Tetrapeptides, as small-sized peptidic inhibitors; synthesis and their inhibitory activity against BACE1

Article abstract of DOI:10.1002/psc.1238

β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is known to be involved in the production of amyloid β-peptide in Alzheimer's disease and is a major target for current drug design. We previously reported substrate-based peptidomimetics, KMI-com

Full text of DOI:10.1002/psc.1238

Rapid Communication
Received: 25 February 2010
Revised: 16 March 2010
Accepted: 17 March 2010
Published online in Wiley Interscience: 7 May 2010
(www.interscience.com) DOI 10.1002/psc.1238
Tetrapeptides, as small-sized peptidic
inhibitors; synthesis and their inhibitory
activity against BACE1
Taeko Kakizawa, Koushi Hidaka, Daisuke Hamada, Ryoji Yamaguchi,
Tsuyoshi Uemura, Hitomi Kitamura, Harichandra D. Tagad,
Takashi Hamada, Zyta Ziora, Yoshio Hamada, Tooru Kimura
and Yoshiaki Kiso∗
β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is known to be involved in the production of amyloid β-peptide in
Alzheimer’s disease and is a major target for current drug design. We previously reported substrate-based peptidomimetics,
KMI-compounds as potent BACE1 inhibitors. In this study, we designed and synthesized tetrapeptides as low molecular-sized
inhibitors. These exhibited high potency against recombinant BACE1, with the highest IC50 value of 34.6 nM from KMI-927.
c
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: Alzheimer’s disease; BACE1 inhibitors; liquid-phase peptide synthesis; tetrapeptides
Introduction
BACE1 inhibitors, a further removal on the prime side amino
acid still remains a challenge. Peptides are believed to possess
good drug candidate properties, i.e. intrinsically potent, orally
bioavailable, metabolically stable, brain penetrant, and safe. In this
report, we describe the synthesis of tetrapeptidic KMI-compounds
and their results on enzymatic assay. Indeed, our synthesized
tetrapeptides exhibited high potency against BACE1, and thus
are proven to be attractive candidates for drug development in
Alzheimer’s disease.
An aspartic protease, BACE1 [the β-site APP (amyloid precursor
protein) cleaving enzyme] is a target protein for developing drugs
against Alzheimer’s disease, because it triggers amyloid β-peptide
(Aβ) formation by cleaving APP at the N-terminus of the Aβ
domain [1,2]. Numerous attempts to synthesize small-molecule
inhibitors of BACE1 focusing on employing peptidomimetics have
been examined [3–5]. Our initial investigation on BACE1 inhibitors
focused on octapeptides that were derived from the sequence of
the Swedish mutant APP [6]. A phenylnorstatine [Pns: (2R,3S)-3-
amino-2-hydroxy-4-phenylbutyric acid] served as the central core
at the P1 position as a substrate transition-state analogue to bind
with BACE1. These peptides were quite large in size encompassing
the P4 –P4ꢁ residues. We embarked on a truncation study that led
to potent and shorter peptidic BACE1 inhibitors, while finding
that removal of the non-prime side residues led to a considerable
decrease in BACE1 affinity [7]. In contrast, removal of the prime
side residues had a mild influence on the loss of inhibitory activity.
After a structure-activity relationship study, we succeeded in
Results and Discussion
Tetrapeptides were prepared by classical approach for
liquid-phase peptide synthesis on Boc chemistry (Boc: tert-
butoxycarbonyl) [10–12]. The structure of exemplary inhibitor
1a and its synthetic route from phenylnorstatine [Pns: (2R,3S)-3-
amino-2-hydroxy-4-phenylbutyric acid] derivative Boc-Pns-OBn
(Bn: benzyl) are summarized in Scheme 1. Boc deprotection
was performed with 4 N HCl/dioxane solution in the pres-
ence of anisole. Coupling reactions with corresponding Boc-
protected amino acids were carried out in the presence of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC·HCl),N-hydroxybenzotriazolemonohydrate(HOBt·H2O),and
ꢁ
ꢁ
removing the P2 –P4 positions to shorten ꢁthe inhibitors down
to pentapeptide size comprising the P4 –P1 residues [8–12]. As
shown in Figure 1, our previous pentapeptide inhibitors, known
as KMI-compounds, possess a modified aromatic ring with acidic
ꢁ
substituents at the P1 position to exhibit a high affinity against
BACE1 [8–13]. To obtain compounds with more potent activity
but with low acidity, our current attempts are mainly focused
on replacing the P3 –P2 residues and modifying the P4 and P1
moieties. However, the overall MWs of these pentapeptides are
>700 Da, which could be unfavorable as drugs. Thus, to develop
more potent but low molecular weight BACE1 inhibitors, current
studies have shifted to the design of less-peptidic molecules
[3,14–20]. Considering the previous approaches for peptide-type
∗
Correspondence to: Prof. Yoshiaki Kiso, Department of Medicinal Chemistry,
ꢁ
Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical
University, Yamashina-ku, Kyoto 607-8412, Japan.
E-mail: kiso@mb.kyoto-phu.ac.jp
Department of Medicinal Chemistry, Center for Frontier Research in Medicinal
Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412,
Japan
c
J. Pept. Sci. 2010; 16: 257–262
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

KAKIZAWA ET AL.
Figure 1. Pentapeptides containing a Pns at P1 position and their BACE1 inhibitory activity.
Scheme 1. Reagents and conditions: (i) anisole, 4 N HCl/dioxane; (ii) Boc-Leu-OH, EDC·HCl, HOBt·H2O, Et3N in DMF; (iii) Boc-Val-OH, EDC·HCl, HOBt·H2O,
Et3Nin DMF;(iv) Boc-Dap(Fmoc)-OH, EDC·HCl, HOBt·H2O, Et3Nin DMF;(v) 20%diethylamine/DMF;(vi) 1H-tetrazole-5-carboxylic acid, EDC·HCl, HOBt·H2O,
Et3N in DMF; (vii) Pd/C, H2 in MeOH; and (viii) preparative HPLC.
Figure 2. The molecular dynamics simulated poses of 2b (KMI-927, orange stick) in the active site of BACE1 (PDB entry, 1FKN): (a) superimposed with
an inhibitor OM99-2 (magenta stick). Catalytic two Asp residues (Asp32 and Asp228) are represented with cyan stick; (b) hydrogen bonding interactions
(dotted line) around C-terminal Pns residue in the enzyme pockets.
Et3N in DMF. After the introduction of Boc-Dap(Fmoc)-OH (Dap:
2,3-diaminopropionic acid, Fmoc: 9-fluorenylmethoxycarbonyl),
Fmoc deprotection was carried out with 20% diethylamine/DMF
solution. The tetrazole ring at the P4 position was introduced with
1H-tetrazole-5-carboxylic acid, EDC·HCl, HOBt·H2O, and Et3N in
DMF. Each coupling reaction or Boc deprotection proceeded in
64–100% yield. The deprotection of benzyl group was performed
by catalytic reduction with Pd/C in MeOH under H2 atmosphere.
After removal of the Boc group, the crude peptides were purified
on preparative HPLC and obtained as a white powder with a purity
of >97% on HPLC (see Supporting Information). The mass-to-
charge analysis showed an expected value (MS: M + Hcalcd: 590.65
Da; M + Hfound: 590.73 Da) to reveal a successful reduction in MW
by ca. 143–211 Da from the parent C-terminal extended inhibitors
depicted in Figure 1.
BACE1 inhibitory activity was determined using previously
published methods [6]. After a reaction with 7 nM recombinant
human BACE1 [21,22] and 25 µM fluorescence resonance energy
transfer (FRET) substrate [23] in an incubation buffer with
2 µM or 0.2 µM inhibitors for 60 min at 37 ◦C, the N-terminal
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 257–262

TETRAPEPTIDES AS BACE1 INHIBITORS
Table 1. Chemical data and BACE1 inhibitory activity of compounds 1a–c and 2a–c
BACE1 inhibition (%)
Compound (KMI No)
R1
R2
Data
at 2 µM
at 0.2 µM
IC50 (nM)
75.0
1a (KMI-750)
Rt: 18.42 mina
92.3
64.3
MALDI-TOF-MS (m/z): 590.73 for [M + H]+
(calcd. 590.65 for C26H40N9O7)
1b (KMI-914)
1c (KMI-915)
Rt: 18.18 mina
94.9
90.7
73.9
52.3
46.0
102
MALDI-TOF-MS (m/z): 650.68 for [M + H]+
(calcd. 649.67 for C29H41FN7O9)
Rt: 19.92 mina
MALDI-TOF-MS (m/z): 630.74 for [M + H]+
(calcd. 630.71 for C31H44N5O9)
2a (KMI-961)
2b (KMI-927)
Rt: 19.95 mina
95.6
95.9
70.0
82.8
63.0
34.6
MALDI-TOF-MS (m/z): 630.05 for [M + H]+
(calcd. 630.72 for C29H44N9O7)
Rt: 19.96 mina
MALDI-TOF-MS (m/z): 690.84 for [M + H]+
(calcd. 690.74 for C32H45FN7O9)
2c (KMI-928)
Rt: 22.36 mina
93.0
63.3
87.6
MALDI-TOF-MS (m/z): 670.79 for [M + H]+
(calcd. 670.77 for C34H48N5O9)
a Retention time on HPLC; linear gradient of CH3CN (0–100% CH3CN for 40 min) in 0.1% aqueous TFA at a flow rate of 0.9 mL min−1 (temperature:
40 ◦C), detected at 230 nm.
cleaved fragment of the substrate was analyzed by RP-HPLC
(see Supporting Information). The activity of 1a was 92.3% at 2 µM
and 64.3% at 0.2 µM, respectively. Our previous pentapeptidic
inhibitory activity at 2 µM, and 52.3–82.8% at 0.2 µM. In spite of
the removal of the P1 position, these tetrapeptides exhibited
ꢁ
moderate BACE1 affinity. Particularly, inhibitors incorporating Cha
at the P2 position showed higher activities than Leu-containing
peptides. IC50 values by enzymatic assay were evaluated for these
compounds, and Cha P2 peptide 2b (KMI-927) showed the highest
potency (IC50 = 34.6 nM).
ꢁ
inhibitors with a modified aromatic ring at P1 position possess
moderate to excellent potency (51–100% inhibitory activity at
2 µM) [6–12]. Compared to these results, inhibitor 1a shows a
relatively high inhibitory activity.
Encouraged by this result, we decided to synthesize more
tetrapeptidic derivatives. In our previous study, the introduc-
tion of 3-cyclohexylalanine (Cha) at P2 and 5-fluoroorotyl or
2,5-dihydroxybenzoyl group at P4 contributed to increase in-
hibitory activity [10]. Thus, we included these moieties into the
tetrapeptides. In an analogous manner as in Scheme 1, P2 Leu
peptides 1b–c and P2 Cha derivatives 2a–c were prepared. Their
structures and obtained data (HPLC retention time [linear gradient
of CH3CN (0–100% for 40 min) in 0.1% aqueous TFA] and MALDI-
TOF-MS) are shown in Table 1. All compounds were obtained with
a purity of >97% on HPLC, and their MS data were consistent with
expected values. The synthesized inhibitors showed 90.7–95.9%
Following removal of all prime side residues, we considered
that modifications of the P1 residue would be interesting because
the C-terminal P1 residue plays a key role in inhibitory activity
by interacting with catalytic Asp residues [8,9]. Thus, as a first
trial, we modified the Pns residue of inhibitors 1a–2c to their
corresponding methyl esters that may be favorable for improving
permeability across the blood–brain barrier [24]. In addition,
easy removals of their methyl group in vivo are expected [24],
so that they are attractive prodrug candidates that release their
corresponding parent compounds shown in Table 1. Starting with
Boc-Pns-OMe, the peptide chain was elongated using similar
procedure shown in Scheme 1 to give peptides 3a–4c. The
c
J. Pept. Sci. 2010; 16: 257–262 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

KAKIZAWA ET AL.
Table 2. Chemical data and BACE1 inhibitory activity of compounds 3a–c and 4a–c
BACE1 inhibition (%)
Compound (KMI No)
R1
R2
Data
at 2 µM
at 0.2 µM
IC50 (nM)
–
3a (KMI-1098)
Rt: 19.36 mina
71.1
–
MALDI-TOF-MS (m/z): 604.82 for [M + H]+
(calcd. 604.68 for C27H42N9O7)
3b (KMI-1100)
3c (KMI-1099)
Rt: 18.88 mina
92.4
63.1
48.1
–
–
MALDI-TOF-MS (m/z): 686.85 for [M + Na]+
(calcd. 686.68 for C30H42FN7O9Na)
Rt: 20.90 mina
–
MALDI-TOF-MS (m/z): 645.02 for [M + H]+
(calcd. 644.74 for C32H45N5O9)
4a (KMI-1078)
4b (KMI-1087)
Rt: 22.72 mina
79.6
88.7
–
–
–
MALDI-TOF-MS (m/z): 644.99 for [M + H]+
(calcd. 644.74 for C30H46N9O7)
Rt: 21.34 mina
39.9
MALDI-TOF-MS (m/z): 704.41 for [M + H]+
(calcd. 704.77 for C33H47FN7O9)
4c (KMI-1079)
Rt: 23.91 mina
74.4
–
–
MALDI-TOF-MS (m/z): 684.79 for [M + H]+
(calcd. 684.80 for C35H50N5O9)
a Retention time on HPLC; linear gradient of CH3CN (0–100% CH3CN for 40 min) in 0.1% aqueous TFA at a flow rate of 0.9 mL−1 (temperature: 40 ◦C),
detected at 230 nm.
obtaineddataaresummarizedinTable 2. Inenzymaticassay, these
were slightly less active than their parent compounds 1a–2c.
At 2 µM of the inhibitors, inhibitory activity was 63.1–92.4%.
Compounds possessing >80% activity at 2 µM were subjected to
lowconcentrationassayat0.2 µM. Fromtheactivityresultsofthese
methylated compounds, it is proven that the introduction of the
5-fluoroorotyl group at the P4 position is favorable for inhibitory
activity as shown in 3b and 4b.
We next turned our attention to the amidation of the
C-terminus of the Pns residue. The amidated peptides 5a–6c
in Table 3 were prepared from Boc-Pns-NH2 [25,26] by similar
procedures described earlier. Through this modification, the
inhibitors showed moderate inhibitory activity that was superior
to the corresponding methylated compounds 3a–4c. From the
resultsof thisseries, itisagainproventhatthe5-fluoroorotylgroup
at P4 position contributes significantly to affinity for BACE1. The
P2 Val and P2 Cha derivatives containing the 5-fluoroorotyl group
(5b and 6b) exhibited 94.0 and 95.1% BACE1 inhibition at 2 µM,
as well as 61.4 and 64.5% at 0.2 µM. Compounds having >50%
inhibitory activity at 0.2 µM (5b and 6b) were subjected for IC50
value evaluation. Compared to the compounds in Tables 1 and
2, the amidated peptides showed moderate potency as BACE1
inhibitors.
To better understand the coordination mode of the tetrapep-
tides toward BACE1, we performed a molecular modeling study
between the structural data of BACE1 and the more potent com-
pound 2b (KMI-927, IC50 = 34.6 nM). The BACE1 binding model
of 2b was simulated using a modeling package (MOE 2008.10,
Chemical Computing Group, Inc., Montreal, Canada). The X-ray
crystal data of OM99-2 complex (PDB ID, 1FKN) [27] was used to
build an initial conformation of the 2b complex. A carboxyl group
of Asp228 was intentionally protonated. Several energy minimiza-
tion processes with an MMFF94x force field were performed with
water molecules, followed by a molecular dynamics simulation
(see Supporting Information). The obtained result is shown in
Figure 2. Inhibitor 2b was stably positioned along the main chain
of N-terminal residues of OM99-2 during the simulation, especially
at the P3 and P4 residues (Figure 2a). On the other hand, the P1
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 257–262

TETRAPEPTIDES AS BACE1 INHIBITORS
Table 3. Chemical data and BACE1 inhibitory activity of compounds 5a–c and 6a–c
BACE1 inhibition (%)
Compound (KMI No)
R1
R2
Data
at 2 µM
at 0.2 µM
IC50 (nM)
–
5a (KMI-1338)
Rt: 17.43 mina MALDI-TOF-MS (m/z): 589.67 for
[M + H]+ (calcd. 589.67 for C26H41N10O6)
75.0
–
5b (KMI-1170)
5c (KMI-1339)
Rt: 17.84 mina MALDI-TOF-MS (m/z): 649.81 for
[M + H]+ (calcd. 649.69 for C29H42FN8O8)
94.0
75.2
61.4
99.4
Rt: 18.80 mina MALDI-TOF-MS (m/z): 629.73 for
[M + H]+ (calcd. 629.72 for C31H45N6O8)
–
–
6a (KMI-1167)
6b (KMI-1169)
Rt: 21.15 mina MALDI-TOF-MS (m/z): 629.65 for
[M + H]+ (calcd. 629.73 for C29H45N10O6)
88.9
95.1
44.3
64.5
–
Rt: 20.35 mina MALDI-TOF-MS (m/z): 689.91 for
[M + H]+ (calcd. 689.76 for C32H46FN8O8)
70.5
6c (KMI-1168)
Rt: 22.62 mina MALDI-TOF-MS (m/z): 669.69 for
[M + H]+ (calcd. 669.79 for C34H49N6O8)
87.8
41.8
–
a Retention time on HPLC; linear gradient of CH3CN (0–100% CH3CN for 40 min) in 0.1% aqueous TFA at a flow rate of 0.9 mL−1 (temperature: 40 ◦C),
detected at 230 nm.
and P2 residues were slightly moved up from the pockets. In our
previous C-terminal extended inhibitors, the modeling study sug-
gested that the Pns residue at P1 interacts with the catalytic two
Asp residues (Asp32 and Asp228). However in 2b, the hydroxyl
group of Pns interacted with the carboxylic side chain of Asp228,
but not with that of Asp32 (Figure 2b). The hydrogen bonding
interactions of the Pns carboxylic group with the side chain of
Arg235 and the amide NH of Thr72 in the flap were observed, and
probably made the positioning of 2b different from that of the
C-terminal extended inhibitors. The modification of the carboxylic
group by methyl ester and amide, compounds 4b and 6b, respec-
tively, would prevent these interactions with Arg235 and Thr72,
reducing the affinity to BACE1.
all prime side residues, the synthesized compounds showed
relatively high BACE1 affinity. We modified the P1 residue of
the synthesized KMI-compounds. In this study, the inhibitors with
a free carboxylic group at the C-terminus exhibited better affinity
than their corresponding methylated or amidated derivatives in
enzymatic assay. The inhibitory effect of P2 Cha derivative 2b (KMI-
927, IC50 = 34.6 nM) was superior to that of other tetrapeptides
reported here.
We have just started investigating tetrapeptidic BACE1 in-
hibitors, and thus our attempts are yet at the early stage of
development. To find more attractive drug candidates, further
study is now in progress.
Acknowledgements
This research was supported in part by the ‘Academic Frontier’
Project for Private Universities: matching fund subsidy from MEXT
(Ministry of Education, Culture, Sports, Science and Technology),
and the 21st Century Center of Excellence Program ‘Development
Conclusion
Tetrapeptidic KMI-compounds were prepared, and their potency
against BACE1 in vitro was examined. In spite of the lack of
c
J. Pept. Sci. 2010; 16: 257–262 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

KAKIZAWA ET AL.
of Drug Discovery Frontier Integrated from Tradition to Proteome’
from MEXT. Mass spectra measurements were performed by H.-O.
Kumada. We thank Dr J.-T. Nguyen for his kind revisions of the
manuscript.
14 Hamada Y, Kiso Y. Recent progress in the drug discovery of non-
peptidic BACE1 inhibitors. Exp. Opin. Drug Discov. 2009; 4: 391–416.
15 Hom RK, Gailunas AF, Mamo S, Fang LY, Tung JS, Walker DE, Davis D,
Thorsett ED, Jewett NE, Moon JB, John V. Design and synthesis
of hydroxyethylene-based peptidomimetic inhibitors of human
β-secretase. J. Med. Chem. 2004; 47: 158–164.
16 Stachel SJ, Coburn CA, Steele TG, Jones KG, Loutzenhiser EF,
Gregro AR, Rajapakse HA, Lai M-T, Crouthamel M-C, Xu M,
Tugusheva K, Lineberger JE, Pietrak BL, Espeseth AS, Shi X-P, Chen-
Dodson E, Holloway MK, Munshi S, Simon AJ, Kuo L, Vacca JP.
Structure-based design of potent and selective cell-permeable
inhibitors of human β-secretase (BACE-1). J. Med. Chem. 2004; 47:
6447–6450.
Supporting Information
Supporting information is available free of charge via the Internet
at www.interscience.wiley.com/journal/jpepsci.
17 Hamada Y, Ohta H, Miyamoto N, Yamaguchi R, Yamani A, Hidaka K,
Kimura T, Saito K, Hayashi Y, Ishiura S, Kiso Y. Novel non-peptidic
and small-sized BACE1 inhibitors. Bioorg. Med. Chem. Lett. 2008; 18:
1654–1658.
18 Barrow JC, Stauffer SR, Rittle KE, Ngo PL, Yang Z, Selnick HG,
Graham SL, Munshi S, McGaughey GB, Holloway MK, Simon AJ,
Price EA, Sankaranarayanan S, Colussi D, Tugusheva K, Lai MT,
Espeseth AS, Xu M, Huang Q, Wolfe A, Pietrak B, Zuck P, Levorse DA,
Hazuda D, Vacca JP. Discovery and X-ray crystallographic analysis of
a spiropiperidine iminohydantoin inhibitor of β-secretase. J. Med.
Chem. 2008; 51: 6259–6262.
References
1 Selkoe DJ. Translating cell biology into therapeutic advances in
Alzheimer’s disease. Nature 1999; 399: A23–A31.
2 Sinha S, Lieberburg I. Cellular mechanisms of β-amyloid production
and secretion. Proc. Natl. Acad. Sci., U. S. A. 1999; 96: 11049–11053.
3 Stachel SJ. Progress toward the development of a viable BACE-1
inhibitor. Drug Dev. Res. 2009; 70: 101–110.
4 Hills ID, Vacca JP. Progress toward a practical BACE-1 inhibitor. Curr.
Opin. Drug Discov. Dev. 2007; 10: 383–391.
19 Ghosh AK, Kumaragurubaran N, Hong L, Kulkarni S, Xu X, Miller HB,
Reddy DS, Weerasena V, Turner R, Chang W, Koelsch G, Tang J.
Potent memapsin 2 (β-secretase) inhibitors: design, synthesis,
protein-ligand X-ray structure, and in vivo evaluation. Bioorg. Med.
Chem. Lett. 2008; 18: 1031–1036.
20 Barazza A, Go¨tz M, Cadamuro SA, Goettig P, Willem M, Steuber H,
Kohler T, Jestel A, Reinemer P, Renner C, Bode W, Moroder L.
Macrocyclic statine-based inhibitors of BACE-1. ChemBioChem 2007;
8: 2078–2091.
21 Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P,
Teplow DB, Ross S, Amarante P, Loeloff R, Luo Y, Fisher S, Fuller J,
Edenson S, Lile J, Jarosinski MA, Biere AL, Curran E, Burgess T, Louis
J-C, Collins F, Treanor J, Rogers G, Citron M. β-Secretase cleavage
of Alzheimer’s amyloid precursor protein by the transmembrane
aspartic protease BACE. Science 1999; 286: 735–741.
22 Yan R, Bienkowski MJ, Shuck ME, Miao H, Tory MC, Pauley AM,
Brashler JR, Stratman NC, Mathews WR, Buhl AE, Carter DB,
Tomasselli AG, Parodi LA, Heinrikson RL, Gurney ME. Membrane-
anchored aspartyl protease with Alzheimer’s disease β-secretase
activity. Nature 1999; 402: 533–537.
5 Ziora Z, Kimura T, Kiso Y. Small-sized BACE1 inhibitors. Drugs Fut.
2006; 31: 53–63.
6 Shuto D, Kasai S, Kimura T, Liu P, Hidaka K, Hamada T, Shibakawa S,
Hayashi Y, Hattori C, Szabo B, Ishiura S, Kiso Y. KMI-008, a novel β-
secretase inhibitor containing a hydroxymethylcarbonyl isostere as
a transition-state mimic: design and synthesis of substrate-based
octapeptides. Bioorg. Med. Chem. Lett. 2003; 13: 4273–4276.
7 Kimura T, Shuto D, Kasai S, Liu P, Hidaka K, Hamada T, Hayashi Y,
Hattori C, Asai M, Kitazume S, Saido TC, Ishiura S, Kiso Y. KMI-358 and
KMI-370, highly potent and small-sized BACE1 inhibitors containing
phenylnorstatine. Bioorg. Med. Chem. Lett. 2004; 14: 1527–1531.
8 Kimura T, Shuto D, Hamada Y, Igawa N, Kasai S, Liu P, Hidaka K,
Hamada T, Hayashi Y, Kiso Y. Design and synthesis of highly active
Alzheimer’s β-secretase (BACE1) inhibitors, KMI-420 and KMI-429,
with enhanced chemical stability. Bioorg. Med. Chem. Lett. 2005; 15:
211–215.
9 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.
Design and synthesis of potent β-secretase (BACE1) inhibitors with
P1ꢁ carboxylic acid bioisosteres. Bioorg. Med. Chem. Lett. 2006; 16:
2380–2386.
10 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. β-Secretase
inhibitors: modification at the P4 position and improvement of
inhibitory activity in cultured cells. Bioorg. Med. Chem. Lett. 2006; 16:
4354–4359.
23 Koike H, Seki H, Kouchi Z, Ito M, Kinouchi T, Tomioka S, Sorimachi H,
Saido TC, Maruyama K, Suzuki K, Ishiura S. Thimet oligopeptidase
cleaves the full-length Alzheimer amyloid precursor protein at a β-
secretase cleavage site in COS cells. J. Biochem. 1999; 126: 235–242.
24 Suzuki M, Noyori R, Langstrom B, Watanabe Y. Molecular design of
prostaglandin probes in brain research: high, specific binding to
a novel prostacyclin receptor in the central nervous system. Bull.
Chem. Soc. Jpn. 2000; 73: 1053–1070.
11 Ziora Z, Kasai S, Hidaka K, Nagamine A, Kimura T, Hayashi Y,
Kiso Y. Design and synthesis of BACE1 inhibitors containing
25 Lee KS, Seon SH, Lee YH, Kim HD, Son MH, Chung BY, Lee JY,
Jin C, Lee YS. Synthesis and biological evaluation of chromone
carboxamides as calpain inhibitors. Bioorg. Med. Chem. Lett. 2005;
15: 2857–2860.
26 Nam DH, Lee KS, Kim SH, Kim SM, Jung SY, Chung SH, Kim HJ,
Kim ND, Jin C, Lee YS. Design and synthesis of 4-quinolinone 2-
carboxamides as calpain inhibitors. Bioorg. Med. Chem. Lett. 2008;
18: 205–209.
a
novel norstatine derivative (2R,3R)-3-amino-2-hydroxy-4-
(phenylthio)butyric acid. Bioorg. Med. Chem. Lett. 2007; 17:
1629–1633.
12 Hamada Y, Abdel-Rahman H, Yamani A, Nguyen J-T, Stochaj M,
Hidaka K, Kimura T, Hayashi Y, Saito K, Ishiura S, Kiso Y. BACE1
inhibitors: optimization by replacing the P1ꢁ residue with non-acidic
moiety. Bioorg. Med. Chem. Lett. 2008; 18: 1649–1653.
13 Asai M, Hattori C, Iwata N, Saido TC, Sasagawa N, Szabo B,
Hashimoto Y, Maruyama K, Tanuma S, Kiso Y, Ishiura S. The novel β-
secretase inhibitor KMI-429 reduces amyloid β peptide production
in amyloid precursor protein transgenic and wild-type mice.
J. Neurochem. 2006; 96: 533–540.
27 Hong L, Koelsch G, Lin X, Wu S, Terzyan S, Ghosh AK, Zhang XC,
Tang J. Structure of the protease domain of memapsin 2 (β-
secretase) complexed with inhibitor. Science 2000; 290: 150–153.
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 257–262