Significance of interactions of BACE1-Arg235 with its ligands and design of BACE1 inhibitors with P2 pyridine scaffold

Article abstract of DOI:10.1016/j.bmcl.2009.03.049

Recently, we reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. Because these inhibitors contained some natural amino acids, we would need to improve their enzymatic stability in vivo and permeability across the blood-brain barrier, so that they become practically useful. Subsequently, non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold, 2,6-pyridenedicarboxylic, chelidamic or chelidonic moiety, at the P₂ position were reported. These inhibitors were designed based on the conformer of docked inhibitor in BACE1. In this study, we discuss the role and significance of interactions between Arg235 of BACE1 and its inhibitor in BACE1 inhibitory mechanism. Moreover, we designed more potent small-sized BACE1 inhibitors with a 2,6-pyridinedicarboxylic scaffold at the P₂ position, that were optimized for the interactions with Arg235 of BACE1.

Full text of DOI:10.1016/j.bmcl.2009.03.049

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2435–2439
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Significance of interactions of BACE1–Arg235 with its ligands
and design of BACE1 inhibitors with P2 pyridine scaffold
Yoshio Hamada a, Hiroko Ohta a, Naoko Miyamoto a, Diganta Sarma a, Takashi Hamada a,
Tomoya Nakanishi a, Moe Yamasaki a, Abdellah Yamani a, Shoichi Ishiura b, Yoshiaki Kiso a,
*
a Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science and 21st Century COE Program, Kyoto Pharmaceutical University,
Yamashina-ku, Kyoto 607-8412, Japan
b Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently, we reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymeth-
ylcarbonyl isostere as a substrate transition-state mimic. Because these inhibitors contained some
natural amino acids, we would need to improve their enzymatic stability in vivo and permeability across
the blood–brain barrier, so that they become practically useful. Subsequently, non-peptidic and small-
sized BACE1 inhibitors possessing a heterocyclic scaffold, 2,6-pyridenedicarboxylic, chelidamic or cheli-
donic moiety, at the P2 position were reported. These inhibitors were designed based on the conformer of
docked inhibitor in BACE1. In this study, we discuss the role and significance of interactions between
Arg235 of BACE1 and its inhibitor in BACE1 inhibitory mechanism. Moreover, we designed more potent
small-sized BACE1 inhibitors with a 2,6-pyridinedicarboxylic scaffold at the P2 position, that were opti-
mized for the interactions with Arg235 of BACE1.
Received 18 February 2009
Accepted 13 March 2009
Available online 18 March 2009
Keywords:
Alzheimer’s disease
BACE1
b-Secretase
BACE1 inhibitor
Ó 2009 Elsevier Ltd. All rights reserved.
Amyloid b (Ab peptide is a main component of senile plaques in
the brains of Alzheimer’s disease (AD) patients that is involved in
AD’s pathologies. According to the amyloid hypothesis,1 b-secre-
tase [BACE1: b-site APP (amyloid precursor protein) cleaving en-
zyme] appears promising as a molecular target for therapeutic
intervention in AD,2–6 because BACE1 triggers Ab peptide forma-
tion by cleaving APP at the N-terminus of the Ab domain.7–12 Pre-
viously, we reported potent peptidic BACE1 inhibitors13–17
(IC50 ꢀ 1.2 nM) with a hydroxymethylcarbonyl (HMC) isostere as
a substrate transition-state mimic.18,19 Among them, KMI-429
exhibited effective inhibition of BACE1 activity in cultured cells,
and significant reduction of Ab production in vivo14b (by direct
administration into the hippocampus of APP transgenic and wild-
type mice). Recently, in order to develop practical anti-AD’s drug,
non-peptidic and small-sized BACE1 inhibitors possessing a 2,6-
pyridinedicarboxylic, chelidamic or chelidonic residue at the P2 po-
sition were synthesized on the basis of in-silico design based on
the conformer of a virtual inhibitor docked in complex with BACE1
(Fig. 1).20 However, these non-peptidic compounds exhibited low-
er BACE1 inhibitory activities than our peptidic KMI-compounds.
In this paper, we discussed the role and significance of the interac-
tions between Arg235 of BACE1 and its inhibitors in BACE1 inhibi-
tion mechanism. Moreover, we found more potent small-sized
BACE1 inhibitors with a 2,6-pyridinedicarboxylic scaffold at the
P2 position that were optimized for interactions with Arg235 of
BACE1.
Although we have been considering the role of interaction be-
tween Arg235 of BACE1 and its ligands since we started this study,
it remained unclear for a long time. The side chain of Arg235 seems
to be peculiar, because it is only found outside of the opening of the
X
N
N
OH
H
N
H
NH
O
N
N
N
N
O
O
O
KMI-1023 (X = -H)
IC50 = 140 nM
KMI-1036 (X = -OSO2CH3) IC50 = 96 nM
O
X
N
N
OH
H
N
H
NH
O
N
N
N
O
O
O
KMI-1030 (X = NH) IC50 = 360 nM
KMI-1027 (X = O) IC50 = 50 nM
* Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591 9900.
E-mail address: kiso@mb.kyoto-phu.ac.jp (Y. Kiso).
Figure 1. Small-sized BACE1 inhibitors.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.049

2436
Y. Hamada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2435–2439
Figure 2. Interactions of BACE1–Arg235 with BACE1 inhibitors in X-ray crystal structures. Space-filling models represent BACE1 inhibitors and BACE1-Arg235. (A) PDB
ID:2P83. (B) PDB ID:2B8L. (C) PDB ID:2IQG. (D) PDB ID:1FKN. (E) PDB ID:1M4H.
active site formed by the flap domain of BACE1. Considering that
most other amino acid side-chains of BACE1, which can interact
with inhibitors or substrates, are found inside the active site be-
tween the flap domain and cleft region of BACE1, Arg235 may play
an important role in the inhibitory mechanism by inhibitors or in
the catalytic action on substrates. Kortum et al.21 suggested that
the amide carbonyl group at the P2 position of their compound
we hypothesized an important key point for considering the role of
interactions with Arg235: the side chain of Arg235 moves in con-
cert with the inhibitor’s size as shown in Figure 3. The guanidino
plane of Arg235 pushes down on the P2 region of the inhibitors,
causing them to be affixed in the active site of BACE1. We specu-
lated that a slightly attractive force such as stacking interactions,
which cannot be accurately estimated by common molecular
mechanics calculations and molecular design programs, played a
significant role for packing down the inhibitors effectively in the
active site of BACE1. Careful attention should be paid to such weak
quantum effects, because softwares tend to calculate all atoms as
rigid bodies. Although strong interactions such as hydrogen bond-
ing might activate the apparent enzyme-catalysis for its substrate
by improving the ‘turn-over’ of the enzyme, such strong attractive
forces is unfavorable for the action of inhibitors. In fact, our KMI-
compounds with hydrogen-bond acceptor or donor, such as aspar-
agine, at the P2 position showed low inhibitory activities.5 The fact
that our KMI-compounds with an asparagine residue, in which a
Swedish mutant APP possessing a high affinity for BACE1 was
coded at the same P2 position, showed disappointing results, seems
to support our hypothesis.
The superimposed views of some crystal structures around the
active site of BACE1 are shown in Figure 3A and B. The side chain of
BACE1’s Arg235 has a restricted range of motion. The surface of the
b sheet structure that consists of four peptide strands behind
BACE1-Arg235 is depicted in Figure 3C, suggesting that the side
chain of BACE1-Arg235 slides sideway, not up and down, along
the wall of this b-sheet structure. We hypothesized that weak
interactions such as stacking halted the sideway motions of
BACE1-Arg235, thereby fixing the BACE1-Arg235 above the P2
region of the inhibitor.
(Pfizer’s inhibitor) interacted by O-
p stacking with Arg235 of
BACE1 from its X-ray crystal structure (PDB ID: 2P83) as shown
in Figure 2A. The Arg235 found in BACE1–Merck’s inhibitor com-
plex (PDB ID:2B8L) showed a similar figure. Arg235 is 2.8 Å from
the N-methyl group at the P2 position of the inhibitor, suggesting
that they may interact by CH-
p stacking as shown in Figure 2B.
The Arg235 in BACE1–Elan’s inhibitor complex (PDB ID:2IGQ) also
assumed a similar pose as shown in Figure 2C. We compared the
publicly-available X-ray crystal structures of BACE1–inhibitor
complexes to understand the role of the interactions between
BACE1–Arg235 and the P2 moieties of inhibitors and found a sur-
prising fact that the guanidino group of Arg235 in these crystal
structures showed the similar figures flopping over the P2 region
of the inhibitors. Moreover, the nearest distances between the gua-
nidino-plane of Arg235 and the P2 region showed similar values of
about 3 Å, in the X-ray crystal structures of most BACE1–inhibitor
complexes (Table 1). However, the Arg235 of the BACE1–OM99-2
complex (PDB ID:IFKN) assumed an exceptionally different pose
from most crystal structures of BACE1 complexes (Fig. 2D). The
side chain of this Arg235 seemed to be repelled by the P2 aspara-
gine residue of OM99-2, because this P2 region interacted with
the P4 glutamic residue by intramolecular hydrogen bonding and
cannot interact with Arg235 by stacking. OM00-3 is an inhibitor
that is structurally similar to OM99-2. However, the Arg235 in a
BACE1 complex (PDB ID:1M4H) with OM00-3 assumed a pose that
is similar to Pfizer’s, Merck’s and Elan’s inhibitors, and not to
OM99-2, because OM00-3 had no intramolecular hydrogen bond
such as OM99-2 as shown in Figure 2E.
We speculated that an electron-rich halogen atom could
interact with the electron-poor guanidino-
p orbital by Coulomb’s
force. If these interactions also involve charge transfer interac-
tions (CT) with directing effects, it is expected that the effects
are similar to stacking interactions with directing effects. Hence,
we envisioned the BACE1 inhibitors with a halogen atom at the
P2 position.
From the fact that the guanidino plane of Arg235 in the crystal
structures of most BACE1 complexes show similar distances from
the P2 region of the inhibitors regardless of their variety and sizes,

Y. Hamada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2435–2439
2437
Table 1
The distances of BACE1–Arg235 from the P2 parts of inhibitors
PDB ID
The distance (Å) from P2 part
The closest P2 atom to guanidino-plane
*
1
Na
Nb
Nc
Plane
*
2
2P83
2B8L
2P8H
2PH6
20AH
2QZL
2P4J
3.2
2.9
2.9
2.7
(2.8)
(3.0)
(2.8)
(2.8)
(2.8)
(2.7)
(2.9)
(2.8)
(3.0)
(2.9)
(2.9)
2.4
=O (CONH2)
*
3
*
3
*
3
4.6 (3.7)
4.8 (3.8)
4.7 (3.8)
4.1 (3.1)
4.5 (3.6)
4.4 (4.0)
4.8 (3.9)
4.8 (3.9)
4.8 (3.9)
4.3 (3.4)
4.3 (4.1)
2.7
4.6 (3.7)
4.7 (3.7)
4.4 (3.4)
4.2 (3.4)
4.5 (3.7)
3.6 (3.3)
4.5 (3.5)
4.5 (3.5)
4.5 (3.7)
4.6 (3.6)
5.1 (3.4)
2.7
3.9 (2.8)
4.1 (2.8)
3.9 (2.8)
3.5 (2.9)
3.7 (2.8)
3.5 (2.9)
4.0 (2.9)
3.9 (2.8)
4.1 (3.0)
3.8 (2.7)
3.9 (3.2)
2.9
N-Methyl
N-Methyl
N-Methyl
N-Methyl*3
N-Methyl*3
N-Methyl*3
N-Methyl*3
2IRZ
2IS0
*
3
*
3
*
3
N-Methyl
N-Methyl
N-Methyl
2QK5
2B8V
1M4H
2HM1
1TQF
1YM2
2G94
2HIZ
1XS7
2IQG
2QK5
2QP8
3CIB
–COOH (OM00-3)
Pyridine ring
4.8
4.2
4.3
2.9
4.8
3.9
3.4
2.6
3.3
3.5
4.3
3.1
2.7
3.5
3.3
2.5
=O(C6H5CH2SO2)
–S–(methionine)
=O(CH3SO2CH2–)
Methylene proton
Carbonyl (amide)
*
4
3.8
3.1
2.6
2.6
*
*
5
4.4 (3.6)
4.8 (3.8)
4.2 (3.1)
4.4 (3.4)
4.3 (3.3)
4.2 (3.1)
4.2 (3.2)
4.2 (3.2)
4.0 (3.0)
3.21
4.9
4.7
4.2
4.3
4.9
4.9
5.6 (4.6)
5.0 (4.1)
3.9 (3.2)
4.5 (3.6)
4.4 (3.6)
4.5 (3.6)
4.4 (3.5)
4.4 (3.5)
4.4 (3.5)
4.3 (3.4)
4.1 (3.4)
4.0
3.7
4.0
4.5
3.7
4.2
4.3
5.1 (3.4)
4.7 (3.7)
4.1 (2.8)
4.1 (3.1)
3.9 (3.0)
4.2 (3.1)
4.0 (3.0)
4.1 (3.0)
4.0 (3.1)
3.9 (2.9)
3.7 (2.8)
4.0
3.9
3.4
3.7
2.9
3.6
3.7
4.3 (3.4)
4.2 (3.5)
(3.0)
(3.1)
(2.8)
(3.0)
(2.9)
(2.9)
(2.9)
(2.8)
(2.7)
3.2
3.1
3.0
3.7
2.6
3.3
3.6
(2.7)
(3.3)
Methyl
Methyl
5
Methyl*5
Methyl*5
Methyl*5
3CIC
*
5
*
5
*
5
*
5
3CID
2QMD
2QMF
2QMG
1W51
2FDP
2VIE
2VJ9
2VIZ
2VNM
2VIJ
2VNN
2PH8
Methyl
Methyl
Methyl
Methyl
Isophthalic ring*6
Isophthalic ring*6
Pyrrolidone ring
Pyrrolidone ring
Pyrrolidone ring
Butanesultan ring
Butanesultan ring
N-Methyl
N-Methyl*3
The values in parenthesis show the distances of BACE1-Arg235 from the nearest protons at the P2 position of the inhibitors.
Na: f-Imino nitrogen atom (flap side).
Nb: f-Imino nitrogen atom (cleft side).
Nc: d-Imino nitrogen atom.
*
1: The distance to guanidino-plane from the P2 region of inhibitor.
*
*
*
*
2: Pfizer’s inhibitor.
3: N-Methyl-N-methanesulfonamide group.
4: Benzyloxycarbonyl group.
5: 5-Methylisophthalamide.
*
6: An isophthalic-ring’s proton.
Figure 3. Locations of BACE1–Arg235s in their respective crystal structures. (A) Ser325, Val332 and Arg235 residues from the four crystal structures were superimposed. (B)
Superimposed view of three crystal structures around the active site of BACE1. The blue surface model represents the flap domain. The space-filling model behind the flap
domain represents BACE1 inhibitor KMI-1023. The blue, yellow and red space-filling models represents the side chain of BACE1–Arg235 in the crystal structures of BACE1,
2B8L, 2IQG and 1W51, respectively. (C) The surface of the b sheet structure behind the BACE1–Arg235 is depicted.
BACE1 inhibitors 1–6 were synthesized to connect in tandem
with the blocks corresponding to the P3–P2 residues and P1–P1 res-
idues, respectively, as shown in Scheme 1. Amide bonds were
formed by common solution-phase synthesis methods using 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimideꢁHCl (EDCꢁHCl) in
the presence of 1-hydroxybenzotriazole (HOBt) as coupling agents.
P3 residues 9a and 9b were synthesized from (S)-phenylglycinol
and N-Boc-4’-fluoro-(S)-phenylglycine methyl ester, respectively,
0

2438
Y. Hamada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2435–2439
Table 2
BACE1 inhibitory activities
X
N
N
OH
N
H
N
NH
H2N
N
X
N
O
OH
Y
O
N
N
OH
H
N
H
N
O
NH
Y
O
N
N
O
N
O
O
8
7a X = Cl, Y = H 7d X = Cl, Y = F
7b X = Br, Y = H 7e X = Br, Y = F
7c X = I, Y = H
O
7f X = I, Y = F
X
N
Compound (KMI-No)
X
Y
BACE1 inhibition %
IC50 (nM)
N
N
OH
H
N
H
N
At 2 nM
At 0.2 nM
NH
O
a
Y
N
KMI-1023
KMI-1036
–H
–OSO2CH3
–H
–H
–H
–H
–H
–F
89
96
98
99
99
99
99
99
62
73
87
88
86
91
93
92
140
96
22
15
24
13
9
N
O
O
1 (KMI-1237)
2 (KMI-1256)
3 (KMI-1293)
4 (KMI-1283)
5 (KMI-1303)
6 (KMI-1302)
–Cl
–Br
–I
–Cl
–Br
–I
O
1 X = Cl, Y = H 4 X = Cl, Y = F
2 X = Br, Y = H 5 X = Br, Y = F
3 X = I, Y = H
–F
–F
6 X = I, Y = F
10
Scheme 1. Reagents: (a) HOBt, EDCꢁHCl/DMF.
as shown in Scheme 2A, and P2 residues 13–15 with a halogen
atom were synthesized from chelidamic acid as shown in Scheme
2B. The P2 and P3 residues were coupled to afford P3–P2 blocks 7a–
0
7f. The P1–P1 block 8 was prepared according to previously re-
ported procedures.13–17,20 All of the inhibitors were purified by
preparative RP–HPLC. BACE1 inhibitory activity of the inhibitors
was determined by enzymatic assay using a recombinant human
BACE1 and FRET (fluorescence resonance energy transfer) sub-
strate as previously reported.5
Synthetic compounds 1–3 showed potent BACE1 inhibitory
activities as shown in Table 2. From our previous study, introduc-
ing a fluorine atom at the P3 benzene ring of inhibitors improves
BACE1 inhibitory activity (data not shown). Hence, we designed
BACE1 inhibitors 4–6 with a fluorine atom at the P3 benzene ring.
These BACE1 inhibitors exhibited more potent BACE1 inhibitory
activities. The fact that an introduction of a halogen atom at the
P2 position in our compounds drastically improved BACE1 inhibi-
tory activity, seems to support our hypothesis.
A
X
H
a
N
OH
O
H2N
9a X = -H
9b X = -F
a
F
F
F
c
b
O
OH
OH
Boc
N
Boc N
H2N
H
H
O
11
10
In conclusion, from the results of our in-silico study using pub-
licly-available crystal structures of BACE1–inhibitor complexes, we
proposed a hypothesis concerning the role and significance of the
interactions of BACE1–Arg235 with the inhibitors in BACE1 inhib-
itory mechanism. Next, we designed more potent and small-sized
BACE1 inhibitors that were optimized for interactions between
the inhibitors and Arg235 of BACE1 based on our hypothesis. More-
over, potent BACE1 inhibitors KMI-1283 (IC50 = 13 nM), KMI-1303
(IC50 = 9 nM) and KNI-1302 (IC50 = 10 nM) were found by introduc-
ing a fluorine atom at the P3 benzene ring. These halogenated
inhibitors with higher lipophilicity are expected improved mem-
brane permeability and bioavailability. Thus our study brings us
one step closer to developing practical anti-AD’s drugs.
B
O
Cl
d
HO
OH
O
O
N
H
N
O
O
O
O
12
Cl
f,e
e
7a,7d
7b,7e
O
OH
N
O
O
13
O
Br
f
g
HO
OH
HO
OH
N
H
Acknowledgements
N
O
O
O
O
O
14
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, Cul-
ture, Sports, Science and Technology) of the Japanese Government,
and grants from MEXT. We thank Dr. Jeffrey-Tri Nguyen for his
help in preparing the manuscript.
Cl
I
f
h
O
O
7c,7f
HO
OH
N
N
O
O
O
12
15
References and notes
Scheme 2. Reagents and conditions: (a) HCHO, water.;(b) LiBH4/MeOH; (c) 4 NꢁHCl/
dioxane; (d) SOCl2, then MeOH; (e) 1 N NaOH/MeOH; (f) 9a or 9b, HOBt, EDCꢁHCl/
DMF; (g) tetra-n-butylammonium bromide, P2O5, THF, reflux; (h) phosphorous acid,
57% HI aq, 80 °C.
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