Acylguanidine inhibitors of β-secretase: Optimization of the pyrrole ring substituents extending into the S1′ substrate binding pocket

Article abstract of DOI:10.1016/j.bmcl.2007.11.043

The proteolytic enzyme β-secretase (BACE-1) produces amyloid β (Aβ) peptide, the primary constituent of neurofibrillary plaques, implicated in Alzheimer's disease, by cleavage of the amyloid precursor protein. A small molecule inhibitor of BACE-1, (diamin

Full text of DOI:10.1016/j.bmcl.2007.11.043

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 767–771
Acylguanidine inhibitors of b-secretase: Optimization of the pyrrole
ring substituents extending into the S01 substrate binding pocket
Lee D. Jennings,a,* Derek C. Cole,a Joseph R. Stock,a Mohani N. Sukhdeo,a
John W. Ellingboe,a Rebecca Cowling,a Guixian Jin,a Eric S. Manas,b Kristi Y. Fan,c
Michael S. Malamas,c Boyd L. Harrison,c Steve Jacobsen,f Rajiv Chopra,d Peter A. Lohse,e
William J. Moore,b Mary-Margaret O’Donnell,e Yun Hu,f Albert J. Robichaud,c
M. James Turner,f Erik Wagnerf and Jonathan Bardf
aChemical & Screening Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
bChemical & Screening Sciences, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
cChemical & Screening Sciences, Wyeth Research, 865 Ridge Road, Monmouth Junction, NJ 08852, USA
dChemical & Screening Sciences, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
eArqule, 19 Presidential Way, Woburn, MA 01801, USA
fDiscovery Neuroscience, Wyeth Research, 865 Ridge Road, Monmouth Junction, NJ 08852, USA
Received 24 September 2007; revised 8 November 2007; accepted 12 November 2007
Available online 17 November 2007
Abstract—The proteolytic enzyme b-secretase (BACE-1) produces amyloid b (Ab) peptide, the primary constituent of neurofibril-
lary plaques, implicated in Alzheimer’s disease, by cleavage of the amyloid precursor protein. A small molecule inhibitor of
BACE-1, (diaminomethylene)-2,5-diphenyl-1H-pyrrole-1-acetamide (1, BACE-1 IC50 = 3.7 lM), was recently described, represent-
ing a new small molecule lead. Initial SAR investigation demonstrated the potential of accessing the nearby S3 and S01 substrate
binding pockets of the BACE-1 enzyme by building substituents off one of the phenyl substituents and guanidinyl functional group.
We report here the optimization of guanidinyl functional group substituents on 1, leading to potent submicromolar BACE-1
inhibitors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is characterized by the deposi-
tion of neuritic plaques on the outside of nerve cells in
areas of the brain related to memory. These plaques
consist predominantly of the abnormal peptide b-amy-
loid (Ab), a protein fragment cleaved from amyloid pre-
cursor protein (APP), during metabolism.1 Although
neuropathologic and epidemiological studies are incon-
clusive as to the pathogenic role of Ab in Alzheimer’s
disease, studies from many laboratories support the
hypothesis that amyloid production plays a key role in
the pathogenesis of Alzheimer’s disease.2,3 As a conse-
quence of the widespread acceptance of this hypothesis,
vigorous efforts in compound screening and medicinal
chemistry have been undertaken to find inhibitors of
the two proteases, b- and c-secretase, responsible for
Ab production.
b-Secretase (BACE-1, Beta Amyloid Cleaving Enzyme
1, also called memapsin 2 and Asp-2) is a membrane-an-
chored aspartyl protease and a member of the pepsin
sub-family of aspartyl proteases. BACE-1 mediates the
primary cleavage of APP at residue 671, generating the
membrane-bound C-terminal APP fragment C99, which
is in turn cleaved by c-secretase, liberating Ab.1,4–6 Pep-
tide-like inhibitors of BACE-1, based on substrate mim-
icry, have been disclosed7 and reviewed.8 Recently,
publications disclosing small molecule BACE-1 inhibi-
tors have started to appear.9 We have recently disclosed
the acylguanidine 1 as an inhibitor of BACE-1 with low
micromolar affinity (BACE-1 IC50 = 3.7 lM, Kd = 2.8
lM) and the key structural features of the enzyme/inhib-
itor complex, as revealed by X-ray crystallography
(Fig. 1).10 An initial round of SAR investigation using
Keywords: BACE-1; Amyloid peptide; Ab peptide; Aspartyl protease
inhibitor; BACE-1 inhibitor; Alzheimer’s disease.
*
Corresponding author. Tel.: +1 845 602 3618; fax: +1 845 602
3045; e-mail: jenninl@wyeth.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.043

768
L. D. Jennings et al. / Bioorg. Med. Chem. Lett. 18 (2008) 767–771
O
HO
N
NH2
NH2
O
R1
NH2
NH2
a
b,c,d
O
N
N
R2
R1
O
R2
O
O
O
N
N
4
5
Cl
R3
1
2
H2N
Boc
HN
N
BACE-1 IC50 = 3.7 μM
BACE-1 IC50 = 0.7 μM
N
H
NH2
N
e,f
O
F
O
R1
N
N
R2
F
R1
R2
H2N
F
NH2
N
HN
6
7
N
O
NH2
N
O
N
Scheme 1. Reagents and conditions: (a) glycine (3 equiv), p-TSA (cat.),
EtOH, reflux, 3d, or glycine (2 equiv), AcOH, reflux, 1–5 h; (b)
TMSCl, MeOH, reflux, 1–2 h; (c) guanidine (5 equiv), DMSO, rt,
1–4 h; (d) Boc2O, DIPEA, CH2Cl2, rt, 1–3d; (e) NaH (3 equiv), alkyl
halide (2.5 equiv), DMF, 60 ꢁC, 16 h; (f) TFA (1 M in CH2Cl2), rt,
16 h.
4
3
43% inhibition @ 2 μM
BACE-1 IC50 = 0.6 μM
Figure 1. Acyl guanidine BACE-1 inhibitors.
H2N
N
parallel synthesis revealed the opportunity to increase
binding potency by adding substituents to the para-posi-
tion of one of the phenyl groups of 1 extending into the
S3 substrate binding pocket. Improved BACE-1 inhibi-
tor 2 was the outcome of this effort.11
HO
N
N
a
N
O
O
R2
R1
R1
R2
N
6
9
H2N
N
Molecular modeling also revealed the potential for add-
ing substituents to the guanidinyl functional group of 1
extending into the S01 pocket, which is occupied by an
Asp side chain of the natural substrate. Inhibitors with
substituents extending into the S01 pocket could take
advantage of differences in the size and relative hydro-
phobicity in the S01 sites of different aspartyl proteases
to enhance selectivity for BACE-1 over cathepsin D
and renin. Peptide mimetic inhibitors of BACE-1 vari-
ously 0do or do not have substituents directed toward
the S1 pocket.8 Stachel9a and Garino9b have separately
reported nonpeptidic BACE-1 inhibitors which have
substituents that extend into the S01 pocket, resulting in
improved selectivity and potency. We report here the re-
sults of our investigation of different substituent groups
extending off the guanidinyl group of 1.
R3
N
H
O
b
N
R2
R1
10
Scheme 2. Reagents and conditions: (a) CDI (5 equiv), 1-H-pyrazole-
1-carboxamidine HCl (5 equiv), DMAP (0.1 equiv), CH2Cl2, rt, 2–5 h;
(b) alkyl amine (3 equiv), DIPEA (3 equiv), rt, 16 h.
Alkylsulfonyl and arylsulfonyl acyl guanidines 13 could
be prepared by the reaction of acyl guanidine intermedi-
ates 12 with the appropriate sulfonyl chloride (Scheme 3).
Substituted acyl guanidine 3 (BACE-1 inhibition 43% at
2 lM) (Fig. 1), prepared at an early point in the project,
Disubstituted guanidines were synthesized by a number
of different routes. In all cases, disubstituted pyrrole
intermediates 6 were prepared following our previously
disclosed route.10 Early on, the substituted acyl
guanidines were prepared by a protection–deprotection
sequence using di-tert-butyl dicarbonate. Alkylation of
the Boc-protected intermediate was carried out using
sodium hydride and was followed by acid mediated
R3
O
H2N
N
H2N
N
Boc
N
H
N
H
O
N
H
a
O
R1
N
R1
R2
N
R2
7
11
deprotection (Scheme 1). Subsequently,
a
more
O
S
NH
O
H2N
N
R3
H2N
N
convenient synthetic procedure was developed allowing
the use of amines as building blocks for side-chain
synthesis (Scheme 2).12 Intermediate 6 was reacted with
carbonyl diimidazole and pyrazole-1-carboxamidine
hydrochloride to afford the iminopyrazole intermediate
9. The pyrazole could be easily displaced by amines to af-
ford the alkyl substituted acyl guanidines. Alkyl ureido
guanidines 11 could be prepared from the Boc-protected
acyl guanidine 7 by reaction with amines in DMSO.
NH2
O
b
O
N
R1
R2
N
R1
R2
12
13
Scheme 3. Reagents and conditions: (a) alkylamine (1.3 equiv),
DMSO, 110 ꢁC, 3 h; (b) alkyl or arylsulfonyl chloride (1.3 equiv),
CH2Cl2, rt.

L. D. Jennings et al. / Bioorg. Med. Chem. Lett. 18 (2008) 767–771
Table 1. BACE-1 inhibition by substituted acyl guanidines 14–29
769
NH2
N
R1
N
O
H
O
N
Cl
Example
R1
BACE-1 inhibition IC50 (lM)
Cathepsin D/BACE-1
Cellular Ab ED50 (lM)
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
trans-4-Carboxy-cyclohexyl
trans-4-Hydroxy-cyclohexyl
3-Carboxy-propyl
0.078
0.125
0.13
7.3
110
45
10.4
1.28
Inactive
nt
2-Carboxyethyl
2-Carboxamido-ethyl
3-Hydroxy-propyl
0.278
0.172
0.121
0.124
0.137
0.203
0.321
0.269
0.241
0.752
0.558
0.203
0.651
>45
55
nt
85
81
1.18
5.27
4.14
5.27
1.23
1.34
3.10
10.7
9.10
5.27
5.45
2,3-Dihydroxy-propyl
2-Acetamidoethyl
70
15
2,2-Dimethyl-3-hydroxy-propyl
3-Methylthio-propyl
2-(1,3-Dioxolan-2-yl)-ethyl
3-(1H-imidazol-1-yl)propyl
2-Thienylmethyl
3.9
13
30
0.7
2.5
0.5
1.0
2-Furylmethyl
2,3,4-Trifluorobenzyl
4-Hydroxy-3-methoxybenzyl
was approximately as potent as the initial lead. An
X-ray crystal structure of the inhibitor bound to
BACE-1 clearly showed that the trifluorophenyl substitu-
ent was accommodated into the S01 substrate binding
pocket. This was almost certainly a sub-optimal pairing
of substituent group with the binding pocket because (1)
the S01 pocket, wherethe Arg235 and Thr329 residues form
part of the pocket wall, was expected to better accommo-
date polar substituent groups, (2) the volume of the 2,3,4-
trifluorobenzyl group was a bit large for the S01 pocket
and, as a consequence, (3) docking experiments indicated
that the benzylic linkage of the bound molecule was some-
what strained. Acting on this hypothesis, a library of ana-
logs was prepared to investigate the effect on binding and
selectivity of different S01 substituents (Table 1). Substitu-
ent groups were selected (based on docking) for size and
polarity complimentarity with the S01 pocket. All of the
analogs made showed a significant (2–9·) boost in po-
tency of BACE-1 inhibition relative to 2. In order to avoid
toxicities caused by inhibition of off-target proteases,
selectivity of inhibitors for BACE-1 over other aspartyl
proteases was a goal of this project. All compounds were
evaluated for inhibition of cathepsin D in a FRET assay.
Compounds with polar substituents, whether having a
cyclic (e.g. 14 and 15) or an acyclic (e.g. 16–25) connection
to the guanidinyl group, showed high levels of BACE-1/
cathepsin D selectivity, whereas analogs with aromatic
substituent groups (e.g. 26–29), while potent, showed
poor selectivity. Reduction of Ab production in CHO
cells recombinantly expressing human wild-type APP10
improved 5-fold in some examples (ED50 2 = 5.9 lM,
LD50 2 > 25 lM).
of sufficient length with hydroxy and cyano groups on the
terminus were preferred. Aromatic substituent groups
also gave potent inhibitors of BACE-1, although
BACE-1/cathepsin D selectivity was diminished. ED50
for Ab production improved in certain examples (31, 38,
and 45) but did not appear to correlate with improving
IC50 for BACE-1 inhibition. Compounds were generally
not toxic to cells (LD50) at doses near the ED50. Com-
pounds with aromatic S10 substituents (41–45) were
exceptional in this regard, suggesting that decreased selec-
tivity was associated with increased toxicity. Acyl guani-
dines with alkyl carbamoyl or alkylsulfonyl substituent
groups (not shown in tables) did not inhibit BACE-1 to
any appreciable extent at the tested concentrations.
The complex of inhibitor 31 with BACE-1 was crystal-
lized and the resulting crystal structure was diffracted
and solved.10 As anticipated, the N-3-hydroxypropyl
group extends into the S10 pocket with little strain,
although not displacing tightly held water molecules
from the pocket (Fig. 2). These tightly held water mol-
ecules effectively become part of the protein with the
inhibitor making hydrogen bonding interactions
through them. Acyl guanidines substituted with alkyl
groups on the guanidine nitrogen provided an improve-
ment in potency of BACE-1 inhibition. A subset of
these analogs, particularly aliphatic substituents with
polar groups at the end of the chain, exhibited promis-
ing levels of selectivity for BACE-1 over cathepsin D,
and activity approaching ꢀ1 lM in the cellular Ab low-
ering assay. Compounds in this series for which absorp-
tion in the Caco-2 drug transport model13 was assessed
(4, 30, and 31) were found to have poor permeability.
The guanidinyl functionality, while involved in impor-
tant hydrogen bonding interactions with the catalytic
aspartic acid residues in the catalytic site, may be
responsible for the poor permeability that characterizes
An analogous SAR study was done using the adamantyl
substituted acyl guanidine inhibitor 4 as a scaffold
(Table 2). Addition of S01 substituents gave a measurable
boost to BACE-1 inhibition potency. Substituent groups

770
L. D. Jennings et al. / Bioorg. Med. Chem. Lett. 18 (2008) 767–771
Table 2. BACE-1 inhibition by substituted acyl guanidines 4, 30–45
NH2
N
R1
N
H
O
N
Example R1
BACE-1 inhibition Cathepsin D Cathepsin
IC50 (lM)
Cellular
Cellular
IC50 (lM)
D/BACE-1 (lM) Ab ED50 (lM) LD50 (lM)
4
H
3-Cyanopropyl
0.6
25
69
17
8.9
4.4
>25
59
35
25
16
31
79
100
88
45
14
50
22
8
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
0.255
0.239
0.68
4.25
19.9
24.7
12.2
6.45
12.5
54.1
73.5
>25
n.t.
3-Hydroxypropyl
2-Hydroxyethyl
2-Cyanoethyl
83
36
1.84
3.62
3.44
4.15
9.51
17.8
21.1
9.16
1.44
18.7
9.48
3.97
2
0.31
39
25
4-Hydroxybutyl
(2R)-2-Hydroxypropyl
(2S)-2-Hydroxypropyl
2,3-Dihydroxypropyl
3-(1H-imidazol-1-yl)propyl
trans-4-Hydroxycyclohexyl
Cyclohexyl
0.255
0.437
0.942
0.536
1.71
28
57
137
>15
0.283
7.2
54.2
3.01
0.144
0.476
1.38
0.599
7.5
4.5
0.5
0.9
1.9
1.1
(5-Methyl-1,3,4-oxadiazol-2-yl)methyl 0.665
(4-Methyl-1,3-thiazol-2-yl)methyl
2-(2-Thienyl)ethyl
3-Aminobenzyl
0.276
0.559
0.744
0.565
3
4.81
1.69
8
2-(3-Thienyl)ethyl
2
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Figure 2. X-ray crystal structure of BACE-1 inhibitor 31 with BACE-1.
Interactions between the hydroxypropyl group and bound water
molecules in the S01 pocket are indicated with yellow dashed lines.
this series. However, an understanding of the effects of
occupation of available substrate binding pockets and
a demonstration of the ability of analogs to exploit
available protein functionality in the substrate binding
pockets for increased BACE-1 potency is an important
first step toward the goal of optimizing the lead for oral
bioavailability, CNS penetration, and a suitable dura-
tion of action. Future publications from these laborato-
ries will disclose the results of further functionalization
of the guanidine group and its replacement with
bioisosteres.
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References and notes
10. Cole, D. C.; Manas, E. S.; Stock, J. R.; Condon, J. S.;
Jennings, L. D.; Aulabaugh, A.; Chopra, R.; Cowling, R.;
Ellingboe, J. W.; Fan, K. Y.; Harrison, B. L.; Hu, Y.;
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1. Selkoe, D. J. Physiol. Rev. 2001, 81, 741.
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