Synthesis, Antiviral and Cytostatic Evaluation of Unsaturated Exomethylene and Keto D-Lyxopyranonucleoside Analogues

Niki Tzioumaki 1, Evangelia Tsoukala 1, Stella Manta 1, George Agelis 2, Jan Balzarini 3, Dimitri Komiotis *

^*Correspondence to Dimitri Komiotis, ¹Department of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, Larissa, Greece. Fax: +30 2410 565-290

Funded by:
Geconcerteerde Onderzoeksacties of the Katholieke Universtiteit Leuven; Grant Number: GOA no. 05/19

Keywords

Cytotoxicity • Exomethylene nucleosides • Ketonucleosides • Unsaturated nucleosides

Abstract

This report describes the synthesis of unsaturated exomethylene lyxopyranonucleoside analogues as potential biologically active agents. Commercially available 1,2,3,4-tetra-O-acetyl--D-lyxopyranose 1 was condensed with silylated thymine and uracil, respectively, deacetylated and acetalated to afford 1-(2,3-O-isopropylidene--D-lyxopyranosyl)thymine 4a and 1-(2,3-O-isopropylidene--D-lyxopyranosyl)uracil 4b. The new derivatives 1-(2,3,4-trideoxy-4-methylene--pent-2-enopyranosyl)thymine 8a and 1-(2,3,4-trideoxy-4-methylene--pent-2-enopyranosyl)uracil 8b were prepared via two different key intermediates, 7a, b and 13a, b in order to elucidate the influence of 2,3-unsaturation and to clarify the difference between the keto and exomethylene group on the biological activity of the target molecules. Compounds 7a, b, 8a, b, and 13a, b were evaluated for their antiviral and cytostatic activity using several virus strains and cell lines. Whereas no marked antiviral activity was noticed, 13a and 13b showed a cytostatic activity that ranged between 7 and 23 M for 13a and 26 and 38 M for 13b against murine leukemia L1210, human lymphocyte Molt4/C8 and CEM cells, and human breast carcinoma MCF7 cells.

Synthesis, structure characterization, and enzyme screening of clenbuterol glucuronides

Two clenbuterol O-glucuronide diastereomers were synthesized by the Koenigs-Knorr reaction. Structures and glucuronidation sites of the glucuronides were characterized by tandem mass spectrometry and nuclear magnetic resonance spectroscopy. The two diastereomers were used as standard compounds in studies of stereoselective glucuronidation of clenbuterol with liver microsomes from different species and with 15 human recombinant UDP-glucuronosyltransferases. In this study, chemical and enzymatic reactions produced only O-glucuronides of clenbuterol, although on the basis of the chemical structure of the aglycone, both O- and N-glucuronides of clenbuterol could be formed. Differences in the production of diastereomers of clenbuterol glucuronides were observed among liver microsomes from the various animals. Dog and bovine liver microsomes were significantly active, and also stereoselective, each producing only one but a different diastereomer. Liver microsomes from rabbit and rat were also rather actively glucuronidating clenbuterol, but human, pig, and moose liver microsomes produced only minor amounts of glucuronides. Human liver microsomes produced only one clenbuterol glucuronide diastereomer, and the same was true of the human UDP-glucuronosyltransferases that were active (formation of glucuronide: 1A9 > 1A10 much greater-than 1A7). The marked differences in the stereoselective glucuronidation of clenbuterol show that UDP-glucuronosyltransferases in the livers of different animals do not have the same functions, activities, or distribution. This needs to be taken into account, particularly in toxicology testing.