Tripeptidic BACE1 inhibitors devised by in-silico conformational structure-based design

Article abstract of DOI:10.1016/j.bmcl.2011.11.102

Previously reported pentapeptidic BACE1 inhibitors, designed using a substrate-based approach, were used as lead compounds for the further design of non-peptidic BACE1 inhibitors. Although these peptidic and non-peptidic inhibitors, with a hydroxymethylcarbonyl isostere as a substrate transition-state mimic, exhibited potent BACE1 inhibitory activities, their molecular-sizes appeared a little too big (molecular weight of >600 daltons) for developing practical anti-Alzheimer's disease drugs. To develop lower weight BACE1 inhibitors, a series of tripeptidic BACE1 inhibitors were devised using a design approach based on the conformation of a virtual inhibitor bound to the BACE1 active site, also called 'in-silico conformational structure-based design'. Although these tripeptidic BACE1 inhibitors contained some natural amino acid residues, they are expected to be useful as lead compounds for developing the next generation BACE1 inhibitors, due to their low molecular size and unique structural features compared with previously reported inhibitors.

Full text of DOI:10.1016/j.bmcl.2011.11.102

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1130–1135
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tripeptidic BACE1 inhibitors devised by in-silico conformational
structure-based design
Yoshio Hamada a,b, , Harichandra D. Tagad b, Yoshinori Nishimura b, Shoichi Ishiura c, Yoshiaki Kiso a,b,d,
⇑
⇑
a Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Minatojima, Chuo-ku, Kobe 650-8586, Japan
b Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
c Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
d Department of Bio-Science, Nagahama Institute of Bio-Science and Technology, Tamura-cho, Nagahama 526-0829, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Previously reported pentapeptidic BACE1 inhibitors, designed using a substrate-based approach, were
used as lead compounds for the further design of non-peptidic BACE1 inhibitors. Although these peptidic
and non-peptidic inhibitors, with a hydroxymethylcarbonyl isostere as a substrate transition-state
mimic, exhibited potent BACE1 inhibitory activities, their molecular-sizes appeared a little too big
(molecular weight of >600 daltons) for developing practical anti-Alzheimer’s disease drugs. To develop
lower weight BACE1 inhibitors, a series of tripeptidic BACE1 inhibitors were devised using a design
approach based on the conformation of a virtual inhibitor bound to the BACE1 active site, also called
‘in-silico conformational structure-based design’. Although these tripeptidic BACE1 inhibitors contained
some natural amino acid residues, they are expected to be useful as lead compounds for developing the
next generation BACE1 inhibitors, due to their low molecular size and unique structural features
compared with previously reported inhibitors.
Received 7 October 2011
Accepted 25 November 2011
Available online 1 December 2011
Keywords:
Alzheimer’s disease
b-Secretase
BACE1
BACE1 inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
Amyloid b peptide (Ab), the main component of senile plaques
in the brains of Alzheimer’s disease (AD) patients,1 is formed by
proteolysis of amyloid precursor protein (APP).2,3 As b-site APP
cleaving enzyme 1 (BACE1, also known as b-secretase) triggers
Ab formation by cleavage at the Ab domain N-terminus,4–7 it is a
molecular target for AD therapeutic intervention.8–11 In recent
reports on pentapeptidic and non-peptidic BACE1 inhibitors,11–16
peptidic inhibitors KMI-420, -429, -684, and -574 (Fig. 1), which
contained phenylnorstatine (Pns: (2R,3S)-3-amino-2-hydroxy-4-
phenylbutyric acid) as a substrate transition-state mimic, showed
potent BACE1 inhibitory activities.17,18 Of these, KMI-429 exhibited
effective activity in cultured cells and a significant reduction of Ab
production in vivo (by direct administration into APP transgenic
and wild-type mice hippocampi).13 However, some natural amino
acid residues in these inhibitors appear necessary for the enzy-
matic stability in vivo and blood–brain barrier permeability
needed to be practical drugs. Accordingly here, non-peptidic BACE1
inhibitors were designed which possessed a heterocyclic scaffold,
such as a chelidamic or 2,5-pyridinedicarboxylic moiety, at the P2
position using ‘in-silico conformational structure-based design’.19
Furthermore, through focusing on interaction with the BACE1-
Arg235 side chain, the non-peptidic inhibitor KMI-1303, possess-
ing a halogen atom on the P2-pyridine scaffold, was designed and
found to be a potent inhibitor (Fig. 2). Although these pentapepti-
dic and non-peptidic BACE1 inhibitors showed potent inhibitory
activities, their molecular weights were considered a little high
(>600 daltons) for practical anti-AD drugs. With the goal of devel-
oping low molecular weight and thus smaller inhibitors, the loca-
tion of two catalytic Asp residues in BACE1 were examined. As all
of the BACE1 active site pockets are created by closure of the flap
domain (Fig. 3A), effective, potential inhibitors might involve fixing
the ‘closed-form’ of the flap domain. Additionally, the two catalytic
Asp residues are located at some distance from the flap domain
(Fig. 3B). Thus, the interactions of the P01 region and substrate tran-
sition-state analogue with the corresponding pockets of BACE1
were hypothesized to be unnecessary for fixing the flap domain.
As most existing BACE1 inhibitors have been designed to optimize
interactions with the flap domain and the two catalytic Asp resi-
dues, they have naturally had high molecular weights to span the
separation. The possible design of an inhibitor lacking the P01 posi-
tion and substrate transition-state analogue was speculated here
such that it could stabilize the folding pose around the inhibitor’s
P1 phenyl group (Fig. 4B). Previously reported from here, a design
tool focused on the conformation of a virtual inhibitor bound in
the BACE1 active site was used to allow the synthesis of non-pep-
tidic BACE1 inhibitors.19 The well known concept that the stability
of a specific conformer of a medicine is crucially important for
proper drug effect has been employed in computational studies
for drug design. In fact, common molecular dynamics calculations
⇑
Corresponding authors. Tel.: +81 749 64 8113; fax: +81 749 64 8140.
E-mail address: y_kiso@nagahama-i-bio.ac.jp (Y. Kiso).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.102

Y. Hamada et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1130–1135
1131
tool for identifying drug candidates. In this study, the proposition
of how to design low molecular weight inhibitors lacking a P01 moi-
ety and substrate transition-state analogue was solved by seeking
to stabilize the folding structure around the P1-phenyl ring of a vir-
tual inhibitor. For steric energy calculations, low molecular weight
model compounds were used to exclude the effect of their intra-
molecular interactions in solution (Fig. 4A), because an inhibitor
docked in the enzyme’s active site generally interacts with the
pocket of enzyme, not within the inhibitor molecule. Dihedral
energy plots were constructed by rotating two covalent bonds, be-
tween the C-terminus amide bond and its side-by-side carbon
atoms, of virtual compounds under an MMFF94x force field, using
MOE software (Chemical Computing Group Inc., Canada). The opti-
mal modification around the P1 region of virtual inhibitors was
then screened using a folding conformer by calculating their steric
energy. A virtual inhibitor with two methyl groups at the P1
position was found that showed a folding structure around the
P1-phenyl ring and a most-stable conformer which overlapped
the red star symbol in the dihedral energy plot chart corresponding
to an inhibitor docked on the enzyme (Fig. 4A); the folding struc-
ture appeared likely to be stabilized by a gem-dimethyl effect. A
series of inhibitors were then designed which had a folding config-
uration around the P1-phenyl ring.
R1
O
O
O
H
H2N
N
N
N
N
R2
H
H
H
O
OH
NH
N
KMI-420 (R1 = H, R2 = COOH) IC50 = 8.2 nM
KMI-429 (R1 = R2 = COOH)
O
NH
IC50 = 3.9 nM
IC50 = 1.2 nM
N
N
N
KMI-684 (R1 = R2
=
)
NH
N
N
O
O
O
H
H2N
N
N
N
N
N
NH
H
H
H
O
OH
N
N
HN
O
F
O
HN
NH
KMI-574 IC50 = 5.6 nM
O
Figure 1. Pentapeptidic BACE1 inhibitor structures.
BACE1 inhibitors 1–38 and 53–57 were synthesized by tradi-
tional solution-phase peptide synthesis methods, similar to previ-
ously reported procedures, from amines corresponding to the P1
residue as starting materials and N-protected amino acids. Peptide
bonds were formed sequentially using 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimideꢀHCl (EDCꢀHCl) in the presence of 1-
hydroxybenzotriazole (HOBt) as a coupling agent.11–16 For exam-
ple, the synthesis of inhibitor 6 began with 2-methyl-1-phenylpro-
pane-2-amine as the P1-amine followed by peptide bond formation
with selected amino acids and then protecting group removal
(Scheme 1). Other P1-amines were prepared from 2-methyl-1-phe-
nylpropane-2-amine 39 or phenylacetic acids 44a–e (Scheme 2). In
particular, amines 41–43 were prepared from amine hydrochloride
40 by bromination, nitration and iodination, respectively, and
amine 52 was prepared from amine 39 by catalytic hydrogenation
using Adams’ catalyst. Inhibitors 11–17, 20, 23–32, 35, and 36 were
synthesized from P2 – P1 blocks 50a–b or 51a–e in a similar man-
ner to above. P2–P1 blocks 50a–b or 51a–e were synthesized from
alcohols 46a–e, previously prepared from phenylacetic acids 44a–
e and nitriles 49a–b. The latter two groups of reactants were
prepared from corresponding Fmoc-protected amino-acid amides
48a–b, using the Ritter reaction and the ensuing Fmoc-deprotec-
Figure 2. BACE1 inhibitor structures with a halogen atom on the P2 aromatic ring.
or docking simulation software produce the potential energy val-
ues of drug molecules using descriptors regarding steric energy.
On the other hand, the design tool used here, ‘in-silico conforma-
tional structure-based design’, is employed as a problem-solving
Figure 3. (A) Superimposed flap domain of ‘open-form’ (PDB ID: 1W50) and ‘close-form’ (PDB ID: 2B8L) of BACE1. (B) KMI-429 docked in BACE1 enzyme (PDB ID: 2B8L).

1132
Y. Hamada et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1130–1135
Figure 4. Design of tripeptidic BACE1 inhibitors. (A) Optimization of virtual inhibitor using in-silico conformational structure-based design, red star indicates configuration of
inhibitor docked in BACE1. (B) Design of tripeptidic BACE1 inhibitor 3 (KMI-1564) from KMI-429 as parent compound.
tion. All inhibitors were purified by preparative reversed phase–
high performance liquid chromatography and their BACE1 inhibi-
tory activity determined by an enzymatic assay using a recombi-
H2N
Boc-Cha-OH
a,b
nant human BACE1 and fluorescence resonance energy transfer
substrate as previously reported.11–16,19,20
a,b
Inhibitor 3 (KMI-1564), containing the same P2–P4 residues as
KMI-429, showed lower BACE1 inhibitory activity (70% inhibition
a,c
Boc-Val-OH
at 2 lM) than KMI-429 (100% inhibition at 2 lM, IC50 = 3.9 nM)
Boc-DAP(Fmoc)-OH
O
a,b
as shown in Table 1. With the reduction of interacting sites be-
tween inhibitor and BACE1 allowed by reducing the molecular
weight (ꢁ70% compared with KMI-429), it appeared that inhibitor
3 and KMI-429 should have had similar affinity for the active site
per unit molecular weight. Furthermore, the tripeptidic BACE1
inhibitor 6 (KMI-1608), designed for optimization at the P4 and
P2 position, was found to be potent.
6 (KMI-1608)
F
HN
O
N
COOH
H
Scheme 1. Reagents and conditions: (a) EDCꢀHCl, HOBt/DMF; (b) 4 N HCl/dioxane;
(c) 20% diethylamine/dioxane.
I
I
R
d
a
H2N
Cl H3N
H2N
H2N
b
39
43
40
c
41 (R = Br)
42 (R = NO2)
O
O
O
R
R
R
e
f
R
HO
HO
i,j
H
AA N
45a (R = p-methyl)
45b (R = o-methyl)
45c (R = p-methoxy)
45d (R = m-methoxy)
45e (R = 3,5-difluoro)
46a (R = p-methyl)
46b (R = o-methyl)
46c (R = p-methoxy)
46d (R = m-methoxy)
46e (R = 3,5-difluoro)
44a (R = p-methyl)
44b (R = o-methyl)
44c (R = p-methoxy)
44d (R = m-methoxy)
44e (R = 3,5-difluoro)
H
50a (AA = Leu, R = p-methyl)
50b (AA = Leu, R = o-methyl)
50c (AA = Leu, R = p-methoxy)
50d (AA = Leu, R = m-methoxy)
50e (AA = Leu, R = 3,5-difluoro)
51a (AA = Cha, R = p-methyl)
51b (AA = Cha, R = o-methyl)
51c (AA = Cha, R = p-methoxy)
51d (AA = Cha, R = m-methoxy)
51e (AA = Cha, R = 3,5-difluoro)
H
g
Fmoc
N
CN
h
Fmoc AA NH2
Fmoc AA OH
47a (AA = Leu)
47b (AA = Cha)
48a (AA = Leu)
48b (AA = Cha)
R
49a (R = isopropyl)
49b (R = cyclohexyl)
k
H2N
H2N
39
52
Scheme 2. Reagents and conditions: (a) 4 N HCl/dioxane; (b) Br2, H2O; (c) HNO3, H2SO4; (d) I2, NaIO4, H2SO4; (e) SOCl2, MeOH; (f) CH3MgI, Et2O; (g) NH4HCO3, EDCꢀHCl, DMF;
(h) (TFA)2O, pyridine, THF; (i) conc. H2SO4, CH3COOH; (j) 20% Et2NH/DMF; (k) H2, PtO2, AcOH.

Y. Hamada et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1130–1135
1133
Table 1
Tripeptidic BACE1 inhibitors
H
AA1 Val AA2 N
H
Compound (KMI-No.)
AA1a
AA2b
BACE1 inhibition % at 2 lM
1 (KMI-1563)
2 (KMI-1606)
3 (KMI-1564)
4 (KMI-1607)
5 (KMI-1565)
6 (KMI-1608)
7 (KMI-1556)
8 (KMI-1609)
Glu
Glu
A
A
B
B
C
C
Leu
Cha
Leu
Cha
Leu
Cha
Leu
Cha
<10
16
70
76
90
92
77
80
a
Amino acids:
O
OH
N
N
F
NH
NH
O
O
O
N
N
O
H
NH
NH
O
NH OH
N
N
N
H
H
H
O
O
Figure 5. Docking simulation illustrations. (A) Substrate transition-state of APP
(wild-type) docked in BACE1. (B) Inhibitor 5 (KMI-1565) docked in BACE1.
A
B
C
b
Cha: L-cyclohexylalanine.
Table 2
Tripeptidic BACE1 inhibitors possessing substituents on the P1-phenyl ring
OH
O
NH OH
Val AA
R
N
H2N
H
O
Compound (KMI-No.)
AA
R
BACE1 inhibition % at 2 lM
9 (KMI-1694)
10 (KMI-1718)
11 (KMI-1724)
12 (KMI-1887)
13 (KMI-1879)
14 (KMI-1866)
15 (KMI-1881)
16 (KMI-1883)
17 (KMI-1885)
18 (KMI-1726)
19 (KMI-1778)
20 (KMI-1897)
Leu
Cha
Cha
Leu
Cha
Leu
Cha
Leu
Cha
Leu
Cha
Leu
p-Nitrophenyl
p-Nitrophenyl
45
80
81
51
54
41
45
46
49
78
88
59
p-Bromophenyl
p-Methylphenyl
o-Methylphenyl
p-Methoxyphenyl
p-Methoxyphenyl
m-Methoxyphenyl
m-Methoxyphenyl
2,4-Diiodophenyl
2,4-Diiodophenyl
3,5-Difluorophenyl
O
F
NH
O
N
O
H
NH
R
Val AA
N
H
H2N
O
21 (KMI-1693)
22 (KMI-1717)
23 (KMI-1719)
24 (KMI-1723)
25 (KMI-1886)
26 (KMI-1895)
27 (KMI-1840)
28 (KMI-1878)
29 (KMI-1865)
30 (KMI-1880)
31 (KMI-1882)
32 (KMI-1884)
Leu
Cha
Leu
Cha
Leu
Cha
Leu
Cha
Leu
Cha
Leu
Cha
p-Nitrophenyl
p-Nitrophenyl
27
89
85
92
86
88
91
84
83
85
84
77
p-Bromophenyl
p-Bromophenyl
p-Methylphenyl
p-Methylphenyl
o-Methylphenyl
o-Methylphenyl
p-Methoxyphenyl
p-Methoxyphenyl
m-Methoxyphenyl
m-Methoxyphenyl
(continued on next page)

1134
Y. Hamada et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1130–1135
Table 2 (continued)
Compound (KMI-No.)
AA
R
BACE1 inhibition % at 2 lM
33 (KMI-1725)
34 (KMI-1777)
35 (KMI-1896)
36 (KMI-1898)
37 (KMI-1842)
38 (KMI-1843)
Leu
Cha
Leu
Cha
Leu
Cha
2,4-Diiodophenyl
2,4-Diiodophenyl
3,5-Difluorophenyl
3,5-Difluorophenyl
Cyclohexyl
91
96
86
91
91
88
Cyclohexyl
Figure 6. (A) Configuration of cyclic amides. (B) Design of tripeptidic BACE1
inhibitor possessing a cyclic amine at the C-terminus.
Figure 7. 3D model of inhibitor 54 (KMI1862) docked in BACE1.
within the molecule. Inhibitor 34 (KMI-1777), with a diiodosubsti-
tuted P1-phenyl ring, exhibited the most potent BACE1 inhibitory
activity; the halogen atoms seemed to have enhanced inhibitory
activity by interacting with the hydrophobic S1 pocket.
Table 3
BAC1 inhibitors with a cyclic amine at the C-terminus
O
Based on the finding that the folding configuration around the
P1 phenyl ring of BACE1 inhibitors was important, an inhibitor
was envisioned which contained a cyclic amine at its C-terminus.
As the configurations of some acetyl cyclic amides examined under
the MMFF94x force field showed that middle-sized cyclic amides
possessed curved structures (Fig. 6A), inhibitors containing a cyclic
amine were expected to have a folded configuration (Fig. 6B). Thus,
tripeptidic BACE1 inhibitors 53–57 were designed with a cyclic
amine at the C-terminus (Table 3), and inhibitor 54 (KMI-1862),
possessing a six-membered cyclic amine, showed the highest
BACE1 inhibitory activity. In the docking simulation model under
the MMFF94x force field, inhibitor 54 appeared situated to interact
effectively with the BACE1 hydrophobic S1–S3 pocket (Fig. 7).
In conclusion, tripeptidic BACE1 inhibitors, designed on the ba-
sis of the conformational structure of inhibitors docked in BACE1,
revealed the presence of two different types of BACE1 inhibitors,
typified by inhibitors 34 (KMI-1777) and 54 (KMI-1862). These
inhibitors showed potent inhibitory activities and had lower
molecular weights (ꢁ500 daltons) than previously reported inhib-
itors (>600 daltons). Although these tripeptidic BACE1 inhibitors
contained some natural amino acid residues, they are expected
to have potential as lead compounds for developing the next gen-
eration BACE1 inhibitors, because of their low molecular weight.
F
NH
O
N
H
O
NH
O
H
n
N
N
H2N
N
H
O
O
Compound (KMI-No.)
n
BACE1 inhibition % at 2 lM
53 (KMI-1861)
54 (KMI-1862)
55 (KMI-1802)
56 (KMI-1803)
57 (KMI-1804)
1
2
3
4
5
88
91
89
85
81
To better understand the observation that inhibitors 3–8
showed potent inhibitory activities, although it lacked a substrate
transition-state analogue, a docking simulation study was per-
formed under the MMFF94x force field using MOE software. In
examining a docked model of the substrate transition-state of
wild-type APP in BACE1 (Fig. 5A), it was apparent that two hydro-
xyl groups on the substrate’s cleavage site interacted with two
catalytic Asp side chains. As one oxygen atom of the cleavage site
of substrate transition-state derives from water in BACE1, there
should be a space corresponding to a water molecule between
the substrate and catalytic site. In the docked model of inhibitor
5 (KMI-1565, Fig. 5B), one of two methyl groups at the C-terminus
of 5 may have bound to the active site by displacing a water mol-
ecule in the catalytic site.
Next, tripeptidic BACE1 inhibitors 9–38 were synthesized with
modifications at the C-terminus (Table 2) and, although most
inhibitors with a substituent on the P1-phenyl ring showed similar
inhibitory activities to their corresponding related inhibitors 5–8,
inhibitor 9 with a nitro group on the P1-phenyl ring and a P2-Leu
residue showed low inhibitory activity. The nitro group of inhibitor
9 might have interacted with the hydrophobic leucine side chain
Acknowledgments
This study was supported in part by the Grants-in-Aid for Scien-
tific Research (A) and (C) from MEXT (Ministry of Education,
Culture, Sports, Science and Technology), Japan. (KAKENHI No.
21249007 and No. 23590137, respectively) We thank T. Hamada
and Dr. J.-T. Nguyen for performing the in vitro enzyme assays
and his help in preparing the manuscript, respectively.
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