Mono- and bis-thiazolium salts have potent antimalarial activity

Article abstract of DOI:10.1021/jm0492608

Three new series comprising 24 novel cationic choline analogues and consisting of mono- or bis (N or C-5-duplicated) thiazolium salts have been synthesized. Bis-thiazolium salts showed potent antimalarial activity (much superior to monothiazoliums). Among them, bis-thiazolium salts 12 and 13 exhibited IC₅₀ values of 2.25 nM and 0.65 nM, respectively, against P. falciparum in vitro. These compounds also demonstrated good in vivo activity (ED₅₀ ≤ 0.22 mg/kg), and low toxicity in mice infected by Plasmodium vinckei.

Full text of DOI:10.1021/jm0492608

J. Med. Chem. 2005, 48, 3639-3643
3639
Brief Articles
Mono- and Bis-Thiazolium Salts Have Potent Antimalarial Activity
Abdallah Hamze´,† Eric Rubi,† Pascal Arnal,† Michel Boisbrun,†,§ Carole Carcel,† Xavier Salom-Roig,†
Marjorie Maynadier,‡ Sharon Wein,‡ Henri Vial,‡ and Miche`le Calas*,†
Laboratoire des Amino acides Peptides et Prote´ines (LAPP) CNRS-UMR 5810, CP 19, Universite´s Montpellier I et II, and
Dynamique Mole´culaire des Interactions membranaires (DMIM) CNRS-UMR 5539, CP 107, Universite´ Montpellier II,
Place E. Bataillon, 34095 Montpellier Cedex 5, France
Received September 13, 2004
Three new series comprising 24 novel cationic choline analogues and consisting of mono- or
bis (N or C-5-duplicated) thiazolium salts have been synthesized. Bis-thiazolium salts showed
potent antimalarial activity (much superior to monothiazoliums). Among them, bis-thiazolium
salts 12 and 13 exhibited IC50 values of 2.25 nM and 0.65 nM, respectively, against P. falciparum
in vitro. These compounds also demonstrated good in vivo activity (ED50 e 0.22 mg/kg), and
low toxicity in mice infected by Plasmodium vinckei.
Introduction
Today approximately 40% of the world’s popula-
tion is at risk of malaria, mostly those living in the
poorest countries.1 Malaria is found throughout the
tropical and subtropical regions of the world and causes
more than 300 million acute illnesses and between 0.7
and 2.7 million deaths annually.2,3 Chloroquine (CQ)
and sulfadoxine/pyrimethamine are two inexpensive
drugs that have become ineffective with the emergence
of drug-resistant parasites.4 Another alternative, arte-
misinin, is of somewhat limited use because of its
relatively high cost and reports of toxicity.5,6 New drugs
with novel mechanisms of action7 and low cost are thus
urgently required. The challenge in malaria chemo-
therapy is to find safe and selective agents whose
potencies will not be compromised by plasmodial resis-
tance. In preceding work our group developed an
original pharmacological model capable of preventing
the reproduction of the parasite, with novel mechanisms
of action.8-10 The compounds were mono- and bis-
ammonium salts, containing a long lipophilic alkyl chain
at the N-position (the linker in bis-ammonium case).
One of the most active compounds against Plasmodium
falciparum was 1 (named G25 in preceding work, Figure
1) with 50% inhibitory concentration (IC50) of 0.65 nM.
These new antimalarial compounds target the plasmo-
dial phospholipid (PL) metabolism.11,12,13 Although 1 is
highly active against P. falciparum strains with preex-
isting resistance against current antimalarials, and
cures without recrudescence P. falciparum-infected Ao-
tus monkeys,10,14 it suffers from limited oral bioavail-
ability and high toxicity. To remedy this problem, we
investigated the replacement of the ammonium group
of bis-cationic compounds by a thiazolium head (Table
1).
Figure 1. General structures of the studied thiazolium salts
and 1 (G25) compound.
Herein, we describe the design, the synthesis, and the
antimalarial activities of two mono-thiazolium salts and
22 bis-thiazolium salts (Figure 1; I, II, and III). The two
polar heads are linked either from the nitrogen atom
(II) or from the C-5 position of the thiazolium ring (III).
The linker is an alkyl chain, with insertion of oxygen
atoms or a phenyl group for some compounds. Our
current aim was to develop a structure-activity rela-
tionship (SAR) study for thiazolium derivatives. After
showing the importance of duplicating the thiazolium
head, we first optimized the linker between the two
thiazoliums, then the substituents of thiazolium rings.
Finally, we investigated the linker position on the
thiazolium ring. All the synthesized derivatives were
evaluated for their in vitro antimalarial activity on P.
falciparum. Among the most active compounds (IC50
)
0.65-5.0 nM), seven were tested for in vivo inhibition
of P. vinkei growth in mice. The two most promising
molecules (ED50 e 0.2 mg/kg) were tested for their
tolerance.
Chemistry
* To whom correspondence should be addressed: Phone: 33 - 4 67
14 38 17. Fax: 33 - 4 67 14 48 66. E-mail: mcalas@univ-montp2.fr.
† LAPP, CNRS-UMR 5810.
Structures of the mono and bis-thiazolium salts are
given in Table 1.
A. Mono-Thiazolium (series I) and N-Duplicated
Thiazolium Salts (series II). In the title series, the
‡ DMIM, CNRS-UMR 5539.
§ Present address: GEVSM, CNRS-UMR 75 65 UHP, Faculte´ de
Pharmacie, 5, rue A. Lebrun, 54001 Nancy Cedex.
10.1021/jm0492608 CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/15/2005

3640 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Brief Articles
Scheme 1a
Table 1. Structure of the Thiazolium Salts and Their in Vitro
Antimalarial Activity against P. falciparum
a Reagents and conditions: (i) EtOH reflux, 72 h; (ii) NaH/THF,
R-I; (iii) NaI, acetone;15 (iv) P2O5/H3PO4/KI;16 (v) CH3CN, reflux,
72 h.
Scheme 2a
a Reagents and conditions: (i) 1,12-diiodododecane, CH3CN,
reflux, 72 h; (ii) methyl 4-chloro-4-oxobutanoate/CH3CN, reflux,
6 h; (iii) SOCl2, reflux, 4 h.
a The shaded cells represent compounds with a C12 linker. b 23
is in the form of a chloride salt.
Scheme 3a
thiazolium head had a methyl or phenyl group at the
C-4 position (R2). Different groups were present at the
C-5 position (R3 ) H, Me, Et, (CH2)2OH, (CH2)2OMe,
(CH2)2Cl, (CH2)2OEt, (CH2)2OiPr, (CH2)2OAc, and (CH2)2-
OCO(CH2)2CO2Me). For 20, the polar head was a
benzothiazolium. The linker between the two thiazolium
heads was generally an alkyl chain varying from 8 to
16 methylene groups. For 31, a phenyl ring was included
in the middle of the chain of eight methylene groups.
The preparation of mono- and bis-thiazolium and ben-
zothiazolium salts was accomplished by alkylating the
N atom of the corresponding thiazole ring with 1-bro-
mododecane in refluxing EtOH or R,ω-diiodoalkanes in
refluxing CH3CN for 3 days (Schemes 1-4). Among the
latter, the C8 and C10 derivatives were commercial. But
1,12-diiodododecane was prepared according to Ains-
cow15 and 1,16-diiodododecane was obtained using
Schultz’s procedure.16
a Reagents and conditions: (i) H2/Pd/C; (ii) 1,12-diiodododecane,
CH3CN, reflux, 72 h.
5-(2-Alkoxyethyl)-4-methylthiazole derivatives 3-5
(Scheme 1) were prepared by O-alkylation of 4-methyl-
5-(2-hydroxyethyl)thiazole 2 with the corresponding
iodoalkane. Compound 21 was prepared from com-
mercial 2-(4-methyl-1,3-thiazol-5-yl)ethyl acetate by N-
alkylation with 1,12-diiodododecane (Scheme 2). Com-
pounds 22 and 23 were generated from 12 by acylation
with methyl-4-chloro-4-oxobutanoate in the case of 22,
and by treatment with SOCl2 under reflux for 23
(Scheme 2). To obtain 25, we used 5-ethyl-4-methylthi-
azole 24 (yielded by hydrogenation of commercial 4-meth-
yl-5-vinylthiazole) and 1,12-diiodododecane (Scheme 3).
Compound 31 was prepared by coupling 3 with 1,4-bis-
(4-iodobutyl)benzene 30, obtained as described in Scheme

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3641
Scheme 4a
precursor 37. Finally, 38 and 39 were generated from
37 by addition of an alkyl halide (R1X) under reflux in
ethanol.
Biological Evaluation and Discussion
A. In Vitro Studies. Mono-Thiazolium Salts (Ser-
ies I). The thiazolium rings in 6 and 7 are both
substituted by a dodecyl group at the nitrogen atom and
a methyl group at the 4-position. Only the R3 substitu-
ent is different, (CH2)2OH for 6, and (CH2)2OCH3 for 7
(Table 1). Biological tests showed significant potent
antimalarial activity for these two drugs, with IC50
values about 70 nM, and thus no significant difference
when the hydroxyl is methylated.
a Reagents and conditions: (i) H2SO4/EtOH;17 (ii) (a) succinic
anhydride/AlCl3; (b) 2 N KOH;17 (iii) NH2NH2,H2O/KOH/TEG;17
(iv) Me2S‚BH3; (v) P2O5/H3PO4/KI; (vi) 3 (4 equiv)/CH3CN, reflux,
72 h.
Scheme 5a
Bis-Thiazolium Salts (Series II and III). Molecule
duplication is often an efficient way to improve the
pharmacological activity.20,21 We thus designed twin
drugs containing two identical thiazolium rings co-
valently combined (Table 1).
(a) Comparison between Mono- and Bis-Thiazo-
lium Salts. The duplication of the polar head of 6 and
7 with a (CH2)12 linker led to a dramatic improvement
of the antimalarial activity. Thus 12 and 13, which are
the duplicated analogues of 6 and 7, are 30 and 110 fold
more potent with IC50 values of 2.25 and 0.65 nM,
respectively. Similar findings were obtained in a previ-
ous SAR study with choline analogues.8,9 These findings
can be associated with the increasing potency of “twin-
drugs”, which can simultaneously fit to both binding
sites of the pharmacological target, increasing the
affinity. In addition, dimerization can also increase
metabolic resistance of the initial compound.22-25 As a
result, we focused our efforts on duplicated molecules.
(b) Optimization of the Linker. To study the
importance of the chain linking the two thiazolium
heads, we synthesized two compounds, bearing the same
cationic heads, equally distant, and differing only by the
nature of the linkers. For 13, the linker is a C12 alkyl
chain, and for 31 a phenyl group is included in a C8 alkyl
chain. 13 (0.65 nM) was 8 times more active than 31
(5.0 nM). Thus, an introduction of a phenyl ring in the
linker slightly reduced the activity. This electron rich
aromatic group should not form additional specific bonds
with the target, unless the rigidification of the linker is
unfavorable. It seems that the linker needs lipophilicity,
but also that lipophilicity needs a strict spatial distribu-
tion.
a Reagents and conditions: (i) DMSO, KOH, 30 min, rt; (ii) R1X,
EtOH, reflux, 48 h.
Scheme 6a
a Reagents and conditions: (i) (a) NaH/THF, 0 °C, 10 min; (b)
BuLi, 0 °C 10 min; (ii) dibromododecane, THF, 1 h; (iii) Br2, CCl4;
(iv) thioformamide, acetone, reflux, 78 h; (v) R1X, EtOH, reflux,
78 h.
4. Ethyl 4-phenylbutanoate (26), γ-(p-succinylphenyl)-
butyric acid (27), and 1,4-bis(γ-carboxypropyl)benzene
(28) were prepared according to Cram’s method.17
Compound 28 was in turn reduced to 1,4-bis(4-hydroxy-
butyl)benzene 29 under the action of Me2S.BH3. Finally,
the replacement of the hydroxyl groups by iodide to give
30 was accomplished by action of P2O5 and KI.18
Then, we studied the influence of the length of the
alkyl chain linker, increasing it from C8 to C16. This
length appears crucial for the antimalarial activity. The
latter increased in an impressive manner when the
length raised from 8 to 12. With the same thiazolium
head, substitution of the C8 by a C10 linker improved
the activity 10-fold (compare 8/10 or 9/11). Changing
from C10 to C12 multiplied the activity by 15 (compare
10/12 or 11/13). The activity did not increase further
by additional lengthening of the chain, as a similar level
of activity was observed when we replaced the C12 by a
C16-linker (13: 0.65 nM to 16: 1.1 nM). The plateau of
activity is reached at this length and thus the C12 linker
appears to be optimal. This linker was thus used for
further optimizations (shaded cells in Table 1).
B. C-5-Duplicated Thiazolium Salts (Series III).
The C5-duplicated derivatives 33, 34 (Scheme 5), which
include two oxygen atoms in the linker, were obtained
by reaction of the thiazole derivative 2 with 1,6-
diiodohexane in the presence of a base to get 32, which
was treated by an alkyl halide (R1X) under reflux in
ethanol. The synthesis of bis-thiazolium salts 38 and
39 is described in Scheme 6. The dianion intermediate
formed by reacting ethyl acetoacetate with NaH in
anhydrous THF, and then by action of BuLi, was treated
with dibromododecane, giving diethyl 3,18-dioxoicosane-
dioate 35. Bromination of 35 according to the procedure
of Svendsen19 gave diethyl 4,17-dibromo-3,18-dioxo-
icosanedioate 36. Treatment of dibromo compound 36
with thioformamide (previously obtained by treatment
of formamide by Lawesson’s reagent) afforded the
(c) Optimization of the Thiazolium Head. In the
series II compounds (N-duplicated thiazolium salts), we

3642 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Brief Articles
Table 2. In Vivo Antimalarial Activity against P. vinckei
to 3.1 mg/kg (Table 2) and are in agreement with the
potent antimalarial in vitro activity against P. falci-
parum. For the seven bis-thiazolium salts of Table 2,
the ED50 values were related with IC50 values. Again,
we observed that the compound with a bulky substitu-
ent at R3 (22, ED50 ) 3.1 mg/kg), and the one with no
substitution at this position (17, ED50 ) 2.6 mg/kg) are
the least active. Compounds bearing a medium size R3
(12, 13, 25) present the highest in vivo activity. For
example, 13 was the most active compound both in vitro
(IC50 ) 0.65 nM) and in vivo (ED50 ) 0.14 mg/kg). This
correlation is not observed in 21, which had a very good
in vivo activity (ED50 ) 0.7 mg/kg) but a moderate in
vitro activity (IC50 ) 3.1 nM). This could be explained
by the in vivo conversion of 21 into 12 under the effect
of serum esterase.
Toxicities were evaluated for 12 and 13, the two most
effective compounds. After ip administration in mice,
both compounds were well tolerated until 10 mg/kg dose
(LD50 > 10 mg/kg), indicating a therapeutic index (TI
) LD50/ED50) > 50. The bis-thiazolium salts, particu-
larly 12 and 13 appeared to be as efficient and less toxic
than the bis-ammonium salt 1 (LD50 ip 1.2 mg/kg, TI )
5) and more efficient than chloroquine (CQ).
antimalarial activity
in vitro IC50, nM
P. falciparum
in vivo ED50, mg/kg
drugs
P. vinckei ip
12
13
17
18
25
21
22
1
2.25
0.65
3
1.5
1.6
3.1
5.0
0.65
20
0.2
0.14
2.6
1
0.4
0.7
3.1
0.22
1.1
CQ
explored the influence of the nature of R3 substituent
at the C-5 position of the thiazolium ring. Molecules
bearing a medium size R3 [Me, 18; Et, 25; (CH2)2OH,
12; (CH2)2OMe, 13; (CH2)2OEt, 14; (IC50 ∼ 0.65-2.25
nM)] are more active than those unsubstituted at this
position (17, 3 nM) or those bearing a bulky R3 sub-
stituent [(CH2)2OiPr, 15; (CH2)2OAc, 21; (CH2)2Cl, 23;
(CH2)2OCO(CH2)2CO2Me, 22; IC50 ∼ 3-10 nM]. The
optimal substituent at R3 position for antimalarial
activity appears to be the methoxyethyl group. With an
equal linker, the presence of OMe (9, 11, 13) compared
with OH (8, 10, 12) in the R3 substituent was favorable
for activity. The methyl group might establish useful
additional hydrophobic interactions, but the target does
not tolerate much more bulky substituent at this
position. As expected, the bulky phenyl group, located
at the C-4 position (19) or fused at C-4 and C-5 (20),
led to a dramatic decrease of the antimalarial activity
(1.8 and 2.3 µM respectively).
(d) Influence of the Linker Position on the
Thiazolium Ring. We designed series III compounds
(33, 34, 38, 39) where the linker is attached to the
5-position of the ring, an alternative anchor point to the
thiazole nitrogen. This linker is either a C12 alkyl chain
(38, 39), or an equivalent length chain but including two
oxygen atoms (33, 34). The nitrogen atoms were alky-
lated either by a methyl or a benzyl group. 38 which
possess the optimized linker (of series II) with 12
methylene groups was 10 to 30-fold less active than its
13 and 14 analogues (series II), with the similar volume
of cationic head. When this volume increased by an
introduction of a phenyl ring on R1 (39, IC50 ) 250 nM),
the activity dropped by 10-fold in comparison to 38.
Compounds 33 and 34 (275 and 36 nM respectively),
whose linker includes two oxygen atoms were slightly
active. As a result, whatever the nature of the linker,
compounds with the linker chain attached to position 5
of the ring are less active than their N-duplicated
analogues. Compounds exhibiting the best activities (12,
13, 17, 18, 25, 21 and 22) were selected for further
evaluation of the antimalarial efficacy in vivo against
P. vinckei.
Conclusion
Twenty four thiazolium salts, divided in three series
(mono, N-bis- and C-5-bis-thiazolium), were designed
and synthesized. Several N-bis-thiazolium salts with a
linker of 12 methylene groups are highly active against
the in vitro growth of P. falciparum (13 molecules
possess IC50s lower than 10 nM). From the seven N-bis-
thiazolium salts selected for in vivo inhibition of P.
vinckei growth in mice, six compounds revealed high
activities with ED50 lower than 3 mg/kg. They can
constitute new potent candidates in the treatment of
malaria. Studies also permitted evaluation of the in vivo
tolerance for two of these molecules. Results are very
encouraging; compounds 12 and 13 (named by us
respectively T3 and T4) were selected for their very
potent in vivo activity (ED50 ) 0.2 and 0.14 mg/kg
respectively), their superiority to chloroquine (ED50
)
1.1 mg/kg), and their good tolerance (therapeutic index
> 50). These two compounds are also effective in vivo
in Aotus monkeys infected by P. falciparum and Rhesus
monkeys infected by P. cynomolgi (unpublished results).
Finally, the presence of the thiazolium ring in these
drugs will enable us to design new neutral prodrugs
(uncharged) starting from these drugs, to improve their
bioavailability by using the same approach as in the
case of another thiazolium salt, vitamin B1.26
Experimental Section
General Procedure A for the Preparation of 5-(2-
Alkoxyethyl)-4-methyl-1,3-thiazoles. A solution of 4-meth-
yl-5-(2-hydroxyethyl)thiazole 2 (12.00 mL, 100.0 mmol) in dry
THF (150 mL) was cooled with an ice bath, and to the solution
was added 5.20 g (130.0 mmol) of NaH (60% suspension in
mineral oil). The mixture was stirred under nitrogen atmo-
sphere for 40 min, and then alkyl iodide (1.3 equiv) was added
dropwise. The suspension was stirred at room temperature
overnight, and 20 mL of distilled water was carefully added.
The mixture was concentrated in vacuo, and EtOAc (300 mL)
was added to the residue. The organic solution was washed
with distilled water (3 × 150 mL) and brine (2 × 150 mL),
B. In Vivo Antimalarial Activity. Among the
thiazolium salts studied herein, seven compounds (Table
2) were evaluated in vivo against P. vinckei in mice.
Compounds were ip administered once daily for 4 days.
All the selected compounds had significant in vivo
antimalarial activities, which occurred over less than 2
log drug concentrations, and the parasitemia clearance
was always complete at the highest dose (data not
shown). ED50 (efficient dose) of the bis-thiazolium salts
are highly encouraging because they ranged from 0.14

Brief Articles
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dried (Na2SO4), and concentrated under reduced pressure to
give a yellow oil which was directly used for the next step.
General Procedure B for the Preparation of Mono-
thiazolium Salts. To a solution of thiazole derivative in EtOH
was added 1-bromododecane, and the mixture was stirred for
72 h at 60 °C. The solvent was evaporated, the residue was
triturated with Et2O, and the mixture was filtered. The
precipitate was crystallized from iPrOH to give the desired
compound.
General Procedure C for the Preparation of N-Dupli-
cated Thiazolium Salts. To a solution of thiazole derivative
(1.0 equiv) in CH3CN was added the appropriate diiodoalkane
(0.25 equiv), and the mixture was refluxed under nitrogen
atmosphere for 72 h. The solvent was evaporated, and the
residue was usually purified by crystallization from iPrOH at
-40 °C for 24 h. Filtration of the crystals, washing with chilled
iPrOH and Et2O, and drying afforded the desired compound.
For those compounds that did not crystallize, washing workup
(sometimes followed by column chromatography) yielded pure
compounds.
In Vitro Antimalarial Activity. In vitro antimalarial
activity was measured using the [3H]-hypoxanthine incorpora-
tion assay with the Nigerian strain of P. falciparum as
previously described.27 Results were expressed as the concen-
tration resulting in 50% inhibition (IC50).
In vivo antimalarial activity was determined against P.
vinckei petteri (279BY) strain in female Swiss mice, according
to a modified version of the 4-day suppressive test of Peters
et al.28 The 50% efficient dose (ED50), which is the dose leading
to 50% parasite growth inhibition compared to the control
(treated with an equal volume of vehicle), was determined on
the day following the last treatment. All results are expressed
as mean ( SEM.
Acknowledgment. These studies were supported by
the European Community (QLK2-CT-2000-01166), Min-
iste`re de l’Education Nationale et de la Recherche
Scientifique (PAL+), and the UNDP/World Bank/WHO
special program for Research and Training in Tropical
Diseases. We thank. Dr Joseph Kappel for helping in
proofreading of the manuscript and Cedric Paniaga for
technical support.
Supporting Information Available: General methods,
experimental procedures details for synthesis, and analytical
data of all compounds. Assessment of biological activity: in
vitro and in vivo antimalarial activity and acute toxicity in
mouse. This material is available free of charge via the
Internet at http://pubs.acs.org.
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