Synthesis and antimalarial activity of new 1,12-bis(N,N′-acetamidinyl)dodecane derivatives

Article abstract of DOI:10.1016/j.bmcl.2006.11.013

Amidoxime and O-substituted derivatives of the bis-alkylamidine 1,12-bis(N,N′-acetamidinyl)dodecane were synthesized and evaluated as in vitro and in vivo antimalarial prodrugs. The bis-O-methylsulfonylamidoxime 8 and the bis-oxadiazolone 9 derivatives sh

Full text of DOI:10.1016/j.bmcl.2006.11.013

Bioorganic & Medicinal Chemistry Letters 17 (2007) 593–596
Synthesis and antimalarial activity of new 1,12-bis-
(N,N0-acetamidinyl)dodecane derivatives
a
d
b
Mahama Ouattara,a,b,c Sharon Wein, Michele Calas, Yen Vo Hoang,
`
Henri Viala and Roger Escaleb,*
aDynamique Mole´culaire des Interactions Membranaires, UMR 5539, CNRS—Universite´ de Montpellier II,
34095 Montpellier, France
bLaboratoire de Chimie Organique Pharmaceutique, Faculte´ de Pharmacie, Universite´ de Montpellier I,
34093 Montpellier, France
c
`
´
´
Laboratoire de Chimie The´rapeutique et Synthese de Medicaments, Faculte de Pharmacie,
Universite´ d’Abidjan-Cocody, BP V 34 Abidjan, France
dLaboratoire des Aminoacides, Peptides et Prote´ines, UMR 5810, CP 22, CNRS—Universite´ de Montpellier II,
34095 Montpellier, France
Received 26 September 2006; revised 3 November 2006; accepted 4 November 2006
Available online 10 November 2006
Abstract—Amidoxime and O-substituted derivatives of the bis-alkylamidine 1,12-bis(N,N0-acetamidinyl)dodecane were synthesized
and evaluated as in vitro and in vivo antimalarial prodrugs. The bis-O-methylsulfonylamidoxime 8 and the bis-oxadiazolone 9
derivatives show relatively potent antimalarial activity after oral administration.
Ó 2006 Elsevier Ltd. All rights reserved.
Malaria has been an ever-present threat to mankind.
The past 10 years have been no exception, as this micro-
bial threat has continued to challenge public health.
Malaria is endemic in about 100 developing countries
and 2.2 billion people are at risk of malaria.1,2 The most
recent estimates indicate that there are more than 500
million clinical cases of malaria annually on the planet,
a number that nearly double previous estimates,3 and
the disease measured in disability adjusted life years
(DALYs) is estimated at 45 million.1,4 The lack of a vac-
cine and the absence of a widely accessible vector con-
trol strategy mean that drugs remain the key control
measure for malaria. However, parasite resistance of
Plasmodium falciparum, the most severe form of malar-
ia, has emerged in all classes of antimalarial drugs except
the artemisinins, and is responsible for this recent in-
crease in malaria-related mortality.5–7 Increased efforts
in antimalarial drug discovery are urgently needed.8,9
We have developed a new strategy of antimalarial treat-
ment targeting the phospholipidic metabolism of the
parasite and have successively designed and evaluated
three generations of compounds that mimic choline,
the polar head of phosphatidylcholine, which accounts
for more than 40% of the total lipid content of the par-
asite.10–12 The first generation consists of bis-quaternary
ammonium salts series.13–15 The lead compound, 1,16-
hexadecamethylenebis(N-methylpyrrolidinium)dibro-
mide G25, efficiently inhibits pharmacoresistant malaria
in vitro16 and was able to cure rodent malaria17 as well
as P. falciparum- and Plasmodium cynomolgi-infected
monkeys at very low dose after intramuscular adminis-
tration.18 However, these ammonium salts are poorly
orally absorbed, due to their permanent positive load,
preventing their use in the field to a large scale.
To remedy the low per os absorption of quaternary
ammonium salts, we then focused on a second genera-
tion of compounds consisting of bioisosteric analogues
(bis-alkylamidine) that exhibit excellent antimalarial
activity.19 These second-generation compounds were as
efficient as the bis-quaternary ammonium salts against
the in vitro growth of the human P. falciparum parasite
(IC50 of = 0.3–2 nM). These amidines present an
Keywords: Antimalarial agents; Bis-alkylamidine; Bis-alkylamidoxime;
O-substituted bis-alkylamidoxime; Prodrugs.
*
Corresponding author. Tel.: +33 4 67 54 86 41; fax: +33 4 67 54 86 25;
e-mail addresses: vial@univ-montp2.fr; roger.escale@univ-montp1.fr
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.013

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M. Ouattara et al. / Bioorg. Med. Chem. Lett. 17 (2007) 593–596
equilibrium between protonated and unprotonated
forms, and thus they can diffuse more easily through
the tissues due to the neutral form. Unfortunately, the
in vivo antimalarial activity after oral administration
was still low (Vial and Calas, data not shown) indicating
that their positive charges are still a drawback for the
oral absorption.
OH, HCl
N
H2N
NH2
C2H5O
+
10
CH3
a
H
H
CH3
N
N
CH3
b
2
3
4
5
H
H
N
10
CH3
N
CH3
c
d
e
f
g
N
N
10
1
N
N
OR
2, R = CH3
OR
i
In the field of various pharmaceutical applications, ami-
dine derivatives appear very potent compounds. Such
therapeutic potential has required neutral surrogate
elaboration to temporarily overcome the basic and ionic
characters of these drugs. During the last decade, pro-
drug strategies have been reported for the aromatic ami-
dine that greatly improve both bioavailability and thus,
activity after oral administration.20–22 Modifications of
the benzamidine group such as benzamidoxime and
derivatives or as carbamate make the prodrugs more
lipophilic and less basic with decreased pKa, overcoming
the low oral bioavailability of benzamidines.22–26 These
modifications have little effect on the half-life elimina-
tion of these compounds22 but improve up to 30-fold
the bioavailability and up to 20-fold the oral activi-
ty.22,26,27 No particular adverse effects have been
observed at comparable benzamidine doses.22,26
Benzamidoxime groups have been shown to be readily
reduced to benzamidine, both in vitro and in vivo, by
a microsomal reductase system present in all mammali-
an species.28
OH
OH
3, R = COCH3
4, R = COC6H5
5, R = CONHC2H5
6, R = CONHC6H5
7, R = PO(OC2H5)2
8, R = SO2CH3
6
7
8
O
O
O
O
h
N
N
N
N
10
CH3
CH3
9
Scheme 1. Synthesis of the bioprecursor compounds 1–9. Reagents
and conditions: (a) NaOHÆEtOH, 80 °C (yield: 46%); (b) CH3I, EtOH/
NaOH 1 N, 5–25 °C (yield: 96%); (c) (CH3CO2)2O (yield: 96%); (d)
ClCOC6H5/CHCl3, TEA, 5–25 °C (yield: 76%); (e) C2H5NCO/CHCl3,
K2CO3, 25 °C (yield: 90%); (f) C6H5NCO/CHCl3, K2CO3, 25 °C (yield:
90%); (g) ClPO(OC2H5)2/DMF, TEA, 0–25 °C (yield: 60%); (h)
CH3SO2Cl/CHCl3, Pyridine, 0–15 °C (yield: 90%); (i) ClCO2CH3/
CHCl3, K2CO3, 25–50°C (yield: 75%).
benzoylamidoxime 4 was synthesized by reacting 1 with
benzoyl chloride and triethylamine in chloroform. A
similar reaction occurs for synthesis of compound 7 with
diethylchlorophosphate in DMF. With methylsulfonyl
chloride in chloroform, pyridine is used to obtain bis-
O-methylsulfonylamidoxime 8. Bis-O-carbamoyl deriva-
tives 5 and 6 were synthesized with the corresponding
isocyanate in heterogeneous medium chloroform–
K2CO3. Under the same conditions, bis-alkylamidoxime
1 with methylchloroformate gives the bis-oxadiazolone
9. At room temperature, the reaction is followed by
TLC and heterocyclisation is completed by gentle heat-
ing at 25–50 °C.
In this paper, we modify the amidine group of the lead
compound 1,12-bis (N,N0-acetamidinyl)dodecane M64
(Fig. 1). This lead compound possesses the linker on
the functional nitrogen atom and had potent antimalar-
ial activity both in vitro against the human P. falciparum
malarial parasites and in vivo against rodent malaria
after ip administration, but not after oral administra-
tion. We describe the synthesis of amidoxime and
O-substituted derivatives of this bis-alkylamidine
(Fig. 1). Biological properties in vitro against P. falcipa-
rum and in vivo against Plasmodium vinckei are reported
and discussed.
In vitro antimalarial activities are evaluated against a
chloroquine-sensitive strain of P. falciparum (Nigerian
strain) and in vivo against the P. vinckei petteri strain
(279BY) in female Swiss mice.29,30 Results are given in
Table 1.
The target compounds were synthesized as outlined in
Scheme 1. Bis-alkylamidoxime 1 was prepared by reac-
tion of 1,12-diaminododecane with ethyl N-hydroxyace-
timidate hydrochloride. It is used as a key intermediate
to obtain compounds 2–9. Experimental reactions were
optimised by varying solvent, base and temperature.
The compounds were characterized by 1H and 13C
NMR, MS (FAB or ESI), FTIR and the data were con-
sistent with the structure. The bis-O-methylamidoxime
derivative 2 was obtained from 1 with methyl iodide
and NaOH 1 N in ethanol. The bis-O-acetylamidoxime
3 was prepared using an acetic anhydride excess as
reagent and solvent of the reaction. The bis-O-
N-hydroxylation of the bis-alkylamidine M64 (IC50
=
2 nM)19 into bis-alkylamidoxime 1 (IC50 = 310 nM)
reduces in vitro antimalarial activity. The heterocyclisa-
tion of 1 in bis-oxadiazolone 9 (IC50 = 7100 nM) or the
O-substitution of 1 by the methyl group (2: IC50
=
4350 nM) and the acetyl group (3: IC50 = 1800 nM) also
strongly decreases in vitro antimalarial activity. Chang-
ing the O-substituted group on the compound 1 by the
phenylcarbamoyl (6: IC50 = 135 nM), ethylcarbamoyl
(5: IC50 = 93 nM) or benzoyl (4: IC50 = 68.7 nM) anti-
malarial activity was gradually improved by a factor
of 2–5 compared to 1. Diethylphosphate (7: IC50
=
9.6 nM) and methylsulfonyl (8: IC50 = 12 nM) substitu-
ents strongly increase antimalarial activity by a factor
of 32 and 26 compared to 1. These derivatives are less
active than the original drug M64. The weaker activity
is in agreement with the fact that the chemical modifica-
tions of the drug lead to less basic prodrugs and that
activity (IC50) of the series of bis-alkylamidine is pKa
H
H
CH3
CH3
NH
N
N
10
NH
M 64
Figure 1. 1,12-bis(N,N0-acetamidinyl)dodecane M64.

M. Ouattara et al. / Bioorg. Med. Chem. Lett. 17 (2007) 593–596
Table 1. In vitro and in vivo antimalarial activity of the compounds 1–9
595
a
IC50 (nM) P. falciparum
b
ED50 (mg/kg)
Compound
P. vinckei
Nigerian strain (CQS)
FCM29 strain (CQR)
W2 strain (CQR)
ip
po
Chloroquine (CQ)
M64
20
2
158
4
143
8.1
3.1
9.2
>10
7.1
>9
>200
90
1
2
3
4
5
6
7
8
9
310
4350
1800
68.7
93
110
>90c
>90
>90
>90
>90
42
>10c
10
135
9.6
>9
12
7100
4.7
>9c
62
a IC50 are means of at least two independent experiments conducted in duplicate.
b Antimalarial activities (Efficient dose 50, ED50) were determined after intraperitoneal (ip) or oral (po) administration of the compounds once daily
for 4 days to infected mice. Range of compound concentrations used to generate data: ip doses 1–9 mg/kg; po doses 10–90 mg/kg.
c Moderate effect at this dose.
dependent.19 However, the in vitro antiplasmodial activ-
ity of 4 (68.7 nM), 5 (93 nM), 6 (135 nM) and in partic-
ular 7 (9.6 nM) or 8 (12 nM) would be consecutive to
their conversion into drugs either chemically or enzy-
matically, by systems that differ from P450 reductases.31
If they do not undergo bioconversion, then they are
probably bioisosteres of M64.
days). No clinical toxic effect was detected during the
tests independent of the used dose.
Based on strategies previously described, we have de-
signed and evaluated, for antimalarial activity, new
modified amidines such as bis-alkylamidoximes,
O-substituted bis-alkylamidoximes and bis-oxadiazol-
ones, able to temporarily reduce the basic character
and to produce orally active bis-alkylamidine drugs. It
is the first time that prodrug strategy is validated for
bis-alkylamidines. Among our compounds, the deriva-
tive bis-O-methylsulfonylamidoxime 8 and the bis-oxa-
diazolone 9 constitute the best prodrug active on
Plasmodium in vivo after oral administration. This strat-
egy applied to bis-alkylamidines linked on the functional
carbon atom is currently being studied.
Compounds were tested for activity in mice infected
with P. vinckei. The mice were treated with compounds
intraperitoneally or orally once daily for four consecu-
tive days (days 1–4 post-infection). The O-protected
derivatives 2, 4 and 7 do not induce any antimalarial ef-
fect after intraperitoneal administration of 9 mg/kg but
compounds 5 and 9 induce a slight antimalarial effect
for dosages higher or equal to 9 mg/kg (data not shown).
A good antimalarial activity by ip is found for com-
pounds 6, 1, 3 and 8, in order of increasing effectiveness.
The last derivative 8 (ED50 ip = 4.7 mg/kg) is as efficient
as its drug M64 (ED50 ip = 3.1 mg/kg).19 Derivatives 2
and 4–7 are not very effective against P. vinckei with
ED50 higher or equal to 10 mg/kg after intraperitoneal
administration and higher or equal to 90 mg/kg after
oral administration. This can probably be attributed to
the low conversion of the drug into the prodrug. On
the other hand, bis-alkylamidoxime 1 and its bis-O-acet-
ylated derivative 3 are effective with ED50 po = 90 mg/
kg. The bis-oxadiazolone 9 (ED50 po = 62 mg/kg) is
even more potent by oral route with an activity at least
3 times more powerful than the corresponding drug
M64. After intraperitoneal administration, the com-
pound bis-O-methylsulfonyl 8 exerts antimalarial activ-
ity (ED50 ip = 4.7 mg/kg) as potent as M64 (ED50
ip = 3.1 mg/kg). Remarkably, after oral administration,
compound 8 showed the most potent antimalarial activ-
ity of all tested prodrugs on the parasitized mice, and
appeared 5 times more effective than its drug M64
(ED50 po = 42 mg/kg).
Acknowledgments
These studies were supported by the European Commis-
sion (QLK2-CT-2000-01166, FP6 Network of Excel-
lence ‘‘BioMalPar’’ and the integrated project
Antimal). We are grateful to Marjorie Maynadier and
Frederic Boudou for their assistance in testing the
compounds.
Supplementary data
1H and 13C NMR, MS (FAB or ESI), FTIR data of new
compounds and biological protocol are given. Supple-
mentary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2006.11.013.
References and notes
1. WHO World Health Report: Changing History; World
Health Organisation: Geneva 2004.
2. Breman, J. G.; Egan, A.; Keusch, G. T. Am. J. Trop. Med.
Hyg. 2001, 64, iv.
Drug M64 and all its bioprecursors are well tolerated by
mice in the in vivo test conditions (intraperitoneal or
oral administration, once daily for four consecutive

596
M. Ouattara et al. / Bioorg. Med. Chem. Lett. 17 (2007) 593–596
3. Snow, R. W.; Guerra, C. A.; Noor, A. M.; Myint, H. Y.;
Hay, S. I. Nature 2005, 434, 214.
18. Wengelnik, K.; Vidal, V.; Ancelin, M. L.; Cathiard, A. M.;
Morgat, J. L.; Kocken, C. H.; Calas, M.; Herrera, S.;
Thomas, A. W.; Vial, H. J. Science 2002, 295, 1311.
19. Vial, H.; Calas, M.; Ancelin, M. L.; Escale, R.; Vidal,
V.; Bressolle, F.; (CNRS, Fr.). Application: FR, 2004,
86 pp.
4. WHO Africa Malaria Report; World Health Organisa-
tion: Geneva 2003, pp WHO/CDS/MAL/2003.1093.
5. Baird, J. K. N. Eng. J. Med. 2005, 352, 1565.
6. Wernsdorfer, W. H.; Payne, D. Pharmacol. Ther. 1991, 50,
95.
20. Clement, B.; Lopian, K. Drug Metab. Dispos. 2003, 31,
645.
7. White, N. J.; Olliaro, P. L. Parasitol. Today 1996, 12,
399.
21. Clement, B. Drug Metab. Rev. 2002, 34, 565.
22. Weller, T.; Alig, L.; Beresini, M.; Blackburn, B.; Bunting,
S.; Hadvary, P.; Muller, M. H.; Knopp, D.; Levet-Trafit,
B.; Lipari, M. T.; Modi, N. B.; Muller, M.; Refino, C. J.;
Schmitt, M.; Schonholzer, P.; Weiss, S.; Steiner, B.
J. Med. Chem. 1996, 39, 3139.
23. Zhou, L.; Lee, K.; Thakker, D. R.; Boykin, D. W.; Tiwell,
R. R.; Hall, J. E. Pharm. Res. 2002, 19, 1689.
24. Kitamura, S. F. H.; Miyawaki, T.; Kawamura, M.;
Terashita, Z.-I.; Naka, T. Chem. Pharm. Bull. 2001, 49,
268.
8. Fidock, D. A.; Rosenthal, P. J.; Croft, S. L.; Brun, R.;
Nwaka, S. Nat. Rev. Drug Disc. 2004, 3, 509.
9. Biagini, G. A.; O’Neill, P. M.; Nzila, A.; Ward, S. A.;
Bray, P. G. Trends Parasitol. 2003, 19, 479.
10. Vial, H.; Mamoun, C. In Plasmodium Lipids: Metabolism
and Function; ASM Press: Washington, DC, 2005; pp 327–
352.
11. Salom-Roig, X. J.; Hamze, A.; Calas, M.; Vial, H. J.
Comb. Chem. High Throughput Screening 2005, 8, 49.
12. Vial, H. J.; Calas, M. Antimalarial Chemother. 2001,
347.
13. Calas, M.; Ancelin, M. L.; Cordina, G.; Portefaix, P.;
Piquet, G.; Vidal-Sailhan, V.; Vial, H. J. Med. Chem.
2000, 43, 505.
14. Ancelin, M. L.; Calas, M.; Bompart, J.; Cordina, G.;
Martin, D.; Ben Bari, M.; Jei, T.; Druilhe, P.; Vial, H. J.
Blood 1998, 91, 1426.
25. Rehse, K.; Bade, S.; Harsdorf, A.; Clement, B. Arch.
Pharm. 1997, 330, 392.
26. Boykin, D. W.; Kumar, A.; Hall, J. E.; Bender, B. C.;
Tidwell, R. R. Bioorg. Med. Chem. Lett. 1996, 6, 3017.
27. Berger, B. J.; Lombardy, R. J.; Marbury, G. D.; Bell, C.
A.; Dykstra, C. C.; Hall, J. E.; Tidwell, R. R. Antimicrob.
Agents Chemother. 1990, 34, 1678.
15. Calas, M.; Cordina, G.; Bompart, J.; Bari, M. B.; Jei, T.;
Ancelin, M. L.; Vial, H. J. Med. Chem. 1997, 40, 3557.
16. Ancelin, M. L.; Calas, M.; Vidal-Sailhan, V.; Herbute, S.;
Ringwald, P.; Vial, H. J. Antimicrob. Agents Chemother.
2003, 47, 2590.
28. Clement, B.; Lomb, R.; Moller, W. J. Biol. Chem. 1997,
272, 19615.
29. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay,
J. D. Antimicrob. Agents Chemother. 1979, 16, 710.
30. Barkan, D.; Ginsburg, H.; Golenser, J. Int. J. Parasitol.
2000, 30, 649.
17. Ancelin, M. L.; Calas, M.; Bonhoure, A.; Herbute, S.;
Vial, H. J. Antimicrob. Agents Chemother. 2003, 47, 2598.
31. Clement, B.; Jung, F. Drug Metab. Dispos. 1994, 22, 486.