Molecular docking simulations were carried out for ligands

Molecular docking simulations were carried out for ligands – in the ACE crystal structure (1O86) and DPP4 crystal structure (2G5P). Comparison of the binding mode of – () with enalaprilat (a) in ACE shows that the phenethyl group (–, enalaprilat) occupies the S1 pocket and the adjacent carboxylic Nemadectin group (–, enalaprilat) lies in the metal binding region. The R groups of – and the methyl group of enalaprilat fit into the S1′ pocket and the pyrrolidine group with the carboxylic acid (–, enalaprilat) positions itself into the S2′ pocket. Comparison of the binding mode of – () with sitagliptin (b) in DPP4 shows that the 2,4,6-triflurophenyl group and the basic amine moiety (–, sitagliptin) binds in the S1 pocket. The S1 pocket is closed and offers tight binding to the docked poses of the ligands, all of which maintain the same alignments and amino acid interactions, as have been reported for other known DPP4 inhibitors.b shows that the S2 pocket is wide enough to accommodate bulkier substitutions. Molecular docking supports this hypothesis and it was observed that the ACE pharmacophore of – binds into the S2 pocket of DPP4 (). The ligands – were synthesized by standard synthetic and peptide coupling methods (). Amine was coupled with β-amino acid using EDCI to afford from which the Cbz group was removed to yield the key intermediate . The proposed DPP4-ACE dual inhibitors – were synthesized from in a simple three-step protocol by coupling the intermediate with – under EDCI coupling conditions followed by sequential deprotection of the Boc and ester groups of –. The homophenylalanine derivatives – were prepared according to a literature procedure. Merged ligands – have high MW (>500) and PSAs (162Å) (). These compounds exhibit gradual increase in cLogPs with increasing alkyl chain length from CH to -Bu (4.23–5.78 respectively). Also, the number of rotatable bonds increases from 14 to 17 on going from to respectively. Next, compounds – were evaluated for their ability to inhibit, ACE and DPP4. For this purpose, plasma of Wistar rat, mouse and human was used as enzyme source for ACE and DPP4. shows the inhibitory activities for compounds – and the reference standards and . As expected compound inhibited ACE potently (IC<11nM) and did not inhibit DPP4 even at concentration of 100μM. Likewise, showed potent inhibition of DPP4 (IC<50nM) across species but showed only weak inhibition potential for ACE enzyme. To our delight, compounds –, incorporating the features of both ACE and DPP4 inhibitors, exhibited dual inhibition of human ACE and DPP4. Compounds (ACE IC=8.6nM; DPP4 IC=57nM) and (ACE IC=51nM; DPP4 IC=67nM) were found to be the most potent dual inhibitors of human ACE and DPP4. Compounds and also displayed dual inhibition of ACE and DPP4 but at relatively higher concentrations (IC>100nM). Compound moderately inhibited human ACE. The inter-species shifts in ACE IC values for compound were not significant (within the range of experimental error). Compound exhibited a very potent inhibition of human ACE (IC=8.9nM). However, showed a considerable shift (12-fold) in mouse ACE inhibition (IC=100nM) and up to 3-fold shift in rat ACE inhibition (IC=24nM). Compounds and also exhibited a considerable loss of inhibitory potential against rat ACE (seventeen- and ten-fold, respectively) and mouse ACE (forty- and eight-fold, respectively). While compound exhibited similar inhibitory potencies against DPP4 across all three species, a progressive loss in rat and mouse DPP4 inhibitory potency was observed for compounds – with the greatest loss being seen for mouse DPP4 (∼2-fold more than that for rat DPP4). The plasma protein binding (PPB) for – was found to increase in human and mouse plasma with increasing length of R () which can be correlated with increasing clogP (); the inherent PPB in mouse plasma being higher than in human plasma. Thus, the upward shift in IC of compound – in mouse ACE and DPP4 (plasma being used as enzyme source) can be explained to a certain extent based on increased PPB in mouse plasma. However, the shift in ICs in rat ACE and DPP4 (plasma being used as enzyme source) cannot be explained based on PPB. These unusual shifts were not noted for the reference compounds and .