Transition state analogue inhibitors of protozoan nucleoside hydrolases

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Abstract

Protozoan parasites are unable to synthesize purines de novo and must rely on purine salvage pathways for their requirements. Nucleoside hydrolases, which are not found in mammals, function as key enzymes in purine salvage in protozoa. Inhibition of these enzymes may disrupt purine supply and specific inhibitors are potential therapeutic agents for the control of protozoan infections. A series of 1,4-dideoxy-1,4-imino-d-ribitols bearing C-bonded aromatic substituents at C-1 have been synthesized, following carbanion additions to the imine Scheme 1, Scheme 3, Scheme 4, and tested as potential nucleoside hydrolase inhibitors. Nucleoside analogues Scheme 2, Scheme 3, Scheme 4 exhibit Ki values in the range 0.2–22 μM against two representative isozymes of protozoan nucleoside hydrolases.

Introduction

Infections due to protozoan parasites are a continuing health problem world-wide with more than two billion people estimated to be infected by these organisms. Approximately two million deaths per year, largely of infants, result from malaria alone.1 Protozoan parasites are devoid of the capacity for de novo purine biosynthesis and rely completely on salvage pathways for their purine requirements.2 Nucleoside hydrolases provide a pathway for obtaining purines by nucleoside salvage in several protozoa whose pathways of purine salvage have been characterized.3 These enzymes are not found in mammals, but they are widely distributed in bacteria, yeast and protozoa.4, 5 Inhibitors for these enzymes have potential as antimicrobials in purine auxotrophs, especially the protozoan parasites.

Two isozymes of protozoan nucleoside hydrolases have been characterized.3, 6, 7, 8, 8, 10, 11, 12 Nonspecific enzymes, the ‘IU-nucleoside hydrolases’5 from Crithidia fasciculata and Leishmania major are examples of the first type. The enzymes catalyze the hydrolysis of all commonly occurring purine and pyrimidine nucleosides, and also p-nitrophenyl β-d-ribofuranoside. Catalysis occurs by promoting the formation of a ribofuranose-based oxocarbenium ion transition state with modest assistance from activation of the leaving group. The ribooxocarbenium ion is promoted by conformational distortion of the sugar using protein contacts and a catalytic site Ca2+ to permit both the ring oxygen and the 5′-hydroxymethyl oxygens' unshared electrons to participate in the cleavage of the sugar-base bonds. The ‘IAG nucleoside hydrolase’ from Trypanosoma brucei brucei is representative of the second isozyme type which comprise purine-specific hydrolases.3, 6, 12 These enzymes obtain a significant part of their catalytic efficiency from protonation of the leaving group. Both classes of hydrolase enzymes utilize mechanisms which involve transition states appreciably stabilized by contributions from the ribofuranose based oxocarbenium ion. This led to the prediction that 1,4-dideoxy-1,4-imino-d-ribitol derivatives 3, which will be partially N-protonated at pH 7 (pKa=6.5),13 would be good transition state analogue inhibitors of the nucleoside hydrolases.

We have reported some preliminary investigations into the synthesis14 and testing15 of iminoribitol derivatives 3 which revealed potent (nM) inhibitors, e.g. 4 and 5 (Scheme 1), of the IU nucleoside hydrolase, but none of the compounds tested had Ki values better than 12 μM against the IAG nucleoside hydrolase. Since this latter class of enzyme has a higher catalytic efficiency in those parasites that have been studied,3, 6, 8 the discovery of more efficient inhibitors of the IAG type of enzyme could be of importance. We present here further attempts at the design and synthesis of compounds to act as inhibitors of nucleoside hydrolases—particularly the IAG class of enzyme. Selected biological data are given; a more detailed analysis is presented elsewhere.16

Section snippets

Results and Discussion

A facile synthesis of some (1S)-1-aryl-1,4-dideoxy-1,4-imino-d-ribitols has been claimed17 following the addition of organometallic C-nucleophiles to 2,3,5-tri-O-substituted-d-ribofuranose derivatives, oxidation of the C-1 and C-4 hydroxy groups in the products, and then reductive amination of the resultant 1,4-dicarbonyl compounds. While further investigations confirmed that these products had the required 1,4-dideoxy-1,4-iminopentitol structures, they also revealed that they had the all-cis β-

Conclusion

A number of (1S)-1-(purin-9-yl)methyl and -aryl-1,4-dideoxy-1,4-imino-d-ribitols have been synthesized and tested against two representative protozoan nucleoside hydrolases. Compounds Scheme 2, Scheme 3, Scheme 4 bind as well as substrate (Ki values 2–5 μM) to the IAG nucleoside hydrolase. It is not obvious why Scheme 2, Scheme 4 are the most potent inhibitors of the IAG nucleoside hydrolase, however both have aglycones that contain sites to accept hydrogen bonds from the enzymatic general

Experimental


NMR spectra were recorded on a Bruker AC-300 instrument at 300 MHz (1H) or 75 MHz (13C) in CDCl3 unless otherwise stated with TMS as internal reference. In D2O acetone was used as internal reference. High-resolution accurate mass determinations were performed by Hort Research Limited on a VG70-250S double focussing magnetic sector mass spectrometer under chemical ionization conditions using isobutane or ammonia as the ionizing gas, or under high-resolution FAB conditions in a glycerol or

Acknowledgements

The authors thank Dr. Robert W. Miles for the unpublished data of Table 1, Table 2, Dr. Herbert Wong for an excellent NMR service and Professor Robin Ferrier for assisting with the preparation of the manuscript. This work was supported by research grants from the National Institutes of Health, USA, and the Foundation for Research Science and Technology, New Zealand.

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