Reverse-benzamidine antimalarial agents: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2010.07.124

In the frame of the development of bis-cationic choline analogs, the RSA of bis-N-alkylamidines were studied and a new series of reverse-benzamidine derivatives was designed. Contrary to the lipophilicity, the basicity of alkylamidine compounds directly influences their antimalarial potencies.

Full text of DOI:10.1016/j.bmcl.2010.07.124

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5815–5817
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Reverse-benzamidine antimalarial agents: Design, synthesis, and
biological evaluation
Olivier Berger a, Sharon Wein b, Jean-Frederic Duckert a, Marjorie Maynadier b, Siham El Fangour a,
Roger Escale a, Thierry Durand a, Henri Vial b, Yen Vo-Hoang a,
*
a Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CNRS—Universités Montpellier I et II, Faculté de Pharmacie, 15 Av. Ch. Flahault, 34093 Montpellier cedex 5, France
b Dynamique Moléculaire des Interactions Membranaires, UMR 5539 CNRS—Université de Montpellier II, Faculté des sciences, Place Eugène Bataillon,
34095 Montpellier cedex 5, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In the frame of the development of bis-cationic choline analogs, the RSA of bis-N-alkylamidines were
studied and a new series of reverse-benzamidine derivatives was designed. Contrary to the lipophilicity,
the basicity of alkylamidine compounds directly influences their antimalarial potencies.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 5 July 2010
Revised 27 July 2010
Accepted 28 July 2010
Available online 3 August 2010
Keywords:
Bis-N-alkylamidines
Antimalarial agents
Total clearance of parasitemia
Chemical design
Reverse-benzamidines
Malaria is an infectious disease widespread in tropical and sub-
tropical regions. The World Health Organization (WHO) estimates
that half the world population is at risk of malaria.1 Although WHO
recommends the use of artemisinin-based combination therapies
(ACTs),2 the malaria control is threatened by the increase of Plas-
modium falciparum strains resistant to most of available antimala-
rials, including artemisinin and derivatives.3,4 To overcome the
parasite multichemoresistance, alkylamidine-based choline ana-
logs have been developed as a potential new chemotherapy against
P. falciparum or P. vivax.5–7 Indeed, Vial and co-workers studied
the parasite’s phospholipidic metabolism and defined the phos-
phatidylcholine de novo biosynthesis as a novel antiplasmodial
target.8
The bis-thiazolium salt T3 is the first choline analog to undergo
phase 3 clinical trials, validating the strategy of using biscationic
antimalarial agents.9–12 Amidines are strong bases (pKa ꢀ13–14)
and exist under physiological conditions mainly as protonated spe-
cies. Bis-alkylamidines are thus bioisosteres of the bis-thiazolium
salts (T3, Fig. 1), sharing the same mechanism of action.
in which the alkyl chain is attached to the functional nitrogen atom
(Fig. 1). Whereas T3 possesses a permanent cationic charge, bis-
alkylamidines exhibit non permanent charges and the amidoxime
(hydroxylated amidine) derivatization temporarily reduces the ba-
sic character of amidine function. The resulting compounds were
able to orally deliver the active bis-N-alkylamidine M64.13 Never-
theless, M64 revealed to be quite unstable in vivo.14
The aim of this work was to investigate structural variations on
the cationic center of the N-alkylamidines. Alkyl or aryl substitu-
ents were introduced on the carbon atom of the amidine function
of N-alkylamidines (1,12-bis-[alkyl(or aryl)imino]-aminododecane
derivatives). Depending on the modulation thus introduced, two
groups can be distinguished. (i) In the usual reversed N-alkylami-
dines (3a–c) an alkyl group is attached to the imino group of the
amidine function. (ii) As opposed to the N-alkylamidines, the com-
pounds whose imino group is attached to an aromatic ring, are re-
-
-
2 Br
2 Cl
Calas et al. previously defined that the duplication of the cat-
ionic heads is necessary for good antiplasmodial activity, and opti-
mized the length of the alkyl linker to 12 methylenes.6 M64 was
the lead compound of the reversed N-alkylamidine compounds,
S
S
H
N
H
N
OH
HO
+
+
N
N
+
10
+
NH2
NH2
10
T3
M64
IC50 = 2.2 nM
IC50 = 2 nM
* Corresponding author. Tel.: +33 467548665; fax: +33 467548036.
E-mail address: yen.vo-hoang@univ-montp1.fr (Y. Vo-Hoang).
Figure 1. Biscationic antiplasmodial compounds.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.124

5816
O. Berger et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5815–5817
Table 1
ferred to as ‘‘reverse-benzamidines” (3d–o). We aimed to improve
the stability of the alkylamidine cationic heads, while maintaining
their in vivo antimalarial potencies.
Evaluation of reverse-benzamidine compounds as new antimalarials
a
c
Compounds
Calculated pKa
P. falciparum
IC50 (nM)
P. vinckei ED50
(mg/kg)
b
Reverse-benzamidines constitute a new series of alkylamidines.
They have been designed to evaluate how the aromatic ring intro-
duced within the cationic heads may influence the antiplasmodial
activity. Besides, we have developed another series of C-alkylami-
dines and reported previously that N-substitutions could improve
their in vivo antimalarial potencies.15 These results may be related
to the resulting pKa and/or lipophilicity. Thus, we have introduced
aromatic substituents and investigated the influence of the lipo-
philicity and the pKa of the resulting reverse-benzamidine com-
pounds on their in vitro and in vivo antimalarial potencies.
Molecules were synthesized as described in Figure 2. The appro-
priate alkyl-/benzo-nitriles 1a–p were treated under Pinner’s con-
ditions (HCl/EtOH) and converted into the ethyl alkyl-/benz-
imidates 2a–p. The late derivatives 2a–p were very unstable. The
crude product reacted immediately with 1,12-dodecanediamine
in the presence of triethylamine. The triethylamine was added to
neutralize the rest of hydrogen chloride that prevented generating
the targeted reversed amidines 3a–p. The different reversed alkyl-/
benz-amidine molecules 3a–p were purified as free amidines by
washing with acetonitrile, water, and diethyl ether. They were
then isolated and conserved as hydrochloride salts (washed with
anhydrous diethyl ether). In the N-alkylamidines, alkyl groups such
as ethyl (3a), iso-propyl (3b), and cyclopropyl (3c) were firstly
introduced, as well as hydroxylated function (3d). The amidines
were obtained starting, respectively, from commercially available
propionitrile, iso-butyronitrile, cyclopropylcyanide or hydrox-
yacetonitrile 1a–d. In the second series of reverse-benzamidines,
the aromatic ring was introduced by starting from the appropriate
benzonitriles.16 Most of the benzamidines were isolated as their
respective hydrochloride salt in good yields, except for 3j that is
more hindered, and for 3o due to problems of purification.
The in vitro antimalarial activities were evaluated against a
chloroquine-sensitive strain of P. falciparum (Nigerian strain).17,18
Results are given in Table 1. Both the N-alkylamidines and the re-
verse-benzamidines possessed potent antimalarial activities, in the
nanomolar range, except 3e, 3g, and 3h. The N-alkylamidine series
is quite homogeneous, with IC50 <20 nM, pKa >12.5, and Log P <5.
The in vitro antimalarial activity was not improved by bulkier alkyl
chains nor by a hydroxyl group introduced to increase the hydro-
solubility (3d). M64 is still the most potent compound among
the N-alkylamidines. Regarding the IC50 and the pKa values, the re-
verse-benzamidines are a more heterogeneous series as compared
to the N-alkylamidines. Indeed, the pKa values ranged between 10
and 14 and the antiplasmodial activities of 3e, 3g, and 3h were
weak (IC50 >450 nM), 3i–k and 3n possessed significantly higher
potencies (60 nM < IC50 < 20 nM), while 3f, 3l, 3m, 3o, and 3p were
the most potent compounds (IC50 <15 nM). The Log P values of the
reverse-benzamidines were higher than in N-alkylamidines group
(Log P >5.5, except for 3k and 3o with Log P ꢀ5). As expected,
the introduction of the phenyl aromatic ring led to increased lipo-
philicity. Since the introduction of the furan dramatically de-
M64d
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
12.65
12.67
12.28
12.67
14.15
11.53
12.08
10.96
10.36
12.67
11.46
10.65
12.41
11.81
11.55
13.53
12.45
2.2
18
10.15
16
9.15
715
13.5
455
520
26.5
24.5
55.5
6.7
3.1
>10
7
5
8.5
ud
>10
ud
ud
>>20
12
12
>5e
5
6.6
39
14
10
5
2.2
3.1
a
pKa were calculated using ACD/pKa DB, version 6.0, Advanced Chemistry
Development Inc.
IC50 against the in vitro growth of P. falciparum are means of at least two
independent experiments conducted in duplicate.
b
c
Antimalarial activities (Efficient Dose 50, ED50) were determined after ip
administration of the compounds once daily for four consecutive days to infected
mice (3 mice/dose).
Compound M64 was previously described.13
d
e
Toxicity is observed at higher doses.
creased antiplasmodial activity (3e: IC50 = 715 nM), we have no
more used a furan ring. In contrast, the compound 3f possessing
a phenyl ring revealed a good potency (IC50 = 13.5 nM). We have
then developed ten substituted reverse-benzamidines 3f–3p vary-
ing their basicity and their lipophilicity. These late constituted a
sufficient panel to establish some RSA studies. We did not point
out any significant relation between the IC50 of N-alkylamidines
nor reverse-benzamidines and their lipophilicity.
As opposed to Log P values, we have noticed the influence of the
basicity of N-alkylamidines and reverse-benzamidines on their
in vitro antiplasmodial activity. The Figure 3 illustrates that the ob-
served IC50 against the human P. falciparum parasite were strongly
related to the calculated pKa values of the compounds. This link is
likely more pronounced in the reverse-benzamidines series. In-
deed, the reverse-benzamidines sharing electro-withdrawing
groups like –CF3 (3g) or –NO2 (3h) possessed decreased basicity.
1000
100
10
H
N
H
N
R
R
O
R
ii
i
R
CN
.2HCl 10
.HCl
NH
NH
NH
1
10
11
12
13
14
1a-p
2a-p
3a-p
calculated pKa
Figure 2. Synthesis of the N-alkylamidines and reverse-benzamidine compounds.
Reagents and conditions: (i) gazeous HCl, EtOH, 20 h, 0 °C to rt; (ii) 1,12-
dodecanediamine, EtOH, Et3N, 24 h, rt. aLog P were calculated using ACD/Log P
DB, Advanced Chemistry Development Inc. bM64 compound was previously
described.6
Figure 3. In vitro antimalarial activity (IC50) of N-alkylamidine (4) and of reverse-
benzamidine (d) as a function of basicity of the cationic heads (calculated pKa). pKa
were calculated using ACD/pKa DB, version 6.0, Advanced Chemistry Development
Inc.

O. Berger et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5815–5817
5817
Their antiplasmodial potencies are thus dramatically decreased,
when compared to 3f. On the other hand, the derivatives with elec-
tro-releasing groups like –NH2 (3o) or –CH3 (3p) presented in-
creased pKa values and revealed good antiplasmodial potencies.
Since their antimalarial activities are strongly linked to their basi-
city, the reverse-benzamidine compounds seem to act in a similar
way as the alkylamidine choline analogs described by Calas et al.6
Indeed, they observed the same correlation between the antiplas-
modial activities and the pKa values of the molecules, suggesting
that the pKa values of alkylamidines reflect their ability to form
strong bonds with the target. This correlation indicates that the
mechanism of action of reverse-benzamidine compounds would
be rather linked to the capacity for these bis-cations to mimic
the choline structure and to inhibit the phospholipidic metabolism,
than associated to the ability of antiparasitic dibenzamidines to
bind DNA as pentamidine or furamidine.19,20
The in vivo antimalarial activities of our compounds were
investigated against the Plasmodium vinckei petteri strain (279BY)
in female Swiss mice.21 The in vitro antiplasmodial activity of 3e,
3g, and 3h being too low, they were not evaluated in vivo. The mice
were infected on day 0 and treated with compounds either intrap-
eritonally (ip) or orally (po) once daily for four consecutive days
(days 1–4 post infection, n = 3 per dose). The parasitemia levels
were monitored in mice at day 5. All the reversed N-alkylamidines
exhibited potent antiplasmodial activities, except 3a (Table 1). But
the modulations performed in N-alkylamidine series did not im-
prove M64 activity. The reverse-benzamidine 3i did not reveal
any antimalarial activity, while a slight antimalarial activity could
be detected with 3f, 3j, 3k, and 3l (ip administration of 5 mg/kg of
3l decreased parasitemia of 40% as compared to control). After ip
administration, the other reverse-benzamidines (3m, 3n, 3o, and
3p) exhibited as potent in vivo antimalarial activities (ED50 ip
<10 mg/kg) as the best N-alkylamidines, while having lower
in vitro antimalarial activities.
ful to Christophe Tran Van Ba for his assistance in testing the
compounds.
Supplementary data
Supplementary data (1H and 13C NMR, MS (FAB or ESI), FTIR
data of new compounds and biological protocol) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2010.07.124.
References and notes
1. In World Malaria Report 2008; WHO Press, 2008, pp 1–190.
2. In WHO Guidelines for the Treatment of Malaria; WHO Press, 2010, pp 1–194.
3. In World Malaria Report 2009; WHO Press, 2009, pp 1–66.
4. Lim, P.; Wongsrichanalai, C.; Chim, P.; Khim, N.; Kim, S.; Chy, S.; Sem, R.; Nhem,
S.; Yi, P.; Duong, S.; Bouth, D. M.; Genton, B.; Beck, H. P.; Gobert, J. G.; Rogers,
W. O.; Coppee, J. Y.; Fandeur, T.; Mercereau-Puijalon, O.; Ringwald, P.; Le Bras,
J.; Ariey, F. Antimicrob. Agents Chemother. 2010, 54, 2135.
5. Vial, H.; Calas, M.; Ancelin, M. L.; Escale, R.; Vidal, V.; Bressolle, F. WO Patent
WO 04/009068, 2004.
6. Calas, M.; Ouattara, M.; Piquet, G.; Ziora, Z.; Bordat, Y.; Ancelin, M. L.; Escale, R.;
Vial, H. J. Med. Chem. 2007, 50, 6307.
7. Salom-Roig, X. J.; Hamze, A.; Calas, M.; Vial, H. J. Comb. Chem. High Throughput
Screen. 2005, 8, 49.
8. Vial, H.; Calas, M.. In Antimalarial Chemotherapy, Mechanism of Action, Modes of
Resistance, and New Directions in Drug Development; Rosenthal, P., Ed.; The
Humana Press: Totowa, 2001; Vol. 347.
9. Vial, H. J.; Wein, S.; Farenc, C.; Kocken, C.; Nicolas, O.; Ancelin, M. L.; Bressolle,
F.; Thomas, A.; Calas, M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15458.
10. Calas, M.; Ancelin, M. L.; Cordina, G.; Portefaix, P.; Piquet, G.; Vidal-Sailhan, V.;
Vial, H. J. Med. Chem. 2000, 43, 505.
11. Hamze, A.; Rubi, E.; Arnal, P.; Boisbrun, M.; Carcel, C.; Salom-Roig, X.;
Maynadier, M.; Wein, S.; Vial, H.; Calas, M. J. Med. Chem. 2005, 48, 3639.
12. 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.
13. Ouattara, M.; Wein, S.; Calas, M.; Vo-Hoang, Y.; Vial, H.; Escale, R. Bioorg. Med.
Chem. Lett. 2007, 17, 593.
14. Results not published.
15. Degardin, M.; Wein, S.; Durand, T.; Escale, R.; Vial, H.; Vo-Hoang, Y. Bioorg. Med.
Chem. Lett. 2009, 19, 5233.
Oral administration of 180 mg/kg of N-alkylamidines or of the re-
verse-benzamidines 3f, 3j, 3k, 3l, and 3p did not reveal any antima-
larial effect. On the other side, significant activities were observed
with the other compounds 3m, 3n, and 3o, but the parasitemiaclear-
ance was not achieved and no ED50 po could be calculated. Thereby,
after administration of 180 mg/kg of 3m, 3n, and 3o, respectively,
42%, 20%, and 58% of decrease of parasitemia could be observed as
compared to control. These compounds appeared more efficient by
oral administration at 180 mg/kg than the N-alkylamidines, which
revealed no significant effects at that concentration. However, fur-
ther studies to improve the bioavailability, as well as pharmacoki-
netics experiments of the best compounds are needed to obtain
compounds suitable for drug development. For example, specific
prodrug strategies might be applied to the most potent compounds
in this new reverse-benzamidine series.
In conclusion, the reverse-benzamidines have been designed as
a new series of antimalarials. The introduction of a phenyl aro-
matic ring within the polar head can lead to molecules with im-
proved in vivo antimalarial activity. Indeed, four reverse-
benzamidine compounds exhibited potent antimalarial activities
(ED50 ip <10 mg/kg) and three of them (3m, 3n, and 3o) led to a de-
crease of parasitemia was detected after oral administration
(180 mg/kg). Furthermore we have shown that the antimalarial po-
tency can be strongly modulated by introducing aromatic substit-
uents that modify the basicity of the reverse-benzamidines.
16. For example, in an oven-dried three-neck flask, p-tolunitrile 1o (2.0 g,
17.07 mmol) was solubilized in 30 ml of dry ethanol and the mixture was
cooled to 0 °C in an ice-water bath. To saturate the reaction medium with
hydrogen chloride, a gaseous hydrogen chloride was bubbled for 20 min and
then, the flask was sealed. The mixture was stirred at room temperature for
20 h. After removal of the solvent under reduced pressure, the imidate 2o was
obtained as a white solid. Ethyl (p-tolu)imidate hydrochloride 2o: 1H (DMSO-
d6, 300 MHz) d: 1.55 (t, 3H, 7.0 Hz); 2.50 (s, 3H); 4.63 (quad, 2H, 7.0 Hz); 7.53
(d, 2H, 8.3 Hz); 8.16 (d, 2H, 8.3 Hz); 12.01 (large s, 2H). In an oven-dried flask,
the imidate 2o (crude, 15.02 mmol) and 1,12-dodecanediamine (1.2 g,
6.01 mmol) was suspended in dry ethanol (40 ml). Then, dry triethylamine
(8.4 ml, 60.1 mmol) was added dropwise. The reaction mixture was stirred at
room temperature and under nitrogen for 24 h. After removal of the solvent
under reduced pressure, the resulting crude was solubilized in an aqueous
solution of sodium hydroxide (1 M) to afford the free base. After stirring for 1 h,
the generated precipitate was washed with acetonitrile, water, and diethyl
ether. In the presence of hydrochloric acid, the amidine hydrochloride 3o was
formed, recovered after removal of the solvent under reduced pressure.
Washing the crude with dry diethyl ether led to a white solid (2.76 g, 80%).
1,12-(p-tolu)amidinedodecane 3o: 1H (DMSO-d6, 300 MHz) d: 1.24 (m, 16H);
1.60 (m, 4H); 2.36 (s, 6H) ; 3.43 (m, 4H); 7.35 (d, 4H, 8.5 Hz); 7.74 (d, 4H,
8.5 Hz); 9.26 (large s, 2H); 9.54 (large s, 2H); 10.01 (large s, 2H). 13C (DMSO-d6,
75 MHz) d: 21.0; 26.0; 27.3; 28.5; 28.8; 42.4; 125.7; 128.0; 129.1; 143.3; 162.0.
FT-IR cmꢁ1: 727; 824; 1377; 1510; 1576; 1618; 1668; 2853; 292þ3; 3041. ES+
SM: 435 [M+H+]; 218 [(M+2H+)/2]. HRMS calcd for C28H39N4O4 435.3488;
found 435.3485. Mp: 80–81 °C (Et2O).
17. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents
Chemother. 1979, 16, 710.
18. Vial, H. J.; Thuet, M. J.; Philippot, J. R. J. Protozool. 1982, 29, 258.
19. Baraldi, P. G.; Bovero, A.; Fruttarolo, F.; Preti, D.; Tabrizi, M. A.; Pavani, M. G.;
Romagnoli, R. Med. Res. Rev. 2004, 24, 475.
20. Boykin, D. W. J. Braz. Chem. Soc. 2002, 13, 763.
21. Barkan, D.; Ginsburg, H.; Golenser, J. Int. J. Parasitol. 2000, 30, 649.
Acknowledgments
These studies were supported by the European Commission
(Antimal integrated project LSHP-CT-2005-018834). We are grate-