Design, Synthesis, and X-ray Structure of Potent Memapsin 2 (β-Secretase) Inhibitors with Isophthalamide Derivatives as the P_2-P₃-Ligands

Abstract

Structure-based design and synthesis of a number of potent and selective memapsin 2 inhibitors are described. These inhibitors were designed based upon the X-ray structure of memapsin 2-bound inhibitor 3 that incorporates methylsulfonyl alanine as the P2-ligand and a substituted pyrazole as the P3-ligand. Of particular importance, we examined the ability of the substituted isophthalic acid amide derivative to mimic the key interactions in the S2-S3 regions of the enzyme active sites of 3-bound memapsin 2. We investigated various substituted phenylethyl, alpha-methylbenzyl, and oxazolylmethyl groups as the P3-ligands. A number of inhibitors exhibited very potent inhibitory activity against mempasin 2 and good selectivity against memapsin 1. Inhibitor 5d has shown low nanomolar enzyme inhibitory potency (Ki=1.1 nM) and very good cellular inhibitory activity (IC50=39 nM). Furthermore, in a preliminary study, inhibitor 5d has shown 30% reduction of Abeta40 production in transgenic mice after a single intraperitoneal administration (8 mg/kg). A protein-ligand X-ray crystal structure of 5d-bound memapsin 2 provided vital molecular insight that can serve as an important guide to further design of novel inhibitors.

Discovery of potent β-secretase (bace-1) inhibitors by the synthesis of isophthalamide-containing hybrids

“…Coburn provided a possible explanation for the unexpected phenomena [23] that the α-methyl of (4-fluorophenyl)ethyl of 3 at the 3-position packs firmly against Ile110 of BACE-1, which orients the 4-fluorophenyl ring toward S 3 and creates a novel S 3 subpocket (S 3 SP ). The subpocket is unique, different from other reported BACE-1 complexed structures, which may make (4-fluorophenyl) ethyl as a suitable substituent as R 3 , even for HE-based inhibitors [14,15] , although it is relatively large for the S 3 pocket. The special orientation of the molecules in the active pockets of BACE-1 is closely associated with the groups of the 5-position of N-terminal isophthalamide, which may result in N-methyl(methylsulfonyl)amino being more preferable than the nitro group at this site when (4-fluorophenyl)ethyl group is selected as R 3 .…”

“…Compounds 10 and 11, which contain the substituent, did show nanomolar activities. The unexpected results pushed us to carefully study the complex crystal structures of 3 and 4 [14,15,23,24] . Coburn provided a possible explanation for the unexpected phenomena [23] that the α-methyl of (4-fluorophenyl)ethyl of 3 at the 3-position packs firmly against Ile110 of BACE-1, which orients the 4-fluorophenyl ring toward S 3 and creates a novel S 3 subpocket (S 3 SP ).…”

“…Compound design The structures of the potent HEbased BACE-1 inhibitors, including compound 5 ( Figure 3) discovered by our group, share the same HE isostere and similar N-terminal isophthalamide scaffold with GRL-7234 [14,28] . These observations promoted us to explore whether potent inhibitors could also be obtained by introducing substituted isopthalamides, discovered by Merck's group and also used by Ghosh and co-workers, into 5.…”

“…Moreover, BACE-1 gene knockout mice showed no Aβ production and appeared healthy, which offered further powerful in vivo evidence for the opinion that BACE-1 was an attractive AD target and inhibition of the protease could effectively reduce Aβ formation, thereby halting the progression of AD [11,12] . Since first discovered in 1999, numerous BACE-1 inhibitors have been designed, synthesized and evaluated the biological activities, in which some molecules caused an in vivo decrease of amyloids when used in animal models (eg, 1, 2) [13][14][15] , especially compound GRL-7234 bearing the HE scaffold, which has been shown to induce a reduction of Aβ 40 production in transgenic mice after a single intraperitoneal administration ( Figure 1) [14] . However, the majority of these compounds are peptide-based analogs [16][17][18][19][20][21] that, if used as drug agents, will face formidable difficulties due to their vulnerability to degradative enzymes, rapid biliary clearance and poor oral absorption [22] .…”

“…Ghosh and co-workers have proven that compounds that incorporate substituted isopthalamides as P 2 -P 3 ligands in combination with the Leu-Ala HE dipeptide isostere can also form direct hydrogen bonds with the catalytic aspartates and display excellent potencies toward BACE-1; this group discovered the most potent compound, GRL-7234, described above [14] . However, this molecule has a large molecular weight and many amide bonds, which may cause problems in further studies.…”