Development of piperazine-tethered heterodimers as potent antimalarials against chloroquine-resistant P. falciparum strains. Synthesis and molecular modeling

Article abstract of DOI:10.1016/j.bmcl.2007.04.077

The design, synthesis, and antiplasmodial activity of antimalarial heterodimers based on the 1,4-bis(3-aminopropyl)piperazine linker is reported. In this series key structural elements derived from quinoline antimalarials were coupled to fragments capable

Full text of DOI:10.1016/j.bmcl.2007.04.077

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3535–3539
Development of piperazine-tethered heterodimers as potent
antimalarials against chloroquine-resistant P. falciparum
strains. Synthesis and molecular modeling
a,b
Sandra Gemma,a,b Gagan Kukreja,a,b Giuseppe Campiani,a,b, Stefania Butini,
*
Matteo Bernetti,a,b Bhupendra P. Joshi,a,b Luisa Savini,a,b Nicoletta Basilico,a,c
Donatella Taramelli,a,c Vanessa Yardley,a,d Alessia Bertamino,a,e Ettore Novellino,a,e
Marco Persico,a,f Bruno Catalanottia,f and Caterina Fattorussoa,f
a
`
European Research Centre for Drug Discovery & Development, Universita di Siena, via Aldo Moro, 53100 Siena, Italy
b
`
Dipartimento Farmaco Chimico Tecnologico, Universita di Siena, via Aldo Moro, 53100 Siena, Italy
`
dDepartment of Infectious and Tropical Diseases, LSHTM, Keppel Street, London WC1E 7HT, UK
c
`
Dipartimento di Sanita Pubblica-Microbiologia-Virologia, Universita di Milano, via Pascal 36, 20133, Milano, Italy
e
`
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita di Napoli Federico II, via D. Montesano 49, 80131 Napoli, Italy
f
`
Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli Federico II, via D. Montesano 49, 80131 Napoli, Italy
Received 23 March 2007; revised 18 April 2007; accepted 19 April 2007
Available online 29 April 2007
Abstract—The design, synthesis, and antiplasmodial activity of antimalarial heterodimers based on the 1,4-bis(3-aminopropyl)piper-
azine linker is reported. In this series key structural elements derived from quinoline antimalarials were coupled to fragments capa-
ble of coordinating metal ions. Biological evaluation included determination of activity against chloroquine-sensitive and
chloroquine-resistant Plasmodium falciparum strains. Some of the novel compounds presented high activity in vitro against chloro-
quine-resistant strains, more potent than chloroquine and clotrimazole. Computational studies revealed that the activity is likely due
to the ability of the compounds to assume a multisite iron coordinating geometry.
Ó 2007 Elsevier Ltd. All rights reserved.
Malaria is a disease caused by Plasmodium falciparum
(Pf) and represents one of the most widespread and
deadly parasitic diseases in man. For over 50 years,
chloroquine (CQ (1), Fig. 1) has been the drug of choice
for malaria chemotherapy, until its efficacy was ham-
pered by the spread of CQ-resistant (CQ-R) Pf strains.
Consequently, new therapeutic strategies are needed.1
During the intraerythrocytic phase of Pf life cycle,
hemoglobin is metabolized within the parasite food vac-
uole (FV) and the released toxic-free heme is detoxified
through formation of hemozoin in the FV or by the glu-
tathione system in the cytoplasm.2,3 Both the widely
used classes of antimalarial drugs, namely 4-aminoquin-
olines and endoperoxides,4 are thought to interfere with
the heme detoxification process killing Plasmodium par-
asites via free-radical intermediates.
Keywords: Malaria; Chloroquine; Iron-complexing agents.
*
Corresponding author. Tel.: +39 0577 234172; fax: +39 0577
234333; e-mail: campiani@unisi.it
Figure 1. Reference and title compounds.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.077

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S. Gemma et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3535–3539
Table 1. Antiplasmodial activity of compounds 4a–h
Compound R1
Prevalent ionic form tautomer Aa Prevalent ionic form tautomer Ba
IC50 (lM)
d
pH 7.4
pH 7.2
pH 5.5
pH 7.4
pH 7.2
pH 5.5
D10b W2c 3D7b K1c
4a
P (56)
DP (48) TP (85)
DP (50) TP (85)
DP (50) TP (85)
DP (79)
DP (73)
TP (94)
26
145
110
663
33 1.6
269 28
98 7.8
495 nt
16
4b
4c
4d
P (56)
DP (81)
DP (81)
DP (73)
DP (73)
TP (68)
TP (94)
TP (94)
TeP (93)
135
447
nt
P (56)
DP (55)
DP (49) TeP (84) TP (67)
4e
P (47) N (47) P (55)
TP (68)
P (69)
P (58)
TP (76)
221
540 40
200
4f
P (51)
DP (51) TP (54)
DP (66)
DP (54)
DP (58)
DP (54)
TP (57)
TP (95)
1610 1440 nt
nt
nt
nt
4g
4h
P (49)
P (44)
TP (85)
407
260
535 nt
207 nt
DP (56)
TP (49) TeP (85) TP (80)
TP (73)
—
TeP (94)
—
2 (CLT)
1 (CQ)
—
—
N (95)
P (92)
N (92)
P (88)
P (81)
—
550
22
490 60
280 10
250
260
DP (87) DP (100) DP (100) DP (100)
nt, not tested.
a TeP, tetraprotonated form; TP, triprotonated form; DP, diprotonated form; P, protonated form; N, neutral form (ACD/pKa DB version 10.00
software (Advanced Chemistry Development Inc., Toronto, Canada)). Percentage of prevalent ionic form in brackets.
b CQ-sensitive clone.
c CQ-resistant clone.
d Values are means of three experiments, standard deviation is within 10% of the mean.
Iron is an essential nutrient for the asexual erythrocytic
phase of the parasite and a considerable number of iron
chelators showed antimalarial activity in vitro, appar-
ently through the mechanism of withholding iron from
vital metabolic pathways of the intraerythrocytic para-
site and through the formation of toxic complexes.
cated and severe malaria in humans.5 A promising
polycyclic pharmacophore to generate antimalarials is
held in clotrimazole (CLT, 2), a well-known antimy-
cotic drug, endowed with low in vitro antimalarial
activity (Table 1). We recently exploited the scaffold
of CLT and developed potent antimalarial agents of
the general structure 3 characterized by specific elec-
tronic features and by improved penetration into the
FV.6
There is evidence that iron chelation therapy with des-
ferrioxamine has clinical activity in both uncompli-

S. Gemma et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3535–3539
3537
In order to identify novel antimalarial lead compounds
with the potential to interfere with iron metabolic and
detoxification pathways, disrupting the peculiar redox
equilibria necessary for Pf survival, we designed a new
series of compounds (4a–h) in which key structural ele-
ments derived from CQ were linked to specific aromatic
residues through a piperazine tether.
overall yield. Alkylation of N1-(7-chloro-4-quinolyl)-
1,4-bis(3-amino propyl)piperazine 810 with the bromo-
derivative 7 provided the desired compound 4a. This
latter was further modified by reduction to 4b and sub-
sequently esterified to 4c by means of methanesulfonyl
chloride. To functionalize the bridged methylene of the
diphenylmethane moiety (4d), compound 7 was trans-
formed to the pyrrolidine derivative 9, which was con-
verted into the chloro derivative 10 by reduction to the
alcohol intermediate followed by chlorination using
thionyl chloride. Finally, compound 10 was coupled to
8 providing the desired final derivative 4d. In Scheme
2 is described the synthesis of compounds 4e–h. Interme-
diate 8 was coupled with the carboxylic acid derivative
13 to afford amide 4e. Compound 13 was in turn
prepared starting from 3-chlorophenyl acetic acid ethyl
ester 11 which was transformed into the imidazole deriv-
ative 12 by exposing 11 first to N-bromosuccinimide and
then to imidazole. Finally, alkaline hydrolysis using
LiOH in aqueous THF afforded acid 13. The synthesis
of 4f and 4h was accomplished by a reductive amination
of the appropriate aldehyde as shown in Scheme 2, by
using sodium borohydride as the reducing agent. Final-
ly, compound 4g was obtained by reacting amine 8 with
2-chlorobenzimidazole in refluxing pentanol.
Previously, quinolyl-1,4-bis(3-aminopropyl)piperazines
were reported to have in vitro and in vivo antimalarial
activity showing a site of action different from that of
CQ (cytoplasm and not FV) although their mechanism
of action was uncertain.7,8 Moreover, during the
preparation of this report Link and co-workers
exploited the same quinolin-1,4-bis(3-aminopro-
pyl)piperazine template for the synthesis of carboxamide
antimalarials.9 In this communication we report the
development of new antimalarial heterodimers, typified
by 4a (Table 1), a potent antiplasmodial agent, specifi-
cally designed to generate multisite iron coordination
antimalarials. The N1-(7-chloro-4-quinolyl)-1,4-bis(3-
aminopropyl)piperazine was the key structural support
of choice to link a panel of aromatic and heteroaromatic
groups, characterized by different metal ion coordina-
tion and protonatability properties (4a–h).
The synthesis of compounds 4a–d is described in Scheme
1. The aldehyde 5 was reacted with the Grignard reagent
obtained from p-bromotoluene 6 to afford the corre-
sponding ketone which was brominated to 7 in good
Compounds 4a–h were tested in vitro against the CQ-S
D10 and the CQ-R W2 Pf strains and selected com-
pounds were also tested against the CQ-S 3D7 and
CQ-R K1 Pf strains.6 The results are reported in
Table 1. Coupling of N1-(7-chloro-4-quinolyl)-1,4-
bis(3-aminopropyl)piperazine to a diphenyl ketone moi-
ety (4a), a reported iron chelating group (ConQuest 1.9,
CSDS), had beneficial effect on the antimalarial potency,
highly improving the activity against both CQ-S
and CQ-R strains with respect to CQ and CLT.
Transformation of the carbonyl group of 4a to the
Scheme 1. Reagents and conditions: (i) a—Mg turnings, THF, reflux,
6 h; b—NBS, AIBN, CCl4, reflux; (ii) pyrrolidine, Et3N, MeCN, 0 °C,
1 h; (iii) a—NaBH4, EtOH, rt, 2 h; b—SOCl2, DCM, 0 to 45 °C, 4 h;
(iv) 8, Et3N, MeCN, 80 °C, 18 h; (v) NaBH4, THF/H2O 2:1, 80 °C, 2 h;
(vi) MeSO2Cl, Et3N, DCM, 0 °C, 2 h.
Scheme 2. Reagents and conditions: (i) a—NBS, AIBN, CCl4, reflux,
4 h; b—imidazole, K2CO3, acetone, reflux, 6 h; (ii) LiOH, H2O/THF,
1:2, reflux, 30 min; (iii) HOBt, NMM, EDC, N,N-DMF, rt, overnight;
˚
(iv) DIEA, n-pentanol, 160 °C, 24 h; (v) Et3N, 3 A molecular sieves,
MeOH, then NaBH4, rt, 6 h.

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S. Gemma et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3535–3539
the diphenylcarbonyl group of 4a, was investigated as
reported below. To analyze structure–activity relation-
ships (SAR), we calculated for all compounds the preva-
lent ionic forms of the two possible tautomers at pH 7.4
(physiological), 7.2 (cytoplasm), and 5.5 (FV) (Table 1)
and, accordingly, performed a comprehensive computa-
tional analysis (Insight2005, Accelrys, San Diego) on the
resulting forms of 4a and 4f, in order to investigate the
role played by electronic and conformational parame-
ters on the antimalarial activity. According to the results
reported by Ryckebusch et al.7, the presence of several
nitrogens protonated at cytoplasmic pH in the 1,4-
bis(3-aminopropyl)piperazine linker of 4a–h (Table 1)
probably prevents the access to the FV. Consequently
the compounds elicit their antimalarial activity at the
cytoplasm level. Taking into account this hypothesis,
we focused on the effect of the imidazole (4f) and poly-
cyclic (4a) terminal fragments on electronic and confor-
mational properties at pH 7.2 (cytoplasm).
At pH 7.2 the presence of the diphenylketone moiety of
4a allows the formation of an intramolecular hydrogen
bond between the N1 of piperazine and the aniline nitro-
gen of the quinoline ring. This, in turn, increases the
electronic density at the quinoline heterocyclic nitrogen
and, consequently, improves its iron coordination prop-
erties, thus reproducing the geometry of a multisite iron
chelator (Fig. 2A) (ConQuest 1.9, CSDS). Moreover the
diphenylketone moiety represents a further iron chela-
tion site (Fig. 2A). On the contrary, the 2-imidazolylm-
ethyl group (4f), protonated at pH 7.2 (Table 1),
establishes hydrogen bond interactions with the quino-
line heterocyclic nitrogen thus decreasing the iron coor-
dination properties of 4f and consequently, its
antimalarial activity (Fig. 2B).
Figure 2. Resulting conformers at pH 7.2 of 4a (A, green) and 4f (B,
magenta) are shown. In (A) 4a was superimposed on the X-ray
structure of iron complexes containing the following moieties: diphe-
nylketone (white; CSD code: VESCEB), piperazine (yellow; CSD code:
HAVNUO), N1,N3-dimethylpropane-1,3-diamine (orange; CSD code:
METGIB), and quinoline (pink; CSD code: FOFROH). The super-
imposition was made fitting the putative iron coordinating atoms. Iron
atoms’ vdW volumes (magenta) are scaled (0.3) for clarity. Displayed
lone pair of piperazine nitrogen is colored in cyan. Heteroatoms are
colored: O, red; N, blue; Fe, magenta; and Cl, light green. All
hydrogens, except those involved in hydrogen bonds (green dashed
lines), have been omitted for clarity.
In summary, we identified novel CQ-based heterodimers
characterized by a 1,4-bis(3-aminopropyl)piperazine lin-
ker as potent antimalarials against CQ-S and CQ-R Pf
strains. Among the compounds presented in this study,
4a was the most potent antimalarial, being 16- and 8-
fold more active than CQ against K1 and W2 strains,
respectively. A putative mechanism of action of 4a was
investigated, paving the way for the development of
new multisite iron complexing antimalarials. Further
studies are in progress to assess the in vivo biological
properties of this class of antimalarial agents.
alcoholic group (4b) or to the corresponding sulfonate
(4c) determined a decrease in activity against CQ-S
and CQ-R strains (4b and 4c vs 4a). In order to define
the role played by iron chelating groups in antimalarial
activity (R1, Table 1), we introduced at R1 aromatic and
heteroaromatic systems endowed with different metal
chelating and protonatability properties. Consequently,
as R1 residue, we selected fragments potentially able to
coordinate iron (ConQuest 1.9, CSDS), such as a frag-
ment of the CLT/3 polycyclic scaffold (4e), the 2-imi-
dazolylmethyl group (4f), and the 2-benzimidazole
bicyclic system (4g); on the other hand, compounds 4d
and 4h were characterized by a pyrrolidinyl(di)phenylm-
ethylene system, two residues unable to chelate iron
(pyrrolidine chelates iron when a carbonyl function is
at C-2—ConQuest 1.9, CSDS). Compounds 4d and 4h
as well as 4e–g resulted less potent than 4a against
D10 and W2 strains, confirming the importance of the
iron chelating properties of the R1 substituent. Recently,
we have reported the potent antimalarial activity against
CQ-S and CQ-R strains of quinolines bearing an imid-
Acknowledgments
This investigation received financial support from
the UNICEF/UNDP/World Bank/WHO Special
Programme for Research and Training in Tropical
Diseases (TDR). Authors thank Intesa S. Paolo bank
for financial support.
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