Synthesis of fluorescent derivatives of praziquantel: Cell-imaging and interaction with Schistosoma japonicum cercariae

Article abstract of DOI:10.1039/c3ob41348a

Schistosomiasis is one of the most burdensome of the neglected tropical diseases. Praziquantel is a recommended drug for treatment against all forms of schistosomiasis. To investigate the interaction between praziquantel and Schistosoma japonicum cercaria

Full text of DOI:10.1039/c3ob41348a

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Biomolecular Chemistry
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Synthesis of fluorescent derivatives of praziquantel:
cell-imaging and interaction with Schistosoma
japonicum cercariae†
Cite this: Org. Biomol. Chem., 2013, 11,
5989
Received 1st July 2013,
Accepted 4th July 2013
Yunzhi Xie,‡ Yibao Li,‡ Yongquan Wu,‡ Chunhua Liu, Xiaokang Li, Xun Li and
Xiaolin Fan*
DOI: 10.1039/c3ob41348a
www.rsc.org/obc
Schistosomiasis is one of the most burdensome of the neglected
The cercarial stage of the schistosome life-cycle is the only
tropical diseases. Praziquantel is a recommended drug for treat- infectious period. The cercarial stage is also the most fragile
ment against all forms of schistosomiasis. To investigate the stage of the life cycle of the schistosome.5 However, many
interaction between praziquantel and Schistosoma japonicum reports focus mainly on the mechanism of praziquantel action
cercariae, two praziquantel derivatives (PZQ-2 and PZQ-3) and one with S. japonicum juvenile worm and adult worm, while few
praziquantel fluorescent derivative (PZQ-5) have been synthesized papers focus on the mechanism of praziquantel interaction
and characterized using nuclear magnetic resonance spectroscopy with S. japonicum cercaria.6–9 Our intention is to use fluo-
(NMR) and MS spectra. The cytotoxicity of PZQ-2, PZQ-3 and rescence imaging technology to elucidate the preliminary
PZQ-5 was measured by performing the methyl thiazolyl tetrazo- interaction between praziquantel derivatives and S. japonicum
lium (MTT) assay. The cell viability for them shows that the three cercariae.
compounds exhibit low cytotoxicity to HeLa cells. Cell imaging
Fluorescence imaging has been used as a powerful tool
experiments demonstrate that PZQ-5 is biocompatible and cell- for the study of medicine and bioscience, including
permeable, which indicates that PZQ-5 is suitable for studying parasitology,10–12 especially for studying the interaction
their interaction. Confocal fluorescence microscopy revealed that between drugs and parasites in vitro and in vivo.13 The abun-
PZQ-5 is mainly located at the cercarial tegument, which leads to dance of fluorescent probes has promoted the development of
the death of cercariae with the increase in time.
fluorescent imaging technology. Currently, the available fluore-
scent dyes are as follows:14–20 (1) organic fluorescent dyes, (2)
phosphorescent metal complexes,16 (3) quantum dots,17 (4)
fluorescent protein,18,19 and (5) up-converting rare-earth nano-
phosphors.20 Charlemagne Gnoula et al.10 used 5(6)-carboxy-
fluorescein diacetate to label nematodes. The result has shown
that only dead nematodes could be labeled. Devon B. Keeney
et al.11 examined the utility of fluorescent fatty acid analog
dyes for labeling larval trematodes to use in experimental
infections. The 4,5-diaminofluorescein-2 diacetate (DAF-2 DA)
was used by Andrea B. Kohn et al.21 to detect NO in living
schistosomes. These reports indicated that fluorescence
imaging technology is suitable for the investigated interaction
between the drug and S. japonicum cercariae.
The main fluorophores of organic fluorescent dyes are
fluorescein, naphthalimide, acridine, rhodamine, coumarins,
etc. N-Hexanoic acid-4-morpholin-1,8-naphthalimide (com-
pound 4) is a good candidate for fluorescent probes with a
large “push–pull” electronic system, good light stability
and chemical stability. Its fluorescence emission wavelength
is moderate (≈520 nm), the fluorescence quantum yield is
relatively high, and the Stokes shift is larger.
1. Introduction
Schistosomiasis is one of the most burdensome of the
neglected tropical diseases.1,2 The WHO estimates that at least
243 million people required treatment for schistosomiasis in
2011. Over 240 million people require treatment for schisto-
somiasis yearly. The number of people infected with schisto-
somiasis increased from 12.4 million in 2006 to 35 million in
2012.3
Praziquantel is a recommended drug for treatment against
all forms of schistosomiasis. It is effective, safe and low-cost.
The WHO strategy for controlling schistosomiasis focuses on
reducing disease through periodic, targeted treatment with
praziquantel.4
Key Laboratory of Organo-pharmaceutical Chemistry, Gannan Normal University,
Ganzhou 341000, P. R. China. E-mail: fanxl2013@gnnu.cn;
Fax: +86 (0)797 8393536
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob41348a
Herein, novel derivatives of praziquantel (Scheme 1, PZQ-2
and PZQ-3) were synthesized by means of nitration and
‡These authors contributed equally to this work.
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3.04–3.11 (m, 1H), 2.84–2.94 (m, 3H), 2.45–2.50 (t, 1H), 1.73
(m, 5H), 1.53 (m, 2H), 1.28 (m, 3H).
2.2.2. Synthesis of PZQ-3. PZQ-2 (0.36 g), ethanol (4 mL),
acetic acid (4 mL), Fe powder (0.26 g) and distilled water
(4 mL) were added to a 100 mL three-neck flask, and finally a
drop of concentrated HCl was added. The ensuing mixture was
heated under reflux for 5 h, and the solvent was removed
under reduced pressure. The water phase was extracted with
CH2Cl2 (20 mL × 3). The combined organic layer was washed
with aqueous sodium bicarbonate and distilled water and
dried over anhydrous Na2SO4, respectively. The solvent was
removed under reduced pressure and the residue was sub-
jected to column chromatography on silica gel. The product
was separated with EA–PE (v/v, 9 : 1), yielding a slightly white
solid. 1H NMR (CDCl3), δ (ppm): 7.00 (d, 1H), 6.75 (s, 1H), 6.70
(d, 1H), 5.04 (d, 1H), 4.70 (m, 2H), 4.42 (d, 1H), 4.05 (d, 1H),
2.84 (m, 3H), 2.66 (d, 1H), 2.46 (m, 1H), 1.72 (m, 5H), 1.52 (m,
2H), 1.27 (m, 3H).
Scheme 1 Synthetic routines of PZQ-5.
reduction reaction. To investigate the interaction between the
drug and S. japonicum cercariae, a novel fluorescent compound
of PZQ-5 was further synthesized by coupling N-hexanoic
acid-4-morpholin-1,8-naphthalimide (compound 4, Scheme 1)
to the praziquantel derivative PZQ-3. Cell imaging and cell
toxicity have been further investigated.
2.2.3. Synthesis of PZQ-5. 4 was synthesized according to a
literature method.23 Compound 4 (0.099 g) was dissolved in
CH2Cl2 (10 mL) in a 50 mL three-neck flask. PZQ-3 (0.85 g),
DCC (0.052 g) and HOBt (0.034 g) were added with stirring.
The ensuing mixture was stirred for 10 h at room temperature.
The solvent was removed under reduced pressure and the
residue was subjected to column chromatography on silica gel.
The product was separated with EA–CH2Cl2 (v/v, 4 : 1), yielding
2. Experimental section
2.1. Materials and instrumentations
All of the starting materials (reagents and solvents) were
obtained from commercial suppliers and used as received. Prazi-
1
a yellow solid. H NMR (CDCl3), δ (ppm): 8.58 (d, 1H), 8.53 (d,
quantel was purchased from Zhejiang Top Medicine Co., Ltd. 1H), 8.43 (d, 1H), 7.71 (t, 1H), 7.64 (s, 1H), 7.26 (m, 2H), 7.13
(d, 1H), 5.05–4.07 (d, 1H), 4.76–4.48 (t, 2H), 4.42–4.46 (d, 1H),
4.15–4.19 (d, 2H), 4.42–4.44 (d, 2H), 3.26–3.28 (d, 2H), 2.89 (m,
(China). The KB cell lines were provided by the Institute of Bio-
chemistry and Cell Biology (China). Infected Oncomelania
hupensis snails were supplied by the Hunan Institute of Para- 3H), 2.72–2.75 (d, 1H), 2.40 (d, 1H), 1.71–1.82 (m, 13H), 1.52
(m, 5H), 1.26 (m, 5H), 0.85–0.88 (m, 3H). 13C NMR (CDCl3), δ
sitic Diseases (WHO collaborating center for schistosomiasis
(ppm): 24.23, 25.04, 25.73, 26.74, 27.52, 28.40, 28.63, 36.54,
control in lakes).
1H NMR and 13C NMR spectra were recorded using a Mer- 39.25, 40.14, 44.48, 48.41, 52.84, 54.25, 59.82, 66.36, 114.41,
115.99, 116.42, 119.02, 122.61, 125.27, 125.49, 129.13, 129.24,
129.54, 130.61, 132.00, 132.53, 136.69, 155.10, 163.41, 163.85,
curyplus spectrometer at 400 MHz and 100 MHz, respectively.
Electrospray ionization mass spectra (ESI-MS) were measured
using a Bruker APEX II FT-ICRMS 4.7 T system. UV-visible 166.11, 170.61, 171.24, 174.16. MS calcd For: C41H47N5O6,
705.84. Found: 728.4 [M + Na]+.
spectra were recorded using a Shimadzu UV-2550 spectro-
meter. Fluorescence spectra were measured using an
Edinburgh LFS920 fluorescence spectrophotometer. Fourier
2.3. Toxicity test
The MTT assay was used to detect the cell survival rate of
PZQ-5. HeLa cells were plated on a 96 well tissue culture plate
transform infrared (FT-IR) spectra were measured using a
Nicolet Nexus 470 spectrometer with KBr pellets. Fluorescence
imaging experiments were performed using an OLYMPUS under an atmosphere of 5% CO2, 95% air at 37 °C to adhere
for 24 h. The cell pellet was mixed with a PZQ-5 solution
(100 μL per well), at the final concentrations of 5, 10, 20, 30,
FV1000 IX81 confocal fluorescence microscope equipped with
a 40× oil-immersion objective lens, excitation at 405 nm was
carried out with a semiconductor laser and emission was and 40 μM, as the experimental group. The cell pellet was
mixed with RPMI 1640 medium containing 0.2% DMSO
(100 μL per well), as the reference group. After the cell culture
was incubated for 24 h, 20 μL of MTT/PBS (5 mg mL−1) was
added to each well and the cell culture was further incubated
for 4 h. After removing the culture medium, 100 μL of DMSO
was added to each well and the absorbance in each well,
collected at 480 to 580 nm. The MTT assay was measured
by means of a Tecan Infinite M200 monochromator-based
multifunction microplate reader.
2.2. Synthesis
2.2.1. Synthesis of PZQ-2. PZQ-2 was synthesized as including the blanks, was measured at 570 nm using a micro-
described previously.22 1H NMR (400 MHz, CDCl3), δ (ppm): titer plate reader. The reference wavelength was 690 nm. The
8.21 (s, 1H), 8.10–8.12 (d, 1H), 7.37–7.39 (d, 1H), 5.25–5.28 (d, detailed mathematical description of the cell survival rate is
1H), 4.87–4.90 (t, 2H), 4.49–4.53 (d, 1H), 4.08–4.12 (d, 1H), given by the following equation: cell survival rate (%) =
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(absorbance of experimental group/absorbance of reference
group) × 100%. The experimental data from the three groups
of parallel experimental data require obtaining the mean and
standard deviation.
2.4. Anti-cercarial activity experiments
The compound PZQ-5 was dissolved in dimethyl sulphoxide
(DMSO), and diluted to different concentrations with distilled
water as follows: 10 μM, 20 μM, 40 μM, and 60 μM. S. japonicum
cercariae were collected from the infected O. hupensis. The
O. hupensis were induced to shed cercariae by exposing them
to bright light for 2 h, and cercariae were then transferred by
metal spatula from the water surface to a plate containing a
dilute solution of the compound PZQ-5, and the activity of the
cercariae was explored by biological microscopy at 2 h, 4 h, 6 h
and 8 h.
Fig. 1 Cell viability (%) estimated using a MTT proliferation test versus incu-
bation concentrations of PZQ-5. HeLa cells were cultured in the presence of
0–40 μmol L−1 PZQ-5 at 37 °C for 24 h.
2.5. Photophysical properties
Table 1 The mortality rate of Schistosoma japonicum cercariae for the cercari-
cide PZQ-5 (25 °C)
The UV-vis and fluorescence emission spectra of PZQ-5 in a
diluted solution of a mixed solvent (PBS–DMSO = 399 : 1)
were studied, and fluorescence quantum yields of PZQ-5 were
determined using quinine sulfate as the standard (yield = 0.53,
in 0.1 M H2SO4, λex: 365 nm).
Mortality (%)
Concentration (μM)
Number of cercariae
2 h
4 h
6 h
8 h
Black
10
19
19
18
13
16
0
0
0
8
19
0
5
28
54
75
0
21
56
77
100
0
84
100
100
100
2.6. Cell and cercaria imaging experiments
20
40
60
HeLa cells were plated on 18 mm glass coverslips under an
atmosphere of 5% CO2, 95% air at 37 °C to adhere for 24 h.
Then the HeLa cells were stained with a 2.5 μM PZQ-5 solution
in DMSO–PBS (v/v, 1 : 399) buffer for 15 min.
Cercariae were suspended in a 200 μL 3 μM solution of
PZQ-5 on a special plate for imaging. Lastly, fluorescence
imaging of cercariae was performed by confocal microscopy at
1 h, 3 h and 4 h, respectively.
The mortality rate of S. japonicum cercariae for the compound
PZQ-5 was high, not only at a high concentration of 60 μM but
also at a low concentration of 10 μM. At a low concentration of
10 μM the mortality reached 84% at 8 h, which implies that
the cercaricide PZQ-5 maintained excellent bioactivity against
cercariae even though the PZQ-5 was further synthesized by
coupling N-hexanoic acid-4-morpholin-1,8-naphthalimide to
the praziquantel derivative PZQ-3 (Table 1).
3. Results and discussion
3.1. Toxicity test
3.3. UV-vis and fluorescence emission spectra
Two praziquantel derivatives (PZQ-2 and PZQ-3) and one
praziquantel fluorescent derivative (PZQ-5) were synthesized.
In order to study their cytotoxicity, the MTT assay was used to
detect the cell survival rate of PZQ-5. After HeLa cells were
incubated with each compound for 24 hours, the cell survival
rate is also over 85% in 40 μM PZQ-5. In addition, the cell
survival rate is about 90% in 40 μM compound 4, and the cell
survival rate is also over 85% in 40 μM PZQ-2 and PZQ-3
(Fig. S3–5†). Herein, no sub-cellular apoptotic changes or sig-
nificant cell death was seen in the cells after incubation with
working concentrations for imaging. In general, PZQ-5 exhibits
low cytotoxicity when used in a certain range of concentrations
and within limited time periods of incubation (Fig. 1).
The photophysical properties of PZQ-5 were investigated
before PZQ-5 was used to label HeLa cells and cercariae. The
UV-vis absorption and fluorescence emission spectra of PZQ-5
in a diluted solution (PBS–DMSO = 399 : 1) were studied
and are shown in Fig. 2. The derivative PZQ-5 exhibits a broad
UV-vis band and maximal absorbance at a wavelength of
383 nm, corresponding to the π–π* transition of fluorophore 4.
Under excitation at 383 nm, PZQ-5 emits green fluorescence at
a maximum wavelength of 510 nm. The fluorescence quantum
yield of PZQ-5 in water was measured to be 0.086 using
quinine sulfate as the standard.
3.4. Cell imaging
3.2. Anti-cercarial activity experiments
In view of its favorable spectroscopic properties, PZQ-5 should
In order to study the anti-cercarial ability of a praziquantel be suitable for fluorescence imaging in living cells. As deter-
fluorescent derivative, anti-cercarial activity experiments were mined using confocal fluorescence microscopy, HeLa cells
performed at different concentrations of the compound PZQ-5. stained with a 2.5 μM solution of PZQ-5 in DMSO–PBS buffer
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Fig. 2 UV-vis absorption (a) and fluorescent emission spectra (b) of PZQ-5
(3 μM) in a mixed solvent (PBS–DMSO = 399 : 1).
Fig. 4 Confocal bright-field, fluorescence and overlay images of cercariae incu-
bated with PZQ-5 (3 μM) in PBS buffer at room temperature for 1 h (a), 3 h (b)
and 4 h (c). All excitation wavelengths were 405 nm, and emission was collected
at 480 to 580 nm. Scale bars = 40 μm.
functions, but also affects biochemical metabolism of schisto-
somes. The S. H. Xiao group has reported on the behavior of
praziquantel acting on cercariae. The cercarial tegument had
swelled 30 min after adding the drug. With extending the
acting time to 2 h, the insect bodies would irregularly shrink
and swell, and local erosion on the cortex also appeared. The
TEM images showed that the cercarial tegument changed after
a half-hour praziquantel treatment. The glycocalyx on the
insect cercarial tegument had also greatly decreased. Two
hours later, the glycocalyx even totally disappeared. The
parenchyma cells on the cercarial body surfaces significantly
changed as well. Some articles showed that praziquantel
mainly destroyed the glycocalyx of the cercarial tegument, thus
affecting the osmotic pressure of insects, making the insects
unable to adapt to lack of isosmotic water.25
Fig. 3 Confocal bright-field image (a), fluorescence image (b) and overlaid
image (c) of HeLa cells incubated with PZQ-5 (2.5 μM) for 15 min at 37 °C. The
excitation wavelength was 405 nm, and emission was collected at 480 to
580 nm. Scale bar = 20 mm.
(v/v, 1 : 399) for 15 min showed significant intracellular fluore-
scence (Fig. 3). The overlay of fluorescence and bright-field
images revealed that the fluorescence signal was mainly
located in the intracellular region. Moreover, quantization of
the fluorescence intensity profile of cells stained with PZQ-5
indicated that an extremely high signal ratio was associated
with the cytoplasm of live cells (Fig. S6†). These results
indicated that PZQ-5 was biocompatible and cell-permeable. It
provides a basis for cercariae imaging.
As a result, no obvious fluorescence signal was detected in
the initial one hour, but the test time was extended, and
a more significant fluorescence signal appeared in 3 h,
especially in the head and tail. With extending the time to 4 h,
the fluorescence of the cercariae tegument continued to
increase. Like the changes mentioned above, we think that in
1 h the accumulation of drugs in the tegument was less as the
drug action time was short, so the fluorescence signal could
not be detected. With an increase of reaction time, the drug in
the cercariae tegument gathered more, and the fluorescence
signal also increased. According to all the results above, fluo-
rescence signals focus mainly on the insects’ epidermis; we
could conclude that praziquantel possibly mainly acts on the
cercarial tegument, thereby causing different morphological
and biological metabolism transformations. Even though
praziquantel has several effects on schistosomes such as on
glycometabolism, ATP suppression and cholinesterase vari-
ation, we cannot obtain the same conclusions as described
above yet.
3.5. Cercaria imaging
Cell imaging experiments demonstrate that PZQ-5 is biocom-
patible and cell-permeable. The praziquantel derivative was
used for labeling cercariae to observe the interaction between
PZQ-5 and cercariae. The interaction between fresh cercariae
and PZQ-5 (3 μM) was in situ observed using confocal fluore-
scence microscopy, as shown in Fig. 4. The PZQ-5 was taken
up by cercariae through the tegument at 1 h, but its
fluorescence intensity was very low (Fig. 4a). Then, the
fluorescence intensity in the cercarial tegument continually
increased during the period from 3 h to 4 h (Fig. 4b and c).
Even if the test time is extended, PZQ-5 is still mainly located
at the cercarial tegument.
According to the reference mentioned,24 the pharmacologi-
cal effects on schistosomes are as follows: excited activity,
insect bodies contracture and cortex damage after praziquantel
acts on them. Praziquantel not only influences physiological
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Acknowledgements
This work was supported by the National Science and Techno-
logy Ministry (2009BAI78B01 and 2009BAI78B02). Financial
support from the Jiangxi Province Education Department
Science Fund (GJJ13664) is also gratefully acknowledged.
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