Discovery of isonicotinamide derived β-secretase inhibitors: In vivo reduction of β-amyloid

Article abstract of DOI:10.1021/jm070272d

β-Secretase inhibition offers an exciting opportunity for therapeutic intervention in the progression of Alzheimer's disease. A series of isonicotinamides derived from traditional aspartyl protease transition state isostere inhibitors has been optimized to yield low nanomolar inhibitors with sufficient penetration across the blood-brain barrier to demonstrate β-amyloid lowering in a murine model.

Full text of DOI:10.1021/jm070272d

J. Med. Chem. 2007, 50, 3431-3433
3431
Discovery of Isonicotinamide Derived â-Secretase
Inhibitors: In Vivo Reduction of â-Amyloid
Matthew G. Stanton,*,† Shaun R. Stauffer,†
Alison R. Gregro,† Melissa Steinbeiser,†
Philippe Nantermet,† Sethu Sankaranarayanan,§
Eric A. Price,§ Guoxin Wu,§ Ming-Chih Crouthamel,§
Joan Ellis,‡ Ming-Tain Lai,§ Amy S. Espeseth,§
Xiao-Ping Shi,§ Lixia Jin,‡ Dennis Colussi,§ Beth Pietrak,§
Qian Huang,§ Min Xu,§ Adam J. Simon,§
Samuel L. Graham,† Joseph P. Vacca,† and Harold Selnick†
Departments of Medicinal Chemistry, Alzheimer’s Research, and
Drug Metabolism, Merck Research Laboratories, Post Office Box 4,
West Point, PennsylVania 19486
Figure 1. Isonicotinamide BACE inhibitor (4) derived from hydroxy-
ethylamine (HEA) 1 (IC50 values from the in vitro ECL assay).
ReceiVed March 10, 2007
Scheme 1. Synthesis of Isonicotinamide â-Secretase Inhibitors
4 and 8a-fa
Abstract: â-Secretase inhibition offers an exciting opportunity for
therapeutic intervention in the progression of Alzheimer’s disease. A
series of isonicotinamides derived from traditional aspartyl protease
transition state isostere inhibitors has been optimized to yield low
nanomolar inhibitors with sufficient penetration across the blood-brain
barrier to demonstrate â-amyloid lowering in a murine model.
Alzheimer’s disease (AD) represents a major unmet medical
need. Despite substantial effort, there exists no disease modify-
ing treatment for AD.1 It has been proposed that the biological
pathway leading to the observed disease pathology is reliant
on the processing of amyloid precursor protein (APP).2 Pro-
teolysis of APP by the secretase enzymes (R, â, and γ) generates
peptide fragments, with the most relevant to AD being Aâ40
and Aâ42. These fragments are derived from the action of both
γ- and â-secretase and are the primary components of the
insoluble amyloid plaques in AD patients. Disruption of this
cascade via γ- and more recently â-secretase inhibition has and
continues to be a central focus of drug discovery efforts.3
Inhibition of the â-secretase (â-site APP cleaving enzyme
or BACE-1) pathway by small molecule interference has been
a goal of the pharmaceutical industry since the identification
and characterization of BACE-1 in 1999.4 Particularly encour-
aging for this strategy was the discovery in 2001 that BACE-1
knockout mice were devoid of â-secretase activity, did not
generate Aâ, and displayed a relatively normal phenotype.5
Previous work in our laboratories revealed 1, a potent
BACE-1 inhibitor derived from a 1,3,5-trisubstituted aromatic
core and containing a traditional aspartyl protease inhibitor
motif, the hydroxyethylamine (HEA) transition state isostere
(Figure 1).6 Interaction of the HEA with the aspartic acid
residues in the catalytic region of the enzyme is critical for
activity. Despite the excellent cellular activity of 1 (sAPPâ )
20 nM), brain penetration of this class of compounds following
i.v. administration in mice is negligible. The alleged culprits
for this lack of CNS penetration are poor permeability and Pgp-
mediated efflux, presumably due to the compound’s multiple
hydrogen bond donors and acceptors.7 With this in mind, the
a Reagents and conditions: (a) CH3NHSO2R1, Pd2(dba)3, Xantphos,
K3PO4, toluene, 100 °C; (b) LiOH, MeOH, THF; (c) R3[CH(CH2)CH]CH2-
NHR2, Pd(t-Bu3P)2, K3PO4, DMF, 120 °C; (d) amine ((2S)-2-amino-3-
phenylpropan-1-ol, (2S)-1-azido-3-phenylpropan-2-amine, 10a,b), BOP,
TEA, DCM; (e) H2, Pd(OH)2, EtOH, TFA; (f) NaOMe (10a) or KF‚HF,
Bu4NF‚2HF (10b); (g) HN3, PPh3, DEAD, THF; (h) EtOAc, HCl.
HEA isostere was truncated to a simple primary alcohol (2).
This led to a substantial loss in BACE-1 inhibitory activity.
Investigations focusing on replacement of the P3 amide and
optimization of the 1,3,5-trisubstituted aromatic core have been
the subject of previous communications.8a,b It was found that
small cyclopropylmethylamine P3 groups in combination with
the isonicotinamide core (3) could provide improved potency
relative to the larger R-methylbenzamide (2). This communica-
tion describes further refinement of the P2 sulfonamide and
optimization of the primary amine aspartyl binding region
leading to 4, a molecule suitable for in vivo evaluation in a
murine model.
Synthesis of isonicotinamide inhibitors 4 and 8a-f began with
methyl 2,6-dichloroisonicotinate (5; Scheme 1). Monosulfona-
mide incorporation according to the procedure described by
Buchwald, followed by saponification, gave isonicotinic acids
6a,b.9 Amination under modified Hartwig conditions generated
the 2-amino-6-sulfonamidoisonicotinic acids 7a-f.10 Coupling
of these acids to either the amino alcohol or the amino azides
derived from phenylalanine, followed by reduction of the azide
and/or removal of the benzyl protecting group (R2 ) benzyl)
gave compounds 4 and 8a-f. Azides 10a,b were prepared by
epoxide opening with the appropriate nucleophile (MeO-, F-)
followed by a Mitsunobu reaction with hydrazoic acid and
deprotection of the amine.
* To whom correspondence should be addressed. Current address: Merck
Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115.
Phone: 617-992-2050. Fax: 617-992-2406. E-mail: matthew_stanton@
merck.com.
† Department of Medicinal Chemistry.
§ Department of Alzheimer’s Research.
‡ Department of Drug Metabolism.
10.1021/jm070272d CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/21/2007

3432 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Letters
Table 1. Binding Affinity, Pgp Efflux, and Brain Penetration for Test Compoundsa
c
cmpdb
R1
R2
R3
R4
X
IC50
sAPPâd
MDR1e
[brain]f
n.d.
1400 ( 1350
n.d.
420 ( 157
1100 ( 492
3080 ( 2400h
560 ( 128h
[brain]/sAPPâ
<0.07
8a
8b
8c
8d
8e
8f
4
CH3
H
H
H
H
H
H
H
OH
OH
OH
NH2
NH2
NH2
NH2
1100
350
14
n.d.
n.d.
3.6
n.d.
36.5
n.d.
4.8
CH(CH3)2
CH(CH3)2
CH3
CH3
CH(CH3)2
CH(CH3)2
CH2CH2CH3
>20 000
>20 000
280 ( 49
173 ( 12
910
H
H
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
g
34
1.5
6.4
3.4
11.4
CH2OCH3
CH2F
CH2F
12 ( 14
18
2 ( 0.2
49 ( 18
>50
a All values lacking standard deviation were measured as single data points. b All compounds were >95% pure by HPLC and characterized by 1H NMR
and HRMS. c Biochemical IC50. Values are reported in nM and were determined via electrochemiluminescence assay. d Cell-based assay. Values are reported
as IC50’s values in nM and were determined via Alpha Screen assay. e B/A-A/B ratio. f Concentrations determined 30 min post 20 mg/kg i.v. dose (n of 3)
and are reported in nM. g Compound exists as a mixture of trans-methyl(cyclopropylmethyl) diastereomers. h Represents concentrations from dosing of
mixture of trans-methyl(cyclopropylmethyl) diastereomers.
Figure 2. Time course study of Aâ reduction in APP-YAC mice upon
Figure 3. Dose response study of Aâ reduction in APP-YAC mice
treatment with 4 (50 mg/kg i.v.).
upon treatment with 4 (3.0 h post i.v. bolus).
Initial efforts in the isonicotinamide series of BACE-1
Table 2. Pharmacokinetic Parameters of Isonicotinamide BACE
inhibitors revealed that truncation of the HEA to a simple
primary alcohol gave reasonable activity for such a simple, low
molecular weight (mw ) 432) compound (8a, Table 1).
Optimization of the P2 sulfonamide (Me to i-Pr) and alkylation
of the P3 amine (NH to NCH2CH2CH3; 8b) gave both improved
biochemical potency and, more importantly, greater passive
permeability and decreased Pgp efflux. These improved proper-
ties were reflected in the in vivo model for brain penetration,
with 8b achieving brain concentrations of 1.4 µM after i.v.
administration (30 min post-dose, 20 mg/kg). Encouraged by
this result, the P3 region of the molecule was further optimized
to the [S,S]-trans-methylcyclopropyl group, imparting further
benefits with respect to potency (8c, 14 nM).8b Despite the low
nanomolar activity of this series, activity in the functional cell-
based assay was not observed (sAPPâ > 20 µM).
Incorporation of an amino residue in place of the hydroxyl
group (alternative aspartyl ligand)8b,c provided molecules with
activity in cell culture (4, 8d-f). Compound 8d achieved CNS
exposure (20 mg/kg i.v. dose) in excess of the molecules cellular
IC50. Optimization of this series was focused on the transition
state isostere. Incorporation of branched alkyl substituents
increased the biochemical potency (4, 8e-f). Attenuation of the
pKa of the amino functionality by incorporation of a fluorom-
ethyl substituent gave improved potency and, in the case of 8f,
reduced Pgp efflux. Analysis of the brain concentrations relative
to the cellular IC50 values revealed compound 4 as a leading
candidate for in vivo studies.
Inhibitors in Rat
b
b
Cla
Vda
(I/kg)
t1/2
Cmax
cmpd
(mg/min/kg)
(hr)
(µM)
%F
8e
8f
4
42.6
59.1
45.8
5.3
4.2
3.9
2.7
1.6
1.6
2.7
0.2
0.3
69
8
13
a 2 mg/kg i.v. dose (solution in 25%DMSO/75%H2O). b 10 mg/kg oral
dose (solution in 1% methylcellulose).
a maximal reduction of Aâ40 (34%) at 3 h (p < 0.01, Tukey-
Kramer HSD) compared with that of vehicle-treated animals,
while a positive control γ-secretase inhibitor lowered brain Aâ40
∼50% (p < 0.001, Tukey-Kramer HSD). Determination of
concentrations of 4 in the brain at these time points indicated a
maximal concentration of 5.8 µM at 0.5 h, with 0.7 µM
remaining at the 4.5 h time point (data not shown).
Having determined a maximal Aâ40 reduction at 3 h post-
dose, a full dose-response study was performed (Figure 3).
The Aâ40 reduction measured in the time-course study was
confirmed in this study with ∼34% reduction of Aâ40 at 50
mg/kg at 3 h after dosing relative to vehicle (p < 0.001, Tukey-
Kramer HSD).12 The Aâ40 reduction was dose proportional, with
doses <25 mg/kg not showing statistical significance. The
concentration of drug in the brain was 1.9 µM and 0.7 µM for
the 50 and 25 mg/kg dose groups, respectively, and below the
limit of quantification in the 12.5 and 6.25 mg/kg groups (data
not shown).
As a result of the ratio of brain concentration to cellular IC50,
compound 4 was chosen as a proof of concept molecule for
studying the effect of inhibition of BACE-1 on brain levels of
Aâ40 (Figure 2). Transgenic mice expressing human WT APP
under the control of a yeast artificial chromosome vector11 were
administered 50 mg/kg of 4 as an i.v. bolus and brain con-
centrations of Aâ40 were measured at 0.5, 1.5, 3, and 4.5 h post-
dose. The results of this study are depicted in Figure 2 and show
The pharmacokinetic parameters of selected isonicotinamide
BACE-1 inhibitors were investigated in rat (Table 2). In general,
compounds from this series displayed high clearance and high
volumes of distribution. The i.v. half-lives were moderate and
the oral bioavailability was poor. The notable exception with
respect to bioavailability is compound 8e, which is 69%
bioavailable, with a good oral maximum concentration of 2.7

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3433
V.; Sherman, M.; Younkin, L.; Younkin, S.; Forster, C.; Sergeant,
N.; Delacourte, A.; Vassar, R.; Citron, M.; Kofuji, P.; Boland, L.
M.; Ashe, K. H. Involvement of b-site APP cleaving enzyme 1
(BACE1) in amyloid precursor protein-mediated enhancement of
memory and activity-dependent synaptic plasticity. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 8167-8172.
µM. The challenge remains to find molecules that combine the
reasonable pk parameters of 8e, with the potency/efficacy of 4.
AD remains one of the more substantial unmet medical needs.
Small molecule interference in the amyloid cascade represents
an attractive therapeutic option. â-Secretase is a particularly
appealing target with knock-out mice demonstrating Aâ reduc-
tion and a relatively normal phenotype.5 The challenges sur-
rounding â-secretase inhibitor design are substantial and are
highlighted by the difficulty in uncovering small molecules that
maintain potency while demonstrating desirable penetration
across the blood-brain barrier. Starting from compounds
containing a known aspartyl protease transition state isostere,
isonicotinamide-based inhibitors were discovered that allowed
for truncation of the HEA isostere to a simple amine. Optimiza-
tion for potency and brain penetration led to 4, a low nanomolar
BACE-1 inhibitor that was effective in reducing Aâ levels in a
murine model in a dose-dependent manner. Issues that remain
to be resolved are Pgp-mediated efflux and poor pharmacoki-
netics. Efforts are currently under way to address these liabilities
and will be the subject of future communications.
(6) (a) Coburn, C. A.; Stachel, S. J.; Li, Y.-M.; Rush, D. M.; Steele, T.
G.; Chen-Dodson, E.; Holloway, M. K.; Xu, M.; Huang, Q.; Lai,
M.-T.; DiMuzio, J.; Crouthamel, M.-C.; Shi, X.-P.; Sardana, V.;
Chen, Z.; Munshi, S.; Kuo, L.; Makara, G. M.; Annis, D. A.;
Tadikonda, P. K.; Nash, H. M.; Vacca, J. P.; Wang, T. Identification
of a small molecule nonpeptide active site â-secretase inhibitor that
displays a nontraditional binding mode for aspartyl proteases. J. Med.
Chem. 2004, 47, 6117-6119. (b) Stachel, S. J.; Coburn, C. A.; Steele,
T. G.; Jones, K. G.; Loutzenhiser, E. F.; Gregro, A. R.; Rajapakse,
H. A.; Lai, M.-T.; Crouthamel, M.-C.; Xu, M.; Tugusheva, K.;
Lineberger, J. E.; Pietrak, B. L.; Espeseth, A. S.; Shi, X.-P.; Chen-
Dodson, E.; Holloway, M. K.; Munshi, S.; Simon, A. J.; Kuo, L.;
Vacca, J. P. Structure-based design of potent and selective cell-
permeable inhibitors of human â-secretase (BACE-1). J. Med. Chem.
2004, 47, 6447-6450. For representative examples of HEA BACE
inhibitors from other laboratories, see: Maillard, M. C.; Hom, R.
K.; Benson, T. E.; Moon, J. B.; Mamo, S.; Bienkowski, M.;
Tomasselli, A. G.; Woods, D. D.; Prince, D. B.; Paddock, D. J.;
Emmons, T. L.; Tucker, J. A.; Dappen, M. S.; Brogley, L.; Thorsett,
E. D.; Jewett, N.; Sinha, S.; John, V. Design, synthesis, and crystal
structure of hydroxyethyl secondary amine-based peptidomimetic
inhibitors of human beta-secretase. J. Med. Chem. 2007, 50, 776-
781. Also see: Hussain, I.; Hawkins, J.; Harrison, D.; Hille, C.;
Wayne, G.; Cutler, L.; Buck, T.; Walter, D.; Demont, E.; Howes,
C.; Naylor, A.; Jeffrey, P.; Gonzalez, M.; Dingwall, C.; Michel, A.;
Redshaw, S.; Davis, J. B. Oral administration of a potent and selective
non-peptidic BACE-1 inhibitor decreases beta-cleavage of amyloid
precursor protein and amyloid-beta production in vivo. J. Neurochem.
2007, 100, 802-809 and references therein.
Supporting Information Available: Experimental procedures
and compound characterization data. This material is available free
of charge via the Internet at http://pubs.acs.org.
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JM070272D