A carbamate-based approach to primaquine prodrugs: Antimalarial activity, chemical stability and enzymatic activation
Graphical abstract
O-Alkyl carbamate derivatives of primaquine were shown to display potent transmission-blocking antimalarial activity.
Introduction
Malaria remains the world’s top-priority tropical disease due to its high death burden, as well as to its economic and social impacts on the development of malaria-endemic countries.1 The emergence and spread of multidrug-resistant Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is still the major obstacle in achieving an effective control of the disease.2 Most antimalarials in clinical use or under development are potent blood-schizontocides, that is they act rapidly against the parasitic forms that invade erythrocytes and cause the usual symptoms to effect a cure from malaria within a reasonable time (ideally 3 days or less).3, 4, 5 However, the ultimate goal of global eradication of malaria parasites from the human population requires the radical cure of all life cycle stages of all malaria species infecting humans.6
Currently, primaquine, 1, is the only available antimalarial that displays a marked activity against gametocytes from all species of parasite causing human malaria, including chloroquine-resistant P. falciparum; it is thus capable of interrupting disease transmission from the host to the mosquito vector.7 Primaquine is also the only antimalarial effective against the latent exoerythrocytic forms (hypnozoites) of Plasmodium vivax responsible for relapsing malaria.7, 8 In addition, primaquine also displays blood schizontocidal activity, particularly against P. vivax and P. falciparum, but at doses that can induce side effects such as methaemoglobinemia.
However, primaquine is rapidly metabolised in mammals to carboxyprimaquine, 2, which is devoid of significant antimalarial activity against the several forms of the parasite.7 After intravenous administration of primaquine to rats, monkeys and humans it was found that the plasma concentration of carboxyprimaquine rapidly exceeded that of the parent drug after 15–30 min.9, 10, 11 The oxidative deamination of the alkyl side chain of primaquine most likely involves three enzymes in a two-step process: first, monoamine oxidase (MAO) or cytochrome P450 systems mediate the oxidation of primaquine to the corresponding aldehyde; second, the aldehyde intermediate is further oxidised to carboxyprimaquine by aldehyde dehydrogenase.7
The synthesis of prodrugs is a widely accepted approach used to overcome metabolic deactivation and bioavailability problems commonly found in amine drugs.12, 13 In the case of primaquine, any prodrug should be activated at a rate adequate to maintain sustained levels of the parent drug while being unable to undergo oxidative deamination prior to the release of primaquine. Since both mechanisms of cytochrome P450- and MAO-catalyzed oxidation of amines appear to involve an initial single electron transfer to form a nitrogen-centred radical cation,14 increasing the ionization potential through nitrogen acylation is expected to reduce significantly the rate of oxidation. For example, acylation of the terminal amino group of primaquine with dipeptides (e.g. 3) blocked the formation of carboxyprimaquine in rat liver homogenates, without affecting the antimalarial activity.15
In the present work we assess carbamates 4 as potential prodrugs for primaquine. The carbamate prodrug approach requires the derivative to be enzymatically activated to a carbamic acid, which rapidly decomposes to the parent amine.16 Carbamates can be activated by esterases or cytochrome P450, and their chemical and enzymatic reactivity can be modulated by appropriately choosing the alcohol carrier.16, 17 We have now synthesized a series of O-alkyl and O-aryl carbamate derivatives 4 of primaquine comprising different alcohol and phenol pro-moieties, in order to determine the impact of these leaving groups on (i) the chemical stability, (ii) rates of activation in human plasma and by rat liver homogenates and (iii) the potency of these derivatives. The target compounds were evaluated in vivo for their gametocytocidal activity.
Section snippets
Synthesis
Primaquine carbamates were synthesised either by reaction of primaquine with the appropriate alkyl or aryl chloroformates or, alternatively, with an equimolar amount of carbonyl diimidazole and the appropriate alcohol (Scheme 1). Carboxyprimaquine, 2, was prepared according to the procedure reported by McChesney et al.18
Hydrolysis in aqueous buffers
The rates of hydrolysis of primaquine carbamates, 4, were investigated in aqueous buffers containing 20% (v/v) of acetonitrile. The observed pseudo-first-order rate constants, k
Conclusions and relevance to prodrug design
The use of carbamates as prodrugs of basic primary amine drugs is still open to discussion mainly due to the lack of in vitro/in vivo prodrug activation data.16 The results presented herein show that primaquine carbamates derived from phenols are readily hydrolysed both in alkaline solutions and human plasma, with chemical and enzymatic reactivities being dependent on the electronic effects on the alcohol moiety. Activation of O-aryl carbamates in human plasma appears to be catalysed by BuChE,
General
Melting points were recorded using a Buchi 510 capillary melting-point apparatus and are uncorrected. 1H NMR spectra were recorded using a Bruker MSX-300 spectrometer. The 1H NMR data are reported as follows: chemical shifts, expressed in ppm, using internal TMS as reference; number of protons; multiplicity (s, singlet; d, doublet; dd, double doublet; t, triplet; q, quartet; m, multiplet; br, broad); and coupling constants, J, quoted in Hertz. The IR spectra were recorded using a Nicolet FTIR
Acknowledgment
The authors are grateful to the Fundação para a Ciência e Tecnologia (PEst-OE/SAU/UI4013/2011) for financial support of this research.
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