A58365A and A58365B, angiotensin converting enzyme inhibitors isolated from the culture filtrate of Streptomyces chromofuscus NRRL15098, are homologous compoundsof molecular formulas C12H13NO6 and C13H15NO6. The molecular similarities of the two inhibitors were established by comparison of their 1H NMR,13C NMR,and UVspectra. Catalytic hydrogenation of A58365A led to a tetrahydro-deoxy derivative, C12Hi7NO5; extensive 1H NMR decoupling studies at 360 MHzallowed all the non-exchangeable protons of the derivative to be connected in a continuous substructure. This fragment was combined with information from other spectroscopic methods to suggest the structures for A58365A(1) and A58365B (2); the conclusions were confirmed by an X-ray crystallographic analysis of A58365A-dimethyl ester.Several inhibitors of angiotensin converting enzyme (ACE) have been discovered from fermentation sources2), many of these inhibitors exert their effect primarily through chelation of the Zn2+ in the metallodipeptidase (aspergillomarasmines A and B, L-681,176, and phenacein). Previous papers from these laboratories have described the development of a high-volume, agar-based screen for the detection of ACEinhibitors produced by fermentations and the discovery of ACEinhibitory activity in the culture broth of Streptomyces chromofuscus NRRL150983), the conditions for the biosynthesis of the ACEinhibitors A58365A and A58365B produced by this microorganism0, and the isolation and characterization of the two ACEinhibitors from A583655). A58365Aand A58365Binhibition of ACEis not reversible by addition of divalent cations3), hence strong chelation is not characteristic of these inhibitors. Furthermore, the structures of these novel fermentation products have provided a unique insight into the stereochemical requirements of captopril-related ACEinhibitors. A58365A has been determined to be 3-carboxy-l ,2,3,5-tetrahydro-8-hydroxy-5-oxo-6-indolizinepropanoic acid (1) ; A58365B is the homologous 4-carboxy-l,3,4,6-tetrahydro-9-hydroxy-6-oxo-2if-quinolizme-7-propanoic acid (2). A58365Ais thus a naturally occurring conformationally restricted analog of 2-methylglutaryl-L-proline (3), which was a part of the structure-activity relationship studies leading to captopril (4)6).Homology of A58365A and A58365B The preliminary characterization of A58365A and A58365B indicated that the two compounds are homologs, having the molecular formulas C12H13NO6 for A58365A (m/z 268.08189 by fast atom Seeref1.

A58365A and A58365B, angiotensin converting enzyme inhibitors, were isolated from the culture filtrate of Streptomyces chromofuscus NRRL 15098. A58365A and A58365B are homologous nitrogen-containing bicyclic structures of molecular formulae C,2H,3NOo and C"H,;NO,.Recently, several inhibitors of angiotensin converting enzyme (ACE) have been discovered from fermentation sources1-11)A previous paper in the present series described the development of a high volume, agar-based screen for the detection of ACE inhibitors produced by fermentations and the discovery of ACE inhibitory activity in the culture broth of Streptomyces chromofuscus NRRL 1509812) (culture A58365.1). The conditions for the biosynthesis of the ACE inhibitors A58365A and A58365Bproduced by this microorganism have also been described". This paper describes the isolation and characterization of the two ACE inhibitors. Materials and Methods Assay of Angiotensin Converting Enzyme Inhibitory ActivityThe qualitative agar-plate method of O'CONNOR and SOMERS12) was employed for monitoring purification of ACE inhibitors. Dried preparations were assayed in the quantitative spectrophotometric assay 12) to determine inhibitor potency.HPLC Assay of A58365A and A58365B HPLC determinations of A58365A and A58365B utilized a 4 mm x 30 cm Waters Associates ,uBondapak C,, reversed phase column with a mobile phase consisting of CH3CN -HCOOH -H2O (6.0: 0.3: 93.7). Broths were adjusted to pH 2.0 and filtered (0.45 um) prior to injection. Partially purified materials required no sample preparation.Detection was by either UV absorption at 325 nm or by fluorescence measurement. For accurate quantitation a Schoeffel model FS970 spectrofluorometer with excitation at 327 nm and a 370 nm emission cutoff filter coupled to a Hewlett-Packard 3390A integrator was used. With a mobile phase flow rate of 2.5 ml/minute, A58365A had a retention time of 4.88 minutes; A58365B, 12.47 minutes.General Methods UV spectra were run on a Cary model 118 spectrophotometer.Fluorescence spectra were obtained with an Amino-Boman Spectrophotofluorometer.IR spectra were recorded on a Nicolet MX-1 FT-IR spectrometer. 1H NMR spectra were obtained with a Bruker model WH-360 NMR