The present study examined the inhibitory eects of N-Hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016) on the renal metabolism of arachidonic acid by cytochrome P450 (CYP) enzymes. HET0016 exhibited a high degree of selectivity in inhibiting the formation of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) in rat renal microsomes. The IC 50 value averaged 35+4 nM, whereas the IC 50 value for inhibition of the formation of epoxyeicosatrienoic acids by HET0016 averaged 2800+300 nM. In human renal microsomes, HET0016 potently inhibited the formation of 20-HETE with an IC 50 value of 8.9+2.7 nM. Higher concentrations of HET0016 also inhibited the CYP2C9, CYP2D6 and CYP3A4-catalysed substrates oxidation with IC 50 values of 3300, 83,900 and 71,000 nM. The IC 50 value for HET0016 on cyclo-oxygenase activity was 2300 nM. These results indicate that HET0016 is a potent and selective inhibitor of CYP enzymes responsible for the formation of 20-HETE in man and rat.

Based on the structure-activity relationship data of BMY-28864 and related pradimicin derivatives, the calcium salt-forming ability and the D-mannopyranoside-speciflc visible absorption maximumshift of BMY-28864 were analysed in the ternary complex formation of BMY-28864 with D-mannopyranoside and calcium. The free C-18 carboxyl group of BMY-28864 was proved to be the sole site for binding to calcium, while no hydroxyl groups of the aglycone were involved in calcium salt formation. The stereospecific D-mannopyranoside-recognizing ability of BMY-28864 was completely abolished by removal of the C-5 disaccharide moiety, and, more particularly, of the C-5 thomosaminemoiety. Close relationship of these findings with the antifungal action was also supported by the in vitro antifungal assay and the potassium leakage induction test.In previous papers1~4), the in vitro antifungal activities of pradimicin and benanomicin derivatives on yeasts were shown to be specifically expressed only in the presence of calcium. Using BMY-28864, a water-soluble pradimicin derivative, specific binding of the pradimicin to yeast cells was proved to depend on the ternary complex formation of BMY-28864with mannan and calcium at a molar ratio of 2 :4 : 15). This highly stereospecific binding of BMY-28864 to the mannose unit (more generally, the specific sugar-recognizing ability of the pradimicin and benanomicin family of antibiotics) is biochemically worth studying, as it is currently unexplicable by the widely accepted concepts of receptor-ligand binding in the light of lectin and carbohydrate sciences. Lectins have been considered to recognize specific sugars based on the intrinsic properties of their peptide components, whereas the pradimicin and benanomicin family of compounds are not peptides. Under these circumstances, it is crucially important and essential to more precisely elucidate the mechanismof ternary complexformation of pradimicins with specific sugars and calcium in critical comparison with lectins. This type of knowledge is not only biochemically useful for receptology, but also clinically important from the viewpoint of selective toxicity of final pradimicin drugs in hosts, as sugars are essential cellular components of host animals to be treated with pradimicin, and assumed to exist ubiquitously at significant concentrations in a variety of forms throughout therapy.In this paper, the structure-activity relationship of BMY-28864 and related pradimicin derivatives is analyzed for identification of the moieties of BMY-28864responsible for binding to D-mannopyranoside and calcium and for induction of the visible absorption maximumshift. In brief, only the free C-18 carboxyl group of BMY-28864serves to bind to calcium as salt, while the C-5 disaccharide moiety is essential for specific recognition of and binding to D-mannopyranoside.

Sequence of reactions in the process of ternary complex formation of BMY-28864 with D-mannopyranosideand calcium was spectrophotometrically determined nnder more strict analytical conditions using metal-free preparations of sugars and the pradimicin derivative at a bandpass slit width of 1 nm. In the first phase of ternary complex formation, BMY-28864stereospecifically recognized and bound to D-mannopyranoside in the absence of calcium, which was revealed by a visible absorption maximumshift of ca. 8 nm. Subsequently, the BMY-28864-D-mannopyranoside conjugate reacted with calcium to yield the ternary complex, which was detected by an additional visible absorption maximumshift of ca. 8 nm. Whenthe three components were mixed at the same time, both phases simultaneously occurred to produce the ternary complex which was accompanied by a visible absorption maximumshift of 16 nm in total. Based on this two-phased reaction sequence, the mechanism of ternary complex formation of BMY-28864with D-mannopyranoside and calcium was reexamined in details. Terminal D-mannopyranoside was confirmed to be essential as BMY-28864-specific sugar receptor by in vitro analysis and animal cell experiments. While calcium, strontium and cadmium behaved similar in the in vitro ternary complex formation, the yeast and animal cell experiments showed that only calcium played a dual role as a base in the ternary complex formation and as an effector in physiological disturbances leading to cell death.In previous papers1~6), the antifungal activities of pradimicin derivatives were positively correlated with their lectin-mimic ternary complex formation by validating the essential roles of the C-18 carboxyl group and the C-5 thomosamine moiety of pradimicin derivatives. In consequence, characterization of the sugar-recognizing ability of pradimicin derivatives in the light of lectin biochemistry using highly-purified metal-free sugar and pradimicin preparations was considered to be informative and indispensable, as calcium and metal impurities in reagents probably interact or compete with the calcium componentof the ternary complex.The findings described in the preceding paper6) that BMS-184497 or BMY-28864methyl ester still retains as ability to showa visible absorption maximumshift of ca. 8nmin spite of the absence of the free C-18 carboxyl group; and that the breadth of the visible absorption peak shift depends on the status of the C-18 carboxyl group {ca. 8nm for the methyl ester and ca. 16nm for the free acid) allowed the hypothesis that the process of ternary complex formation might be divided into the sugar-recognization phase and the calcium salt formation phase. Therefore, the authors have attempted to elucidate the sequence of events in the visible absorption peak shift which seemed to be parallel with the process of ternary Correspondence should be addressed to Jun Okumura, Bristol-Myers

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