Iron-containing metallocenes as active site directed inhibitors of the proteinase that cleaves the NH2-terminal propeptides from type I procollagen

Derivatives of ferrocene (dicyclopentadienyliron) (Fc) were examined as active site directed inhibitors of type I procollagen N-proteinase, the enzyme that cleaves the NH2-terminal propeptides from type I procollagen. The compounds were shown here to be reversible, competitive inhibitors of the enzyme. The effectiveness of the Fc inhibitors varied with modification of the cyclopentadienyl (cp) rings. The monocarboxylic acid (I) and the 1,1'-dicarboxylic acid (II) derivatives of Fc inhibited 50% of the enzymic activity (I50) at concentrations of 1.0 and 0.5 mM, respectively. The Ki values were 0.3 mM for both I and II. Derivatization of the carbonyl alpha to the cp ring of compound I (FcCOCH2CH2COOH, III) increased the inhibitory activity (I50 = 0.100 mM; Ki = 0.065 mM). Removal of the carbonyl alpha to the cp ring of III did not improve inhibitory activity: FcCH2CH2COOH, I50 = 2 mM; FcCH = CHCOOH, I50 = 1.5 mM. The active inhibitory species apparently contained iron in the 3+ valence state since two ferrocenium derivatives were very effective inhibitors: ferrocenium tetrachloroferrate, IV (I50 = 0.030 mM; Ki = 0.004 mM), and carboxyferrocenium hexafluorophosphate, V (I50 less than 0.1 mM; Ki less than 0.05 mM). In addition, reduction of III with ascorbic acid abolished its inhibitory activity. Compounds I and III stabilized the enzyme to heat denaturation in the absence of exogenous calcium; compound IV did not stabilize the enzyme. Further observations indicated that Fc derivatives were specific inhibitors of procollagen N-proteinase.