Thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyliglycine is a highly potent inhibitor (K; = 3.5 nM) of "enkephalinase," a metalloendopeptidase cleaving the Gly-Phe bond (positions 3 and 4) of enkephalins in brain tissue. In accordance with this property, thiorphan displays antinociceptive activity after systemic administration. However, thiorphan also inhibits to a lesser extent (K; = 140 nM) the widely distributed angiotensin-converting enzyme, a carboxydipeptidase implicated in blood pressure regulation. Therefore, in view of an eventual clinical use of enkephalinase inhibitors, it was very important to develop fully specific compounds. Such derivatives were obtained taking into account that N-methylation of the ultimate amide bond of dipeptides strongly decreases enkephalinase affinity without affecting angiotension-converting enzyme recognition, whereas retro-inversion of the amide bond leads to the inverse effect. Thus, the retro-inverso dipeptide (R)-H2N-CH(CH24)-NHCO-CH2-CO2H exhibits an inhibitory potency on enkephalinase (IC50 12 jM) close to that of the natural dipeptide L-Phe-Gly (IC50 3 jIM). This result shows the topological analogy between the crucial components involved in enkephalinase recognition both in active dipeptides and structurally related retro-inverso isomers. Taking into account these observations, retro-thiorphan, (R,S)-HS-CH2-CH-(CH20)-NHCO-CH2-COOH, was prepared. As compared to thiorphan, the retro isomer is 50% as potent (Ki = 6 nM) on enkephalinase but displays a drastic loss of potency on angiotension-converting enzyme (IC50 > 10,000 nM). This specificity was interpreted as a consequence of differences in the stereochemical constraints involving enzyme-inhibitor hydrogen bonding. This hypothesis is supported by reported crystallographic studies on related enzymes such as thermolysin and carboxypeptidase A. As expected, retro-thiorphan exhibits about the same analgesic potency as thiorphan on the hot plate and writhing tests in mice. Therefore, the topological concept of retro-inverso isomers could be extended to other enkephalinase inhibitors, allowing the design of potent and highly selective compounds occurring as new classes of analgesic and psychoactive agents.An important approach in the search for antinociceptive agents is to prevent degradation of the endogenous morphine-like peptides, enkephalins (1, 2), at the synaptic level. The characterization of a membrane.bound metalloendopeptidase called enkephalinase (3)(4)(5), which seems to be specifically involved in cleavage of the Gly-Phe (positions 3 and 4) bond of [Met]enkephalin (Tyr-Gly-Gly-Phe-Met) or [Leu]enkephalin (Tyr-GlyGly-Phe-Leu), made this approach possible. A highly potent enkephalinase inhibitor, thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyl]glycine [Ki = 3.5 nM (6, 7)], was rationally designed taking into account the occurrence in this peptidase of (i) a Zn atom in its catalytic site able to coordinate a thiol group with a strong affinity (7, 8); (ii) a Sj' subsite exhibiting a specificity for aro...

A stereospecific synthesis of thiorphan [N-[2(RS)-(mercaptomethyl)-1-oxo-3-phenylpropyl]glycine] and retro-thiorphan [3-[[1(RS)-(mercaptomethyl)-2-phenylethyl]amino]-3-oxopropanoic acid], two highly potent inhibitors of enkephalinase, a neutral endopeptidase involved in enkephalin metabolism, is reported. Due to a rapid isomerization process, derivatives of retro-thiorphan, which contains a 2-substituted malonyl moiety, cannot be separated by classical methods. However, a separation of the diastereoisomeric mixtures of these retro-thiorphan derivatives was achieved by HPLC. The absolute configuration of each isomer was determined by using an NMR configurational correlation. The inhibitory potency of the various inhibitors indicates that, in the thiorphan series, the affinity for enkephalinase is independent of the stereochemistry of the 2-(mercaptomethyl)-1-oxo-3-phenylpropyl moiety. In contrast, in the retro-thiorphan series a 100-fold difference in the inhibitory activity of the two enantiomers is observed. This indicates that there are large differences in the conformational behavior of the two series of inhibitors at the active site of the enzyme.

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