Boom in the development of non-peptidic β-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease

Boom in the Development ofNon-Peptidic b-Secretase (BACE1)Inhibitors for theTreatment of

Alzheimer’s Disease

Romano Silvestri

Istituto Pasteur, Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco,

Sapienza Universita di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italy

Published online 23 July 2008 in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/med.20132

!

Abstract: b-Amyloid cleaving enzyme-1 (BACE1) has become a significant target for the therapy

of Alzheimer’s disease. After the discovery of the first non-peptidomimetic b-secretase inhibitorsby Takeda Chemicals in 2001, several research teams focused on SAR development of these

agents. The non-peptidic BACE1 inhibitors may potentially overcome the classical problems

associated with the peptide structure of first generation, such as blood–brain barrier crossing,

poor oral bioavailability and susceptibility to P-glycoprotein transport. In the past 6 years a

boom in research of non-peptidic BACE1 inhibitors has disseminated findings over hundreds

of publications and patents. The rapidly growing literature has been reviewed with particular

emphasis on literature of pharmaceutical companies. � 2008 Wiley Periodicals, Inc. Med Res Rev, 29,

No. 2, 295–338, 2009

Key words: Alzheimer’s disease; b-secretase; BACE1; non-peptidic inhibitors

1 . I N T R O D U C T I O N

Alzheimer’s disease (AD) is a degenerative brain syndrome first described by Alois Alzheimer in

1906, that affectsmore than 37million peopleworldwide.1 Social cognition is an early impairment of

AD. Dementia of the Alzheimer type leads to a progressive decline in memory with concomitant

abatement of thinking, comprehension, and learning capabilities.2 AD accounts for most cases of

dementia that are diagnosed after the age of 60.3,4

Two main theories are invoked for AD treatment. The ‘‘cholinergic hypothesis’’ relates the

neurodegeneration with the loss of cholinergic neurotransmission; the ‘‘amyloid hypothesis’’

Correspondence to:RomanoSilvestri,DipartimentodiChimicaeTecnologiedel Farmaco,SapienzaUniversita' di Roma,Piazzale

AldoMoro 5, I-00185 Roma, Italy.E-mail: [email protected]

Medicinal Research Reviews, Vol. 29, No. 2, 295^338, 2009

� 2008 Wiley Periodicals, Inc.

correlates the formation of amyloid b (Ab) peptides with the progression of the disease. According tothe ‘‘cholinergic hypothesis,’’ the deterioration of cholinergic neurons in the basal forebrain and the

associated loss of cholinergic neurotransmission in the cerebral cortex and other areas, significantly

contribute to the neurodegeneration in AD.5 On the other hand, the involvement of the amyloid

cascade in the progression of AD is supported by many evidences. The b-amyloid precursor protein

(APP) is a multifunctional protein associated with the viability, growth and functions of the central

neuronal cells. According to the ‘‘amyloid hypothesis,’’ disruption of APP would be correlated to

the emergence of AD.6,7 a-Secretase enzyme usually cleaves APP to form sAPPa and CTF83

membrane-bound fragment; then g-secretase enzyme cleaves CTF83 into peptide p3 and CTFg. ThesAPPa fragment activates NF-kB which is correlated to a neuroprotective effect (Fig. 1). In AD

pathological condition, APP is first cleaved by b-secretase to form sAPPb and membrane-bound

Figure 1. The cholinergichypothesis andamyloidhypothesis in AD.

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Medicinal Research Reviews DOI 10.1002/med

CTF99 fragments. The CTF99 fragment is then cleaved by g-secretase to form soluble Ab peptides.

In hydrophilic medium the hydrophobic Ab peptides spontaneously polymerize into neurotoxic

oligomers.8 Ab40 is the major secreted fragment, but the longer Ab42 aggregates more readily to

toxic oligomers and is increased by Familial ADmutations suggesting that it is the pathological form;

some oligomeric strands are involved in the hyperphosphorylation of tau protein.9,10 The effect of

AChon tau phosphorylation at specific siteswas recognized.11Mutant types at positions 31–23 ofAbfragments are associated with the higher incidence in developingAD.12 Interference of the oligomers

with neurotransmission at the synapses results in cell death and memory loss.13–17

Aggregation of Ab peptides leads to neuritic plaques made of a central oligomeric core and

surrounded by dystrophic neurities, activated microglia and reactive astrocytes.18 The poorly soluble

plaques would trigger the neurodegenerative cascade responsible of AD.5,19

A. AD Therapies

In the past few years many research laboratories evaluated potential drugs for AD. However,

currently effective drugs for the treatment of AD are not available.2 Historically, acetylcholinesterase

inhibitors (AChEIs) were the first group of drugs marketed for AD treatment. AChEIs increase both

concentration and duration of action of acetylcholine (ACh) in the synaptic cleft. Consequently, the

cholinergic receptors decreased inAD are stimulated, and the growing ofAb aggregates is reduced.20

The AChEIs tacrine (First Horizon Pharmaceuticals*), donepezil (Eisai/Pfizer), rivastigmine

(Novartis), and galantamine (Johnson& Johnson) are on themarket for the symptomatic treatment of

AD,21 and their patent expiration is upcoming.1 New generation (also dual binding site) AChEIs

(phenserine, cymserine, and also AP2238 and tolserine, and the APP metabolism modulators

TV3326 and its enantiomer TV3279) and ACh release enhancers (coluracetam) are currently under

investigation.6,22–25 There is a correlation between ‘‘cholinergic’’ and ‘‘amyloid hypothesis.’’ Rapid

Ab deposition and consequent development of plaques occur in the presence of AChE (AChE acts as

mediator of plaque formation via peripheral anionic side (PAS)/Ab interaction).26,27 An Ab AChE-

cycle would favor Ab deposition in AD through expression of AChE.28 Accordingly, AchE and Abwould be co-localized in neuritic plaques where they boost transformation of Ab into fibrils. Both

cholinergic agonists and AChEIs can stimulate the non-amyloidogenic pathway and reduce the

biosynthesis of amyloidogenic compounds in the brain.21,29,30

2 . A D I N T E R V E N T I O N S T R A T E G I E S

Ab peptides and senile plaques derived from oligomers, as well as neurofibrillary tangles composed

of tau protein,31 are involved in AD pathogenesis.32 The b-amyloid production/deposit may be

potentially lowered using copper-zinc chelating agents (clioquinol),33 Ab-aggregation blockers,

disassembling amyloid fibrils agents (melatonin, AZD-103)34,35 and monoclonal antibody favor the

peripheral clearance of Ab from the brain.36 Tramiprosate is glycosaminoglycan mimetic that binds

to Ab peptides, thereby stopping the amyloid plaque growth.37

The characteristic hallmarks of AD include neuritic plaques and neurofibrillary tangles (NFTs).

NFTs are composed of tau protein in an abnormally hyperphosphorylated form. The pathological

hyperphosphorylation of tau protein is an early event in AD-related neurofibrillary changes.38 The

inhibition of tau-related neurofibrillary degeneration is a novel promising approach in AD therapy.

The tau protein inhibition may be achieved by (i) targeting one or more tau kinase(s) (GSK3-b,CDK5, ERK2, MARK, PKA, SAPK, calmodulin, casein kinase inhibitors), (ii) increasing the

activity of protein phosphatase 2A (PP2A), (iii) inhibiting the presumed toxic properties of

pathological tau proteins (huntingtin).33

*Fordetailed informationoncompaniesmentioned in this article, see the Appendix.

DEVELOPMENT OF NON-PEPTIDIC b-Secretase * 297


Protein kinase C may induce a-secretase activation in the non-amyloidogenic pathway.39

MuscarinicM1-agonists (talsaclidine, NGX267) andM3-antagonists can activate protein kinaseC.40

Interestingly, M1-agonists can enhance the ACh concentration at synaptic level, thereby reinforcing

the cholinergic system. Ab plaques were reduced using the enzyme adamalysin-10 which stimulates

the expression of a-secretase.41 3-Hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) inhibitors

(lovastatine, simvastatine) proved to activate adamalysin-10.42–44

The g-secretase enzyme is a transmembrane complex made mainly by presenilin-1.45

g-Secretase inhibitors are generally hydrophobic non-transition-state analogues. Inhibitors of

g-secretase should have little effect on notch activity.46,47 Bristol-Myers Squibb Co., Merck and

Schering applied for sulfone and sulfone-related g-secretase inhibitors, Eli Lilly, DuPont and Merck

for benzodiazepines or benzocaprolactams.44,48–50 Currently three g-secretase inhibitors are

ongoing Phase II (LY450139, Eli Lilly; MK 0752, Merck) or Phase I (E2012, Eisai) clinical trials.1

The development of the amyloid vaccine AN-1792 (Elan/Wyeth) was discontinued. A newer

vaccine (ACC-001, Elan/Wyeth) with an improved safety profile is now in a Phase I trial. Three anti-

Ab monoclonal antibodies (mAbs) against various domains of Ab are currently ongoing Phase II

(bapineuzumab as symptom-management drug, Elan/Wyeth; LY2062430, Eli Lilly) or Phase I

(RN1219, Pfizer) clinical trials. Neuronal nicotinic ACh receptor agonists in Phase II trials are

AZD3480 (AstraZeneca/Targacept), MEM 3454 (Roche/Memory Pharmaceuticals), and GTS-21

(CoMentis) symptom-management drugs.1

A. bb-Secretase Inhibitors

b-Amyloid cleaving enzyme-1 (BACE1, Asp2 or memapsin) and BACE2 (Asp1) are the two

isoforms of the amyloid b-secretase identified so far. BACE1 belongs to the aspartyl protease family

and is the major isoform present in the brain; BACE2 plays a marginal role in AD.51 The early

discovery of BACE1 inhibitors of the Swedish mutant APP which significantly reduced Ab40 and

Ab42 levels, triggered the search for new BACE1 inhibitors for AD treatment.52 Several academic

(i.e., Ghosh and Tang, Shuto, Kimura, Fujii, Hu, and Hanessian) and industrial (i.e., Elan, Pfizer, Eli

Lilly, Merck, and Bachem) teams synthesized new peptidomimetic b-secretase inhibitors. This

strategy was based on the design of truncated polypetides bearing non-cleavable transition state

mimicking groups. Elan Pharmaceuticals discovered one of the early potent BACE1 inhibitors by

replacing the P1 leucine with a statin moiety.53 Ghosh and Tang used X-ray crystallographic data of

OM99-2 in complex with recombinant enzyme54 to develop a peptidomimetic b-secretase inhibitorbinding pocket that served as basis to design more potent BACE1 inhibitors (OM00-3, GT-1017).55

To overcome the problem of BACE1/BACE2 selectivity56 this team designed potent cyclic

derivatives based on the crystallographic data of OM99-2.57 Shuto and co-workers synthesized

hydroxymethylcarbonyl and phenylnorstatin transition state analogues and Manzenrieder et al.

incorporated a phosphino dipeptide isostere, all strategies leading to remarkable BACE1 inhibitory

activity.58–60 The not-isomerizable tetrazolyl derivative KMI-429 showed improved stability and

potent enzyme inhibitory activity.61 KMI-429 reduced the level of soluble and insoluble Ab40, andsoluble and insoluble Ab42, in both APP transgenic and wild-type (WT) mice (2 mg injected into thehippocampus of a WT mouse led to 30–40% reduction of Ab levels).62 Kimura and co-workers63

obtained potent BACE1 inhibitors (i.e., KMI-570 and KMI-684) by bioisosteric replacement of the

carboxylic group. Following the statin approach, Pfizer,64 Eli Lilly,65 and Elan Pharmaceuticals66

discovered highly potent BACE1 inhibitors. Merck and Bachem improved BACE1 selectivity by

designing a derivative capable to interact with the Arg235 residue.67 The development of BACE1

inhibitors was summarized by recent excellent reviews.68–72

Inhibitors based on the peptidomimetic strategy suffer from well-known difficulties

associated with polypeptides, such as blood–brain barrier crossing, poor oral bioavailability and

susceptibility to P-glycoprotein transport.73 Efforts to overcome these problems led to design new

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non-peptidomimetic b-secretase inhibitors. The first non-peptidomimetic inhibitors were discovered

by Takeda Chemicals in 2001.74,75 In the subsequent 5 years, a boom in research work on non-

peptidomimetic BACE1 inhibitors has disseminated findings over hundreds of publications and

patents. We here attempted to review this rapidly growing literature.

3 . D E V E L O P M E N T O F N O N - P E P T I D I C bb- S E C R E T A S E I N H I B I T O R S

In 2001, Takeda Chem. Ind. reported the first non-peptidomimetic BACE1 inhibitors.74,76 Derivative

5was found themost potent inhibitor among a series of tetralines 1–9, and showed IC50 ¼ 0.349 mMas determined by a fluorescence assay. Compounds 3, 6, 7, and 9 also showed IC50 values in the

submicromolar range of concentration (Table I). Afterwards, Takeda have discontinued publishing

non-peptidomimetic inhibitors and applied for peptidic BACE inhibitors.77,78

Neurologic, Inc. described non-peptidomimetic phosphinylmethyl and phosphorylmethyl

succinic and glutaric acid analogues 10–12 along with some phosphorous-containing tetrapep-

tides.79,80 Noteworthy, the b-secretase inhibitory activity of 10–12was indirectly associated throughmeasurements of the compounds ability to increase sAPPa (it was expected to increase the pool

of non-amyloidogenic derivatives). The activity of 12 (LQ b-3) on sAPPa secretion was dose-

proportional at � 1 nM concentration (Chart 1).

Table I. BACE1 Inhibitory Activity of Derivatives 1–9 (Takeda)

Chart 1. Structure ofcompounds10–12 (Neurologic).



In 2002, Vertex Pharm., Inc. disclosed hundreds of different heterocyclic inhibitors.81 The

associated Ki values for BACE inhibition were given as a range of concentration; the most potent

compounds inhibited BACE1 with Kis < 3 mM. Some selected examples 13–35 are shown in

Table II.

Vertex proposed a 3-D pharmacophore map of BACE to guide the design of new inhibitors. The

compounds described in the invention showed hydrogen-bonding moiety interactions (HB) with the

active site and other key residues of BACE, and hydrophobic interactions (HPB) with BACE subsites

Table II. Compounds 13–35 Endowed With BACE1 Ki < 3 mM (Vertex Pharm., Inc.)

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(no crystallographics is provided). Five different BACE/inhibitor binding modes were hypothesized

involving four, five, six, six and an additional H-bond, or seven features of the inhibitor, respectively.

As an example of these binding modes, the seven features binding mode with BACE is shown in

Figure 2.

A. Isophthalamide-Based Inhibitors

1. Optimization of N-Terminus

Elan Pharm., Inc. found that the peptidic nature of potent and selective BACE inhibitors limited their

utility for drug development.82–84 In an effort to overcome this problem, Elan moved away from the

statin-like transition mimic, and selected the hydroxyethylene (HE) isostere because of fewer

peptidic bonds (Chart 2).85

Replacing the statin moiety with the HE core in a series of heptapeptide analogues resulted in a

10-fold increase of BACE inhibitory activity. Based on these findings, Elan synthesized a variety of

HEs bearing the optimal isophthalamideN-terminus of the statin series and different C termini at the

P1 0 substituent. Molecular modeling studies revealed that HE- and statin-based peptidic inhibitors

showed nearly identical binding modes for the N-terminal residues. All the peptidic amide groups of

the HE dipeptide isosteres formed H-bonds to the enzyme. However, the shorter dipeptide backbone

led to divergent conformations between the two series at the C terminus and showed the inability of

the statins to fill the S1 0 subsite. Structure andBACE1 inhibitory activity of selected examples 36–41

are shown in Table III.

Figure 2. Seven featuresbindingmodewith BACE.

Chart 2. The statinandhydroxyethylene cores (Elan).



The HEs were more cell permeable than the statins. Compound 42 (BACE1 IC50 ¼ 0.03 mM)

was less potent than the best statin analogue 43 toward BACE, but demonstrated a ratio of cell to

enzyme activity (EC50/IC50) of 100 (10 times better than two of the most potent statins 43 and 44)

(Chart 3).85

Table III. BACE1 Inhibitory Activity of Derivatives 36–41 (Elan)

Chart 3. Structure ofcompounds 42–44 (Elan).

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Elan improved the unfavorable cell-to-enzyme potency ratios (C/E� 100) of statins and

HE transition state isostere (TSI)-based inhibitors.86 The hydroxyethyl secondary amine (HEA)

isostere was an alternative of HE because of the strict preference of BACE for acyclic residues

at the S1 0 subsite, and considering that the majority of CNS drugs contain a basic amine.87 HE

inhibitor 45 was converted into the HEA inhibitors 46 and 47 by insertion of a secondary

nitrogen between the methylene and the P1 0 substituent. The preferred configuration for the CH–OHfunctionalitywas opposite to the stereochemistry of otherBACETSIs previously described85 (the (R)

isomer was 1,000-fold more active than (S) isomer) (Chart 4, the differences in the structures

are highlighted in bold). Compound 47 was a potent cellular inhibitor of Ab production

(more potent than the statin and the HE BACE inhibitors with comparable enzyme potency).

Compound 51 bearing a C-terminal benzyl amine exhibited submicromolar BACE enzyme activity

and cell-to-enzyme ratio (C/E) of 0.3. The meta-iodo analog 52 was 25-fold more potent than the

parent analog 51. Compounds 52 and 53were enzymatically more active than 45; their improved cell

potency was correlated to the reduced number of amide bonds and introduction of a basic amine

(Table IV).87

Chart 4. Comparisonof HEandHEAcores (Elan).



The bindingmode of 52was explained usingX-ray crystallography. Compound 52 bounds to the

BACE active site between the catalytic aspartates (residues 32 and 228) and the flap-residues 66–76,

and forms seven key H-bonds with the protein (Fig. 3).

Elan applied for b-secretase inhibitors (i.e., 54–57) characterized by either HE or HEA motif.

Representative examples are shown in Chart 5.88–94 Compound 57was administered at 100mg/kg to

rats, and reduced Ab levels by 17% in brain cortex and by 47% in plasma.95

Table IV. BACE1 Inhibitory Activity and Cellular Inhibition of Ab Production of 47–53 (Elan)

Figure 3. Interactionbetween52 andhBACE.

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Elan and also Elan in collaboration with Pharmacia & Upjohn applied for b-secretase inhibitorshaving different structures (some representative examples are shown in Chart 6): 3,4-disubstituted

piperidines,96 azahydroxylated ethylamines.97 macrocycles (58),98,99 aminodiols100–103 (59),

bicyclo compounds,104 diaminodiols105 (60), N-(3-amino-2-hydroxy-propyl) substituted alkyla-

mides (61),106 benzamide 2-hydroxy-3-diaminoalkanes,107 substituted amines,108 hydroxypropyl-

amines,109 allylamides,110 hydroxy substituted indanylamides (62),111 1,3-diamino-2-

hydroxypropanes,112,113 substituted hydroxyethylamines114 (63), acetyl 2-hydroxy-1,3-diaminoal-

kanes (64),115 phenacyl 2-hydroxy-3-diaminoalkanes (65),116 piperidines and piperazines,117

hydroxyethylamines (66),118 aminocarboxamides,119 amino-alkanoic acid amides and alkanoic acid

diamides,120 aryl alkanoic acid amides,121 hydroxyaminopropyl amides,122,123 ureas (67) and

carbamates,124,125 acetyl 1,3-diamino-2-hydroxyspirocyclohexanes,126 bicyclic compounds,127

2-amino- and 2-thio-substituted 1,3-diaminopropanes (68),128 oximes,129,130 ethanolcyclic-

amines,131 and cyclopropyl derivatives (69).132

2. Introduction of Sulfonates at P2 Position

Merck Research Laboratories set up a research project to identify non-peptide inhibitors of

b-secretase which might overcome the historical problems associated with peptide-like structures.

High throughput screening (HTS) of a multimillion compound library led to identify the N-(5-

aminopentyl)oxyacetamide (70). Compound 70 proved to be a reversible and selective BACE1

inhibitor, and reproducibly inhibited b-secretase in five different enzymatic assays at IC50 ¼25 mM.133,134 Replacing the tertiary amide group with either 2-cyanophenyl group (71) or the

sulfonate ester (72) resulted in improvement of inhibitory activity. The (R)-stereochemistry at the

chiral methyl group was essential for good enzymatic activity. In an electrochemiluminescence

(ECL) enzyme inhibition assay compound 74 displayed IC50 ¼ 1.4 mM (Table V).

Crystallographic examination of the BACE1/inhibitor complex revealed that 74 occupied

the S4–S1 subsites of the enzyme without any direct contact with the catalytic aspartic acids.

Compound 74 formed a H-bond between the oxyacetamide NH and a water molecule situated

between the aspartyl dyad. The authors observed that this water-mediated binding mode is almost

unique among aspartyl protease inhibitors. The P3 R-methyl group packs firmly against Ile110,

while unexpectedly orienting the para-fluorophenyl ring toward S3 creating a novel S3 subpocket

(S3 sp) (Fig. 4).

Chart 5. Structure ofcompounds 54–57 (Elan, and Elanand Pharmacia &Upjohn).



Chart 6. Structures 58–69 frompatents (Elan, and ElanandPharmacia &Upjohn).

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Compound 74 showed as a notable feature the lack of a direct interaction between the inhibitor

and the catalytic aspartate dyad.133 Chemical modifcation of 74 to interact directly with the catalyticdyad was expected to increase the inhibitory activity. The HEA isostere was chosen among potential

HE motifs because of its lower molecular weight and one less amide bond. In addition, the sulfonate

ester present in 72–74 was replaced with a sulfonamide group. A series of isophthalamides were

potent and selective BACE1 inhibitors.135,136 Compound 75 displayed excellent activity in both

enzymatic and cell-based assays.

Compound 75 (IC50 ¼ 15 nM) exhibited 100-fold enhanced potency compared with 74.

However, compounds 75–82 showed moderate BACE1/BACE2 selectivity (Table VI).135 Stachel

and co-workers developed an optimized cell-based assay: HEK293 cells were stably transfected

with a mutant form of APP containing the synthetic BACE1 cleavage sequence NFEV at the site

of proteolysis. The secretion of the BACE1 dependent N-terminal fragment of APP _NFEV

(sAPP _NF) was increased dramatically, thus providing a direct measure of BACE1 activity in

cells.135 Compounds 75–82 significantly reduced sAPP _NF formation in this assay, with several of

the inhibitors exhibiting low nanomolar potency (82 displayed an IC50 of 9 nM). Isophthalamides

75–82 exhibited remarkable enzyme selectivity and showed little to no inhibition of renin up to

500 mM. Against cathepsin D (CathD) 75 demonstrated approximately 500-fold selectivity. The

binding of P2 sulfonamide with Asn233 and Arg235 residues, and the interaction of the P1 0 groupwith the S1 0 site were believed factors imparting the selctivity. Asn233 is changed to Leu in BACE2

Table V. Structure and BACE1 Inhibitory Activity of Oxacetamides 70–74 (Merck)

Figure 4. H-bonding inside the BACE1/74 complex.



and cathD, and to Tyr in renin; Arg235 is changed to Val in cathD and Ser in renin. The more

hydrophilic S1 0 site of BACE1 better accommodates a small hydrophobic group, whereas cathD and

renin have longer, primarily hydrophobic S1 0 regions. The difference between BACE1 and BACE2consists of only two amino acid differences (Gln326 to Pro and Thr329 toAsn). Thus, this regionmay

primarily affect selectivity over cathD and renin rather than differentiate between BACE1 and

BACE2 activity.135

An overlay of 74with 75 showed that they occupy the samegeneral space in the active site: (i) the

(R)-a-methylbenzamide occupies the S3 subpocket andR-stereochemistry at themethyl is preferred;

(ii) the sulfonamide of 75 occupies the S2 site as the sulfonate ester of 74, and in both series each

sulfone oxygenmakes important H-bonding interactions with the enzyme; (iii) the alkyl group on the

nitrogen of the sulfonamide serves mainly to orient the binding conformation; (iv) in 75 the

phenylalanyl group occupies the S1 pocket in amanner similar to that of known peptide inhibitors (the

hydroxylamine motif is involved in direct interactions with the catalytic dyad); (v) the cyclopropyl

group of 75 is oriented toward the S 01 site of the enzyme.

The observation that the P2/P3 amide group of 75 serves mainly to orient the peptide backbone,

suggested the synthesis of the S3-truncated analogues.137 The feasibility of removing the

S3 amide bond was investigated by synthesis of the truncated analogues 83 and 84. The only

twofold loss of activity of 84 versus 83 (Table VII) demonstrated that amide removal was tolerated,

without significant loss of activity. Modeling studies showed that the Z-alkenyl compound

95would orient itself in the S3 pocket, thusmimicking the s-strand amide bond geometry. 95 showed

excellent binding activity. The apparent permeability (Papp) associated with 75 was low

(0.6� 10�6 cm/sec) and P-glycoprotein (P-gp) efflux values were indeterminable. In contrast, while

the olefine congeners (95) were still found to be moderate substrates for P-gp efflux, the associated

Papps (14� 10�6 cm/sec) were significantly increased as to permit measurement. The hydroxyethyl

amine moiety, amide bond, and sulfonamide of 95were thought to be all contributors to P-gp efflux.

This study demonstrated that analogues of 75 with both lower polar surface area and less H-bond

donors and acceptors possess better pharmacodynamic properties.

Table VI. Structure and BACE1 Inhibitory Activity of Isophthalamide 75–82 (Merck)

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Amides 97 and 98, truncated analogues of 77 devoid of any prime-side binding elements, were

weak BACE1 inhibitors (Chart 7). Compound 98 was elaborated with the prime-side binding

elements contained in 99 to produce thec[CH2NH]-reduced amide 100. This inhibitor showed potentactivity against BACE1 (IC50 ¼ 117 nM) but was less effective in the cell culture assay (sAPPb-NF;IC50 ¼ 3.7 mM).138,139

Table VII. Structure and BACE1 Inhibitory Activity of Compounds 83–96 (Merck)

Chart 7. Designofc[CH2NH]-reducedamide isostere100 (Merck).


Either small alkyl groups (101–103) or longer hydrophilic substituents at the P1 0

region produced compounds with enhanced inhibitory activities (Table VIII). Compounds

101–103 also showed a dramatic increase in cellular potency and exhibited IC50 values in

the 17–22 nM range of concentration. Inhibitor 104 bearing a polar substituent showed

diminished cell-based activity as a result of the lower cell permeability. Derivatives 100–

104 showed some selectivity over BACE2 (5- to 10-fold) and the IC50 values versus human

renin were greater than 20 mM. Compound 105 was potent inhibitor in both the enzyme and

cell-based assays (BACE1; IC50 ¼ 20 nM; sAPPb _ NF; IC50 ¼ 23 nM). This modification

also improved the cell permeability of the series (Papp ¼ 24� 10�6 cm/sec) when compared to

either HEA inhibitor 77 (Papp ¼ 0.6� 10�6 cm/sec) or reduced amide 101 (Papp ¼ 10�10�6 cm/sec). However, both compounds were susceptible to P-gp transport. SAR at the P2 0

region showed that small alkyl, cycloalkyl and benzyl groups were tolerated but only at the

expense of both cellular and enzymatic potency (106–109). The presence of a secondary NH

group was also needed to align the H-bonding network between the enzyme and the inhibitor

(Table IX).

X-ray structure of 101 at 1.8 A resolution showed that when compared to compound 77,

both inhibitors occupied the same general space in the active site with a few key differences

(Fig. 5). The binding mode of 101 was consistent with the known binding properties of

c[CH2NH]-pseudopeptides to Asp proteases. The nitrogen atom at the scissile bond of

reduced amide 101 shared an H-bond with only half of the dyad, specifically Asp228 (typically,

the hydroxyl and/or amino group of transition state analogues is in close contact with both

catalytic Asps).

3. Macrocyclization

X-ray crystallographic data of the 75/BACE1 complex revealed the presence of space in proximity of

the P1 and P3 groups. This observation prompted the design of macrocycles that would link these

Table VIII. Structure and BACE1 Inhibitory Activity for P1 0 Derivatives 100–105 (Merck)

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groups in an isophthalamide-based inhibitor. Macrocyclic 111 (IC50 ¼ 2.9 mM) showed higher

affinity for BACE1 than the corresponding seco variant 110 (IC50> 100 mM). Macrocyclic

compound 112 also incorporated the c[CH2NH]-reduced amide bioisosteric group present in

102 (Chart 8).140,141

Table IX. Structure and BACE1 Inhibitory Activity for P2 0 Derivatives 106–109 (Merck)

Figure 5. H-bondsofcompound101.



The high potency exhibited by macrocycle 112 (IC50 ¼ 32 nM, assayed as 1:1 diastereomeric

mixture) was correlated to the prime-side binding domain; however, in the cell-based assay 112wassignificantly less potent (IC50 ¼ 5.4 mM). New analogues deprived of the amide bond were

synthesized to improve the physico-chemical properties. The ring-contracted 14-membered

macrocycle 113 showed potency of 90 nM against BACE1, but in the cell-based and apparent

permeability assays 113was superior to 112. Efforts to improve potency culminated in the synthesis

of macrocycle 114 as a single diastereomer. As BACE1 inhibitor, 114 (IC50 ¼ 4 nM) was eightfold

more potent than 112. Apparent permeability of 114 compared with 112was significantly improved

(Papp ¼ 13� 10�6 cm/sec). Macrocycle 114 was dosed at 100 mg/kg in the APP-YAC mouse. The

brain/plasma ratio of compound concentration was determined to be 20–30% (compound

concentrations of 114 in the brain ¼ �1100 nM). Analysis of the diethanolamine (DEA) brain

extracts APP-YACmice (100 mg/kg as an intravenous bolus of 114, and euthanized after 1 and 3 hr)

demonstrated a robust decrease in Ab40 levels (25% less thanvehicle; however, by the 3 hr timepoint,

only a 10% reduction was measurable). Analysis of the concentration of 114 in the brain correlated

well with reduction in Ab levels, and a statistically significant reduction was observed when brain

concentrations of >700 nM were achieved.

B. Isonicotinamides

Merck Research Laboratories evaluated docking/scoring protocols which could be used to design

new BACE1 inhibitors in a drug discovery program. The simple scoring methods PLP1 score and

MMFFs interaction energy (Einter) performed as well or better than more computationally intensive

methods for a series of substrate-based BACE1 inhibitors. In particular, MMFFs Einter performed

well for a series of HEA BACE1 inhibitors.142

Chart 8. Transformationof110 into112 via macrocyclizationc[CH2NH]-reducedamide isosterism (Merck).

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In an effort to improve CNS penetration and pharmacokinetic stability of BACE1 inhibitors,

Merck discovered a series of highly potent and selective isonicotinamides.143 The improvement in

potency was due to the incorporation of a trans-methylcyclopropane P3 that could be responsible of

enhanced van derWaals contacts within the context of a 10s-loop down BACE1 conformation. Non-

capped compound 115 displayed potent activity in both BACE1 (IC50 ¼ 11 nM) and sAPP _NF

cellular (IC50 ¼ 38 nM) assays. The 115/BACE1 complex revealed the inhibitor occupied the S1–S3

sites, and the primary amine engaged both catalytic aspartateswith H-bonding contacts (Fig. 6).With

respect to S3, 115 bound and stabilized the 10s-loop down conformation (defining the size of the

S3pocket). The presence of amethyl capping group (NMevs.NH) generally resulted in a reduction in

intrinsic and cellular inhibition. In contrast, capping group had a positive affect in reducing

susceptibility to P-gp efflux. For example, in a cell-line expressing human P-gp, 117was a moderate

substrate for P-gp efflux, while a closely related non-capped analog, 116, exhibited significant efflux.The methoxyethyl was an alternative capping group; compound 118 was 4- and 12-fold more

potent than 117 in terms of enzymatic and cell-inhibition, and in the cell-based assay was the only

sub-100 nM BACE1 inhibitor within the ‘‘capped’’ series.

Further refinement of the P2 sulfonamide and optimization of the primary amine aspartyl

binding region led to the discovery of 119, a molecule suitable for in vivo evaluation in a murine

model. Transgenic mice expressing human WT APP were administered 50 mg/kg of 119 as

an intravenous bolus. A maximal reduction of Ab40 (34%) was observed at 3 hr compared

with that of vehicle-treated animals (concentration of 119 in the brain was of 5.8 mM at 0.5 hr

(maximal concentration), and 0.7 mM at 4.5 hr). A full dose-response study confirmed a dose

proportional reduction of Ab40 at 50 mg/kg at 3 hr (the concentration of drug in the brain was 1.9 and

Figure 6. Non-capped (115,116) andcapped (117,118) isonicotinamides.



0.7 mM for the 50 and 25 mg/kg dose groups, respectively). In general, the pharmacokinetic

parameters of the isonicotinamides displayed high clearance and high volumes of distribution. The

intravenous half-lives were moderate and the oral bioavailability was poor. Notable exception,

compound 120 was 69% bioavailable, with a good oral maximum concentration of 2.7 mM(Chart 9).144

C. Heterocyclic Derivatives

In 2004–2005,Merck registered a number of heterocyclic derivatives as BACE1 inhibitors.145–148 In

2006 and 2007, Merck also in collaboration with Sunesis Pharm., Inc. applied for pyrrolidinyl

compounds,149 spiropiperidine compounds,150,153,154 2-aminopyridines,151 and 2-aminomethylox-

adiazole compounds.152 Unfortunately, the inhibitory activities against BACE1 are not reported.

Representative compounds (121–138) selected from claims are shown in Charts 10.

Amgen applied for 2-hydroxy-1,3-diaminoalkanes, also including spiro-substituted chromans,

as b-secretase modulators. Compounds 139,155 140156 and 141157 displayed IC50 < 5 mM in both

FRET and cell-based assay (Chart 11).

In 2003 Smithkline Beecham disclosed hydroxyethylene compounds (i.e., 142–150) endowedwith effective inhibitory activity against BACE1. Themost active compound 142 inhibited BACE1 at

180 nM (Table X).158 Further development of this series led to the disclosure of new analogues which

inhibited BACE1 in the low nanomolar range of concentration159,160

Cyclic analogues characterized by a 3-(1,1-dioxotetrahydro-1,2-thiazin-2-yl) or 3-(1,

1-dioxoisothiazolidin-2-yl)benzamido moietey (compounds 151–154) inhibited BACE1 and CathD

in the 0.2–0.001 mM and 3–0.1 mM range of concentration, respectively (Chart 12).161,162

Glaxo Group Ltd registered a series of tricyclic indolecarboxamides (i.e., 155–166) which

showed BACE1 inhibition <1 mM and >100-fold selectivity for BACE1 over CathD.163,164

Compounds 167 and 168 were prepared by replacing the hydroxyethylamino motif with an

aminomethylcarbonyl group.165–167 Afterwards, new tricyclic derivatives (i.e., 169–172) charac-

terized by both the azaoxathiazepine dioxide ring and hydroxyethylaminomotif were synthesized.168

Compounds 169–172 inhibited BACE1 at <10 mM and showed >10-fold selectivity for BACE1

over CathD (Chart 13).

Bristol-Myers Squibb synthesized pyrrolidinone (i.e., 173)169 and azepinone170,171 (i.e., 174)

compounds as b-secretase inhibitors. Aminopyrrolidinone 173 inhibited b-secretase at 0.1 mM.170

Isophthalamides 175–178 inhibited BACE1 in the 0.1–1.0 mM range (175) or at concentration<0.1 mM (176–178)172 (Chart 14).

Chart 9. Structure of isonicotinamides119 and120 (Merck).

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Chart 10. Pyrrolidinyl compounds121–125, spiropiperidine compounds126–130, 2-aminopyridines 131–134, and 2-aminome-thyloxadiazole compounds135–138 (Merck).



D. Guanidines

Indoleacetic acid acyl guanidines, and isoxazolylcarbonyl- and isothiazolylcarbonylguanidines

were also reported as b-secretase inhibitors. Compounds 179–182 inhibited BACE at <0.1 mMconcentration (Chart 15).173,174

In 2005 Wyeth Research applied for diphenylimidazopyrimidine and imidazole amine

compounds which were found surprisingly effective and selective BACE1 inhibitors. Several

compounds inhibited BACE1 at submicromolar concentration. The BACE1 and BACE2 inhibitory

activity of examples 183–194 is shown in Table XI.175,176

Amino 5,5-diphenylimidazolone derivatives 195–202 were developed as potent inhibitors of

b-secretase; that is, derivatives198–202 inhibitedBACE1 in the 0.01–0.1mMrange of concentration

and were >100-fold selective for BACE1 over CathD177 (Chart 16).

Chart 11. Structure ofcompounds139–141 (Amgen).

Table X. BACE1 and BACE2 Inhibitory Activity of Derivatives 142–150 (Smithkline & Beecham)

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2-Aminopyridine derivatives (i.e., 203–205) inhibited BACE1 in the 0.01–0.1 mM range of

concentration178 (Chart 17).

High-throughput screening (HTS) of Wyeth’s compound library by means of a FRET assay

identified the acylguanidine 206 (BACE1 IC50 ¼ 3.7 mM) as a prototype of a new class of BACE1

inhibitors.179 X-ray crystal structure of the BACE1/206 complex revealed that two nitrogen groups

on the acylguanidine moiety formed H-bonds with the catalytic aspartic acids Asp32 and Asp228.

The third nitrogen faced away from the catalytic residues toward the S1 0 pocket. In contrast

to complexes with peptidomimetic inhibitors, the flap region adopted an ‘‘open conformation’’ that

hosted the diarylpyrrole portion of the inhibitor (such a conformation would be stabilized by

p-stacking interactions between the pyrrole and the phenyl ring of Tyr71). The two aryl groups of theBACE1/206 complex extended into the S1 (large and spherical) and S2 0 pockets. The P2 0-phenyl wasinvolved in p-stacking interaction with Trp76 (Fig. 7)

Wyeth synthesized analogues of 206.179 Compound 207 (IC50 ¼ 0.6 mM) bearing the more

spherical adamantyl group was sixfold more potent than 206 (Table XII). BACE1/207 cocrystal

structure confirmed that the adamantyl group occupied the S1 pocket. The cocrystal structure of

208 (IC50 ¼ 160 nM) showed that the 4-(4-acetoxyphenoxy)phenyl group extended through the S1pocket into the S3 pocket as predicted by the models. Substitution of the guanidine nitrogen with a

3-propanol group gave 209 (IC50 ¼ 240 nM), which was approximately 2.5-fold more potent

than the corresponding unsubstituted acylguanidine 207. Compound 210 bearing the para-

propyloxyphenyl group extending from S1-S3 also showed potent BACE1 inhibitory activity

(IC50 ¼ 110 nM), and demonstrated that a large group was not required for an optimal S3 pocket

binding. These compoundswere generally highly selective for BACE1 overCathD (>50-fold, except

206which is 16-fold) and pepsin (IC50> 50 mM). Compounds with diaryl substituents on the pyrrole

generally had lower BACE1/BACE2 selectivity, while compounds with the adamantyl substituent

show greater BACE1 over BACE2 selectivity. Substitution on the guanidine nitrogen led to lower

BACE1/BACE2 selectivity. Several compounds had low micromolar activity in the cellular Abtotallowering ELISA assay.

Wyeth applied for thienyl (211–214) and terphenyl acylguanidines (215–218) structurally

correlated with compounds 206–210, which inhibited the BACE1 in the submicromolar range of

concentration (Chart 18).180–182

AstraZeneca andAstex applied for 2-aminopyrimidin-4-ones (219),183–185 2-aminopyrimidines

(220),186 2-aminoimidazol-4-ones (221a),187,188 and dihydroisoquinolin-1-amines (221b)189 for the

treatment of Alzheimer’s disease and related pathologies. As a common structural feature, the

heterocyclic moieties of 219–221a show a guanidino motif (Chart 19).

Janssen Pharm. applied for 2-aminoquinazoline derivatives which inhibited b-secretase in threedifferent assays (i.e., compounds 222–225, Table XIII.190–192

Chart 12. Dioxothiazobenzamides151–154 (GlaxoGroup Ltd).



Chart 13. Tricyclic indolecarboxamides155–172 (GlaxoGroup Ltd).

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Chart 14. Structure ofcompounds173–178 (Bristol-Myers Squibb).

Chart 15. Structure ofcompounds179–182 (Bristol-Myers Squibb).



Table XI. Structure, BACE1 and BACE2 Inhibitory Activity of Derivatives 183–194 (Wyeth)

Chart 16. 2-Amino-5,5-diphenylimidazolone derivatives195–202 (Wyeth).

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Chart 17. 2-Aminopyridines 203–205.

Figure 7. Bonding interactions of the BACE1/206 complex.

Table XII. BACE1, BACE2 and Related asp Proteases Inhibitory Activity of 206–210 (Wyeth)

Pepsin: IC50> 50mMforderivatives206^210.



In a BACE1 inhibition assay a series of bioisosteric 2-amino-3,4-dihydropyrido[3,

4-d]pyrimidines (i.e., compounds 226–229) showed Ki values in the submicromolar range of

concentration (Chart 20).193

Johnson & Johnson Pharm. focused SAR development on 2-amino-3,4-dihydroquinazoline

230 (BACE1 Ki ¼ 900 nM).205 An X-ray structure of BACE1/230 obtained in collaboration with

Astex Ther. showed that the side chain bent back onto itself in a hairpin turn orientation, allowing the

N-cyclohexyl substituent to occupy the S1 binding pocket, while the flap region of the protein adopted an

‘‘open’’ structure. Compound 230 formed H-bonds with the catalytic aspartates as shown in Chart 21.

Compound 231 incorporated a 1,3-disubstituted phenyl ring in the side chain and 6-phenoxy substitution

instead of the 6-benzoyl group was sixfold more potent than 230 (BACE1 Ki ¼ 158 nM). To fill the S1 0

pocket a cyclohexyl was introduced on the (R)-a-carbon of the side chain. The (S) enantiomer

233 inhibited BACE1 with Ki ¼ 11 nM (racemate 232Ki ¼ 30 nM). Compound 233 displayed modest

selectivity for BACE-1 over renin and CathD as well as the hERG channel. Compound 233 exhibited

excellent potency in a cellular assay, which measures the inhibition of Ab1–40 secretion in CHO cells

transfected with the Swedish familial AD mutant APP (K670N/M671L). Additionally, 233 lowered

b-amyloid1–40 in plasma by 40–70% in rats after oral administration (30 mg/kg), 3 hr post-dosing.194

Chart 18. Substituted thienyl (211–214) and terphenyl guanidines (215–218) (Wyeth).

Chart 19. Structure ofcompounds 219–221 (AstraZenecaandAstex).

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E. Others

Eli Lilly synthesized potent amides 234 and 235 which in BACE1 mcaFRET assay showed

IC50s ¼ 1 and 3 nM, respectively. In HEK293 cells 234 and 235 exhibited EC50s ¼ 51 and 45 nM,

Table XIII. BACE1 Inhibitory Activity of Compounds 222–225 (Janssen)

aBACEassay-1 (MCA-substrate);

bBACEassay-2 (FS1-substrate);

c% Inhibition (FS1-substrate):% inhibitionvalues>100%were indistiguishable

from100%withintheerrorofmeasurement.dCompound222 registeredbyJanssenPharm.192 isidenticaltoderivative232publishedbyJohnson&

JohnsonPharm.194

Chart 20. BACE1inhibitoryactivityofcompounds 226–229 (Janssen).



respectively (the activity of BACE1 in HEK293 was determined by measuring the concentration of

sAPPb released in the conditioned media following the compound treatment).195 As a further

development, either isoquinoline (234) or piperazine (235) nucleus were replaced by a pyrrolidine

(236) or morpholine (237) core to give potent inhibitors in both BACE1mcaFRETassay (IC50 ¼ 3.9

and 78 nM) and HEK293 cells assay (EC50 ¼ 13 and 95 nM).196,197 Compound 237 was

administered at 100 mg/kg (subcutaneous injection) to growth factor promoter-expressing mice,

and led to 39–40% reduction of Ab levels in the plasma and cerebrospinal fluids, respectively

(Chart 22).197

Novartis searched for new inhibitors of BACE1 that would be also capable to inhibit the

generation of b-amyloid and subsequent aggregation into oligomers and fibrils.198,199 The lactam

compounds (i.e., 238 and 239,199 and 240200) were evaluated as inhibitors of BACE1 and BACE2,

human CathD, and cellular release of amyloid peptide 1–40. In at least one of the above-indicated

tests, the agents showed activity at concentration below 20 mM. Somemacrocyclic lactams were also

reported for treatment of neurological or vascular disorders related to b-amyloid generation and/or

aggregation200–202 (i.e., 239200) (Chart 23).

Chart 21. Structure ofcompounds 230–233 (Jonhson& Johnson).

Chart 22. Structure ofcompounds 234–237 (Eli Lilly).

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Astex Ther. used the Pyramid fragment screening methodology to BACE1 to identify by X-ray

crystallography three distinct chemotypes (aminoheterocycle, piperidine and aliphatic hydroxyl

group) binding to the catalytic aspartates. The fragment hits were endowed with weak BACE1

inhibitory activity but most of them displayed relatively good ligand efficiency. Virtual screening

around the aminoheterocycle recognition motif identified an aminopyridine with increased

potency. By means of Pyramid nonpeptidic fragments were identified useful for the design of new

(nonpeptidic) BACE1 inhibitor.203

The 2-aminoquinoline (241) ligand bounds to the active site of BACE1 forming interactions

between the charged aminopyridine motif and the two catalytic aspartates of the enzyme.203 The

observation that the fused phenyl ring of 241 was adjacent to the S1 pocket, suggested the design of

6-substituted phenylethyl-2-aminopyridine (242).204 Crystal structure indicated that the amino-

pyridine of 242 retained the bidentate binding to the catalytic diad Asp32 and Asp228 and, as

expected, the phenyl substituent occupied the S1 pocket. The indole-substituted aminopyridine

243was designed to better occupy the S1 pocket by incorporating a larger bicyclic ring system in the

hydrophobic pocket. The NH of indole formed a new interaction with the carbonyl of Gly230 on

the edge of the S3 pocket. A consideration that larger hydrophobic groups should be tolerated

in the S1–S3 region indicated to replace the indole of 243 with a 3 0-methoxybiphenyl-3-yl

group (244) (Chart 24).

Chart 23. Structure ofcompounds 238–240 (Novartis).

Chart 24. Developmentof 6-substitued 2-aminopyridines 241–244 (Astex).



The 2,3-diaminopyridine 245 was identified as a hit in silico by virtual screening of

aminopyridines.204 Compound 245 formed the charged bidentate interaction with the catalytic diad;

however, it changed its orientation relative to the fragment 241, and formed an additional H-bond

between the 3-amino group and Asp32. The switch in orientation of the aminopyridine ring allowed

the N-benzyl group to occupy the S1 pocket. Introduction of a pyridin-3-yl ring at position 3 of the

benzyl group provided ligands 246–248 which formed favorable contacts with the binding pocket

and showed improved aqueous solubility. Interestingly, the pyridyl nitrogen of 246 formed anH-bond

with a water molecule at the bottom of the S3 pocket. The oxygen of the methoxy group of

247 displaced this water molecule, and the alkyl group of 248 was positioned within the narrow

opening at the bottom of the S3 pocket. The phenyl ring of 249 bounds the S1 pocket with

concomitant small rotation of the 2,3-diaminopyridine moiety without loss of interaction with the

catalytic diad. The indolyl group occupied S3 pocket as expected, and formed a new interaction

between the NH of the heterocycle and the backbone carbonyl of Gly230. Compounds 250 and

251 were prepared to explore under ‘‘flap’’ toward the S2 0 region. Compound 251 occupied the S2 0

pocket of the enzyme, making hydrophobic contacts and a H-bond between the side chain NH of

Trp76 and the pyridyl nitrogen atom (Chart 25).

Sunesis Pharm., Inc. considered the primary amine group as a possible bioisostere of the

hydroxyl group present in HE-containing BACE1 inhibitors.205 Replacement of the HE motif of

252with the aminoethylene (AE) bioisostere afforded compound 253 endowedwith slightly reduced

enzymatic inhibitory activity but distinctly improved cell potency; the (S) isomer was preferred for

activity (compare 253 with 254) (Table XIV). Replacement of the benzylvaline amide group with a

4-fluoroaniline (compounds 255 and 256) caused loss of activity. Incorporation of a benzyl R1

substituent (257 and 258) resulted in a neat increase in inhibitory potency relative to the isobutyl

analogue. The three-dimensional cocrystal structure of 258 in complex with BACE1 showed that the

AE isostere had similar binding affinity for the catalytic site as the HE counterpart. The cell-based

activity of AE inhibitors wasmarkedly better than the HE-containing compounds. This result may be

due to themore hydrophilic character of theAE inhibitors at neutral pH and to themore acidic cellular

compartments in which BACE1 is localized.

Chart 25. Developmentof 3-substitued 2-aminopyridines 245–251 (Astex).

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4 . C O N C L U S I O N S

The aspartyl protease BACE1 has become a very attractive approach to develop new effective agents

for the treatment of Alzheimer’s disease. The research towards the discovery of non-peptidic BACE1

inhibitors has received a great impulse in the past 6 years. A wide number of structurally different

classes of compounds were found to be potent BACE1 inhibitors; however, some patents did not

disclose the BACE1 inhibitory concentrations. Isophthalamide-based compounds displayed

remarkable BACE1 inhibitory activity; chemical modification of this scaffold by introduction of a

sulfonate, macrocycle, isonicotinamide, guanidine and heterocycle (indole, imidazole, piperidine,

morpholine, isoquinoline) moiety provided BACE1 inhibitors which showed IC50s < 10 nM

concentration. The non-peptidic inhibitors showed high selectivity over BACE2 (BACE1/BACE2

selectivity>100) and other human proteases (cathD, pepsin, and renin). Their weak or non-peptidic

character favors CNS penetration and oral bioavailability. The non-peptidic structures showed potent

activity in both an in vitro assay and in a cellular assay (HEK2983 cells). In experiments in animal

models, the non-peptidic inhibitors (i.e., 57, 114, 225, and 258) led to signficant reductions of Ablevels in animal models. These inhibitors are valuable drug-candidates and might become first-line

drugs for the treatment of Alzheimer’s disease in the near future.

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A P P E N D I X

Bristol-Myers Squibb, PO Box 4000, Route 206 and Provinceline road, Princeton, NJ 08453-4000

(US)

CoMentis, Inc., 280 Utah Avenue, Suite 275, South San Francisco, CA 94080 (US)

Eisai R&D Management Co., Ltd, 6-10, Koishikawa 4-chome, Bunkyo-ku, Tokyo, 1128088 (JP)

Elan Pharmaceuticals, Inc., 800 Gateway Boulevard, So. San Francisco, CA 94080 (US)

Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 (US)

First Horizon Pharmaceutical Corporation, 6195 Shiloh Road, Alpharetta, Georgia 30005 (US)

Glaxo Group Limited, Glaxo Wellcome House, Berkeley Avenue, Greenford, Middlesex UB6 0NN

(UK)

Johnson & Johnson Pharmaceutical Research and DeVelopment, LLC, Spring House, Pennsylvania

19477 (US), and Turnhoutseweg 30, 2340 Beerse, Belgium

Memory Pharmaceuticals Corporation, 100 Philips Parkway, Montvale, New Jersey 07645 (US)

Merck & Co., Inc., 126 East Lincoln Avenue, Rahway, NJ 07065-0907 (US)

Neurologic, Inc., 15010 Broschart Road, Suite 200, Rockville, MD 20850 (US)

Roche Laboratories, 340 Kingsland Street, Nutley, New Jersey, 07110 (US)

Sunesis Pharmaceuticals, Incorporated, 341Oyster Point Boulevard, South San Francisco, California

94080 (US)

Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, MA 02139-4242 (US)



Romano Silvestri is an Associate Professor in Medicinal Chemistry at the Dipartimento di Chimica e

Tecnologie del Farmaco (Department of Medicinal Chemistry and Technologies) of the Faculty of Pharmacy at

the Sapienza University of Rome (Italy). He was born in 1959 in Revo (Trento, Italy). He studied medicinal

chemistry (1983, Degree in Pharmacy) at the Faculty of Pharmacy in Rome. From 1986 to 1989 he worked on

his PhD inMedicinal Chemistry. From 1994 to1997 he was an Assistant Professor. He is the author of more than

130 publications and 6 patents in the field of antiviral agents (discovery of novel classes of HIV NNRTI classes:

IAS, PAS, PBTD, DAMNI), antifungal agents (analogues of bifonazole (Ph.D. thesis), ketoconazole and

miconazole), antitumor agents (antimitotic agents, ellipticine andCC-1065 analogues), antiinflammatory drugs

(analogues of lonazolac, tolmetin, lefetamine, and sulindac), CNS agents (antidepressant drugs: mianserine

analogues and MAO inhibitors), and heterocyclic chemistry (pyrrole, indole, tri- and tetracyclic systems).

338 * SILVESTRI


Documents

Boom in the development of non-peptidic β-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease