In 1997, Biot and coworkers reported the synthesis of four ferrocenyl analogues of chloroquine.29 In each case, the ferrocene ring is inserted in place of two methylene groups in the butyl side chain of chloroquine. The terminal amino group was varied to give a dimethylamino (10), diethylamino (11), piperidinyl (12) or N-methylpiperazinyl (13) analogue. The dimethylamino analogue was found to be the most active in mice infected with chloroquine-sensitive and -resistant strains of Plasmodium.29,30 When evaluated against a drug resistant strain of the human P. falciparum, its activity against the life stages of the plasmodium in the red blood cell was found to be about 22 times that of chloroquine.29 Compound 10 was subsequently called ferrochloroquine. Further tests showed that ferrochloroquine had a curative effect on murine P. vinckei and protected mice from lethal infection at a daily dose of 8.4mg/kg when given for 4 days subcutaneously or orally.30 Furthermore, ferrochloroquine was approximately 300-800-fold more selective against plasmodia than mammalian L5178Y lymphoma cells. Acute toxicity tests in mice showed that ferrochloroquine was more toxic in starved animals.31 In vitro susceptibility tests against P. falciparum isolates from Libreville and the Haut-Ogooue region in Gabon confirmed that ferrochloroquine has significant activity against chloroquine-resistant strains.32,33


Various structure-activity studies have been reported in the literature. Delhaes and co-workers investigated the importance of chirality to activity.34 The ferrocene ring in ferrochloroquine is 1,2 substituted. Thus ferrochloroquine has planar chirality and exists in two enantiomeric forms. Both enantiomers (+) (1'R) ferrochloroquine (14) and (—) (1'S) ferrochloroquine (15) were synthesized and found to be equivalent in anti-plasmodial activity as well as in cytotoxicity against mouse lymphoma cells. When the enantiomers were compared to the racemate, equivalent activity was detected in vitro but the racemate had a higher curative effect in infected mice. In view of this observation, racemate ferrochloroquine is the formulation of choice.

"N 15

"N 15

Biot and coworkers investigated the attachment of the ferrocene ring to different positions of chloroquine.35 The attachment of the ferrocene ring to the quinoline ring (16) or to the end of the terminal amino function (17) caused a decrease in activity. Compound 16 in which ferrocene is linked to the quinolinyl nitrogen gave rise to a salt that is completely inactive, but compound 17 retained activity against chloroquine-sensitive strains. It would seem that the position of ferrocene in ferrochloroquine is optimal.

Phase I metabolism of chloroquine proceeds by dealkylation of the terminal amino function to give less active mono-desethyl and di-desethyl metabolites. Thus, it was of interest to investigate the activity of equivalent dealkylated derivatives of ferrochloroquine.36 The desmethyl (18) and di-desmethyl (19) analogues of ferrochloroquine were synthesized and found to be comparable to ferrochloroquine.

They were more active than chloroquine against sensitive and resistant plasmodia, with 19 being less active than 18 against resistant plasmodia. Thus, if dealkylated compounds like 18 and 19 are formed as metabolites of ferrochloroquine, anti-plasmodial activity is likely to be retained even on metabolism.

In ferrochloroquine, the quinoline ring and the terminal basic side chain are attached to the same cyclopentadienyl ring of ferrocene, i.e. the substitution pattern is 1,2-. Beagley and co-workers investigated the effect of attaching the substituents to different cyclopentadienyl rings (i.e. 1,1'-substitution as shown in 20).37 Compound 20 was as active as ferrochloroquine against a chloroquine-sensitive strain (D10) of P. falciparum but was slightly less active against a resistant plasmodial strain (K1). Another pair of 1,2- and 1,1'-substituted ferrochloroquine analogues (21, 22) showed the reverse trend (1,2-substituted 21 > 1,1'-substituted 22) but both compounds were still more active than chloroquine against resistant plasmodia. Unlike ferrochloroquine, there is an additional basic nitrogen atom in the side chain of 21 and 22. The presence of this additional basic centre may have influenced anti-plasmodial activity so that a comparison between ferrochloroquine and its 1,2-isomer 20, and that of 21 and 22 need not result in a similar trend. Thus, the effects of 1,1'- versus 1,2-substitution are probably dependent on the nature of the side chain.


Extending the methylene chain in 21 (n = 2) to give homologues 23-25 (n = 3,4,6) caused a gradual decrease in activity against the chloroquine-sensitive D10 strain, with only 21 retaining the same level of activity as chloroquine.38 When an amide group is inserted into the secondary amino group of the side chain of 21 and 23-25 to give ureas 26-29 respectively, a rebound in activity was observed (Table 10.1). The increase in activity was consistently observed for all the ureas 26-29.

However, a different trend is observed when the activity was evaluated against the chloroquine-resistant K1 strain. Here, extending the methylene chain did not have a clear-cut effect on the activities of 21 and 23-25. The analogue 23 (n = 3) is about three times more active than chloroquine while 24 (n = 4) is comparable with chloroquine. Among the ureas 26-29, activity decreased as the methylene chain was extended (26 > 27 > 28 > 29), a trend not observed for the amines.



26: n = 2 27: n = 3 28: n = 4 29: n = 6
Table 10.1 Anti-plasmodial activity and half-wave potential (E/2) of compounds 21, amines 23-25 and ureas 26-2938



IC50 D10 (nM)

IC50 K1 (nM)

E1/2 (mV)














































*As shown in structures 21, 23-25 and 26-29.

*As shown in structures 21, 23-25 and 26-29.

When an amide moiety is attached to the secondary amino function of 21 and 23-25, a urea is obtained. This has the effect of reducing the basicity of the amino function, as well as diminishing its hydrogen bond donor and acceptor abilities. Bulk is also increased in the urea. These changes in physicochemical properties appear to enhance activity against chloroquine-sensitive plasmodia (26-29 > 21, 23-25) but not against resistant plasmodia.

The results in Table 10.1 also emphasize the importance of the methylene side chain for activity. The length of the methylene spacer in 7-substituted 4-aminoquinolines is a major determinant of activity against chloroquine-resistant P. falciparum.39 The same appears to be true for the ferrocene ureas 26-29. A longer methylene side chain resulted in lower activity against the chloroquine-resistant strain K1 (26 > 27 > 28 > 29).

The authors also noted a progressive decrease in the half-wave potential of the ferrocene analogues as the methylene chain is lengthened from n = 2 to n = 4.38 The half-wave potential measures the ease with which Fe2+ in ferrocene undergoes oxidation. Comparing the ureas 26 and 28, the more active 26, which has a shorter methylene chain (n = 2), is associated with a larger half-wave potential, indicating that Fe2+ is more resistant to oxidation. The significance of this observation was not discussed further by the authors but it may signal a role for Fe2+ in anti-plasmodial activity. Not withstanding these observations, Beagley etal., in another study, found equivalent anti-plasmodial activities for ferrochloroquine and its phenyl analogue 30 and concluded that the primary role of ferrocene in ferrochlor-oquine is that of a hydrophobic spacer.37

The mechanism of action of ferrochloroquine in malaria is still under investigation. Ferrochloroquine is a weaker base than chloroquine with pKa values of 6.99 and 8.19 for the quinoline nitrogen and the tertiary amine, respectively.31 The mechanism of action of chloroquine has been widely reviewed.40

The basic properties of chloroquine are believed to contribute in a significant way to its anti-plasmodial activity. It is hypothesized that the diprotonated form of chloroquine is trapped in the acidic food vacuole of the plasmodia (pKa of chloroquine = 7.94 and 10.0331). Because of the poor permeability of the protonated species, exceptionally high concentrations of chloroquine build up in the food vacuole where it is able to disrupt critical processes like the disposal of heme. This mechanism may not apply to ferrochloroquine as it is a weaker base than chloroquine and is also more lipophilic. Ferrochloroquine was originally designed with the intention that the presence of Fe2+ would reduce chloroquine resistance.30 This is indeed found to be true for ferrochloroquine but the question of how, or even if, the ferrous ion contributes to the activity of ferrochloroquine remains unanswered.

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