 2.26 (t, J = 11.3 Hz, 1H), 1.76 (s, 3H),
1.64 (d, J = 12.8 Hz, 4H), 1.54–1.37 (m, 3H), 1.22
(s, 2H).
General procedure for the synthesis of
compounds 6a–h
N-(2,2-Dimethoxyethyl)-N-(2-oxo-2-(2-phenethylamino)ethyl)
cyclohexanecarboxamide 6a–h (0.1 mmol) was added
portionwise to methanesulfonic acid (1.6 mmol) at 0 °C.
After heating to 70 °C for 6 h, the reaction mixture was
poured into an ice–water mixture (5.0 mL), and the pH
was adjusted to 8 by addition of aqueous solution 2 N
NaOH. The solution was extracted with CH2Cl2
(3 9 10 mL). The combined organic layer was washed
with brine (2 9 10 mL) and dried over anhydrous
magnesium sulfate. After concentration, the crude material
N-(2,2-dimethoxyethyl-N-(2-oxo-2-(2-(3-methoxyphenyl)
ethylamino) ethylcyclohexanecarboxamide 4d. 91%
yield. 1H NMR (400 MHz, CDCl3) d 7.20 (s, 1H), 6.98 (s,
1H), 6.75 (s, 2H), 6.74–6.70 (m, 1H), 6.47 (s, 1H), 4.57 (s,
1H), 4.39 (s, 1H), 3.98 (d, J = 5.3 Hz, 2H), 3.78 (s, 3H),
3.54 (d, J = 5.8 Hz, 2H), 3.49–3.45 (m, 1H), 3.41 (d,
218
Chem Biol Drug Des 2013; 82: 216–225

Praziquantel Derivatives with Anti-Schistosomal Activity
was purified by column chromatography (petroleum ether/
ethyl acetate = 2:1) to afford the final product compounds
6a–h.
(t, J = 10.7 Hz, 3H), 2.47 (s, 1H), 1.76 (d, J = 21.8 Hz,
5H), 1.62–1.41 (m, 2H), 1.28 (s, 3H). 13C NMR (75 MHz,
CDCl3)
d 174.71, 164.11, 135.37, 134.51, 131.47,
128.07, 125.60, 124.58, 54.78, 48.91, 45.01, 40.68,
38.67, 29.38, 29.17, 28.97, 25.65. LCMS (ESI+) m/
z:391.1022 (M + H+) (calcd for C19H23BrN2O2 391.1016)
error: 1.5 ppm.
8-Methoxylpraziquantel 6a. 38% yield. 1H NMR
(400 MHz, CDCl3) d 7.22 (s, 1H), 6.89 (d, J = 7.7 Hz, 1H),
6.78 (d, J = 7.9 Hz, 1H), 5.16 (d, J = 12.7 Hz, 1H), 4.88
(d, J = 9.5 Hz, 1H), 4.79 (d, J = 7.0 Hz, 1H), 4.46 (d,
J = 17.5 Hz, 1H), 4.05 (d, J = 17.5 Hz, 1H), 3.84 (s, 3H),
2.93 (d, J = 16.3 Hz, 1H), 2.76 (q, J = 11.8 Hz, 2H), 2.66
(d, J = 12.4 Hz, 1H), 2.46 (t, J = 11.0 Hz, 1H), 1.87–1.66
(m, 5H), 1.55 (d, J = 12.9 Hz, 2H), 1.27 (s, 3H).13C NMR
9-Methoxypraziquantel
6d. Compound
6d
was
obtained using general procedure for preparing com-
pound 6 from compound 4d in 53% yield. 1H NMR
(400 MHz, CDCl3) d 7.20 (d, J = 8.5 Hz, 1H), 6.83 (d,
J = 7.6 Hz, 1H), 6.70 (s, 1H), 5.12 (d, J = 12.4 Hz, 1H),
4.82–4.72 (m, 2H), 4.46 (d, J = 17.4 Hz, 1H), 4.07 (d,
J = 17.4 Hz, 1H), 3.91 (d, J = 13.3 Hz, 1H), 3.80 (s, 3H),
2.91 (dd, J = 24.1, 12.3 Hz, 3H), 2.76 (t, J = 13.8 Hz,
2H), 2.46 (t, J = 11.2 Hz, 1H), 1.88–1.68 (m, 7H), 1.31
(75 MHz, CDCl3)
d 174.73, 164.25, 157.02, 133.91,
127.46, 123.78, 117.34, 108.46, 55.41, 54.81, 48.96,
45.18, 40.76, 38.49, 29.22, 28.98, 25.69, 22.50. LCMS
(ESI+) m/z: (M + H+) (calcd for C20H26N2O3 343.2016),
found: 343.2018. Error: 0.6 ppm.
(d, J = 20.4 Hz, 4H). 13C NMR (75 MHz, CDCl3)
d
10-Bromopraziquantel 6b. 40% yield. 1H NMR
(300 MHz, CDCl3) d 7.44 (s, 1H), 7.40–7.33 (m, 1H), 7.07
(s, 1H), 5.13 (s, 1H), 4.83 (s, 2H), 4.44 (s, 1H), 4.10 (s,
2H), 2.88 (s, 3H), 2.79–2.69 (m, 2H), 2.53–2.38 (m, 1H),
1.72 (s, 3H), 1.27 (s, 6H), 0.88 (s, 1H). The 1H NMR data
were consistent with reported literature (8).
174.72, 164.38, 158.60, 136.13, 126.57, 124.84, 113.88,
113.03, 108.68, 55.46, 55.29, 54.57, 48.96, 45.22,
40.75, 39.04, 29.21, 28.96, 25.90, 25.69. LCMS (ESI+)
m/z:343.2002 (M + H+) (calcd for C20H26N2O3 343.2016)
error: 4.1 ppm.
9-Bromopraziquantel 6e. Compound 6e was obtained
using general procedure for preparing compound 6 from
compound 4e in 50% yield. 1H NMR (300 MHz, CDCl3) d
7.51 (s, 1H), 7.16 (s, 1H), 5.11 (s, 1H), 4.85 (d,
J = 23.3 Hz, 2H), 4.47 (d, J = 17.8 Hz, 1H), 4.05 (d,
J = 17.8 Hz, 1H), 2.97 (s, 1H), 2.80 (s, 3H), 2.45 (s, 1H),
1.76 (d, J = 21.5 Hz, 6H), 1.59–1.39 (m, 4H).13C NMR
Synthesis of compound 5c. To
a solution of 4c
(48 mg, 0.11 mmol) in 2.0 mL CH2Cl2 was added p-tolu-
enesulfonic acidÁH2O (71 mg, 0.37 mmol) at 0 °C. The
reaction was stirred at reflux for 12 h and quenched by
addition of 5 mL saturated NaHCO3 to adjust the pH to 8.
The organic layer was separated, washed with water
(2 9 10 mL) and brine (1 9 10 mL), dried over sodium
sulfate, and evaporated. The crude material was purified
by column chromatography (petroleum ether/ethyl ace-
(75 MHz, CDCl3)
d 174.75, 164.31, 137.01, 132.08,
131.78, 130.09, 127.11, 121.35, 54.68, 48.96, 44.78,
40.72, 38.77, 29.18, 28.96, 28.46, 25.65. LCMS (ESI+) m/
z: 391.1000 (M + H+) (calcd for C19H23BrN2O2 391.1016)
error: 4.1 ppm.
1
tate = 4:1) to give 5c as a white powder (20 mg, 46%). H
NMR (400 MHz, CDCl3) d 7.53 (d, J = 7.9 Hz, 1H), 7.22
(s, 2H), 7.14–7.08 (m, 1H), 6.10 (d, J = 5.9 Hz, 1H), 5.40
(d, J = 5.9 Hz, 1H), 4.32 (s, 2H), 3.76 (t, J = 7.3 Hz, 2H),
3.05 (t, J = 7.0 Hz, 2H), 2.45 (t, J = 11.5 Hz, 1H), 1.76
(dd, J = 28.4, 14.7 Hz, 6H), 1.55–1.44 (m, 3H), 1.27 (d,
J = 9.7 Hz, 4H).
10-Methylpraziquantel 6f. Compound 6f was obtained
using general procedure for preparing compound 6 from
compound 4f in 63% yield.1H NMR (400 MHz, CDCl3) d
7.08 (d, J = 15.6 Hz, 3H), 5.16 (d, J = 13.7 Hz, 1H), 4.82
(d, J = 10.8 Hz, 1H), 4.75 (d, J = 7.4 Hz, 1H), 4.47 (d,
J = 17.4 Hz, 1H), 4.08 (d, J = 17.5 Hz, 1H), 2.88 (dd,
J = 25.7, 13.2 Hz, 3H), 2.75 (t, J = 12.5 Hz, 2H), 2.51–
2.41 (m, 1H), 2.33 (s, 3H), 1.81 (s, 3H), 1.74 (d,
J = 9.5 Hz, 3H), 1.30 (d, J = 19.6 Hz, 4H).13C NMR
Synthesis of 8-bromopraziquantel 6c. A solution of
5c (577 mg, 1.47 mmol) in concentrated H2SO4 (2.0 mL)
was stirred at 0 °C. After 30 min, the reaction was
warmed up to room temperature and the reaction mixture
was stirred for 4 h, quenched by addition of 10% aqueous
NaOH at 0 °C to adjust the pH to 8. The mixture was
extracted with CH2Cl2 (3 9 30 mL). The combined
organic layer was separated and washed with brine
(2 9 10 mL), dried over sodium sulfate, and concentrated.
The crude material was purified by column chromatogra-
phy (petroleum ether/ethyl acetate = 4:1) to give com-
pound 6c as white powder (560 mg, 97%).1H NMR
(300 MHz, CDCl3) d 7.52 (d, J = 6.9 Hz, 1H), 7.33–7.23
(m, 1H), 7.16 (s, 1H), 5.13 (d, J = 12.9 Hz, 1H), 4.85 (dd,
J = 26.0, 7.6 Hz, 2H), 4.47 (d, J = 17.2 Hz, 1H), 4.06 (d,
J = 17.1 Hz, 1H), 2.98 (d, J = 11.1 Hz, 1H), 2.84
(75 MHz, CDCl3)
d 174.76, 164.39, 136.63, 132.41,
131.56, 129.11, 128.25, 126.06, 54.97, 48.98, 45.35,
40.77, 39.14, 29.23, 28.97, 28.32, 25.70, 21.11. LCMS
(ESI+) m/z: 327.2072 (M + H+) (calcd for C20H26N2O2
327.2067) error: 1.5 ppm.
9-Methylpraziquantel 6g. Compound 6g was obtained
using general procedure for preparing compound 6 from
1
compound 4g in 56% yield. H NMR (300 MHz, CDCl3): d
7.12 (d, J = 19.1 Hz, 2H), 7.00 (d, J = 11.5 Hz, 1H), 5.13
(d, J = 13.1 Hz, 1H), 4.78 (d, J = 8.8 Hz, 2H), 4.46 (d,
J = 17.6 Hz, 1H), 4.06 (d, J = 17.6 Hz, 1H), 2.89
Chem Biol Drug Des 2013; 82: 216–225
219

Wang et al.
(t, J = 15.4 Hz, 2H), 2.75 (t, J = 13.8 Hz, 2H), 2.47 (d,
J = 11.4 Hz, 1H), 2.31 (s, 3H), 1.75 (d, J = 22.1 Hz, 5H),
1.50 (d, J = 11.3 Hz, 1H), 1.24 (s, 4H). 13C NMR
(75 MHz, CDCl3): d 174.77, 164.44, 137.22, 134.52,
129.77, 127.78, 125.34, 54.82, 48.99, 45.21, 40.78,
39.14, 29.23, 28.98, 28.65, 25.70, 20.97. LCMS (ESI+) m/
z: 327.2071 (M + H+) (calcd for C20H26N2O2 327.2067)
error: 1.2 ppm.
2H), 4.47 (d, J = 17.5 Hz, 1H), 4.07 (d, J = 17.8 Hz, 1H),
2.94–2.72 (m, 4H), 2.45 (s, 1H), 2.30 (s, 3H), 1.76 (d,
J = 35.0 Hz, 7H), 1.54 (d, J = 11.8 Hz, 2H), 1.26 (d,
J = 7.7 Hz, 3H).13C NMR (100 MHz, CDCl3) d 174.84,
169.60, 164.47, 149.46, 134.04, 132.37, 130.38, 121.10,
118.82, 55.72, 54.90, 49.09, 44.98, 40.84, 39.14, 29.79,
29.30, 29.07, 28.31, 25.78, 21.13. LCMS (ESI+) m/z:
371.1966 (M + H+) (calcd for C21H26N2O4 371.1965) error:
0.3 ppm.
8-Methylpraziquantel 6h. Compound 6h was obtained
using general procedure for preparing compound 6 from
compound 4h, in 44% yield. 1H NMR (300 MHz,CDCl3) d
7.09 (d, J = 39.2 Hz, 3H), 5.15 (d, J = 13.4 Hz, 1H),
4.83 (d, J = 21.9 Hz, 2H), 4.46 (d, J = 17.5 Hz, 1H),
4.04 (d, J = 17.4 Hz, 1H), 2.93–2.64 (m, 4H), 2.46 (s,
1H), 2.27 (s, 3H), 1.75 (d, J = 24.1 Hz, 5H), 1.52 (s,
3H), 1.26 (s, 2H). 13C NMR (75 MHz, CDCl3) d 174.42,
163.96, 136.49, 132.93, 132.48, 128.46, 126.25,
122.84, 54.79, 48.68, 45.05, 40.47, 38.41, 28.93, 28.70,
25.65, 25.41, 19.08. LCMS (ESI+) m/z: 327.2070
(M + H+) (calcd for C20H26N2O2 327.2067) error:
0.9 ppm.
Infection and collection of juvenile and adult
Schistosoma japonicum
Female ICR mice were percutaneously infected with 100
S. japonicum cercariae via shaved abdominal skin (11).
Mice were killed at 14 or 42 days after infection, and
then, juvenile and adult Schistosoma were collected by
perfusion with ice-cold Hank’s balanced salt solution
(HBSS) from mesenteric vein and livers. Groups of four
couples of adult Schistosoma were washed and placed
in a 12-well Falcon plate containing 4 mL RPMI 1640
medium and stored at 37 °C in an atmosphere of 5%
CO2 in air. Praziquantel derivatives were added to
achieve a series of diluted concentrations 10–50 lM, and
PZQ was used as reference drug. Compound activity
was assessed by survival and vitality rate (%) within
72 h.
Synthesis of 10-methoxylpraziquantel 6i. 10-Hydroxy
PZQ (114 mg, 0.35 mmol) in DMF (2.5 mL) was methylat-
ed with iodomethane (566 mg, 3.99 mmol) in the pres-
ence of potassium carbonate (504 mg, 3.64 mmol) and
tetra-butyl ammonium iodide (11.0 mg) at room tempera-
ture overnight (9). Analysis by TLC indicated a complete
reaction after 48 h. The reaction mixture was quenched
with brine and extracted with Et2O (3 9 5 mL). The
organic fractions were washed with brine, combined, dried
over Na2SO4, filtered, and concentrated in vacuum. Purifi-
cation by flash chromatography (petroleum ether/ethyl
acetate = 3:1) gave 6i as white powder (100 mg, 86%).1H
NMR (400 MHz, CDCl3) d 7.09 (s, 1H), 6.79 (s, 2H), 5.12
(s, 1H), 4.82 (s, 2H), 4.45 (s, 1H), 4.10 (s, 1H), 3.79 (s,
3H), 2.88 (s, 3H), 2.70 (s, 1H), 2.53–2.41 (m, 1H), 1.82 (s,
3H), 1.72 (s, 3H), 1.28 (s, 4H).13C NMR (75 MHz, CDCl3)
Schistosome incubation in vitro
RPMI 1640 medium supplemented with 20% newborn calf
serum, 100 U/mL penicillin, 100 U/mL streptomycin, and
0.5 lg/mL amphotericin B was used to maintain the
Schistosoma in vitro (6). The volume of medium added to
each of the 12 wells of a Falcon plate was 3.98–4.00 mL,
and then, four pairs of worms (four male and four female
worms) were placed in each well. For juvenile incubation,
50 lL of 14-day-old schistosomula suspended in the
medium was added to each well of the plate. The plate
was incubated at 37 °C in 95% air + 5% CO2 overnight
before addition of drugs or PZQ at different concentra-
tions. The final volume in each well was 4.0 mL. Control
wells contained the worms and medium only or 0.4%
dimethyl sulfoxide (DMSO) alone. After addition of the
aforementioned drugs, the plates were incubated
continuously for 72 h, and each test with juvenile or adult
schistosomes was repeated three times. During 72 h,
compound activity was assessed by percentage of sur-
vival and vitality each 24 h. The score of worm vitality is
illustrated as follows: 3 points, male schistosomes oral
sucker attached to the plate or worms moved very natu-
rally and actively and the body was transparent just as
observed in the control group during the observation
period; 2 points, worms acted weakened obviously all
over the body, with the body curled or swelling with blebs
with various sizes emerged along the tegument; 1 point,
worms acted very weak with oral sucker or tail moving
occasionally; 0 point, worms were dead without any
d
174.37, 163.98, 158.11, 133.36, 129.85, 126.28,
113.60, 109.73, 55.03, 54.64, 48.61, 44.78, 40.34, 38.98,
29.31, 28.86, 28.64, 27.52, 25.37, 25.32. LCMS (ESI+) m/
z:343.2015 (M + H+) (calcd for C20H26N2O3 343.2016)
error: 0.3 ppm.
Synthesis of 10-acetylpraziquantel 6j. Acetic anhy-
dride (0.4 mL, 2.44 mmol) was added dropwise to a
cooled solution of 10-hydroxy PZQ (200 mg, 0.61 mmol)
in pyridine (5 mL) while keeping the reaction temperature
below 5 °C (10). After addition, the reaction mixture was
then stirred at room temperature for 6 h. The mixture was
treated with 15 mL 10% aqueous NaHCO3, washed with
water (2 9 10 mL), dried by MgSO4, and concentrated to
afford the crude product that was further purified by col-
umn chromatography (petroleum ether/EtOAc = 2:1) to
give compound 6j as white powder (190 mg, 84%).1H
NMR (400 MHz, CDCl3) d 7.19 (d, J = 6.5 Hz, 1H), 7.01
(s, 2H), 5.11 (d, J = 12.9 Hz, 1H), 4.81 (d, J = 17.3 Hz,
220
Chem Biol Drug Des 2013; 82: 216–225

Praziquantel Derivatives with Anti-Schistosomal Activity
movement. The average score of 4 worms was counted
for in vitro test.
Tek microplate reader (Winooski, VT, USA). Survival ratios
are expressed in percentages with respect to untreated
cells. Values were determined from replicates of three
wells from at least three independent experiments.
Schistosoma japonicum incubation in vivo
Thirty ICR mice were divided into six groups. Each rat was
infected with 100 S. japonicum cercariae via shaved
abdominal skin (6). Forty-two days after infection, the mice
were administered a single 250 mg/kg oral doses of
compounds 6a, 6c, 6i, 6j, and PZQ, respectively. The
compounds were prepared as suspensions in 7% (vol/vol)
Tween 80 and 3% (vol/vol) ethanol before oral administra-
tion. Untreated mice (same amount of solvent only, without
compounds) served as controls (Each group include five
mice). Animals were killed at 30 day after infection, and
S. japonicum was perfused with ice-cold HBSS (pH 7.2)
from mesenteric vein and liver.
Results and Discussions
Chemistry
We initially attempted Kim group reported method to
achieve PZQ derivatives (12), which involved a key reaction
intermediate amide acetal. This unstable and highly hygro-
scopic intermediate makes this route far from an efficient
way to provide a diversity of PZQ analogs. We then
resorted to the Ugi multicomponent reaction (Scheme 1)
(7,13). The common reaction intermediate isocyanide 3
was prepared by treating 2 with POCl3 and Et3N (14).
Compounds 2a–2e were readily prepared by a reaction of
the corresponding amine with HCOOEt. Then, compounds
3a–3e reacted with paraformaldehyde, cyclohexanecarb-
oxylic acid, and aminoacetaldehyde dimethyl acetal to
yield the advanced precursor 4a–4e quantitatively (13).
Finally, 4a–4b and 4d–4h undergo the Pictet–Spengler
reaction under acidic conditions to yield PZQ derivatives
6a–6b and 6d–6h in high yield (15). Accordingly, 4c was
first converted to 5c using pyridinium p-toluenesulfonate
(pTSA) in 50% yield, which undergoes intramolecular cycli-
zation to provide 6c in concentrated H2SO4. 10-Hydroxy
PZQ was prepared according to the previously reported
method (6). It is subsequently treated with CH3I and
K2CO3, Bu4NI, in DMF (9,16) to yield compound 6i. Mean-
while, compound 6j was prepared by treating 10-hydroxy
Cytotoxicity
The antiproliferative activities of chalcone thiosemicarbaz-
ide derivatives were determined using a standard (MTT)-
based colorimetric assay (Sigma, St. Louis, MO, USA)
(10). Briefly, cell lines were seeded at a density of 5 9 103
cells/well in 96-well microtiter plates (Costar, New York,
NY, USA). After 24 h, exponentially growing cells were
exposed to the indicated compounds at final concentra-
tions of 50 lM. After 48 h, cell survival was determined by
the addition of an MTT solution (20 lL of 5 mg/mL MTT in
PBS). After 6 h, the medium was removed by aspiration.
The cells were dissolved in 150 lL DMSO, and optical
absorbance was measured at 570 nm on an Elx800 Bio-
Scheme 1: Synthesis
route
of
praziquantel (PZQ) derivative 6a–6j.
Chem Biol Drug Des 2013; 82: 216–225
221

Wang et al.
Table 1: Effects of compounds 6a–6j on 42-day adult Schistosoma japonicum in vitro culture
24 h
48 h
72 h
Survival
(%)
Vitality
(%)
Survival
(%)
Vitality
(%)
Survival
(%)
Vitality
(%)
Compounds
Control
Concentrations (lM)
Worms
25
4♂
4♀
4♂
4♀
6♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
4♂
4♀
100
100
0
100
100
0
8.3
50
67
56
58
0
100
100
0
25
50
100
50
100
0
100
100
0
8.3
16.7
67
16.7
58
0
100
100
0
25
40
40
50
100
0
0
0
0
0
100
100
0
8.3
13
13
16.7
42
0
0
0
0
0
6a
25
10
25
25
10
5
25
100
100
100
100
0
25
0
75
100
100
50
100
0
100
100
100
100
100
33
100
73
100
50
75
67
6b
6c
8.3
0
0
0
0
0
25
33
67
33
50
0
58
50
67
33
44
11
67
25
33
17
25
22
42
0
33
50
61
0
17
0
8.3
16.7
25
25
50
100
50
100
0
100
50
100
25
100
33
100
75
100
0
75
33
75
0
50
75
67
0
8.3
16.7
67
33
33
0
56
25
58
8.3
33
11
33
25
33
0
25
11
25
0
17
25
33
0
100
50
100
0
60
33
33
0
6d
6e
25
25
10
25
25
25
25
10
25
10
25
10
5
100
20
100
25
67
33
100
25
100
0
50
0
25
0
0
0
55
6.7
33
8.3
22
11
33
8.3
33
0
11
0
8.3
0
0
0
6f
6g
6h
6i
100
0
100
100
100
0
50
0
25
50
6j
17
0
0
0
0
5.6
0
0
0
0
PZQ
25
0
0
25
75
8.3
0
0
8.3
25
25
25
8.3
8.3
75
PZQ, praziquantel.
PZQ with Ac2O in pyridine (17). All compounds were puri-
fied by silica gel column and identified by 1H and 13C
NMR and HRMS.
mented with 20% newborn calf serum, 100 U/mL penicil-
lin, 100 lg/mL streptomycin, and 0.5 lg/mL amphotericin
B. Cultures were kept at 37 °C in an atmosphere of 5%
CO2 in air and were observed under a Leica MZ 12.5 ste-
reomicroscope (Houston, TX, USA). Worms of S. japoni-
cum were exposed overnight (16 h) to various
compounds, washed, and subsequently cultured in drug-
free medium. Juvenile worms were obtained after day 14
infection. In contrast, adult worms were obtained at day
42 infection. Compounds were then added from 5.0 mM
DMSO stock solutions to achieve a series of diluted con-
centrations 10–50 lM. Compound activity was assessed
by percentage of survival and vitality within 72 h. The
In vitro efficacy of compounds 6a–6j against
Schistosoma japonicum
All prepared analogs were first evaluated for their ability
against adult schistosomes J. in vitro. According to previ-
ously described method (18), four male/female worms
obtained from rat infections were distributed in duplicate
tissue culture dishes (3.5 cm) in Dulbecco’s modified
minimum Eagle’s medium (bicarbonate buffered) supple-
222
Chem Biol Drug Des 2013; 82: 216–225

Praziquantel Derivatives with Anti-Schistosomal Activity
Table 2: Effects of compounds 6a, 6c, 6i, and 6j on 14-day juvenile Schistosoma japonicum in vitro culture
24 h
48 h
72 h
Survival
(%)
Vitality
(%)
Survival
(%)
Vitality
(%)
Survival
(%)
Vitality
(%)
Compounds
Concentrations (lM)
Worms
Control
6a
6c
–
25
25
25
25
25
8
15
13
8
11
8
100
73
46
69
0
100
49
15
23
0
100
73
27
62
0
100
42
9
21
0
100
60
18
62
0
100
24
6
21
0
6i
6j
PZQ
37.5
12.5
25
8.3
25
8.3
PZQ, praziquantel.
Table 3: Effect of single 250 mg/kg oral dose of compounds 6a,
6c, 6i, and 6j administered to mice harboring 42-day-old adult
Schistosoma japonicum infection
score of worm vitality is illustrated as follows: 3 scores: the
highest score, as observed in the control group during the
observation period. Worms moved actively and softly, and
the body was transparent. 2 scores: worms acted all over
the body, but stiffly and slowly, with the body translucent.
1 score: parasites moved partially with opaque appear-
ance. 0 score: the worm remained contracted, did not
resume movements, and we could deem it ‘dead’. The
average score of 8 worms was counted for in vitro test.
Data representative of repeated experiments were shown
in Table 1 (for adult worm).
Number of detected worms/
Total worm
ꢁ
Compounds mice (X Æ s)
reduction (%)
Control
6a
6c
71.25 Æ 1.64
39.0 Æ 4.64
37.75 Æ 8.0
59.5 Æ 2.06
61.33 Æ 1.70
26.33 Æ 8.5
0
44.3
54.3
12.8
13.6
62.4
6i
6j
PZQ
PZQ, praziquantel.
For all compounds tested against adult schistosomiasis. J
(Table 1), compounds 6a, 6i, and 6j shown worm killing
activity close to PZQ. Compounds 6b, 6d, 6f, 6g, and 6h
had comparable activity; no worm was killed at concentra-
tion up to 25 lM, but the worm vitality score reduced
significantly. The potency of compound 6e is marginally
higher than these five weakest compounds. At 10 lM, the
worm viability is reduced to 6.7% and 33% for male and
female worms, respectively, which is obviously less potent
compared with PZQ at the same concentration. The result
of particular significance is for 6c. At concentration as low
as 10 lM, this compound killed 100% worm, which is the
same as PZQ. At 5 lM, the efficacy of compound 6c is
slightly better than PZQ against male worm, but much less
efficient against female worm compared with PZQ. Nota-
bly, all PZQ derivatives demonstrated higher capability
against male than female worm. Overall, the in vitro effect
of these compounds revealed that modification of the 8
and 10 position in the aromatic ring is more tolerated, sub-
stitution at position 9 reduced the compound worm killing
ability significantly; halogenation of the aromatic ring in
position 8 retained the compound capability, bigger sized
substitutes, like methoxy, is not favorable to the com-
pound activity.
6c and 6j, also displayed considerable effect against
juvenile worm. Compound 6j displayed highest potency,
killed all tested worms within 24 h.
Effect against adult schistosomes in rats
For the in vivo study, female ICR mice were infected with
ca. 100 schistosomiasis. J cercariae on day 0 followed by
administration of 250 mg/kg oral doses of test compounds
suspended in distilled water to groups of 10 mice on day
42 post-infection (adult stage). At 21 days post-treatment,
animals were killed and dissected to assess total worm
reduction as described in detail (19,20).
For those compounds shown significant effect against
both adult and juvenile worm in vitro, the worm reduction
capability was evaluated in vivo. The result is shown in
Table 3. Two of the tested compounds 6a and 6c
displayed obvious worm killing capability. Especially, com-
pound 6c demonstrated 54.3% worm reduction ability,
which is close to the reference compound. Compound 6j
displayed 13.6% worm reduction capability, which is very
close to 10-OH PZQ (6), suggesting 6j might be hydro-
lyzed in vivo and 10-OH PZQ could be released. Although
6i exhibited good worm killing activity in vitro, the worm
reduction capability is not satisfactory in vivo. This could
be related to the increased lipophilicity by introducing a
methoxy group or the intolerance of substitutes in the
aromatic ring at the PZQ binding site of the target protein.
Based on the adult worm killing ability, compounds dis-
played significant potency at 25 lM were further evaluated
for their ability against juvenile worm (Table 2) (11). Again,
compounds 6c and 6j demonstrated significant worm
killing activity, reduced worm vitality to <10% in 72 h at
25 lM. Compounds 6a and 6i, although less potent than
Chem Biol Drug Des 2013; 82: 216–225
223

Wang et al.
Table 4: Effect of compounds 6a, 6c, 6i, and 6j on the morbidity of HL-60 cancer and HL-7702 normal cell lines
Percentage of morbidity at 50 lM (%)
6a
6c
6i
6j
PZQ
HL-60
HL-7702
14.4 Æ 6.1
À5.7 Æ 4.1
18.7 Æ 7.9
À14.5 Æ 6.5
22.3 Æ 6.6
À14.3 Æ 8.4
37.2 Æ 3.4
À18.5 Æ 6.5
4.1 Æ 0.1
À20.0 Æ 10.9
PZQ, praziquantel.
Determination of compound cytotoxicity
the preparation thereof. German Patent DT 2362539,
Merck, 1975 (US patent 4001411, 1977).
An excellent antischistosomal drug should be supposed
to kill selectively the schistosomes without or to a signifi-
cantly less extent, being harmful to the host (21,22). The
four excellent compounds (6a, 6c, 6i, 6j) displaying high
activity against adult S. japonicum in vitro were tested on
HL-60 and a normal liver cell line HL-7702 for their
cytotoxic potential at 50 lM. Praziquantel was used as
reference drugs, and data are summarized in Table 4. As
PZQ, all tested compounds are not toxic to normal liver
cell line and to a small extent, toxic to HL-60 cancer
cell.
4. Seubert J. (1976) German Patent DT 2504250, Merck.
5. Ronketti F., Ramana V., Xia C.M., Pica-Mattoccia L.,
Cioli D., Todd M.H. (2007) Praziquantel derivatives I:
modification of the aromatic ring. Bioorg Med Chem
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6. Duan W.W., Qiu S.J., Zhao Y., Sun H., Qiao C.H., Xia
C.-M. (2012) Praziquantel derivatives exhibit activity
against both juvenile and adult Schistosoma japoni-
cum. Bioorg Med Chem Lett;22:1587–1590.
7. Liu H., William S., Herdtweck E., Botros S., Domling A.
(2012) MCR synthesis of praziquantel derivatives.
Chem Biol Drug Des;79:470–477.
Conclusions
8. Kim C.S., Min D.Y. (1998) Synthesis of praziquantel
derivatives and their in vitro activity against clonorchis
sinensis [J]. Arch Pharm Res;21:744–748.
9. Mathew P.L., Hejaz H.A.M., Mary F.M., Simon P.N.,
Atul P., Michael J.R., Barry V.L.P. (2005) A ring substi-
tuted estrogen-3-O-sulfamates: potent multitarget anti-
cancer agents. J Med Chem;48:5243–5256.
10. Li H.Q., Yang J., Ma S.H., Qiao C.H. (2012) Structure-
based design of rhodamine-based acylsulfonamide
derivatives as antagonist of the anti-apoptotic Bcl-2
protein. Bioorg Med Chem;20:4194–4200.
11. Pica-Mattoccia L., Valle C., Basso A., Troiani A.R., Vig-
orosi F., Liberti P., Festucci A., Cioli D. (2007) Cyto-
chalasin D abolishes the schistosomicidal activity of
praziquantel. Exp Parasitol;115:344–351.
In summary, we have explored the structure–activity rela-
tionship in PZQ introducing different substitutes to the
phenyl ring of the parent compound. We identified that
introduction of a bromine substitute at 8- of PZQ leads to
compound 6c with higher potency against adult S. japoni-
cum than PZQ in vitro, and acetyl 10-OH PZQ displayed
significant capability to kill juvenile worm in vitro. Our in
vivo study demonstrated that compound 6c shown com-
parable worm reduction ability to PZQ. However, none of
these compounds established superior worm killing ability
to PZQ. The compound cytotoxicity toward cancer and
normal cell line revealed that all prepared PZQ derivatives
were not cytotoxic. Nonetheless, we have provided
experimental details for the PZQ aromatic ring SAR study.
12. Joong H.K., Yong S.L. (1998) Formation of pyrazinois-
quinoline ring system by the tandem amidoalkylation
and N-acyliminium ion cycliation: an efficient synthesis
of praziquantel. Tetrahedron;54:7395–7400.
Acknowledgments
13. Cao H.P., Liu H.X., Domling A. (2010) Efficient multi-
component reaction synthesis of the schistosomiasis
drug praziquantel. Chem Eur J;16:12296–12298.
14. Ambra A.G., Valeria P., Maria G.C., Simone D.M.,
Antonella V., Armando A.G., Pier L.C., Giuseppe B.,
Gian C.T., Giovanni S., Tracey P. (2009) Synthesis,
biological evaluation, and molecular docking of
ugi product containing a zinc-chelating moiety as
This project is funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions.
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