Nitrocatechol derivatives as inhibitors of catechol-O-methyltransferase.

Catechol-0-methyltransferase (EC 2.1.1.6; COMT) catalyses Sadenosylmethionine-dependent methylation of a variety of catechols and plays a role in terminating the activities of the catecholamine neurotransmitters dopamine and noradrenaline. Two isoenzymes, the soluble and membrane-bound forms (S-COMT and MB-COMT) are present in most tissues and although the former is more abundant, their different kinetic parameters suggest that the MB-form may play the dominant role in inactivating the catecholamines [I]. Studies on the inhibition of COMT have been extensive since specific and effective inhibitors could help elucidate the physiological role and properties of the enzyme. Furthermore, development of effective COMT inhibitors could be promising for therapeutic use both in Parkinson's disease and in diseases connected to an aberrant catecholamine metabolism. Early COMT inhibitors, e.g. tropolone, were not suitable for use in vivo (see review [2]). It was observed that catechol compounds with electron withdrawing substituents, particularly NO2 groups, greatly enhanced inhibitory activity [3] and this gave rise to a second generation of potent and selective COMT inhibitors [4,5]. Nitrocatechol was the key structure in the majority of these compounds (see Fig. 1) and these had inhibition constant (Ki) values in the nanomolar range [4,5]. The aims of this work were to study the efficacy of some nitrocatechol derivatives as inhibitors of both forms of COMT, especially the MB-form. Most previous studies have concentrated on the soluble enzyme and since the MB-form may be more important at low, physiological concentrations of substrate, it is possible that this form may have different affinities for inhibitors of the enzyme. Human S-COMT and MB-COMT cloned and expressed in Escherichiu coli [6] were used as enzyme sources. COMT activity was assayed with using a slight modification of a direct solvent-extraction assay which determined the amount of radioactive guaiacol formed 171. A series of fixed inhibitor concentrations was used (see Table l ) , while the concentration of catechol was varied between approximately half to several times the K, concentration for each form of the enzyme and meth~l [~H]labelled AdoMet was held at 390 pM. Representative doublereciprocal plots are also shown in Figs. 2 (a) and (b) for the inhibition of S-COMT and MB-COMT by 4,5-dinitrocatechol with respect to catechol. The Ki values were determined for the nitrocatechol derivatives by plotting slopes of the double reciprocal plots against inhibitor concentration and results for both forms of the enzyme are shown in Table 1. .