A carbamate-based approach to primaquine prodrugs: Antimalarial activity, chemical stability and enzymatic activation

https://doi.org/10.1016/j.bmc.2011.11.059Get rights and content

Abstract

O-Alkyl and O-aryl carbamate derivatives of the antimalarial drug primaquine were synthesised as potential prodrugs that prevent oxidative deamination to the inactive metabolite carboxyprimaquine. Both O-alkyl and O-aryl carbamates undergo hydrolysis in alkaline and pH 7.4 phosphate buffers to the parent drug, with O-aryl carbamates being ca. 106–1010 more reactive than their O-alkyl counterparts. In human plasma O-alkyl carbamates were stable, whereas in contrast their O-aryl counterparts rapidly released the corresponding phenol product, with primaquine being released only slowly over longer incubation periods. Activation of the O-aryl carbamates in human plasma appears to be catalysed by butyrylcholinesterase (BuChE), which leads to carbamoylation of the catalytic serine of the enzyme followed by subsequent slow enzyme reactivation and release of parent drug. Most of the O-aryl and O-alkyl carbamates are activated in rat liver homogenates with half-lives ranging from 9 to 15 h, while the 4-nitrophenyl carbamate was hydrolysed too rapidly to determine an accurate rate constant. Antimalarial activity was studied using a model consisting of Plasmodium berghei, Balb C mice and Anopheles stephensi mosquitoes. When compared to controls, ethyl and n-hexyl carbamates were able to significantly reduce the percentage of infected mosquitos as well as the mean number of oocysts per infected mosquito, thus indicating that O-alkyl carbamates of primaquine have the potential to be developed as transmission-blocking antimalarial agents.

Graphical abstract

O-Alkyl carbamate derivatives of primaquine were shown to display potent transmission-blocking antimalarial activity.

  1. Download : Download full-size image

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.

References and notes (41)

  • N. Vale et al.

    Eur. J. Med. Chem.

    (2009)
  • T.N.C. Wells et al.

    Trends Parasitol.

    (2010)
  • A.M. Clark et al.

    J. Pharm. Sci.

    (1984)
  • B. Li et al.

    Biochem. Pharmacol.

    (2005)
  • I.B. Wilson et al.

    J. Biol. Chem.

    (1961)
  • J.C. Verheijen et al.

    Bioorg. Med. Chem. Lett.

    (2009)
  • F. Lopes et al.

    Bioorg. Med. Chem.

    (2000)
  • L. Constantino et al.

    Exp. Toxicol. Phatol.

    (1999)
  • E. Gangl et al.

    J. Chromatogr., A

    (2002)
  • N. Yumibe et al.

    Biochem. Pharmacol.

    (1996)
  • K.J.P. Yoon et al.

    Bioorg. Med. Chem.

    (2003)
  • WHO, World Malaria Report...
  • T. Rodrigues et al.

    Future Med. Chem.

    (2011)
  • P. Olliaro et al.

    Clin. Pharmacol. Ther.

    (2009)
  • T.N.C. Wells et al.

    Nat. Rev. Drug Disc.

    (2009)
  • F.J. Gamo et al.

    Nature

    (2010)
  • P.L. Alonso et al.

    PLoS Med.

    (2011)
  • J.K. Baker et al.

    Pharm. Res.

    (1984)
  • G.W. Mihaly et al.

    Br. J. Clin. Pharmacol.

    (1984)
  • B. Testa et al.

    Chem. Biodivers.

    (2009)
  • Cited by (0)

    View full text