Exploration of potential prodrug approach of the bis-thiazolium salts T3 and T4 for orally delivered antimalarials

Article abstract of DOI:10.1016/j.bmcl.2010.05.001

We report here the synthesis and biological evaluation of a series of 37 compounds as precursors of potent antimalarial bis-thiazolium salts (T3 and T4). These prodrugs were either thioester, thiocarbonate or thiocarbamate type and were synthesized in one

Full text of DOI:10.1016/j.bmcl.2010.05.001

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3953–3956
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploration of potential prodrug approach of the bis-thiazolium salts
T3 and T4 for orally delivered antimalarials
Sergio A. Caldarelli a, Michel Boisbrun a, , Karine Alarcon a, Abdallah Hamzé a, Mahama Ouattara a,
Xavier Salom-Roig a, Marjorie Maynadier b, Sharon Wein b, Suzanne Peyrottes a, Alain Pellet c,
Michèle Calas a,à, Henri Vial b,à,
*
a Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France
b Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR 5235, Université de Montpellier 2, CC 107, Place Eugène Bataillon,
34095 Montpellier Cedex 5, France
c Sanofi-Aventis, Research & Development, 195 route d’Espagne, BP 13669, 31036 Toulouse Cedex 1, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here the synthesis and biological evaluation of a series of 37 compounds as precursors of
potent antimalarial bis-thiazolium salts (T3 and T4). These prodrugs were either thioester, thiocarbonate
or thiocarbamate type and were synthesized in one step by reaction of an alkaline solution of the parent
drug with the appropriate activated acyl group. Structural variations affecting physicochemical proper-
ties were made in order to improve oral activity. Twenty-five of them exhibited potent antimalarial activ-
ity with IC50 lower than 7 nM against Plasmodium falciparum in vitro. Notably, 3 and 22 showed IC50 = 2.2
and 1.8 nM, respectively. After oral administration 22 was the most potent compound clearing the par-
asitemia in Plasmodium vinckei infected mice with a dose of 1.3 mg/kg.
Received 10 March 2010
Revised 30 April 2010
Accepted 1 May 2010
Available online 10 May 2010
Keywords:
Antimalarials
Choline analogues
Prodrug
Ó 2010 Elsevier Ltd. All rights reserved.
Today, approximately 40% of the world’s population is at risk of
malaria and 109 countries are endemic for malaria. Every year, 300
million malaria cases cause at least a million deaths, mostly of chil-
dren under the age of 5.1 Malaria is caused by protozoan parasites
of the genus Plasmodium. It is nowadays well described that para-
site resistance of Plasmodium falciparum, the most severe form of
malaria, is responsible for malaria-related mortality and morbid-
ity,2–4 and constitute a major obstacle for an eradication of malar-
ia.5 Because of the lack of a vaccine, it is thus clear that the
discovery of new treatments based on innovative mechanisms of
action is really needed.6,7
In the past decade, our group has developed a new approach
that targets the lipid peculiarities of P. falciparum. During its intra-
erythrocytic development, the parasite synthesizes considerable
amounts of membranes, from phospholipid precursors scavenged
from the human host. Among those, phosphatidylcholine (PC) is
the most abundant and its content increases sixfold after infec-
tion.8–10 Consequently, the phospholipid biosynthesis has been
viewed as an ideal target for the development of new antimalarial
drugs.11 The essential role of PC for the parasite survival has been
demonstrated by using choline analogues, which had been ratio-
nally designed and optimised for their ability to inhibit PC biosyn-
thesis in Plasmodium and to block parasite proliferation in the low
nanomolar range. In this context, compounds containing a duplica-
tion of the cationic group (i.e., bisquaternary ammonium salts)
separated by a long lipophilic alkyl chain had been designed and
evaluated.12–16 We reported the synthesis and antimalarial activi-
ties of a series of bis-thiazolium salts (Fig. 1).17,18 T3 and T4, the
lead compounds in this series, showed powerful in vitro and
in vivo biological activities (IC50 = 2.2 and 0.65 nM on P. falciparum,
and ED50 = 0.2 and 0.14 mg/kg in mice infected by Plasmodium
vinckei by intraperitoneal mode). Compound T3 (SAR97276; 1,12-
bis[5-(2-hydroxyethyl)-4-methyl-1,3-thiazol-3-ium]dodecane di-
bromide) is a new antimalarial drug, which is currently being eval-
uated in clinical trials (phase II) for severe malaria.
However, the presence of permanently charged cationic groups,
a crucial requirement for antimalarial activity of choline analogues,
Br
Br
S
+
S
+
N
(CH2)12
N
R1O
OR1
* Corresponding author. Tel.: +33 4 67143745; fax: +33 4 67144286.
E-mail address: vial@univ-montp2.fr (H. Vial).
R1 = H
1 = CH3 T4
T3
Present address: Groupe SUCRES, UMR 7565 Nancy-Université, CNRS, BP70239,
54506 Vandœuvre-lès-Nancy Cedex, France.
R
à
These authors contributed equally to the work.
Figure 1. T3 and T4, lead choline analogues.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.001

3954
S. A. Caldarelli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3953–3956
O
O
is detrimental to oral absorption. As oral administration is essential
for treatment in dispensaries in endemic countries and for prophy-
lactic or curative treatment for travellers, this led us to develop
neutral precursor of bis-thiazolium salts.19 These prodrugs incor-
porate thioester, thiocarbonate or thiocarbamate pro-moieties
(TE series), which in vivo are expected to revert back to thiazolium
salts after enzymatic transformation by plasmatic esterases
(Fig. 2a). A similar prodrug approach has been applied to improve
oral delivery of Thiamin (vitamin B1), a thiazolium salt which is
also poorly absorbed.20–23
O
H
H
O
H
O
-OH
-OH
OR1
H
H
R2
CH2Cl2
OR1
X
S-
S
N
S
N
S
N
R2
OR1
N
+
H2O
OR1
T
ET-
T°
TE
X = leaving group
Figure 3. Drug–prodrug conversion mechanism.
Table 1
Preliminary studies on TE prodrugs 1–3 (Fig. 2b) demonstrated
that such molecules lead to a complete protection in murine mod-
els using short-course treatment. Furthermore, in Rhesus monkeys
prodrug 3 was responsible for a complete cure of Plasmodium cyno-
molgi infection without recrudescence, showing valuable pharma-
cokinetic properties.19 Thus, bioprecursor 3 appeared to be stable
in saline medium, whereas a rapid conversion into T3 with an ini-
tial conversion half-life of ꢀ5 min and nearly complete bioconver-
sion (recovery >85%) was observed in plasma after 8 h.24 From
these encouraging results, we developed a program to improve
the delivery of our thiazolium drugs based on neutral bio- precur-
sors and we synthesized a library of 37 neutral prodrugs of the par-
ent drugs T3 and T4.17 Structural variations of the pro-moieties
were designed to affect lipophilicity, molecular weight and stabil-
ity towards enzymes. Ideally, prodrugs should be quantitatively
and rapidly converted in vivo to the active parent drug after cross-
ing the gastrointestinal barrier.
The synthetic pathway developed takes advantage of the partic-
ular reactivity of thiazolium ring (T) which may be opened in basic
aqueous solution and re-closed upon acidification (Fig. 3). Thus,
thiazolium ions show a pH-dependent equilibrium which favours
the quaternary cation T at low pH and a ring-opened amido enethi-
olate (ETÀ) at high pH, with the hemithioacetal T° being the most
plausible intermediate. Trapping of the ETÀ intermediate with an
acylating agent leads to a neutral thioester, thiocarbonate or thio-
carbamate (TE) structure according to the method reported by
Matsukawa et al.20 for S-acylated thiamine (Fig. 3).
Thioester derivatives (Table 1) were obtained upon treatment
of T4 with an excess of sodium hydroxide aqueous solution under
ice cooling, followed by slow addition (to the in situ formed thio-
late) of the appropriate commercial or freshly prepared (from the
corresponding acid and thionyl chloride) acyloyl, aryloyl chloride
or activated carboxylic acid in dichloromethane. Compound 18
was prepared from levulinic acid activated by isobutyl chlorofor-
mate (IBCF) and derivative 2 required the prior preparation of the
corresponding substituted-benzoyl chloride.25 Derivative 20 was
obtained in two steps from Boc-proline-OH activated with IBCF
followed by cleavage of the tertbutyloxycarbonyl amino-protect-
ing group with TFA. Thiocarbonate compounds (Table 2) were
synthesized from commercially available alkyl chloroformates.
Fluorinated derivative 30 required the previous preparation of
2-fluoroethylchloroformate by reacting 2-fluoroethanol with
In vitro (against P. falciparum) and in vivo (against P. vinckei) antimalarial activities of
the thioester prodrugs and the corresponding drug T4
O
H
O
N
S
(CH2)6
R2
OMe
2
Compd
R2
Yield (%)
IC50 (nM)
ED50 (mg/kg)
ip
po
T4a
4c
5
—
-Me
-Et
-iPr
-cycloPr
-iBu
-tBu
-cycloBu
-cycloPentyl
CH3
—
0.65
1.1
4.3
1.1
2
2.4
6.7
3.6
8.2
0.14
nd
ꢁ0.1
0.12
nd
nd
nd
nd
0.65
8.1
nd
12
11
16
16
nd
nd
17
69
74
91
77
96
72
88
90
1c
6
7
8c
9
10
11
17
3.3
nd
16
12c
13
14
15
16
17
18
19
-Ph
70
76
86
15
76
78
43
1.7
<0.5
0.25
>2.5
1.9
nd
0.4
15
24
>90
80
14
10
3
-Ph-2-Me
-Ph-2-CF3
–CH2OMe
2-Furfuryl
(CH2)2CO2Me
(CH2)2COMe
C(CH3)2OCOMe
110 (7.5)b
2200 (1600)b
2.3
1.6
5.9
1.6
15.6
0.15
60.3
57
N
2c
80
1.5
2.8
2.1
3.4
nd
nd
90
O
H
N
20
21
46d
22
17
N
<30
a
b
c
Activity of this compound has already been reported.17
After pre-incubation.
Activity of these compounds has already been reported.18,19
Total yield over two steps.
d
phosgene. Thiocarbamate 32 was synthesized from 4-nitrophenyl
2-oxopyrrolidine-1-carboxylate prepared beforehand from pyrr-
olidin-2-one, chlorotrimethylsilane and 4-nitrophenyl chloro-
formate.
Similarly, addition of an acyloyl chloride to an alkaline aque-
ous solution of T3 led to the chemically unstable thioester which
underwent an intramolecular trans-acylation with the free hydro-
xyl group, yielding after re-closing of thiazolium ring to an
O-acylated T3 analogue (Fig. 4). Thus, intramolecular acyl migra-
tion was avoided by trapping the free hydroxyl group of the thi-
oester intermediate with an excess of acyloyl chloride. This
protocol provided O- and S-diacylated T3 prodrugs 33–36
(Fig. 5a, Table 3). Finally, T3 cyclic thiocarbonate prodrug 3
(TE3)19 and its thio-analogue 37 were obtained by treating an
alkaline solution of T3 with either 4-nitrophenyl chloroformate
(Fig. 5b, Table 3) or thiocarbonyldiimidazole.
(a)
O
N
H
O
+
N
(CH2)6
S
S
esterase
(CH2)6
+
R'COOH
R2
OR1
OR1
T
TE
1: R2 = iPr
2: R2
2
2
O
N
(b)
H
S
O
(CH2)6
N
O
O
=
All new compounds were characterised by 1H NMR (purity
P95%) and MS (ESI) and the data were consistent with the
structures.
2
3
Figure 2. Bioprecursors (TE) of thiazolium salts (T).

S. A. Caldarelli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3953–3956
3955
Table 2
O
N
Br
+
In vitro (against P. falciparum) and in vivo (against P. vinckei) antimalarial activities of
thiocarbonate and thiocarbamate prodrugs and the corresponding drug T4
H
O
S
(CH2)6
N
(CH2)6
S
1) NaOH, H2O
a)
b)
R2
2) R2COCl, TEA, CH2Cl2
O
OH
H
O
OCOR2
T3
33-36
2
2
N
S
(CH2)6
R2
O
N
Br
+
H
O
O
OMe
1) NaOH, H2O
2
S
(CH2)6
N
(CH2)6
S
O
O
2)
Compd
R2
Yield (%)
IC50 (nM)
ED50 (mg/kg)
Cl
NO2
OH
T3
2
ip
po
TE3 (3)
TEA, CH2Cl2
2
53%
T4a
22
23
24
25
26
—
—
0.65
1.85
2.9
6.2
5.6
0.14
0.072
0.21
<0.1
nd
8.1
1.3
6.3
6.8
16
–OCH3
–OEt
–OiPr
OBu
88
71
70
82
82
Figure 5. TE prodrug synthesis of bis thiazolium salts T3.
Table 3
–OPh
3.5
0.3
24
In vitro (against P. falciparum) and in vivo (against P. vinckei) antimalarial activities of
the thioester prodrugs and the corresponding parent drug T3
O
NO2
27
50
3.1
nd
>>30
13
O
H
O
Cl
N
S
28
95
10.6
0.11
(CH2)6
R2
O
CH3
Br
29
30
81
77
2.7
2.2
nd
13
O
O
OCOR2
2
F
0.1
3.8
Compd R2
Yield (%) IC50 (nM)
ED50 (mg/kg)
N
31
32
15
40
310
>5
>90
12
ip
po
O
T3a
33
34
35
36
—
-iPr
—
—
74
44
52
2.2
7
0.2
1.2
0.5
1.8
13
60
13
>60
>180
29.5
0.9
N
(CH2)2CO2Me
Ph-2,3,4-(OCH3)3
Ph-2-Me
48
16.5 (6.5)b
Activity of this compound has already been reported.17
a
3500 (570)b >20
CHO
O
O
S
(CH2)6
(CH2)6
N
3c
45
22
2.2
0.25
5
The powerful in vitro activities of the bis-thiazolium salts T3
and T417 and prodrugs 1–318,19 (Tables 1 and 3) have already been
reported. IC50 values are 2.2 and 0.65 nM for T3 and T4, respec-
tively. Compounds 1, 2 and 3 exhibited IC50 in the same low nano-
molar range values as the parent drugs of 1.1, 1.5 and 2.2 nM,
respectively, indicating a quantitative conversion.
2
CHO
S
S
N
37
4400
>30
O
2
Activity of these compounds has already been reported.17
After pre-incubation.
a
b
c
In vivo T3, T4 and their respective bioprecursors (1 and 3)
showed outstanding antimalarial activities after intraperitoneal
administration (ED50 of 0.1–0.25 mg/kg; Tables 1 and 3). Com-
pound 2 had somewhat lower activity (ED50 of 3.4 mg/kg). After
oral administration, T3 and T4 showed 13 and 8.1 mg/kg ED50 val-
ues and neutral bioprecursors 1, 2 and 3 exhibited 11, 90 and 5 mg/
kg respectively.
On the basis of these results new neutral thioester prodrugs of
T4 bis-thiazolium salt (compounds 4–21, Table 1) were tested
in vitro and in vivo for their antimalarial activity. In comparison
with prodrugs 1–3 and the parent drug, the introduction of lipo-
philic alkyl or cycloalkyl chain as acyl substituents (R2, com-
pounds 4–11, Table 1) led to high in vitro active compounds
(IC50 of 1.1–8.2 nM) probably due to a fast prodrug conversion.
However, the oral effective dose was in all cases slightly higher
than for parent drug T4 (ED50 values ranging from 11 to 17 mg/
kg). A phenyl group as R2 residue was tolerated (12) but the pres-
ence of hindered substituents contiguous to the thioester function
dramatically increased IC50. Thus, compounds 13 and 14 were,
respectively, 65 and 1300-fold less active than 12. As these com-
pounds showed lower IC50 values after incubation, this lack of
Activity of this compound has already been reported.18,19
activity was attributed to a lower prodrug–drug conversion rate.
Moreover, in vivo activities correlate with in vitro ones (ED50 of
24 and >90 mg/kg, respectively). We then examined the effect
of decreasing the pro-moiety lipophilicity. For this purpose, polar
functional groups were firstly introduced as ether, ketone or ester
(see Table 1, compounds 15 to 19). The best result was obtained
with compound 18 which showed an ED50 value of 3 mg/kg after
oral administration. Secondly, we introduced
nitrogen in the R2 substituent. Thus, compounds 2, 20 and 21
containing morpholino, pyrrolidine and pyridine moieties,
a protonatable
a
respectively, exhibited powerful IC50 (1.5–2.8 nM). However, oral
activities were lower than T4 parent drug (ED50 >17 mg/kg).
The introduction of thiocarbonate and thiocarbamate as enzy-
mo-labile pro-moieties was also studied (Table 2). Alkyl (22–25),
aromatic (26 and 27) and halo alkyl (28–30) derivatives presented
IC50 values ranging from 1.85 to 10.6 nM. The most potent in vivo
O
Br
Br
O
+
H
S
+
Trans
acylation
1) OH-
R2
(CH2)6
(CH2)6
N
N
S
S
(CH2)6
N
OH
2) R2COCl
OH
OC(O)R2
O-acylated T3
T3
2
2
2
Figure 4. Trans-acylation of T3 thioester prodrugs.

3956
S. A. Caldarelli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3953–3956
activities were obtained for the methylthiocarbonate prodrug 22
and for the 2-fluoroethyl derivative 30. Irrespective of the mode
of administration, both derivatives were more potent in vivo than
the parent drug T4 (oral ED50 = 1.3 and 3.8 mg/kg, respectively).
Pyrrolidine thiocarbamate derivatives 31 and 32 were also evalu-
ated. 31 showed low in vitro and in vivo activities. The presence
of the electron withdrawing carbonyl group in 32 probably en-
hances the rate of the thiocarbonate bond hydrolysis, resulting in
better IC50 and quite good in vivo activity.
(22, 6.45; 30, 7.04; 18, 6.68 and 3, 6.20)26 compared to the weaker
active ones.
The thiocarbonate derivative 22 appears to be the most power-
ful. Indeed, it is 3–4 times more potent than previously reported
TE3 prodrug (3). To our knowledge, this class of antimalarials rep-
resents one of the most potent in the rodent model tested yet, and
preliminary studies on efficacy and tolerance of these compounds
in mice and primates are very promising, indicating that this ap-
proach may be applicable to the human infection.24
Then six O-acylated T3 prodrugs were evaluated (Table 3). Com-
pound 33 (R2 = isopropyl) exhibited the highest in vitro activity
within the series (7 nM). However, this prodrug showed a poor
ED50 probably due to its high lipophilic character and to a fast pro-
drug–drug conversion rate. The introduction of a polar function
within the R2 moiety led to derivative 34 which showed lower
in vitro activities but interestingly oral activities in the range of
the parent T3 drug. In order to decrease the converting rate, we
introduced a 3,4,5-trimethoxyphenyl group (compound 35), with
donor electronic properties or a steric demanding 2-methylphenyl
substituents (compound 36). Both compounds showed high IC50
values, which decreased after pre-incubation, and low in vivo
activities (ED50 >60 mg/kg). These results may be associated to
an incomplete prodrug–drug transformation.
Acknowledgements
These studies were supported by the European Commission
(AntiMal integrated project LSHP-CT-2005-018834). We are grate-
ful to Yann Bordat for assistance in testing the compounds.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.001.
References and notes
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In order to get a low molecular weight prodrug the cyclic thio-
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