Design, synthesis, and antimalarial activity of structural chimeras of thiosemicarbazone and ferroquine analogues

Article abstract of DOI:10.1016/j.bmcl.2007.10.003

The design, synthesis, and antimalarial activity of chimeras of thiosemicarbazones (TSC) and ferroquine (FQ) is reported. Key structural elements derived from FQ were coupled to fragments capable of coordinating metal ions. Biological evaluation was conducted against four strains of the malaria parasite Plasmodium falciparum and against the parasitic cysteine protease falcipain-2. To establish the role of the ferrocenyl moiety in the antiplasmodial activity of this series, purely organic parent compounds were also synthesized and tested. The presence of the aminoquinoline structure, allowing transport of the compounds to the food vacuole of the parasite, seems to be the major contributor to antimalarial activity.

Full text of DOI:10.1016/j.bmcl.2007.10.003

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Bioorganic & Medicinal Chemistry Letters 17 (2007) 6434–6438
Design, synthesis, and antimalarial activity of structural
chimeras of thiosemicarbazone and ferroquine analogues
b
a
c
Christophe Biot,a, Bruno Pradines, Marie-Helene Sergeant, Jiri Gut,
*
´ `
Philip J. Rosenthalc and Kelly Chibaled,e
a
ˆ
Universite´ des Sciences et Technologies, Unite´ de Catalyse et de Chimie du Solide—UMR CNRS 8181, ENSCL, Batiment C7,
BP 90108, 59652 Villeneuve d’Ascq Cedex, France
bInstitut de Me´decine Tropicale du Service de Sante des Arme´es, Unite´ de Recherche en Biologie et Epide´miologie Parasitaires,
Bd Charles Livon, Parc le Pharo, BP 46, 13998 Marseille Arme´es, France
cDepartment of Medicine, San Francisco General Hospital, University of California, San Francisco,
Box 0811, San Francisco, CA 94143, USA
dDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
eInstitute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
Received 10 September 2007; revised 2 October 2007; accepted 2 October 2007
Available online 5 October 2007
Abstract—The design, synthesis, and antimalarial activity of chimeras of thiosemicarbazones (TSC) and ferroquine (FQ) is reported.
Key structural elements derived from FQ were coupled to fragments capable of coordinating metal ions. Biological evaluation was
conducted against four strains of the malaria parasite Plasmodium falciparum and against the parasitic cysteine protease falcipain-2.
To establish the role of the ferrocenyl moiety in the antiplasmodial activity of this series, purely organic parent compounds were also
synthesized and tested. The presence of the aminoquinoline structure, allowing transport of the compounds to the food vacuole of
the parasite, seems to be the major contributor to antimalarial activity.
Ó 2007 Elsevier Ltd. All rights reserved.
Following the discovery of the antimalarial drug candi-
R1
N
CH3
date ferroquine (FQ, SR97193, Fig. 1),1,2 there has been
an intense interest in the synthesis and evaluation of its
analogues and/or derivatives.3–5 For the last several
years, we have been working in this bioorganometallic
area with the aim of developing ‘‘back-up’’ 4-
aminoquinolines.6
CH3
HN
N
N
H
N
R2
N
CH3
Fe
+
S
X
Cl
Y
TSC
FQ
S
On the other hand, derivatives of thiosemicarbazones
(TSC, Fig. 1) have shown potent antimalarial activi-
ties.7,8 Due to their intrinsic metal (e.g. iron) chelating
properties, the mechanism of action of TSCs is believed
to result from the generation of reactive oxygen radi-
cals.9 Nevertheless, the toxicity of the TSCs in Plasmo-
dium berghei infected mice precluded their
development as antimalarial therapeutics.10 Renewed
interest in TSCs has arisen following the discovery of
N
CH3
HN
N
N
N
H
H
Fe
Cl
Y
X
Figure 1. Development of structural chimeras based on ferroquine
(FQ) and thiosemicarbazones (TSC). The dashed circles indicate the
merged groups.
Keywords: Malaria; Antimalarial agents; Chimeras; Ferroquine; Thio-
semicarbazones; Falcipain-2; Bioorganometallics.
new leads that kill species of protozoan parasites
through the inhibition of cysteine proteases as well as
other novel targets.11,12
*
Corresponding author. Tel.: +33
christophe.biot@ensc-lille.fr
3
20 43 48 93; e-mail:
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.003

C. Biot et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6434–6438
6435
Ar
O
CH3
N
H
H
N
H3C
N
SCH3
SCH3
NH2
+
+
CS2 CH3I
H2N
H2N
Ar
R
CH3OH
S
S
2 and 3
1
CH3OH
NH2
CH3
N
H
N
H
N
R
Ar
S
4a-e and 5a-e
Scheme 1. Typical synthetic procedure of thiosemicarbazone-ferrocene conjugates. Ar is 1-(3,4-dichlorophenyl)ethanone or 1-(4-bro-
mophenyl)ethanone.
Table 1. Chemical structures of the hybrid molecules and reference
compounds
In this study, a chimeric ligand approach (Fig. 1) was
used to combine the properties of FQ (accumulation
in the food vacuole of the parasite via the 4-aminoquin-
oline nucleus and high antimalarial activity due to the
metallocenic fragment) and of the selected TSC.
Compound
Ar
R
Yields (%)
X
Y
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
Cl Cl
Cl Cl
Cl Cl
Cl Cl
Cl Cl
62
18
15
94
97
75
37
15
99
99
Fe
In principle this strategy is aimed at either preventing or
slowing down the emergence of drug resistance and is
based on covalent binding between two active frag-
ments. Potent antimalarial activities have been reported
for hybrid molecules based on trioxanes and chloro-
quine,13,14 amodiaquine and naphthoquinone,15,16 triflu-
oromethylartemisinin and mefloquine,17 as well as
artemisinin and quinine.18
H3C
N
H3C
Fe
HN
Fe
In order to compare the contribution of each fragment,
the corresponding ferrocenic derivatives 4a, 4b and 5a,
5b (without the 4-aminoquinoline moiety) and the
purely organic compounds 4d–e and 5d–e were also
synthesized.
Cl
N
HN
Cl
Cl
N
HN
The target molecules were synthesized according to the
general synthetic procedure as shown in Scheme 1.19
The methyl hydrazinecarbodithioate 1 was synthesized
from commercially available hydrated hydrazine, car-
bon disulfide, and methyl iodide.7 Condensation of 1
with the appropriate acetophenone in methanol afforded
the thiosemicarbazone thioesters 2 and 3. The choice of
the acetophenone was based on a previous study.20
Finally, nucleophilic substitution with the primary
amines R-NH2 under conditions previously reported21
provided the thiosemicarbazones 4a–e and 5a–e (Table 1).
N
Br
Br
Br
Br
Br
H
H
H
H
H
Fe
H3C
N
H3C
Fe
HN
The new compounds were evaluated in vitro against
four different Plasmodium falciparum strains (Table 2)
from different geographical and antimalarial resistance
backgrounds (3D7 originates from Africa, W2 from
Indochina, FCR3 from Gambia, and Bre1 from
Brazil).22
Fe
Cl
N
HN
Cl
Cl
N
In order to determine potential targets for this class of
compounds in P. falciparum, selected compounds were
also tested against the parasitic cysteine protease falci-
pain-2 (Table 3).23 As described previously, potent
inhibitors of falcipain-2 block hemoglobin hydrolysis,
HN
N

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C. Biot et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6434–6438
Table 2. In vitro susceptibilities of P. falciparum strains in nM (gray) and a lM (white)
aSD, standard deviation.
Table 3. Effects on the activity of falcipain-2 and FV of P. falciparum
W2 strain
potency against falcipain-2 (see Table 3), which did
not correlate with the antimalarial activity, suggests that
other mechanisms of inhibition are involved, as previ-
ously suggested.25
Compound
falcipain-2
IC50 (nM)
SDa
Food vacuole
abnormality
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
8130
14,250
2415
1307
877
ND
ND
—
As expected, chimeras of TSC and FQ analogues (4c
and 5c) were the most active derivatives of this series
against the different strains of P. falciparum. Never-
theless, the corresponding purely organic derivatives
(4d, 4e, 5d, and 5e) showed comparable potency.
Contrary to previous results,1–6,26 introduction of
the ferrocene moiety did not increase antimalarial
activity.
212
1870
4416
741
—
>20,000
>20,000
4250
—
—
1628
95
1072
12,932
>20,000
—
—
5697
—
a SD, standard deviation.
Interestingly, the rigid metallocenic compounds (4c and
5c) derived from FQ showed better inhibition of falci-
pain-2 compared to the corresponding flexible alkyl ana-
logues (4d, 4e, 5d, and 5e).
leading to swollen, dark staining parasite food
vacuoles.24
Compounds 4b–e and 5b–e were much more active than
the first ferrocenyl TSC hits,20 4a and 5a, with activity in
the low micromolar range. Introduction of the amino
side chain, in compounds 4b and 5b compared to com-
pounds 4a and 5a, slightly increased the antimalarial
activity. These data suggest that the basic amino group
may add potency, probably by assisting transport to the
acidic food vacuole of the parasite. The low inhibitory
It is intriguing that the typical food vacuole abnormality
associated with inhibitors of falcipain-2 was observed
with only compound 4d. These results suggest that the
inhibitors may have two different modes of action, one
hitting falcipain-2 and another acting independently.
At this point, we can speculate that these compounds
preferentially concentrate in the food vacuole, explain-
ing better parasite than enzyme IC50s.

C. Biot et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6434–6438
6437
19. Details of the typical synthetic procedure and spectro-
scopic data of the representative conjugates 4c and 5c are
displayed as follows. The thiosemicarbazone thioester 2
(76 mg, 0.18 mM) (or 3, 173 mg, 0.57 mM) was refluxed
with the corresponding amine (R-NH2) in methanol
(5 mL) for 4 h. After cooling, the solvent was removed
under reduced pressure. N-{2-[(7-Chloroquinolin-4-ylami-
no)methyl]ferrocenyl}-2-[1-(3,4-dichlorophenyl)ethylidene]-
hydrazinecarbothioamide 4c. The crude product was
purified by silica-gel chromatography (6:2:1, ethyl ace-
tate:petroleum ether: triethylamine) to give 4c (18 mg,
0.03 mM, 15%) as a yellow solid. Mp 98–102 °C. 1H NMR
(300 MHz; CDCl3) d 8.43 (d, 1H, J = 5.37 Hz), 7.83 (d,
1H, J = 1.98 Hz), 7.74 (d, 1H, J = 2.13 Hz), 7.60 (d, 1H,
J = 8.85 Hz), 7.40 (d. 1H, J = 8.46 Hz), 7.25 (d, 1H, J =
8.55 Hz), 7.06 (dd, 1H, J = 2.01 and 8.97 Hz), 6.45 (d, 1H,
J = 5.43 Hz), 4.40 (m, 1H), 4.32 (m, 1H), 4.18 (br s, 6H).
4.15 (m, 1H). 4.14 (m, 1H), 3.41 (s, 2H). 2.01 (s, 3H); 13C
NMR (75 MHz; CDCl3) d 208.0, 176.7, 145.2, 136.8,
134.8, 133.9, 132.8, 130.4, 127.9, 125.3, 124.9, 121.8 (2C),
117.0, 98.9, 83.1 (2C), 70.7, 70.3, 69.4 (5C), 67.4, 41.1,
29.7, 13.4; HRMS (electrospray) m/z 649: M°+
35Cl35Cl35Cl, 650: MH+ 35Cl35Cl35Cl, 651: M°+
35Cl35Cl37Cl, 652: MH+ 35Cl35Cl37Cl, 653: M°+ 35Cl37
Cl37Cl, 655: M°+37Cl37Cl37Cl. N-{2-[(7-Chloroquinolin-4-
ylamino)methyl]ferrocenyl}-1-[1-(3-bromophenyl)ethylidene]-
hydrazinecarbothioamide 5c. The crude product was
purified by silica-gel chromatography (6:2:1. ethyl ace-
tate:petroleum ether: triethylamine) to give 5c (60 mg,
0.09 mM, 15%) as a yellow solid. Mp 103–105 °C.
1HNMR (300 MHz; CDCl3) d 8.40 (d, 1H, J = 4.99 Hz),
7.78 (d, 1H, J = 2.34 Hz), 7.66 (m, 1H), 7.61 (s, 1H), 7.60
(d, 1H, J = 7.51 Hz), 7.40 (d, 1H, J = 7.99 Hz), 7.32 (d,
1H, J = 7.52 Hz), 7.09 (dd, 1H, J = 1.88 and 9.45 Hz), 6.45
(d, 1H, J = 5.30 Hz), 4.46 (m, 1H). 4.30 (m, 1H), 4.16 (br s,
6H), 4.15 (m, 1H), 4.14 (m, 1H), 3.65. (s, 2H), 2.01 (s, 3H);
13CNMR (75 MHz; CDCl3) d 211.7,176.7 (2C), 151.8,
149.1 (2C), 146.1, 138.9, 132.7, 130.0, 129.1, 124.9, 122.7,
121.7, 117.0, 99.0, 83.3 (2C), 70.6, 70.1, 69.5 (5C), 67.4,
46.1, 41.0, 29.7, 13.5; HRMS (electrospray) m/z 659: M°+
35Cl79Br, 661: M°+ 35Cl81Br and 37Cl79Br, 663: M°+
37Cl81Br.
Previously, it has been demonstrated that FQ has re-
duced affinity for mutant forms of the P. falciparum
chloroquine resistance transporter (PfCRT), a protein
located in the parasite digestive vacuole and involved
in drug transport and chloroquine resistance.27–29 Spe-
cific mutations in PfCRT mediate resistance to chloro-
quine.30 However, once again, there was no significant
difference in the activity of ferrocenyl compounds be-
tween chloroquine susceptible and chloroquine-resistant
parasites.
Acknowledgments
The authors thank R. Amalvict, E. Baret, and J. Mos-
nier for technical support. This work was supported in
part by grants from the National Institutes of Health,
USA. P.J.R. is a Doris Duke Charitable Foundation
Distinguished Clinical Scientist.
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6438
C. Biot et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6434–6438
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Harvester; Perkin-Elmer). Filter microplates were dried,
and 25 lL of scintillation cocktail (Microscint O; Perkin-
Elmer) was placed in each well. Radioactivity incorpo-
rated by the parasites was measured with a scintillation
counter (Top Count; Perkin-Elmer). The IC50 (the drug
concentration leading to 50% of the uptake of [3H]hypo-
xanthine by parasites in drug-free control wells) was
determined by nonlinear regression analysis of log-dose/
response curves.
23. Assays of recombinant falcipain-2 activity and of abnor-
mal food vacuole morphology were performed as previ-
ously described. Shenai, B. R.; Lee, B. J.; Alvarez-