Structure-activity relationship study of BACE1 inhibitors possessing a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P2 position

Article abstract of DOI:10.1016/j.bmcl.2013.12.007

We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited potent activities in enzymatic and cellular assays (KMI-4

Full text of DOI:10.1016/j.bmcl.2013.12.007

Bioorganic & Medicinal Chemistry Letters 24 (2014) 618–623
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study of BACE1 inhibitors possessing
a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P2 position
b
b
b
b
Yoshio Hamada a,b, , Kenji Suzuki , Tomoya Nakanishi , Diganta Sarma , Hiroko Ohta ,
⇑
Ryoji Yamaguchi b, Moe Yamasaki b, Koushi Hidaka 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 Laboratory of Peptide 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:
We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a
hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited
potent activities in enzymatic and cellular assays (KMI-429 in particular inhibited Ab production
in vivo), these inhibitors contained some natural amino acids that seemed to be required to improve
enzymatic stability in vivo and permeability across the blood–brain barrier, so as to be practical drug.
Recently, we synthesized non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaf-
fold at the P2 position. Herein we report the SAR study of BACE1 inhibitors possessing this heterocyclic
scaffold, a chelidonic or 2,6-pyridinedicarboxylic moiety.
Received 8 August 2013
Revised 27 November 2013
Accepted 2 December 2013
Available online 8 December 2013
Keywords:
Alzheimer’s disease
b-Secretase
Ó 2013 Elsevier Ltd. All rights reserved.
BACE1
BACE1 inhibitor
R
N
b-Secretase, also called BACE1 [b-site APP (amyloid precursor
protein) cleaving enzyme 1], is a potential molecular target for
drugs against Alzheimer’s disease (AD),1–5 because BACE1 triggers
amyloid b (Ab) peptide formation by cleaving APP at the N-termi-
nus of the Ab domain.6–11 Recently, we reported potent pentapep-
tidic BACE1 inhibitors,12–18 KMI-420, -429 and -684, possessing
phenylnorstatine [Pns: (2R,3S)-3-amino-2-hydroxy-4-phenylbu-
tyric acid] as a substrate transition-state mimic. Among them,
KMI-42913 exhibited effective inhibition of BACE1 activity in cul-
tured cells, and significant reduction of Ab production in vivo (by
direct administration into the hippocampus of APP transgenic
and wild-type mice).13b However, these inhibitors contained some
natural amino acids that seemed to be required to improve enzy-
matic stability in vivo and permeability across the blood–brain
barrier, so as to be practical drug. Hence, we designed and reported
about the non-peptidic and small-sized BACE1 inhibitors KMI-
1036 and KMI-1027 that possess a heterocyclic scaffold19 at the
P2 position, as shown in Figure 1. These inhibitors were designed
using KMI-429 as a lead compound, and optimized on the basis
of the design approach focused on the conformer of a docked inhib-
itor in BACE1, the so called ‘in silico conformational structure-based
design’. Additionally, we found significant interaction between the
P2 region of the inhibitor and BACE1-Arg235. Specifically,
comparison of 36 publicly available X-ray crystal structures of
N
N
OH
H
N
H
N
NH
O
N
N
O
O
O
KMI-1023 (R = H)
KMI-1036 (R = OSO2CH3) IC50 = 96 nM
IC50 = 140 nM
O
X
N
N
OH
H
N
H
N
NH
O
N
N
O
O
O
KMI-1027 (X = O) IC50 = 50 nM
KMI-1030 (X = N) IC50 = 360 nM
Figure 1. BACE1 inhibitors with a P2 heterocyclic scaffold.
BACE1-inhibitor complexes indicated that a slightly attractive
force between the guanidino-plane of Arg235 and the P2 region
of inhibitors by a quantum effect, such as stacking or
interaction, played a key role in the BACE1 inhibitory mecha-
nism.20 Hence we designed a potent BACE1 inhibitor, KMI-1303,
possessing an electron-rich halogen atom at the P2 position, which
r–p
seemed to interact with the electron-poor guanidino-
p orbital by
⇑
Corresponding authors.
E-mail address: pynden@gmail.com (Y. Hamada).
Coulomb’s force20 as shown in Figure 2. This paper describes the
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.007

Y. Hamada et al. / Bioorg. Med. Chem. Lett. 24 (2014) 618–623
619
using phenylmethanesulfonyl chloride in the presence of triethyl-
amine. The P1–P10 residues (40 in Scheme 1) were synthesized from
Boc-Apns-OH 39 [Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbu-
0
tyric acid] and P1 -aromatic amines. For the synthesis of inhibitor
2, the 2S-enantiomaer of Boc-Apns-OH, namely Boc-Pns-OH, was
0
used. The P1 -aromatic amine corresponding to compound 4 was
prepared according to previously reported methods.14 Eventually,
0
the P3–P2 blocks and P1–P1 blocks were coupled, affording the de-
sired BACE1 inhibitors. The BACE1 inhibitor 3 was synthesized
from its methyl ester, which was prepared from methyl 3-amino-
benzoate as a starting material, by alkaline hydrolysis. The BACE1
inhibitor 34 was synthesized from compound 33 by catalytic
hydrogenation using 5% Pd-C. All the inhibitors were purified by
preparative RP-HPLC. Their BACE1 inhibitory activities were deter-
mined by enzymatic assay using a recombinant human BACE1 and
FRET (fluorescence resonance energy transfer) substrate as previ-
ously reported.4,12–18
Figure 2. BACE1 inhibitors with a halogen atom on the P2 aromatic ring.
For the SAR study, the BACE1 inhibitors 1–36 were compared
with the previously reported19,20 KMI-compounds shown in Fig-
ures 1 and 2. First, we checked the stereochemistry of P1 position
corresponding to a transition-state analogue of inhibitors. While
inhibitor 1 (possessing an Apns residue at the P1 position) exhib-
ited moderate BACE1 inhibitory activity, compound 2 (possessing
its 2S-enantiomer, Pns, at the same position) showed no BACE1
activity, as shown in Table 1. On the basis of this finding, we
adopted the Apns residue at the P1 position of BACE1 inhibitors
3–36. Next, a panel of compounds possessing a chelidonic residue
structure–activity relationship (SAR) study of non-peptidic and
small-sized BACE1 inhibitors possessing a chelidonic or 2,6-pyri-
dinedicarboxylic scaffold at the P2 position.
BACE1 inhibitors 1–33, 35, and 36 were synthesized to connect
in tandem with the blocks corresponding to the P3–P2 residues and
0
P1–P1 residues, respectively. Amide bonds were formed by com-
mon solution-phase synthesis methods using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimideꢀHCl (EDCꢀHCl) in the presence
of 1-hydroxybenzotriazole (HOBt) as coupling agents. As an exam-
ple, the synthesis of inhibitor 28 is outlined in Scheme 1. Briefly, P3
amines were coupled with monomethyl esters, which were pre-
pared from the dimethylester of 2,6-pyridinedicarboxylic acids,
and then hydrolyzed by an alkaline to afford the P3–P2 residues
(38 in the case of Scheme 1). The P3–P2 residues possessing a cheli-
donic residue were prepared by condensation between the P3
amine and chelidonic acid, because of their alkaline-instability.
(S)-4-Phenyloxazolidine, a P3-amine, was prepared from (S)-2-phe-
nylglycinol according to previously reported methods.19–21 Other
P3-amines were acquired commercially. The dimethylesters (42,
43, 45, 48–51 and 53) of 5-substituted 2,6-pyridinedicarboxylic
acids corresponding to the P2 units were prepared from chelidamic
acid 41, as shown in Scheme 2. The P2 unit 56 of inhibitor 36 was
prepared from diethyl 4-bromo-2,6-pyridinedicarboxylate 54 by
stannylation and the Stille cross coupling reaction. Compound 47
corresponding to the P3–P2 unit of inhibitor 21 was prepared from
compound 41 by tautomerization and subsequent sulfonylation
0
at the P2 position and diverse residues at the P1 position were syn-
thesized and their BACE1 inhibitory activities were estimated.
Most of the compounds without the 5-(3-aminophenyl)tetrazole
0
as a P1 residue exhibited no BACE1 inhibitory activity; compounds
3 and 4 are shown in this paper as examples. The inhibitor KMI-
870 possessing a benzylamino-type residue at the P3 position
exhibited potent BACE1 inhibitory activity. Therefore, the inhibi-
tors 5–20 were synthesized with P3-benzylamino-type residues
and a P2-chelidonic scaffold as shown in Table 2. Comparisons of
the compounds 5, 6, KMI-870, and KMI-900 suggested that a-sub-
stituents and the stereochemistry of the benzyl-type groups at the
P3 position were important for their BACE1 inhibitory activity.
Notably, since KMI-900 exhibited potent inhibitory activity, we
hypothesized that the
a,a-dimethyl group of KMI-900 stabilized
the folding conformer between the P2 ring and the P3 benzene ring
of the docked inhibitor in BACE1 via the gem-dimethyl effect. The
5-membered ring at the P3 position of KMI-1027 that exhibited
O
O
a,b,c
O
OH
O
O
N
N
O
N
O
O
O
N
N
OH
O
H
N
H
N
P3-P2 unit
NH
37
O
38
N
N
f
N
O
O
O
N
N
OH
OH
H
28 (KMI-1262)
H
N
NH
H2N
N
Boc
OH
N
d,e
O
O
P1-P1' unit
39
40
Scheme 1. Reagents and conditions: (a) 1 N NaOH 1.3 equiv/MeOH, rt; (b) (S)-4-phenyloxazolidine, EDCꢀHCl, HOBt/DMF; (c) 1 N NaOH 6 equiv/MeOH; (d) 5-(3-
aminophenyl)tetrazole, EDCꢀHCl, HOBt/DMF; (e) 4 N HCl/dioxane; (f) EDCꢀHCl, HOBt/DMF.

620
Y. Hamada et al. / Bioorg. Med. Chem. Lett. 24 (2014) 618–623
O
OCH3
O
O
b
a
O
O
HO
OH
O
O
O
O
N
H
N
N
H
N
O
O
O
O
O
O
O
O
c
41
44
43
42
d
SO2CH2Ph
f
O
O
N
O
N
H
N
e
H
N
OH
OH
O
O
O
N
H
O
O
O
O
O
46
47
45
S
N
Cl
N
SCH3
N
h
g
O
O
O
O
O
O
O
O
O
O
O
O
O
48
49
50
i
SO2CH3
N3
NH2
N
N
N
j
k,l
O
O
O
O
O
O
O
O
N
O
O
O
O
51
52
53
SnBu3
Br
N
CH3
m
n
O
O
O
O
O
N
N
O
O
O
O
O
O
55
54
56
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, reflux, 1 week (b) diethylsulfate, K2CO3/DMF, rt; (c) dipropylsulfate, K2CO3/DMF, rt; (d) (R)-1-phenylethylamine,
EDCꢀHCl, HOBt/DMF, rt; (e) phenylmethanesulfonyl chloride, Et3N/DMF, rt; (f) SOCl2, then MeOH; (g) CH3SNa/DMF, 60–80 °C; (h) isopropylamine, K2CO3/DMF, 60–80 °C; (i)
NaN3/DMF, 60 °C; (j) H2, Pd-C/MeOH, rt; (k) methanesulfonyl chloride, K2CO3/acetone, rt; (l) MeI, K2CO3/acetone, rt; (m) (Bu3Sn)2, Pd(PPh3)4/toluene (n) Pd2 (dba)3, P(o-
CH3C6H4)3, CuCl, K2CO3/DMF, 60 °C.
Table 1
BACE1 inhibitors possessing a chelidonic scaffold at the P2 position
O
OH
H
N
H
N
N
R
*
O
O
O
O
Compound
(KMI-No)
⁄Configuration
R
BACE1 inhibition
% at 2 lM
1 (KMI-765)
2 (KMI-789)
3 (KMI-848)
R
S
R
Tetrazol-5-yl
Tetrazol-5-yl
–COOH
60
6
10
O
N
4 (KMI-876)
R
O
23
N
H
potent BACE1 inhibitory activity evidently fixes this folding struc-
ture by covalent bonding within this 5-membered ring. Compound
7, possessing a cyclohexyl group at the P3 position, exhibited low
BACE1 inhibitory activity, suggesting that a plane structure is
preferred as a P3 residue of the inhibitor. By comparison, in
compounds 10–16, p-substitution on the P3 benzene ring of inhib-
itors seems to be preferable for inhibitory activity, particularly
with respect to inhibitor 14 (containing a p-fluorophenyl group),

Y. Hamada et al. / Bioorg. Med. Chem. Lett. 24 (2014) 618–623
621
Table 2
BACE1 inhibitors possessing a chelidonic scaffold at the P2 position
O
O
N
N
OH
H
N
H
N
NH
R
O
N
O
O
Compound
(KMI-No)
R
BACE1
inhibition
ClogP*1
Compound
(KMI-No)
R
BACE1
inhibition
ClogP
% at 2
lM
% at 2 lM
5 (KMI-925)
KMI-870
6 (KMI-1037)
KMI-900
Benzylamino
(R)-1-Phenylethylamino
(S)-1-Phenylethylamino
25
70
25
81
3.05
3.40
3.40
3.75
11 (KMI-923)
12 (KMI-887)
13 (KMI-898)
14 (KMI-902)
(R)-1-(4-Methoxyphenyl)ethylamino
(R)-1-(3-Methoxyphenyl)ethylamino
(R)-1-(4-Methylphenyl)ethylamino
(R)-1-(4-Fluorophenyl)ethylamino
70
48
54
76
3.31
3.31
3.86
3.45
a,a-Dimethylbenzylamino
(cumylamino)
KMI-1027
(See Fig. 1)
96
26
29
13
51
2.63
4.16
5.35
4.83
3.13
15 (KMI-922)
16 (KMI-911)
17 (KMI-903)
18 (KMI-871)
19 (KMI-991)
20 (KMI-926)
(R)-1-(3-Fluorophenyl)ethylamino
(R)-1-(4-Chlorophenyl)ethylamino
(R)-1-Phenylpropylamino
[(1R)-2-Hydroxy-1-phenyl–ethyl]amino
2-Methyl-2-phenyl-hydrazino
N-Methyl-N-benzylamino
62
61
32
11
21
67
3.45
3.99
3.93
2.39
3.31
3.57
7 (KMI-899)
8 (KMI-827)
9 (KMI-1040)
10 (KMI-886)
(R)-1-Cyclohexylethylamino
N,N-Dibenzylamino
Benzhydrylamino
(R)-1-(4-Nitrophenyl)ethylamino
*1
Calculated values of LogP using ACD/LogP software (Advanced Chemistry Development Inc., Canada).
Figure 3. BACE1 inhibitors KMI-870 and KMI-902 (14) docked in BACE1.
which exhibited the highest inhibitory activity. In order to investi-
gate this improved inhibitory activity by p-substitution on the P3
benzene ring, computational docking simulations were performed
under the MMFF94x force field using the MOE software (Chemical
Computing Group Inc., Canada). Stereo views of the docked inhib-
itors KMI-870 and 14 (KMI-902) in BACE1 (PDB ID: 2B8L) are
shown in Figure 3. Both models show that the P3 benzene ring
tightly occupies the S3 sub-pocket of BACE1. Particularly, the fluo-
rine atom of KMI-902 seems to interact with the side chains of the
hydrophobic amino acids Ala335 and Tyr14, more tightly than does
KMI-870. Comparisons among compounds 17–20 suggested that
which could enhance BACE1 inhibitory activity. However, on the
basis of the fact that compound 5 exhibited low inhibitory activity,
we considered that the substitution at this position seems to affect
the inhibitory activity for reasons related to the stability of the
docked conformer in BACE1 rather than the hydrophobic interac-
tion with BACE1; an
a-methyl group at the P3-benzyl group of
KMI-900 faces the opening of BACE1’s flap domain and does not
interact with any BACE1 residues. (see Fig. 4)
We then synthesized a series of BACE1 inhibitors possessing
2,6-pyridinedicarboxylic scaffolds for SAR study at the P2 position
(Table 3). The inhibitors with (S)-4-phenyloxazolidine among the
P3-amines (A, B, and C in Table 3) exhibited the highest BACE1
inhibitory activities, as was the case for inhibitors such as KMI-
the substitution at the
a-position of the P3-benzyl group was pref-
erably hydrophobic and small-sized groups, such as methyl groups,

622
Y. Hamada et al. / Bioorg. Med. Chem. Lett. 24 (2014) 618–623
of Arg235-BACE1 via a weak quantum effect, exhibited potent
BACE1 inhibitory activity.20 These small-sized groups on the
P2-pyridine ring might also interact with the guanidino-plane of
Arg235-BACE1 via a weak quantum attractive force, in addition
to interacting with the halogen atom.
The non-peptidic BACE1 inhibitors were expected to exhibit im-
proved enzymatic stability in vivo and membrane permeability
across the blood–brain barrier. Hence we evaluated the BACE1
inhibitory activities of non-peptidic KMI-1023, -1030, -1027, and
-1036 in cultured cells, and compared the results with those from
previously reported analyses of peptidic KMI-compounds.15 As
shown in Figure 5. BACE1 inhibitory assay using cultured cells evi-
dently reflects the membrane permeability of inhibitors to a de-
gree, because endogenous BACE1 is localized predominantly in
the later Golgi and trans-Golgi network,22,23 and thought to cleave
mainly at the b-site of APP on the trans-Golgi network.22 BACE1
inhibitory activities in cultured cells were determined as previ-
ously reported.13b,15 Briefly, HEK293 cells stably expressing human
BACE1 enzyme (BACE1-HEK293) were incubated with or without
KMI-compounds (100 lM) for 6 h. The soluble APPb (sAPPb) pep-
tide, the N-terminus domain released from APP upon cleavage by
BACE1, was then quantified by western blotting using polyclonal
anti-sAPPb antibody. BACE1 inhibitory activities in cultured cells
were calculated from sAPPb levels, and compared with in vitro
enzymatic inhibitory activities. Contrary to our expectation, non-
peptidic inhibitors (black circles in Fig. 5, ClogP = 2.30–2.63) are
found on the same line as peptidic inhibitors (open circles,
ClogP = 1.60–2.34) in the correlation graph, suggesting that the
membrane permeability of non-peptidic and peptidic KMI-com-
pounds is in the same range.
In conclusion, BACE1 inhibitors possessing a chelidonic or 2,6-
pyridinedicarboxylic scaffold at the P2 position were synthesized
for SAR studies. We found that with regard to substituents at the
P2 and P3 positions, small-sized and hydrophobic residues were
preferable for BACE1 inhibition, by way of interaction with BACE1’s
corresponding pockets and the stability of the docked inhibitor’s
conformer in BACE1. Unfortunately, assays using cultured cells
suggested that the membrane permeability of our non-peptidic
BACE1 inhibitors is similar to that of peptidic KMI-compounds.
However, non-peptidic KMI-compounds are expected to improve
Figure 4. Inhibitor KMI-1256 docked in BACE1 (PDB ID: 2B8L) The inhibitor and
Arg235 were depicted by space-filling modeling. The cartoon and stick models
indicate the BACE1 enzyme.
1027 containing a P2-chelidonic scaffold. KMI-1319 containing a
methanesulofonyloxy group on the P2-pyridine ring, exhibited
potent BACE1 inhibitory activity. In contrast, compound 21, con-
taining a phenylmethanesulofonyloxy group at the same position,
showed lower inhibitory activity, suggesting that a large-sized
group on the P2-pyridine ring might be undesirable for BACE1 inhi-
bition. The fact that the BACE1 inhibitors with a methoxy (22, 25
and 28), methylmercapto (24, 27 and 31), azide (33) or methyl
(36) group exhibited potent BACE1 inhibitory activity suggests that
small-sized and hydrophobic substituents on the P2-pyridine scaf-
fold of the inhibitor are preferable for inhibitory activity We have
previously reported that BACE1 inhibitors with a halogen atom at
the P2 position, which may interact with the guanidino-
p orbital
Table 3
BACE1 inhibitors possessing the 2,6-pyridinedicarboxylic scaffold at the P2 position
X
N
N
OH
H
N
H
N
NH
A = (R)-1-phenylethylamino
B = cumylamino
R
N
N
O
O
O
R = A, B or C
N
C =
O
Compound
(KMI-No)
R
X
BACE1 inhibition
% at 2 lM
ClogP
Compound
(KMI-No)
R
X
BACE1 inhibition %
IC50 (nM)
ClogP
at 2
lM
at 0.2 lM
KMI-918*1
A
B
C
A
A
A
A
A
B
B
B
B
–H
–H
–H
66
69
89
80
29
73
46
82
84
74
42
85
4.01
4.36
2.37
4.08
5.71
4.78
5.31
4.65
4.43
5.13
5.66
5.00
KMI-1036*1
C
C
C
C
C
C
C
C
C
C
C
–OSO2CH3
–OCH3
–OEt
–OPr
–SCH3
–SCH(CH3)2
–N3
–NH2
–N(CH3)SO2CH3
–CH3
–Br
96
91
79
64
95
72
95
78
75
95
99
73
53
—
—
66
—
68
53
—
68
88
96
151
—
—
89
—
2.44
3.54
4.07
4.60
3.01
3.89
2.86
2.52
1.62
2.83
3.63
KMI-941*1
28 (KMI-1262)
29 (KMI-1270)
30 (KMI-1331)
31 (KMI-1284)
32 (KMI-1318)
33 (KMI-1316)
34 (KMI-1315)
35 (KMI-1300)
36 (KMI-1320)
KMI-1256*2
KMI-1023*1
KMI-1319*1
–OSO2CH3
21 (KMI-943)
22 (KMI-1263)
23 (KMI-1271)
24 (KMI-1285)
KMI-942*1
25 (KMI-1264)
26 (KMI-1272)
27 (KMI-1286)
–OSO2CH2Ph
–OCH3
–OEt
79
–SCH3
–OSO2CH3
–OCH3
–OEt
—
—
15
–SCH3
*1,*2
See the Refs. 18 and 19, respectively.

Y. Hamada et al. / Bioorg. Med. Chem. Lett. 24 (2014) 618–623
623
5. (a) Hamada, Y.; Kiso, Y. Expert Opin. Drug Discov. 2013, 8, 709; (b) Hamada, Y.;
Kiso, Y. Expert Opin. Drug Discov. 2012, 7, 903; (c) Hamada, Y.; Kiso, Y. Expert
Opin. Drug Discov. 2009, 4, 391; (d) Ziora, Z.; Kimura, T.; Kiso, Y. Drugs Future
2006, 31, 53; (e) Nguyen, J.-T.; Yamani, A.; Kiso, Y. Curr. Pharm. Design 2006, 12,
4309.
6. Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E. A.; Denis, P.;
Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.;
Edenson, S.; Lile, J.; Jarosinski, M. A.; Biere, A. L.; Curran, E.; Burgess, T.; Louis, J.
C.; Collins, F.; Treanor, J.; Rogers, G.; Citron, M. Science 1999, 286, 735.
7. Yan, R.; Bienkowski, M. J.; Shuck, M. E.; Miao, H.; Tory, M. C.; Pauley, A. M.;
Brashier, J. R.; Stratman, N. C.; Mathews, W. R.; Buhl, A. E.; Carter, D. B.;
Tomasselli, A. G.; Parodi, L. A.; Heinrikson, R. L.; Gurney, M. E. Nature 1999, 402,
533.
8. Sinha, S.; Anderson, J. P.; Barbour, R.; Basi, G. S.; Caccavello, R.; Davis, D.; Doan,
M.; Dovey, H. F.; Frigon, N.; Hong, J.; Jacobson-Croak, K.; Jewett, N.; Keim, P.;
Knops, J.; Lieberburg, I.; Power, M.; Tan, H.; Tatsuno, G.; Tung, J.; Schenk, D.;
Seubert, P.; Suomensaari, S. M.; Wang, S.; Walker, D.; Zhao, J.; McConlogue, L.;
John, V. Nature 1999, 402, 537.
9. Hussain, I.; Powell, D.; Howlett, D. R.; Tew, D. G.; Meek, T. D.; Chapman, C.;
Gloger, I. S.; Murphy, K. E.; Southan, C. D.; Ryan, D. M.; Smith, T. S.; Simmons, D.
L.; Walsh, F. S.; Dingwall, C.; Christie, G. Mol. Cell. Neurosci. 1999, 14, 419.
10. Selkoe, D. J. Nature 1999, 399, A23.
11. Sinha, S.; Lieberburg, I. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11049.
12. Kimura, T.; Shuto, D.; Kasai, S.; Liu, P.; Hidaka, K.; Hamada, T.; Hayashi, Y.;
Hattori, C.; Asai, M.; Kitazume, S.; Saido, T. C.; Ishiura, S.; Kiso, Y. Bioorg. Med.
Chem. Lett. 2004, 14, 1527.
Figure 5. The correlation of BACE1 inhibition (%) by KMI-compounds as deter-
mined by the cultured cell assay against inhibition determined by the in vitro
enzymatic assay. The open circles and black circles indicate peptidic and non-
peptidic BACE1 inhibitors, respectively.
13. (a) Kimura, T.; Shuto, D.; Hamada, Y.; Igawa, N.; Kasai, S.; Liu, P.; Hidaka, K.;
Hamada, T.; Hayashi, Y.; Kiso, Y. Bioorg. Med. Chem. Lett. 2005, 15, 211; (b) Asai,
M.; Hattori, C.; Iwata, N.; Saido, T. C.; Sasagawa, N.; Szabó, B.; Hashimoto, Y.;
Maruyama, K.; Tanuma, S.; Kiso, Y.; Ishiura, S. J. Neurochem. 2006, 96, 533.
14. Kimura, T.; Hamada, Y.; Stochaj, M.; Ikari, H.; Nagamine, A.; Abdel-Rahman, H.;
Igawa, N.; Hidaka, K.; Nguyen, J.-T.; Saito, K.; Hayashi, Y.; Kiso, Y. Bioorg. Med.
Chem. Lett. 2006, 16, 23–80.
15. Hamada, Y.; Igawa, N.; Ikari, H.; Ziora, Z.; Nguyen, J.-T.; Yamani, A.; Hidaka, K.;
Kimura, T.; Saito, K.; Hayashi, Y.; Ebina, M.; Ishiura, S.; Kiso, Y. Bioorg. Med.
Chem. Lett. 2006, 16, 4354.
16. Hamada, Y.; Abdel-Rahman, H.; Yamani, A.; Nguyen, J.-T.; Stochaj, M.; Hidaka,
K.; Kimura, T.; Hayashi, Y.; Saito, K.; Ishiura, S.; Kiso, Y. Bioorg. Med. Chem. Lett.
2008, 18, 1649.
17. Hamada, Y.; Tagad, H. D.; Nishimura, Y.; Ishiura, S.; Kiso, Y. Bioorg. Med. Chem.
Lett. 2012, 22, 1130.
18. Hamada, Y.; Ishiura, S.; Kiso, Y. ACS Med. Chem. Lett. 2012, 3, 193.
19. Hamada, Y.; Ohta, H.; Miyamoto, N.; Yamaguchi, R.; Yamani, A.; Hidaka, K.;
Kimura, T.; Saito, K.; Hayashi, Y.; Ishiura, S.; Kiso, Y. Bioorg. Med. Chem. Lett.
2008, 18, 1654.
20. Hamada, Y.; Ohta, H.; Miyamoto, N.; Sarma, D.; Hamada, T.; Nakanishi, T.;
Yamasaki, M.; Yamani, A.; Ishiura, S.; Kiso, Y. Bioorg. Med. Chem. Lett. 2009, 19,
2435.
21. Hamada, Y.; Nakanishi, T.; Suzuki, K.; Yamaguchi, R.; Hamada, T.; Hidaka, K.;
Ishiura, S.; Kiso, Y. Bioorg. Med. Chem. Lett. 2012, 22, 4640.
22. Yan, R.; Han, P.; Miao, H.; Greengard, P.; Xu, H. J. Biol. Chem. 2001, 276, 36788.
23. Huse, J. T.; Liu, K.; Pijak, D. S.; Carlin, D.; Lee, V. M.-Y.; Doms, R. W. J. Biol. Chem.
2002, 277, 16278.
the bio-availability, due to their enhanced enzymatic stability
in vivo.
Acknowledgments
This study was supported in part by the Grants-in-Aid for Scien-
tific Research (A) and (C) from MEXT (Ministry of Education, Cul-
ture, 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.
References and notes
1. (a) Ghosh, A. K.; Shin, D.; Downs, D.; Koelsch, G.; Lin, X.; Ermolieff, J.; Tang, J. J.
Am. Chem. Soc. 2000, 122, 3522; (b) Ghosh, A. K.; Bilcer, G.; Harwood, C.;
Kawahara, R.; Shin, D.; Hussain, K. A.; Hong, L.; Loy, J. A.; Nguyen, C.; Koelsch,
G.; Ermolieff, J.; Tang, J. J. Med. Chem. 2001, 44, 2865.
2. Tung, J. S.; Davis, D. L.; Anderson, J. P.; Walker, D. E.; Mamo, S.; Jewett, N.; Hom,
R. K.; Sinha, S.; Thorsett, E. D.; John, V. J. Med. Chem. 2002, 45, 259.
3. Tamamura, H.; Kato, T.; Otaka, A.; Fujii, N. Org. Biomol. Chem. 2003, 1, 2468.
4. Shuto, D.; Kasai, S.; Kimura, T.; Liu, P.; Hidaka, K.; Hamada, T.; Shibakawa, S.;
Hayashi, Y.; Hattori, C.; Szabo, B.; Ishiura, S.; Kiso, Y. Bioorg. Med. Chem. Lett.
2003, 13, 4273.