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. 2012 Jul 12;3(11):897–902. doi: 10.1021/ml3001165

Discovery of an Orally Available, Brain Penetrant BACE1 Inhibitor That Affords Robust CNS Aβ Reduction

Andrew W Stamford †,*, Jack D Scott †, Sarah W Li †, Suresh Babu ‡, Dawit Tadesse ‡, Rachael Hunter ‡, Yusheng Wu †, Jeffrey Misiaszek †, Jared N Cumming †, Eric J Gilbert †, Chunli Huang †, Brian A McKittrick †, Liwu Hong †, Tao Guo ‡, Zhaoning Zhu †, Corey Strickland #, Peter Orth #, Johannes H Voigt #, Matthew E Kennedy §, Xia Chen §, Reshma Kuvelkar §, Robert Hodgson §, Lynn A Hyde §, Kathleen Cox ±, Leonard Favreau ±, Eric M Parker §, William J Greenlee †
PMCID: PMC3568987  NIHMSID: NIHMS396965  PMID: 23412139

Abstract

graphic file with name ml-2012-001165_0009.jpg

Inhibition of BACE1 to prevent brain Aβ peptide formation is a potential disease-modifying approach to the treatment of Alzheimer’s disease. Despite over a decade of drug discovery efforts, the identification of brain-penetrant BACE1 inhibitors that substantially lower CNS Aβ levels following systemic administration remains challenging. In this report we describe structure-based optimization of a series of brain-penetrant BACE1 inhibitors derived from an iminopyrimidinone scaffold. Application of structure-based design in tandem with control of physicochemical properties culminated in the discovery of compound 16, which potently reduced cortex and CSF Aβ40 levels when administered orally to rats.

Keywords: BACE1, inhibitor, Alzheimer’s disease, Aβ40, iminopyrimidinone, X-ray crystallography

Alzheimer’s disease (AD) is a chronic neurodegenerative disease and is now the sixth leading cause of death in the Unites States.1 Current treatments of AD are palliative and only transiently effective, and they do not impact disease progression.2 Due to the progressive and debilitating nature of the disease and the lack of disease-modifying therapeutic agents, novel treatments that slow or halt disease progression are urgently sought.

Neuropathological hallmarks of AD are the accumulation of β-amyloid as extracellular neuritic plaques and the appearance of intracellular neurofibrillary tangles comprised of hyperphosphorylated tau protein.3 Although the cause of AD remains poorly understood, several lines of evidence suggest that accumulation of β-amyloid is an underlying cause of neuronal cell death.3−5 The amyloid peptides are produced from amyloid precursor protein (APP) in two proteolytic steps, the first of which is mediated by the aspartyl protease β-site APP cleaving enzyme-1 (BACE1)6 to produce a C-terminal fragment that is subsequently processed by γ-secretase to yield Aβ peptides of 36–43 amino acids. While Aβ40 is the most abundant species formed, Aβ42 is more prone to aggregation and formation of oligomeric neurotoxic species.7−9 BACE1 inhibition to prevent Aβ peptide formation is considered to be a highly attractive amyloid-lowering approach to a potentially disease-modifying AD treatment. In this regard, BACE1 knockout mice do not produce β-amyloid, demonstrating that BACE1 is necessary for Aβ production in vivo.10−12 Furthermore, BACE1 knockout mice do not exhibit overt abnormalities, displaying a moderate peripheral nerve hypomyelination caused by loss of BACE1-mediated processing of Neuregulin-1.13,14

For more than a decade, there have been intense efforts to identify potent, selective BACE1 inhibitors that effectively inhibit Aβ peptide production in the CNS of preclinical animal models. Although substantial work has been carried out on peptidomimetic inhibitors, this has met with limited success, as such inhibitors tend to be Pgp substrates and suffer restricted brain penetration.15 Nonpeptidic BACE1 inhibitors have been reported by us16−18 and others,19−23 and they offer the most promising avenue to the discovery of orally bioavailable, brain penetrant inhibitors. A recent report of a nonpeptidic BACE1 inhibitor that produced substantial reductions in central Aβ levels in preclinical species and in healthy volunteers confirms the validity of this approach.24

We had previously reported the discovery of the potent iminohydantoin BACE1 inhibitor 1 that was rationally designed from fragment-based screening hits.16,17 In the course of lead optimization, the identification of 1 was an important milestone en route to the discovery of the highly ligand efficient iminohydantoin BACE1 inhibitor 2 (Chart 1).18 Upon oral administration to rats at the dose of 30 mg/kg, 2 acutely lowered CSF Aβ40 by approximately 50% 3 h postdose relative to vehicle-treated controls. While the ability of 2 to elicit CSF Aβ reduction represented a significant advance in our efforts to develop centrally active BACE1 inhibitors, 2 had a significant IC50 shift in a cell-based Aβ40 assay relative to its BACE1 Ki and limited brain penetration. Consequently, in order to develop inhibitors with improved in vivo potency, concurrent optimization efforts focused on the identification of inhibitors with improved enzymatic/cellular potency shifts and brain exposures. Toward this goal, we report herein the design and optimization of a series of BACE1 inhibitors based on the homologous iminopyrimidinone scaffold 3 that also evolved from the lead iminohydantoin 1 (Chart 1).

Chart 1. Iminoheterocycle BACE1 Inhibitor Design.

Chart 1

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An X-ray cocrystal structure of 1 bound to BACE1 revealed that the guanidine functionality of 1 forms intricate H-bond donor–acceptor interactions with Asp32 and Asp228 at the BACE1 active-site, the methoxybiphenyl moiety occupies the contiguous S1 and S3 pockets, and the remaining phenyl substituent occupies space in the vicinity of S2′ (Figure 1). In order to maximize the probability of identifying inhibitors with high CNS exposure, our optimization strategy was to adhere to the design principles of maintaining or improving LE while limiting MW, restricting cLogP in a desirable range (≤4), and avoiding additional H-bond donors to minimize Pgp activity and polar surface area.25 In accordance with this approach, we pursued focused modifications of the iminoheterocyclic core and of the biaryl motif that occupies the S1 and S3 pockets of the enzyme.

Figure 1.

Figure 1

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X-ray cocrystal structure of BACE1 and 1 (blue) superimposed with docked 4 (yellow).

Ring expansion of the core to a six-membered iminopyrimidinone 3 was expected to have a significant impact on physicochemical properties and conformation. In comparison to the iminohydantoin core, the iminopyrimidinone is predicted to be more basic.26 We reasoned that enhanced basicity could increase cell potency by favoring partitioning to the acidic intracellular compartment where BACE1-mediated APP processing is operative. A second major design consideration centered on conformational differences between the five- and six-membered ring iminoheterocycle scaffolds. The iminopyrimidinone ring differs from the planar iminohydantoin core in that the additional sp3 center imparts a significant pucker to the ring. Consequently, the docked pose of the iminopyrimidinone 4 (Chart 1) overlaid with iminohydantoin 1 places the S1 phenyl substituent and the amidine in a similar location; however, the 6-methyl substituent has a trajectory that diverges from that of its iminohydantoin counterpart, projecting toward Ile118 (Figure 1).27 Based on this analysis, it was hypothesized that substituents at C6 smaller than phenyl projecting toward the prime-site might be necessary in order to avoid unfavorable steric interactions with the enzyme.

To test these hypotheses, targeted sets of iminopyrimidinones 4–6 and 7–9 were prepared that differed in their substitution at C6 of the iminopyrimidinone ring (Table 1). As predicted by docking experiments, the 6-methyl iminopyrimidinones 4–6 had BACE1 affinities that were substantially higher than those of their 6-phenyl counterparts 7–9 and were comparable to that of the iminohydantoin 1. Also noteworthy was the observation that the cell Aβ40 IC50 values of the iminopyrimidinones 4–6 were minimally shifted (2–3-fold) relative to their Ki values, in contrast to the cases of the corresponding 5,5-diphenyl iminohydantoins represented by 1.18 Comparison of iminopyrimidinones 4–6 in a rat oral pharmacokinetic screen demonstrated that cyanophenyl derivative 5 had superior rat oral pharmacokinetic properties. Compound 5 adminstered at a dose of 10 mg/kg afforded a plasma AUC0–6 h of 8.6 μM·h compared to corresponding values of <1 μM·h for 4 and 6 administered at the same dose.28

Table 1. BACE1 Affinity, Cell Activity, and Selectivity of Iminopyrimidinone Derivatives 3.

graphic file with name ml-2012-001165_0005.jpg

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a

BACE1 Ki and cell IC50 values are the average of a minimum of two independent determinations.

b

Selectivity ratios were derived from cathepsin D Ki values (average of a minimum of two independent determinations).

c

Not determined.

An X-ray cocrystal structure of the 6-methyl iminopyrimidinone derivative 4 and BACE1 was in close agreement with the predicted binding mode (Figure 2).29 Notably, the bound conformation of the iminopyrimidinone ring projects the C6 methyl group to within 3.6 Å of Ile118, consistent with a favorable hydrophobic interaction. As expected, the biaryl motif of 4 binds in the contiguous S1–S3 pockets, with the methoxyl group projecting toward the S3 subpocket. Well-defined density for the flap was observed, with the side-chain of Tyr71 positioned <4 Å away from the methylene of the iminopyrimidinone core.

Figure 2.

Figure 2

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Comparison of 4 (yellow) docked to BACE1 and the X-ray cocrystal structure of 4 (green).

Given the encouraging results obtained with the 6-methyl iminopyrimidinones 4–6, we next explored a wide range of biaryl and heterobiaryl motifs that could bind in S1–S3. The most productive of these was replacement of the proximal phenyl ring with the bioisosteric thiophenyl group. In exploring this modification, distal (3-cyano)phenyl substitution was chosen because of the superior pharmacokinetic properties conferred by this substituent. Of the three possible thiophenyl regioisomers 10–12 that maintain the iminopyrimidinone core and 3-cyanophenyl substituent in a 1,3-relationship, we were gratified to find that the affinities and LEs of the 2,4-thiophenyl and 2,5-thiophenyl regioisomers 10 and 11 were significantly improved compared to those of the corresponding biphenyl derivative 5 (Table 1). This improvement in affinity also translated to improved cell potency, with the 2,4 regioisomer 10 (Ki 57 nM; cell IC50 68 nM) exhibiting Ki and cell IC50 values of <100 nM.

Replacement of the 3-cyanophenyl moiety by the 3-prop-1-ynyl-5-pyridyl group as in 2, analogs 13–15, resulted in further improved BACE1 affinity and cell activity, with 13 (Ki 7.8 nM; cell IC50 13 nM) achieving single-digit nanomolar affinity and a cell potency shift of only 2-fold. A further gain of affinity could be realized by incorporation of chloro substitution at the thiophene 4-position of 14 to afford 16, which had a Ki value of 1.7 nM. Similar to the case of 16, chloro substitution at the thiophene 5-position of 15 gave 17 with enhanced affinity. The increased lipophilicity conferred by chloro substitution resulted in only modestly increased cell potency shifts, with 16 maintaining good cell activity (IC50 11 nM).

The selectivities of the iminopyrimidinone inhibitors for BACE1 versus the closely related aspartyl protease cathepsin D ranged from 61- to 130-fold for the biphenyl derivatives 4–6 and the 2,4-thiophenyl analogs 10 and 13, and they were generally somewhat decreased for the 2,5- and 3,5-thiophenyl analogs (e.g., 11, 14–17). In addition to selectivity over cathepsin D (21-fold; Table 1), 16 was profiled against other human aspartyl proteases. Compound 16 is not selective for BACE1 over the close homologue BACE2 (Ki < 1 nM) but exhibits good selectivity over cathepsin E (350×) and pepsin (260×) and excellent selectivity over renin (>10,000×).

An X-ray cocrystal structure showed that 16 bound to BACE1 in a mode highly consistent with 4, but it was notable in two respects that may contribute to its higher affinity (Figure 3). First, the propynyl group of 16 binds deep in the S3 subpocket where it is positioned to engage in a hydrophobic interaction with Ala335, as previously reported for 2.18 Second, in comparison to the cocrystal structure of 4, the flap is in a more closed conformation, forming a pocket which accommodates the chloro substituent while positioning the side-chain of flap Tyr71 in close proximity to the methylene of the iminopyrimidinone core.

Figure 3.

Figure 3

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X-ray cocrystal structure of 16 (blue) and BACE1.

In Caco-2 cells, 16 exhibited good permeability (Papp 153 nm·s–1) and a low efflux ratio (AP/BL 2.4), suggesting that the compound does not possess an overt human Pgp liability. In rat and human hepatocytes, 16 had low intrinsic clearance (4.9 and 2.7 μL·min–1·106 cells, respectively), reflecting low metabolic turnover, and displayed significant free fractions of 0.02 and 0.04, respectively, in rat and human plasma protein binding assays. These in vitro parameters translated to favorable rat pharmacokinetic properties, with 16 displaying high oral bioavailability (69%) and plasma exposure, a plasma half-life of 2.8 h, and an average brain/plasma ratio of 3.

Based on its excellent in vitro and rat pharmocokinetic profiles, 16 was subjected to pharmacodynamic profiling in rats. In a dose titration study, 16 elicited a robust reduction in CSF and cortex Aβ40 levels with ED50 values of 4 and 6 mg/kg in the respective compartments 3 h after oral administration (Figure 4). As shown in Figure 4, plasma and brain exposures increased in a relatively dose-proportional manner across the dose range of 0.3 to 30 mg/kg.

Figure 4.

Figure 4

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Dose response of 16 on CSF (□; collected from the cisterna magna) and cortex (●; Aβ40 3 h postdose in Sprague–Dawley rats (PO, formulated in 20% hydroxypropyl-β-cyclodextrin, 5 mL/kg). Doses ≥3 mg/kg were significantly different from vehicle, p < 0.02, Dunnett’s posthoc test. Dose dependent increase in plasma concentration (▲) of 16; brain/plasma ratios ranged from 2.4 to 4.2.

Optimization of 1 to inhibitor 16 achieved a significant gain in BACE1 affinity without concomitant increase in number of H-bond donors, cLogP, polar surface area, or MW, as reflected by the large increase in LE from 0.35 kcal·mol–1 to 0.48 kcal·mol–1. In addition to good solubility (200 μM, pH 7.4), the measured physical properties of 16, including a pKa value of 7.5 and a log D value of 1.8, fall within CNS druglike ranges.30

The synthesis of 16 (Scheme 1) employed an Ellman asymmetric aldol condensation to assemble the β-amino acid subunit and set the stereochemistry of the quaternary center.31 Thus, addition of the enolate of methyl acetate to (R)-tert-butylsulfinamide 19, derived from 1-(3-chlorothiophen-2-yl)ethanone 18, in the presence of chlorotitanium triisopropoxide afforded the (S)-β-aminoester derivative 20 in a >99:1 diastereomeric ratio. Acidic cleavage of the sulfinamide of 20 and coupling of the derived amine to tert-butyl N-[(methylamino)-thioxomethyl]carbamate proceeded with concomitant intramolecular cyclization to give the iminopyrimidinone derivative 21. Bromination of the thiophene afforded 22, which was subjected to sequential Suzuki coupling with (5-(prop-1-ynyl)pyridin-3-yl)boronic acid and Boc deprotection to give 16.

Scheme 1. Synthesis of Compound 16.

Scheme 1

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Reagents and conditions: (i) (R)-2-methylpropane-2-sulfinamide, Ti(OEt)4, THF, reflux; (ii) LDA, methyl acetate, ClTi(OiPr)3, THF, −78 °C; (iii) HCl, 1,4-dioxane; (iv) EDCI, N-[(methylamino)thioxomethyl]-tert-butylcarbamate, iPr2NEt, THF; (v) NBS, DMF, 50 °C; (vi) (5-(prop-1-ynyl)pyridin-3-yl)boronic acid, 2 M Na2CO3, PdCl2(dppf)·CH2Cl2, 1,4-dioxane, 65 °C; (vii) CF3COOH, CH2Cl2.

In conclusion, application of structure-based design with simultaneous control of physicochemical parameters enabled discovery of the iminopyrimidinone BACE1 inhibitor 16 starting from prototype iminohydantoin 1. The robust reduction of CNS Aβ levels that was achieved following oral administration of 16 to rats demonstrates the potential of the iminoheterocycle class of BACE1 inhibitors to deliver agents that could test the therapeutic utility of BACE1 inhibition in clinical trials.

Acknowledgments

We thank Thomas Novak and Charles Ross for HRMS spectra, and James Cuff for optical rotation measurements. This work is based upon research conducted at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation and the National Institutes of Health/National Institute of General Medical Sciences under NSF award DMR-0936384, using the Macromolecular Diffraction at CHESS (MacCHESS) facility, which is supported by award GM103485 from the National Institutes of Health, through its National Institute of General Medical Sciences.

Glossary

Abbreviations

AD

Alzheimer’s disease

APP

amyloid precursor protein

BACE1

β-site APP cleaving enzyme-1

CNS

central nervous system

CSF

cerebrospinal fluid

Pgp

P-glycoprotein

LE

ligand efficiency

CatD

cathepsin D

Supporting Information Available

Synthetic methods and characterization data for compounds 1–17, and methods for in vitro, in vivo, and pharmacokinetic assays. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Present Address

△ Merck Research Laboratories, 126 E. Lincoln Ave., Rahway, NJ 07065, United States.

The authors declare no competing financial interest.

Funding Statement

National Institutes of Health, United States

Supplementary Material

ml3001165_si_001.pdf (304.8KB, pdf)

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Associated Data

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Supplementary Materials

ml3001165_si_001.pdf (304.8KB, pdf)

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