High affinity, bioavailable 3-amino-1,4-benzodiazepine-based γ-secretase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.07.031

In this paper, we describe the development of a novel series of high affinity, orally bioavailable 3-amino-1,4 benzodiazepine-based γ-secretase inhibitors for the potential treatment of Alzheimer's disease. We disclose structure-activity relationships based around the 1, 3 and 5 positions of the benzodiazepine core structure.

Full text of DOI:10.1016/j.bmcl.2003.07.031

Bioorganic& Medicinal Chemistry Letters 13 (2003) 4143–4145
High Affinity, Bioavailable 3-Amino-1,4-benzodiazepine-Based
ꢀ-Secretase Inhibitors
Andrew P. Owens,a,* Alan Nadin,a Adam C. Talbot,a Earl E. Clarke,b
Timothy Harrison,a Huw D. Lewis,b Michael Reilly,a Jonathan D. J. Wrigleyb
and Jose L. Castroa
aDepartment of Medicinal Chemistry, Merck Sharp & Dohme Research Laboratories, The Neuroscience Research Centre,
Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK
bDepartment of Biochemistry and Molecular Biology, Merck Sharp & Dohme Research Laboratories,
The Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK
Received 13 May 2003; revised 10 June 2003; accepted 4 July 2003
Abstract—In this paper, we describe the development of a novel series of high affinity, orally bioavailable 3-amino-1,4 benzodi-
azepine-based ꢀ-secretase inhibitors for the potential treatment of Alzheimer’s disease. We disclose structure–activity relationships
based around the 1, 3 and 5 positions of the benzodiazepine core structure.
# 2003 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a common neurodegenera-
tive disease that is clinically characterized by a pro-
gressive cognitive and memory decline. It has been
thought for some time that the deposition and accumu-
lation of amyloid-b (Ab) peptides into extracellular
proteinaceous plaques may be responsible for this
neurodegeneration.1 These Ab peptides are produced by
sequential cleavage of membrane-bound amyloid-b pre-
cursor protein (bAPP) by two proteases known as b-
and g-secretases. One particularly attractive approach
to the treatment of AD may therefore be to inhibit
either b- or g-secretase, thereby reducing the formation
of Ab peptides and consequently their aggregation into
amyloid plaques. Based on this hypothesis, we
embarked on a g-secretase inhibitor discovery program
to find an orally bioavailable, high potency compound.
Recently, we reported identification of a novel series of
ꢀ-secretase inhibitors based on the screening lead 1 and
described some initial structure–activity relationships
(SARs) based on changes to the aromatic ring and car-
bon backbone of the hydrocinnamoyl C3 side chain.2
The original synthesis of 1 involved a 16-step synth-
esis2,3 which was not ideal for the preparation of analo-
gues to investigate SARs. A recent publication from our
laboratories4 has described a novel strategy for the
synthesis of 3-amino-1,4-benzodiazepines based on
reactions of the chloroimidate (imidoyl chloride) 2,
thereby allowing convergent construction of structures
such as 1 from a central intermediate. Herein, we
describe an improved synthesis of 1 and present the in
vitro potencies of a range of analogues at the C5, C3,
and N1 positions.
A new route for the synthesis of 1 is shown in Scheme 1.
Methylation of 2 using sodium hydride and methyl
iodide in dimethylformamide gave 3 in excellent yield.
Palladium-catalyzed cross-coupling of 3 with 4-carbox-
amidophenyl boronicaidc under standard aqueous
conditions gave 4. Removal of the BOC protecting
group with trifluoroacetic acid and acylation with 3-
(2,4-dichlorophenyl)propionic acid gave 1 (IC50 33
nM).2,5 A range of homochiral 2-(S)-methyl 3-phenyl-
propionicaicds (prepared via benzylation of Evans’
homochiral
were reacted with the amine derived from 4. It was then
4-benzyl-3-propionyl-2-oxazolidinones)6
*Corresponding author. Tel.: +44-1279-440000; fax: +44-1279-
440390; e-mail: andrew_owens@merck.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.07.031

4144
A. P. Owens et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4143–4145
Table 1. Structure–activity relationships of C5 substituted enzodi-
azepines
Entry
Ar
IC50 (nM)
Entry
Ar
IC50 (nM)
7a
0.3
7f
2.2
7b
7c
7d
7e
188.4
7g
7h
7i
1.2
4.7
0.2
100.6
13.2
28.0
3.6
7j
Scheme 1. (i) ICH3, NaH, DMF, 87%; (ii) Pd(PPh3)4, Na2CO3, H2O–
DME, 4-(CONH2)–C6H4B(OH)2, 62%; (iii) TFA, DCM, 100%;
(iv) EDC, HOBT, RCO2H, separation of diastereomers, 70%;
(v) Pd(PPh3)4, Na2CO3, H2O–DME, ArB(OH)2 or ArB(–OCMe2-
CMe2O–), 52–93%.
This study revealed some interesting SARs. For exam-
ple, tethering the carboxamide to the phenyl ring resul-
ted in identification of 7a and 7c, compounds with
comparable in vitro potency to 5, whereas the isomer 7d
was much less potent. Compound 7a was evaluated in
vivo, but was found to have very low exposure in rat
possible to separate the mixtures into homochiral single
diastereomers by flash silica chromatography.7 Incor-
poration of the homochiral 2-(S)-methyl-3-(3,4-
difluorophenyl)propionic acid side chain, identified in a
previous publication,2 resulted in compound 5, which
showed a hundred fold increase in potency (IC50=0.49
nM) compared to 1. However, neither compound had
significant systemic exposure in rat.
(systemicAUC
<0.004 mM h, Cmax<0.002 mM, 1
0À04 h
mg/kg po). Our first approach to address this issue was
to modify the polarity of the C5 substituent. Methyla-
tion of 7a to give 7b, resulted in a 600-fold decrease in
activity which initially led us to believe in the essential
requirement of a hydrogen bond donating group in the
C5 region. However, further investigation of SAR by
deletion of the nitrogen atom, changing ring size and
polarity (compounds 7e–j) offered evidence to negate
this theory, resulting in compounds retaining high
inhibitory potency.
We next went on to investigate changes to the 4-car-
boxamidophenyl region of compound 5. These com-
pounds were synthesized from the intermediate 3 by
palladium-catalyzed cross-coupling with commercially
available aryl boronicacids, or borate esters (prepared
by reaction of bis(pinacolato)diboron with the corre-
sponding bromides)8 under standard aqueous Suzuki
conditions to give intermediates 6a–j (Scheme 1). Sub-
sequent BOC deprotection followed by acylation with
homochiral 2-(S)-methyl-3-(3,4-difluorophenyl)propionic
acid and separation of diastereomers gave 7a–j (Table 1).
Evaluation of these compounds in vivo showed that all
had low systemicexposure in rat with the exception of
compound 7i (AUC0À4 h=0.09 mM h, Cmax 0.04 mM, 5
mg/kg po). Although systemicexposure of 7i remained
poor, we reasoned that increasing polarity of the mol-
ecule by modification of the substituent at the N1

A. P. Owens et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4143–4145
4145
offered excellent potency (IC50 1.2 nM) together with
improved systemicexposure (AUC h=2.9 mM h,
0À4
Cmax 0.9 mM, 5 mg/kg po). Further iv studies showed
that compound 10b had rat bioavailability of 22% and
t1/2=1.4 h. Furthermore, compound 10b entered the
brain, although levels were relatively low (AUC0À4h
=0.17 mM h, 5 mg/kg po).
In conclusion, we have demonstrated a practical appli-
cation of a new approach to the synthesis of 3-amino-
1,4-benzodiazepines from a single intermediate 2, which
allowed rapid evaluation of a range of benzodiazepines
optionally substituted at N1, C3 and C5. This approach
resulted in identification of 10b, a potent g-secretase
inhibitor, with reasonable oral bioavailability.
References and Notes
1. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353. Moore,
C. L.; Wolfe, M. S. Exp. Opin. Ther. Pat. 1999, 9, 135. Jhee,
S.; Shiovitz, T.; Crawford, A. W.; Cutler, N. R. Exp. Opin.
Invest. Drugs 2001, 10, 593. Conde, S. Exp. Opin. Ther. Pat.
2002, 12, 503. Dominguez, D. I.; De Strooper, B.; Annaert, W.
Amyloid 2001, 8, 124.
2. Churcher, I.; Ashton, K.; Butcher, J. W.; Clarke, E. E.;
Harrison, T.; Lewis, H. D.; Owens, A. P.; Teall, M. R.; Wil-
liams, S.; Wrigley, J. D. J. Bioorg. Med. Chem. Lett. 2003, 13,
179.
3. Churcher, I.; Nadin, A. J.; Owens, A. P. PCT Int. Appl.
WO 0230912, 2002.
Scheme 2. (i) NaH or Cs2CO3, R1-X (X=Br, I), DMF, 86–94%; (ii)
HBr–AcOH, 90–96%; (iii) EDC, HOBT, RCO2H, DMF, separation
of diastereomers, 70%; (iv) NH2Me or NHMe2, sealed tube, MeOH,
80–90%; (v) LiOH, dioxane–H2O, 48%; Z=CO2CH2Ph.
Table 2. Structure–activity relationships of N1-substituted benzo-
diazepines
4. Nadin, A.; Sanchez Lopez, J. M.; Owens, A. P.; Howells,
D. M.; Talbot, A. C.; Harrison, T. J. Org. Chem. 2003, 68,
2844.
Entry
R1
IC50 (nM)
5. SH-SY5Y cells stably overexpressing the bAPP C-term-
inal fragment SPA4CT (Dyrks T.; Dyrks E.; Monning U.;
Urmoneit B.; Turner J.; Beyreuther K. FEBS Lett. 1993
Nov 29;335:89) are induced with sodium butyrate prior to
plating. Compounds are added at a range of concentrations
after 2 h and incubated overnight. Aliquots of conditioned
media are removed for analysis by a Homogeneous Time
Resolved Fluorescence (HTRF) assay (Clarke, E. E.; Shear-
man, M. S. J. Neurosci. Methods 2000, 102, 61). All IC50
data shown are the geometricmean of minimum of three
10a
7i
H
Me
1.8
13.2
1.2
12.7
1.8
10b
10c
10d
10e
10f
10g
CH2CONH2
CH2CO2Me
CH2CONHMe
CH2CONMe2
CH2CH2OAc3.7
CH2CH2OH
5.5
0.9
independent results. Cell viability is measured by a colori-
TM
position may improve absorption. The synthesis of
compounds 10a–10g, starting from known benzo-
diÀazepine 8,9 is shown in Scheme 2.
metricecll proliferation assay (CellTitre 96
Promega).
AQ assay,
6. Evans, D. A.; Ennis, M. D.; Mathre, D. J. J. Am. Chem.
Soc. 1982, 104, 1737.
7. All compounds shown containing an a-methyl substituent
are single diastereomers with an (S) stereo conformation at the
C3 benzodiazepine centre.
8. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem.
1995, 60, 7508.
9. Castro Pineiro, J. L.; Churcher, I.; Guiblin, A. R.; Harri-
son, T.; Kerrad, S.; Madin, A.; Nadin, A. J.; Owens, A. P.;
Sparey, T. J.; Teall, M. R.; Williams, S. PCT Int. Appl. WO
0190084, 2002.
Evaluation of the in vitro potencies of 10a–10g revealed
some interesting observations. Deletion of the N1
methyl substituent gave compound 10a, which had an 8-
fold improvement in potency (Table 2). We then intro-
duced a range of polar groups, compounds 10b–10g, the
majority of which, although tolerated, offered no sig-
nificant improvement in exposure in rat. However, the
carboxamidomethyl moiety of compound 10b uniquely