(S)-N-(5-Chlorothiophene-2-sulfonyl)-β,β-diethylalaninol a Notch-1-sparing γ-secretase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2008.11.116

Accumulation of beta-amyloid (Aβ), produced by the proteolytic cleavage of amyloid precursor protein (APP) by β- and γ-secretase, is widely believed to be associated with Alzheimer's disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) γ-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-β,β-diethylalaninol 7.b.2 (Aβ _40/42 EC₅₀ = 28 nM), which is efficacious in reduction of Aβ production in vivo.

Full text of DOI:10.1016/j.bmcl.2008.11.116

Bioorganic & Medicinal Chemistry Letters 19 (2009) 926–929
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
(S)-N-(5-Chlorothiophene-2-sulfonyl)-b,b-diethylalaninol a Notch-1-sparing
c-secretase inhibitor
a
b
b
c
Derek C. Cole a, , Joseph R. Stock , Anthony F. Kreft , Madelene Antane , Suzan H. Aschmies ,
Kevin P. Atchison c, David S. Casebier f, Thomas A. Comery c, George Diamantidis b, John W. Ellingboe a,
Boyd L. Harrison b, Yun Hu c, Mei Jin c, Dennis M. Kubrak b, Peimin Lu c, Charles W. Mann b,
Robert L. Martone c, William J. Moore e, Aram Oganesian d, David R. Riddell c, June Sonnenberg-Reines c,
Shaiu-Ching Sun c, Erik Wagner c, Zheng Wang b, Kevin R. Woller f, Zheng Xu c,
Hua Zhou c, J. Steven Jacobsen c
*
a Chemical and Screening Sciences, Wyeth Research, Pearl River, NY 10965, USA
b Chemical and Screening Sciences, Wyeth Research, CN 8000, Princeton, NJ 08543, USA
c Discovery Neuroscience, Wyeth Research, CN 8000, Princeton, NJ 08543, USA
d Drug Safety and Metabolism, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
e Chemical and Screening Science, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
f ArQule, Inc., 19 Presidential Way, Woburn, MA 01801, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Accumulation of beta-amyloid (Ab), produced by the proteolytic cleavage of amyloid precursor protein
Received 2 September 2008
Revised 24 November 2008
Accepted 26 November 2008
Available online 6 December 2008
(APP) by b- and
around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification
of the Notch-1-sparing (9.5-fold) -secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-b,b-dieth-
ylalaninol 7.b.2 (Ab
c-secretase, is widely believed to be associated with Alzheimer’s disease (AD). Research
c
EC50 = 28 nM), which is efficacious in reduction of Ab production in vivo.
40/42
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Alzheimer’s disease
c
-Secretase inhibitor
Sulfonamide
Amyloid b-peptide (b-amyloid, Ab) plaques found in the brains
of Alzheimer’s disease (AD) patients are formed by aggregation of
Ab peptide, produced by sequential cleavage of amyloid precursor
protein (APP), by two aspartic proteases. Initial cleavage by
b-secretase (BACE-1) generates the membrane bound b-C-terminal
fragment (b-CTF or C99). Subsequent cleavage within the trans-
entered clinical trials. However,
modulate the processing of other
c
c
-secretase inhibitors which also
-secretase substrates, in partic-
ular Notch-1, have been shown to alter cell differentiation in tis-
sues and cause goblet cell hyperplasia in intestinal epithelium.9
membrane region by c-secretase, a complex of proteins including,
presenilin-1 (PS1), nicastrin, anterior parynx defective-1 (Aph-1),
and presenilin enhancer-2 (Pen-2), results in formation of Ab frag-
ments, predominantly Ab40 and the more aggregatory Ab42.1 Meth-
ods of lowering Ab levels in brain are currently sought as novel
disease modifying therapies for treatment of AD.2 Despite the mul-
titude of X-ray crystal co-structures determined for BACE-1 and its
inhibitors, it has proven difficult to obtain potent inhibitors with
F
CO2H
O
OH
Cl
H
N
N
N
H
F
N
S
O
O
O
O
F
BMS-299897
LY-450139
suitable physical properties to allow brain penetration.3
c-Secre-
tase on the other hand has proven more amenable to discovery
of inhibitors and BMS-299897,4,5 LY4501396,7 and MK-07528 have
O
O
O
O
S
OH
S
OH
S
N
H
N
Cl
H
* Corresponding author. Tel.: +1 845 602 4619; fax: +1 845 602 5561.
E-mail address: coledc@wyeth.com (D.C. Cole).
Cl
1a
7.b.2
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.116

D. C. Cole et al. / Bioorg. Med. Chem. Lett. 19 (2009) 926–929
927
High-throughput screening of Wyeth and ArQule compound
R
R
a, b
O
O
collections using a cell based Ab40/42 lowering assay, which uses
a sandwich ELISA with 6E10 antibody for Ab capture and Ab40
and Ab42 C-terminal specific antibodies for detection10 led to the
identification of (S)-4-chlorophenylsulfonyl isoleucinol 1a.11 Com-
pound 1a inhibits formation of both Ab40 and Ab42 almost equally
S
OH
S
OH
N
H2N
Cl
H
O
Scheme 2. Reagents and conditions: (a) LiAlH4, THF, 60 °C; (b) 5-chlorothiophene-
2-sulfonyl chloride, Et3N, THF.
(average Ab40/42 EC50 = 5.5
lM) while increasing b-CTF in a dose
dependent manner without effecting APP, consistent with a
c
-
secretase inhibition mechanism.12 Initial research of these leads
by varying substitution on the benzene ring led to small increases
in potency.11 Methylation of the nitrogen (NH to N-Me) and the
alcohol (OH to OMe), replacement of the sulfonamide with amide,
urea and amine linkers all led to loss of activity, while conversion
of the primary alcohol to secondary and tertiary alcohols also de-
creased activity.11
the amino alcohol had significantly lower activity, while the (
valinol analog 2b with smaller methyl groups at the b-position
was approximately 5-fold less active (EC50 = 820 nM) and the (
tert-butyl glycinol analog 2c was 45-times less potent. The (
allo-isoleucinol analog 2d with opposite (R)-stereochemistry at
the b-center had similar activity to 1f. Taken together these results
suggest a requirement for two alkyl substitutents at the amino
alcohol b-center, with chirality of this center having little effect.
L)-
L
)-
)-
L
The focus of this manuscript is on the continued research on
In contrast, the (
at the -center is much less active (EC50 > 7.6
strict requirement for the correct (S)-stereochemistry at the
D
)-isoleucinol analog 2e with (R)-stereochemistry
M) indicating the
-cen-
-center led to a slight increase
this
c-secretase inhibitor by varying the sulfonamide aryl group
a
l
and the amino alcohol side chain. The initial array was prepared
by reaction of sulfonyl chlorides with (S)-isoleucinol (Scheme 1).
The 4-bromophenyl analog 1b was about 2-fold more active, while
the unsubstituted phenyl analog 1c had equivalent potency (Table
1). While, the unsubstituted thiophen-2-yl analog 1d had compa-
rable activity to the phenyl analog 1c, the 5-substituted thio-
phene-2-sulfonyl amides 1e, 1f and 1g were 5- to 10-fold more
potent than the corresponding 4-substituted phenyl analogs (1a
and 1b). Additional substitution on the thiophene ring in the 3-
or 4-position as in 1h and 1i led to at least 6-fold loss in potency.
The 5-chlorofuran-2-yl analog (1j) was considerably less active, as
were other more polar aryl ring systems and aliphatic sulfona-
mides (data not shown).
a
ter. Additional substitution at the
a
in potency, compare 2b and 2f.
In order to gain a more thorough understanding of the require-
ment for two substituent at the b-center while maintaining the
center in the S-configuration, we used the asymmetric amino acid
synthesis protocol developed by Hruby to prepare an array of b-
branched amino alcohols (Scheme 3).13 Cinnamic acids were cou-
pled with (R)-4-benzyl-2-oxazolidinone to give the alkenoyloxaz-
olidin-2-one products 3. Cuprate reagents prepared in situ by
addition of Grignard reagents to copper bromide dimethylsulfide
under carefully controlled temperature conditions, underwent Mi-
a
-
In order to vary the amino alcohol portion of the molecule 5-
chlorothiophene-2-sulfonyl chloride was reacted with a set of ami-
no alcohols, prepared by lithium aluminum hydride reduction of
Table 2
Mean Ab40/42 EC50 values for 5-chlorothiophene-2-sulfonyl amino alcohol derivatives
the corresponding amino acids (Scheme 2). Compared to the (
isoleucinol analog (1f; EC50 = 170 nM, Table 1) the ( )-leucinol ami-
no alcohol (2a, Table 2) with a single substituent on the b-carbon of
L)-
L
R
O
O
S
OH
S
N
Cl
H
Compound
Amino alcohol
Amino acid
EC50 (nM)a
>30,000
O
O
a
OH
S
OH
2a
L
L
L
L
-Leucine
H2N
Ar
N
H
OH
H2N
Scheme 1. Reagents: (a) ArSO2Cl, Et3N, THF.
2b
2c
2d
2e
-Valine
820
7500
140
OH
H2N
Table 1
Mean Ab40/42 EC50 values for arylsulfonyl (S)-isoleucinol derivatives
-tert-Leucine
-allo-Isoleucine
OH
H2N
O
O
S
OH
Ar
N
H
a
OH
Compound
Ar
EC50 (nM)
H2N
1a
1b
1c
1d
1e
1f
1g
1h
1i
4-Chlorophenyl-
4-Bromophenyl-
Phenyl-
5500
2200
4700
7000
460
170
160
1050
>30,000
>30,000
D
-Isoleucine
7600
490
Thiophen-2-yl-
OH
5-Fluorothiophen-2-yl-
5-Chlorothiophen-2-yl-
5-Bromothiophen-2-yl-
4,5-Dichlorothiophen-2-yl-
3-Bromo-5-chlorothiophen-2-yl-
5-Chlorofuran-2-yl-
H2N
2f
L-a-Methyl-valine
OH
H2N
1j
a
a
Values are the average of Ab40 and Ab42 inhibition (typically with <30%
Values are the average of Ab40 and Ab42 inhibition (typically with <30%
difference).
difference).

928
D. C. Cole et al. / Bioorg. Med. Chem. Lett. 19 (2009) 926–929
Table 4
O
O
O
O
Mean Ab40/42 EC50 values for cyclic aminoalcohol replacements
a
b
Ra-h
Ra-h
d
OH
Ra-h
N
O
R
O
O
S
OH
S
N
Bn
Cl
3
H
R1-7
R1-7
Ra-h
O
O
O
c
Compound Amino alcohol
Amino acid
EC50
(nM)a
O
O
N
N
Br
N3
Bn
Bn
8a
8b
L
-Cyclopentylglycine
-Cyclohexylglycine
580
4
5
Ra-h R1-7
OH
OH
Ra-h R1-7
OH
H2N
H2N
O
O
e
OH
S
S
N
H
Cl
H2N
L
820
7
6
H
Scheme 3. Reagents and conditions: (a) i—(R)-4-benzyl-2-oxazolidinone,
(CH3)3CCOCl, Et3N, THF, (ii) n-BuLi, THF; (b) i—R1-7MgBr, CuBr.DMS, THF ꢁ40 to
ꢁ15 °C; ii—NBS, ꢁ78 °C; (c) N,N,N0,N0-tetramethylguanidinium azide, ACN, rt; (d)
LiAlH4, THF, 60 °C; (e) 5-chlorothiophene-2-sulfonyl chloride, Et3N, THF.
N
8c
(S)-2-Amino-2-(piperidin-4-yl)acetic
>30,000
acid
OH
H2N
chael addition and the anion was trapped with N-bromosuccini-
mide to give the a
-bromo derivatives 4.14 Displacement of the bro-
a
Values are the average of Ab40 and Ab42 inhibition (typically with <30% differ-
ence) and mean of three experiments for each protein with a standard deviation of
<20%.
mide with N,N,N0,N0-tetramethylguanidinium azide yielded the
corresponding azides 5.13 Simultaneous reduction of amide and
azide moieties by treatment with lithium aluminum hydride
yielded the corresponding amino alcohols 6, which were reacted
with 5-chlorothiophene-2-sulfonyl chloride to give the desired 5-
chlorothiophene-2-sulfonyl amino alcohols 7.
The b,b-diethyl analog 7.b.2 was the most active (EC50 = 28 nM,
Table 3). While one larger alkyl group was tolerated especially in
the pro-S side chain, for example, compare 7.b.6 versus 7.g.2.
The presence of a phenyl (see 7.c.7 or 7.h.1) or two larger alkyl
substituents (see 7.e.4) was detrimental to activity.
110
100
90
80
70
60
Compound 7.b.2 was further profiled for selectivity in a Notch-1
cleavage assay12 and found to have an EC50 = 266 nM or 9.5-fold
selectivity ratio between b-CTF and Notch-1 cleavage. Compound
7.b.2 had good solubility (37 ng/mL at pH 7.4) permeability
(13 ꢀ 10ꢁ6 m/s at pH 7.4) and did not inhibit the cytochrome
50
40
0
5
10
15
20
Dose (mg/kg)
P450 enzymes (2C9, 3A4, 2D6) at 50 lM. Unfortunately, 7.b.2 was
Figure 1. Reduction of Ab40 (N) and Ab42 (j) levels in Tg2576 mouse by 7.b.2 dosed
rapidly metabolized in rat, mouse and human liver microsomes
with half-life of 1, 2 and 8 min, respectively, due to rapid hydrox-
ylation of the methyl groups.
sub-cutaneously.
In an attempt to prevent rapid metabolism of the alkyl chains, a
set of 5-chlorothiophene-2-sulfonyl amino alcohols with cyclic
side chains were prepared using the procedure outlined in Scheme
2. However, all these amino alcohol derivatives had significantly
weaker activity relative to the b,b-diethyl analog 7.b.2. The cyclo-
pentyl and cyclohexylglycinol analogs 8a and 8b had EC50 = 580
and 820 nM, respectively (Table 4), while the 4-piperidine glycinol
Table 3
Mean Ab40/42 EC50 values for 5-chlorothiophene-2-sulfonyl b,b-disubstituted amino
alcohol derivatives
Ra-h R1-7
O
O
S
OH
S
analog 8c was greater than 30 lM.
N
Cl
H
Poor oral bioavailability of 7.b.2 precluded oral testing, however
when dosed 20 mg/kg sub-cutaneously in Tg2576 mouse 7.b.2 led
to a 55% reduction in brain Ab40 levels (Fig. 1). Sub-chronic dosing
EC50 (nM)a R1 = Me R2 = Et R3 = n-
R4 = n-
R5 = i-
R6 = n-
Hexyl
R7 = Ph
Pr
Bu
Bu
of 1 mg/kg sub-cutaneously for 5 days resulted in 25 and 15%
R
R
a = Me
b = Et
115 212
28 43
253 826
71
12
reduction in brain Ab40 and Ab42
.
28
29
In summary, a series of 5-chlorothiophene-2-sulfonylamido
alcohols were synthesized based on the HTS hit 1a. Research vary-
ing the aryl and aminoalcohol moieties of the series using iterative
parallel synthesis lead to 7.b.2, with 28 nM cellular potency in the
Rc = n-Pr
103
264
111
4055
R
R
d = i-Pr
e = n-Bu
4180
Rf = n-
72
20,708 >30,000
>30,000
Pentyl
c-secretase inhibition assay. We have demonstrated the require-
Rg = n-
429
ments for (S)-configuration at the -center and bis-substitution
a
Hexyl
Rh = Ph
3906
>
with small alkyl groups at the b-center. Despite its poor oral PK,
7.b.2 was efficacious in Ab lowering experiments in vivo. Future
publications will focus on more detailed in vivo characterization
of 7.b.2 and on improving the microsomal stability and oral PK
30,000
a
Values are the average of Ab40 and Ab42 inhibition (typically with <30% differ-
ence) and mean of three experiments for each protein with a standard deviation of
<20%.
of this very attractive series of c-secretase inhibitors.

D. C. Cole et al. / Bioorg. Med. Chem. Lett. 19 (2009) 926–929
929
D.; Lu, P.; Lundquist, J.; Mann, C.; Martone, R. L.; Moore, W.; Oganessian, A.;
Porte, A.; Riddell, D.; Sonnenberg-Reines, J.; Stock, J. R.; Sun, S.-C.; Wagner, E.;
Woller, K.; Xu, Z.; Zhou, H.; Jacobsen, J. S. Bioorg. Med. Chem. Lett. 2008, 18,
4232.
References and notes
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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.
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Ereshefsky, L.; Solomon, R.; Winchell, G.; Shearman, M.; Murphy, M.;
Gottesdiener, K. Abstract of Papers, 10th International Conference on
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14. Representative procedure for synthesis of (R)-4-benzyl-3-(2-bromo-3-ethyl-
pentanoyl)-oxazolidin-2-one. To CuBr.DMS complex (34.5 g, 168 mmol,
1.2 equiv) in THF/DMS (2:1, 2 L), cooled to ꢁ40 °C was added EtMgBr
(112 mL 3 M solution in THF, 336 mmol, 2.4 equiv). The solution was
allowed to warm to ꢁ15 °C, stirred for 15 min, then re-cooled to ꢁ40 °C.
(Note: it is important that the temperature does not go above ꢁ10 °C). A
solution
of
(R)-4-benzyl-3-pent-2-enoyl-oxazolidin-2-one
(36.27 g,
140 mmol) in THF (280 mL) was added. The solution was allowed to
warm to room temperature and stirred for 8–16 h. The solution was re-
cooled to ꢁ78 °C and
1.2 equiv) in THF (280 mL) was added. The solution was allowed to warm to
0 °C and stirred at 0 °C for 3 h, then quenched with 1:1 solution of
a slurry of N-bromosuccinimide (31 g, 168 mmol,
a
saturated ammonium carbonate and 0.5 N potassium bisulfate (1 L). The
organic phase was decanted off, dried and concentrated. The product was
purified by flash chromatography on silica gel with 20% ethyl acetate in
hexanes to give (2R,4R)-4-benzyl-3-(2-bromo-3-ethyl-pentanoyl)-oxazolidin-
2-one (29.5 g) as slightly green oil almost exclusively as a single isomer by
NMR.
11. Kreft, A. F.; Harrison, B.; Aschmies, S.; Atchison, K. P.; Casebier, D.; Cole, D. C.;
Diamantidis, G.; Ellingboe, J. W.; Hauze, D.; Hu, Y.; Huryn, D.; Jin, M.; Kubrak,