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rarily mask the basic character allowing oral delivery of the bis-C-
alkylamidine drug M34.
the N-substituted bis-alkylamidine drugs 3b–e, two reduction con-
ditions were developed. The compounds 3c–e were synthesized by
hydrogenating 1c–e in the presence of catalytic 10% Pd/C, in a
methanolic solution of 3 N hydrochloric acid. To prevent debenzy-
lation, the derivative 3b was generated as an hydrochlorate salt
using a Zinc powder suspension in a methanolic solution of acetic
acid,20 followed by treatment with 37% aqueous hydrochloric acid
solution in methanol. It is noteworthy that methylated 3c and
ethylated 3d drugs could be obtained by this method, where we
did not succeed using Pinner’s conditions.28 Indeed, because of
their basic nature and their facile hydrolysis to the corresponding
amides, amidines often necessitate suitable protections to facilitate
their synthesis and their purification.20,21 The reduction of oxygen-
ated derivatives was used several times to generate amidines,29
and oxadiazolones have been reported as suitable amidines protec-
tions.21,22,30,31 Thus, Moormann et al. described a versatile oxa-
diazolone synthon for the synthesis of acetamidine derivatives.20
The compounds were characterized by 1H and 13C NMR, MS (ESI),
FTIR and the data were consistent with the structure.
The in vitro antimalarial activities were evaluated against a
chloroquine-sensitive strain of P. falciparum (Nigerian strain).32,33
Results are given in Table 2. All C-alkylamidine drugs, including
M34 and N-substituted compounds 3b–e, presented potent
in vitro antimalarial activities, with IC50 in the very low nanomolar
range. The introduction of N-substituents did not alter the antima-
larial potency of alkylamidine drugs. On the other hand, their ami-
doxime 3b–e or oxadiazolone 1b–e prodrug derivatives revealed
very weak antimalarial activities. Since these molecules are not
protonated in physiological conditions, they were not able to act
as choline analogs.
The in vivo antimalarial activities of the compounds were inves-
tigated against the Plasmodium vinckei petteri strain (279BY) in fe-
male Swiss mice.34 The mice were infected on day 0 and were
treated by ip or oral (po) administration of compounds once daily
for four consecutive days (days 1–4 post infection). The parasite-
mia levels were monitored in mice after ip or po administration
of 3 appropriate doses (n = 3 per dose). No antimalarial effect
was observed after ip administration of 10 mg/kg of M34, 3b and
3c, even though these compounds possess potent in vitro antima-
larial activities. On the other hand, the N-substituted C-alkylami-
dine drugs 3d and 3e revealed potent activity after ip
administration with complete clearance of parasitemia (without
recrudescence in the following 28 days) and ED50 of 6.3 and
8 mg/kg. Only ethyl and methoxyethyl N-monosubstitutions of
alkylamidine drugs lead to improved antimalarial in vivo activity.
The amidoxime derivative of compound 2b possessed no signif-
icant in vivo antimalarial activity. On the other hand, the N-substi-
tuted C-alkylamidoximes 2c, 2d and 2e revealed significant ip
antimalarial activity with 5 mg/kg of 2c, 2d and 2e reducing para-
sitemia by 44%, 76% and 33% respectively, compared to control.
When amidoximes were administered orally, no antimalarial activ-
ity could be detected for 2c. The only oral effect that could be ob-
served was a 20% and 16% reduction of parasitemia with 180 mg of
compounds 2d and 2e respectively. Thus, N-substituted amidox-
ime derivatives could not be considered as efficient prodrugs.
Except for one compound, the N-substituted oxadiazolone
derivatives were not active against the malaria parasite after ip
or oral administration to mice at the dose indicated in Table 2.
Problems of very poor water-solubility were encountered with
these very lipophilic derivatives. Indeed, 2b, 2c and 2d could not
be solubilised for testing at higher doses than 90, 45 and 20 mg/
kg respectively. Among the O-modulations needed to improve
the oral antimalarial activity of amidoximes, the oxadiazolone
derivatization could not be applied to N-substituted C-alkylami-
doximes, due to the formation of insoluble compounds. The only
soluble N-substituted C-alkyloxadiazolone was 1e and oral admin-
It is noteworthy that the M34 alkylamidine drug presents excel-
lent growth inhibition of the virulent P. falciparum parasite with an
IC50 in the sub-nanomolar range while having no in vivo activity
against Plasmodium vinckei after intraperitoneal (ip) administration.
The introduction of N-substituents on the amidine function might
improve in vivo antimalarial activity. Indeed, these modulations
influence both the pKa17 and, above all, the lipophilicity of amidines
drugs. Thus, the application of the amidoxime-based prodrug strat-
egy may improve the oral activity of the resulting drugs. The oxa-
diazolone derivatization was one of the O-substituents needed to
obtain molecules with a relevant oral antimalarial activity in alky-
lamidine series.16,18 In the same way, Kitamura et al. described oxa-
diazolone derivatives of platelet aggregation inhibitors.19 The
derivatizations of the benzamidino group resulted in prodrugs with
improved oral bioavailability.
The aim of this study was to develop a convenient route to N-
substitutedbis-C-alkylamidinedrugs, the corresponding C-alkylam-
idoximes and C-alkyloxadiazolones. The antimalarial activity of
these compounds was evaluated to see the ability of the N-substitu-
ents to improve the potency of C-alkylamidine drugs and the oral
activity of the amidoxime and oxadiazolone prodrug candidates.
The target compounds were synthesized as outlined in Scheme 1.
The bis-alkyloxadiazolone 1a was prepared in three steps.16 1a was
used as a key intermediate to obtain the N-substituted bis-
alkyloxadiazolones 1b–e. The main assays to optimise the conditions
of N-alkylation upon NMR conversion rate are reported in Table 1.
The mild conditions used for oxadiazolone alkylation20–25 did
not succeed due to the insolubility of the starting oxadiazolone
1a in solvents as acetone or methanol.26 Thus, dimethylformamide
(DMF) was preferred, leading to the N-alkylated bis-oxadiazolones.
The best results were obtained with the use of sodium methoxide
as a base. N-substitutions were performed using activated halides
such as benzyl bromide or non hindered halides like methyl, ethyl
and methylethyl ether halide with satisfying yields (61–70% for
1b–e, Scheme 1).27
Our first attempts to reach the corresponding N-substituted bis-
alkylamidoximes 2b–e using classical basic conditions (NaOH 5%)
failed.20,24 However, the N-substituted bis-alkylamidoxime deriva-
tives 2b–e were obtained from 1b–e using sodium methoxide in
anhydrous methanol with acceptable yields (58–83%). To prepare
N
N
( )10
O
O
NH
NH
1a
O
O
i
a : R = H
N
N
( )10
b : R = Bn
O
O
c : R = Me
N
N
d : R = Et
R
R
e : R = (CH2)2OMe
O
O
1b-e
iii
ii
H
N
H
N
H
H
N
N
R
( )10
R
( )10
R
R
+NH2
NH2
Cl-
+ Cl-
N
N
HO
OH
2b-e
3b-e
Scheme 1. Synthesis of the drugs and prodrug candidates. Reagents and condi-
tions: (i) RX/DMF, MeONa, 25 °C, overnight (yields 1b–e: 61–70%; (ii) MeONa/
MeOH, reflux, 40 h (yields 2b–e: 58–83%); (iii) H2/Pd/C 10%, MeOH, HCl 3 N, rt, 4 h
(yields 3c–e: 70–82%) or Zn, MeOH/AcOH, 60 °C, 3 h (yield 3b: 85%).
Degardin, Melissa
