Design and synthesis of molecular probes for the determination of the target of the anthelmintic drug praziquantel

Article abstract of DOI:10.1016/j.bmcl.2014.04.014

Schistosomiasis is a highly prevalent neglected tropical disease caused by blood-dwelling helminths of the genus Schistosoma. Praziquantel (PZQ) is the only drug available widely for the treatment of this disease and is administered in racemic form, even

Full text of DOI:10.1016/j.bmcl.2014.04.014

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2469–2472
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of molecular probes for the determination of
the target of the anthelmintic drug praziquantel
Lalit Kumar Sharma a, , Pauline M. Cupit b, ,à, Tino Goronga a, , Thomas R. Webb a,§, Charles Cunningham b,
⇑
a Department of Chemical Biology & Therapeutics, St Jude Children’s Research Hospital, Memphis, TN 38205, USA
b Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 February 2014
Revised 2 April 2014
Accepted 4 April 2014
Available online 13 April 2014
Schistosomiasis is a highly prevalent neglected tropical disease caused by blood-dwelling helminths of
the genus Schistosoma. Praziquantel (PZQ) is the only drug available widely for the treatment of this dis-
ease and is administered in racemic form, even though only the (R)-isomer has significant anthelmintic
activity. Progress towards the development of a second generation of anthelmintics is hampered by a lack
of understanding of the mechanism of action of PZQ. In this Letter, we report an efficient protocol for the
small-scale separation of enantiomers of 2 (hydrolyzed PZQ) using supercritical fluid chromatography
(SFC). The enantiopure 2 was then used to develop several molecular probes, which can potentially be
used to help identify the protein target of PZQ and study its mode of action.
Keywords:
Schistosomiasis
Praziquantel
Supercritical fluid chromatography (SFC)
Molecular probes
Anthelmintic
Ó 2014 Elsevier Ltd. All rights reserved.
Schistosomiasis is an intravascular parasitic infection caused by
trematode worms of the genus Schistosoma. The disease affects
approximately 249 million people including 114 million school-
age children, 90% of whom live on the African continent where
Schistosoma mansoni and Schistosoma haematobium are the pre-
dominant causative agents.1 Praziquantel (PZQ, 1) is the least
expensive, easiest to use and most readily available of all current
anti-schistosomal drugs.2,3 It is highly effective against all schisto-
some species that are known to infect humans and is well-toler-
ated, making it suitable for mass treatment campaigns. Although
PZQ efficacy is high, reported cure rates range from 60% to
95%.4,5 There are at least two explanations for this phenomenon.
First, PZQ does not kill juvenile schistosomes readily between 2
and 5 weeks after infection of the definitive host.6–9 Without rigor-
ous follow-up treatment, this can leave a reservoir of surviving
worms to continue the cycle of infection. A second potential prob-
lem is the presence of drug resistance traits in laboratory and nat-
ural populations of worms.10–14
mainly with the (R)-stereo isomer.15–19 The inactive (S)-isomer is
responsible for its side effects,18 extremely bitter taste20 and large
pill size.21 The classical resolution of the PZQ intermediate 2 has
recently been reported and has the potential to lend itself to the
preparation of more optically pure versions of the drug on a com-
mercial scale.22 Although the molecular target of PZQ remains
unknown, a number of lines of evidence suggests that it disrupts
cellular Ca2+ homeostasis by affecting voltage gated Ca2+ channel
function.23–26 The lack of an efficient general small-scale method
for developing molecular probes of exceptional high optical purity
limits our ability to readily prepare specific molecular probes
based on the active and inactive soluble configuration of the PZQ
pharmacophore. Molecular probes of this type may lead to an
improved understanding of the mode of action of PZQ on schisto-
somes. Here, we report an efficient method to obtain highly puri-
fied samples of each of the two enantiomers of hydrolyzed PZQ
(2, see Scheme 1) on gram scales with supercritical fluid chroma-
tography (SFC). Enantiopure (R)-2 and (S)-2 were used to synthe-
size (R)-PZQ, (S)-PZQ and other PZQ-based molecular probes.
These molecular tools can potentially be used to identify the
molecular target and site of localization of this drug.
Currently, PZQ (1) is synthesized and employed as a racemic
mixture; however, the anthelmintic activity of PZQ is associated
Our synthesis of new molecular probes starts with racemic 2,
which is generated from (rac)-PZQ (1) using the precedented
hydrolysis of 1 on a 20 g scale under acidic conditions (Scheme 1).22
The reaction provided 2 in 77% isolated yield. Recently, SFC has
⇑
Corresponding author. Tel.: +1 (505) 277 5578; fax: +1 (505) 277 0304.
E-mail address: ccunnin@unm.edu (C. Cunningham).
These authors contributed equally to this work.
Current address: Center for Discovery and Innovation in Parasitic Diseases,
à
Department of Pathology, University of California San Francisco, San Francisco, CA
94158, USA.
gained acceptance as
a valuable chromatographic technique
because of improved resolution and a shorter analysis time
§
Current address: SRI International, 333 Ravenswood Avenue, Menlo Park, CA
compared to high performance liquid chromatography (HPLC).27
94025-3493, USA.
http://dx.doi.org/10.1016/j.bmcl.2014.04.014
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2470
L. K. Sharma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2469–2472
injection value duration 45 s and total elution time 7 min. The SFC
N
N
O
preparative-scale system worked efficiently to separate the enanti-
omers of 2 (on a gram scale) as shown by the SFC analysis of (R)-2
and (S)-2 in Figure 1B and C, respectively. The identity of (R)-2 and
(S)-2 was determined by comparing their optical rotation values
with the literature.22 In addition to optical rotation, the absolute
stereochemistry of (R)-2 was further confirmed by X-ray analysis
(CCDC 994262) as shown in Figure 2.
N
O
HCl, reflux
N
H
O
(rac)-PZQamine
(rac)-PZQ
2
1
N
O
N
O
SFC
Once the protocol for obtaining the enantiopure (R)-2 and (S)-2
was established, they were then used for the synthesis of several
molecular probes (Fig. 3). First, (R)-2 and (S)-2 were coupled with
cyclohexanoyl chloride under basic conditions that provided enan-
tiopure (R)-PZQ and (S)-PZQ, respectively.22 Using a procedure we
previously developed for the racemic 2,7 (R)-2 and (S)-2 were used
to obtain the optically pure fluorescently labeled probes: (R)- and
(S)-PZQ-Bodipy (compounds (R)-3 and (S)-3). Additionally, we
designed and synthesized a ‘short-arm’ PZQ molecular probe (com-
pound 4) containing the diazirine functionality for photoaffinity
labeling studies, as well as an alkyne moiety making 4 suitable
for click chemistry. Using a similar approach, we also prepared
another (R)-PZQ-click probe having a longer photoreactive arm
(compound 5). Details of the experimental procedures for the syn-
thesis of compounds shown in Figure 3 are in the supplementary
information section. While a different method of preparation of
PZQ enantiomers has been reported previously,22 to the best of
our knowledge the synthesis of molecular probes (R)-3, (S)-3, 4
and 5 in an enantiomerically pure form has not.
H
H
+
N
H
N
H
(R)-PZQamine
(S)-PZQamine
(S)-2
(R)-2
Scheme 1. Synthesis of racemic 2 and separation of the two enantiomers (R)-2 and
(S)-2 using supercritical fluid chromatography (SFC).
Additionally, SFC generally uses supercritical CO2 in combination
with a polar organic solvent (e.g., alcohols) as the mobile phase;
making it a greener and cost-effective alternative to HPLC and
reverse phase liquid chromatography (RPLC). Given the advantages
of SFC, we investigated its ability to separate the enantiomers of 2.
The preliminary conditions for separation were determined using
the Berger SFC analytical system, ProNTo. Interestingly, a recent
study on HPLC analysis of (rac)-2 showed the inefficiency of both
Chiralcel AD-H and OD-H columns to separate the enantiomers
of 2.22 Our initial study using SFC indicated that the Chiralcel
AD-H column worked more efficiently compared to the Chiralcel
OD-H column in separating the enantiomers of 2. Figure 1A shows
the chromatogram for the separation of (rac)-2 on the chiral AD-H
column using 35% methanol as the mobile phase. The retention
time was 3.7 0.1 min for (R)-2 and 4.3 0.1 min for (S)-2.
Next, we evaluated the activity of the synthesized compounds
against sexually mature male S. mansoni PR-1 worms in vitro.
Worms were incubated overnight in the presence of different con-
centrations of each compound after which they were washed and
maintained for 7 days in drug-free media. The EC50 for each PZQ
molecule at the end of this 7 day period is shown in Table 1. The
Based on the above data, the following conditions were selected
for subsequent work using Berger SFC preparative-scale system:
Chiralcel AD-H column, methanol/supercritical CO2 solvent system
(35:65), amount per injection 7 mg, total flow 55, cycle time 3 min,
EC50 of commercially available racemic PZQ (2.5
to that reported by Pica-Mattoccia and Cioli (1.6
of (R)-1 was significantly less than that of the racemic PZQ (1)
l
l
M) was similar
M).9 The EC50
Figure 1. SFC analysis of (A) (rac)-2; (B) (R)-2 and (C) (S)-2. Chiralcel AD-H column; solvent system: methanol/supercritical CO2 (35:65).

L. K. Sharma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2469–2472
2471
of the racemic PZQ (1). Andrews (1985) reported that (S)-PZQ was
approximately 60 and 100-fold less effective than the racemic PZQ
and (R)-PZQ, respectively using an in vitro scoring of worm death
based on contraction after 10 min and vesiculation after 18 h.28
No immediate contraction or paralysis was observed when the
schistosomes were exposed to (R)-PZQ-Bodipy ((R)-3). After both
overnight incubation in the presence of the drug and 7 days after
incubation in drug-free media, the worms were contracted at con-
centrations >40
lM with almost no movement. The EC50 was
Figure 2. X-ray structure of (R)-2 highlighting stereochemistry at C6 (crystallo-
graphic numbering). Nitrogen atoms are shown in blue, and oxygen atom is shown
in red.
53.3 M and likely reflects the reduced ability of this larger mole-
l
cule to penetrate the worm tegument. This decrease in activity of
(R)-3 compared to (R)-1 may also be attributed to the effect of
derivatization at the cyclohexane position.29,30 The EC50 of the
(S)-Bodipy was not calculated as this was unlikely to provide infor-
mative data due to the relative insolubility of the molecule and the
greatly reduced anthelmintic activity associated with (S)-isomers.
Probes 4 and 5 were designed to meet all the structural require-
ments for retention of anthelmintic activity.29,30 The short-arm
N
N
O
N
N
O
H3C
N
N
O
F
N
B
F
CH3
N
O
O
O
(R)-PZQ-click probe (4) was only partially soluble at 300 lM in
(R)-PZQ
(S)-PZQ
(R)-PZQ-Bodipy
1% DMSO. When exposed to the probe, the worms did not immedi-
ately contract or shorten. After both overnight incubation and
7 days after the probe was washed out, worms were loosely curled
(R)-1
(S)-1
(R)-3
N
N
O
with little movement at concentrations >100
lM. The EC50 was
H3C
F
B
N
O
132.1 M, approximately 100-fold higher than that of (R)-PZQ
l
and again likely reflects a lack of ability to cross the worm tegu-
ment due to the increased molecular weight. The long-arm (R)-
PZQ-click probe (5) was also only partially soluble in 1% DMSO at
N
O
N
O
N
O
F
CH3
N
F
F
F
>300 lM and did not kill schistosomes, even at this relatively high
(S)-PZQ-Bodipy
N
N
concentration. The longer photoreactive arm increases the hydro-
phobicity of the probe and its relative insolubility also likely ren-
ders it less active and less effective. Thus, while we believe that
(R)-1 and 4 may be employed in the identification of PZQ target(s)
using live S. mansoni or the S. mansoni proteome, 5 is only likely to
be useful with the latter. Further studies using these molecular
probes are being pursued in our laboratories.
In summary, this protocol provides an efficient method for
rapid small-scale synthesis of compounds and probes derived from
enantiopure (R)-2 and (S)-2 by taking advantage of SFC. We have
also reported the initial evaluation of the biological activity of
these probes on schistosomes. The probes based on enantiopure
(R)-2 may have profound advantages in the study of the mecha-
nism of action of PZQ against schistosomes.
(S)-3
short-arm (R)-PZQ-clickprobe
4
N
N
O
O
O
N
H
O
N
N
N
long arm (R)-PZQ-click probe
5
Figure 3. Compounds and probes synthesized from enantiopure (R)-2 and (S)-2.
Table 1
Summary of in vitro EC50 values for sexually mature S. mansoni PR-1 worms to PZQ
molecules and probes
Acknowledgments
95% CIb
(lM)
a
Compound
EC50
(
lM)
1
2.5
0.5
1.9–3.3
0.5–0.5
72.3–84.4
48.6–58.6
ND
We would like to thank Mr. K.C. Lim, University of California
San Francisco, for his assistance with the parasite life-cycle and
animal perfusions. We would also like to thank J. Bollinger, St. Jude
Children’s Research Hospital for his determination of the single
crystal X-ray structures of compounds (R)-2. NIH NIAID grants,
R56AI087807 and 1R01AI087807-01A1 and awards from the
American Lebanese Syrian Associated Charities (ALSAC) and St Jude
Children’s Research Hospital (SJCRH) supported this study.
(R)-1
(S)-1
(R)-3
(S)-3
4
78.1
53.3
ND
132.1
>300
114–153.1
ND
5
Worms were exposed overnight to different drug concentrations then cultured in
drug-free media for 7 days.
a
Dose–response curves with the mean and SEM from three replicates were
calculated by plotting the percentage worms alive at day 7 against the log of the
drug concentration (nM) using GraphPad Prism 6. The R-squared values were
always >0.9.
Supplementary data
b
CI = confidence intervals. ND = not determined.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2014.04.014.
reflecting the increased purity of the active enantiomer. Worm
contraction and shortening was observed immediately when either
the racemic 1 or (R)-1 was added to the worms. Seven days after
the drugs were washed out, worms remained contracted at con-
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