Novel non-peptidic and small-sized BACE1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.01.056

Recently, we reported substrate-based β-secretase (BACE1) inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. These inhibitors showed potent BACE1 inhibitory activities (～1.2 nM IC₅₀). In order to improve in vivo enzymatic stability and permeability across the blood-brain barrier, these penta-peptidic inhibitors would need to be further optimized. On the other hand, non-peptidic inhibitors possessing isophthalic residue at the P₂ position were reported from other research groups. We selected isophthalic-type aromatic residues at the P₂ position and an HMC isostere at the P₁ position as lead compounds. On the basis of the design approach focused on the conformer of docked inhibitor in BACE1, we found novel non-peptidic and small-sized BACE1 inhibitors possessing a 2,6-pyridinedicarboxylic, chelidamic or chelidonic residue at the P₂ position.

Full text of DOI:10.1016/j.bmcl.2008.01.056

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1654–1658
Novel non-peptidic and small-sized BACE1 inhibitors
Yoshio Hamada,a Hiroko Ohta,a Naoko Miyamoto,a Ryoji Yamaguchi,a
Abdellah Yamani,a Koushi Hidaka,a Tooru Kimura,a Kazuki Saito,b Yoshio Hayashi,a
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 substrate-based b-secretase (BACE1) inhibitors with a hydroxymethylcarbonyl (HMC) isostere as
a substrate transition-state mimic. These inhibitors showed potent BACE1 inhibitory activities (ꢀ1.2 nM IC50). In order to improve
in vivo enzymatic stability and permeability across the blood–brain barrier, these penta-peptidic inhibitors would need to be further
optimized. On the other hand, non-peptidic inhibitors possessing isophthalic residue at the P2 position were reported from other
research groups. We selected isophthalic-type aromatic residues at the P2 position and an HMC isostere at the P1 position as lead
compounds. On the basis of the design approach focused on the conformer of docked inhibitor in BACE1, we found novel non-
peptidic and small-sized BACE1 inhibitors possessing a 2,6-pyridinedicarboxylic, chelidamic or chelidonic residue at the P2 position.
Ó 2008 Elsevier Ltd. All rights reserved.
Amyloid b (Ab) peptide is a main component of senile
plaques in the brains of Alzheimer’s disease (AD) pa-
R1
tients, that seems to cause ADs pathologies. According
to the amyloid hypothesis,1 b-secretase [BACE1: b-site
APP (amyloid precursor protein) cleaving enzyme] ap-
pears promising as a molecular target for therapeutic
intervention in AD,2–6 because BACE1 triggers Ab pep-
tide formation by cleaving APP at the N-terminus of the
Ab domain.7–12 Recently, we reported potent penta-pep-
tidic BACE1 inhibitors13–16 containing a hydroxymeth-
ylcarbonyl isostere (HMC) as a substrate transition-
state mimic17 as shown in Figure 1. Among them,
KMI-42914 exhibited effective inhibition of BACE1
activity in cultured cells, and significant reduction of
Ab production in vivo (by direct administration into
the hippocampuses of APP transgenic and wild-type mi-
ce).14b However, these inhibitors contained some natural
amino acids that seemed to be required to improve enzy-
matic stability in vivo and permeability across the
O
O
O
H
N
H2N
R2
N
H
N
H
N
H
O
OH
NH
N
O
NH
N
N
KMI-420 (R1 = -H,
R2 = -COOH) IC50 = 8.2 nM
KMI-429 (R1 = -COOH, R2 = -COOH) IC50 = 3.9 nM
KMI-570 (R1 = -H,
R2
=
)
IC50 = 4.8 nM
) IC50 = 1.2 nM
N
NH
N
N
N
N
KMI-684 (R1
=
, R2
=
NH
NH
N
N
N
N
Figure 1. BACE1 inhibitors consisting of penta-peptides.
blood–brain barrier, so as to be practical anti-ADs
drugs. Non-peptidic BACE1 inhibitors possessing iso-
phthalic residue such as Elan’s and Merck’s inhibitors
as shown in Figure 2A, were reported from other re-
search groups.18,19 Herein, we selected isophthalic-type
aromatic residues at the P2 position and an HMC isoste-
Keywords: Alzheimer’s disease; BACE1; b-Secretase; Non-peptidic B-
ACE1 inhibitor.
*
Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591
9900; e-mail: kiso@mb.kyoto-phu.ac.jp
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.056

Y. Hamada et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1654–1658
1655
0
and P1–P1 residues, respectively. Amide bonds were
formed by traditional solution-phase synthesis methods
using
OH CH3
O
H
N
H
N
N
1-ethyl-3-(3-dimethylaminopropyl)carbodiim-
O
O
ideÆHCl (EDCÆHCl) in the presence of 1-hydroxybenzo-
triazole (HOBt) as coupling agents. As shown in Scheme
1, after amide-bond formation, inhibitor 2 was synthe-
sized by deprotection using alkaline hydrolysis. Synthe-
sis of the blocks for the preparation of inhibitors 3–13 is
shown in Scheme 2. 4-Methanesulfonyloxypyridine-2,5-
dicarboxlic derivatives 18c and 20b were synthesized
from chelidamic derivatives 19c and 21a by tautomeriza-
tion and an ensuing methanesulfonation using methane-
sulfonyl chloride in the presence of triethylamine.
Blocks 20–21 possessing a 5-membered ring were syn-
thesized from 17 that was prepared from (S)-2-phenyl-
glycinol by cyclization with formaldehyde. As an
example, the synthesis of inhibitor 13 is shown in
Scheme 3. All of the inhibitors were purified by prepara-
tive RP-HPLC.
F
F
Elan inhibitor IC50 = 30 nM
O
O
S
N
OH
H
H
H
N
N
N
O
O
Merck inhibitor IC50 = 15 nM
dihedral angle
O
N
R
O
N
N
R
BACE1 inhibitory activity of the inhibitors was deter-
mined by enzymatic assay using a recombinant human
H
O
O
1
2
OH
BACE1-bound type: 46
unbound type: 76
H
N
H
N
COOH
R =
a,b
O
O
O
N
H2N
HCl
N
Boc
N
H
OH
NH
H
OH
N
N
OH
16
X
Figure 2. (A) Non-peptidic BACE1 inhibitors. (B) Design of small-
sized BACE1 inhibitor 2 possessing heterocyclic ring at the P2 position.
H
N
OH
re at the P1 position as lead compounds. Based on the
design approach that a conformer of docked inhibitor
in BACE1 was stabilized, we designed novel BACE1
inhibitors.
N
H3C
18a R = H, X = H
18b R = CH3, X = H
R
O
O
c
18c R = CH3, X = OSO2CH3
NH2
O
H3C
R
c
BACE1 inhibitors 2–13 were synthesized to connect in
tandem the blocks corresponding to the P3–P2 residues
d
H
(R = H or CH3)
N
OH
Y
H3C
R
O
O
19a R = H, Y = NH
19b R = H, Y = O
19c R = CH3, Y = NH
19d R = CH3, Y = O
X
N
O
OH
O
OH
H2N
N
e
O
20a X = H
20b X = OSO2CH3
c
O
d
c
O
OH
Y
NH
N
O
O
21a Y = NH
21b Y = O
O
17
Scheme 2. Reagents and conditions: (a) 5-(3-aminophenyl)tetrazole,
EDCÆHCl, HOBt/DMF, 83%; (b) anisole, 4 N HCl/dioxane, 99%; (c)
2,6-pyridinedicarboxylic acid, chelidamic acid or chelidonic acid,
EDCÆHCl, HOBt/DMF, 55–65%; (d) methanesulfonyl chloride Et3N/
THF, 60–78%; (e) HCHO, water, 86%.
Scheme 1. Reagents and conditions: (a) methyl 3-aminobenzoate,
EDCÆHCl, HOBt/DMF, 85%; (b) anisole, 4 N HCl/dioxane, 99%; (c)
dipropylamine, EDCÆHCl, HOBt/DMF, 55%; (d) EDCÆHCl, HOBt/
DMF, 91%; (e) 1 N NaOH/MeOH, 99% (crude).

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Y. Hamada et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1654–1658
norstatine [Apns: (2S,3S)-3-amino-2-hydroxy-4-phenyl
O
O
butyric acid], with syn-stereochemistry similar to
Merck’s BACE1 inhibitor (Fig. 2A), at the P1 position.
3–5 showed moderate BACE1 inhibitory activity. We
noticed the importance of the a-methyl group at the
P3 position for BACE1 inhibitory activity. Inhibitors
6–9 possessing an a,a-dimethylbenzylamino group at
the P3 position were designed and showed improved
inhibitory activity (Table 1). According to the crystal
structure of isophthalic-type Merck’s inhibitor (PDB
ID: 2B8L, Fig. 3B) and the coordinates of docked inhib-
itors in BACE1 by our docking simulation study, inhib-
itors possessing a P3 benzylamino-type residue displayed
O
OH
O
N
N
O
H2N
HCl
N
H
NH
OH
N
N
O
21b
16
O
O
N
N
OH
H
H
NH
O
N
N
a
N
N
O
O
O
13
8
Scheme 3. Reagents: (a) EDCÆHCl, HOBt/DMF, 90%.
6
BACE1 and FRET (fluorescence resonance energy
transfer) substrate as previously reported.5
Docked inhibitor
in BACE1
4
We first envisioned virtual inhibitor 1 possessing an iso-
phthalic residue at the P2 position and an HMC isostere,
phenylnorstatine [Pns: (2R,3S)-3-amino-2-hydroxy-4-
phenylbutyric acid], at the P1 position. A stable confor-
mation docked in BACE1 (PDB ID: 1W51) was calcu-
lated using docking simulation software, MOE
(Chemical Computing Group Inc., Canada). Second, a
stable conformation of unbound inhibitor 1 was searched
by calculating the steric energies of conformers around
the bond of the P3 amide and P2 isophthalic residue in
MM2 force field. The ideal dihedral-angles between the
P3 amide and P2 isophthalic residue of inhibitor 1 in the
BACE1-bound and unbound conformers were found to
be quite different (BACE1-bound type: 46°, unbound
type: 76°). We thought that high-active inhibitors could
be found by resolving this variation. Herein, we designed
inhibitor 2 possessing a P2 heterocyclic residue, which
lacks a hydrogen atom that would induce steric hindrance
between the P3 moiety and P2 aromatic ring, as shown in
Figure 2B. However, synthesized inhibitor 2 showed no
BACE1 inhibitory activity.
2
0
—180 —120
—60
0
60
120
180
Dihedral Angle (degree)
O
N
H
O
10'
O
N
N
O
Dihedral angle
10
Next, we designed inhibitors 3–5 possessing (R)-a-meth-
ylbenzylamino group at the P3 position and allophenyl-
Figure 4. Conformational study of BACE1 inhibitor 10 (KMI-1023).
100 is virtual isophthalic analogue of inhibitor 10.
Figure 3. (A) Inhibitor 13 (KMI-1027, space-filling model) docked in BACE1. Molecular surface model indicates BACE1. The yellow transparent
part in the center indicates a flap domain. (B) Stereoview of superimposed inhibitor 10 (KMI-1023, red lines) and Merck inhibitor (yellow lines) in
BACE1(PDB ID: 2B8L). White dashed lines and red balls indicate hydrogen bond interactions and water molecules, respectively.

Y. Hamada et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1654–1658
1657
folding conformers between the P2 ring and P3 benzene
rings to bind to the S3 sub-pocket19 in BACE1. We as-
sumed that the two methyl groups stabilized this folding
conformer by gem-dimethyl effect. However, a,a-dim-
ethylbenzylamino group is labile under acidic condi-
tions20 similar to trityl group. Interestingly, HIV
protease inhibitors with a,a-dimethylbenzyl amide
showed low oral bioavailability, because of their acid-
instability.21 We designed inhibitors 10–13 introducing
a 5-membered ring at the P3 position, in order to im-
prove acid-resistance and fix the folding pose between
the P2 and P3 rings. Inhibitors 10–13 showed potent
BACE1 inhibitory activity (Table 2). Modeled structures
of docked inhibitors 10 and 13 in BACE1 (PDB ID:
2B8L) by docking simulation using MOE are shown in
Figure 3. The P3 benzene ring was confirmed to take
Table 1. BACE1 inhibitors possessing benzylamino-type groups at the P3 position and their BACE1 inhibitory activities
X
N
N
OH
H
N
H
N
H
N
NH
N
N
R1 R2
O
O
O
R1
R2
X
BACE1 inhibition % at 2 lM
3
6
7
CH3
CH3
CH3
H
H
66%
69%
84%
CH3
CH3
H
OSO2CH3
4
5
8
9
CH3
CH3
CH3
CH3
H
NH
O
51%
70%
65%
81%
H
CH3
CH3
NH
O
Table 2. BACE1 inhibitors with a 5-membered ring at the P3 position and their BACE1 inhibitory activities
X
N
N
OH
H
N
H
N
NH
O
N
N
N
O
O
O
Compound (KMI No.)
X
H
BACE1 inhibition %
at 0.2 lM
IC50 (nM)
at 2 lM
10 (KMI-1023)
11 (KMI-1036)
93%
96%
63%
73%
140
96
OSO2CH3
12 (KMI-1030)
13 (KMI-1027)
NH
O
83%
96%
36%
78%
360
50

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Y. Hamada et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1654–1658
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.
8. 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.
9. 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.; Suo-
mensaari, S. M.; Wang, S.; Walker, D.; Zhao, J.;
McConlogue, L.; John, V. Nature 1999, 402, 537.
10. 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.
11. Selkoe, D. J. Nature 1999, 399, A23.
12. Sinha, S.; Lieberburg, I. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 11049.
13. Kimura, T.; Shuto, D.; Kasai, S.; Liu, P.; Hidaka, K.;
Hamada, T.; Hayashi, Y.; Hattori, C.; Asai, M.; Kitaz-
ume, S.; Saido, T. C.; Ishiura, S.; Kiso, Y. Bioorg. Med.
Chem. Lett. 2004, 14, 1527.
14. (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.;
the form of a folding conformer against the P2 ring and
tightly occupy the S3 sub-pocket of BACE1 (Fig. 3A). In
addition, both ideal conformers around the bond of the
P3 amide and P2 ring of unbound and BACE1-bound
inhibitor 10 were confirmed to coincide. Interestingly,
virtual isophthalic analogue 100 in BACE1 showed high
steric energy state caused by the interaction of the P3
moiety and ring’s hydrogen atom (Fig. 4).
In conclusion, on the basis of the design approach fo-
cused on the conformer of a docked inhibitor in complex
with BACE1, BACE1 inhibitors possessing a heterocy-
clic ring at the P2 position and 5-membered ring at the
P3 position were designed. We found novel small-sized
and non-peptidic BACE1 inhibitors. These inhibitors
are expected improved membrane permeability and bio-
availability as practical anti-ADs drugs. This design ap-
proach based on a conformer of enzyme-/receptor-
bound inhibitor/ligand, which was predicted in silico,
was showed the usefulness of the drug discovery re-
search, in contrast to exhaustive combinatorial
synthesis.
Acknowledgments
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.
´
Hattori, C.; Iwata, N.; Saido, T. C.; Sasagawa, N.; Szabo,
B.; Hashimoto, Y.; Maruyama, K.; Tanuma, S.; Kiso, Y.;
Ishiura, S. J. Neurochem. 2006, 96, 533.
15. Kimura, T.; Hamada, Y.; Stochaj, M.; Ikari, H.; Nag-
amine, A.; Abdel-Rahman, H.; Igawa, N.; Hidaka, K.;
Nguyen, J.-T.; Saito, K.; Hayashi, Y.; Kiso, Y. Bioorg.
Med. Chem. Lett. 2006, 16, 2380.
16. 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.
17. (a) Mimoto, T.; Imai, J.; Tanaka, S.; Hattori, N.;
Takahashi, O.; Kisanuki, S.; Nagano, Y.; Shintani, M.;
Hayashi, H.; Akaji, K.; Kiso, Y. Chem. Pharm. Bull. 1991,
39, 2465; (b) Mimoto, T.; Imai, J.; Tanaka, S.; Hattori, N.;
Kisanuki, S.; Akaji, K.; Kiso, Y. Chem. Pharm. Bull. 1991,
39, 3088.
18. Hom, R. K.; Gailunas, A. F.; Mamo, S.; Fang, L. Y.;
Tung, J. S.; Walker, D. E.; Davis, D.; Thorsett, E. D.;
Jewett, N. E.; Moon, J. B.; John, V. J. Med. Chem. 2004,
47, 158.
19. Stachel, S. J.; Coburn, C. A.; Steele, T. G.; Jones, K. G.;
Loutzenhiser, E. F.; Gregro, A. R.; Rajapakse, H. A.; Lai,
M.-T.; Crouthamel, M.-C.; Xu, M.; Tugusheva, K.;
Lineberger, J. E.; Pietrak, B. L.; Espeseth, A. S.; Shi, X.-
P.; Chen-Dodson, E.; Holloway, M. K.; Munshi, S.;
Simon, A. J.; Kuo, L.; Vacca, J. P. J. Med. Chem. 2004,
47, 6447.
References and notes
1. (a) Hong, L.; Koelsch, G.; Lin, X.; Wu, S.; Terzyan, S.;
Ghosh, A. K.; Zhang, X. C.; Tang, J. Science 2000, 290,
150; (b) Sipe, J. D. Annu. Rev. Biochem. 1992, 61, 947; (c)
Selkoe, D. J. Ann. N. Y. Acad. Sci. 2000, 924, 17; (d)
Steiner, H.; Capell, A.; Leimer, U.; Haass, C. Eur. Arch.
Psychiatry Clin. Neurosci. 1999, 249, 266; (e) Selkoe, D. J.
Ann. Med. 1989, 21, 73.
2. (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.
3. 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.
4. Tamamura, H.; Kato, T.; Otaka, A.; Fujii, N. Org.
Biomol. Chem. 2003, 1, 2468.
20. a,a-Dimethylbenzyl (cumyl) group is recognized as a N-
protecting group of amides that is cleavable by acid, such as
TFA or formic acid. e.g. ‘Wuts, P. G. M.; Greene, T. W.
Greene’s PROTECTIVE GROUPS in ORGANIC SYS-
THESIS, 4th ed., John Wiley & Sons, Inc, 2007, p. 907.
21. Reich, S.; Melnick, M.; Davies, J. F., II; Appelt, K.;
Lewis, K. K.; Fuhry, M. A.; Pino, M.; Trippe, A. J.;
Nguyen, D.; Dawson, H.; Wu, B.-W.; Musick, L.; Kosa,
M.; Kahil, D.; Webber, S.; Gehlhaar, D. K.; Andrada,
D.; Shetty, B. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
3298.
5. 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.
6. (a) Ziora, Z.; Kimura, T.; Kiso, Y. Drugs Future 2006, 31,
53; (b) Nguyen, J.-T.; Yamani, A.; Kiso, Y. . Curr. Pharm.
Des. 2006, 12, 4309.
7. 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.;