Design and synthesis of amidoxime derivatives for orally potent C-alkylamidine-based antimalarial agents

Article abstract of DOI:10.1016/j.bmcl.2008.12.058

Within the frame of the design of prodrug candidates to deliver a C-alkylamidine antimalarial agent, we showed that specific O-substitutions were needed on the alkylamidoxime structure. Among the newly synthesized molecules, bis-oxadiazolone and bis-O-met

Full text of DOI:10.1016/j.bmcl.2008.12.058

Bioorganic & Medicinal Chemistry Letters 19 (2009) 624–626
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of amidoxime derivatives for orally potent
C-alkylamidine-based antimalarial agents
Mahama Ouattara a, , Sharon Wein b, Séverine Denoyelle a, Stéphanie Ortial a, Thierry Durand a,
Roger Escale a, Henri Vial b, Yen Vo-Hoang a,
*
a Institut des Biomolécules Max Mousseron, UMR 5247, CNRS – Université Montpellier I – Université Montpellier II, 34093 Montpellier, France
b Dynamique Moléculaire des Interactions Membranaires, UMR 5235, CNRS – Université de Montpellier II, CP 107, 34095 Montpellier, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Within the frame of the design of prodrug candidates to deliver a C-alkylamidine antimalarial agent, we
showed that specific O-substitutions were needed on the alkylamidoxime structure. Among the newly
synthesized molecules, bis-oxadiazolone and bis-O-methylsulfonylamidoxime derivatives induced a
complete clearance of parasitemia in mice after oral administration.
Received 5 December 2008
Accepted 14 December 2008
Available online 24 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
C-Alkylamidine
Design of prodrug
Specific O-substitutions
Alkylamidoxime
Clearance of parasitemia
Oral antimalarial agent
Malaria is a problem of public health, with 300 to 500 million
people worldwide affected by this potentially lethal disease. More-
over, Plasmodium falciparum parasites become resistant to most
marketed antimalarials, except to artemisinin and its derivatives.1
Cocktails of antimalarials are now recommended to prevent che-
moresistances. Unfortunately, the number of drug classes is very
restricted. This emphasizes that alternative drugs with a novel
mechanism of action need to be urgently developed.
Vial and coll. first developed bis-quaternary ammonium salts as
new antimalarial chemotherapeutic agents. These are bis-cationic
choline analogues targeting the phospholipidic metabolism.2,3
These agents were able to inhibit plasmodial phosphatidylcholine
de novo biosynthesis in infected erythrocytes.4–9 This strategy
was then optimized with bis-thiazolium salts active against rodent
and non-human primate malaria (T3, Fig. 1).10,11 The test subjects
displayed rapid and complete healing without recrudescence.
However, these potent molecules contain permanent cationic
charges, which may be the cause of their low oral absorption. Thus,
bis-alkylamidine derivatives were designed as bioisosteric ana-
logues, which do not possess any permanent cationic charges.12 In-
deed, amidines are strong bases (pKa ꢀ13–14) and exist under
physiological conditions mainly as protonated species. Two chem-
ical series of bis-cationic agents have been developed: N-alkylam-
idines, as M64, and C-alkylamidines, as M34 (Fig. 1). These
compounds also mimic choline and inhibit the P. falciparum para-
site’s multiplication in the low nanomolar range, similarly to
T3.12,13
The antimalarial activity of these bis-alkylamidines relies on the
cationic character of the hydrophilic head,12 this property may also
prevent traversal of the intestinal barrier. This limitation presents a
compelling target for a prodrug strategy. The amidoxime prodrug
principle was originally developed by Clement and coll. for pent-
amidine,14,15 and later applied to other benzamidines such as
-
2 Br
S
S
OH
HO
+
+
N
N
10
T3
IC50 = 2.2 nM
NH2
NH2
NH
H
H
N
N
HN
10
10
NH
NH
M64
IC50 = 2 nM
M34
IC50 = 0.3 nM
* Corresponding author. Tel.: +334 67548665; fax: +33 467548036.
E-mail address: yen.vo-hoang@univ-montp1.fr (Y. Vo-Hoang).
Present address: Laboratoire de Chimie Thérapeutique et Synthèse de Médica-
ments, Faculté de Pharmacie, Université d’Abidjan-Cocody, BP V 34 Abidjan, France.
Figure 1. Bis-cationic antiplasmodial compounds.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.058

M. Ouattara et al. / Bioorg. Med. Chem. Lett. 19 (2009) 624–626
625
sibafiban16 and melagatran17 and to furamidine, an anti-protozoan
agent.18 In this approach, amidoxime functions mask the positive
charge of amidines, which enables these compounds to be effi-
ciently absorbed from the gastrointestinal tract while inhibiting
their biological activity. This biological activity is recovered by
the in vivo reduction of inactive prodrugs into active bis-cationic
drugs.19 The clinical application of this concept led to the oral
use of the amidoxime ximelagatran.20,21 Although the amidoxime
prodrug strategy was widely studied for benzamidine drugs, little
is known about its application to alkylamidine antimalarial drugs.
We previously reported the successful use of amidoxime deriva-
tives to temporarily reduce the basic character and to produce or-
ally active bis-N-alkylamidine M64.22
The aim of this study was to extend these results to M34, the
lead compound of bis-C-alkylamidines. We were interested in
developing M34 prodrugs because this drug revealed higher
in vitro activity than M64. Moreover, the M34 bis-C-alkylamidine
structure allows a broader range of modulations, in particular it
enables the synthesis of 1,2,4-oxadiazole derivatives,23 which is
not possible for M64.
The targeted compounds were obtained as outlined in Scheme
1. Bis-alkylamidoxime 1 was prepared by heating 1,12-dicyanod-
odecane with an excess of hydroxylamine hydrochloride in an eth-
anolic solution of NaOH 1 M and was used as a key-intermediate
for compounds 2–12. The 1,2,4-oxadiazole 2 was generated by
heating 1 with oxalylethyl chloride and pyridine in chloroform.
In the presence of ammonia in ethanol, 2 was converted into the
corresponding carbamoyl-oxadiazole 3. The cyanooxadiazole 4
was prepared from 3 using trifluoroacetic anhydride and pyridine
in dioxane. The bis-O-acetylamidoxime 5 was obtained from bis-
alkylamidoxine 1 using an excess of acetic anhydride. The bis-O-
benzoylamidoxime 6 was synthesized in the presence of benzoyl
chloride and triethylamine in chloroform. Bis-O-carbamoyl deriva-
tives 7 and 8 were prepared with the corresponding isocyanate in
heterogeneous conditions using chloroform–K2CO3. The bis-O-
methylamidoxime derivative 9 was obtained from 1 with dimeth-
ylsulfate and NaOH 1 M in ethanol. Bis-O-methylsulfonyl-amidox-
ime 10 was synthesized with methylsulfonyl chloride in
chloroform in the presence of pyridine. The thiadiazolone 11 was
obtained from 1 using thiocarbonyldiimidazole (TCDI) in tetrahy-
drofuran (THF), followed by diethylether trifluoroborate in THF.
The bis-oxadiazolone 12 was generated in two steps: 1 reacted
with ethylchloroformiate in the presence of triethylamine, and
then, the intermediate compound was cyclized by heating in xy-
lene. These reactions occurred chemoselectively at the OH group
of amidoxime 1.24 Indeed, in amidoximes, the –NH2 group is elec-
tron deficient as observed in amides.25 All structures were fully
characterized by 1H and 13C NMR, MS (FAB or ESI) and in particular
by FTIR.26 The two characteristic bands at 3490–3400 and 3374–
3315 cmꢁ1 were observed and corresponded to symmetrical and
asymmetrical NH2 stretching modes.27
The in vitro antimalarial activities (Table 1) were evaluated
against a chloroquine-sensitive strain of P. falciparum (Nigerian
strain).28 Since the introduction of either an oxygen or a sulfur
atom leads to the loss of the compounds’ basicity, the compounds
1–12 should not be able to act as choline analogues. The neutral
compounds 1, 3–9, and 12 exhibited very weak intrinsic antiplas-
modial activity compared to M34. Contrary to the previous com-
pounds, the methylsulfonate derivative 10 and, to a lesser extent,
the thiadiazolone 11 were the only compounds to have in vitro
antimalarial activities occurring in the same nanomolar range as
the parent alkylamidine M34. Indeed, these molecules revealed
IC50 lower than 20 nM (10: IC50 = 2.5 nM and 11: IC50 = 17.5 nM).
Since they do not possess any cationic character, their in vitro anti-
malarial activity raises the question if they are metabolized into
active drugs or if they possess intrinsic antimalarial activity.
The in vivo antimalarial activities of our compounds were
investigated against the Plasmodium vinckei petteri strain (279BY)
in female Swiss mice.29 The mice were treated with compounds
either intraperitoneally (ip) or orally (po) once daily for four con-
secutive days (days 1–4 post-infection). After ip administration
(20 mg/kg), no antimalarial effect could be detected with most of
the O-protected derivatives 1, 3–9, and 12. However, with the thia-
diazolone 11, parasitemia was reduced to 50% compared to control
and a total clearance of parasitemia was observed with the meth-
ylsulfonate derivative 10 (ED50 ip = 7 mg/kg). Since the compounds
10 and 11 exert their antimalarial activity at the same nanomolar
range (in vitro) or with the same ED50 ip (in vivo) as bis-cationic
bis-alkylamidines, these prodrug candidates are likely efficiently
converted into M34 drug either by chemical way or eventually
by enzymatic systems differing from cytochrome P450 reductases.
No antimalarial activities could be detected after either ip or
oral administration of compounds 3–7 at the tested doses, which
N
N
NH2
NH2
10
N
N
RO
10
OR
a
e
f
5
6
7
8
5, R = COCH3
6, R = COC6H5
7, R = CONHC6H5
8, R = CONHC2H5
9, R = CH3
Table 1
NH2
NH2
In vitro and in vivo antiplasmodial activities of compounds 1–12
g
h
i
N
N
N
Compound
Plasmodium falciparum
Plasmodium vinckei
HO
OH
10
IC50 (nM)a
ED50 (mg/kg)b
1
ip
po
j
9
10
10, R = SO2CH3
b, c, d
N
M34
1
3
4
5
6
7
8
9
0.3
>10
nd
>100
120
nd
R1
k
l
R1
3500
7000
100,000
1400
1100
nd
180
6800
2.5
N
11
12
O
>20
>20
>10
>10
>20
>20
>20
7
O
X
H
N
O
O
nd
HN
N
>90
>90
>180
90
180
40
10
X
N
N
2, R1 = CO2C2H5
3, R1 = CONH2
4, R1 = CN
10
11, X = S
12, X = O
10
11
12
Scheme 1. Synthesis of the bioprecursor compounds 1–12. Reagents and condi-
tions: (a) NH2OH.HCl, EtOH/NaOH 1 M, 5–25 °C (96%); (b) ClCOCO2C2H5/Pyridine,
CHCl3, 85 °C (62%); (c) EtOH/NH3 10%, 25 °C (91%); (d) (CF3CO)2O/Pyridine, dioxane,
25 °C (61%); (e) (CH3CO)2O, NaOH 2 M (90%); (f) ClCOC6H5/CHCl3, TEA, 5–25 °C
(87%); (g) C6H5NCO/CHCl3, K2CO3, 25 °C (81%); (h) C2H5NCO/CHCl3, K2CO3, 25 °C
(74%); (i) (CH3)2SO4, EtOH/NaOH 1 M, 5–25 °C (48%); (j) CH3SO2Cl/Pyridine, CHCl3,
0–15 °C (92%); (k) TCDI/THF, then BF3.OEt2/THF, 25 °C (63%); (l) 1—ClCOOEt, TEA,
CHCl3, 25 °C (75%); 2—xylene, reflux (95%).
17.5
9200
20
>20
>120
100
a
IC50 values are means of at least two independent experiments, each of them
were conducted in duplicate. (nd, not determined.)
b
Antimalarial activities (efficient dose 50, ED50) were determined after intra-
peritoneal (ip) or oral (po) administration of the compounds once daily for 4 days to
infected mice.

626
M. Ouattara et al. / Bioorg. Med. Chem. Lett. 19 (2009) 624–626
Compound 10 (ip)
Compound 10 (po)
Compound 12 (po)
Acknowledgments
100
80
60
40
20
0
These studies were supported by the European Commission
(Antimalintegrated projectLSHP-CT-2005-018834). We are grateful
to Marjorie Maynadier and Frederic Boudou for their assistance in
testing the compounds. We thank Aaron Gabriel (Harvard Medical
School, Cambridge, USA) for a critical reading of the manuscript.
Supplementary data
1H and 13C NMR, MS (FAB or ESI), FTIR data of new compounds
and biological protocol are given. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2008.12.058.
1
10
100
Dose (mg/kg)
Figure 2. In vivo antimalarial activity of compounds 10 (j, N) and 12 (s) against
P. vinckei-infected mice after ip (dashed line) or oral (straight line) treatment. Mice
were iv-infected with 107 parasites, leading to parasitemia of 0.5% on day 1.
Treatment consisted in one daily administration for four consecutives days (days 1–
4 post-infection). Parasitemia were checked on day 5 and expressed in % of control.
Results are the mean of three mice per dosage SEM. After ip administration,
compound 12 do not significantly affect parasitemia until 20 mg/kg.
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were limited by the weak solubility of the compounds. The appli-
cation of the validated prodrug strategies including acetylated
amidoxime30 and oxadiazole,23 did not lead to any in vivo antima-
larial activity. On the other hand, molecules 1 and 8–12 revealed
significant activities against P. vinckei after oral administration. Gi-
ven orally, 120 mg of the bis-alkylamidoxime 1, and 180 mg of the
bis-O-methylamidoxime 9 reduced parasitemia to 50% compared
to control. These compounds appeared more efficient by oral
administration at 90 mg/kg than the parent drug M34, which pos-
sessed no significant effects at that concentration. According to
these results, the prodrug strategies using the simple amidoxime
(1) or the O-methylamidoxime (9), that were validated for ben-
zamidine drugs (melagatran17 and furamidine18), appear less effec-
tive at improving the oral bioavailability of C-alkylamidine M34.
The specific enzymes may not recognize alkylamidoxime struc-
tures whereas benzamidoxime prodrugs of melagatran and of
furamidine are recognized. No data are yet available on biotrans-
formations of alkylamidoximes. Oral administration of 120 mg/kg
of the thiadiazolone derivative 11 and of bis-O-ethyl-car-
bamoylamidoxime 8 led to, respectively, 25% and 80% decreases
of parasitemia as compared to control. Contrary to the previous
compounds, the two derivatives, methylsulfonate 10 and oxa-
diazolone 12, were successful, since a total clearance of parasite-
mia was obtained with 120 mg/kg of both compounds (Fig. 2).
Since the oxadiazolone 12 possesses weak in vitro activity, it is
likely that the significant oral antimalarial activity of oxadiazolone
12 is linked to its in vivo conversion into M34. The results obtained
with antithrombotic agents and M64 confirm this hypothesis.22,23
Remarkably, the methylsulfonyle substituent greatly increased
in vivo antimalarial activity. The bis-O-methylsulfonyle compound
10 displays the most potent oral antiplasmodial activity (ED50
po = 40 mg/kg) of all tested compounds, totally inhibiting P. vinckei
growth in infected mice.
19. Clement, B. Drug Metab. Rev. 2002, 34, 565.
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In conclusion, we were challenged to develop prodrugs of M34
that displayed oral antimalarial activity, since this drug exhibited
no in vivo antimalarial activity. Strategies developed for benzami-
dines do not apply to alkylamidine drugs and specific O-substitu-
ents are necessary to obtain molecules with relevant oral
antimalarial activity. In C-alkylamidine series, the oxadiazolone
12 and the O-methylsulfonate 10 exhibited potent antimalarial
activities when given orally. These prodrug candidates may be effi-
ciently converted into active drug. They induce the fall of parasite-
mia within two log of concentration and the complete clearance of
the blood parasite after one daily dose for 4 days.
26. NMR and FTIR were in full accord with the assigned structure. For example,
1,12-bis-(N,N0-methylsulfonyloxy amidinyl)-dodecane 10: mp 107–108 °C
(CHCl3); 1H (DMSO-d6, 300 MHz) d: 1.23 (s, 16H); 1.51 (m, 4H); 2.03 (t, 4H);
3.06 (s, 6H); 6.59 (s, 4H). 13C (DMSO-d6, 75 MHz) d: 26.4; 28.3; 28.6; 28.9; 29.0;
30.0; 35.6; 160.5. FTIR cmꢁ1: 1166; 1332; 1619; 3373; and 3497. ES+ SM: 443
[M+H+]; 222 [(M+2H+)/2]; 885 [2M+H+].
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