As the catalytic apparatus of β-secretase is virtually the same as those in HIV protease and renin, it was assumed from the beginning that the principles of inhibitor design for other aspartic protease drugs may be employed for the development of β-secretase inhibitors. From the precedence of drug development for HIV protease and renin, it is likely that successful β-secretase inhibitor drugs will mimic the conformation of substrates at transition state. The resulting transition-state inhibitors typically exhibit high potency (Ghosh et al. 2008a). β-Secretase hydrolyzes APP and generates Aβ primarily within the endosomes of brain neurons. Therefore, a clinically effective β-secretase inhibitor must have the ability to penetrate the blood–brain barrier (BBB) and the neuronal membranes. The upper limit of molecular size that cross BBB is around 550 Da. In addition, such inhibitors should possess good drug-like absorption, distribution, metabolism and excretion (ADME) properties. For developing selectivity, potency of the inhibitors against β-secretase is often compared with that against two other human aspartic proteases: memapsin 1 (BACE-2), as it is the closest homolog of β-secretase, and against cathepsin D, the most abundant aspartic protease in human cells. Since the discovery of β-secretase, both academic and industrial laboratories have devoted much effort toward the development of drug-like β-secretase inhibitors. More than 400 publications and patents focusing on β-secretase inhibitors have now appeared in the last eight years (Ghosh 2010). Herein, we will attempt to summarize the major developments in this field.
Pseudopeptide β-secretase inhibitors
The first highly potent inhibitor, OM99-2 (Ghosh et al. 2000) was created based upon a β-secretase substrate where the scissile peptide bond was replaced by a Leu-Ala hydroxyethylene transition-state isostere. The X-ray crystal structures of β-secretase complexes of OM99-2 (Hong et al. 2000) provided detailed information on the extensive interactions in each of the eight subsites of the protease. Subsequently, replacement of subsite ligands led to a variety of potent β-secretase inhibitors (Ghosh et al., 2001). A statine-derived cell permeable β-secretase inhibitor 1 is shown in Fig. 1. The active diastereomer (IC50 = 0.12 μM) displayed β-secretase selective inhibition and was effective in inhibiting Aβ formation in transfected human embryonic kidney (HEK-293) cells (EC50 = 4.0 μM) (Hom et al. 2003).
Kimura et al. (2005) have reported inhibitor 2 (IC50 = 8.2 nM) containing a phenylnorstatine moiety as the transition-state isostere (TS-isostere). Subsequently, they reported phenylnorstatine-based compounds 3 and 4 as potent β-secretase inhibitors (3, IC50 = 4.8 nM; 4, IC50 = 1.2 nM), in which tetrazole rings were demonstrated to be an appropriate bioisosteric replacement for the carboxylic acids at both P4 and P1′ positions (Kimura et al. 2006). In an attempt to develop pharmaceutically useful compounds, the same group started an investigation of bioisosteres of the acidic tetrazole ring. Introduction of a 5-fluoroorotyl group at the P4 position and l-cyclohexylalanine residue at the P2 position resulted in inhibitor 5, which maintained optimal enzyme inhibitory activity (IC50 = 5.6 nM) while displaying 84%β-secretase inhibition in cultured cells at a concentration of 100 μM/L (Hamada et al. 2006).
Larhed and co-workers reported a series of new tert-alcohol containing β-secretase inhibitors utilizing α-phenylnorstatine or α-benzylnorstatine as the central core. The most potent inhibitor, 6 (IC50 = 0.19 μM), was co-crystallized with β-secretase. A novel binding mode for this class of inhibitors was identified, in which the N-terminal amine and not the tert-hydroxy group served as the TS-isostere (Wångsell et al. 2011). Larhed and co-workers, used a masked tert-hydroxy central core in combination with a substituted isophthalamide containing an inverted amide bond, to design potent inhibitor 7 (IC50 = 0.23 μM). This inhibitor was selective towards cathepsin D, but showed low cell permeability. The epimers at the quaternary center were equally potent suggesting that in this series of inhibitors the absolute stereochemistry of that carbon is of minor importance (Wångsell et al. 2009).
Maillard and co-workers reported a series of hydroxyethylamine (HEA)-based inhibitors combining the isophtalamide moiety with an HEA isostere bearing R stereochemistry at the TS hydroxyl and a 3,5-difluorophenyl fragment as the P1 aryl group. Compound 12 (Fig. 3, IC50 = 20 nM and EC50 = 15 nM) represents a potent and cell permeable peptidomimetic inhibitor of the human β-secretase (Maillard et al. 2007). Similarly, high enzymatic potency was observed for inhibitor 13 in which the C-5 position of the isophthalamide ring was functionalized with a polar primary amide to increase affinity for β-secretase and selectivity over cathepsin D (Kortum et al. 2007).
Poor metabolic stability due to microsomal N-debenzylation and N-depropylation (Freskos et al. 2007a) prompted the replacement of the isophthalate N-terminus by acyclic sulfones. The authors first identified the racemic Cbz-derivative 14 as a lead compound endowed with good enzymatic activity, but more potent against cathepsin D (IC50 = 67 nM) (Freskos et al. 2007). Subsequently, structure-based design resulted in the synthesis of derivative 15 with highly improved enzymatic inhibitory activity (IC50 = 2 nM) and cellular potency (1 nM). The X-ray crystal structure of 15-bound β-secretase highlighted a close association between the pyridyl nitrogen and Arg235 in the S2 site. The authors suggested that selectivity (cathepsin D, IC50 = 474 nM) could be due to the higher lipophilicity of the cathepsin D S2 pocket (Freskos et al. 2007a).
Inhibitor 16 (GSK188909) was described as an orally bioavailable β-secretase inhibitor capable of lowering brain Aβ in APP transgenic mice (Hussain et al. 2007). The studies which led to the discovery of this orally active hydroxyethylamino isostere-based inhibitor have been reported (Beswick et al. 2008; Clarke et al. 2008a,b). GSK188909 inhibited β-secretase activity with an IC50 of 4 nM, while showing good selectivity with respect to BACE-2, renin and cathepsin D. It caused a decrease in Aβ40 and Aβ42 production in cell based assays expressing both wild type and Swedish variant APP sequences (IC50 = 5 and 30 nM, respectively).
Subsequently, molecular modeling suggested that the key non-primed side interactions of 16 could be mimicked by a tricyclic indole derivative. It was reasoned that constraining the active conformation in this way would make binding to the protein much more efficient while also potentially reducing or eliminating the propensity for N-dealkylation of aniline moiety. They examined a variety of heteroaryl P2′ groups, representative compound 17 with a 4-pyranyl amine (IC50 = 20 nM, EC50 = 16 nM) showed improved permeability (b/p = 0.37) and clearance, resulting in an increased bioavailability in rats and dogs (Charrier et al. 2008). Ghosh et al. (2008b) reported a series of potent and selective inhibitors incorporating the HEA isostere. Representative compound 18 (GRL-8234) has exhibited excellent in vitro potency (Ki = 1.8 nM, Cell IC50 = 1 nM) and modest selectivity against cathepsin D (IC50 = 79 nM) and BACE-2 (IC50 = 138 nM). This compound inhibited Aβ production in mice (Ghosh et al. 2008b). An IP administration of 18 at a dose of 8 mg/kg to Tg 2576 mice resulted in 65% reduction of Aβ production in plasma after 3 h. Furthermore, GRL-8234 was shown to rescue age-related cognitive decline in APP transgenic mice (Chang 2011).
Iserloh et al. (2008a) investigated conformationally constrained versions of the HEA motif found in many aspartyl protease inhibitors. They developed 4-benzyloxypyrrolidine and 4-phenoxypyrrolidine containing inhibitors 19 and 20 shown in Fig. 4. These inhibitors exhibited good in vitro potency (5 and 3 nM, respectively) although with low cellular activity (150 and 165 nM, respectively) and modest selectivity against other human aspartyl proteases. In addition, both inhibitors exhibited insufficient pharmacokinetic properties in rats as evidenced by low plasma levels following oral dosing (Iserloh et al. 2008b). Subsequently, replacement of the N,N-dipropylamide present in 19 and 20 with a 2-(R)-methoxymethylpyrrolidine amide resulted in compound 21 with a marked improvement in cellular potency (Ki = 0.7 nM, cell IC50 = 21 nM), while maintaining good selectivity over related human aspartyl proteases such as cathepsin D, cathepsin E and renin (Iserloh et al. 2008a).
Based upon X-ray crystallography and molecular modeling, a series of novel, potent piperazinone and imidazolidinone-based peptidomimetic β-secretase inhibitors were developed. Piperazinones in particular are tolerant of a wide diversity of modifications to their non-prime side. Compound 22 (IC50 = 3 nM, cell IC50 = 300 nM), produced modest inhibition of peripheral Aβ40 in a transgenic mouse model with a single dose (Cumming et al. 2008).
The m-tolyl sulfonamide moiety, combined with the optimal methoxymethyl pyrrolidine isophthalamide group on the non-prime side, led to the extremely potent inhibitor 23 (Ki = 0.18 nM, cell IC50 = 7 nM). This inhibitor has resulted in a robust and persistent lowering of peripheral Aβ in a transgenic mouse model following a single subcutaneous dose. However, this compound, as with many peptidomimetics, is a substrate for Pgp, and this liability limits its oral bioavailability, brain penetration, and ultimately its central efficacy (Cumming et al. 2010).
Sealy et al. (2009) reported β-secretase inhibitor 24 (IC50 = 47 nM and cell ED50 = 17 nM). The X-ray crystal structure of β-secretase with 24 illustrated that difluoroaryl, cyclohexyl, and tert-butyl substituents occupy the S1, S1′, and S2′ pockets, respectively. Compound 24 did not possess good pharmacokinetic parameters. Further work led to highly permeable HEAs with very low levels of Pgp liability which translated into an orally efficacious inhibitor in the wildtype pre-clinical guinea pig animal model. Compound 25 has shown a cell IC50 of 26 nM but only a 230 nM value in the biochemical assay (Truong et al. 2010).
Rajapakse et al. (2006) described a series of tertiary carbinamine-derived inhibitors in which the primary amine is reported to interact with the catalytic Asp of β-secretase. Inhibitor 26 (Fig. 5) displayed high potency in enzymatic and cellular assays (IC50 = 12 and 65 nM, respectively) and good selectivity toward both renin and cathepsin D, while showing only moderate selectivity towards BACE-2 (IC50 = 620 nM). A series of interesting inhibitors based on a 2,6-diamino-isonicotinamide core coupled to a truncated reduced amino isostere as the aspartate binding element has been developed. Compound 27 (Stauffer et al. 2007), displayed a cellular IC50 of 49 nM and in vivo activity in transgenic mice expressing human wild-type APP. After i.v. administration of a 50 mg/kg dose of inhibitor 27, a maximal reduction of Aβ40 (34%) at 3 h from dosing was observed and the concentration of drug in the brain was 1.9 μM (Stanton et al. 2007). The combination of the isonicotinic core containing a P3 methylcyclopropyl group with the oxadiazolyl tertiary carbinamine resulted in compound 28. It turned out to be a very potent inhibitor with good functional activity (IC50 = 0.4 nM; sAPPβ_NF IC50 = 40 nM) along with minimal Pgp susceptibility suggesting good potential for brain penetration (BA/AB = 1.9, Papp=22 × 10−6 cm/s) (Nantermet et al. 2009). However, significant pharmacokinetic liabilities were associated with 28 as it exhibited poor oral bioavailability in multiple species. IP dosing in transgenic mice at high doses showed reduction of brain Aβ levels. The co-crystal structure of 28 with β-secretase revealed that the inhibitor occupied the S1–S3 sites. The benzyl group occupied the S1 pocket. Optimization of the P1 substituent was subsequently explored. Incorporation of a 4-fluoro substituent gave compound 29 a 2-fold improvement in both in vitro and cell-based assays as compared to 28. Unfortunately, the modest improvement of in vitro potency did not result in a superior pharmacodynamic response for compound 29 (Zhu et al. 2010).
The macrocyclization strategy is an established method to pre-organize and stabilize bioactive conformations. Highlighting the open nature of the S1–S3 subsites, many macrocyclic β-secretase inhibitors have appeared in the literature (Ghosh et al. 2005). The close spatial proximity between the P1 aryl group and the P3 methyl of carbinamine-based inhibitors suggested the possibility of increasing potency by stabilizing the bioactive conformation as well as the potential of improving the physicochemical liabilities of the acyclic series with the preparation of macrocyclicethers and macrolactones.
As shown in Fig. 6, macrolactone 30 exhibited good potency (IC50 = 2 nM; sAPPβ_NF IC50 = 5 nM), and was found to be not only hydrolytically stable at physiological pH but also stable in rat and human plasma and in microsomal preparations in the absence of NADPH (Lindsley et al. 2007). Inhibitor 31 incorporated an isophthalamide scaffold coupled to a reduced amide isostere. This compound presented an enzyme IC50 of 4 nM, a cellular IC50 of 76 nM and, most importantly, improved membrane permeability and reduced Pgp susceptibility. When i.v. administered in a mouse model at a dose of 100 mg/kg, it produced a decrease in Aβ40 levels of 25% in brain extracts (Stachel et al. 2004, 2006). A series of macrocyclic peptidic β-secretase inhibitors was recently designed by Lerchner and co-workers. Representative compound 32 has shown an IC50 of 9 nM. The introduction of the cyclopropyl into HEA-bearing β-secretase inhibitors led to a loss of selectivity over the closely related aspartyl proteases cathepsins D and E (Lerchner et al. 2010).
A low molecular weight acylguanidine inhibitor was discovered by using high-throughput screening (HTS) at Wyeth. Optimization of the hit using structure-based design led to compound 33 (Fig. 7, IC50 = 110 nM). The X-ray structure of β-secretase complexed with a closely related analogue of 33 revealed that the N-acylguanidine moiety forms hydrogen bonding interactions with the key catalytic aspartates while the substituents on the acylguanidine nitrogen extend into the S1′ pocket, forming hydrogen-bonding interactions with Arg235 and Thr329 via bridging water molecules. The p-propyloxyphenyl group extends from the S1 to the S3 pocket with minimal strain and the pyrrole ring forms a π-stacking interaction with the flap Tyr71. Moreover, the crystal structure revealed that the inhibitor stabilizes the enzyme in an open conformation. This is different to most peptidomimetic inhibitors which bind to β-secretase in a closed-flap form (Cole et al. 2006).
Preliminary structure–activity relationship (SAR) investigations as well as the bioisosteric replacement of the guanidyl functionality resulted in an only modest improvement of β-secretase inhibitory potency. However, poor selectivity over BACE-2 enzyme and poor permeability, as assessed in a Caco-2 drug transport model, remain the main drawbacks for this class of compounds (Cole et al. 2008; Jennings et al. 2008).
Aminoimidazole and aminohydantoin-based inhibitors
Aminoimidazole-based inhibitor 34 was derived from the addition of either a pyridine or a pyrimidine ring on a previously identified lead at Wyeth. The extension towards the S3 region of the β-secretase binding pocket led to an IC50 value for β-secretase of 20 nM. Furthermore, 34 showed a cellular activity of 90 nM, and more than 100-fold selectivity over the other structurally related aspartyl proteases BACE-2, cathepsin D, renin, and pepsin. Acute oral administration of the R isomer of 34 at 30 mg/kg resulted in a significant 71% reduction of plasma Aβ40 measured at the 6 h time point in a Tg2576 mouse model (p < 0.001) (Malamas et al. 2009). An optimized derivative 35 (S was the active enantiomer), displayed an IC50 value for β-secretase of 10 nM, cellular EC50 activity of 20 nM, and more than 80-fold selectivity over the other tested aspartyl proteases. Acute oral administration of 35 at 100 mg/kg resulted in a 69% reduction of plasma Aβ40 at 8 h in a Tg2576 mouse (p < 0.001) (Malamas et al. 2010).
Miscellaneous non-peptidic scaffolds
Using virtual screening, Moka and co-workers discovered a novel non-peptidic inhibitor (Fig. 9) of β-secretase based on an isatin motif (compound 39, IC50 = 2.4 μM). The relatively poor solubility of the present series of compounds coupled with the presence of the nitro and the phenolic functionalities are the main drawbacks for this series of compounds (Moka et al. 2009). Virtual screening in combination with bioassay also resulted in the identification of multiple novel non-peptide inhibitors. The most potent molecule, compound 40 (IC50 = 2.8 μM) has a benzothiazole ring which docks into the S1 pocket of the enzyme and spans the interaction through almost all the subsites of β-secretase (Xu et al. 2010b).
An in silico virtual screening of the commercial database SPECS chemical library led to the identification of compound 41 as a sub-micromolar inhibitor (IC50 = 0.53 μM) with low cytotoxicity (Xu et al. 2010a). Starting from peptidomimetic β-secretase inhibitors, Hanessian and co-workers developed a series of inhibitors where the P2 amino acid, including the P2/P3 peptide bond was replaced with a rigid 3-aminomethyl cyclohexane carboxylic acid. Compound 42 was the most active compound of the series (IC50 = 2.5 nM), with a 50-fold selectivity over BACE-2 and cathepsin D, and good cellular activity. Co-crystallization revealed an unexpected binding mode with the P3/P4 amide bond placed into the S3 pocket resulting in a new hydrogen bond interaction pattern. Unfortunately, testing of compound 42 in an in vitro model (cells stably transfected with the gene for the human Pgp transporter), resulted in a high efflux ratio (BA/AB = 97). Therefore, the modification of the P2/P3 amide region did not overcome this challenging problem (Hanessian et al. 2010).
Recently, Sasaki et al. found that amentoflavone-type biflavonoids have significant neuroprotective effects and β-secretase inhibitory activity. Among these compounds, 2,3-dihydroamentoflavone 43 and 2,3-dihydro-6-methylginkgetin 44 exhibited potent inhibitory effects with IC50 values of 0.75 and 0.35 μM, respectively (Sasaki et al. 2010). Macchia and co-workers, using a HTS fluorescence assay, identified compound 45 (IC50 = 0.50 μM) as a promising new lead (Asso et al. 2008).
A multi-target-directed ligand (MTDL), the goal of which is to enhance efficacy and improve safety, is rationally designed to hit multiple targets for a particular disease. The complexity and multiple etiologies of AD render the MTDL approach a potentially effective strategy for AD treatment. In the last few years, many MTDLs have been reported with improved pharmacological profiles, such as β-secretase inhibitors bearing acetylcholinesterase (AChE) inhibitory activity or metal chelating properties.
Melchiorre and co-workers (Cavalli et al. 2007) designed compound 46, incorporating a 1,4-benzoquinone functionality as a radical scavenger into the polyamine skeleton series of cholinergic derivatives. The biological profile of 46 (Fig. 10) was then explored in detail by means of both in vitro and in vivo assays to assess its therapeutic potential for combating AD. Compound 46 was found to be a potent inhibitor of the activity of AChE (IC50 = 1.55 nM) and it also inhibited β-secretase activity in a concentration-dependent manner (IC50 = 108 nM). Moreover, when tested at an equimolar concentration (50 μM) with Aβ42, 46 was able to inhibit fibril formation (95.5 ± 0.4%) and it also showed activity against the formation of reactive oxygen species.
Recently, a series of dual inhibitors of AChE and β-secretase were designed based on the MTDL strategy. Among them, inhibitor 47 exhibited modest dual potency in an enzyme inhibitory potency assay (β-secretase: IC50 = 0.567 μM; AChE: IC50 = 1.83 μM), and also showed good inhibitory effects on Aβ production of APP transfected HEK-293 cells (IC50 = 98.7 nM) (Zhu et al. 2009). Considering the crucial roles of β-secretase and metal ions in AD pathology, Huang and colleagues designed a series of novel 1,3-diphenylurea derivatives by hybridizing the metal chelator LR-90 with a β-secretase inhibitor. Compound 48 (IC50 = 27.85 μM) was the most effective inhibitor. All compounds in this series showed the ability to chelate metal ions (Huang et al. 2010).