Conformational restriction approach to BACE1 inhibitors II: SAR study of the isocytosine derivatives fixed with a cis-cyclopropane ring

Article abstract of DOI:10.1016/j.bmcl.2013.03.056

To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.

Full text of DOI:10.1016/j.bmcl.2013.03.056

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Conformational restriction approach to BACE1 inhibitors II: SAR
study of the isocytosine derivatives fixed with a cis-cyclopropane
ring
b
b
b
b
Shuji Yonezawa a,d, , Hidekuni Yamakawa , Chie Muto , Motoko Hosono , Takahiko Yamamoto ,
⇑
Kazunari Hattori b, Masahiro Sakagami b, Hiroko Togame c, Yoshikazu Tanaka a, Toru Nakano a,
Hiroshi Takemoto b, Mitsuhiro Arisawa d, Satoshi Shuto d,
⇑
a Shionogi Innovation Center for Drug Discovery, Shionogi & Co., Ltd, Kita-21 Nishi-11 Kita-ku, Sapporo 001-0021, Japan
b Medicinal Research Laboratories, and Innovative Drug Discovery Research Laboratories, Shionogi & Co., Ltd, 1-1 Futaba-cho 3-chome, Toyonaka, Osaka 561-0825, Japan
c Quality, Safety and Regulatory Affairs Management Division, Shionogi & Co., Ltd, 1-8 Doshomachi 3-chome, Chuo-ku, Osaka 541-0045, Japan
d Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications
were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion
of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an
ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while
the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed
an interesting effect of a conformational restriction with a cyclopropane ring.
Received 19 February 2013
Revised 13 March 2013
Accepted 15 March 2013
Available online xxxx
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Alzheimer’s disease
b-Secretase (BACE1)
Conformational restriction
Cyclopropane
In the amyloid cascade hypothesis, b-secretase (BACE1, Beta-
Amyloid Converting Enzyme 1) is currently regarded as an attrac-
tive target for the treatment of Alzheimer’s disease (AD).1 BACE1
first cleaves the amyloid precursor protein (APP), and mature Ab
peptides of various lengths, including the most pathogenic Ab42,
dine-type inhibitors; it was induced by a CH–p interaction be-
tween the rigid cyclopropane ring and the Tyr71 side chain.
Based on the unique binding mode, we performed derivatization
studies. While 3 which contains a non-substituted phenyl ring
showed diminished activity, we discovered that derivative 4 which
bears a m-methoxyphenyl group at the meta position of the termi-
nal benzene ring shows a potent IC50 value. Binding of 4 to BACE1
was accompanied by the further induced fit of the protein, a flip of
Leu30, which suggested the importance of a hydrogen bond
network including methoxy oxygen, water and the main chain of
Ser229 (Fig. 1). To improve the activity of these meta-substituted
derivatives, we attempted further structural modification and re-
port our results here.
Compound design was done using two different strategies
( Fig. 2): (a) modification of the terminal aromatic ring and (b)
insertion of spacers between the aromatic rings. We hypothesized
that replacement by another functional groups or positional
change of the methoxy group of 4, which works as the key hydro-
gen bond acceptor, might lead to strengthening of the interaction
between the ligand and BACE1. We also investigated the effects
of introducing heteroaromatic rings.
are eventually produced by the action of c
-secretase.2 Knockdown
of BACE1 expression in mice leads to a decrease in the production
of all forms of Ab, without any significant dysfunction symptoms.3
There have been numerous reports on the BACE1 inhibitors by
many pharmaceutical companies,4 and some compounds are al-
ready in clinical trials.5
In a previous Letter, we reported the results of our investigation
on the cyclopropane-based conformational restriction approach
using a known BACE1 inhibitor 16 bearing an isocytosine moiety
as a lead. Among the four stereoisomers synthesized, cis-(1S,2R)
isomer 2 was found to be the most effective.7
The X-ray crystallographic structure analysis of 2 complexed
with BACE1 (PDB code no. 3VV6) revealed an unexpected binding
mode that was clearly different from those of the known ami-
⇑
Corresponding authors. Tel.: +81 11 700 4702; fax: +81 11 700 4714 (S.Y.);
tel./fax: +81 11 706 3769 (S.S.).
In the preceding Letter, we reported that diminished activity of
3 would result from the steric clash of the terminal substituent
E-mail addresses: shuji.yonezawa@shionogi.co.jp (S. Yonezawa), shu@pharm.
hokudai.ac.jp (S. Shuto).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.056
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2
S. Yonezawa et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
a)
)
Asp228
(b)
WATER
Ser229
H
O
Ser229
WATER
O
H
Asp32
S3
OMe
O
O
HO
H
H
N
O
N
H
Asp32
Asp228
OH
N
Leu30
O
Tyr71
S1
Leu30
Tyr71
Figure 1. (a) X-ray structure of 3 complexed with BACE1 (PDB code no. 3VV7); (b) schematic view of the interaction.
Modification of
X
the terminal Ar ring
Ar
R
N
NH2
O
N
N
2
1
N
O
NH2
Me
Strategy a)
Strategy b)
Me
(R) (S)
N
O
NH2
Me
N
5
O
N
N
NH2
1
Insertion of
a spacer
2 [cis-(1S, 2R)]
IC50: 157 µM LE = 0.29
IC50: 171 µM LE = 0.30
3 (R= H) IC50: 614 µM LE = 0.18
4 (R = OMe) IC50: 6.7µM LE = 0.27
R
N
NH2
N
O
6
Figure 2. Strategies of compound design.
with BACE1.7 Through docking studies based on the second strat-
egy, using the X-ray structure of compound 2 complexed with
BACE1, we found that insertion of a spacer between the two aro-
matic rings of 3, an ethylene group for instance, may circumvent
the steric obstacle, and enable us to expand a substituent at the
meta position (Fig. 3). Therefore, we hypothesized that introducing
another substituent into this position might make it possible to de-
velop a new type of inhibitors.
meta-Substituted biaryl derivatives 8–15 and compound 19
were prepared by Suzuki–Miyaura coupling of 77 with the corre-
sponding boronic acids or esters in good yields (Scheme 1). We also
obtained the spacer-included derivatives 16–18 and 20–24 by
cross coupling with alkyl trifluoroborates or Sonogashira coupling
in the same manner. Some commercially unavailable potassium al-
kyl trifluoroborates for the coupling reactions were prepared using
a known method8,9 (for details, see Supplementary data).
Br
N
O
NBoc2
N
O
NH2
Me
Ar
a
N
N
Me
8-15
7
)
b (Y = -(CH2)n-) c (Y =
)
d (Y =
N
O
NH2
Me
Y
Ar
N
16-24
Scheme 1. Cross coupling reactions. Reagents and conditions: (a) Ar-B(OH)2 or Ar-
Bpin, K2CO3, PdCl2(PPh3)2, dioxane, H2O, 150 °C under microwave, 30 min; (b) (i)
Ar-Y-BF3K, PdCl2(dppf), Cs2CO3, THF, H2O, 80 °C, (ii) TFA, CH2Cl2; (c) (compound 19)
styrylboronic acid, K2CO3, PdCl2(PPh3)2, dioxane, H2O, 100 °C; (d) (compound 20) (i)
phenylacetylene, PdCl2(PPh3)2, CuI, 80 °C, then 150 °C under microwave, (ii) TFA,
CH2Cl2.
Modification of the substituent on the terminal benzene ring of
3 was investigated (Table 1). Extending the length of the alkoxy
chain was not effective (compound 8), which suggests that there
was not enough space to accommodate a substituent larger than
a methoxy group at this position. When the methoxy group was
displaced with a simple methyl group, the IC50 value decreased
(compound 9). Introducing a sulfur or nitrogen substituent or fluo-
rine atom with hydrogen bond acceptor abilities (compounds 10–
13), or migration of the substitution position (compound 14) from
the meta to the para did not improve the activity nor ligand effi-
ciency. These findings strongly suggest that such modifications
lead to loss of the hydrogen bond network using the oxygen atom
of the meta-methoxy group, which allows effective interaction
with the active site of BACE1. In fact, the binding activity severely
diminished when the terminal benzene ring was transformed into
a heteroaromatic ring, pyrazine ring for example (compound 15),
probably due to the decreased electron density of the oxygen atom
leading to the decline of its hydrogen acceptor ability. It is also as-
sumed that the hydrophilic pyrazine ring did not meet the intrinsic
hydrophobic nature of the S3 pocket.
N
O
NH2
2
N
Me
Figure 3 suggests that insertion of a spacer between the two
aromatic rings might be effective, leading us to investigate the
structure-activity relationship with regard to the type of spacer
(Table 2). Compounds with a simple single-bonded spacer with
1–3 carbon atoms (16–18) were prepared, and their inhibitory ef-
fects were evaluated. We found that 17 with a spacer of two carbon
atoms exhibited BACE1 inhibition. Its inhibitory activity and ligand
S3
S1
Figure 3. Docking studies of compound 17, which bears phenethyl group at the
meta position.
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S. Yonezawa et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
Table 1
SAR of the meta-substituted biaryl derivatives
R1
R2
N
O
NH2
Me
N
Compd
R1
R2
BACE1 IC50
(
lM)
Ligand efficiency
3
4
8
9
10
11
12
13
14
H
H
H
H
H
H
H
H
H
614a
0.18a
0.27a
0.22
0.23
0.23
—
—
0.23
0.24
OMe
OEt
Me
SMe
NMe2
NH2
F
6.7a
39.4
74.9
47.5
>200
>200
72.4
30.8
H
OMe
OMe
N
N
N
O
NH2
Me
15
84.9
0.21
N
a
Data taken from Ref. 7.
seems to indicate that there is little space for further structural
modification around the terminal benzene ring of 17.
Table 2
SAR of the meta-substituted spacer-inserted derivatives
For comparison, we synthesized a compound with an ethylene
linker (compound 25) which corresponds to the unrestricted ana-
log of 17, and found that it exhibited no activity. As described in
the preceding Letter,7 changing the ethylene moiety between the
isocytosine ring and the central benzene ring in compound 25 for
a cyclopropane ring is likely to have altered the binding mode with
BACE1, enabling incorporation of an ethylene spacer between two
benzene rings, and eventually resulting in inhibition of the activity.
Induced-fit due to the cyclopropane-based conformational
N
NH2
Me
Y
N
O
Compd
Y
BACE1 IC50
(l
M)
Ligand efficiency
16
17
18
19
20
–CH2–
>200
22.2
>200
>200
>200
—
0.25
—
—
—
–CH2CH2–
–CH2CH2CH2–
(E)-CH@CH–
–C„C–
restriction driven by CH–p interaction of the ligand with the aro-
matic ring of the protein, which was typically observed in the X-
ray structure of the complex of 3 and BACE1, would cause a bind-
ing mode different from that of the original unrestricted com-
pound, leading to the development of structurally novel
isocytosine-type inhibitors.
In conclusion, through the derivatization study of meta-substi-
tuted derivatives based on the X-ray structure of conformationally
restricted compound 2 and BACE1, we found another type of inhib-
itor 17 bearing a spacer between the two aromatic rings, while the
corresponding unrestricted ethylene linker compound 25 showed
none of the inhibitory activity. This is another good example of
efficiency were superior to those of the biaryl-based parent com-
pound 3. With shorter or longer spacers (compounds 16 or 18),
the activity completely disappeared. Insertion of a double or triple
bond spacer was also ineffective (compounds 19 or 20), probably
due to the conformational restriction of the terminal aromatic ring
to an orientation unsuitable for the binding.
We also investigated the substitution on the terminal benzene
ring of 17 (Table 3). However, the inhibitory effects of all the com-
pounds synthesized were significantly weaker than that of 17. This
Table 3
SAR results of the substitution on the terminal ring of 17
R2
R3
R1
N
O
NH2
Me
N
Compd
R1
R2
R3
BACE1 IC50 (lM)
Ligand efficiency
17
21
22
23
24
H
Me
H
H
H
H
H
Me
H
OMe
H
H
H
Me
H
22.2
120
>200
>200
100
0.25
0.20
—
—
0.20
O
N
Me
N
25
>200
—
NH2
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4
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Acknowledgments
We thank Mr. Rikio Ikenishi (Shionogi Techno Advance Re-
search Co., Ltd) for elemental analysis, Ms. Izumi Fujisaka (Shionogi
Techno Advance Research Co., Ltd) for measuring the optical rota-
tion, the members of the Instrumental Analysis Division, Equip-
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Hokkaido University for the high-resolution mass spectroscopy
analysis. This investigation was supported by a Grant-in-Aid for
Scientific Research (21390028) from the Japan Society for the Pro-
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Supplementary data
Supplementary data (all experimental information) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2013.03.056.
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Please cite this article in press as: Yonezawa, S.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.03.056