Novel γ-secretase inhibitors discovered by library screening of in-house synthetic natural product intermediates

Article abstract of DOI:10.1016/j.bmcl.2006.04.025

Screening of our in-house compound library comprised of intermediates of natural product synthesis projects resulted in discovering two novel γ-secretase inhibitors, which coincidently had similar moieties, that is, cyclohexenone and two aryl groups arran

Full text of DOI:10.1016/j.bmcl.2006.04.025

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3813–3816
Novel c-secretase inhibitors discovered by library screening
of in-house synthetic natural product intermediates
Yasuko Takahashi,a Haruhiko Fuwa,b Akane Kaneko,b Makoto Sasaki,b
Satoshi Yokoshima,a Hifumi Koizumi,a Tohru Takebe,a Toshiyuki Kan,c
a
a,
a,
*
Takeshi Iwatsubo,a,* Taisuke Tomita, Hideaki Natsugari and Tohru Fukuyama
*
aGraduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bGraduate School of Life Sciences, Tohoku University, 1-1 Tsutsumidoori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
cSchool of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
Received 7 March 2006; accepted 11 April 2006
Available online 8 May 2006
Abstract—Screening of our in-house compound library comprised of intermediates of natural product synthesis projects resulted in
discovering two novel c-secretase inhibitors, which coincidently had similar moieties, that is, cyclohexenone and two aryl groups
arranged on the core six-membered ring. Structure–activity relationship studies of these compounds were also developed.
Ó 2006 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD), a dementing neurodegenera-
tive disorder, is currently a serious public health prob-
lem in aging society and is predicted to affect 20
million people worldwide within 20 years.1 AD is char-
acterized pathologically by neuronal loss in the cerebral
cortex accompanied by massive deposition of amyloid-b
peptides (Ab) as senile plaques. A body of evidence sug-
gests that amyloid deposits are strongly implicated in
the pathogenesis of AD. Therefore, regulation of the
Ab levels is considered as a mechanism-based therapeu-
tics for AD. Ab is produced from amyloid precursor
protein (APP) through sequential proteolytic processing
by two membrane associated aspartic proteases termed
b- and c-secretases. Thus, these secretases are predicted
to be prime molecular targets toward prevention and
cure of AD.
5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide (LY411575),4
and 2-[(1R)-1-[[(4-chlorophenyl)sulfonyl](2,5-difluoro-
phenyl)amino]ethyl]-5-fluorobenzenepropanoic acid
(BMS-299897))5 are reported to reduce Ab levels in
mouse brains and biological fluids by oral administra-
tion. However, several c-inhibitors have been shown to
affect Notch processing pathway, thereby causing
unwanted side effects.6–8 Thus, discovery and develop-
ment of new inhibitors without side effects have been
continuously challenging and demanding tasks for
medicinal chemists. Herein we provide our new findings
that cyclohexenone derivatives are novel potent c-secre-
tase inhibitors.
We began our search for c-secretase inhibitors by
screening our in-house compound library. The library
consisted of intermediates of several natural product
synthesis projects in our group (e.g., ecteinascidin
743,9 vinblastine,10 antascomicin,11 etc.), and thus, high
structural diversity of the members of the library is at-
tained. The ability of compounds to inhibit Ab forma-
tion was evaluated by an in vitro assay using
recombinant C-terminal fragment of APP as a sub-
strate.12 After an intensive screening, we discovered
two compounds with potent c-secretase inhibitory activ-
ity among about 600 selected compounds (Fig. 1).
Though these compounds were intermediates in the dif-
ferent targets of natural products, coincidentally they
have similar structures, six-membered cyclic enone, on
A number of c-secretase inhibitors have been reported
so far.2 In fact, some potent inhibitors (e.g., N-[N-(3,5-
difluorophenacetyl)-L-alanyl]-S-phenylglycine tert-butyl
ester
(DAPT),3
N2-[(2S)-2-(3,5-difluorophenyl)-2-
hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-
Keywords: c-Secretase; Inhibitor; Structure–activity relationships;
Cyclohexenone.
*
Corresponding authors. Tel.: +81 3 5841 4899; fax: +81 3 5841 4708
(T.I.); tel./fax: +81 3 5841 4775 (H.N.); tel.: +81 3 5841 4777; fax: +81
3 5802 8694 (T.F.); e-mail addresses: iwatsubo@mol.f.u-tokyo.ac.jp;
natsu@mol.f.u-tokyo.ac.jp; fukuyama@mol.f.u-tokyo.ac.jp
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.025

3814
Y. Takahashi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3813–3816
O
THPO
THPO
O
CHO
OH
Ph
Ph
O
CbzHN
,c
HO
N
O
CbzHN
7
8
O
O
OH
Ph
OAc
Ph
GS155 (1
S416 (2)
THPO
HN
HO HN
Figure 1. Compounds with potent inhibitory activity toward c-
secretase.
d,e
f
Ns
Ns
9
10
OAc
Ph
which two aromatic rings are arranged. We paid atten-
tion to the common moieties of these compounds, and
synthesized various derivatives to explore the struc-
ture–activity relationships.
OH
Ph
N
g,h
i
N
H
Ns
11
12
GS155 (1) had been synthesized from furfral 3 by means
of aza-Achmatowicz reaction (Scheme 1). Furfral was
transformed into the corresponding cyanohydrin. Alkyl-
ation followed by cleavage of the silyl ether afforded ke-
tone 4, which was reduced with sodium borohydride
to furnish alcohol 5. Nitrogen was introduced
using N-benzyloxycarbonyl-2-nitorobenzenesulfonam-
ide (NsNHCbz)13 by means of Mitsunobu reaction,14
and the Ns group was removed to produce 6. Upon
treatment with m-chloroperbenzoic acid (m-CPBA), 6
underwent aza-Achmatowicz reaction to form GS155
(1).15
OH
Ph
O
O
Ph
Ph
N
R
N
N
R
,l
R
13a-d
14a-
5a-d
Scheme 2. Reagents and conditions: (a) THPOCH2CCH, n-BuLi,
THF, À78 °C, 99%; (b) Pd–C, H2, MeOH; (c) NsCl, aq Na2CO3,
CH2Cl2, 0 °C, 67% (two steps); (d) Ac2O, pyridine; (e) CSA, MeOH,
0 °C; (f) DEAD, Ph3P, toluene, 96% (three steps); (g) K2CO3, MeOH,
0 °C, 96%; (h) PhSH, Cs2CO3, CH3CN; (i) RCl, aq K2CO3, CH2Cl2,
84–91% (two steps); (j) DMP, CH2Cl2, 0 °C, 71–80%; (k) TMSCl,
LHMDS, THF, À78 °C; (l) Pd(OAc)2, CH3CN.
For the derivatization of GS155, we planned to remove
the hemiaminal moiety of GS155 because the hemiami-
nal moiety seemed labile. The derivatives were synthe-
sized as shown in Scheme 2. Addition of alkyne to DL-
phenylalaninal (7)16 furnished a diastereomeric mixture
of alcohol 8. Hydrogenolysis of 8 induced saturation
of the triple bond and cleavage of the benzyloxycarbon-
yl group (Cbz) to afford aminoalcohol. After introduc-
tion of 2-nitrobenzenesulfonyl group (Ns) onto the
amino group selectively, sequential protective group
manipulation furnished 10, which was cyclized by means
of Mitsunobu reaction to afford 11. After removal of the
Ns group under standard conditions, acylation of the
amine with various acyl chlorides afforded 13a–d. The
remaining secondary alcohol was oxidized to afford ke-
tone 14a–d, which was transformed into enone 15a–d
according to the Ito-Saegusa’s method.17
On the other hand, GS416 (2) had been prepared from
tri-O-acetyl-D-glucal via catalytic Ferrier rearrange-
ment,18 and the synthesis has been already reported.11
According to the similar procedure, derivatives of 2 were
synthesized as shown in Scheme 3. Deprotection of the
acetyl groups of methyl glucoside 16 derived from tri-
O-acetyl-D-glucal followed by selective acetalization of
the resultant 1,3-diol moiety gave benzylidene acetal
17a,b. Alkylation of the remaining 3-hydroxyl group
(introduction of R1) was followed by cleavage of the
benzylidene acetal, and subsequent selective protection
of the primary alcohol with trityl group afforded
18a,b. The second alkyl group (R2) was introduced onto
the remaining secondary hydroxyl group. After removal
of the trityl group, the resulting alcohol was converted
into enol ether according to the known procedure. That
is, the primary alcohol was converted into iodide 20a,b,
which was treated with sodium hydride to afford 21a,b.
The enol ether 21a,b was then subjected to the condi-
tions of catalytic Ferrier rearrangement to furnish
hydroxyketone 22a,b. Mesylation of the alcohol induced
b-elimination to produce enone 23a,b. The results of
hydrogenation depended on the source of Pd–C. Using
10% Pd–C purchased from Wako Pure Chemical Indus-
tries saturated the double bond of 23a,b selectively
(24a,b). Similarly GS416 (2) was reduced to give 24e.
In contrast, 10% Pd–C purchased from Aldrich Chemi-
cal Company afforded debenzylated products (24c,d).
O
O
CHO
O
a,b
c
O
OH
3
4
5
O
O
O
NH
HO
N
O
d,e
f
O
O
6
GS155 (1)
Scheme 1. Reagents and conditions: (a) TMSCN, CH3CN, sealed
tube, 120 °C, 95%; (b) LDA, BnBr, THF, À78 °C; TBAF, rt, 54%; (c)
NaBH4, MeOH, 0 °C to rt, 67%; (d) NsNHCbz, DEAD, Ph3P,
toluene–THF, 0 °C to rt, 68%; (e) PhSH, K2CO3, DMF, 53%; (f) m-
CPBA, CH2Cl2, 69%.
The results of c-secretase inhibition assay of these deriv-
atives are shown in Tables 1 and 2. Removal of the

Y. Takahashi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3813–3816
3815
H
H
bered ring by intramolecular hydrogen bonding to in-
crease its activity. Saturation of the double bond
caused loss of the inhibitory activity (14a,b). The aryl
group connected to the nitrogen atom was important
for the inhibition. The methoxycarbonyl derivative 15b
had weaker activity, and benzoyl derivative 15c was
slightly stronger. In the case of GS416 (2) saturation
of the double bond also caused a complete loss of activ-
ity, while 24b retained activity. Though the benzyl group
closer to the ketone was more important for the activity,
the presence of both benzyl groups was needed for main-
taining the high activity. These results were parallel to
the SAR of 1.
MeO
O
MeO
O
O
OAc
OAc
a-c
O
Ph
d,e
OR1
OAc
16
17a,b
H
H
MeO
O
MeO
O
OTr
OH
OTr
OR2
g,h
f
OR1
OR1
18a,b
19a,b
H
MeO
O
MeO
O
I
OR2
OR2
i
j
These results suggested that the enone moiety in each
derivative is the most important structure for the c-se-
cretase inhibition, while some compounds such as 24b
retained activity. The enone moiety could be necessary
to fix the molecule to the active conformation in the
GS155 derivatives, while not necessary in the GS416
derivatives. It is known that enone moiety tends to react
with enzyme nonspecifically by means of Michael
addition. However, inhibitory activities of 1 and 2
against c-secretase were retained in the presence of 2-
mercaptoethanol, under which conditions Michael addi-
tion of 1 or 2 with proteins is thought to be suppressed
(data not shown). Moreover, these compounds had no
inhibitory effect on b-galactosidase activity (data not
shown). Thus, we could rule out the possibility of non-
specific inhibition and/or crosslink of c-secretase by
these compounds.
OR1
OR1
20a,b
21a,b
HO
O
O
OR2
O
OR2
OR2
OR1
OR1
OR1
22a: R1 = Bn, R2 = Me
22b: R1 = Me, R2 = Bn
23a: R1 = Bn, R2 = Me
23b: R1 = Me, R2 = Bn
24a: R1 = Bn, R2 = Me
24b: R1 = Me, R2 = Bn
24c: R1 = H, R2 = Me
24d: R1 = Me, R2 = H
24e: R1 = Bn, R2 = Bn
GS416 (2) : R1 = Bn, R2 = Bn
Scheme 3. Reagent and conditions: (a) NaOMe, MeOH; (b)
PhCH(OMe)2, CSA, DMF, 73% (two steps); (c) NaH, R1X, DMF,
0–50 °C; (d) CSA, MeOH, CH2Cl2, 96–97% (two steps); (e) TrCl,
pyridine, CH2Cl2, 62–88%; (f) R2X, NaH, DMF; (g) CSA, MeOH, 79–
91% (two steps); (h) I2, Ph3P, imidazole, THF, 85–90%; (i) NaH,
DMF, 86–91%; (j) Hg(OCOCF3)2, acetone–H2O, 96–98%; (k) MsCl,
Et3N, CH2Cl2, 0 °C to rt, 79–83%; (l) H2, Pd–C, EtOAc, 95–100%.
In summary, we discovered novel cyclohexenone c-se-
cretase inhibitors from our in-house library comprised
of intermediates of natural product synthesis projects.
The SAR study of these compound suggested the enone
moiety and the two aryl groups were important for the
potent c-secretase inhibitory activity. Synthesis of
molecular probes for functional analysis of c-secretase19
as well as structural development studies using 1 and 2
are currently underway and will be reported in due
course.
Table 1. Inhibitory activities of GS155 (1) and its derivatives
Compound R–
IC50 (Ab40, lM) IC50 (Ab42, lM)
13a
13b
14a
BnOCO– >100
MeOCO– >100
>100
>100
>100
BnOCO–
21% inhibition
at 100 lM
MeOCO– >100
BnOCO– 4.6
MeOCO– 7.2
14b
15a
>100
7.4
15b
15c
9.8
2.2
PhCO–
PhSO2–
—
1.0
5.9
0.5
15d
GS155 (1)
12.3
0.7
Acknowledgments
This work was financially supported in part by a Grant
for the 21st Century COE Program and Scientific
Research on Priority Areas from The Ministry of
Education, Culture, Sports, Science and Technology
(MEXT) and by the Program for Promotion of Funda-
mental Studies in Health Sciences of the National Insti-
tute of Biomedical Innovation (NIBIO), Japan. Y.T.
and H.F. are a research fellow and a postdoctoral fel-
low, respectively, of Japan Society for the Promotion
of Science (JSPS).
Table 2. Inhibitory activities of GS416 (2) and its derivatives
Compound R1 R2
IC50 (Ab40, lM) IC50 (Ab42, lM)
Me >100
Me Bn >100
Bn Me 33.9
Me Bn
22a
Bn
>100
>100
22b
23a
26.0
10.2
>100
13.9
>100
23b
6.9
4.9
24a
24b
Bn Me >100
Me Bn
24c
24d
H
Me >100
Me
Bn
—
H
>100
>100
1.6
>100
>100
24e
GS416 (2)
Bn
—
1.1
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