Design and synthesis of BACE1 inhibitors containing a novel norstatine derivative (2R,3R)-3-amino-2-hydroxy-4-(phenylthio)butyric acid

Article abstract of DOI:10.1016/j.bmcl.2006.12.097

A novel norstatine derivative, phenylthionorstatine [(2R,3R)-3-amino-2-hydroxy-4-(phenylthio)butyric acid; Ptns], containing a hydroxymethylcarbonyl (HMC) isostere was designed, synthesized, and stereochemically determined. Then, Ptns was introduced into

Full text of DOI:10.1016/j.bmcl.2006.12.097

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1629–1633
Design and synthesis of BACE1 inhibitors containing a novel
norstatine derivative (2R,3R)-3-amino-2-hydroxy-4-
(phenylthio)butyric acid
Zyta Ziora, Soko Kasai, Koushi Hidaka, Ayaka Nagamine, Tooru Kimura,
Yoshio Hayashi and Yoshiaki Kiso*
Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, 21st Century COE Program,
Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
Received 11 October 2006; revised 21 December 2006; accepted 23 December 2006
Available online 8 January 2007
Abstract—A novel norstatine derivative, phenylthionorstatine [(2R,3R)-3-amino-2-hydroxy-4-(phenylthio)butyric acid; Ptns], con-
taining a hydroxymethylcarbonyl (HMC) isostere was designed, synthesized, and stereochemically determined. Then, Ptns was
introduced into the structure of BACE1 inhibitors at the P1 position. Finally, Ptns was found as a suitable P1 moiety for potent
BACE1 inhibitor design.
Ó 2007 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is the most common neuro-
degenerative disease, and the accumulation of amyloid
b peptide (Ab) is a major factor in the pathogenesis of
Alzheimer’s disease.1 Ab is formed by proteolytic
processing of amyloid precursor protein (APP). Two
enzymes, b-secretase (b-site APP cleaving enzyme,
BACE1) and c-secretase, are responsible for the sequen-
tial processing of APP.2 Since the cleavage of APP by
b-secretase is the first step in Ab formation, BACE1
plays a critical role in the progression of AD. Therefore,
the development of BACE1 inhibitors is valuable in the
elucidation of AD pathology. BACE1 was identified as a
novel membrane-bound aspartic protease and the crys-
tal structure of its catalytical domain was also deter-
mined. Based on the common enzymatic mechanism of
aspartic proteases, substrate transition-state mimics
have been proposed and are currently widely used for
the design of highly potent aspartic protease inhibitors.3
protease, and BACE1.7 We also described the impor-
tance of the stereochemistry of the transition-state
mimetic hydroxyl group for the inhibitory activity.4b,c,7
Through the study of the stereochemical preference of
the P1 position, introducing Pns [phenylnorstatine:
(2R,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] or its
(2S,3S)-diastereomer, Apns, as a transition-state mimic,
we found that (2R)-hydroxyl group of HMC, in Pns,
was better for efficient inhibitory activity against
BACE1.7
In order to develop more active compounds, we have fo-
cused on the P1 phenylalanine derivative, since inhibi-
tors containing Pns exhibited higher BACE1 inhibitory
activity than those with norstatine [(2R,3S)-3-amino-2-
hydroxy-5-methylhexanoic acid; Nst], a leucine mimetic
from the sequence of Swedish mutant APP (P1 ꢀ P01:
L*D). The phenyl group of Pns enhanced the interaction
between inhibitor and protease at the S1 site.7 In the se-
quence of the wild type APP, b-secretase recognizes Met
at S1, what could be a starting point for another mimetic
design (P1 ꢀ P01: M*D). Noteworthy is the SAR study of
HIV-1 protease, plasmepsin II, and cathepsin D inhibi-
tors containing HMC, demonstrating the important role
of the lipophilic P1 aromatic ring system, which fits into
the S1 hydrophobic pocket.8 In Nelfinavir (nelfinavir
mesylate, nonpeptidic inhibitor of HIV-1 protease) thi-
ophenyl moiety was introduced at P1 site and showed
In our previous study, we applied a hydroxymethylcar-
bonyl isostere (HMC) at P1 position of potent inhibitors
toward several human disease-related aspartic proteases
such as renin, HIV-1protease,4 plasmepsin II,5 HTLV-I6
Keywords: Alzheimer’s disease; BACE1 inhibitor; Norstatine; Hydroxy-
methylcarbonyl isostere.
*
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 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.097

1630
Z. Ziora et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1629–1633
10-fold greater affinity than a phenyl analogue.9 These
all observations stimulated us to modify the side chain
of Pns.
OH
OH
O
O
a
b, c
OH
N
O
O
O
N
H
O
N
O
H
O
O
3
4
Thus, we designed phenylthionorstatine (Ptns) [(2R,3R)-
3-amino-2-hydroxy-4-(phenylthio)butyric acid] and its
(2S,3R)-diastereomer, and then we have focused our de-
sign on Ptns containing peptide inhibitors (2, Fig. 1).
S
S
O
O
e
d
H
O
N
O
N
N
H
O
H
We have synthesized Ptns starting from readily available
N-benzyloxycarbonyl-L-serine (3, Scheme 1), and after
protecting carboxylic group by Weinreb amide, 4 was
transformed into its mesylate and then treated at 0 °C
with sodium thiophenolate prepared in situ in DMF to
give the phenylsulfide (5).10 Reduction of 5 with LiAlH4
resulted in aldehyde 6.11 The key intermediate for the
Ptns preparation is the cyanohydrin derivative 7 pro-
duced in the next step. Several methods were reported
for the synthesis of hydroxymethylcarbonyl units,
involving aqueous hydrolysis of cyanohydrin, obtained
from protected aldehyde by treatment with potassium
cyanide,12 or a one-pot procedure consisting of reaction
of protected aldehyde with (trimethylsilyl)cyanide.13 In
our case, we used acetone cyanohydrin and trimethyl-
aluminum as a reaction accelerator in chloroform at
0 °C.14 Compound 7 was directly transformed into the
methyl esters 8 and 9 by treatment with dry methanolic
hydrogen chloride, followed by in situ hydrolysis of the
intermediate imidate hydrochloride.13
O
S
O
5
6
S
O
O
N
f
N
H
O
N
H
O
OH
OH
7
8
+
S
O
O
O
N
H
O
OH
9
Scheme 1. Reagents and conditions: (a) MeNH(OMe)ÆHCl, EDCÆHCl,
NMM, CH2Cl2, ꢀ10 °C, 2 h, 100%; (b) MsCl, Et3N, CHCl3, 0 °C,
0.5 h; (c) PhSNa, DMF, 0 °C to rt, 24 h, over 2 steps 95%; (d) LiAlH4,
THF, 0 °C, 0.5 h; (e) Me2C(OH)CN, Me3Al, CHCl3, 0 °C to rt, 10 h,
75% over 2 steps; (f) 4 N HCl/dioxane, MeOH, 4 °C, 24 h, H2O, 4 °C,
24 h, 52%, ratio 8:9 was 1.8:1; separation by flash chromatography
(hexane:AcOEt, 2:1).
To determine the stereochemistry of esters 8 and 9, first,
these esters were separated by flash chromatography
and then were converted to the corresponding oxazolid-
inones (10 and 11, respectively), by treating the
esters with 6 N NaOH in DMF13 (Scheme 2). The C-2
configuration of these compounds was unequivocally
S
1
S
determined by the H NMR spectrum of the oxazolidi-
Hb
COOH
O
O
Ha
HN
nones. Thus, 8 gave 10 with an Ha, Hb trans disposition,
as indicated by their J value of 3.9 Hz, while 9 gave 11
with JHa,Hb = 8.7 Hz, a cis configuration.15
O
N
H
O
O
OH
8
10
O
S
O
OH
O
Hb
COOH
S
Ha
HN
OH
O
O
O
O
O
O
O
H
N
H
H
N
H2N
N
O
N
H
N
H
N
N
H
OH
O
N
H
O
H
11
O
OH
Pns
O
O
OH
O
9
OH
Scheme 2. Reagents and conditions: 6 N NaOH, DMF, rt, 2 h; 1H
NMR analysis: 10, JHa,Hb = 3.9 Hz; 11, JHa,Hb = 8.7 Hz.
O
1
(KMI-008)
Esters 8 (2R,3R) and 9 (2S,3R) were saponified (1 N
NaOH in DMF) to provide corresponding acids. After
the removal of benzyloxycarbonyl group by TFA with
dimethylsulfide (40 equiv) and anisole (5 equiv),16 Ptns
and its diastereomer, Aptns [(2S,3R)-3-amino-2-hy-
droxy-4-(phenylthio)butyric acid; allophenylthionorsta-
tine], were obtained. Aptns, as a (2S,3R)-diastereomer
that is unfavorable for the design of BACE1 pentapep-
tidic inhibitors, could be used for the design of the other
aspartyl protease inhibitors. While Ptns, the (2R,3R)-di-
O
OH
O
S
OH
O
O
O
O
O
H
N
H
H
N
H2N
N
N
H
N
H
N
N
H
OH
H
O
OH
Ptns
O
O
2
OH
O
(KMI-172)
Figure 1. Structure of BACE1 inhibitors containing Pns (1, KMI-008)
and Ptns (2, KMI-172).

Z. Ziora et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1629–1633
Table 1. BACE1 inhibitory activity, compounds 1–2 and 12–17
1631
astereomer, was finally N-protected by Fmoc masking
fragment (Fmoc-OSu in THF/H2O, standard method)
and then applied to SPPS (solid phase peptide synthesis)
for the BACE1 inhibitor synthesis.
Compound (KMI no.)
HMC type
BACE1 inhibition (%)
At 2 lM
At 0.2 lM
1 (KMI-008)
2 (KMI-172)
Pns
90a
82
32
34
Ptns
Pns
Compounds 1, 2, and 12–17 (Figs. 2 and 3) were synthe-
sized by the Fmoc based solid phase method, described
previously.7,17a These compounds were adopted to enzy-
matic assay using a recombinant human BACE1. Inhib-
itory activity was determined based on the decrease% of
the cleaved substrate by the enzyme7 (Table 1).
12 (KMI-122)
13 (KMI-366)
14 (KMI-260)
15 (KMI-536)
16d (KMI-494)
17 (KMI-538)
61b
69
Ptns
Pns
82b
96
44
57
80
86
Ptns
Pns
98
99
Ptns
a Data from Ref. 7.
b Data from Ref. 16a.
Our attempts to replace Pns by its thio-derivative in
octapeptide type structure led us to the analogue 2 (Ta-
ble 1, compounds 1 and 2 at 2 lM concentration). Then,
we reduced the size of inhibitors from octapeptides to
pentapeptides. Compound 12 was a lead compound
for pentapeptide series in our previous study.17a Its Ptns
counterpart 13 possessed potency similar to Pns. After
modification at P01 position, where Asp was replaced
by 3-aminobenzoic acid, compound 15 exhibited better
inhibitory activity than previously reported inhibitor
1417a (Fig. 2) (Table 1, compounds 12–15).
O
R
O
O
O
OH
OH
H
N
H2N
N
H
N
N
H
H
O
OH
O
O
OH
Then we have focused on the N-terminal optimization
of the peptides. We already reported many BACE1
inhibitors with various hydrogen bond acceptor groups
at the P4 position.17a,d Through the SAR study we iden-
tified also a few potent peptides bearing isophthalic moi-
ety at P4 (peptides 16a–d, Fig. 3). In that short series of
inhibitors with Pns at P1, compounds containing addi-
tional amino group (3-aminoisophthalyl fragment)
showed higher activity, and the acidic form (16d) was
more potent than ester (16c). We decided to synthesize
an inhibitor with Ptns replacing Pns, and with 3-aminoi-
sophthalic moiety at N-terminal. The Ptns counterpart
17 (KMI-538) exhibited slightly higher activity than
16d (Table 1.). As we demonstrate here, the modification
of P1 position by introducing Ptns improved the BACE1
inhibitory activity.
12
13
(KMI-122)
R: Ph
(KMI-366)
R: S-Ph
R
O
O
O
H
N
H2N
OH
N
H
N
N
H
H
O
OH
O
O
OH
14
15
(KMI-260)
R: Ph
(KMI-536)
R: S-Ph
Figure 2. Modification of P01 position. Structures of pentapeptides
containing Pns (12 and 14) and Ptns (13 and 15).
O
O
O
H
H2N
N
OH
The idea to modify the P1 site by elongation of side
chain (thiophenyl fragment instead phenyl) was con-
firmed by computational simulations. Inhibitor 17 was
docked in BACE1 by using a modeling package (MOE
2005.06, Chemical Group, Inc., Montreal, Canada).
N
N
H
N
H
H
O
OH
O
NH
R1
16a (KMI-562), R1: H, R2: Me
(52% BACE1 inhibition at 2 μM)
16b (KMI-545), R1: H,
O
2
R : H
OR2
(84% BACE1 inhibition at 2 μM)
O
Additional energy minimization processes with
MMFF94x force field were performed.
a
16c (KMI-555), R1: NH ,
2
R : Me
2
(83% BACE1 inhibition at 2 μM)
16d (KMI-494), R1: NH ,
2
R : H
2
Conformation of compound 17, presented in Figure 4,
resulted in almost the same pose as a OM99-2.18 The
exception is the P01 position. The C-terminal of 17 nicely
fixed the pocket, while in OM99-2 the P01 residue did not
reach the S01 site. The important hydrogen bond interac-
tions between the Ptns anchor and Asp32 and Asp228
are shown in Figure 4B. It is in excellent agreement with
our previous observations for inhibitors with Pns.7,17b,c
In this model, other hydrogen bond interactions with
Gly 11, Arg 307, and Arg 235 were also observed, and
those coincided with the previously reported modeling
study of KMI-429,17a KMI-684,17c and KMI-574.17d
The presence of additional amino group at P4 (3-amino
group from aminoisophthalic residue) resulted in new
hydrogen bond interaction with Ser 325 (Fig. 4C), and
(98% BACE1 inhibition at 2 μM)
S
O
O
O
H
N
H2N
OH
N
N
N
H
H
H
O
OH
O
NH
NH2
O
17
(KMI-538)
OH
O
Figure 3. Modification of isophthalic moiety at P4 position and
introduction of Ptns.

1632
Z. Ziora et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1629–1633
We are grateful to Mr. T. Hamada for performing in vitro
enzymatic assay.
References and notes
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Figure 4. Modeled Structure of 17 (KMI-538, green sticks) in the
active site of BACE1 (PDB entry, 1FKN). (A) 17 is superimposed with
an inhibitor OM99-2 (magenta line). Catalytic two Asp residues are
represented with light blue stick. Close-up views of P1 position (B) and
P4 position (C) with possible hydrogen bonds (dotted lines) and
molecular surface of the enzyme pocket (blue).
that is reasonable explanation of enhanced BACE1
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In conclusion, we present for the first time, phenylthio-
norstatine and its synthesis with full stereochemical deter-
mination. The Fmoc-Ptns was applied for the design and
synthesis of BACE1 inhibitors. Inhibitor 17 (KMI-538)
containing Ptns demonstrated potent inhibitory activity.
These results show the possibility of further design of
BACE1 inhibitors with Ptns at P1 position. Our efforts
are also directed toward the study of other aspartic prote-
ase, and the application of peptides containing Ptns, or its
diastereomer Aptns, as inhibitors.
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15. Selected physical data: (a) compound 8: yield = 33%;
25
1
½aꢁD ¼ ꢀ54:2 (c = 0.054 in CHCl3); H NMR (400 MHz,
CDCl3): d = 7.44–7.19 (m, 10H), 5.11 (d, 3J = 10.3 Hz, 1H,
NH), 5.07 (s, 2H, CH2), 4.60 (dd, 3J = 4.0 and 1.8 Hz, 1H,
CH), 4.24–4.20 (m, 1H, CH), 3.73 (s, 3H, CH3), 3.11 (d,
3J = 4.0 Hz, 1H, OH), 3.23, 3.07 (2dd, 2J = 13.8 Hz,
3J = 9.1 and 5.9 Hz, 2H, CH2); 13C NMR (100 MHz,
CDCl3): d = 173.78, 155.64, 136.14, 135.03, 129.38, 129.06,
128.46, 128.11, 127.92, 126.44, 69.77, 66.89, 52.98, 52.73,
34.92; HRMS (FAB): calcd for C19H22O5NS [M+H]+
376.1219, found 376.1223; purity was higher than 97%
(HPLC analysis at 230 nm); (b) compound 9: Yield = 18%;
Acknowledgments
This research was supported in part by The Frontier Re-
search Program and 21st Century COE program of The
Ministry of Education, Culture, Sports, Science and
Technology of Japan (MEXT), and grants from MEXT.
25
1
½aꢁD ¼ ꢀ11:9 (c = 0.043 in CHCl3); H NMR (400 MHz,
3
CDCl3): d = 7.37–7.19 (m, 10H), 5.39 (d, J = 9.2 Hz, 1H,

Z. Ziora et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1629–1633
1633
NH), 5.09 (s, 2H, CH2), 4.35 (br s, 1 H, CH), 4.26–4.22 (m,
1H, CH), 3.66 (s, 3H, OCH3), 3.48–3.45 (br s, 1H, OH),
3.09, 3.04 (2dd, 2J = 13.9 Hz, 3J = 5.9 and 7.5 Hz, 2H,
CH2); 13C NMR (100 MHz, CDCl3): d = 172.74, 155.91,
136.14, 135.14, 129.75, 128.99, 128.46, 128.12, 128.06,
126.58, 72.02, 66.96, 52.91, 52.71, 33.91; HRMS (FAB):
calcd for C19H22O5NS [M+H]+ 376.1219, found 376.1224;
purity was higher than 95% (HPLC analysis at 230 nm);
(c) compound 10: 1H NMR (300 MHz, CD3OD):
d = 7.46–7.23 (m, 5H), 4.85 (d, 3J = 3.9 Hz, 1H), 3.97
(ddd, 3J = 6.0, 6.0 and 3.9 Hz, 1H), 3.21, 3.12 (2dd,
2J = 14.1 Hz, 3J = 6.0 and 6.0 Hz, 2H, CH2); LRMS
(FAB): 254 [M+H]+, 253 [M+Å]; HRMS (FAB): calcd for
C11H11O4NS [M+Å] 253.0409, found 253.0405; (d) com-
pound 11: 1H NMR (300 MHz, CD3OD): d = 7.43–7.22
16. (a) Kiso, Y.; Ukawa, K.; Nakamura, S.; Ito, K.; Akita, T.
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3
(m, 5H), 5.14 (d,3J = 8.7 Hz, 1H), 4.21 (ddd, J = 8.7, 8.7
and 4.2 Hz, 1H), 3.24, 2.90 (2dd, 2J = 13.7 Hz, 3J = 8.7
and 4.2 Hz, 2H, CH2); LRMS (FAB): 254 [M+H]+, 253
[M+Å]; HRMS (FAB): calcd for C11H11O4NS [M+Å]
253.0409, found 253.0403.
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Ermolieff, J.; Tang, J. J. Am. Chem. Soc. 2000, 122, 3522;;
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