Investigation of 7-benzylidenenaltrexone derivatives as resistance reverser for chloroquine-resistant Plasmodium chabaudi

Article abstract of DOI:10.1016/j.bmcl.2012.06.085

Derivatives of 7-benzylidenenaltrexone (BNTX), which was recently reported to be an effective chloroquine (CQ)-resistance reverser, were synthesized and evaluated for their CQ-resistance reversing activities. The synthesized derivatives showed CQ-resistan

Full text of DOI:10.1016/j.bmcl.2012.06.085

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5174–5176
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Investigation of 7-benzylidenenaltrexone derivatives as resistance reverser
for chloroquine-resistant Plasmodium chabaudi
a,b
a
a
b
b,
Yoshinori Miyata , Hideaki Fujii , Yuka Uenohara , Seiki Kobayashi , Tsutomu Takeuchi
Hiroshi Nagase
a
,
a,
⇑
School of Pharmacy, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo 108-8641, Japan
School of Medicine, Keio University, 35, Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Derivatives of 7-benzylidenenaltrexone (BNTX), which was recently reported to be an effective chloro-
quine (CQ)-resistance reverser, were synthesized and evaluated for their CQ-resistance reversing activi-
ties. The synthesized derivatives showed CQ-resistance reversing effects. They also reacted with
glutathione (GSH) both enzymatically and chemically, and inhibited glutathione reductase activity.
Received 19 May 2012
Revised 19 June 2012
Accepted 26 June 2012
Available online 1 July 2012
7-Benzyl derivative, which was obtained by reduction of the olefin group in a,b-unsaturated ketone
structure of BNTX, also exhibited CQ-resistance reversing effect, but its potency was significantly lower
than that of BNTX. These outcomes suggested that the decrease in GSH level could be one of the mech-
anisms of CQ-resistance reversing effects induced by BNTX derivatives.
Keywords:
Malaria
Chloroquine-resistance
Resistance reversing effect
Glutathione
Ó 2012 Elsevier Ltd. All rights reserved.
Inhibition of glutathione reductase
Malaria is a major cause of illness and death in children and
adults in tropical countries. Despite the intense Roll Back Malaria
campaign for malaria control by the World Health Organization
sitized by CQ-resistant P. chabaudi. Herein, we report the synthesis
of BNTX derivatives and evaluate their CQ-resistance reversing ef-
fects. We also describe a possible mechanism for action of CQ-
resistance reversing effects induced by BNTX derivatives.
(
WHO), the worldwide estimated levels of morbidity and mortality
still remain high with over 200 million incidents of disease and
around 655,000 deaths per year. The spread of resistance to avail-
BNTX derivatives 2 were prepared by aldol condensation of nal-
trexone (1) with the corresponding aldehydes (Scheme 1). Basic
reaction conditions were reported to provide the objective BNTX
1
able antimalarial drugs such as chloroquine (CQ, Fig. 1) is a major
threat to malaria control. The introduction of artemisinin and its
derivatives like artesunate (Fig. 1) as antimalarial drugs provided
a great impact on malaria therapy. WHO presently recommends
artemisinin-based combination therapy (ACT) as a first-line ther-
apy for Plasmodium falciparum malaria.1 However, the emergence
1
6
and dimeric byproducts, resulting in low yield of BNTX. On the
other hand, acidic reaction conditions prevented formation of di-
mers and, as a result, the yields of BNTX derivatives 2 were im-
proved. Indeed, the yield of 2a under acidic conditions was 77% as
compared to 58% under the basic conditions. 7-Benzyl derivative 3
was obtained by catalytic hydrogenation of BNTX (2a) (Scheme 2).
We evaluated the CQ-resistance reversing effects of BNTX deriv-
2
of artemisinin-resistant malaria has been recently reported.
Therefore, it is important to develop not only new antimalarial
3
–12
13,14
15
drugs
but also novel agents that can reverse the resistance.
atives 2 were evaluated using the method that previously reported
We recently found that the d
1
opioid receptor antagonist 7-ben-
(Fig. 2). All the tested compounds exhibited CQ-resistance reversing
effects, and the activities of BNTX (2a) was the most potent among
them. However, there was no clear indication of the influence of
the substituent R for reversal of CQ-resistance.
zylidenenaltrexone (BNTX, Fig. 1) exhibited resistance reversing ef-
fect for CQ-resistant Plasmodium chabaudi.15 The CQ-resistance
reversing effect of BNTX was more potent than that of verapamil
(
Fig. 1) which was the first drug reported to show resistance
As the parasites are vulnerable to oxidative stress within
erythrocytes, efficient antioxidative systems are required for their
reversing effect in CQ-resistant P. falciparum. Moreover, BNTX
administered perorally was effective in the assay using mice para-
1
7
survival and development.
Reduced glutathione (GSH) is a
well-known tripeptide playing an important role as an intracellular
redox buffer.
⇑
Corresponding author.
E-mail address: nagaseh@pharm.kitasato-u.ac.jp (H. Nagase).
Present address: Institute of Tropical Medicine (NEKKEN), Nagasaki University,1-
18
Moreover, it has been reported that GSH is utilized for detox-
17,19,20
ification of heme
and that the increase in intracellular GSH
1
8
21,22
levels was responsible for the development of CQ-resistance.
1
2-4, Sakamoto, Nagasaki 852-8523, Japan.
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.085

Y. Miyata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5174–5176
5175
NEt2
OH
N
NH
N
O
O
Cl
OH
Chloroquine (CQ)
BNTX
O
O
O
O
Figure 2. CQ-resistance reversing activities of BNTX derivatives 2 in mice at day 4
after infection with P. chabaudi. BNTX derivatives 2 (10 mg/kg) or CQ (3 mg/kg)
were administered perorally or intraperitoneally, respectively. The statistical
significance of differences between the groups was assessed with one-way ANOVA
H
H
X
H
Artemisinin (X=O)
Artesunate (S configuration, X=H, O(C=O)CH CH CO H)
⁄
followed by Dunnett’s test. p <0.05 versus CQ treatment group.
2
2
2
MeO
MeO
OMe
OMe
with GSH under both enzymatic conditions (in the presence of glu-
tathione S-transferase (GST)) and chemical conditions (in the ab-
sence of GST). BNTX derivatives 2 also showed GR inhibitory
N
CN
Me
i-Pr
Verapamil
activities
(inhibition
activities,
2a:
38.96 ± 8.28%,
2b:
-
Cl
3
4.74 ± 9.29%, 2c: 65.58 ± 4.80%). On the other hand, 7-benzyl
Me N
S
NMe2
2
derivative 3, which lacks a Michael acceptor structure, showed
the CQ-resistance reversing effect (Fig. 3), but its effect was signif-
icantly less potent than that of BNTX (2a) which possesses an
N
Methylene blue
a,b-unsaturated structure. No inhibition activity of 3 for GR was
Figure 1. Structures of chloroquine, BNTX, artemisinin, artesunate, verapamil, and
methylene blue.
observed. These outcomes suggested that the mechanism of action
of CQ-resistance reversing effects by BNTX derivatives 2 could in-
clude the depletion of the GSH levels. Methylene blue (Fig. 1),
which is a non-competitive inhibitor of GR, has been reported to
2
6
R
act as a CQ-sensitizer. However, the potency of the CQ-resistance
reversing activities (Fig. 2) did not appear to correlate with the
reactivities with GSH (Table 1) or the GR inhibitory activities. No
clear relationship between the potency of the CQ-resistance
reversing activities and the reactivities with GSH or GR inhibition
could be established. The results of our studies indicate that the
CQ-resistance reversing effects induced by BNTX derivatives 2
must follow a mechanism that involves processes other than a de-
OH
OH
basic
N
N
conditions
O
or
O
O
O
acidic
conditions
2
7
OH
naltrexone (1)
OH
crease in the GSH level. This contention would be supported by
the result that the CQ-resistance reversing effect of 7-benzyl deriv-
ative 3 persisted despite the observation that 3 has neither GR
inhibition activity nor a Michael acceptor moiety which can react
with GSH. In general, the transporter proteins like P. falciparum
CQ-resistance transporter (PfCRT), P. falciparum multidrug resis-
tance 1 protein, and P. falciparum multidrug resistance-associated
2a-c
R = H (a), F (b), OMe (c)
Scheme 1. Synthesis of BNTX derivatives 2. Basic conditions: ArCHO, KOH, MeOH,
1
00 °C. Acidic conditions: ArCHO, PhCO
2
H, i-Pr
2
EtN, Tol/DMF = 5/1, 130 °C. Com-
pound 2a: 77%, 2b: 54%, 2c: 93%.
OH
N
OH
N
H2
Pd/C
O
MeOH ,rt
68%
O
O
O
OH
OH
2
a
3
Scheme 2. Synthesis of 7-benzyl derivative 3.
In view of the possible importance of GSH and because BNTX has a
Michael acceptor structure, we attempted to evaluate the in vitro
Figure 3. CQ-resistance reversing activities of BNTX (2a) and 7-benzyl derivative 3
in mice at day 4 after infection with P. chabaudi. BNTX (2a) and 7-benzyl derivative
3 (10 mg/kg) or CQ (3 mg/kg) were administered perorally or intraperitoneally,
23,24
reactivities of the BNTX derivatives 2 with GSH
and the inhibi-
tion of glutathione reductase (GR)25 by the 2 analogs. Both the
respectively. The statistical significance of differences between the groups was
reaction with GSH and inhibition of GR should induce a decrease
in the free GSH levels which would lead to an unfavorable environ-
ment for the malaria parasites. All the BNTX derivatives 2 reacted
⁄⁄⁄
assessed with one-way ANOVA followed by Dunnett’s test.
p <0.01 versus CQ
treatment group. or Student’s t-test. #p <0.05 versus CQ+BNTX (2a) treatment
group.

5
176
Y. Miyata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5174–5176
Table 1
Residual ratio (%) of GSH in the reaction of BNTX derivatives 2 with GSH
11. Wu, T.; Nagle, A. S.; Chatterjee, A. K. Curr. Med. Chem. 2011, 18, 853.
12. Rodrigues, T.; Prudêncio, M.; Moreira, R.; Mota, M. M.; Lopes, F. J. Med. Chem.
2012, 55, 995.
Compd
R
Residual GSH (%) in
the presence of GST
Residual GSH (%) in
the absence of GST
13. Henry, M.; Alibert, S.; Orlandi-Pradines, E.; Bogreau, H.; Fusai, T.; Rogier, C.;
Barbe, J.; Pradines, B. Curr. Drug Targets 2006, 7, 935.
1
1
4. Egan, T. J.; Kaschula, C. H. Curr. Opin. Infect. Dis. 2007, 20, 598.
5. Miyata, Y.; Fujii, H.; Osa, Y.; Kobayashi, S.; Takeuchi, T.; Nagase, H. Bioorg. Med.
Chem. Lett. 2011, 21, 4710.
6. Portoghese, P. S.; Garzon-Aburbeh, A.; Nagase, H.; Lin, C. E.; Takemori, A. E. J.
Med. Chem. 1991, 34, 1292.
2
2
2
a
b
c
H
F
OMe
40.06 ± 0.41
39.62 ± 1.34
25.63 ± 1.18
31.82 ± 2.33
33.09 ± 1.47
18.03 ± 2.03
1
1
1
7. Müller, S.; Gilberger, T. W.; Krnajski, Z.; Lüersen, K.; Meierjohann, S.; Walter, R.
D. Protoplasma 2001, 217, 43.
8. Although glutathiones exist in cells at relatively high concentration, most
amounts of them are in not the reduced form (GSH) but the oxidized form
(GSSG). It is believed that the malaria parasites could convert GSSG into GSH
using GR on demand and utilize GSH for the decrease in the oxidative stress
level in erythrocytes. Therefore, the parasites are thought to be very
susceptible to the GSH level in erythrocytes.
protein (PfMRP) were said to play a central role in eliciting the CQ-
resistance, and intense investigations of these transporters have
been carried out. However, these studies showed that the entire
process of CQ-resistance could not be fully explained by the activ-
ities of these transporters alone.28 For example, mutations in the
Plasmodium vivax ortholog of CRT did not correlate with observed
O
NH
2
H
N
HO C
N
H
CO H
2
9
2
2
CQ-resistance, and in a series of CQ-resistant strains, P. chabaudi
O
O
3
0
S
S
CRT was not altered. Moreover, although PfMRP has been re-
ported to play a role in parasite response to antimalarial drugs
such as CQ and artemisinin, experiments using parasites with dis-
rupted PfMRP suggest that PfMRP might not be the key component
that can convert a clinically sensitive parasite into a resistant par-
asite. Our results imply that in addition to the effects of some
transporter proteins, GSH levels may also play an important role
in eliciting CQ-resistance.
In conclusion, we synthesized BNTX derivatives 2 and evaluated
their CQ-resistance reversing activities. BNTX derivatives 2 showed
CQ-resistance reversing effects. They also reacted with GSH, show-
ing both enzymatic and chemical effects, and inhibited GR activity.
H
N
HO C
CO H
2
2
N
H
O
NH2
3
1
oxidized glutathione (GSSG)
NADPH
GR
NADP
O
H
NH
2
7
-Benzyl derivative 3, which lacks a Michael acceptor structure,
N
exhibited CQ-resistance reversing effect, but its potency was sig-
nificantly lower than that of BNTX (2a). These outcomes suggest
that a decrease in GSH levels could be one of the mechanisms of
CQ-resistance reversing effects induced by BNTX derivatives.
HO C
N
H
CO H
2
2
O
HS
reduced glutathione (GSH)
19. Ginsburg, H.; Famin, O.; Zhang, J.; Krugliak, M. Biochem. Pharmacol. 1998, 56,
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