New amine and urea analogs of ferrochloroquine: Synthesis, antimalarial activity in vitro and electrochemical studies

Article abstract of DOI:10.1016/S0040-4039(00)01036-4

Amine and urea analogs of ferrochloroquine with varying methylene spacer lengths were synthesised, studied by cyclic voltammetry and evaluated in vitro against a sensitive (D10) and resistant (K1) strain of Plasmodium falciparum. Most analogs were found to be more active than chloroquine in both strains. In D10 ureas were more active than amines and antimalarial activity in this strain correlated well with the length of the methylene spacer and redox potentials. The length of the methylene spacer was a major determinant of antimalarial activity in K1. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01036-4

Tetrahedron Letters 41 (2000) 6231±6235
New amine and urea analogs of ferrochloroquine: synthesis,
antimalarial activity in vitro and electrochemical studies
Kelly Chibale,a,* John R. Moss,a Margaret Blackie,a Donelly van Schalkwykb
and Peter J. Smithb
aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
bDepartment of Pharmacology, University of Cape Town Medical School, Observatory 7925, South Africa
Received 9 May 2000; accepted 20 June 2000
Abstract
Amine and urea analogs of ferrochloroquine with varying methylene spacer lengths were synthesised,
studied by cyclic voltammetry and evaluated in vitro against a sensitive (D10) and resistant (K1) strain of
Plasmodium falciparum. Most analogs were found to be more active than chloroquine in both strains. In
D10 ureas were more active than amines and antimalarial activity in this strain correlated well with the
length of the methylene spacer and redox potentials. The length of the methylene spacer was a major
determinant of antimalarial activity in K1. # 2000 Elsevier Science Ltd. All rights reserved.
Malaria is a parasitic disease that a‚icts 300±500 million people and kills up to 2 million
worldwide. The chemotherapy of malaria has been undermined by the widespread development
of resistance of the most dangerous form of the causative agent, Plasmodium falciparum, to
clinically used antimalarial drugs such as chloroquine (CQ), me¯oquine, quinine and sulfadoxine-
pyrimethamine. As a result drugs such as CQ 1 (Fig. 1) have been rendered virtually useless and
malaria is impossible to treat in some parts of the world. There is thus a clear need for new drugs
with structures and mechanisms of action di€erent from those that are presently used.
Figure 1. Chemical structures of chloroquine (1), ferrochloroquine (2) and new analogs 3a and 3b
* Corresponding author. Fax: +27 21 689 7499; e-mail: chibale@psipsy.uct.ac.za
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01036-4

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It has been shown throughout history that incorporation of metals into drugs can have a
profound e€ect on the biological activity. Within the context of malaria, coordination of metal
complexes to CQ is a relatively new approach to new antimalarial agents. So far, this approach
has shown great potential with demonstrable enhancement of the ecacy of CQ against both
sensitive and resistant strains of Plasmodium falciparum by coordination to ruthenium and gold
complexes.1,2 The disclosure of ferrochloroquine (FQ) 2 (Fig. 1) in which the carbon chain of CQ
is replaced by the hydrophobic ferrocenyl group has been a welcome addition to organometallic
antimalarial drugs.3,4 This compound has been shown to be superior to CQ in both in vitro and
in vivo tests. Related analogs of 2, including its potential metabolites have also recently been
reported.5,6
The recent6 ®nding that unlike CQ metabolites monodesethylchloroquine (DECQ) and
didesethylchloroquine (di-DECQ), potential metabolites of FQ are more active than CQ against
Plasmodium falciparum suggests that the ferrocenyl moiety in FQ and related analogues plays an
important role in the observed antimalarial activity. These compounds can thus be regarded as
useful lead compounds which can serve as a basis for the design of further analogues and mimics.
In this preliminary communication, we report on the synthesis, in vitro antimalarial activities and
electrochemical studies of new amine (3a) and urea (3b) ferrocenic analogues of FQ.
The rationale behind the design of these new analogues and subsequent electrochemical studies
is as follows. First, in order to explore extensive and detailed structure±activity relationships
within the FQ class of organometallic antimalarial agents, we reasoned that new analogs (such as
3, R=H) of FQ that would serve as sca€olds for the parallel synthesis of chemical libraries were
required. We envisaged introducing chemical diversity at the more reactive nucleophilic
secondary amino group adjacent to the ferrocenylmethyl moiety. In designing these new analogs
we also took into account the recent disclosure that in 7-substituted 4-aminoquinolines, the
length of the methylene spacer between two nitrogens in the side chain of CQ analogues is a
major determinant of activity against CQ-resistant Plasmodium falciparum.7,8 Second, we
reasoned that although the ferrocenyl moiety is chemically stable in many media, the possibility
exists that under appropriate conditions, in the presence of the malaria parasite in vitro and/or in
vivo, the iron metal centre in 2 may undergo redox reactions. We reasoned that the ease of
oxidation of Fe(II) in the ferrocenyl moiety of 3a and 3b might be in¯uenced by chemical groups
in the vicinity of this moiety. We thus wanted to explore a possible correlation between the redox
potentials of initial compounds 3a and 3b and their in vitro antimalarial activities.
Derivatives 3a and 3b were prepared from commercially available 4,7-dichloroquinoline 4 and
the corresponding diamines. Reaction of the resulting products 5 with ferrocenyl aldehyde 63
gave compounds 3a after reduction of the imine formed in situ using polymer-supported
borohydride in moderate chemical yields. Reaction of compounds 3a with benzyl isocyanate gave
the corresponding urea derivatives 3b in low to high yields, Scheme 1. All new compounds gave
spectroscopic and analytical data consistent with their structures.
The antimalarial activities of derivatives 3a and 3b in vitro against both the CQ-sensitive (D10)
and CQ-resistant (K1) strains of Plasmodium falciparum as well as their redox potentials are
presented in Table 1.9 Data for CQ are included for comparison purposes. Antimalarial activities
of compounds 3a were comparable to CQ in D10. Further in this series antimalarial activities in
D10 correlated well with the chain length and ease of oxidation of the ferrocenyl group. The
longer the chain length the greater the ease of oxidation and the lower the antimalarial activity as
10
indicated by the decreasing values of the half-wave potential (E1/2) and increasing IC50 values,
respectively, on moving from 3a (n=2) to 3a (n=6). In K1 no such correlation was revealed.

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Scheme 1. Reagents and conditions: (a) 4.5 equiv. of H2N(CH2)nNH2, 80ꢀC, 1 h, 82±90%; (b) 4.0 equiv. of Amberlite
IRA-400 borohydride resin, 25ꢀC, 18 h, 60±65%; (c) 1.2 equiv. of PhCH2NCO, CH2Cl2, 25ꢀC, 3 h, 34±75%
Table 1
Antimalarial activity in vitro and electrochemical data of compounds 3a and 3b9
However, it is noteworthy that good activity against K1 within the 3a series was only observed
for the compound with a 3-carbon methylene spacer (n=3) which was found to be 3 times more
active than CQ. This ®nding lends further support to earlier observations that in 7-substituted
4-aminoquinolines, the length of the methylene spacer between two nitrogens in the side chain of
CQ analogues is a major determinant of activity against CQ-resistant Plasmodium falciparum.7,8
Generally speaking there was an improvement (up to fourfold in some cases) in the potency on
moving from amines 3a to the benzyl ureas 3b. This was especially the case in D10. While no
correlation was revealed between antimalarial activity, length of the methylene spacer and ease of
oxidation in D10 within the urea series, a correlation was observed in K1 between the length of
the methylene spacer and the antimalarial activity as evidenced by an increase (albeit modest) in
IC50 values with increase in the length of the methylene spacer. With the exception of the 6-carbon
methylene spacer, the ease of oxidation as evidenced by the decrease in the E1/2 values
correlated well with the antimalarial activity and length of the methylene spacer. In compounds

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3b the length of the methylene spacer between the two secondary amine nitrogens in the side chain
of CQ analogues is also a determinant of activity against CQ-resistant Plasmodium falciparum.7,8
Higher E1/2 values for the ureas relative to the amine precursors suggests that the presence of the
electron-withdrawing urea moiety in close proximity to the cyclopentadiene system makes the
urea compounds more dicult to oxidize.
In summary we have shown a correlation in D10 between in vitro antimalarial activity of FQ
analogues and their redox potentials. Our results further support earlier ®ndings that in 7-substituted
4-aminoquinolines, the length of the methylene spacer between two nitrogens in the side chain of
CQ analogues is a major determinant of activity against CQ-resistant Plasmodium falciparum.7,8
We have further demonstrated the potential bene®ts of utilizing analogs such as 3a as sca€olds
for the synthesis of new analogs exempli®ed by 3b. Coupled with the simplicity of the chemistry,
our approach to new analogs of FQ can potentially give rise to a large number of compounds for
exploration of structure±activity relationships within this series of compounds in view of the
commercial availability of a wide range of electrophilic reagents including acid and sulfonyl
chlorides. This work is now in progress.
Acknowledgements
We thank Dr Alan Hutton for help with cyclic voltammetric studies. Financial support from
the Wellcome Trust (grant no. 052075/Z/97, to K.C.), the University of Cape Town and National
Research Foundation (South Africa) is gratefully acknowledged.
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the assay was the unparasitized red blood cells without drug. The parasites were incubated at a 1% haematocrit
and a 2±3% parasitaemia in a volume of 200 ml along with the particular drug at the appropriate de®ned
concentration in DMSO. Each drug concentration was measured in duplicate. The parasites were incubated for 2
days in the plate in dessicator cabinets under a CO2/air gas mixture. Once the incubation was complete, 100 ml of
Malstat Reagent and 25 ml of NBT/PES solution containing equal volumes of Nitro Blue Tetrazolium (1.6 mg/ml)
and phenazine etho sulphate (0.08 mg/ml) were added to each well of a new ¯at bottomed 96-well microtitre plate.
The culture in each of the wells of the original plate was resuspended and 10 ml of the culture was taken from each

6235
well and added to the corresponding well of the Malstat plate, thus initiating the lactate dehydrogenase reaction.
Colour development was monitored at 620 nm as the reaction proceeded. The percentage viabilities were
calculated using a SigmaPlot (Jandel Scienti®c) transformation. The dose±response curves were generated using
a non-linear regression in Prism (Graphpad Software). Statistical analyses were performed also using Prism.
10. Cyclic voltammetry was performed on a BAS 100B Electrochemical Analyser using a three electrode system
comprising a platinum disk working electrode, a platinum wire auxiliary electrode and a Ag/Ag+ reference
electrode (0.01 M AgNO3 and 0.1 M Bu4NClO4 in acetonitrile). Unless other stated, all measurements were
made on acetonitrile solutions which were 1±2 mM in sample and 0.1 M Bu4NClO4 at a scan rate of 100 mV s^1.
Under these conditions the ferrocene/ferrocenium couple, which was used as a reference, had an E1/2 value of 0.08
V. All solutions were purged with argon and voltammograms were recorded under a blanket of argon.