Journal of Antimicrobial Chemotherapy (2000) 46, 625-628
© 2000 The British Society for Antimicrobial Chemotherapy
Brief report |
Simple 1,2-aminoalcohols as strain-specific antimalarial agents
School of Chemical Sciences, Dublin City University, Dublin 9, Ireland
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Abstract |
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We report here the in vitro activity of a selection of 1,2-aminoalcohol-containing compounds against cloned strains of human Plasmodium falciparum. These compounds exhibit moderate antimalarial activity but a high degree of strain specificity, preferentially inhibiting a chloroquine-resistant strain of the organism.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Malaria threatens millions of people annually with infection and death.1 The parasite responsible for infection demonstrates remarkable drug resistance and few new chemotherapies are available. The need for new antimalarial agents has never been greater, as the range and scope of the disease continue to grow, with malaria reaching and re-infecting areas once thought clear from the disease.
Enzymes, particularly protease enzymes within the malarial parasite, are prime potential chemotherapeutic targets. Protease inhibitors should theoretically disrupt the life cycle of the malarial parasite through inhibiting enzymes used in the degradation of host haemoglobin. One well-known inhibitor is bestatin (2S,3R-3-amino-2-hydroxyphenylbutanyl-l-leucine), a dipeptide isolated from culture filtrates of Streptomyces oliveticuli. Bestatin contains a core 1,2-(S,R)-aminoalcohol moiety which, it has been postulated, is essential for aminopeptidase activity. Bestatin is an active inhibitor of most aminopeptidases, specifically aminopeptidase B and leucine aminopeptidase.2,3 Its broad range of application stems from the presence of two structural features, the amide linkage and the l-leucine side chain. These moieties make the molecule a target for proteolytic enzymes. Bestatin exhibits numerous biological activities, notably acting as an immunomodifier4 and as a potent analgesic.5 It also inhibits tumour growth.6 Most aminopeptidases are metalloenzymes, the majority of which require zinc as a metal cofactor for activity. The mode of inhibition of bestatin is not fully understood; it has been postulated that the 1,2-aminoalcohol moiety is coordinated with zinc in the enzyme, as the enzyme attempts to cleave the peptide bond,7 and later it was suggested that the carbonyl group along with the hydroxy group underwent this coordination.8 However the molecule binds to the protein, it is generally accepted that the 2(S)-hydroxyl group is vital for tight binding to the enzyme and subsequent inhibition activity.9 Given that we knew bestatin to be a protease inhibitor with antimalarial activity, and that the compound's anti-enzymic properties stemmed from the presence of the 1,2-aminoalcohol system, we decided to synthesize a series of 1,2-aminoalcohol-containing species loosely based on the structure of bestatin, and to test these molecules as antimalarial agents and protease inhibitors.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Chemistry
A series of 18 1,2-aminoalcohols was prepared according to the synthetic route previously published.10 In this route, a simple aldehyde is treated with trimethylsilylcyanide (TMSCN) to yield an oxygen–trimethylsilyl (O-TMS) protected cyanohydrin ether. This cyanohydrin species is subsequently treated with a simple Grignard reagent to give an intermediate iminium salt. The iminium moiety is reduced by treatment with sodium borohydride (NaBH4), yielding the desired free amine species. The desired 1,2-aminoalcohol is produced by deprotecting the oxygen, removing the trimethylsilyl group by treating with weak aqueous acid solution and subsequent work-up.
By incorporating different aldehydes and Grignard reagents in this synthetic route, the nature of the end product can be varied. We designed a series of inhibitors that, through changing the nature of the aldehyde and Grignard reagent used, would serve as a type of homologous series of inhibitory compounds. This series of non-peptidyl analogues of bestatin should give direct information as to the nature of those groups and molecular parameters that are essential for protease and antimalarial activity.
Biochemistry
The compounds were tested in vitro against the blood stages of two cloned strains of human Plasmodium falciparum parasite, one chloroquine resistant (W2) and one chloroquine sensitive (D6). The inhibition studies were carried out four times for each compound. In tandem with the in vitro antimalarial testing, a simple, efficient in-house leucine aminopeptidase (LAP) enzyme assay was prepared based on the existing LAP quality control test procedure advocated by Sigma–Aldrich. The assay is colorimetric and utilizes continuous spectrophotometric rate determination based on the principle that the active enzyme acts on a specific aqueous substrate, l-leucine-p-nitroanilide, to yield l-leucine and the highly coloured compound p-nitroaniline. All 18 aminoalcohols synthesized were tested at a concentration of 10 mM for their inhibitory activity against the enzyme.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The six antimalarial active compounds are illustrated in the Figure
. Of the 18 aminoalcohols assayed, six demonstrated antimalarial activity (see Table for IC50s). The effects on LAP of the series as a whole lie outside the scope of this report, but the results for those compounds demonstrating in vitro antimalarial activity are illustrated in the Table.
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This series of compounds preferentially inhibited the chloroquine-resistant strain. Three of the six compounds demonstrated activity against both strains, but in these cases there is still higher inhibition against the chloroquine-resistant strain, with a specificity ratio of almost 1:4 recorded for the most active compound, 6. Of the six antimalarials, only two, compounds 4 and 5, inhibited LAP. This suggests that the antimalarial activity of the other four active antimalarial species does not stem from their action against LAP. In each of these cases, for compounds 1–3 and 6, we see that they act as enzyme activators, enhancing the activity of leucine aminopeptidase at the 10 mM concentration tested. What is most interesting from a design point of view is that compound 6, which is structurally (in terms of electron distribution and molecular size) the closest relative of bestatin, activated LAP but had antimalarial activity approximately twice that of bestatin in vitro. The active species may still be working as protease inhibitors in their antimalarial role, but inhibiting a parasitic enzyme other than LAP, with a degree of specificity, judging from the more potent activity exhibited against the W2 strain.
Given that the design of the entire 1,2-aminoalcohol series was initially based on the activity of the enzyme inhibitor bestatin and the clear demonstration that the series as a whole does not exhibit specific potent inhibition of LAP, we must conclude from the available evidence that the 1,2-aminoalcohol series has a different mode of antimalarial activity from that of bestatin, and a high degree of specificity against a chloroquine-resistant strain of the disease which warrants further investigation.
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Acknowledgments |
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The authors express their thanks to Dr Rob Ridley and his staff in the Tropical Disease Research division of the World Health Organization, particularly Nep Castillon, for the initial in vitro evaluation of the compounds described in this work.
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Notes |
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*Corresponding author. Tel: +353-1-7045312; Fax: +353-1-7045503; E-mail: howarthj{at}ccmail.dcu.ie
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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1 . Ridley, R. G. & Hudson, A. T. (1998). Chemotherapy of malaria. Current Opinion in Infectious Diseases 11, 691–705.[Medline]
2 . Umezawa, H. (1981). Small Molecular Immunomodifiers of Microbial Origin, pp. 1–16. Japan Scientific Societies Press, Tokyo.
3 . Taylor, A., Peltier, C. Z., Torre, F. J. & Hakamian, N. (1992). Inhibition of bovine lens leucine aminopeptidase by bestatin: number of binding sites and slow binding of this inhibitor. Biochemistry 32, 784–90.
4 . Bruley-Rosset, M., Florentin, L., Kiger, N., Schulz, J. & Mathe, G. (1979). Restoration of impaired immune functions of aged animals by chronic bestatin treatment. Immunology 38, 75–83.[Web of Science][Medline]
5 . Chaillet, P., Marcais-Collado, H., Costentin, J., Yi, C., De la Baume, S. & Schwartz, J. C. (1983). Inhibition of enkephalin metabolism by, and antinociceptive activity of bestatin, an aminopeptidase inhibitor. European Journal of Pharmacology 86, 329–36.[Medline]
6 . Umezawa, H. (1980). Low molecular weight immunomodulators produced by microorganisms. Biotechnology and Bioengineering 22, Suppl. 1, 99–110.
7 . Nishizawa, R., Saino, T., Takita, T., Suda, H. & Aoyagi, T. (1977). Synthesis and structure–activity relationships of bestatin analogues, inhibitors of aminopeptidase B. Journal of Medicinal Chemistry 20, 510–5.[Medline]
8 . Nishino, N. & Powers, J. C. (1979). Design of potent reversible inhibitors for thermolysin. Peptides containing zinc coordinating ligands and their use in affinity chromatography. Biochemistry 18, 4340–7.[Medline]
9 . Rich, D. H., Moon, B. J. & Harbeson, S. (1984). Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effects of inhibitor structure on slow binding processes. Journal of Medicinal Chemistry 27, 417–22.[Web of Science][Medline]
10 . Howarth, J., Lloyd, D. G. & McCormac, P. (1998). A convenient one-pot synthesis of 1,2-aminoalcohols. Synthetic Communications 28, 2751–9.
Received 1 November 1999; returned 23 February 2000; revised 31 March 2000; accepted 19 May 2000
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