BACE1 inhibitors: Optimization by replacing the P1′ residue with non-acidic moiety

Article abstract of DOI:10.1016/j.bmcl.2008.01.058

Recently, we reported potent BACE1 inhibitors KMI-429, -684, and -574 possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell assays, especially, KMI-429 was confirmed to significantly inhibit Aβ production in vivo. However, acidic moieties at the P₄ and P₁^′ positions of KMI-compounds were thought to be unfavorable for membrane permeability across the blood-brain barrier. Herein, we replaced acidic moieties at the P₄ position with other hydrogen bond acceptor groups, and these inhibitors exhibited improved BACE1 inhibitory activities in cultured cells. In this study, we replaced the acidic moieties at the P₁^′ position with non-acidic and low molecular sized moieties.

Full text of DOI:10.1016/j.bmcl.2008.01.058

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1649–1653
0
BACE1 inhibitors: Optimization by replacing the P1 residue
with non-acidic moiety
Yoshio Hamada,a Hamdy Abdel-Rahman,a Abdellah Yamani,a Jeffrey-Tri Nguyen,a
Monika Stochaj,a Koushi Hidaka,a Tooru Kimura,a Yoshio Hayashi,a Kazuki Saito,b
Shoichi Ishiurac and Yoshiaki Kisoa,*
aDepartment of Medicinal Chemistry, Center for Frontier Research in Medicinal Science and 21st Century COE Program,
Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
bLaboratory of Proteomic Sciences, 21st Century COE Program, Kyoto Pharmaceutical University, Yamashina-ku,
Kyoto 607-8412, Japan
cDepartment of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
Received 26 November 2007; revised 8 January 2008; accepted 15 January 2008
Available online 19 January 2008
Abstract—Recently, we reported potent BACE1 inhibitors KMI-429, -684, and -574 possessing a hydroxymethylcarbonyl isostere as
a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell0assays, especially, KMI-
429 was confirmed to significantly inhibit Ab production in vivo. However, acidic moieties at the P4 and P1 positions of KMI-com-
pounds were thought to be unfavorable for membrane permeability across the blood–brain barrier. Herein, we replaced acidic moi-
eties at the P4 position with other hydrogen bond acceptor groups, and these 0inhibitors exhibited improved BACE1 inhibitory
activities in cultured cells. In this study, we replaced the acidic moieties at the P1 position with non-acidic and low molecular sized
moieties.
Ó 2008 Elsevier Ltd. All rights reserved.
b-Secretase, also called BACE1 [b-site APP (amyloid
R1
precursor protein) cleaving enzyme], is a molecular tar-
get for developing drugs against Alzheimer’s disease
(AD),1–5 because BACE1 triggers amyloid b (Ab) pep-
tide formation by cleaving APP at the N-terminus of
the Ab domain.6–11 Recently, we reported potent
BACE1 inhibitors12–15 KMI-420, -429, and -684
(Fig. 1) possessing phenylnorstatine [Pns: (2R,3S)-3-
amino-2-hydroxy-4-phenylbutyric acid] as a substrate
transition-state mimic.16 Specifically, KMI-429 exhib-
ited effective inhibition of BACE1 activity in cultured
cells, and significant reduction of Ab production
in vivo (APP transgenic and wild-type mice).13b Accord-
ing to structure-activity relationships studies0 on KMI-
compounds, an acidic moiety at the P4 and P1 positions
is required for improving BACE1 inhibitory activity.
O
O
O
H
N
H2N
R2
N
N
N
H
H
H
O
OH
NH
N
KMI-420 (R1 = H, R2 = COOH) IC50 = 8.2 nM
KMI-429 (R1 = R2 = COOH)
KMI-684 (R1 = R2
O
NH
N
IC50 = 3.9 nM
N
N
=
)
IC50 = 1.2 nM
NH
N
N
O
F
O
O
H
N
H2N
N
N
N
N
NH
H
H
H
O
OH
N
N
HN
O
0
However, acidic moieties at the P4 and P1 positions
O
KMI-574 IC50 = 5.6 nM
HN
NH
Keywords: Alzheimer’s disease; BACE1; b-Secretase; BACE1 inhibitor.
Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591
9900; e-mail: kiso@mb.kyoto-phu.ac.jp
O
*
Figure 1. BACE1 inhibitors consisting of penta-peptides.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.058

1650
Y. Hamada et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1649–1653
were thought to be unfavorable for membrane perme-
ability across the blood–brain barrier. In order to im-
prove membrane permeability of KMI-compounds, we
replaced the acidic moieties at the P4 position with other
hydrogen bond acceptor groups, and these inhibitors
(e.g., KMI-574 in Fig. 1) exhibited improved BACE1
inhibitory activities in cultured cells.15 This paper de-
scribes the BACE1 inhibitors, in which the acidic moie-
0
ties at the P1 position were replaced with non-acidic and
low molecular-sized moieties. In the subsequent paper,
we report the small-sized BACE1 inhibitors, in which
the P2 residue was replaced with non-peptidic aromatic
moieties.
A
N
BACE1 inhibitors 1–19 were synthesized by traditional
solution-phase peptide synthesis methods in a similar
manner as previously reported procedures,13a as shown
in Scheme 1. From amines corresponding to the P1 res-
idue of the inhibitors as starting materials and N-pro-
tected amino acids, peptide bonds were formed
N
COOH
HN
N
a,b
0
13
(KMI-758)
Boc-DAP(Fmoc)-OH
a,c
Boc-Val-OH
a,b
Boc-Leu-OH
sequentially
using
1-ethyl-3-(3-dimethylaminopro-
a,b
Boc-Pns-OH
pyl)carbodiimideÆHCl (EDCÆHCl) in the presence of
1-hydroxybenzotriazole (HOBt) as coupling agents.
Aromatic amines 24 and 25 were synthesized from triflu-
oroacetophenone and 3-(N-tert-butyloxy)aminobenzoic
acid, respectively, as illustrated in Scheme 2. Other
amines were commercially available. Previously, we re-
ported that N-protected a-hydroxy-b-amino acids, such
as Boc–Pns-OH, induced the corresponding homobis-
lactones by dimerization of acids via a condensation
reaction with a poor-nucleophilic amine.17 From this
observation, inhibitors 11, 18, and 19 were synthesized
according to the procedure shown in Scheme 1B.
Namely, after the hydroxyl group of Boc–Pns-OH was
protected by an introduced 1,3-oxazolidine ring,18 an
amide bond was formed using EDCÆHCl in the presence
of 4-dimethylaminopyridine (DMAP) or 1-ethoxycar-
a,b
H2N
Cl
Cl
B
O
d
O
Boc
N
OH
Boc
N
OH
O
H
OH
(Boc-Pns-OH)
O
20
e
O
b
H
Pns NHSO2CF3
Boc
N
NHSO2CF3
22
O
bonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as a
coupling agent. Inhibitor 4 was synthesized from ethyl
21
O
2-amino-4-methylthiazole-5-carboxylate corresponding
0
to the P1 moiety as starting material, followed by a sub-
N
N
COOH
sequent alkaline hydrolysis. In the case of inhibitors
1–12, Boc–Glu(O-tert-butyl)-OH were used as an N-ter-
minal amino acid moiety. After the elongation of the
peptide chain, all inhibitors were synthesized by depro-
tection using 4 N HCl in dioxane and purified by
preparative RP-HPLC.
HN
N
a,b
19
Boc-DAP(Fmoc)-OH
Boc-Val-OH
(KMI-880)
a,c
a,b
Boc-Leu-OH
a,b
H Pns NHSO2CF3
BACE1 inhibitory activity of the inhibitors was deter-
mined by enzymatic assay using a recombinant human
BACE1 and FRET (fluorescence resonance energy
transfer) substrate as previously reported.4 Recently,
we reported from a computer-assisted docking simula-
tion study of inhibito0r KMI-684 docked in BACE1
study14 that the two P1 tetrazole rings were found near
the hydrophobic amino acids of BACE1, and that the
hydrophobic regions above and below these tetrazole
rings might interact favorable with the partially hydro-
22
Scheme 1. Reagents: (a) EDCÆHCl, HOBt/DMF; (b) anisole, 4 N HCl/
dioxane; (c) 20% diethylamine/DMF; (d) p-anisaldehyde dimethyl
acetal, pyridinium p-toluenesulfonate/toluene, reflux; (e) NH2SO2CF3,
EDCÆHCl, DMAP/DMF.
a
b
CF3
CF3
CF3
O2N
H2N
0
phobic inner wall of the S1 pocket, which consisted of
Tyr71, Pro70, Tyr198, Ile126, Ser35 (a and b-carbons),
Thr329, Ile226, and Val332. Hence, we considered
O
O
O
24
23
0
replacing the P1 acidic moieties with hydrophobic
groups, that could interact with the hydrophobic regions
c,d
0
Boc
N
COOH
H2N
CONHSO2CF3
of BACE1. To screen for non-acidic P1 moiety, we se-
HCl
H
lected previously reported inhibitor KMI-260 (see Table
1) containing a P4 glutamic acid 0residue as the reference
compound, and replaced its P1 -containing carboxylic
acid group with diverse moieties listed in Table 1. Com-
25
Scheme 2. Reagents: (a) NaNO3/concd. H2SO4; (b) H2, Pd–carbon/
MeOH; (c) NH2SO2CF3, EDCÆHCl, DMAP/DMF; (d) anisole, 4 N
HCl/dioxane.
0
pounds 1–3, in which the P1 carboxylic acid groups

Y. Hamada et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1649–1653
1651
0
Table 1. Modification at the P1 position of BACE1 inhibitor KMI-
260 possessing P4 glutamic acid
were removed or replaced by a phenolic hydroxyl group,
exhibited low BACE1 inhibitory activity. Replacing the
0
P1 benzene ring with other aromatic ring, 4 possessing a
carboxylic acid group exhibited potent BACE1 inhibi-
0
tory activity, although 5 with no acidic P1 moiety exhib-
ited low activity. Second, we introduced hydrophobic
O
O
O
H
H2N
N
R
N
H
N
N
0
groups (halogen or nitro group) on the P1 benzene ring.
Compounds 6–8 and 10 exhibited higher BACE1 inhib-
itory activity. However, 9 and 11, which possessed an
H
H
O
OH
O
OH
0
elongated side chain or two nitro groups at the P1 ben-
zene ring, respectively, exhibited moderate BACE1
Compound
(KMI No)
R
BACE1 inhibition
(%) at 2 lM
0
Table 2. Modification at the P1 position of BACE1 inhibitors KMI-
420 and KMI-429
KMI-260
84
53
54
COOH
1 (KMI-517)
2 (KMI-541)
O
O
O
H
H2N
N
R
N
N
N
H
H
H
O
OH
NH
N
OH
O
NH
N
N
3 (KMI-749)
4 (KMI-701)
60
94
Compound (KMI No)
R
BACE1 inhibition
(%) at
HO
COOH
CH3
2 lM
0.2 lM
87
S
N
KMI-420
KMI-429
See Figure 1
See Figure 1
Cl
99
COOEt
100
99
98
84
S
N
5 (KMI-694)
6 (KMI-491)
66
78
CH3
Cl
13 (KMI-758)
14 (KMI-761)
Cl
Cl
Cl
CF3
95
68
7 (KMI-477)
80
74
OH
CF3
Br
Cl
CF3
8 (KMI-735)
9 (KMI-752)
15 (KMI-811)
16 (KMI-818)
17 (KMI-773)
18 (KMI-819)
19 (KMI-880)
92
93
96
96
98
57
72
70
75
81
CF3
CF3
51
71
60
86
CF3
O
CF3
O
10 (KMI-806)
11 (KMI-817)
12 (KMI-845)
NO2
NO2
NO2
NO2
NO2
NO2
SO2CF3
CONHSO2CF3

1652
Y. Hamada et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1649–1653
inhibitory activity, suggesting that the spatial location of
0
0
Arg307
Gly11
Arg307
Gly11
the P1 moiety interacting with the S1 hydrophobic
regions of BACE1 might be important for improving
BACE1 inhibitory activity. On the other hand, com-
pound 12 possessing N-sulfonylcarboxamide group at
Lys321
Lys321
Thr232
Thr232
0
the P1 position showed higher BACE1 inhibitory activ-
ity. The sulfonylamino and carbonyl groups were
thought to form hydrogen bond interactions with the
Arg235
Arg235
Gly230
Asp32
Gly230
Asp32
Asp228
Asp228
0
S1 binding sites of BACE1. Moreover, the N-sulfonyl-
carboxamide group has a hydrophilic property, and
might function as a bioisostere of carboxylic acid.
Gln73
Gln73
Thr72
Thr72
Tyr198
Thr329
Tyr198
Thr329
0
From the above results, we replaced the P1 carboxylic
Arg128
Arg128
acid groups of inhibitors KMI-420 and -429 with halo-
gen atoms or nitro groups. As shown in Table 2, inhib-
itors 13–19 showed potent BACE1 inhibitory activities.
Inhibitor 13 showed especially potent BACE1 inhibitory
activities (BACE1 inhibition IC50: 14.0 nM). These re-
sults were consistent with our previous findings that
Figure 3. Stereoscopic view of superimposed inhibitors 13 (KMI-758,
yellow lines) and KMI-684 (red lines) in BACE1 (PDB ID: 1W51).
0
hydrophobic regions existed around the hydrophilic S1
Lys321
Arg307
Lys321
Arg307
binding sites of BACE1, as shown in Figure 2. Then,
we performed docking simulation experiments using a
BACE1 crystal structure (PDB ID: 1W51) and previ-
ously reported method.14,15 As shown in Figure 3, the
3D-structure of 13 docked in BACE1 was similar to that
of the docked structure of KMI-684, suggesting that
these halog0en atoms interacted with the hydrophobic re-
gions of S1 pocket.
Ser325
Ser325
Arg235
Arg235
Thr232
Thr232
Thr329
Thr329
Asp228
Gly11
Gly11
Asp228
Gly230
Thr72
Gly230
Thr72
Gln73
Gln73
Asp32
Asp32
0
We noted that Thr72 was near the P1 benzene ring, and
0
Tyr71
Tyr71
the P1 benzene ring could be replaced by other residues
0
(as exemplified by compounds 4 and 5). Hence, the P1
phenyl ring could be replaced by a hydrogen bond
acceptor that could form hydrogen bonds with Thr72.
BACE1 inhibitor 19 (KMI-880) in which we replaced
the P1 benzene ring with a trifluoromethanesulfonyl
group exhibited potent BACE1 inhibitory activity as
shown in Table 2 (BACE1 inhibition IC50: 29.9 nM).
Figure 4. Stereo view of docked inhibitor 19 (KMI-880, colored ball-
and-stick model) in BACE1 (PDB ID: 1W51, colored skeleton model).
0
A modeled structure of the docked inhibitor 19 in
BACE1 is shown in Figure 4. Hence, the important role
0
of the P1 sulfonyl group and P1 residues as a bridge
from Thr72 of the flap domain to the active site
(Asp32 and Asp228) on the cleft of BACE1 was
confirmed.
Lys321
Gly11
Gly264
Arg302
Thr329
Val312
Asn233
O
N
H2N
O
Arg235
Lys224
Ile226
Tyr198
N
Thr72
N
H
N
0
N
In conclusion, BACE1 inhibitors modified at the P1 po-
sition were designed, and we found potent BACE1
NH
Thr232
Gly230
0
S3
inhibitors 13 (KMI-758) having hydrophobic P1 moie-
0
O
pocket
ties and 19 (KMI-880) with a P1 sma0ll-sized residue.
HN
Gln73
These inhibitors with non-acidic P1 moieties are
thought to be a further step toward a practical anti-
AD’s drug.
Asp228
O
NH
Asp32
OH
N
Tyr71
Pro70
H
N
N
O
N
S1
pocket
N
Tyr198
Acknowledgments
Thr72
N
N
N
Arg128
N
Ser35
Ile126
This study was supported in part by the ‘Academic
Frontier’ Project for Private Universities, matching fund
subsidy and the 21st Century COE Program from
MEXT (Ministry of Education, Culture, Sports, Science
and Technology) of the Japanese Government, and
grants from MEXT. We thank T. Hamada for perform-
ing in vitro enzymatic assay.
KMI-684
Figure 2. A cartoon depiction of KMI-684 docked in BACE1 and a
co0mputer-assisted docking model of the vicinity of the inhibitor’s two
P1 tetrazole rings. Meshed surface model indicates BACE1 (green
mesh. hydrophobic surface; red mesh, hydrophilic surface).

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