Synthesis of biotinylated photoaffinity probes based on arylsulfonamide γ-secretase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.05.091

Synthesis and biological evaluation of an arylsulfonamide class of γ-secretase inhibitors are described. Design, synthesis, and biological evaluation of multifunctional molecular probes harboring a benzophenone photophore as a cross-linking group and a biotin tag are also reported.

Full text of DOI:10.1016/j.bmcl.2006.05.091

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4184–4189
Synthesis of biotinylated photoaffinity probes based
on arylsulfonamide c-secretase inhibitors
Haruhiko Fuwa,* Kenichi Hiromoto, Yasuko Takahashi, Satoshi Yokoshima,
Toshiyuki Kan,ꢀ Tohru Fukuyama, Takeshi Iwatsubo,
Taisuke Tomita and Hideaki Natsugari*
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received 19 April 2006; revised 9 May 2006; accepted 29 May 2006
Available online 12 June 2006
Abstract—Synthesis and biological evaluation of an arylsulfonamide class of c-secretase inhibitors are described. Design, synthesis,
and biological evaluation of multifunctional molecular probes harboring a benzophenone photophore as a cross-linking group and a
biotin tag are also reported.
Ó 2006 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a most common neurode-
generative disorder currently being a serious public
health problem in the aging society. The accumulation
of 40–42 residue amyloid b-protein (Ab) in brain
regions serving memory and cognition is a central
pathogenic feature of AD. Ab is generated through
proteolysis of amyloid precursor protein (APP)1 by
two types of membrane associated aspartic proteases
termed b- and c-secretase, both of which have been sig-
nificant therapeutic targets toward prevention and
treatment of AD.2 c-Secretase, a macromolecular com-
plex comprised of Presenilin-1 (PS1), Pen-2, Aph-1,
and Nicastrin, is known to endoproteolyze several
transmembrane proteins other than APP. Pharmaco-
logical regulation of the substrate selectivity of the
proteolytic processing mediated by c-secretase is,
therefore, an important issue toward the development
of c-secretase-targeted therapeutics without any severe
adverse effects. To date, a number of small-molecule
c-secretase inhibitors, including BMS-299,897 (1) and
related arylsulfonamides3 (Fig. 1), have been discov-
ered. Recent in vivo studies using transgenic mice have
shown that BMS-299,897 effectively reduced brain Ab
levels without causing Notch mediated toxicity,4 which
is the major plausible adverse effect of other dipeptidic
inhibitors such as LY411575 (N2-[(2S)-2-(3,5-difluoro-
phenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-
6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide)
and related compounds.5 These intriguing results com-
bined with the distinct structural feature suggested that
the biological mode of action(s) of the arylsulfonamide
inhibitors would be different from those of the previ-
ously known dipeptidic class of derivatives. However,
using a photoaffinity probe DAP-BpB (4) designed
based on DAPT (3: N-[N-(3,5-difluorophenylacetyl)-L-
alanyl]-(S)-phenylglycine tert-butylester),6,7 our recent
studies have revealed that an analogue of 1 competi-
tively inhibits labeling of the C-terminal fragment of
PS1 (PS1-CTF) by DAP-BpB in a dose-dependent
manner.7 Our finding implies the possibility that the
arylsulfonamides and DAPT derivatives interact with
the same region of PS1-CTF. However, we cannot rule
out another possibility that the arylsulfonamides affect
allosterically the binding of DAPT to PS1-CTF. To
clearly address this complicated issue, we set out to
synthesize arylsulfonamide-based photoaffinity probes,
which will provide direct evidence for the molecular
target(s) and mode of action(s) of the arylsulfonamide
inhibitors.8 Herein we report our investigation of struc-
ture–activity relationships of 1 and related analogues,
and the design and synthesis of molecular probes based
on 1.
Keywords: Structure–activity relationships; Photoaffinity probes; Aryl-
sulfonamides; c-Secretase inhibitors.
*
Corresponding authors. At present address: Laboratory of Biostruc-
tural Chemistry, Graduate School of Life Sciences, Tohoku Univer-
sity, 1-1 Tsutsumidoori-Amamiya, Aoba-ku, Sendai 981-8555,
Japan. Tel.: +81 3 5841 4775; fax: +81 3 5841 4775; e-mail addresses:
hfuwa@bios.tohoku.ac.jp; natsu@mol.f.u-tokyo.ac.jp
ꢀ
Present address: School of Pharmaceutical Sciences, University of
Shizuoka, Yada, Suruga-ku, Shizuoka 422-8526, Japan.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.091

H. Fuwa et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4184–4189
4185
F
F
O
N
CO2H
Cl
C
A
B
O
H
Cl
F
N
O
N
N
F
N
H
O
O
S
S
O
F
O
O O
F
F
2
3: DAPT
1: BMS-299897
O
O
NH
HN
H
H
O
H
H
H
N
H
F
N
N
N
S
N
H
8
O
O
O
4: DAP-BpB
F
Figure 1. Structures of BMS-299897 (1), its analogue 2, DAPT (3), and DAP-BpB (4).
Table 1. Ab40 production inhibitory activity for compounds 2 and
10–12
To design probes maintaining sufficient potency, the
structure–activity relationships of the arylsulfonamide
inhibitors had to be first established. Since our prelimin-
ary attempts to modify 1 into the corresponding amide
derivatives resulted in complete loss of potency, com-
pound 2 was instead selected as a starting point. To
probe structure–activity relationships of the appendage
of the A ring,9 a series of analogues were investigated,
which could be prepared conveniently from 2-hydroxy-
acetophenone 5 (Scheme 1). Thus, Mitsunobu reaction10
of 5 with 3-bromopropanol followed by reduction affor-
ded alcohol 6. Sulfonamide 8 prepared from 2,5-difluo-
roaniline 7 was alkylated with 6 under the Mitsunobu
conditions to give rise to bromide 9. A set of side chain
modified analogues could be readily accessed from 9;
treatment of 9 with various amines led to the tertiary
amines 2 and 10–12. Their ability to inhibit Ab produc-
tion was evaluated by the cell-free in vitro assay using
O
X
Cl
F
N
S
O
O
F
Compound
X
Ab40 IC50 (lM)
N
2
10
11
2.1
2.0
4.4
N
N
O
recombinant C-terminal fragment of APP as
a
substrate,11 and the results are summarized in Table 1.
Tertiary amines 2 and 10–12 showed good inhibitory
12
1.0
N
OH
O
O
Br
activity against Ab40 production with IC50 values
around the micromolar range. In contrast, other deriva-
tives such as amides, nitriles, halides, azides and carbox-
ylic acids (not shown) were only weakly active or
inactive at 10 lM, suggesting the importance of a basic
nitrogen for potency and the difficulty of drastic struc-
tural modification such as installation of a cross-linking
group and/or a biotin tag at this position. Since the
pyrrolidine derivative 10 (previously described as
HF14057)7d was easily accessible from 5 in just five steps
with high overall yield and was almost equipotent to the
racemic 1 (IC50 0.86 lM),12 we selected 10 as a lead for
further examination of structure–activity relationships.
a,b
OH
6
5
Cl
H
F
NH2
F
N
c
S
O
O
F
F
8
7
O
N
R
O
N
Br
Cl
Cl
d
e
+
6
8
F
F
S
S
O
F
O
O O
F
We next investigated the effect of modification of the B
ring of 10. We elaborated compounds 13–19 from the
respective anilines by successive sulfonylation, Mitsun-
obu alkylation with alcohol 4, and treatment with
pyrrolidine (Scheme 2). Their inhibitory activities are
shown in Table 2. Interestingly, the unsubstituted
2, 10-12: R = amine
9
Scheme 1. Reagents and conditions: (a) 3-bromopropanol, PPh3,
DEAD, toluene, 60 °C; (b) NaBH4, MeOH/CH2Cl2, 0 °C, 50% (two
steps); (c) p-ClC6H4SO2Cl, pyridine, CH2Cl2, rt, 80%; (d) PPh3,
DEAD, toluene, 0 °C to rt, 74%; (e) secondary amine, rt, 76–95%.

4186
H. Fuwa et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4184–4189
that the modification of the C ring moiety induces loss
of activity.
O
N
Cl
NH2
b,c
a
H
Cl
N
S
O
With the preliminary structure–activity relationships in
mind, we set out on the design and synthesis of multi-
functional molecular probes based on arylsulfonamides.
We planned to utilize a benzophenone group as a pho-
tophore. To establish a suitable modification position
for the incorporation of a photophore, we planned to
synthesize five compounds (24–28), each of which has
a benzoyl group at the A or B ring (Fig. 2).
N
O
X
S
O
B
X
O
X
13-19
Scheme 2. Reagents and conditions: (a) p-ClC6H4SO2Cl, pyridine,
CH2Cl2, rt, 67–93%; (b) compound 6, PPh3, DEAD, toluene, 0 °C to
rt, 62–87%; (c) pyrrolidine, rt, 64–94%.
Table 2. Ab40 production inhibitory activity for compounds 10 and
13–19
Synthesis of compounds 24 and 25 was commenced with
commercially available acid 29 (Scheme 4). Conversion
of 29 to the corresponding amide 30 was followed by
Compound
X
Ab40 IC50 (lM)
Relative potencya
10
13
14
15
16
17
18
19
2,5-F2
o-Cl
m-Cl
2.0
1
4.0
3.8
1.5
4.3
3.8
1.8
200
0.49
0.53
1.3
p-Cl
o-OMe
m-OMe
p-OMe
H
0.47
0.53
1.1
O
N
N
O
N
N
O
Cl
Cl
0.01
F
a Values are calculated by dividing the IC50 value of compound 10 with
that of each compound.
S
S
O O
O O
F
X
26: X = o-COPh
27: X = m-COPh
28: X = p-COPh
derivative 19 displayed dramatically enhanced inhibito-
ry potency, being approximately 100-fold active com-
pared to the parent 10. Also noteworthy is that
compounds 13–18 exhibited potent inhibitory activity,
suggesting that the modification of the substituents of
the B ring would be well tolerated.
24: X = F
25: X = H
Figure 2. Structures of benzophenone-embedded analogues 24–28.
OMe
OMe
N
N
HO2C
OH
Structure–activity relationships of the C ring substituent
were briefly investigated. Alcohol 6 could readily be
alkylated with a series of sulfonamides under the stan-
dard Mitsunobu conditions, and ensuing treatment with
pyrrolidine afforded compounds 20–23 (Scheme 3).
Their inhibitory activities were found to be 10- to 100-
fold less potent than that of 19 (Table 3). It seems likely
c
b
a
OH
OH
O
O
NO2
NO2
NH2
29
OMe
30
31
OMe
OMe
N
O
N
N
OH
I
OH
e
d
O
Br
O
O
32
SiMe3
OMe
SiMe3
33
34
O
N
X
OMe
N
NH2
N
a
H
b,c
X
O
Br
g
O
O
Br
N
h
f
O
O
S
C
N
O
O
S
O
O
OH
36
35
20-23
Scheme 3. Reagents and conditions: (a) arylsulfonyl chloride, pyri-
dine, CH2Cl2, rt, 82–100%; (b) compound 6, PPh3, DEAD, toluene,
0 °C to rt, 46–60%; (c) pyrrolidine, rt, 64–86%.
O
Br
O
N
i,j
O
O
24: X = F
25: X = H
Cl
OH
X
N
S
O
X
37
Table 3. Ab40 production inhibitory activity for compounds 19–23
O
Compound
X
Ab40 IC50 (lM)
Relative potencya
Scheme 4. Reagents and conditions: (a) MeONHMeÆHCl, EDCIÆHCl,
HOBtÆH2O, Et3N, CH2Cl2, rt, 90%; (b) H2, 5% Pd/C, MeOH, rt, 99%;
(c) NaNO2, aq HCl, 0 °C then KI, rt, 68%; (d) trimethylsilylacetylene,
PdCl2(PPh3)2, PPh3, CuI, Et3N, THF, rt, 98%; (e) 3-bromopropanol,
PPh3, DEAD, toluene, 0 °C to rt, 89%; (f) HCO2H, 60 °C, 70%; (g)
NaBH4, MeOH, 0 °C, 97%; (h) PhMgBr, THF, À78 °C to rt, 98%; (i)
compound 8 or N-phenyl-p-chlorobenzenesulfonamide, PPh3, DEAD,
toluene, 0 °C to rt; (j) pyrrolidine, rt, 80% (24), 78% (25).
19
20
21
22
23
Cl
F
0.01
0.35
0.40
1.9
1
0.03
0.03
0.005
0.01
CF3
Br
H
0.69
a Values are calculated by dividing the IC50 value of compound 19 with
that of each compound.

H. Fuwa et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4184–4189
4187
reduction of the nitro group to give amine 31, which was
Cl
H
transformed to the iodide 32 via diazotization. Under
modified Sonogashira conditions,13,14 iodide 32 was
coupled with trimethylsilylacetylene to afford the cou-
pling product 33. Mitsunobu alkylation of 33 with 3-
bromopropanol led to alkyne 34, which was hydrated
with formic acid to furnish acetophenone 35. Reduction
of 35 with NaBH4 afforded alcohol 36, which was react-
ed with PhMgBr to give rise to alcohol 37. Mitsunobu
coupling of 37 with appropriate sulfonamide followed
by treatment with pyrrolidine furnished 24 and 25. On
the other hand, compounds 26–28 were prepared from
the respective aminobenzophenones as described for
the other B ring analogues. Evaluation of these benzo-
phenone analogues revealed that incorporation of a ben-
zoyl group to the A ring was unexpectedly favorable for
increasing potency, as compound 24 displayed ca. 10-
fold potent inhibitory activity compared to the parent
10 (IC50 0.13 lM), while the B ring modified analogues
26–28 were almost equipotent to 10 (Table 4). Interest-
ingly, in the case of these benzophenone analogues,
elimination of two fluorine atoms from the B ring was
detrimental for potency. Therefore, incorporation of
(+)-biotin as a reporter group to 24 was next explored.
F
NO2
F
F
NO2
F
a
b,c
F
N
S
O
O
F
O
HO
O
CO2t-Bu
38
CO2t-Bu
40
39
Cl
O
Br
O
H
F
N
Cl
S
O
g,h
f
d,e
O
F
N
S
O
F
O
F
O
41
CO2allyl
O
42
CO2allyl
O
N
N
O
N
Br
O
O
Cl
Cl
i,j
F
F
S
S
O
O
F
O
O
F
O
O
O
NH
H
HN
O
H
CO2pfp
43
H
HN
N
S
We designed two types of biotinylated photoaffinity
probes, in which (+)-biotin was attached at the B ring
(Scheme 5, 44a,b) or at the end of the benzoyl group
(Scheme 6, 49a,b). The synthesis of 44a,b is illustrated in
Scheme 5. The known phenol 3815 was alkylated with
tert-butyl bromoacetate to give 39. The nitro group was
reduced and subsequent sulfonylation of the derived aro-
matic amine furnished sulfonamide 40. At this stage, the
tert-butyl group was removed and the resultant acid was
converted to allyl ester 41. Mitsunobu alkylation of 41
with 37 afforded the coupling product 42. Deprotection
of the allyl ester of 42 [Pd(PPh3)4/pyrrolidine] was fol-
lowed by condensation with pentafluorophenol (PfpOH)
to afford the activated pfp ester 43. Coupling of 43 with
appropriate biotinylated amines16,17 followed by treat-
ment with pyrrolidine afforded the targeted probes 44a,b.
n
O
44a: n = 1
44b: n = 4
Scheme 5. Reagents and conditions: (a) BrCH2CO2t-Bu, K2CO3,
DMF, rt, 96%; (b) H2, 5% Pd/C, MeOH, rt; (c) p-ClC6H4SO2Cl,
pyridine, CH2Cl2, rt, 86%; (d) TFA, CH2Cl2, 0 °C to rt; (e) allyl
alcohol, EDCIÆHCl, HOBtÆH2O, Et3N, DMF/THF, rt, 85%; (f)
compound 37, PPh3, DEAD, toluene, 0 °C to rt, 81%; (g) Pd(PPh3)4,
pyrrolidine, THF, rt; (h) pentafluorophenol, EDCIÆHCl, CH2Cl2, rt,
54%; (i) (5-biotinamido)pentylamine or (8-biotinamido)octylamine,
Et3N, DMF, rt; (j) pyrrolidine, rt, 35% for 44a (two steps), 33% for 44b
(two steps).
OTBS
OTBS
OTBS
a
b
36
On the other hand, the synthesis of 49a,b is summarized in
Scheme 6. The benzophenone moiety was efficiently con-
structed by treatment of iodoarene 4518 with i-PrMgBr19
followed by coupling with the Weinreb amide 36 to fur-
nish benzophenone 46 in 71% yield. After coupling with
8 under Mitsunobu conditions to give 47, the silyl group
was removed by aqueous HF to afford 48. Activation of
the resultant hydroxyl group as the corresponding mixed
O
Br
O
MgBr
I
45
O
46
OH
OP
NH
HN
H
H
H
H
O
N
N
S
n
3
O
O
O
Br
O
d,e,f
Cl
O
N
Table 4. Ab40 production inhibitory activity for compounds 10 and
24–28
F
N
F
O
S
Cl
O
O
Compound
Ab40 IC50 (lM)
Relative potencya
F
N
F
S
49a: n = 3
49b: n = 6
47: P = TBS
48: P = H
c
10
24
25
26
27
28
2.0
0.13
17.0
1.2
1
15
O
O
0.1
1.7
0.7
0.6
Scheme 6. Reagents and conditions: (a) i-PrMgBr, THF, À20 °C; then
36, À78 to 0 °C, 71%; (b) compound 8, PPh3, DEAD, toluene, 0 °C to
rt, 82%; (c) HF, CH3CN/H2O, 0 °C, 95%; (d) p-NO2C6H4OCOCl,
pyridine, THF/CH3CN, rt, 97%; (e) (5-biotinamido)pentylamine or
(8-biotinamido)octylamine, Et3N, DMF, rt; (f) pyrrolidine, rt, 61%
for 49a (two steps), 53% for 49b (two steps).
3.0
3.2
a Values are calculated by dividing the IC50 value of compound 10 with
that of each compound.

4188
H. Fuwa et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4184–4189
3. (a) Smith, D. W.; Munoz, B.; Srinivasan, K.; Bergstrom,
C. P.; Chaturvedula, P. V.; Deshpande, M. S.; Keavy, D.
J.; Lau, W. Y.; Parker, M. F.; Sloan, C. P.; Wallace, O. B.;
Wang, H. H. PCT Int. Appl. WO0050391; (b) Lewis, S. J.;
Smith, A. L.; Neduvelil, J. G.; Stevenson, G. I.; Lindon,
M. J.; Jones, A. B.; Shearman, M. S.; Beher, D.; Clarke,
E.; Best, J. D.; Peachey, J. E.; Harrison, T.; Castro, J. L.
Bioorg. Med. Chem. Lett. 2005, 15, 373; (c) Rishton, G.
M.; Retz, D. M.; Tempest, P. A.; Novotny, J.; Kahn, S.;
Treanor, J. J. S.; Lile, J. D.; Citron, M. J. Med. Chem.
2000, 43, 2297.
Table 5. Ab40 production inhibitory activity for compounds 10, 44a,b,
and 49a,b
Compound
Ab40 IC50 (lM)
Relative potencya
10
2.0
0.01
1
200
100
690
328
44a
44b
49a
49b
0.02
0.0029
0.0061
a Values are calculated by dividing the IC50 value of compound 10 with
that of each compound.
4. (a) Barten, D. M.; Guss, V. L.; Corsa, J. A.; Loo, A.;
Hansel, S. B.; Zheng, M.; Munoz, B.; Srinivasan, K.;
Wang, B.; Robertson, B. J.; Polson, C. T.; Wang, J.;
Roberts, S. B.; Hendrick, J. P.; Anderson, J. J.; Loy, J. K.;
Denton, R.; Verdoorn, T. A.; Smith, D. W.; Felsenstein,
K. M. J. Pharmacol. Exp. Ther. 2005, 312, 635; (b)
Anderson, J. J.; Holtz, G.; Baskin, P. P.; Turner, M.;
Rowe, B.; Wang, B.; Kounnas, M. Z.; Lamb, B. T.;
Barten, D.; Felsenstein, K.; McDonald, I.; Srinivasan, K.;
Munoz, B.; Wagner, S. L. Biochem. Pharmacol. 2005, 69,
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Brown, L.; Pognan, F.; Gadient, R.; Jacobs, R. T.; Zacco,
A.; Greenberg, B.; Ciaccio, P. J. Toxicol. Sci. 2004, 82,
341.
carbonate (p-nitrophenyl chloroformate, and pyridine),
coupling with appropriate biotinylated amine, and ensu-
ing displacement of the bromide with pyrrolidine fur-
nished the targeted probe 49a,b.
The biotinylated photoaffinity probes 44a,b and 49a,b
thus generated were evaluated and the results are sum-
marized in Table 5. To our delight, all compounds dis-
played significant inhibitory activities against Ab40
production, suggesting that these photoprobes maintain
sufficient affinity toward c-secretase.
5. (a) Hadland, B. K.; Manley, N. R.; Su, D.-M.; Longmore,
G. D.; Moore, C. L.; Wolfe, M. S.; Schroeter, E. H.;
Kopan, R. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7487;
(b) Doerfler, P.; Shearman, M. S.; Perlmutter, R. M. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 9312; (c) Geling, A.;
Steiner, H.; Willem, M.; Bally-Cuif, L.; Haass, C. EMBO
Rep. 2002, 3, 688; (d) Searfoss, G. H.; Jordan, W. H.;
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In conclusion, we have investigated structure–activity
relationships of an arylsulfonamide class of c-secretase
inhibitors. Examination of the appendage of the A ring re-
vealed that the readily accessible tertiary amine deriva-
tives (2 and 10–12) were almost equipotent to the parent
compound 1. Modification of the substituents of the B
ring was well tolerated, but replacement of the chlorine
atom of the C ring with another group was detrimental
to potency. Introduction of a benzoyl group to the appro-
priate position of the A ring was unexpectedly favorable
for increasing potency. The preliminary structure–activi-
ty relationships led us to the design and synthesis of bio-
tinylated photoaffinity probes 44a,b and 49a,b that were
proved to display excellent inhibitory activity against
Ab production in our cell-free in vitro assay. Photoaffinity
labeling experiment using 44a,b and 49a,b to elucidate
molecular target(s) of arylsulfonamide c-secretase inhibi-
tors is ongoing in our laboratories and the results will be
reported shortly.
´
6. (a) Dorman, G.; Prestwich, G. D. Biochemistry 1994, 33,
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Angew. Chem., Int. Ed. Engl. 1995, 34, 1296; (c) Dorman,
G.; Prestwich, G. D. Trends Biotechnol. 2000, 18, 64.
7. For relevant reports from our laboratories, see: (a) Kan,
T.; Tominari, Y.; Morohashi, Y.; Natsugari, H.; Tomita,
T.; Iwatsubo, T.; Fukuyama, T. Chem. Commun. 2003,
2244; (b) Fuwa, H.; Okamura, Y.; Morohashi, Y.; Tomita,
T.; Iwatsubo, T.; Kan, T.; Fukuyama, T.; Natsugari, H.
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Acknowledgments
This work was financially supported in part by 21st cen-
tury COE program, by Scientific Research on Priority
Areas ‘Creation of Biologically Functional Molecules’
from the Ministry of Education, Culture, Sports, Sci-
ence and Technology (MEXT), and by the Program
for Promotion of Fundamental Studies in Health Sci-
ences of the National Institute of Biomedical Innovation
(NIBIO), Japan. Y.T. is a research fellow of Japan Soci-
ety for the Promotion of Science (JSPS). Continuous
support by Takeda Pharmaceutical Company, Ltd is
gratefully acknowledged.
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