Evaluation of amidoxime derivatives as prodrug candidates of potent bis-cationic antimalarials

Article abstract of DOI:10.1016/j.bmcl.2019.06.045

Plasmodium falciparum is responsible for most of the cases of malaria and its resistance to established antimalarial drugs is a major issue. Thus, new chemotherapies are needed to fight the emerging multi-drug resistance of P. falciparum malaria, like cho

Full text of DOI:10.1016/j.bmcl.2019.06.045

Accepted Manuscript
Evaluation of amidoxime derivatives as prodrug candidates of potent bis-cati-
onic antimalarials
Olivier Berger, Stéphanie Ortial, Sharon Wein, Séverine Denoyelle, Françoise
Bressolle, Thierry Durand, Roger Escale, Henri Vial, Yen Vo-Hoang
PII:
S0960-894X(19)30427-5
https://doi.org/10.1016/j.bmcl.2019.06.045
BMCL 26523
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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18 June 2019
22 June 2019
Please cite this article as: Berger, O., Ortial, S., Wein, S., Denoyelle, S., Bressolle, F., Durand, T., Escale, R., Vial,
H., Vo-Hoang, Y., Evaluation of amidoxime derivatives as prodrug candidates of potent bis-cationic antimalarials,
Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.06.045
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Amidoxime prodrugs
Leave this area blank for abstract info.
R1 = primary or N-substituted amine
NOH
X
NOH
R2 = H or Me
Br
NOH
X
NOH
X
NOH
R1
R1
R1
N
R3
R3
S
bioconversion studies
OR2
NH
NH
R3 = H or mono- or di-substituents
X = Alkyl or phenoxy-alkyl chain
R1
X
R1

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Evaluation of amidoxime derivatives as prodrug candidates of potent bis-
cationic antimalarials
Olivier Bergera , Stéphanie Ortiala , Sharon Weinb , Séverine Denoyellea , Françoise Bressollec, †, Thierry
Duranda , Roger Escalea , Henri Vialb and Yen Vo-Hoang a, 
a Institut des Biomolecules Max Mousseron, UMR 5247, Université de Montpellier, CNRS, ENSCM, Faculté des Sciences Pharmaceutiques et Biologiques, 15
avenue Charles Flahault, 34093 Montpellier, France.
b Dynamique Moléculaire des Interactions Membranaires Normales et Pathologiques, UMR 5235 CNRS-UM, Place Eugène Bataillon, 34095 Montpellier,
France.
c Pharmacocinetique Clinique, EA4215, Faculté des Sciences Pharmaceutiques et Biologiques, 15 Avenue Charles Flahault, 34093 Montpellier, France.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Plasmodium falciparum is responsible for most of the cases of malaria and its resistance to
established antimalarial drugs is a major issue. Thus, new chemotherapies are needed to fight the
emerging multi-drug resistance of P. falciparum malaria, like choline analogues targeting
plasmodial phospholipidic metabolism. Here we describe the synthesis of amidoxime derivatives
as prodrug candidates of reverse-benzamidines and hybrid compounds able to mimic choline, as
well as the design of a new series of asymmetrical bis-cationic compounds. Bioconversion
studies were conducted on amidoximes in asymmetrical series and showed that amidoxime
prodrug strategy could be applied on C-alkylamidine moieties, like benzamidines and that N-
substituents did not alter the bioconversion of amidoximes. The antimalarial activity of the three
series of compounds was evaluated in vitro against P. falciparum and in vivo against P. vinckei
petteri in mice.
Keywords:
oral antimalarial activity
amidoximes
prodrugs
biotransformations
choline analogues
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: 33-475119539; fax: +33-475119641; e-mail: yen.vo-hoang@umontpellier.fr
† Present address : R&D Department, Pharmacometrica, Longcol, La Fouillade, France.

reported as well as the bioconversion studies of the asymmetrical
amidoxime prodrug candidates into the desired bis-cationic
drugs.
Malaria is a widespread life-threatening disease caused by
Plasmodium parasites, responsible for 219 million cases
worldwide in 2017 and 435 000 deaths.1 Moreover, multidrug
resistance, including artemisinin resistance of P. falciparum has
been reported.2 To counter chemo-resistance, choline analogues
have been developed as a new class of antimalarial drugs.3 Not
only are they structurally different from existing agents, but they
also exhibit an innovative mechanism of action, as demonstrated
with the bisthiazolium salt T3 (Figure 1).4 Indeed, they condemn
the parasite to death by multiple ways:5 i) T3 highly and
specifically accumulates inside P. falciparum infected
erythrocytes;6 ii) T3 competitively inhibits choline transport and
enzymes of the phosphatidylcholine de novo biosynthesis
pathway of Plasmodium.7 Unfortunately,T3 [named albitiazolium
(INN)] will not be further clinically developed due to its low oral
bioavailability and high clearance in children.8 Bis-alkylamidines
were also developed as choline analogues,9 as well as reverse-
benzamidines (e.g. compound 1)10 and hybrid bis-cationic
compounds (e.g. compound 2).11
The synthesis of amidoxime derivatives of reverse-
benzamidines is presented in Scheme 1. Bis-benzamides 3a-g
were prepared from appropriate benzoyl chlorides and 1,12-
diaminododecane in dichloromethane (DCM), in the presence of
triethylamine (TEA), at room temperature (RT). By reaction with
Lawesson’s reagent, 3a-g afforded bis-thioamides, which reacted
with either hydroxylamine or methoxyamine hydrochlorides in
the presence of mercury (II) oxide (HgO) and pyridine (Pyr) in
tetrahydrofuran (THF) to provide the targeted bis-
benzamidoximes 4a-f and bis-O-methyl benzamidoximes 4g-m.
)
H2N
NH2
(
10
a
: R1 = H
3a
3b
3c
3d
3e
3f
: R1 = p-OMe
: R1 = m-OMe
: R1 = p-CH3
: R1 = 3,5-OMe
: R1 = 3,4-OMe
: R1 = o-OMe
O
O
)
N
H
(
N
10
R1
R1
H
2 Br -
3g
3a-g
HO
OH
R1
H
R2 = H
4a
R2 = Me
4g
S
S
N
N
b, c
8
p-OMe
m-OMe
p-CH3
4b
4h
NOR2
NOR2
T3
albithiazolium :
4c
4i
IC50 = 2.2 nM
4d
4j
)
N
H
(
N
10
R1
R1
3,5-OMe
3,4-OMe
o-OMe
4e
4f
4k
H
2 Br -
NH
NH
4l
H2N
4a-m
4m
N
N
N
S
8
H
H
10
NH2
OH
Scheme 1. Synthesis of amidoximes in reverse-benzamidine series. Reagents
and conditions: a) benzoyl chloride, TEA, DCM, RT, 2 h (80-100%); b)
Lawesson’s reagent, toluene, reflux, 16 h (60-95%); c) NH2OH.HCl or
NH2OMe.HCl, Pyr/HgO, THF, RT, 24 h (69-95%).
2 HCl
OMe
OMe
1
hybrid alkylamidine 2
IC50 = 2.2 nM
reverse-benzamidine
IC50 = 6.6 nM
H
H
O
O
5
Amidoxime prodrug candidates of hybrid bis-cations were
obtained as described in Scheme 2. The nitriles 5a-d, prepared
according to a previous report,11 reacted with hydroxylamine
hydrochloride in the presence of sodium hydroxide in ethanol
(EtOH) to generate the amidoximes 6a-d.
N
N
12
XN
NX
NH2
NX
NX
NH2
M64
bis-N-alkylamidine
bis-N-alkylamidoxime
X = H
X = OH
pentamidine X = H
pentamidoxime X = OH
M64-AH
Figure 1. Chemical structures of described drugs and amidoxime prodrugs.
Br
Br
H2N
Over the past decade, we focused our effort on the design of
orally available bis-alkylamidine-based compounds. Indeed, their
bis-cationic character under physiological conditions is necessary
for their antimalarial activity but prevents absorption from the
gastrointestinal tract. To circumvent the too low oral
bioavailability of amidines, the approach “amidoximes instead of
amidine drugs” was developed12 and succeeded for many
benzamidine-type compounds, including antiparasitic and
antibacterial agents,13 thrombin inhibitors,14 and anticancer
agent,15 as well as for antiviral guanidine drug.16 Therefore, we
applied the amidoxime prodrug strategy to bis-N-alkylamidines,17
bis-C-alkylamidine18–20 and bis-N-alkylguanidines.21 The
bioconversion of pentamidoxime prodrug (Figure 1) into the
corresponding pentamidine drug was described, as well as other
benzamidoximes22 and N-hydroxyguanidine or acetamidoxime
prodrugs.16 Similarly, the bis-N-alkylamidoxime M64-AH could
be converted into M64 drug (Figure 1).23 Apart from this work on
bis-N-alkylamidine series and to the best of our knowledge, no
data is available on the metabolism of either bis-C-
alkylamidoxime or N-substituted alkyl/benz-amidoximes.
)
NC
N
(
)
10
(
N
10
a
S
S
NOH
OH
OH
5a
6a
Br
S
Br
O
N
)
(
O
N
)
n
(
n
S
a
OR3
OR3
CN
H2N
NOH
5b-d
6b-d
: n = 9, R3 = CH3
: n = 9, R3 = H
: n = 6, R3 = H
b
c
d
Scheme 2. Synthesis of hybrid prodrug candidates. Reagents and conditions: a)
NH2OH, EtOH, RT, 4 days (14-53%).
At last, asymmetrical amidines and amidoximes were
synthesized according to Scheme 3. Compounds 7a,b11 reacted
with potassium cyanide to generate the nitriles 8a,b, which were
converted under Pinner’s conditions into unstable ethyl
imidates,24 and reacted with appropriate amines to provide the
corresponding amidines 9, 10a-c. The amidoxime 11a was
obtained from the nitrile 8b by reaction with hydroxylamine
In the present study, we describe the synthesis of amidoxime
prodrug candidates of reverse-benzamidines and hybrid
compounds as well as the development of a novel series of
asymmetrical drugs and their potential prodrugs. In vivo and in
vitro antimalarial activities of the synthesized compounds are

hydrochloride and sodium hydroxide. For N-substituted
amidoximes 11b-c, the nitrile 8a was reduced with
diisobutylaluminium hydride (DIBAL-H). The resulting
against the Nigerian strain of P. falciparum26,27 and in vivo
against the P. vinckei petteri strain (279BY) in female Swiss
mice according to a modified version of the four-day suppressive
test.28 Since the antiplasmodial activity is strongly related to the
basicity of the amidines,9 the bis-amidoximes 4a-m and 11a-c
were expected to possess very moderate in vitro antiplasmodial
activity (micromolar range). This assumption was verified by
testing in vitro one compound in each series, i.e. 4b: IC50 = 24
µM, 11a: IC50 = 20 µM (concentration required to inhibit the
parasite viability by 50%). In vivo, the tested amidoximes in
reverse-benzamidine series did not reveal any significant activity
at the tested doses.29
dialdehyde
reacted
immediately
with
hydroxylamine
hydrochloride and Pyr to generate the dioxime 12. By reaction
with N-chlorosuccinimide (NCS) in dimethylformamide (DMF),
12 formed dihydroxamoyle chloride 13, which reacted
immediately with appropriate amines to provide the targeted
1
amidoximes 11b-c. All the compounds were characterized by H
and 13C NMR, MS (ESI), FTIR and the data were consistent with
the structures.25
R4
C
)
O
C
(
n
CN
Table 1 reports the IC50 and the ED50 (dose required to inhibit
the parasitemia by 50%) after intraperitoneal (i.p.) and oral (p.o.)
administrations of the amidoxime prodrug candidates of the
hybrid drugs (compounds 6a-d). In vitro, the antiplasmodial
activities of the amidoxime derivatives of the hybrid drugs were
moderate (IC50 > 200 nM). Indeed, since the amidoxime polar
head is not protonated under physiological conditions, the
molecules 6a-d may not act as bis-cations, but as mono-
thiazolium salts, about 100 fold less potent as compared with the
duplicated analogues.30,31 In vivo, compound 6c did not reveal
any antimalarial effect at the tested doses while a significant
antimalarial activity was detected after i.p. administration of 6a
and 6b (ED50 of 20 and 8.8 mg/kg respectively). Unfortunately,
6a and 6b did not reveal any oral antimalarial activity at the
tested doses. On the contrary, i.p. administration of 20 mg/kg of
compound 6d did not result in a clearance of parasitemia, but oral
administration of 6d revealed a significant antimalarial activity
(ED50 = 85 mg/kg) with a complete clearance of parasitemia
(without recrudescence in the following 28 days).32 This
noteworthy oral antimalarial effect may be related to the nature
of the linker of 6d. Indeed, longer linker (12 or 11 methylene
units for 6a or 6c respectively) may hamper oral bioavailability.
On the contrary, the shorter alkyl chain of 6d decreased
molecular flexibility (less rotatable bonds), an important
characteristic for good oral bioavailability.33 In addition, the
aromatic ring facilitated the oral efficiency of 6d11 and
maintained the desired distance between the cationic heads.30
)
(
O
Br
n
)
(
O
n
NH2
b, c
a
2 Cl-
R4
NH2
CN
CN
4
7a:
7b:
n = 9
n = 6
8a:
8b:
n = 9
n = 6
9
: n = 9, R = NH2
: n = 6, R4 = NH2
: n = 6, R4 = NH-iPr
: n = 6, R4 = pyrrolidine
10a
10b
10c
e, f
H
d
NH2
C
)
(
O
C
6
C
)
(
O
C
6
NOH
NOH
11a
H
NOH
H2N
NOH
12
R4
Cl
g
C
C
O
C
)
(
6
)
(
O
C
6
NOH
NOH
h
11b:
11c:
R = NH-iPr
R = pyrrolidine
R4
NOH
Cl
NOH
13
Scheme 3. Synthesis of asymmetrical bis-cationic drugs and amidoxime prodrug
candidates. Reagents and conditions: a) KCN, EtOH/H2O, reflux, 24 h (81-85%);
b) gaseous HCl, anhydrous EtOH/Et2O, RT, 24 h; c) NH3, isopropylamine or
pyrrolidine in anhydrous EtOH, RT, 48 h (53-92%, two steps); d) NH2OH.HCl,
NaOH, EtOH/H2O, reflux, 72 h (76%); e) DIBAL-H, CH2Cl2, -78°C to -40 °C, 1 h;
f) NH2OH.HCl, Pyr, EtOH, reflux, 24h (46%, two steps); g) NCS, DMF, RT, 2 h;
h) isopropylamine or pyrrolidine, TEA, Et2O, RT, 48h (15-30%, two steps).
Among these three series, seventeen compounds were
evaluated for antimalarial activity. They were tested in vitro
Table 1. In vitro and in vivo evaluation of antimalarial activity of hybrid amidoxime prodrug candidates 6a-d produced via
Scheme 2.
Linker
ED50 [mg/kg] b
IC50 [[nM] a
Compounds
R3
P. vinckei
P. falciparum
n
Aromatic ring
i.p.
1.2
p.o.
-
Artesunate
3.8
23
Chloroquine
1.1
20
3.4
> 180
> 90
> 90
85
6a
6b
6c
6d
H
CH3
H
10
9
-
240 c
530
315
360
Phenyl-O
Phenyl-O
Phenyl-O
8.8
9
> 20
> 20
H
6
a IC50 are means of at least two independent experiments conducted in duplicate.
b Antimalarial activities (Efficient dose 50, ED50) were determined after i.p. or p.o. administration of the compounds once daily for 4 days to infected mice.
b Single value determined in duplicate.
.

Table 2. In vitro and in vivo evaluation of antimalarial activity of asymmetrical drugs 9, 10a-c and prodrug candidates 11a-c
produced via Scheme 3.
Calculated values
ED50 [mg/kg] b
IC50 [nM] a
Compounds
n
R4
P. vinckei
P. falciparum
pKa
Log P
4.23
i.p.
2.2
p.o.
9
9
6
6
6
6
6
6
NH2
NH2
11.66
12.40
12.30
13.00
6.41
195
51.5
9.5
n.d. c
10a
10b
10c
11a
11b
11c
2.88
5.24
5.36
3.31
5.63
5.76
9
> 90
110
51
NHiPr
2.8
pyrrolidine
NH2
16
1.4
2000
n.d. c
n.d. c
> 10
> 20
10
> 180
> 180
> 100
NHiPr
3.30
pyrrolidine
2.21
a pKa and Log P values were calculated using ACD/pKa DB, version 6.0, Advanced Chemistry Development Inc.
b IC50 are means of at least two independent experiments conducted in duplicate.
c Antimalarial activities (Efficient dose 50, ED50) were determined after i.p. or p.o. administration of the compounds once daily for 4 days to infected mice.
d n.d. means not determined.
Regarding these latter interesting results, we consequently
designed an original series of asymmetrical drugs/prodrugs
sharing simultaneously benz-amidine/-amidoxime and alkyl-
amidine/-amidoxime moieties as polar heads, both being possibly
N-substituted. The evaluation of their antimalarial activity is
reported in Table 2. Concerning drugs, compound 9 exhibited
weaker in vitro antimalarial activity than compounds 10a-c (IC50
in nanomolar range), maybe because of its too long alkyl linker.30
Nevertheless, when tested in vivo, all the amidine drugs 9, 10a-c
showed potent i.p. antimalarial activities (close to values
Artesunate and chloroquine reported in Table 1). Remarkably, a
complete clearance of parasitemia could be recorded after oral
administration of the two amidine drugs 10b and 10c (ED50 of
110 and 51 mg/kg). Therefore, pyrrolidine and isopropylamine
introduced on amidine moieties led to favourable
pharmacological profile. Since 9 had the weakest in vitro activity,
we focused our effort on testing amidoximes 11a-c with 8
methylene bridges. Unfortunately, the asymmetrical amidoximes
11a-c did not reveal any antimalarial activity at the tested doses
in vivo, whether after i.p. or p.o. administration, except 11c, with
ED50 i.p. of 10 mg/kg. It is worth mentioning that all these new
compounds in hybrid (6a-d) and asymmetrical series (9, 10a-c
and 11a-c) were well tolerated by mice in the herein reported
experiments. No sign of clinical toxicity was observed after i.p.
or p.o. administration of drugs once daily for four consecutive
days.
as predominant metabolites (from 82% to 90 % of the amidoxime
disappearance depending on the prodrugs).
Table 3. Metabolism of pentamidoxime and asymmetrical
amidoximes 11a-c by human liver microsomes.
Compounds
tR (min) [a]
Rate of the total prodrug
disappearance [b]
Pentamidoxime
Monoamidine
Pentamidine
11a
29.0
27.1
25.1
15.6
16.5
17.3
13.3
12.1
10.0
12.3
11.8
3%
15%
82%
7%
Monoamidine
10a
7%
86%
6%
11b
Monoamidine
10b
4%
90%
14%
86%
11c
10c
a Retention time (tR) of metabolites analyzed by HPLC-UV (for 11a and 11b)
or by LC-MS (11c) after incubation of pentamidoxime,11a, 11b, or 11c with
human liver microsomes.
b The prodrug disappearance is expressed in % of control.
In conclusion, we have designed a new series of asymmetrical
compounds sharing a C-alkylamidine cationic head and a
benzamidine cationic head as well as an optimal length of the
alkyl chain comprised of eight methylene linked to an aromatic
ring. We could reach highly potent compounds 10b-c.
Amidoxime derivatives were synthesized in three series of
compounds. Disappointingly, amidoximes did not reveal any oral
antimalarial activity at the tested doses in reverse-benzamidine
series and in asymmetrical series. Nevertheless, asymmetrical
amidoximes 11a-c were efficiently converted into the
corresponding amidine drugs 10a-c by liver microsomes.
Noteworthy, the conversion was not altered using C-akylamidine
function nor N-substituents. The observed lack of oral activity of
amidoximes (reverse benzamidine or asymmetrical series) might
be solved by specific O-substituents. However, the amidoxime
prodrug approach improved significantly oral antimalarial
activity of one new hybrid amidoxime 6d, with an i.p./p.o. index
> 0.23, paving the way to finding orally potent prodrugs.
In addition, high pressure liquid chromatographic (HPLC)
methods were developed to detect and determine each amidine
(10a-c) and amidoxime (11a-c) in human matrices. The stability
of amidoximes 11a-c in human plasma and blood was confirmed
by using different conditions (see SD for details). The
bioconversion of pentamidoxime (used as reference) and
amidoximes 11a-c into pentamidine and amidines 10a-c,
respectively was studied using a modified version of the
conditions described by Clement and co-workers.22,34 Briefly,
human liver microsomal preparations containing amidoximes
(0.5 mm) as substrate plus NADH,H+ cofactor solution were
incubated for two hours at 37 °C, then assayed by HPLC. The
disappearance of the prodrug candidates and the formation of
biotransformation products were monitored. The results are
presented in Table 3. Liver microsomes effectively reduced both
amidoxime functions of pentamidoxime, 11a, 11b and 11c,
mainly leading to respectively pentamidine,35 10a, 10b and 10c

ximelagatran and its active metabolite melagatran: a mini-review.
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Acknowledgements
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cancer agent upamostat by the mARC enzyme system.
This work was supported by the European Community
Integrated Project AntiMal (No. IP-018834). S.D. and S. O. are
grateful to Sanofi-Aventis for postdoctoral fellowships. We are
grateful to Christophe Tran Van Ba for his assistance in testing
compounds.
Xenobiotica.
https://doi.org/10.3109/00498254.2013.767481.
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17. Ouattara M, Wein S, Calas M, Vo-Hoang Y, Vial H, Escale R.
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Supplementary data
1
13
H and C NMR, MS (FAB or ESI), FTIR data of new
compounds and biological protocol are given.
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