Dual inhibition of ACE and NEP as a strategy for
Dual inhibition of ACE and NEP as a strategy for treating hypertension has been extensively investigated, including contributions from these laboratories through the discovery of Sampatrilat . Given that both targets are related zinc metalloproteinases, dual enzyme inhibition can be achieved within a single pharmacophore possessing a modified peptide backbone linked through to a zinc ion chelator (carboxylic acid) (). Selectivity for either ACE or NEP is highly influenced by the nature of the additional functionality present in the S2′ subsite. Selective NEP inhibitors do not always require an α-amino MLN2238 at the C-terminus (e.g., Candoxatrilat , ), however both ACE and NEP can tolerate biarylalanine substituents as disclosed by Zambon in their dual ACE/NEP inhibitor (). Exploration of the S2′ subsite of NEP within the Candoxatrilat template may enable identification of a selective NEP inhibitor with enhanced properties. This letter explores the lipophilic binding pocket at S2′ with conformationally restricted heteroarylalanines in place of the tyrosine in Sampatrilat and the biphenyl group of compound . The preferred natural amino acid stereochemistry was employed, enabling the design and synthesis of all analogues to be derived from -aspartic acid. Oxazoles were synthesised by peptide coupling of the appropriately substituted aminoethanol with N-BocAsp-OBn , to give the corresponding amides . Oxidation of the alcohol with Dess–Martin periodinane and cyclisation using either iodine and triphenylphosphine or dibromotetrachloroethane and triphenylphosphine, followed by final N-Boc deprotection with trifluoroacetic acid (TFA) furnished the desired amino esters – expressing the heteroaryl alanine (). 1,2,4-oxadiazole was obtained by acylating N-hydroxybenzamidine with to afford acylamidoxime , followed by cyclisation as a melt. TFA deprotection furnished the desired amino ester intermediate as a second class of heterocyclic variants (). Isomeric oxazoles – were synthesised via N-CbzAspOEt , which was obtained according to literature precedent from Cbz-aspartic acid anhydride. Amide bond formation with the appropriate aminoketone followed by POCl cyclisation produced the desired heterocycles. N-Cbz deprotection with HBr/AcOH furnished the amino esters (). A similar protocol was employed to obtain the isomeric 1,3,4-oxadiazole . Benzoyl hydrazine was reacted with the acid chloride of in good yield, followed by cyclisation in the presence of chlorodimethylimidazolinium tetrafluoroborate. N-Cbz deprotection under palladium catalysed hydrogenolysis furnished the desired amino ester (). Amide bond formation of the synthesised amino esters with carboxylic acids and followed by a two step deprotection strategy (TFA removal of the Bu ester followed by base hydrolysis of the ethyl ester) completed the synthesis of the novel heterocyclic diacids (). These compounds were tested for their ability to inhibit both ACE and NEP activity in vitro. The experimental details for the pharmacological assays have been previously described. The results are summarised in . shows the structure–activity relationship for the conformationally restricted heteroarylalanine analogues . Within the 4-substituted oxazoles –, we found that increasing lipophilicity through to the 4-phenyloxazole increased NEP inhibition; however selectivity over ACE was modest. Co-crystallisation of with human NEP shows the binding mode of the P2′ phenyl oxazole in the deep, lipophilic S2′ pocket (). Key interactions include the left hand acid (as drawn) zinc coordination, and hydrogen bonding between the inhibitor amide group and Arg 717 and Asn 542. The natural amino acid stereochemistry enables the right-hand side acid to interact with key Arg 110 and 102 residues in the active site, orientating the phenyl oxazole deep into the S2′ cavity. The subsite is fully occupied and replacement of the pendant phenyl in P2′ for a less lipophilic alkyl substituent such as ethyl and isobutyl (, ) only served to lower NEP activity.