Discovery and evaluation of BMS-708163, a potent, selective and orally bioavailable γ-secretase inhibitor

Article abstract of DOI:10.1021/ml1000239

During the course of our research efforts to develop a potent and selective γ-secretase inhibitor for the treatment of Alzheimer's disease, we investigated a series of carboxamide-substituted sulfonamides. Optimization based on potency, Notch/amyloid-β precursor protein selectivity, and brain efficacy after oral dosing led to the discovery of 4 (BMS-708163). Compound 4 is a potent inhibitor of γ-secretase (Aβ40 IC₅₀ = 0.30 nM), demonstrating a 193-fold selectivity against Notch. Oral administration of 4 significantly reduced Aβ40 levels for sustained periods in brain, plasma, and cerebrospinal fluid in rats and dogs.

Full text of DOI:10.1021/ml1000239

pubs.acs.org/acsmedchemlett
Discovery and Evaluation of BMS-708163, a Potent,
Selective and Orally Bioavailable γ-Secretase Inhibitor
Kevin W. Gillman,*,† John E. Starrett, Jr.,*,† Michael F. Parker,† Kai Xie,† Joanne J. Bronson,†
Lawrence R. Marcin,† Kate E. McElhone,† Carl P. Bergstrom,† Robert A. Mate,†
Richard Williams,‡ Jere E. Meredith, Jr.,§ Catherine R. Burton,§ Donna M. Barten,§
Jeremy H. Toyn,§ Susan B. Roberts,§ Kimberley A. Lentz, John G. Houston,§ Robert Zaczek,§
Charles F. Albright,§ Carl P. Decicco,† John E. Macor,† and Richard E. Olson†
†Neuroscience Discovery Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford,
Connecticut 06492, §Neuroscience Discovery Biology, Bristol-Myers Squibb, 5 Research Parkway, Wallingford,
Connecticut 06492, Metabolism and Pharmacokinetics, Bristol-Myers Squibb, 5 Research Parkway, Wallingford,
Connecticut 06492, and ‡Medicinal Chemistry Department, Albany Molecular Research Incorporated, 26 Corporate Circle,
Albany, New York 15098
ABSTRACT During the course of our research efforts to develop a potent and
selective γ-secretase inhibitor for the treatment of Alzheimer's disease, we in-
vestigated a series of carboxamide-substituted sulfonamides. Optimization based
on potency, Notch/amyloid-β precursor protein selectivity, and brain efficacy after
oral dosing led to the discovery of 4 (BMS-708163). Compound 4 is a potent
inhibitor of γ-secretase (Aβ40 IC50 = 0.30 nM), demonstrating a 193-fold selecti-
vity against Notch. Oral administration of 4 significantly reduced Aβ40 levels for
sustained periods in brain, plasma, and cerebrospinal fluid in rats and dogs.
KEYWORDS γ-Secretase, brain penetrant, oxadiazole, Alzheimer's, amyloid, clini-
cal candidate
istopathological and genetic evidence suggest that
the amyloid-β (Aβ) peptide plays a key causative role
in Alzheimer's disease (AD).1 Aβ is generated by the
recently reported preclinical5 results with the γ-secretase
inhibitor begacestat, and Eli Lilly presented clinical evidence
indicating reductions in plasma Aβ40 with the small mole-
cule γ-secretase inhibitor LY450139.6 The phase I clinical
trials with 1 provided specific information to guide our
efforts to develop an improved second-generation com-
pound for clinical evaluation. Compound 1 demonstrated
significant human Pregnane-X Receptor (PXR) transactivation
(EC60 = 2.4 uM), indicating potential for autoinduction in man.
In addition, 1 had a modest brain to plasma ratio (B/P = 0.2) in
Tg2576 mice. We reasoned that improvement in the brain to
plasma ratio should increase central exposure and efficacy and
decrease side effects associated with peripheral exposure such
as autoinduction and Notch-related toxicities. Furthermore, 1
showed limited ability to lower brain Aβ40 in rats, which may
be related to lower levels of APP-derived secretase substrate in
this model.7
H
proteolytic processing of the amyloid-β precursor protein
(APP) through consecutive cleavages by β-site APP-cleaving
enzyme (BACE) and γ-secretase. APP is cleaved by BACE to
form a β-secretase-derived C-terminal fragment of APP
(βCTF), which undergoes further cleavage by γ-secretase to
form Aβ.2 Most Aβ isoforms contain a common N terminus,
formed by BACE cleavage, while heterologous cleavage by γ-
secretase creates Aβ isoforms ranging from 37 (Aβ37) to 42
(Aβ42) amino acid residues. Aβ40 is the most abundant
isoform, while Aβ42 is most tightly linked with AD patho-
genesis.
In our laboratories, one approach for the treatment of AD
has been to identify potent and selective γ-secretase inhibi-
tors to lower the production of Aβ in the brains of affected
and at-risk patients. Because of the role of γ-secretase in
Notch processing, the identity of secretase inhibitors with
minimal impact on Notch signaling was also an important
feature of our strategy. Our first γ-secretase clinical candi-
date, the 4-chloro-N-(2,5-difluorophenyl)phenylsulfonamide
1 (Figure 1),3 progressed to phase I clinical trials. However,
clinical progression of 1 was halted due to significant phar-
macokinetic liabilities and lack of significant Aβ lowering in
both single-ascending and multiple-ascending dose studies
in man.4 In addition to our efforts in the clinic, Wyeth
The discovery of a second-generation compound with
improved pharmacokinetic properties commenced with
the identification of the novel 2-oxoazepan-3-yl-benzenesul-
fonamide 2 (Aβ42 IC50 = 9.0 nM) from a high-throughput
screen of the BMS corporate compound deck.8
Received Date: January 29, 2010
Accepted Date: February 28, 2010
Published on Web Date: March 22, 2010
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for Aβ40 reduction in the brain. Consistent with the discre-
pancy between central and peripheral efficacy, analysis of
plasma and brain exposures of 3 revealed that the brain to
plasma ratio was very low (brainconcn/plasmaconcn = 0.01).
In addition, 3 had poor in vitro microsomal stability [%
remaining after 10 min of incubation in liver microsomes =
19% (rat) and 44% (human)],10 indicating the potential for
rapid clearance in vivo. In an effort to improve brain
penetration and metabolic stability, a variety of substituents
on the benzenecarboxamide nitrogen were tested.11
1,2,4-Oxadiazoles have been successfully employed as
amide and ester bioisosteres for the optimization of brain-
penetrant molecules.12-14 We utilized this approach for
optimizing 3 and prepared a number of oxadiazole ana-
logues of 3. From this effort, the novel oxadiazole (R)-2-(4-
chloro-N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfo-
namido)-5,5,5-trifluoropentanamide (4; BMS-708163)15 was
synthesized and subsequently evaluated in vitro and in vivo.
The Cytochrome P-450 (CYP) inhibition in human recombi-
nant CYP enzymes for compound 2 was reported to be in the
nanomolar range for CYP isoforms 3A4 and 2C19.8 SAR was
not performed on CYP inhibition per se; however, this liability
was continually monitored in subsequent analogues. Com-
pound 4 was evaluated in vitro as an inhibitor of human
recombinant CYPs. Weak inhibition (IC50 = 20 μM) was ob-
served for CYP2C19. No inhibition was observed for the other
isoforms up to the maximum tested concentration of 40 μM.
The synthesis of 4 was initiated via an asymmetric
Strecker reaction with trifluorobutyraldehyde 5 and (R)-R-
methylbenzylamine,16 affording nitrile 6 as a 3.65:1 mixture
of diastereomers (Scheme 1). Subsequent hydrolysis of
nitrile 6 directly to the carboxamide 7, followed by fractional
crystallization of the HCl salt, afforded 8 in a >99:1 ratio of
the desired (R,R) vs the (S,R) diastereomer. Removal of the
chiral auxiliary under hydrogenolysis conditions with cata-
lytic palladium hydroxide gave the amine hydrochloride salt
9, followed by conversion of the amine to the corresponding
sulfonamide under standard conditions afforded 10 in 84%
yield over the two steps. Alkylation of 10 with cesium
carbonate and 4-cyano-2-fluorobenzyl bromide 11 afforded
12 in 68% yield with no loss of stereochemical purity as
determined by chiral high-performance liquid chromato-
graphy. Lastly, conversion of 12 to oxadiazole 4 was accomp-
lished via a two-step procedure starting with the addition of
hydroxylamine to the arylnitrile, affording the intermediate
amide-oxime 13. Annulation of the amide-oxime 13 with
triethyl orthoformate and a catalytic amount of boron tri-
fluoride etherate gave the oxadiazole 4 in 69% yield over
two steps.17 The overall conversion of aldehyde 5 to oxa-
diazole 4 was amenable to scale-up as needed for in vivo
testing.
Figure 1. Novel sulfonamide-based γ-secretase inhibitors.
Table 1. Summary of GS H4-8 SW1-40 IC50 Values and Notch
(mNotch1-ΔE) IC50 Values for Selected Sulfonamide-Based
γ-Secretase Inhibitorsa
IC50 ( SD (nM)
compound
Aβ
Notch
Notch/APP ratio
1
2
3
4
1.4 ( 1.2
4.1 ( 1.2b
0.14 ( 0.05
0.30 ( 0.15
540 ( 230
960 ( 420
26 ( 9.1
390
230
190
193
58 ( 23
a IC50 and standard deviation values were calculated on the basis of a
minimum of three independent experiments. b IC50 values measured
using a GS H4-8 SW1-42 assay (n = 3).
Early structure-activity relationship (SAR) exploration
identified an improvement in Aβ40 in vitro potency by
deannulation of the caprolactam ring of 2 (Table 1), which
afforded a variety of acyclic amino acid amide analogues
that were initially optimized based on potency, selectivity,
and desirable physicochemical properties. The (R)-derived
amino acid sulfonamides consistently provided higher
potency. Further exploration into the SAR of the acyclic
carboxamide series led to N-methyl-substituted benzene-
carboxamide 3, which was identified as potent inhibitor of
Aβ40 formation (IC50 = 0.14 nM).
Compound 3 was tested in female wild-type rats to
determine central and peripheral efficacy following oral
administration at 1, 3, 10, and 30 mg/kg. Animals were
sacrificed at 5 h postdose, and brain and plasma samples
were collected for analyses of both drug exposure and Aβ40
levels.9 A single dose of compound 3 at 30 mg/kg signifi-
cantly reduced both plasma Aβ40 and brain Aβ40 to levels of
14 ( 21 and 59 ( 12% (% of vehicle control ( SEM),
respectively. However, no significant reductions in brain
Aβ40 were observed at 1, 3, and 10 mg/kg. Although we
were encouraged by this preliminary finding, this series
required further optimization to reduce the dose necessary
In vitro, 4 demonstrated an improved metabolic stability
profile relative to 3 across species [% remaining after 10 min
of incubation = 32% (rat), 75% (dog), and 97% (human)],
as well as a significantly diminished PXR transactivation
profile (EC60 > 16.7uM) relative to compound 1. Compound
4 potently inhibited the formation of Aβ40 and Aβ42 in
H4-8Sw cells. Analysis of multiple experiments yielded an
IC50 = 0.30 ( 0.15 nM (mean ( SD, n = 50) for Aβ40
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Scheme 1. Synthesis of Compound 4a
a Reagents and conditions: (a) NaCN, AcOH, MeOH, 3.65:1 de, 89%. (b) H2SO4, CH2Cl2. (c) HCl, Et2O/MeOH recrystallization, >99% de, 23%. (d)
Pd(OH)2, H2 (50 psi), EtOH/H2O. (e) 4-Chlorobenzene-1-sulfonyl chloride, DIPEA, CH2Cl2, 84% (two steps). (f) Cs2CO3, DMF, 68%. (g) NH2OH, EtOH.
(h) BF3:OEt2, CH(OMe)3, CH2Cl2 69% (two steps).
Table 2. Single-Dose Pharmacokinetic Parameters of 4 in Rats and Dogsa
dose
AUC (0-24 h)
(μM h)
species
route
(mg/kg)
C
max (μM)
T
max (h)
T
1/2 (h)
CLTp (mL/min/kg)
11.7 ( 2.48
V
ss (L/kg)
F (%)
rat (female)
dog (male)
IV
PO
1
10
2.60 ( 0.42
27.2 ( 7.39
6.90 ( 2.20
11.4 ( 1.76
5.34 ( 0.74
3.87 ( 0.56
2.29 ( 1.1
14.0 ( 9.2
105
42
IV
PO
1
2.5
6.04 ( 1.12
6.35 ( 2.07
4.20 ( 0.93
0.58 ( 0.24
0.75 ( 0.25
a Mean ( SD. Compound 4 (10 μM) was 97.0 ( 0.7% bound to human serum proteins and 96.3-97.9% bound to serum proteins in the animal
species studied.
inhibition and an IC50 = 0.27 ( 0.12 nM (mean ( SD, n =
45) for Aβ42 inhibition.
an IV dose, plasma concentrations exhibited a multiexpo-
nential decline. The total body clearance of 4 was low in both
species (rat =11.7 ( 2.48 mL/min/kg; dog = 4.20 ( 0.93
mL/min/kg). The volume of distribution at steady state (Vss)
was high, indicating extensive extravascular distribution
(rat = 5.34 L/kg; dog = 3.87 L/kg). Oral bioavailability from
solution formulations of 4 was 105 and 42% in rats and dogs,
respectively. In addition, 4 demonstrated a high brain to
plasma ratio (brainconcn/plasmaconcn = 2.4) in dogs.
On the basis of favorable oral bioavailability and sufficient
half-life for QD dosing, 4 was dosed orally in female rats and
analyzed for its potential to reduce Aβ40 in plasma and brain
over a time course of 24 h at doses of 1, 10, and 100 mg/kg.
Compound 4 significantly reduced both plasma and brain
Aβ40 levels relative to control at 10 and 100 mg/kg for the
entire dosing interval (Figure 2A,B). The increase in plasma
Aβ levels observed at the 1 mg/kg dose is related to an
intrinsic characteristic of γ-secretase pharmacology and is
In addition to APP processing, γ-secretase activity is
required for signaling by the Notch family of transmem-
brane receptors.18,19 Because inhibition of Notch signaling
causes undesired, mechanism-based side effects, a cellular
assay for Notch1-ΔE signaling was used to counterscreen
γ-secretase inhibitors.20-22 Compound 4 exhibited weaker po-
tency for inhibition of Notch processing, IC50 = 58 ( 23 nM
(mean ( SD, n = 58), as compared to its inhibition potency
for APP cleavage. On the basis of the cellular potencies for
inhibiting Aβ40 generation (0.30 nM) and Notch signaling
(58 nM) in these assays, 4 demonstrated a Notch/APP selectivity
ratio of 193X (95% CI = 163-232). With compound 4, there
were no dose-limiting effects in dogs with QD/PO dosing for
6 months at 3 mg/kg.
Table 2 illustrates the pharmacokinetic parameters of 4 in
female rats and male dogs.23 In both rats and dogs following
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Figure 2. Plasma and brain Aβ40 following a single dose of 4 in female rats. The time course of changes in (A) plasma Aβ40 and (B) brain
Aβ40. Mean ( SEM (n = 3 animals) for each time point shown.
Figure 3. (A) Brain and CSFAβ40 % reduction and (B) plasma concentration by 4 dosed PO 2 mg/kg in male dogs. Mean ( SEM (n = 1 or 2
animals) for each time point shown.
observed with low levels of γ-secretase inhibition (Figure 2A).7
In brain, 4 demonstrated significant Aβ40 lowering for 8 h
after an oral dose of 1 mg/kg (Figure 2B). In a separate study,
when measured 5 h after single oral doses ranging from 3 to
100 mg/kg, 4 significantly lowered CSF Aβ40 levels in rats
(data not shown).
as well as incorporation of a trifluoromethyl group24 in the
side chain improved brain penetration and significantly in-
creased the ability of this class of compounds to lower brain
Aβ40. Compound4 iscurrentlyundergoing clinicalevaluation
to determine its potential to act as a disease-modifying agent
for the treatment of Alzheimer's disease. Results from these
studies and a more detailed SAR summary surrounding the
discovery of 4 will be presented in due course.
In addition to the in vivo studies performed in rats, 4 was
administered orally to male beagle dogs to measure Aβ40
lowering in brain and CSF. In this study, dogs were adminis-
tered a single oral dose of 2 mg/kg of 4 and euthanized 2, 3, 5,
10, 16, and 24 h postdose (n = 1 or 2 per time point). Aβ40
measurements from the frontal cortex and CSF showed a
rapid and sustained reduction in brain and CSF Aβ40 levels
(Figure 3). In this experiment, CSFAβ reductions measured in
the dog correlated with Aβ reductions measured in the frontal
cortex. Because it is not possible to directly measure human
brain Aβ, a pharmacologic proof of concept of Aβ reductions
in humans will rely primarily on measurement of a surrogate
biomarker(s). On the basis of the correlation observed be-
tween brain and CSF Aβ40 lowering activity in dogs, our
results suggest that CSF Aβ40 may serve as a surrogate
biomarker for brain Aβ40 levels in humans.
SUPPORTING INFORMATION AVAILABLE Experimental
details for synthetic procedures and associated chemical data for
compounds 3-13 as well as experimental details on the Aβ40 and
mNotch1-ΔE assays. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. (K.W.G.) Tel: 203-677-6785. Fax: 203-677-7884. E-mail:
Kevin.Gillman@bms.com. (J.E.S.) Tel: 203-677-6306. Fax: 203-
677-7884. E-mail: John.Starrett@bms.com.
ACKNOWLEDGMENT We thank Rex Denton, and Gary Pilcher
for evaluation of the in vivo toxicology studies of 4, Mark
Thompson, Sam Varma, Carol Krause, Victoria Wong, and Harvey
Ferguson for contributions to in vitro studies, and Nina Hoque, Jason
Corsa, Tracey Fiedler, Maria Pierdomenico, Valerie Guss, Wendy
Clarke and Sarah J. Taylor for contributions to in vivo studies.
In summary, we designed and synthesized the novel
oxadiazole-substituted sulfonamide 4 as a potent and
selective γ-secretase inhibitor for the potential treatment
of Alzheimer's disease. Replacement of the benzamide
functionality in compound 3 with a bioisosteric oxadiazole
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