Design of pentapeptidic BACE1 inhibitors with carboxylic acid bioisosteres at P1′ and P4 positions

Article abstract of DOI:10.1016/j.bmc.2010.03.032

We previously reported potent BACE1 inhibitors KMI-420 and KMI-570 possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. Acidic moieties at the P₁^′ and P₄ positions of KMI inhibitors are thought to be unfavorable in terms of membrane permeability across the blood-brain barrier. Herein, we replaced acidic moieties at the P₄ position with hydrogen bond accepting groups and acidic moieties at the P₁^′ position with less acidic and similar molecular-size moieties (carboxylic acid or tetrazole bioisosteres). These inhibitors exhibited improved BACE1 inhibitory activities and a thorough quantitative structure-activity relationship study was performed.

Full text of DOI:10.1016/j.bmc.2010.03.032

Bioorganic & Medicinal Chemistry 18 (2010) 3175–3186
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design of pentapeptidic BACE1 inhibitors with carboxylic acid bioisosteres
at P0 and P4 positions
1
Harichandra D. Tagad, Yoshio Hamada, Jeffrey-Tri Nguyen, Takashi Hamada, Hamdy Abdel-Rahman,
Abdellah Yamani, Ayaka Nagamine, Hayato Ikari, Naoto Igawa, Koushi Hidaka, Youhei Sohma,
*
Tooru Kimura, Yoshiaki Kiso
Department of Medicinal Chemistry, Centre Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-Ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported potent BACE1 inhibitors KMI-420 and KMI-570 possessing a hydroxymethylcar-
bonyl isostere as a substrate transition-state mimic. Acidic moieties at the P01 and P4 positions of KMI
inhibitors are thought to be unfavorable in terms of membrane permeability across the blood–brain bar-
rier. Herein, we replaced acidic moieties at the P4 position with hydrogen bond accepting groups and
acidic moieties at the P10 position with less acidic and similar molecular-size moieties (carboxylic acid
or tetrazole bioisosteres). These inhibitors exhibited improved BACE1 inhibitory activities and a thorough
quantitative structure–activity relationship study was performed.
Received 17 February 2010
Revised 12 March 2010
Accepted 13 March 2010
Available online 21 March 2010
Keywords:
Alzheimer disease
b-Secretase
Ó 2010 Elsevier Ltd. All rights reserved.
Inhibitors
Bioisosteres
Structure–activity relationship
1. Introduction
itors is invaluable to elucidate the pathology of AD. Several transi-
tion-state analogues of BACE1 inhibitors modeled on the b-
Alzheimer’s disease (AD) is a neurodegenerative disorder lead-
ing to the loss of memory and other intellectual abilities.1,2 The
diagnosis of AD can be confirmed by the presence of amyloid pla-
ques,3 neurofibrillary tangles (NFTs), synaptic loss and atrophy in
specific areas of the brain.4 Although the pathogenesis of AD is
highly complex, several pathological traits characterize this dis-
ease such as amyloid plaques composed of clusters of b-amyloid
peptide on the blood vessels and the outside surface of neurons
in the brain. The generation of 40- and 42-residue amyloid b (Ab)
peptides in the human brain by proteolysis of the membrane an-
chored b-amyloid precursor protein (APP) is a key event in the pro-
secretase cleaving sequence have been reported with relatively
low IC50 values, although these inhibitors are often too large to
be good drug candidates.16,17
Recently, we reported small-size BACE1 inhibitors with acidic
moieties at the P01 position and tetrazolic acid at the P4 position
using hydroxymethylcarbonyl isosteres as a transition-state mimic
(Fig. 1).18–22 Although these inhibitors exhibited potent inhibitory
activity, acidic moieties that are similar to carboxylic groups often
P4
P3
P2
P1
P1'
gression of Alzheimer’s disease. Two proteases, called b- and
c-
secretases, cleave APP in the amyloidogenic pathway.5–9 Ab genesis
is initiated by BACE1 (b-site APP Cleaving Enzyme type 1) cleavage
of APP before the Asp residue of the Ab sequence to form an N-ter-
minus of the Ab peptide.10–13
O
O
O
H
H2N
N
N
N
N
R
H
H
H
O
OH
This scission librates two cleavage fragments: a secreted APP
ectodomain (APPsb) and membrane bound carboxyl terminal frag-
NH
N
O
ment (CTF), C-99, which is subsequently cleaved by
c-secretase to
NH
N
N
generate the C-terminus of the Ab peptide and an APP intracellular
domain (AICD). As BACE1 plays a critical role as a rate determining
step in the progression of AD,14,15 the development of BACE1 inhib-
KMI- 420 ( R = COOH ) IC50 = 8.2 nM
N
NH
KMI- 570 ( R =
) IC50 = 1.2 nM
N
N
* 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. Pentapeptidic BACE1 inhibitors.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.032

3176
H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
possess low membrane permeability across the blood–brain bar-
rier. Hence, there is a need to design and synthesize inhibitors with
non- or less-acidic moieties or bioisosteres. Herein, we synthesize
inhibitors with carboxylic acid bioisosteres. KMI inhibitors with
tetrazole ring as a carboxylic acid bioisostere led to enhancement
in BACE1 inhibitory activity. Our structure–activity relationship
(SAR) study on KMI compounds showed that an acidic moiety at
the P01 as well as P4 position are essential for improving BACE1
inhibitory activity. Previous reports from our laboratory showed
that replacement of the carboxylic acid group found in the P01 by
less acidic and similar molecular-size carboxylic acid bioisosteres
like tetrazole, oxadiazole, thiadiazole or triazole-type heterocycles
showed high inhibitory activity.23 Moreover, inhibitors with aro-
matic hydrogen bond acceptors such as tetrazole-5-carbonyl, 2,5-
dihydroxybenzoyl or 5-fluoroorotyl groups at the P4 position
exhibited higher BACE1 inhibitory activity.24 Herein, we designed
and synthesized pentapeptidic KMI inhibitors with carboxylic acid
bioisosteres such as triazole or thiazole derivatives at the P01 posi-
tion, and 2,5-dihydroxybenzoyl or 5-fluoroorotyl groups at the P4
position that exhibited IC50 values in the low nanomolar range
(Fig. 2).
2. Chemistry
Acid bioisosteres corresponding to the P01 position of KMI inhib-
itors were synthesized. Heterocycles 2, 4–6 were synthesized from
3-nitrobenzonitrile. Compound 2 was synthesized from 3-nitro-
benzonitrile which was first converted into 3-nitrotetrazole by so-
dium azide mediated cyclization followed by reduction of the nitro
group under catalytic hydrogenation condition using 10% Pd/C to
give 3-(1H-tetrazol-5-yl)benzenamine 2. On the other hand tert-
butyl 3-amidinophenylcarbamate (aldoxime) 3 was synthesized
from 3-aminobenzonitrile in a two-step protocol reported by Naka
and co-workers.25,26 Initially, the amino group was protected using
di-tert-butyl dicarbonate [(Boc)2O] and then converted into carba-
mate 3 using hydroxylamine in DMSO, and then was further con-
verted into corresponding amino derivatives 4–6 as shown in
Scheme 1. Carbamate 3 was reacted with 2-ethylhexylchlorofor-
mate and the obtained intermediate was cyclized by refluxing in
xylene to afford 4. 5-Oxo-1,2,4-thiadiazole 5 was synthesized by
reacting 1,10-thiocarbonyldiimidazole (TCDI) with aldoxime 3 fol-
lowed by treatment with boron trifluoride diethyl etherate
(BF3ꢀOEt2). The synthesis of 5-thioxo-1,2,4-oxodiazole
6 was
accomplished by treatment of aldoxime with TCDI and 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (DBU). The obtained Boc-protected deriv-
atives were further converted to their respective free amine 5 and
6. Analogues 927,28 and 1029–31 were synthesized from the N-Boc
protected methyl ester of 3-amino benzoic acid 7b. Amino hydra-
zide derivative 8 was prepared by refluxing tert-butyl-3-(methoxy-
carbonyl)phenylcarbamate and hydrazine hydrate (N2H4ꢀH2O) in
tetrahydrofuran. The reaction of hydrazide 8 with carbon disulfide
and KOH in ethanol followed by deprotection of the tert-butyloxy-
carbonyl afforded 5-(3-aminophenyl)-1,3,4-oxadiazole-2(3H)-thi-
one 9. Reacting the same benzoyl hydrazide 8 with methyl
isothiocyanate (MITC) gave a thiosemicarbazide derivative which
on heating in the presence 2–3 drops of Et3N in EtOH or 1 N NaOH
(aq) underwent smooth cyclization through dehydration to afford
the tert-butyl-3-(4,5-dihydro-4-methyl-5-thioxo-1H-1,2,4-triazol-
3-yl)phenylcarbamate. It was further exposed to 4 N HCl/dioxane
to effectively remove the Boc group to give 10 (Scheme 2). The syn-
thesis of the peptides was performed by traditional solution-phase
peptide synthetic method, starting from N-Boc protected Pns [phe-
nylnorstatine: (2R,3S)-3-amino-2-hydroxy-4-phenylbutyric acid]
using appropriate amino acids. Peptide bond formation was carried
out with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideꢀHCl
(EDCꢀHCl) and 1-hydroxybenzotriazole (HOBt). Boc (tert-butyloxy-
carbonyl) and Fmoc (9-fluorenylmethoxycarbonyl) protected
amines were cleaved with a cocktail of anisole/4 N HCl/dioxane
P4
P1'
O
O
O
H
H2N
N
OH
N
H
N
H
N
H
O
OH
O
NH
O
R
H
O
S
N
O
S
O
N
N
N
N
OH
O
HN
HN
N
CH3
HO
H
O
N
N
N
S
S
N
F
N
HN
HN
O
NH
HN
O
Figure 2. BACE1 inhibitors with carboxylic acid bioisostere at P0 and P4 positions.
1
e, f, g
N
H2N
O
HN
4
O
b, c
d
Boc
h, i, g
Boc
N
N
H
CN
O2N
CN
N
OH
N
H
1a
a, b
1b
H2N
H2N
3
NH2
S
5
6
HN
HN
O
j, g
H
N
N
H2N
O
S
N
2
N
N
Scheme 1. Reagents and conditions: (a) NaN3, NH4Cl, DMF, reflux; (b) H2, 10% Pd/C, MeOH; (c) (Boc)2O, Et3N, THF, rt, 46 h; (d) NH2OHꢀHCl, Et3N, DMSO, 75 °C, 15 h; (e)
pyridine, 2-ethylhexyl chloroformate, THF, 0 °C, 30 min; (f) xylene, reflux, 2 h; (g) anisole, 4 N HCl/dioxane, rt, 1 h; (h) TCDI, THF, rt, 1 h; (i) BF3ꢀOEt2, THF, rt, 1 h; (j) TCDI, DBU,
MeCN, rt,1 h.

H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
3177
c, d
N
N
H2N
NH
S
O
N
9
H
b
R
O
Boc
N
N
N
NH2
H
H
O
O
7
8
e, d
R = H, 7a
H2N
a
NH
S
R = Boc, 7b
10
Scheme 2. Reagents and conditions: (a) (Boc)2O, Et3N, THF, rt, 48 h; (b) N2H4ꢀH2O, THF, reflux, 46 h; (c) CS2, KOH, EtOH, reflux, 12 h; (d) anisole, 4 N HCl/dioxane, rt, 1 h; (e)
methyl isothiocyanate, THF, rt, 6 h then aq 1 N NaOH, 45 °C, 3 h.
acter of the P4 side chain. Simultaneously, we observed the effect of
Cha and Leu on BACE1 inhibitory activity at the P2 position. The P2
position’s Cha seemed to display a slight enhancement in activity
compare to that of Leu. We selected P4 glutamic acid inhibitors
with similar molecular-size, less acidic aniline derivatives at the
P01 position as shown in Table 2.
R1-COOH
Boc-DAP(Fmoc)-OH
Boc-Val-OH
a, b
27 or 28
a, c
a, b
Boc-Leu-OH
a, b
Inhibitors 18–21 showed similar inhibitory activity as that of
tetrazole analogue 17. From these previous outcomes as shown
Boc-Pns-OH
a, b
in Tables 1 and 2, we fixed the P0 and P4 position residues with less
1
6
acidic and aromatic hydrogen bond acceptor carboxylic acid bio-
isosteres, respectively, and a P2 Leu residue. The pentapeptides
were synthesized starting from Pns with carboxylic acid isosteres
at the P01 position and aromatic hydrogen bond acceptor heterocy-
cles (2,5-dihydroxybenzoic acid and 5-fluoroorotic acid) at the P4
position as shown in Fig. 2. The BACE1 inhibitory activity for inhib-
itors 23–32 with P01 position modifications is summarized in Table
3 alongside 11 and 13 tetrazole derivatives as references. With
regards to the heterocyclic rings, 5-oxo-1,2,4-oxadiazole, 5-oxo-
1,2,4-thiadiazole, 5-thioxo-1,2,4-oxadiazole and 2-thioxo-1,3,4-
oxadiazole derivatives were found to be as potent as the tetrazole
derivatives, suggesting that the spatial location of the P01 moiety
interacting with the S01 hydrophobic regions of the BACE1 is impor-
tant for improving BACE1 inhibitory activity. We synthesized com-
pounds with 2,5-dihydroxybenzoic acid and 5-fluoroorotic acid at
the P4 position and varied the P01 position tetrazole residue with
its bioisosteres like 5-oxo-1,2,4-oxadiazole, 5-oxo-1,2,4-thiadia-
zole, 5-thioxo-1,2,4-oxadiazole, 2-thioxo-1,3,4-oxadiazole and 2-
methyl-3-thioxo-1,2,4-triazole derivatives. These five member ring
heterocycles having similar size as that of tetrazole and possessing
delocalized negative charges around the ring system32 acted as
hydrogen bond acceptors. An anionic group or hydrogen bond
acceptor at the P01 carboxylic acids coordinated improved inhibitor
site-interactions with the S01 pocket. In addition, these heterocycles
possessed partial hydrophobic regions above and below the ring’s
plane that might interact favorably with the hydrophobic inner
walls of the S01 pocket and led to enhancement in the inhibitory
activity of compounds 11, 13, 17–30 (Tables 2 and 3). However
inhibitors 22, 31 and 32 which contained N-methyl heterocycles
(2-methyl-3-thioxo-1,2,4-triazole) at the P01 position, resulted in a
Scheme 3. Reagents: (a) EDCꢀHCl, HOBtꢀH2O, DMF; (b) anisole, 4 N HCl/dioxane; (c)
20% piperidine/DMF.
and 20% piperidine in N,N-dimethylformamide (DMF), respec-
tively, as shown in Scheme 3. The obtained crude peptides were
further purified by column chromatography and finally purified
by preparative RP-HPLC to afford pure peptides with >95% purity.
3. Results and discussion
3.1. BACE1 inhibition data
Previously, we reported compounds with 2,5-dihydroxybenzoic
acid, 5-fluoroorotic acid or tetrazolic acid at the P4 position that
exhibited potent BACE1 inhibitory activity, and these moieties
could be used as carboxylic acid bioisosteres in pentapeptidic
inhibitors. Moreover Tables 1 and 2 shows pKa data for P01 and P4
moieties obtained from Advanced Chemistry Development (ACD/
Labs) software for Solaris at 25 °C. Table 1 shows that the P4 2,5-di-
hydroxybenzoyl (pKa = 9.08) and 5-fluoroorotyl (pKa = 6.02) amide
moieties are less acidic than the P01 tetrazole (pKa = 4.84) thus the
latter one may contribute more to the overall acidity (pKa = 4.25)
of peptides 11–14. Whereas the P4 tetrazole derivatives having
higher pKa = 2.70 than the P0 position tetrazole residue lead to high
1
acidity (pKa = 2.86) in peptides 15 and 16. From Table 2, we ob-
served that P01 aniline derivatives 4–6, 9, 10 have much less acidity
compared to that of tetrazole analogue 2 (pKa = 4.84) and hence
can be used as a bioisosteres. Thus from Tables 1 and 2, we decided
to replace the 2,5-dihydroxybenzoyl and 5-fluoroorotyl moieties at
the P4 position, and used the aniline derivatives (4–6, 9, 10) at the
P01 position to minimize the overall acidity of the pentapeptides.
These inhibitors were synthesized from unnatural amino acid
fall in BACE1 inhibitory activity at 0.2 lM, suggesting that the free
amine functionality might be involved in hydrogen bond interac-
tions and its N-methyl derivatives prevented hydrogen bond for-
mation with the S01 pocket of BACE1.
When the inhibitory activity of compounds 17–21 was compared
to that of 11, 13, 23–30, the latter ones were more potent, suggesting
a strong involvement of the P4 position residue. In other words, both
the P01 and P4 are important for improving the BACE1 inhibitory
L
-cyclohexyl-alanine (Cha) or natural amino acid Leu at the P2 po-
sition, and their respective inhibitory activity is shown in Table 1.
The 2,5-dihydroxybenzoic acid analogues, 11 and 12, with Leu
or Cha at the P2 position, respectively, exhibited similar inhibitory
activity. The high inhibitory activity of inhibitors 13 and 14 was
due to the presence of a strong electron withdrawing fluorine atom
in 5-fluoroorotic acid that led to a boost in the electrophilicity of
the aromatic ring and enhanced the hydrogen bond accepting char-
activity. Compounds 11, 13, 23–30, in which both P0 and P4 positions
1
were replaced by their respective carboxylic acid bioisosteres,
showed potent BACE1 inhibitory activity. Considering that tetrazole
displayed higher inhibitory activity when placed at the P4 position,

3178
H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
Table 1
Pentapeptidic BACE1 inhibitors with modifications at P2 and P4 positions
O
O
O
H
H2N
N
N
N
N
N
NH
H
H
H
O
OH
N
N
R2
NH
O
R1
a
b
Compound (KMI No.)
R1
R2
BACE1 inhibition (%)
Predicted pKa
at 2
l
M
at 0.2
lM
IC50 (nM)
Most acidic
P4 moietiesc
9.08
HO
11 (KMI-573)
12 (KMI-575)
13 (KMI-572)
99
93
—
4.25
OH
OH
HO
99
92
98
97
—
4.25
4.25
4.25
9.08
6.02
6.02
F
O
NH
>99
>99
6.5
5.6
HN
O
O
F
NH
O
14 (KMI-574)
15 (KMI-570)
HN
N
NH
>99
>99
98
98
4.8
3.3
2.86
2.86
2.70
2.70
N
N
N
N
16 (KMI-571)
NH
N
a
b
c
See Ref. 24.
Data from ACD/Labs software for Solaris 25 °C.
Correspond to the pKa of R1CONH2.
this position of the pentapeptide was modified with nitrogen con-
taining heterocycles such as imidazole and triazole with Cha or
tion equation (r2 = 0.92) between percent inhibitions at 2
lM and
0.2 lM, from compounds, 11–21, 23–36. We examined each posi-
Leu at the P2 position and tetrazole at the P0 position as shown in Ta-
tion where moieties were varied, namely the P0 , P2 and P4 residues.
1
1
ble 4. Synthesized inhibitors 33–36 having imidazole or triazole at
the P4 position showed a large loss of potency as compared to that
of tetrazole analogues 15 and 16 which might be due to the differ-
ence in the number of binding regions between imidazole and tria-
zole as compare to that of the tetrazole analogues. On the other
hand, by increasing the number of nitrogen atoms in five member
ring system, BACE1 inhibitory activity also increased, thus suggest-
ing the involvement of hydrogen bond interactions.
The spread of inhibitory activity values was small (63–74%) for
compounds that only differed at the P01 residue (17–21).
This narrow range suggested that, other than the N-meth-
ylthiooxotriazole outlier 22, these moieties are effective bioisoster-
es of the tetrazole ring and that any parameterization for the P10
residue would contribute very little to the QSAR equation. Simi-
larly, having a P2 residue as either L-Leu or L-cyclohexyl-alanine
led to inhibitors with only slight improvement in BACE1 inhibitory
activity (cf. 11 and 12, 13 and 14, 15 and 16, 33 and 34, 35 and 36).
This suggested that the S2 subsite could accommodate different
hydrophobic moieties.
3.2. Quantitative structure–activity relationship
To correlate chemical structure with BACE1 inhibition and sta-
tistically support our previous structure–activity relationship spec-
BACE ¼ ꢁð5:447 AccP4 ꢁ 0:079 Acc2 Þ ꢁ ð5:064 DonP4
P4
ꢁ 0:382 Don2P4Þ þ 163:175
ulations,
a formal quantitative structure–activity relationship
n ¼ 11; r2 ¼ 0:85; F ¼ 9; p ¼ 0:01
ð1Þ
(QSAR) study was performed. To increase the spread of data, that
is, the BACE1 inhibition range, the inhibitory percentage at
AccP4 and DonP4 are the approximation to the sum of VDW sur-
face areas (Å2) of pure hydrogen bond acceptors and donors,
respectively, on the P4 side chain.
There is a hydrogen bonding region in the S4 subsite that consti-
tutes of Asn233, Arg235, Gly264, Arg307 and Lys321. Because the
0.2
0.2
l
l
M was considered instead of 2
M was not determined for compound 22 because of its mod-
M. To include compound 22 in our quantita-
tive structure–activity relationship study, we extrapolated its
percent inhibition at 0.2 M to 6% from a well-fitted linear correla-
lM. However, inhibition at
erate inhibition at 2
l
l

H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
3179
Table 2
BACE1 inhibitory activity of P4 glutamic compounds
O
O
O
H
H2N
N
N
H
N
N
R
H
H
O
OH
O
OH
Compounda (KMI No.)
R
BACE1 inhibition (%)
at 0.2
Predicted pKa
b
P01 moietiesc
at 2
92
lM
l
M
Most acidic
4.24
N
17 (KMI-569)
18 (KMI-596)
NH
74
66
4.84
7.14
N
N
N
O
O
94
92
95
92
74
4.24
4.24
4.24
4.24
4.24
HN
N
S
19 (KMI-683)
20 (KMI-879)
21 (KMI-666)
63
68
72
—
7.31
5.54
6.58
8.89
HN
HN
O
O
N
O
S
N
NH
S
N
NH
22 (KMI-686)
N
H3C
S
a
b
c
See Ref. 23.
Data from ACD/Labs software for Solaris at 25 °C.
Correspond to the pKa of RPhNH2.
S4 subsite has hydrogen accepting and donating potentials, we ob-
served a good correlation (Eq. 1) between BACE1 inhibition and the
approximation to the sum of van der Waals (VDW) surface areas of
pure hydrogen bond acceptors and donors when compounds 11–
17, 33–36 were evaluated with the software Molecular Operating
Environment (MOE, ver. 2008.10, Chemical Computing Group Inc.,
Canada). The well-fitted (r2 = 0.85) and statistically significant
(p = 0.01) equation was validated by leave-one-out cross-valida-
tion (r2 range: 0.84–0.93).
Each descriptor was expressed in quadratic form such that the
optimal or worst value for the descriptor could be calculated from
the vertex of the parabola and a range of vertices could be obtained
from cross-validation. The worst VDW surface areas of hydrogen
bond acceptors and donors for the P4 moiety were calculated to
approximately 34–35 Å2 and 6–7 Å2, respectively, with both
descriptors contributing almost equally to the equation.
P0 , P2 and P4 residues in our inhibitors. Because the change in
1
the P2 residue did not greatly affect BACE1 inhibition, the final
QSAR equation would be composed of only the properties of
the P01 and P4 residues found in compounds 11–36. Adopting a
naïve assumption that the interactions at the S01 subsite would
not interfere with the S4 subsite, we derived Eq. 2 in which
the physicochemical features of the P01 and P4 residues were
additive. In other words, Eq. 2 considered the less desired impact
of the N-methylthioxotriazole in the P01 moiety, as well as the
VDW surface areas of hydrogen bond acceptors and donors in
the P4 side chain. As with the previous equation, the derived
well-fitted (r2 = 0.82) and statistically significant equation (p
<0.01) was validated by leave-one-out cross-validation (r2 range:
0.81–0.89).
Each descriptor contributed almost equally to the equation
(MP1 , 25%; AccP4, 38%; DonP4, 36%) which meant that the P0 moiety
0
1
contributed less to BACE1 inhibition than the P4 moiety within this
BACE ¼ 39:520 MP1 ꢁ ð5:868 AccP4 ꢁ 0:084 Acc2P4Þ
0
series of compounds. Eq. 2 revealed the worst VDW surface areas
for AccP4 and DonP4 as around 35 Å2 and 3–6 Å2, respectively,
which were in agreement with the respective approximations ob-
tained from Eq. 1. Hence, with these QSAR equations, we could ob-
tain a more thorough understanding on the physicochemical
ꢂ ð2:616 DonP4 ꢁ 0:286 Acc2P4Þ þ 130:284
n ¼ 26; r2 ¼ 0:82; F ¼ 18; p 6 0:01
ð2Þ
MP1 represents the P01 presence of the less effective N-meth-
ylthioxotriazole (0) or other more effective tetrazole bioisosteres
(1).
0
features in the P0 , P2 and P4 residues of a generic inhibitor that
1
would be beneficial or detrimental to BACE1 inhibitory activity,
while being able to statistically ascertain the reliability of our pre-
dictions (Fig.3).
We set out to derive a QSAR equation that correlates BACE1
inhibition at 0.2 lM with the physiochemical properties of the

3180
H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
Table 3
BACE1 inhibitory activity of pentapeptide with P01 modifications
O
O
O
H
N
H2N
N
N
N
R2
H
H
H
O
OH
NH
O
R1
Compound (KMI No.)
13 (KMI-572)
11 (KMI-573)
23 (KMI-707)
24 (KMI-708)
25 (KMI-1494)
26 (KMI-1436)
27 (KMI-809)
28 (KMI-810)
29 (KMI-1542)
30 (KMI-1495)
31 (KMI-1543)
32 (KMI-1524)
R1
R2
BACE1 inhibition (%)
at 2
>99
l
M
at 0.2
lM
IC50 (nM)
6.5
F
O
N
NH
N
NH
O
N
98
HN
HO
N
NH
N
N
99
93
96
92
93
92
95
93
95
89
92
64
—
OH
O
F
N
N
N
O
O
HN
HN
HN
HN
HN
HN
O
NH
O
>99
>99
99
7.9
10.0
—
HN
HO
O
O
OH
O
F
S
NH
O
HN
O
HO
N
N
N
N
N
S
99
—
O
OH
O
F
O
S
NH
O
>99
>99
>99
99
10.2
10.6
—
HN
HO
O
S
OH
O
F
NH
S
NH
O
HN
HO
NH
S
O
—
OH
O
F
N
NH
N
N
NH
O
99
—
H3C
H3C
HN
S
HO
N
NH
S
94
—
OH

H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
3181
Table 4
BACE1 inhibitory activity of pentapeptidic inhibitors with modifications at P2 and P4 positions
O
O
O
H
N
H2N
N
N
N
N
NH
H
H
H
O
OH
N
N
R2
NH
O
R1
Compound (KMI No.)
R1
R2
BACE1 inhibition (%)
at 2
87
lM
at 0.2
lM
IC50 (nM)
N
33 (KMI-1544)
34 (KMI-1437)
50
—
—
N
H
N
83
45
N
H
N
35 (KMI-1545)
36 (KMI-1541)
15 (KMI-570)
16 (KMI-571)
91
58
69
98
98
—
NH
N
N
N
NH
94
—
N
NH
NH
>99
>99
4.8
3.3
N
N
N
N
N
around the active site of BACE1 are shown in Figure 5A. The side
chain of BACE1-Arg moves to accommodate the inhibitor’s size
along the wall of the b-sheet structure, which consists of four pep-
tide strands behind BACE1-Arg235. The guanidine plane of BACE1-
Arg235 pushes down on the P2 part of the inhibitor causing affixing
it in the active site of BACE1. Because the occupied area of inhibitor
16 in the S2 pocket of BACE1 is in proximity of the inhibitor in the
crystal structure of PDB 2B8L, the 3D-models in Figures 4 and 5B
were depicted using the coordinates from PDB 2B8L. As shown in
Figure 5B, the guanidine plane of BACE1-Arg235 is effectively pack-
ing down inhibitor 16 in the active site of BACE1.
4. Conclusions
In conclusion, pentapeptidic BACE1 inhibitors were designed
and synthesized using various similar molecular-size lipophilic,
hydrogen bond acceptor groups at the P01 position and polar groups
at the P4 position. From structure–activity relationship study,
non-carboxylic acid residues at the P01 position and aromatic polar
residues at the P4 position were as effective as acidic moieties in
promoting potent BACE1 inhibitory activity as tetrazole deriva-
tives. Hence, these heterocycles act as bioisosteres of carboxylic
acids as well as tetrazole ring at the P01 and at P4 position.
Figure 3. Comparative chart between calculated and observed BACE1 inhibition
from Eq. 2.
3.3. Computer-assisted docking study
5. Experimental section
5.1. Materials
Replacing the Leu residue at the P2 position slightly improved
BACE1 inhibitory activity despite its big molecular size. Hence,
we performed an in silico docking simulation using the software
MOE as shown in Figure 4. Previously, we reported on the signifi-
cant role of interactions between BACE1-Arg235 and a P2 part of
the inhibitor in the inhibition mechanism. The superimposed
views of three crystal structures (PDB ID: 2B8L, 2IQG and 1W51)
Reagents and solvents were purchased from Wako Pure Chem-
ical Ind., Ltd (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), Aldrich
Chemical Co. Inc. (Milwaukee, WI, USA), and Tokyo Kasei Kogyo
Co., Ltd (Tokyo, Japan) and used without further purification.

3182
H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
Figure 4. 3D View of docked inhibitor KMI-571 (16).
Figure 5. (A) Configurations of the Arg235-side chain in BACE1-inhibitor complexes. Red, blue and yellow models are derived from X-ray crystal structures, 2B8L, 2IQG and
1W51 in PDB ID, respectively. Color-coded space filling models indicate the side chain of BACE1-Arg235. Color-coded stick models indicate the inhibitors docked in BACE1. (B)
Interactions between BACE1-Arg235 and the P2 part of KMI-571 (16).
254-Column chromatography was performed on Merck 107734
Silica Gel 60 (70–230 mesh). Preparative HPLC was carried out
on a C18 reverse phase column (250 ꢂ 20 mm; YMC-Pack-ODS-
AM) with a binary solvent system: a linear gradient of CH3CN in
compounds. The purified target compounds were immediately
lyophilized to afford their respective amorphous powders.
5.1.2. General procedure for Boc-deprotection (B)
0.1% aqueous trifluoroacetic acid (TFA) with
a
flow rate of
Anisole, 4 N HCl in 1,4-dioxane was added to the afforded Boc-
protected peptide/amine (ca. 1.0 mmol) at 0 °C and stirred for 1 h
at room temperature. The solution was evaporated in vacuo and
used directly to the next step without purification. A small portion
of the product was purified by preparative HPLC. The desired frac-
tions were collected and immediately lyophilized to afford white
amorphous powders, compounds 2, 4–6, 9, 10, 11, 13, 15, 16 and
23–36.
5.0 mL/min and detection at 230 nm. 1H NMR spectra were ob-
tained on a JEOL AL300 (300 MHz) and Varian Unity Inova 400NB
(400 MHz) spectrometer with TMS as an internal standard. Mass
spectra (electrospray ionization, MeOH as the mobile phase) were
analyzed with SHIMADZU LCMS-2010 spectrometer. MALDI-TOF-
MS was performed on a Voyager-DE RP spectrometer (Per Septive
Biosystems, Inc.). Analytical HPLC was performed using a C18 re-
verse phase column (4.6 ꢂ 150 mm; YMC-Pack-ODS AM-302) with
a binary solvent system: linear gradient of CH3CN 0–100% in 0.1%
aqueous TFA in 40 min at a flow rate of 0.9 mL/min, detected at
230 nm. The purity of the desired compounds were >95% pure.
5.2. Synthesis of aniline derivatives for P01 position
5.2.1. tert-Butyl 3-cyanophenylcarbamate (1b)
A mixture of 3-nitrobenzonitrile (5.00 g, 33.8 mmol) and 10% Pd/C
(0.50 g) in dry MeOH was stirred at room temperature under H2 at
1 atm pressure for 3 h. The reaction mixture was filtered and solvent
was evaporated in vacuo to give 3-aminobenzonitrile (3.48 g). To a
solution of3-aminobenzonitrile (2.35 g, 20 mmol)intetrahydrofuran
(THF), Et3N (2.76 mL, 20 mmol), (Boc2)O (4.80 g, 22 mmol) were
added.Thereactionmixturewasstirredfor46 hatroomtemperature.
The solvent was evaporated in vacuo and obtained residue was dis-
solved in ethyl acetate, extracted with 10% citric acid, the organic
layer was washed with brine, dried over Na2SO4, evaporated to give
desired compound which was further purified by column chromatog-
raphy. Yield: 3.29 g (75%). Purity >98% by analytical HPLC
5.1.1. General procedure for coupling reaction (A)
To a solution of amine (1.0 mmol) in DMF were added Boc-AA-OH
orcarboxylicacid(1.0 mmol),thereactionmixturewascooledto0 °C,
EDCꢀHCl (1.2 mmol) and HOBtꢀH2O (1.2 mmol) and Et3N (2 mmol)
were added and the reaction was stirred for 12 h at room tempera-
ture. After removal of the solvent in vacuo, the residue was dissolved
in ethyl acetate (20 mL), washed with 10% citric acid aq (3 ꢂ 5 mL),
10% NaHCO3 aq (3 ꢂ 5 mL) and brine (1 ꢂ 5 mL), dried over anhy-
drous sodium sulfate (Na2SO4), evaporated to give the crude product
which wasfurtherpurifiedbycolumnchromatographyandsubjected
to next step, or purified by preparative HPLC in the case of the target

H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
3183
(tR = 25.22 min). ESI-MS m/z 217.05 for [Mꢁ1] (calcd 218.25 for
C12H14N2O2). 1H NMR (300 MHz, CDCl3) d (ppm): 1.52 (s, 9H), 7.26
(s, 1H), 7.29–7.32 (m, 1H), 7.37 (dd, J = 7.8, 1H), 7.49–7.53 (m, 1H).
quantitative yield (over all two steps). Purity >97% by analytical
HPLC (tR = 12.06 min). ESI-MS m/z 191.95 for [Mꢁ1] (calcd
193.23 for C8H7N3OS). 1H NMR (300 MHz, CD3OD) d (ppm) 7.52–
7.55 (m, 1H), 7.67 (dd, J = 7.89, 1H), 7.88 (m, 1H), 7.98 (dd,
J = 3.67, 1H).
5.2.2. 3-(1H-Tetrazol-5-yl)benzenamine (2)
A mixture of 3-nitrobenzonitrile (1a, 0.700 g, 2.10 mmol), so-
dium azide (0.411 g, 6.30 mmol) and ammonium chloride
(0.337 g, 6.30 mmol) in DMF (10 ml) was stirred at 120 °C for
24 h, poured into water and extracted with ethyl acetate. The ex-
tract was washed with water, dried over Na2SO4, and concentrated
in vacuo to give 5-(3-nitrophenyl)-1H-tetrazole which was further
converted into title compound (3) by catalytic hydrogenation con-
dition using 20 wt % of Pd/C under atmospheric pressure in dry
MeOH at room temperature. Yield: 78% (over two steps). Purity
>99% by analytical HPLC (tR = 6.41 min). ESI-MS m/z 160 for
[Mꢁ1] (calcd 161.16 for C7H7N5). 1H NMR (300 MHz, CD3OD) d
(ppm): 6.88–6.91 (m, 1H), 7.26 (dd, J = 3.67, 1H), 7.28 (s, 1H),
7.32–7.34 (m, 1H).
5.2.6. 3-(3-Aminophenyl)-1,2,4-oxadiazole-5(4H)-thione (6)
To a solution of aldoxime 3 (3.37 g, 13.43 mmol) in acetonitrile
(12 ml), TCDI (3.53 g, 20.14 mmol), DBU (7.26 g, 53.72 mmol) was
added and reaction was stirred at room temperature for 1 h. The
reaction mixture was diluted with water; its pH was adjusted to
4 by using 1 N HCl and extracted with ethyl acetate. The extract
was washed with water and dried over Na2SO4. The solvent was
evaporated in vacuo; the residue obtained was directly subjected
for Boc-deprotection (2.91 g). The obtained crude product was fur-
ther purified by column chromatography to give compound 6.
Yield: 1.3 g (68%). Purity >96% by analytical HPLC (tR = 13.47 min).
ESI-MS m/z 192 for [Mꢁ1] (calcd 193.23 for C8H7N3OS). 1H NMR
(300 MHz, CD3OD) d (ppm) 7.63–7.66 (m, 1H), 7.75 (dd, J = 7.89,
1H), 7.87 (dd, J = 1.38, 1H), 7.88–7.92 (m, 1H).
5.2.3. tert-Butyl 3-amidinophenylcarbamate (3)
The Et3N (6.18 g, 61.10 mmol) was added to a suspension of
hydroxylamine hydrochloride (4.24 g, 61 mmol) in DMSO
(20 mL). An insoluble material was filtered off and washed with
THF. The filtrate was concentrated in vacuo to remove THF and
tert-butyl-3-cynophenylcarbamate (1b) (5.00 g, 12.20 mmol) was
added to the DMSO solution of hydroxylamine. After stirring at
75 °C for 15 h, the reaction mixture was diluted with water and ex-
tracted with ethyl acetate. The organic phase was extracted with
1 N HCl (25 mL). The aqueous solution was adjusted to pH 10 with
1 N NaOH and extracted with ethyl acetate. The organic layer was
washed with water and dried over Na2SO4. The solvent was evap-
orated in vacuo and the obtained crude product was recrystallized
from ethyl acetate–MeOH–hexane to give 3 Yield: 5.54 g (96%).
Purity >97% by analytical HPLC (tR = 16.26 min). ESI-MS m/z
250.10 for [Mꢁ1] (calcd 251.28 for C12H17N3O3). 1H NMR
(300 MHz, DMSO) d (ppm) 1.47 (s, 9H), 5.9 (br s, 2H), 7.22 (m,
2H), 7.42 (m, 1H), 7.82 (s, 1H), 9.59 (br s, 1H).
5.2.7. tert-Butyl 3-(methoxycarbonyl)phenylcarbamate (7b)
To a solution of 3-aminomethylbenzoate (0.100 g, 0.66 mmol)
in THF (5 mL), Et3N (0.100 g, 0.993 mmol) and di-tert-butyl dicar-
bonate (0.159 g, 0.728 mmol) was added at 0 °C. The reaction mix-
ture was stirred at room temperature for 48 h. The solvent was
evaporated in vacuo. The residue was dissolved in ethyl acetate,
extracted with NaHCO3 followed by washed with brine. The organ-
ic layer was dried over Na2SO4, evaporated to give desired product.
The obtained crude product was purified by column chromatogra-
phy. Yield: 0.106 g (64%). Purity >99% by analytical HPLC
(tR = 25.62 min). ESI-MS m/z 274 for [M+Na] (calcd 251.28 for
C13H17NO4). 1H NMR (300 MHz, CDCl3) d (ppm) 1.52 (s, 1H), 3.9
(s, 3H), 7.36 (dd, J = 3.85, 1H), 7.69 (dd, J = 2.75, 1H), 7.72 (dd,
J = 1.38, 1H), 7.97 (dd, J = 1.93, 1H).
5.2.8. tert-Butyl 3-aminobenzohydrazide (8)
The ester 7b (0.450 g, 1.79 mmol) and 85% hydrazine hydrate
(0.87 mL, 17.9 mmol) in 20 mL of THF were refluxed for 46 h. The
excess solvent was removed in vacuo. The obtained solid was
recrystallize by using hexane to furnish colorless crystalline ben-
zoyl hydrazide intermediate (8). Yield: 0.414 g (92%). Purity >99%
by analytical HPLC (tR = 17.03 min). ESI-MS m/z 274 for [M+Na]
(calcd 251.28 for C12H17N3O3). 1H NMR (300 MHz, CD3OD) d
(ppm) 1.47 (s, 9H), 7.28 (m, 1H), 7.35 (d, J = 8.99, 1H), 7.51 (d,
J = 7.16, 1H), 7.93 (s, 1H).
5.2.4. 3-(3-Aminophenyl)-1,2,4-oxadiazol-5(4H)-one (4)
2-Ethylhexyl chloroformate (0.38 g, 0.20 mmol) was added
dropwise to an ice-cooled mixture of 3 (0.50 g, 2.0 mmol) and pyr-
idine (0.79 g, 2.1 mmol) in DMF (5 mL). The resulting mixture was
stirred at 0 °C for 30 min, diluted with water, and extracted with
ethyl acetate. The extract was washed with water and dried over
Na2SO4. The solvent was evaporated in vacuo, and the residue
was dissolved in xylene (5 mL). The solution was heated under re-
flux for 2 h. The solvent was evaporated in vacuo, the residue
(0.255 g) obtained was directly subjected for tert-butylcarbamate
(Boc) deprotection to give 4. Yield: 0.103 g (62% overall two steps).
Purity >97% by analytical HPLC (tR = 11.92 min). ESI-MS m/z 175.95
for [Mꢁ1] (calcd 177.16 for C8H7N3O2). 1H NMR (300 MHz, CD3OD)
d (ppm) 7.31 (d, J = 7.34, 1H), 7.46–7.54 (m, 3H).
5.2.9. 5-(3-Aminophenyl)-1,3,4-oxadiazole-2(3H)-thione (9)
To a solution of compound 8 (0.423 g, 1.68 mmol) in ethanol (10 mL)
was added carbon disulfide (slightly more than one equivalent)and KOH
(0.094 g, 1.68 mmol) at 0 °C. The resulting solution was refluxed for 12 h
or until most of the hydrogen sulfide had evolved. The solvent was evap-
orated and the residue obtained dissolved in water and acidified with di-
luted solution of HCl. The solid obtained was filtered and recrystallized
from MeOH and subjected for Boc-deprotection. Yield: 0.190 g (78%,
overall two steps). Purity >95% by analytical HPLC (tR = 12.59 min).
ESI-MS m/z 192 for [Mꢁ1] (calcd 193.23 for C8H7N3OS). 1H NMR
(300 MHz, CD3OD) d (ppm) 7.61–7.67 (m, 1H), 7.74 (dd, J = 8.82, 1H),
7.93–7.95 (m, 1H), 8.01–8.06 (m, 1H).
5.2.5. 3-(3-Aminophenyl)-1,2,4-thiadiazol-5(4H)-one (5)
A mixture of aldoxime 3 (3.00 g, 11.95 mmol) and TCDI (2.55 g,
14.34 mmol) in THF (25 mL) was stirred at room temperature for
1 h. It was diluted with water and extracted by ethyl acetate and
the organic layer was washed with water, dried over Na2SO4. The
solvent was evaporated in vacuo. The obtained residue was dis-
solved in THF (10 mL) and then BF3ꢀOEt2 (7.5 mL, 59.76 mmol)
was added at 0 °C. The reaction mixture was stirred at room tem-
perature for 1 h, diluted with water, extracted with ethyl acetate,
washed with 1 N HCl, dried over Na2SO4. The solvent was evapo-
rated in vacuo. The obtained crude product was washed with ethyl
acetate to remove non-polar impurities. The obtained pure product
was directly subjected for Boc-deprotection to give compound 5 in
5.2.10. 5-(3-Aminophenyl)-4-methyl-2H-1,2,4-triazole-3(4H)-
thione (10)
To a solution of benzoyl hydrazide 8 (0.500 g, 1.99 mmol) in
THF (12 mL), methyl isothiocyanate (0.146 g, 1.99 mmol) and 2–3
drops of Et3N were added and reaction mixture was stirred at room
temperature for 6 h. Water was added and the precipitate obtained

3184
H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
was filter off, washed with water and dried well. The obtained
product was dissolved in 4% aqueous NaOH (49.75 mL, 4.97 mmol)
and stirred at 45 °C for 3 h. Resulting mixture was filter off and fil-
trate was acidified by 10% HCl until ꢃpH 3. Obtained precipitate
was filtered, washed with water and dried well to give desire prod-
uct, which was recrystallized from methanol and exposed for ani-
sole, 4 N HCl/dioxane for Boc-deprotection. Yield: 62% over all
three steps. Purity >99% by analytical HPLC (tR = 12.48 min). ESI-
MS m/z 205 for [Mꢁ1] (calcd 206.27 for C9H10N4S). 1H NMR
(300 MHz, DMSO) d (ppm) 2.51 (s, 3H), 7.45–7.48 (m, 1H), 7.58–
7.65 (m, 3H), 14.01 (s, 1H).
1H), 3.98 (m, 1H), 4.19 (m, 1H), 4.33 (m, 2H), 7.23 (m, 1H), 7.28
(m, 4H), 7.50 (t, 1H), 7.68 (d, J = 7.6, 1H), 7.79 (d, J = 8, 1H), 7.86
(d, J = 8.8, 1H), 8.03 (d, J = 7.6, 1H), 8.49 (s, 1H), 8.54 (d, J = 8, 1H).
5.3.4. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(2H-Tetrazol-5-yl)phe
nylcarbamoyl)-1-hydroxy-3-phenylpropan-2-ylcarbamoyl)-3-
cyclohexylpropylcarbamoyl)-2-amino-6-methyl-3-oxoheptyl)-
2H-tetrazole-5-carboxamide (16)
Compound 16 was prepared from amine 2 and tetrazolic acid by
using General Method C. Yield: 49%. Purity >98% by analytical HPLC
(tR = 21.95 min). TOF-MS m/z 773.82 for [M+1] (calcd 772.39 for
C36H48N14O6). 1H NMR (300 MHz, CD3OD) d (ppm) 0.82 (m,
11H+3H), 1.07 (m, 1H), 1.28 (m, 3H), 1.50 (d, J = 9.0, 1H), 1.94
(m, 1H), 2.74 (m, 1H), 2.88 (m, 1H), 3.55 (m, 2H), 3.95 (s, 1H),
4.09–4.28 (m, 3H), 4.37 (m, 1H), 7.27 (m, 5H), 7.49 (m, 1H), 7.67
(d, J = 7.5, 1H), 7.85 (d, J = 8.7, 1H), 8.01 (d, J = 8.4, 1H), 8.45 (s,
1H), 8.57 (d, J = 10.5, 1H).
5.3. General experimental procedure for synthesis of KMI
compounds (C)
Toanice-cooled solutionof amine(1.0 mmol)inDMFwere added
Boc-AA-OH or carboxylic acid (1.0 mmol), followed by EDCꢀHCl
(1.2 mmol), HOBtꢀH2O (1.2 mmol) and Et3N (2 mmol) were added.
The reaction mixture was stirred for 12 h at room temperature. After
removal of the solvent in vacuo, the residue was dissolved in ethyl
acetate (20 mL), extracted with 10% citric acid aq (3 ꢂ 5 mL), 10%
NaHCO3 aq (3 ꢂ 5 mL) and finally washed with brine (1 ꢂ 5 mL),
then dried over Na2SO4, evaporated to give the crude product which
was further purified by column chromatography and then subjected
for tert-butyloxycarbamate deprotection by using General Proce-
dure B. The obtained product was subjected to the next step or puri-
fied by preparative HPLC in the case of the target compound. The
purified target compounds were immediately lyophilized to afford
their respective amorphous powders.
5.3.5. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(4,5-Dihydro-5-oxo-1,2,4-
oxadiazol-3-yl)phenylcarbamoyl)-1-hydroxy-3-phenylpropan-
2-ylcarbamoyl)-3-methylbutylcarbamoyl)-2-amino-6-methyl-
3-oxoheptyl)-5-fluoro-1,2,3,6-tetrahydro-2,6-dioxopyrimidine-
4-carboxamide (23)
Compound 23 was prepared from amine 4 and 5-fluoroorotic
acid by using General Method C. Yield: 42%. Purity >99% by analyt-
ical HPLC (tR = 19.78 min). ESI/TOF-MS m/z 807.05 for [Mꢁ1] (calcd
808.81 for C37H45FN10O10). 1H NMR (300 MHz, CD3OD) d (ppm)
0.79–0.86 (m, 13H), 1.34 (m, 2H), 2.62–2.89 (m, 5H), 3.58 (m,
2H), 3.96 (d, J = 2.94, 1H), 4.28 (d, 1H), 4.21 (d, 1H), 7.17–7.27
(m, 6H), 7.38–7.56 (m, 1H), 7.70–7.81 (d, J = 2.94, 1H), 8.10 (d,
J = 8.81 1H).
5.3.1. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(2H-Tetrazol-5-yl)phenylc
arbamoyl)-1-hydroxy-3-phenylpropan-2-ylcarbamoyl)-3-meth
ylbutylcarbamoyl)-2-amino-6-methyl-3-oxoheptyl)-2,5-dihydr
oxybenzamide (11)
Compound 11 was prepared from amine 2 and 2,5-dihydroxy-
benzoic acid by using General Method C. Yield: 47%. Purity >99%
by analytical HPLC (tR = 19.96 min). ESI/TOF-MS m/z 771.05 for
[Mꢁ1] (calcd 772.37 for C38H48N10O8). 1H NMR (300 MHz, DMSO)
d (ppm) 0.54 (d, J = 6.61, 3H), 0.59 (d, J = 6.06, 3H), 0.76–0.89 (m,
7H), 1.03 (d, J = 4.95, 2H), 1.23–1.30 (m, 3H), 1.94–1.99 (m, 1H),
2.25–2.28 (m, 1H), 2.74 (m, 1H), 2.83–2.93 (m, 2H), 4.26–4.37
(m, 2H), 6.76 (d, J = 8.62, 1H), 6.85 (dd, J = 5.78, 1H), 7.20–7.31
(m, 7H), 7.58–7.66 (m, 2H), 8.05 (dd, J = 4.14, 1H), 8.20 (s, 1H).
5.3.6. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(4,5-Dihydro-5-oxo-1,2,4-
oxadiazol-3-yl)phenylcarbamoyl)-1-hydroxy-3-phenylpropan-
2-ylcarbamoyl)-3-methylbutylcarbamoyl)-2-amino-6-methyl-
3-oxoheptyl)-2,5-dihydroxybenzamide (24)
Compound 24 was prepared from amine 4 and 2,5-dihydroxyben-
zoic acid by using General Method C. Yield: 51%. Purity >99% by ana-
lyticalHPLC(tR = 20.84 min). ESI/TOF-MSm/z787.54 for[Mꢁ1](calcd
788.35 for C39H48N8O10). 1H NMR (400 MHz, CD3OD) d (ppm) 0.59 (d,
J = 6.4, 3H), 0.78–0.97 (m, 9H), 1.23–1.44 (m, 3H), 2.12 (m, 1H), 2.85–
2.99 (m, 3H), 3.92–3.96 (m, 3H), 4.15 (d, J = 6, 1H), 4.11 (d, 1H), 4.17–
4.22 (m, 1H), 6.76 (d, J = 8.8, 1H), 6.91–6.94 (dd, J = 8.8, 1H), 7.17 (m,
1H), 7.18–7.26 (m, 5H), 7.32(d, J = 3.2, 1H), 7.38–7.42(m, 1H), 7.62(d,
J = 8.8, 1H), 7.89 (d, J = 8, 1H), 8.06 (s, 1H).
5.3.2. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(2H-Tetrazol-5-yl)phenylc
arbamoyl)-1-hydroxy-3-phenylpropan-2-ylcarbamoyl)-3-meth
ylbutylcarbamoyl)-2-amino-6-methyl-3-oxoheptyl)-5-fluoro-
1,2,3,6-tetrahydro-2,6-dioxopyrimidine-4-carboxamide (13)
Compound 13 was prepared from amine 2 and 5-fluoroorotic
acid by using General Method C. Yield: 61%. Purity >99% by analyt-
ical HPLC (tR = 18.23 min). ESI/TOF-MS m/z 791.35 for [Mꢁ1] (calcd
792.82 for C36H45FN12O8). 1H NMR (300 MHz, CD3OD) d (ppm)
0.91–0.97 (m, 6H), 1.28–1.45 (m, 9H), 2.81–3.1 (m, 5H), 3.5 (m,
1H), 3.65 (m, 2H), 3.72 (m, 1H), 4.21 (d, J = 5.69, 1H), 7.48–7.64
(m, 5H), 7.76 (d, J = 7.34, 2H), 7.89 (d, J = 8.26, 2H), 8.17 (s, 1H).
5.3.7. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(4,5-Dihydro-5-oxo-1,2,4-
thiadiazol-3-yl)phenylcarbamoyl)-1-hydroxy-3-phenylpropan-
2-ylcarbamoyl)-3-methylbutylcarbamoyl)-2-amino-6-methyl-
3-oxoheptyl)-5-fluoro-1,2,3,6-tetrahydro-2,6-dioxopyrimidine-
4-carboxamide (25)
Compound 25 was prepared from amine 5 and 5-fluoroorotic acid
byusingGeneralMethodC. Yield:41%.Purity>99%byanalyticalHPLC
(tR = 19.96 min). ESI/TOF-MS m/z 823 for [Mꢁ1] (calcd 824.88 for
C37H45FN10O9S). 1H NMR (300 MHz, CD3OD) d (ppm) 0.54 (d, J = 6.6,
3H), 0.60 (d, J = 6.6, 3H), 0.78 (d, J = 6.6, 6H), 0.87 (m, 1H), 1.24–1.37
(m, 5H), 2.73 (m, 2H), 3.93 (m, 1H), 4.14 (m, 1H), 4.17 (t, J = 6.6, 1H),
4.30 (d, J = 7.8, 2H), 7.27–7.40 (m, 6H), 7.58 (d, J = 8.7, 1H), 7.67 (d,
J = 7.2, 1H), 7.86 (d, J = 8.7, 1H), 8.26 (s, 1H).
5.3.3. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(2H-Tetrazol-5-yl)phe
nylcarbamoyl)-1-hydroxy-3-phenylpropan-2-ylcarbamoyl)-3-
methylbutylcarbamoyl)-2-amino-6-methyl-3-oxoheptyl)-2H-
tetrazole-5-carboxamide (15)
Compound 15 was prepared from amine 2 and tetrazolic acid by
using general method C. Yield: 45%. Purity >98% by analytical HPLC
(tR = 20.26 min). TOF-MS m/z 733.98 for [M+1] (calcd 732.36 for
C33H44N14O6). 1H NMR (400 MHz, DMSO) d (ppm) 0.48 (d, J = 6.8,
3H), 0.55 (d, J = 6.4, 3H), 0.83 (m, 6H), 1.19 (m, 2H), 1.59 (m, 1H),
1.98 (m, 1H), 2.77 (m, 1H), 2.89 (m, 1H), 3.59 (m, 2H), 3.96 (br s,
5.3.8. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(4,5-Dihydro-5-oxo-1,2,4-
thiadiazol-3-yl)phenylcarbamoyl)-1-hydroxy-3-phenylpropan-
2-ylcarbamoyl)-3-methylbutylcarbamoyl)-2-amino-6-methyl-
3-oxoheptyl)-2,5-dihydroxybenzamide (26)
Compound 26 was prepared from amine 5 and 2,5-dihydroxyben-
zoic acid by using General Method C. Yield: 89%. Purity >99% by ana-

H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
3185
lyticalHPLC(tR = 21.50 min). ESI/TOF-MSm/z803.03for[Mꢁ1] (calcd
804.91 for C39H48N8O9S). 1H NMR (300 MHz, CD3OD) d (ppm) 0.53 (d,
J = 5.8, 3H), 0.85–0.97 (m, 9H), 1.1 (m, 2H), 1.25–1.40 (m, 3H), 2.92 (m,
2H), 3.56(m, 1H), 4.09(d, J = 4.5, 1H), 4.14 (d, J = 3.9, 2H), 4.21 (d, J = 6,
2H), 6.76 (d, J = 7.5, 1H), 6.97 (dd, J = 4.2, 1H), 7.18–7.26 (m, 7H), 7.32
(m, 1H), 7.76 (d, J = 8.9, 1H), 7.94 (m, 2H).
5.3.13. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(4,5-Dihydro-4-methyl-
5-thioxo-1H-1,2,4-triazol-3-yl)phenylcarbamoyl)-1-hydroxy-3-
phenylpropan-2-ylcarbamoyl)-3-methylbutylcarbamoyl)-2-am
ino-6-methyl-3-oxoheptyl)-5-fluoro-1,2,3,6-tetrahydro-2,6-
dioxopyrimidine-4-carboxamide (31)
Compound 31 was prepared from amine 10 and 5-fluoroorotic
acid by using General Method C. Yield: 75%. Purity >99% by analyt-
ical HPLC (tR = 19.34 min). ESI/TOF-MS m/z 836.52 for [Mꢁ1] (calcd
837.34 for C38H48FN11O8S). 1H NMR (300 MHz, CD3OD) d (ppm)
0.58–0.65 (m, 6H), 0.89–0.97 (m, 6H), 1.12 (m, 2H), 1.28–1.35
(m, 5H), 2.89–2.95 (m, 1H), 3.63 (d, J = 7.5, 2H), 3.89–3.96 (m,
2H), 4.07–4.10 (m, 1H), 4.19–4.21 (m, 2H), 4.60 (d, J = 5.7, 1H),
7.20 (d, J = 4.5, 1H), 7.27 (m, 5H), 7.40–7.47 (m, 1H), 7.79–7.82
(m, 2H), 8.08 (s, 1H).
5.3.9. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(4,5-Dihydro-5-thioxo-
1,2,4-oxadiazol-3-yl)phenylcarbamoyl)-1-hydroxy-3-phenylpro
pan-2-ylcarbamoyl)-3-methylbutylcarbamoyl)-2-amino-6-met
hyl-3-oxoheptyl)-5-fluoro-1,2,3,6-tetrahydro-2,6-dioxopyrimi
dine-4-carboxamide (27)
Compound 27 was prepared from amine 6 and 5-fluoroorotic
acid by using General Method C. Yield: 52%. Purity >99% by analyt-
ical HPLC (tR = 21.70 min). ESI-MS (TOF-MS) m/z 822.95 (824.62)
(calcd 824.31 for C37H45FN10O9S). 1H NMR (300 MHz, CD3OD) d
(ppm) 0.62 (d, J = 5.87, 3H), 0.66 (d, J = 5.87, 3H), 0.90 (d, J = 6.97,
6H), 1.17 (m, 2H), 2.21 (d, 2H), 2.73 (d, 2H), 3.07 (t, J = 6, 1H),
3.53 (t, J = 8.7, 1H), 4.08 (d, J = 2.1, 2H), 4.14 (d, J = 6.6, 1H), 4.57
(d, 2H), 7.24–7.30 (m, 6H), 7.37 (m, 1H), 7.52 (d, J = 8.26, 1H),
7.91 (d, 1H), 8.20 (s, 1H).
5.3.14. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(4,5-Dihydro-4-methyl-
5-thioxo-1H-1,2,4-triazol-3-yl)phenylcarbamoyl)-1-hydroxy-3-
phenylpropan-2-ylcarbamoyl)-3-methylbutylcarbamoyl)-2-am
ino-6-methyl-3-oxoheptyl)-2,5-dihydroxybenzamide (32)
Compound 32 was prepared from amine 10 and 2,5-dihydroxy-
benzoic acid by using General Method C. Yield: 67%. Purity >99% by
analytical HPLC (tR = 20.78 min). ESI/TOF-MS m/z 816.56 for [Mꢁ1]
(calcd 817.36 for C40H51N9O8S). 1H NMR (300 MHz, DMSO) d (ppm)
0.53 (d, J = 6.61, 3H), 0.59 (d, J = 6.61, 3H), 0.77 (m, 6H), 0.85–0.87
(m, 2H), 1.18–1.28 (m, 3H), 1.9 (s, 3H), 2.78 (m, 2H), 2.87 (m, 1H),
3.95 (m, 1H), 4.14–4.18 (m, 2H), 4.29 (d, J = 7.71, 2H), 6.72 (d,
J = 8.81, 1H), 6.83 (dd, J = 5.78, 1H), 7.22–7.28 (m, 6H), 7.39–7.48
(m, 2H), 7.71 (m, 1H), 7.83 (d, J = 8.62, 1H), 8.03 (s, 1H).
5.3.10. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(4,5-Dihydro-5-thioxo-
1,2,4-oxadiazol-3-yl)phenylcarbamoyl)-1-hydroxy-3-phenylpro
pan-2-ylcarbamoyl)-3-methylbutylcarbamoyl)-2-amino-6-met
hyl-3-oxoheptyl)-2,5-dihydroxybenzamide (28)
Compound 28 was prepared from amine 6 and 2,5-dihydroxy-
benzoic acid by using General Method C. Yield: 47%. Purity >99%
by analytical HPLC (tR = 19.78 min). ESI/TOF-MS m/z 803.87 for
[Mꢁ1] (calcd 804.33 for C39H48N8O9S). 1H NMR (300 MHz, CD3OD)
d (ppm) 0.56 (d, J = 6.42, 3H), 0.60 (d, J = 6.79, 3H), 0.94–1.0 (m,
9H), 2.11–2.19 (m, 2H), 2.85–2.99 (m, 3H), 3.63–3.78 (m, 2H),
4.13–4.19 (m, 3H), 6.77 (d, J = 8.62, 1H), 6.95 (dd, J = 5.87, 1H),
7.15–7.30 (m, 7H), 7.37 (d, J = 2.75, 1H), 7.52 (s, 1H), 7.76 (m,
1H), 8.23 (s, 1H).
5.3.15. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(2H-Tetrazol-5-yl)phe
nylcarbamoyl)-1-hydroxy-3-phenylpropan-2-ylcarbamoyl)-3-
methylbutylcarbamoyl)-2-amino-6-methyl-3-oxoheptyl)-1H-
imidazole-4-carboxamide (33)
Compound 33 was prepared from amine 2 and 4-imidazole car-
boxylic acid by using General Method C. Yield: 62%. Purity >98% by
analytical HPLC (tR = 18.32 min). ESI/TOF-MS m/z 729.59 for [Mꢁ1]
(calcd 730.37 for C35H46N12O6). 1H NMR (300 MHz, CD3OD) d
(ppm) 0.58 (d, J = 6.24, 3H), 0.64 (d, J = 6.24, 3H), 0.99 (d, J = 6.61,
6H), 1.29 (m, 2H), 2.79 (m, 1H), 2.76–3.01 (m, 3H), 3.56–3.60 (m,
1H), 3.66–3.72 (m, 2H), 4.22 (d, J = 8.99, 3H), 4.33 (m, 1H), 7.24–
7.29 (m, 6H), 7.50 (d, J = 5.1, 1H), 7.71 (dd, J = 10.92, 2H), 8.19 (s,
1H), 8.45 (s, 1H), 8.99 (s, 1H).
5.3.11. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(4,5-Dihydro-5-thioxo-
1,3,4-oxadiazol-2-yl)phenylcarbamoyl)-1-hydroxy-3-phenylpro
pan-2-ylcarbamoyl)-3-methylbutylcarbamoyl)-2-amino-6-met
hyl-3-oxoheptyl)-5-fluoro-1,2,3,6-tetrahydro-2,6-dioxopyrimi
dine-4-carboxamide(29)
5.3.16. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(2H-Tetrazol-5-yl)phe
nylcarbamoyl)-1-hydroxy-3-phenylpropan-2-ylcarbamoyl)-3-
cyclohexylpropylcarbamoyl)-2-amino-6-methyl-3-oxoheptyl)-
1H-imidazole-4-carboxamide (34)
Compound 34 was prepared from amine 2 and 4-imidazole car-
boxylic acid by using General Method C. Yield: 73%. Purity >98% by
analytical HPLC (tR = 19.98 min). ESI/TOF-MS m/z 769.25 for [Mꢁ1]
(calcd 770.40 for C38H50N12O6). 1H NMR (300 MHz, CD3OD) d
(ppm) 0.74 (d, J = 6.9, 3H), 0.88 (d, J = 6.6, 3H), 0.74 (d, J = 7.5,
2H), 1.12 (m, 1H), 1.23–1.55 (m, 10H), 1.89 (m, 2H), 2.75 (m,
2H), 2.95 (m, 1H), 3.92 (m, 1H), 4.19 (m, 2H), 4.31 (m, 2H), 7.24
(m, 6H), 7.64 (d, J = 8.4, 1H), 7.85 (d, J = 8.1, 1H), 8.03 (d, J = 7.8,
1H), 8.23 (s, 2H).
Compound 29 was prepared from amine 9 and 5-fluoroorotic
acid by using General Method C. Yield: 24%. Purity >99% by analyt-
ical HPLC (tR = 20.48 min). ESI/TOF-MS m/z 823.90 for [Mꢁ1] (calcd
824.88 for C37H45FN10O9S). 1H NMR (300 MHz, CD3OD) d (ppm)
0.69 (d, J = 6.3, 3H), 0.78 (d, J = 6.3, 3H), 0.86 (d, J = 6.6, 3H), 0.91
(d, J = 6.6, 3H), 1.26–1.29 (m, 2H), 2.74 (m, 2H), 3.01 (m, 2H),
3.82–3.89 (m, 2H), 3.98 (d, J = 6.8, 1H), 4.19 (d, J = 6.9, 2H), 4.31–
4.33 (m, 2H), 7.27 (m, 5H), 7.46–7.7.51 (m, 2H), 7.75 (d, J = 9.9,
1H), 7.88 (d, J = 10.2, 1H), 8.18 (s, 1H).
5.3.12. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(4,5-Dihydro-5-oxo-
1,3,4-oxadiazol-2-yl)phenylcarbamoyl)-1-hydroxy-3-phenylpro
pan-2-ylcarbamoyl)-3-methylbutylcarbamoyl)-2-amino-6-met
hyl-3-oxoheptyl)-2,5-dihydroxybenzamide (30)
Compound 30 was prepared from amine 9 and 2,5-dihydroxy-
benzoic acid by using General Method C. Yield: 79%. Purity >99%
by analytical HPLC (tR = 22.06 min). ESI/TOF-MS m/z 803.50 for
[Mꢁ1] (calcd 804.33 for C39H48N8O9S). 1H NMR (300 MHz, CD3OD)
d (ppm) 0.55–0.60 (m, 6H), 0.81–0.98 (m, 7H), 1.24 (m, 1H), 1.61
(m, 3H), 2.88 (m, 2H), 3.61 (m, 1H), 4.10–4.25 (m, 5H), 6.77 (d,
J = 8.99, 1H), 6.94 (dd, J = 5.87, 1H), 7.25 (m, 7H), 7.41 (m, 1H),
7.62 (d, J = 10.09, 1H), 7.85 (d, J = 8.26, 1H), 8.19 (s, 1H).
5.3.17. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(2H-Tetrazol-5-yl)phe
nylcarbamoyl)-1-hydroxy-3-phenylpropan-2-ylcarbamoyl)-3-
methylbutylcarbamoyl)-2-amino-6-methyl-3-oxoheptyl)-1H-
1,2,4-triazole-3-carboxamide (35)
Compound 35 was prepared from amine 2 and 4-triazole car-
boxylic acid by using General Method C. Yield: 61%. Purity >98%
by analytical HPLC (tR = 18.84 min). ESI/TOF-MS m/z 730.49 for
[Mꢁ1] (calcd 731.8 for C34H45N13O6). 1H NMR (300 MHz, CD3OD)
d (ppm) 0.47–0.73 (m, 6H), 0.79–1.03 (m, 6H), 1.21–1.53 (m, 2H),

3186
H. D. Tagad et al. / Bioorg. Med. Chem. 18 (2010) 3175–3186
Tomasselli, A. G.; Parodi, L. A.; Heinrikson, R. L.; Gurney, M. Nature 1999, 402,
533.
5. Hong, L.; Koelsch, G.; Lin, X.; Wu, S.; Terzyan, S.; Ghosh, A. K.; Zhang, X. C.;
Tang, J. Science 2000, 290, 150.
6. Sipe, J. D. Annu. Rev. Biochem. 1992, 61, 947.
7. Selkoe, D. J. Ann. N.Y. Acad. Sci. 2000, 924, 17.
2.03–2.11 (m, 1H), 2.82–3.05 (m, 2H), 3.55–3.73 (m, 3H), 3.88 (q,
J = 7.2, 1H), 4.07–4.27 (m, 2H), 4.30–4.38 (m, 1H), 4.61–4.70 (m,
1H), 7.13–7.34 (m, 6H), 7.45–7.55 (m, 1H), 7.71–7.79 (dd, J = 8.1,
2H), 8.39 (s, 1H), 8.54 (s, 1H).
8. Steiner, H.; Capell, A.; Leimer, U.; Haass, C. Eur. Arch. Psychiatry Clin. Neurosci.
1999, 249, 266.
9. Selkoe, D. J. Ann. Med. 1989, 21, 73.
5.3.18. N-((2S,5S)-5-((S)-1-((1R,2S)-1-(3-(2H-Tetrazol-5-yl)phe
nylcarbamoyl)-1-hydroxy-3-phenylpropan-2-ylcarbamoyl)-3-
cyclohexylpropylcarbamoyl)-2-amino-6-methyl-3-oxoheptyl)-
1H-1,2,4-triazole-3-carboxamide (36)
Compound 36 was prepared from amine 2 and 4-triazole car-
boxylic acid by using General Method C. Yield: 72%. Purity >97%
by analytical HPLC (tR = 20.66 min). ESI/TOF-MS m/z 770.60 for
[Mꢁ1] (calcd 771.39 for C37H49N13O6). 1H NMR (300 MHz, CD3OD)
d (ppm) 0.73–0.79 (m, 6H), 0.85–0.90 (q, J = 5.1 2H), 1.03 (d, J = 5.4,
1H), 1.17–1.28 (m, 11H), 1.50 (m, 1H), 1.92–1.99 (m, 1H), 2.71–
2.78 (q, J = 6.9, 2H), 2.84–2.92 (q, J = 7.8, 1H), 3.93 (m, 1H), 4.20
(m, 2H), 4.28–4.38 (m, 2H), 7.22–7.38 (m, 6H), 7.57 (d, J = 7.8,
1H), 7.64 (d, J = 8.1, 1H), 7.83 (d, J = 8.7, 1H), 8.23 (s, 1H).
10. Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, 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.
11. 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.
12. Hussain, I.; Powell, D.; Howlett, D. R.; Tew, D. G.; Meek, T. D.; Chapman, C.;
Gloger, I. S.; Murphy, K. E.; Southa, C. D.; Ryan, D. M.; Smith, T. S.; Simmons, D.
L.; Walsh, F. S.; Dingwall, C.; Christie, G. Mol. Cell. Neurosci. 1999, 14, 419.
13. 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.
14. Stachel, S. J. Drug Dev. Res. 2009, 70, 101.
15. Silvestri, R. Med. Res. Rev. 2009, 29, 295.
16. 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.
5.4. BACE1 assay
17. Ghosh, A. K.; Shin, D.; Downs, D.; Koelsch, G.; Lin, X.; Ermolieff, J.; Tang, J. J. Am.
Chem. Soc. 2000, 122, 3522.
18. 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.
19. 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.
20. Asai, M.; 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.
21. 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.
BACE1 inhibitory activity of the inhibitors was determined by
enzymatic assay using recombinant human BACE1 and FRET (fluo-
rescence resonance energy transfer) substrate using previously re-
ported methods.16 After the enzymatic reaction with BACE1 and
FRET substrate, (7-methoxycoumarin-4-yl)acetyl-Ser-Glu-Val-
Leu Asp-Ala-Glu-Phe-Arg-Lys(2,4-dinitrophenyl)-Arg-Arg-NH2, in
incubation buffer with 2 or 0.2 lM KMI-compounds, the N-termi-
*
nal cleavage fragment of substrate was analyzed by fluorescence
detection using RP-HPLC.
22. Mimoto, T.; Imai, J.; Tanaka, S.; Hattori, N.; Kisanuki, S.; Akaji, K.; Kiso, Y. Chem.
Pharm. Bull. 1991, 39, 3088.
23. 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, 2380.
24. 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.
25. Kohara, Y.; Imamiya, E.; Kubo, K.; Wada, T.; Inada, Y.; Naka, T. Bioorg. Med.
Chem. Lett. 1995, 5, 1903.
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 Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of the Japanese Govern-
ment, and grants from MEXT. H.D.T. thanks JASSO for the award
of doctoral scholarship.
26. Kohara, Y.; Kubo, K.; Imamiya, E.; Wada, T.; Inada, Y.; Naka, T. J. Med. Chem.
1996, 39, 5228.
27. Charistos, D. A.; Vagenas, G. V.; Tzavellas, L. C.; Tosleridis, C. A.; Rodios, N. A. J.
Heterocycl. Chem. 1994, 31, 1593.
28. Young, R. W.; Wood, K. H. J. Am. Chem. Soc. 1955, 77, 400.
29. Omar, M. T. Arch. Pharm. Res. 1997, 20, 602.
30. Kane, J. M.; Dalton, C. R.; Staeger, M. A.; Huber, E. W. J. Heterocycl. Chem. 1995,
32, 183.
31. Godefroi, E. F.; Wittle, E. L. J. Org. Chem. 1956, 21, 1163.
32. Herr, R. J. Bioorg. Med. Chem. 2002, 10, 3379.
References and notes
1. Selkoe, D. J. Nature 1999, 399, A23.
2. Sinha, S.; Lieberburg, I. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11049.
3. Selkoe, D. J. Neuron 1991, 6, 487.
4. Yan, R.; Bienkowski, M. J.; Shuck, M. E.; Miao, H.; Tory, M. C.; Pauley, A. M.;
Brashier, J. R.; Stratman, N. C.; Mathews, R. W.; Buhl, A. E.; Carter, D. B.;