Zlatina Naydenova

Natural and synthetic inhibitors of UDP-glucuronosyltransferase.

Glucuronidation is a major detoxification pathway in vertebrates. The reaction is catalyzed by a family of UDP-glucuronosyltransferases (UGTs) and involves conjugation of many endobiotic and xenobiotic substances with glucuronic acid, forming inactive water-soluble glucuronides. UGT prevents the accumulation of potentially toxic compounds and/or their subsequent bioactivation to more toxic intermediates, although biologically active glucuronides are also known. Impairment of UGTs may have important toxicological consequences. Substances found to inhibit or down-regulate UGT activity include endogenous compounds, a wide range of clinically used drugs, environmental contaminants, and natural toxic substances present in the diet. The development of selective, active-site-directed UGT inhibitors greatly enables the study of various UGT isoenzymes. A promising approach offers the design of transition-state analogs of the glucuronidation reaction.

Synthesis of some 5′-O-amino acid derivatives of uridine as potential inhibitors ofUDP-glucuronosyltransferase

SummaryIn an attempt to develop potential inhibitors ofUDP-glucuronosyltransferase, some 5′-O-amino acid derivatives of uridine were synthesized. N-protectedL-amino acids were coupled at the 5′-O-position of 2′,3′-O-isopropylideneuridine by esterification employing the method of symmetrical anhydrides in presence of 4-dimethylaminopyridine, 5′-O-(N-benzyloxycarbonyl-O-tert.butyl-L-threonl)-2′3′-O-isopropylideneuridine (1), 5′-O-(N-tert.butyloxycarbonyl-O-benzyl-L-seryl)-2′,3′-O-isopropylideneuridine and (2), 5′-O-(N-tert.butyloxycarbonyl-L-valyl)-2′,3′-O-isopropylideneuridine (3), and 5′-O-(N-tert.butyloxycarbonyl-L-valyl)-2′,3′-O-isopropylideneuridine (4) were obtained in good yield after column chromatography on silica gel. The treatment of2 withTFA/CH2Cl2 (6:1) at room temperature for 30 min led to a selective removal of theBoc group without deblocking of the 2′,3′-O-isopropylidene group of uridine. Treatment of2 withTFA/H2O (5:1) at room temperature for 1 h, however, released bothBoc and 2′,3′-isopropylidene groups. TheZ group of1 was deprotected by catalytic hydrogenolysis over 10% Pd/C/ammonium formate.ZusammenfassungIn einem Versuch, potentielle Inhibitoren derUDP-Glukuronosyl-Transferase zu entwickeln, wurden einige 5′-O-Aminosäurederivate des Uridins synthetisiert. N-GeschützteL-Aminosäuren wurden durch Veresterung mit der 5′-O-Position des 2′,3′-isopropylidenuridins gekuppelt (Methode der symmetrischen Anhydride in der Gegenwart von 5-Dimethylaminopyridin). Solcherweise wurden 5′-O-(N-Benzyloxycarbonyl-O-tert.butyl-L-threonly)-2′,3′-O-isopropylidenuridin (1), 5′-O-(N-tert.Butyloxycarbonyl-O-benzyl-L-seryl)-2′,3′-O-isopropylidenuridin (2), 5′-O-(N-tert.Butyloxycarbonyl-L-leucyl)-2′,3′-O-isopropylidenuridin (3) und 5′-O-(N-tert.Butyloxycarbonyl-L-valyl)-2′,3′-O-isopropylidenuridine (4) nach Säulenchromatographie (Kieselgel) in guter Ausbeute hergestellt. Die Behandlung von2 mitTFA/CH2Cl2 (6:1) bei Zimmertemperatur (30 min) führte zu einer selektiven Abspaltung derBoc-Gruppe ohne Deblockierung der 2′,3′-O-Isopropylidengruppe des Uridins. Eine Behandlung von2 mitTFA/H2O (5:1) bei Zimmertemperatur für 1 Stunde führte hingegen zur Abspaltung sowohl derBoc als auch der 2′,3′-O-Isopropylidengruppe. DieZ-Gruppe von1 wurde durch katalytische Hydrogenolyse auf 10% Pd/C/Ammoniumformiat abgespalten.

Synthesis of 5′-O-Oligopeptide Derivatives of Uridine as Inhibitors of UDP-glucuronosyltransferase

Summary. In order to design potential inhibitors of UDP-glucuronosyltransferase, the synthesis of some 5′-O-oligopeptide derivatives of uridine is presented. 5′-O-(N-tert.Butyloxycarbonyl-O-benzyl-L-seryl-L-valyl)-2′,3′-O-isopropylideneuridine (1) was synthesized by the DCC/HOBt method from N-tert.butyloxycarbonyl-O-benzyl-L-serine and 5′-O-L-valyl-2′,3′-O-isopropylideneuridine in 95% yield. In a similar way, 5′-O-(N-tert.butyloxycarbonyl-L-valyl-O-benzyl-L-seryl)-2′,3′-O-isopropylideneuridine (2) was obtained from N-tert.butyloxycarbonyl-L-valine and 5′-O-(O-benzyl-L-seryl)-2′,3′-O-isopropylideneuridine in 93% yield. Treatment of 1 and 2 with HCl/EtOAc at room temperature for 30 min led to removal of both Boc and 2′,3′-O-isopropylidene groups. 5′-O-(O-Benzyl-L-seryl-L-valyl)-uridine (3) and 5′-O-(L-valyl-O-benzyl-L-seryl)-uridine (4) were obtained in 94% and 91% yields, respectively.Zusammenfassung. Die Synthese von 5′-O-Oligopeptidderivaten des Uridins als Inhibitoren der UDP-Glucuronosyltransferase wird beschrieben. 5′-O-(N-tert.Butyloxycarbonyl-O-benzyl-L-seryl-L-valyl)-2′-,3′-O-isopropylidenuridin (1) wurde nach der DCC/HOBt-Methode aus N-tert.Butyloxycarbonyl-O-benzyl-L-serin und 5′-O-L-Valyl-2′-,3′-O-isopropylidenuridin in 95%iger Ausbeute hergestellt. Auf ähnliche Weise erhielt man aus N-tert.Butyloxycarbonyl-L-valin und 5′-O-(O-Benzyl-L-seryl)-2′,3′-O-isopropylidenuridin in 93%iger Ausbeute 5′-O-(N-tert.Butyloxycarbonyl-L-valyl-O-benzyl-L-seryl)-2′,3′-O-isopropylidenuridin (2). Beide Schutzgruppen –Boc und 2′,3′-O-Isopropyliden – wurden mit HCl/EtOAc bei Zimmertemperatur (30 min) abgespalten. 5′-O-(O-Benzyl-L-seryl-L-valyl)-uridin (3) and 5′-O-(L-Valyl-O-benzyl-L-seryl)-uridin (4) entstanden in Ausbeuten von 94 bzw. 91%.

INHIBITION OF UDP-GLUCURONYLTRANSFERASE ACTIVITY IN RAT LIVER MICROSOMES BY PYRIMIDINE DERIVATIVES

Thirty-one differently substituted pyrimidine bases were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol and phenolphthalein by rat liver microsomes. 5-Nitrouracil (compound 1) and its isomer 4,6-dihydroxy-5-nitropyrimidine (compound 2) were the most potent and selective inhibitors of 4-nitrophenol glucuronidation, without any effect on the phenolphthalein conjugating activity of UDP-glucuronyltransferase (UGT). Kinetic studies with compound 1 revealed a mixed type of inhibition toward the acceptor substrate 4-nitrophenol and an atypical competitive type of inhibition toward UDP-glucuronic acid, with apparent Ki values of 0.11 and 0.2 mM, respectively. Two benzylamino-substituted pyrimidines (compounds 10 and 12) and an orotic acid derivative (compound 25) inhibited both 4-nitrophenol and phenolphthalein UGT activities.

Effect of s-triazine and phenoxyalkanoic acid herbicides on UDP-glucuronosyltransferase in rat liver microsomes

Twelve herbicides, representatives of two chemical groups, substituted phenoxyalkanoic acids and s-triazines, were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol (4-NP), phenolphthalein (PPh) and 4-methylumbelliferone (4-MU) by rat liver microsomes. One millimole MCPA, ametryn and cyanazine significantly decreased PPh UDP-glucuronosyltransferase (UGT) activity, while propazine was found to be a most potent inhibitor of 4-NP glucuronidation. Concentrations of 0.1 mM dichlorprop and cyanazine were still inhibitory against PPh-UGT. The inhibition of 4-MU glucuronidation by the herbicides was low and not specific. As a whole, s-triazine derivatives were more inhibitory than the substituted phenoxyalkanoic acids. Kinetic studies with propazine revealed a non-competitive type of inhibition towards the acceptor substrate 4-NP, with an apparent Ki value of 0.540 mM. With ametryn, an uncompetitive type of inhibition against PPh and a mixed type of inhibition towards UDPGA were found, with apparent Ki values of 0.330 mM and 0.380 mM, respectively. © 1999 Society of Chemical Industry

Inhibition of UDP-glucuronosyltransferase by 5'-O-amino Acid and Oligopeptide Derivatives of Uridine: Structure-Activity Relationships

Abstract The inhibitory effect of a series of 5′-O-amino acid and oligopeptide derivatives of uridine on rat liver UDP-glucuronosyltransferase (UGT) activities was investigated using two assay systems. A quantitative structure-activity relationship (QSAR) study was performed. The compounds include a lipophilic residue linked to the nucleoside by a variable spacer. More over, half of the derivatives have two spacers linked to the uridine moiety. Compound 1, a serine derivative of isopropylideneuridine, was found to be the most potent inhibitor of both 4-nitrophenol (4-NP) and phenolphthalein (PPh) glucuronidation, with an I50 of 0.45 mᴍ and 0.22 mᴍ , respectively. Kinetic studies with this substance revealed a mixed type of inhibition towards 4-NP and UDP-glucuronic acid, with apparent Ki values of 150 μᴍ and 120 μᴍ , respectively. The dipeptide derivatives 11-14 exhibited a low activity against 4-NP conjuga tion. However, a marked suppression of PPh glucuronidation was found with compounds 11 and 13. Generally, compounds with two spacers are more inhibitory against the UGT activities studied. The QSAR analysis outlined the significance of the spacers with a minimum length of 5 atoms and lipophilic residues linked to them for the inhibitory effect of the compounds. The most significant contribution to this effect is given by the six-atom spacer for both, 4-NP and PPh substrates. 4-NP converting UGT isoforms seem to respond more specifically to the inhibitors: a five-atom for the first and a six-atom for the second spacer enhance binding to both 4-NP and PPh conjugating isoenzymes, while a long second spacer contributes to inhibitor binding to UGT isoforms only converting PPh.

Influence of Pesticides on UDP-glucuronosyltransferase in Rat Liver Microsomes

Fifteen pesticides, representatives of different chemical groups, were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol (4-NP) and phenolphthalein (PPh) by rat liver microsomes. Three herbicides (simazine, chlorsulfuron, tribenuron-methyl), two insecticides (dioxacarb, carbaryl) and one fungicide (zineb) significantly decreased the UDP-glucuronosyltransferase (UDPGT) activity. The carbamate insecticide dioxacarb was found to be the most potent inhibitor, at 1 mM concentration suppressing 4-NP-UDPGT activity completely, and reducing by 55% the activity associated with the conjugation of PPh. One millimole simazine and carbaryl affected only 4-NP glucuronidation, while chlorsulfuron and zineb exerted a marked inhibition of both 4-NP and PPh conversion. Concentrations of 0.1 mM carbaryl, dioxacarb and zineb were still inhibitory against 4-NP-UDPGT, with zineb producing 40% inhibition of PPh glucuronidation. As a whole, UDPGT isoforms conjugating PPh were less sensitive to the agrochemicals tested. Kinetic studies with dioxacarb, chlorsulfuron and carbaryl revealed a mixed type of inhibition with respect to the acceptor substrate 4-NP, with apparent K i values of 70 μM, 120 μM and 160 μM, respectively.