Piperazine sulfonamide BACE1 inhibitors: Design, synthesis, and in vivo characterization

Article abstract of DOI:10.1016/j.bmcl.2010.03.050

With collaboration between chemistry, X-ray crystallography, and molecular modeling, we designed and synthesized a series of novel piperazine sulfonamide BACE1 inhibitors. Iterative exploration of the non-prime side and S2′ sub-pocket of the enzyme culmin

Full text of DOI:10.1016/j.bmcl.2010.03.050

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2837–2842
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Piperazine sulfonamide BACE1 inhibitors: Design, synthesis,
and in vivo characterization
b, ,
a
b
a
a
Jared Cumming a, , Suresh Babu
, Ying Huang , Carolyn Carrol , Xia Chen , Leonard Favreau ,
*
*
William Greenlee a, Tao Guo b, Matthew Kennedy a, Reshma Kuvelkar a, Thuy Le b, Guoqing Li a,
Nansie McHugh a, Peter Orth a, Lynne Ozgur b, Eric Parker a, Kurt Saionz b, Andrew Stamford a,
Corey Strickland a, Dawit Tadesse b, Johannes Voigt a, Lili Zhang a, Qi Zhang a
a Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Ligand Pharmaceuticals, Inc., 3000 Eastpark Boulevard, Cranbury, NJ 08512, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
With collaboration between chemistry, X-ray crystallography, and molecular modeling, we designed and
synthesized a series of novel piperazine sulfonamide BACE1 inhibitors. Iterative exploration of the non-
prime side and S20 sub-pocket of the enzyme culminated in identification of an analog that potently low-
ers peripheral Ab40 in transgenic mice with a single subcutaneous dose.
Received 5 February 2010
Revised 8 March 2010
Accepted 9 March 2010
Available online 12 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Alzheimer’s disease
b-Secretase
BACE
Aspartyl protease inhibitors
Peptidomimetics
Alzheimer’s disease (AD)1 has become the sixth leading cause of
death in the United States and is the most common form of demen-
tia in the elderly. Despite significant research efforts in both indus-
try and academia, there are currently no disease modifying
therapies available to treat this illness. Significant evidence sug-
on the second ring nitrogen, and (3) hydrogen bonding to Thr72
NH of the flap from the ring carbonyl. X-ray crystal structures of
the piperazinones bound to BACE18 suggested that a similar hydro-
gen bond to the flap could be achieved if the ring carbonyl were
gests that the pathology of AD is linked to generation of b-amyloid
2
peptides (Ab40,42
)
through proteolytic processing of amyloid pre-
cursor protein (APP), first by b-secretase (BACE1)3 and subse-
OH
H
N
H
N
Pr2N
S3
quently by
c-secretase. BACE1 in particular offers an attractive
R = Ph
1
drug target,4 since BACE1 knockout mice are viable5 and produce
a significant reduction in disease pathology when crossed with
transgenic AD mice.6
Ar = 3,5-F2Ph
O
O
O
N
Ar
BACE1 Ki = 3 nM
R
S2
S1
As part of our peptidomimetic BACE1 inhibitor efforts, we have
previously reported on a series of novel piperazinone structures 1
(Fig. 1).7 These cyclic amines achieved excellent BACE1 potency
through a three point interaction in the active site: (1) requisite
binding to the Asp32/Asp228 catalytic diad through the basic
amine and hydroxyl group, (2) occupation of S20 with substituents
Thr72 NH
S2'
(flap)
OH
OH
H
H
N
N
N
X
N
O
S
(O)n
R
R
O
* Corresponding authors. Tel.: +1 908 740 2515; fax: +1 908 740 7152 (J.C.); tel.:
+1 908 912 9180 (S.B.).
S2'
2
3
S2'
E-mail addresses: jared.cumming@merck.com (J. Cumming), sbabu@ptcbio.com
(S. Babu).
Present address: PTC Therapeutics, 100 Corporate Court, South Plainfield, NJ
07080, USA.
Figure 1. Initial design concepts of piperazine sulfonamide analogs 3 from earlier
piperazinone series 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.050

2838
J. Cumming et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2837–2842
O
OH
Boc
N
Boc
Bn2N
F
OH
OH
Bn2N
Ar
N
H
N
H
N
H
a
Pr2N
a
F
+
10
11
12
O
N
Bn
5
O
N
Bn
O
O
O
N
Ar
4
6
Ph
Ph
OH
OH
Boc
Boc
H2N
Ar
N
H2N
Ar
N
b
d
c
H
N
H
N
Pr2N
Pr2N
N
Bn
b,a
a
O
N
13
14
Bn
O
O
N
Ar
7
8
O
OH
H
Boc
OH
10
: R = Bn
: R = H
H
N
H
N
Pr2N
N
N
b
O
O
O
11
N
R
N
H
Ar
Ar
Scheme 2. Synthesis of reference BACE1 inhibitors 12–14 from intermediates 10
and 11 (Ar = 3,5-difluorophenyl). Reagents: (a) TFA, ꢁ80%; (b) PhC(O)Cl, DIEA.
OH
H
N
H
N
Pr2N
e,f
3a
O
O
N
S
O
Ar
deleted and an oxo moiety introduced on the substituent at the
4-position of the ring (e.g., structure 2, Fig. 1). From this initial con-
cept, with extensive input from molecular modeling,9 we designed
piperazine sulfonamides 3. The sulfonyl moiety in these analogs
was intended to serve two functions. First, one of the sulfonyl oxy-
gens would hydrogen bond with the Thr72 NH, and second, the sp3
nature of the sulfonyl itself would provide the correct trajectory for
the pendant aryl group to occupy the S20 subsite.
O
Ph
Scheme 1. Synthesis of BACE1 inhibitor 3a as
a representative example for
generation of piperazine sulfonamide analogs 3 (Ar = 3,5-difluorophenyl). Reagents
and conditions: (a) LDA + 5 then 4, ꢀ78 °C, 41%; (b) H2, Pd(OH)2, HOAc, EtOH, ꢁ90%;
(c) BH3ꢂSMe2, THF, reflux, 78%; (d) 9, EDCI, DIEA 81%; (e) RSO2Cl, DIEA, 60–90%; (f)
TFA, DCM, ꢁ80%.
OH
H
H
N
Pr2N
O
N
O
N
X
Ar
(
)
O
R
n
BACE1
IC50 (nM)
Cmpd
Piperazine core
OH
H
N
3a
5
N
O2S
Ph
OH
H
N
12
120
N
Ph
OH
H
N
13
14
1150
490
N
O
Ph
OH
H
N
N
H
Figure 2. Initial SAR scan14 of various piperazine core derivatives (Ar = 3,5-difluorophenyl), along with overlay of their X-ray crystal structures in BACE1. Only sulfonamide 3a
(green) simultaneously occupies S20 and has a hydrogen bond to Thr72 NH of the flap (grey). N-Benzyl derivative 12 (yellow) only occupies S20, and benzamide 13 (blue) only
has the hydrogen bond to the flap.

J. Cumming et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2837–2842
2839
Synthesis of these novel inhibitors began with a stereoselective
Aldol addition of the conjugate base of piperazinone 5 to (S)- -ami-
structures (Fig. 2),15 clearly show the synergy of simultaneous con-
tact with the S20 pocket and the flap. Sulfonamide 3a is the only
analog that can achieve both of these interactions and is by far
the most active compound. It is 24-fold more potent than benzyl
analog 12 that only occupies S20, albeit with excellent overlap. In
addition, sulfonamide 3a is 230-fold more potent than benzamide
13 that has a hydrogen bond to the flap but, due to the sp2 nature
of the amide linker, cannot occupy S20.
With the piperazine sulfonamide core validated, we turned our
attention to optimization of the prime and non-prime side substi-
tution patterns of these inhibitors. On the prime side, there was a
relatively steep SAR around substitution on or replacement of the
S20 phenyl group, reflecting the relatively defined and rigid nature
of that sub-site. In fact, most substitutions of the phenyl group
caused a loss in BACE1 activity (Table 1) relative to unsubstituted
phenyl 3a. The exceptions to this general trend, with BACE1 Ki
values <10 nM, were ortho-tolyl, meta-tolyl, and meta-chloro-
phenyl inhibitors, 3b, 3c and 3f. The meta-tolyl compound 3c in
particular gave the best overall Ki and cell potency of these analogs.
a
no aldehyde 410 (Scheme 1). Adduct 6 was the major product of this
reaction. The desired absolute and relative stereochemistry of the
newly formed hydroxyl group was set under non-chelation control
enforced by the N,N-dibenzyl groups on the amino aldehyde.11,12
Debenzylation of adduct 6 via palladium-mediated hydrogenation
proceeded smoothly to give primary amine 7 that was then sub-
jected to borane reduction to give piperazine 8. The primary amine
of intermediate 8 was coupled to 5-methyl-N,N-dipropylisophthalic
acid (9)13 in the presence of EDCI and HOBt to give amide 10. The
piperazine N-benzyl group was removed with a second hydrogena-
tion to give core 11. Sulfonylation of this key intermediate followed
by removal of the Boc group gave BACE1 inhibitors 3, with phenyl-
sulfonamide 3a shown as a representative example.
In addition to sulfonamides, we also made analogous benzyl,
benzamide, and N-unsubstituted analogs 12–14 (Scheme 2) for
baseline comparison to sulfonamide 3a. The BACE1 potencies14 of
these four compounds, in conjunction with their X-ray crystal
Table 1
SAR of the piperazine sulfonamide S20 substituents with the N,N-dipropyl isophthalamide non-prime side binding motif (Ar = 3,5-difluorophenyl)14
OH
H
N
H
N
Pr2N
O
O
Ar
N
S
O
O
R
Compd
R
BACE1 Ki (nM)
Cell IC50 (nM)
120
Compd
R
BACE1 Ki (nM)
Cell IC50 (nM)
57
N
3a
3
3m
3
N
N
3b
5
64
3n
7
100
N
S
3c
1
48
3o
3p
8
83
3d
60
1300
10
400
N
Cl
N
3e
3f
400
7
3q
3r
3s
53
240
970
2900
O
Cl
S
N
240
N
3g
42
1700
Cl
N
OMe
3h
20
440
3t
1200
N
O
N
3i
120
310
73
3u
3v
5800
73
OMe
3j
Me
Pr
2300
1800
CN
Cl
3k
1500
1300
3w
95
Cl
OMe
3l
45
OMe

2840
J. Cumming et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2837–2842
Table 3
All para substitution (examples 3d, 3g, 3i, 3j) produced a drop in
potency of at least 10-fold, and addition of a meta substituent to
these analogs to give 3,4-disubstitution (compounds 3k, 3l) did
not significantly rescue the lost activity.
SAR of the sulfonamide substituents occupying the S20 subsite with the methoxy-
methyl pyrrolidine non-prime side binding motif (Ar = 3,5-difluorophenyl)14
MeO
Further expanding the scope of S20 SAR, several heterocycles
(Table 1, 3m–3p) gave BACE1 Ki values 610 nM, though did not of-
fer improvements in cell potency over meta-tolyl analog 3c despite
their increased polarity. A variety of other heterocycles were less
well tolerated (3q–3u, BACE1 Ki >50 nM). Finally, small alkyl sul-
fonamides 3v and 3w that did not display significant lipophilicity
to S20 showed only modest potency improvements (ꢁ5-fold) over
unsubstituted piperazine 14.
OH
H
N
H
N
N
O
O
Ar
N
S
O
O
R
Compd
R
BACE1 Ki (nM)
Cell IC50 (nM)
13
Using the optimized meta-tolyl sulfonamide piperazine core, we
next examined a number of isophthalamide variations that have
found wide use in the literature as non-prime side binding motifs
(Table 2). In particular, methoxymethyl pyrrolidine amide 3x16 and
sultam 3z17 were both very potent against BACE1 (Ki ꢁ1 nM) with
exceptional cellular activity (IC50 <10 nM). While some truncation
of these moieties was tolerated in vitro (e.g., analogs 3y, 3aa,18
3ee
1
Cl
3ff
1
22
N
3gg
3hh
2
1
70
68
S
N
Cl
Table 2
SAR of non-prime side isophthalamide variations with the meta-tolyl sulfonamide
core (Ar = 3,5-difluorophenyl)14
3ii
2
1
53
N
OH
H
N
H
N
R
3jj
14
O
N
O S
O
3kk
3ll
c-Pr
Me
7
13
100
220
Ar
3bb,19 BACE Ki 6 3 nM), these modifications all resulted in at least
fourfold loss of cellular potency (IC50 P 38 nM). Further reduction
in the size of the non-prime side group, as exemplified by lactam
3cc and simple acetamide 3dd, resulted in larger losses of both
in vitro and cellular potency.
Of the two potent lead sulfonamides 3x and 3z identified above,
the former was chosen for additional SAR development based on
its superior physico-chemical properties, specifically its lower
molecular weight and fewer hydrogen bond donors and accep-
tors.20 With this optimal methoxymethyl pyrrolidine isophthala-
mide group on the non-prime side, re-examination of the S20
pocket showed that a wide variety of aryl sulfonamide substituents
3ee–3jj (Table 3) gave excellent BACE1 activity (Ki 6 3 nM) and
good cellular potencies (IC50 <70 nM). Truncated alkysulfonamides
3kk and 3ll were also now fairly well tolerated, as opposed to the
earlier S20 SAR (c.f. analog 3ll in Table 3 vs analog 3v in Table 1).
Compd
R
BACE1 Ki (nM)
Cell IC50 (nM)
3c
1
1
48
Pr2N
O
O
MeO
3x
3y
7
N
MeO
3
1
270
N
O
SO2
N
Table 4
3z
8
H
N
In vitro, counterscreening, and permeability profile of piperazine sulfonamide 3x
(Ar = 3,5-difluorophenyl)
Ph
O
N
MeO
OH
SO2
SO2
H
N
H
N
N
3aa
3bb
1
1
62
38
O
O
N
O S
O
Ar
N
H
N
Assay
BACE1 Ki (nM)
0.8
BACE2 Ki (nM)
Cell IC50 (nM)
5.4
7.0
N
H
Cathepsin D Ki (nM)
Cathepsin E Ki (nM)
Pepsin Ki (nM)
200
86
73
O
3cc
14
1350
4350
BuN
Caco-2 AP-BL (efflux ratio)
1 (177)
3dd
Me
365

J. Cumming et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2837–2842
2841
In conclusion, using an iterative structure-based approach, we
have designed a series of novel peptidomimetic piperazine sulfon-
amide BACE1 inhibitors. The sulfonamide portion of these struc-
tures achieves both occupancy of S20 and capture of a key
hydrogen bond to the flap of the active site. These interactions,
in conjunction with optimal non-prime side binding motifs, gave
compounds with excellent BACE1 potency, both in vitro and in
cells. In addition, analog 3x produced a robust and persistent low-
ering of peripheral Ab in a transgenic mouse model following a sin-
gle subcutaneous dose. However, these compounds, as with many
peptidomimetics, are substrates for P-pg, and this liability limits
their oral bioavailability, brain penetration, and ultimately their
central efficacy. Based on these observations, we have directed sig-
nificant effort toward identification of non-peptidic BACE1 inhibi-
tors, and results will be reported in due course.25
60
50
40
30
20
10
0
-87%
30
-92%
-92%
300
VEH
100
mg/kg, s.c. (3h)
Dose
(mg/kg)
Brain
(ng/g)
Plasma
(ng/mL)
B/P
30
133
248
657
615
0.2
0.2
0.5
100
1506
300
1432
Acknowledgments
Figure 3. Acute dose–response of piperazinone sulfonamide 3x in 6-week-old pre-
plaque CRND8 mice along with average compound levels measured in brain and
plasma for each dose (eight animals per dose, administered subcutaneously, Ab40
and compound levels measured at 3 h post-dose. Changes in plasma Ab40 are
expressed as mean standard error as a percent of vehicle. For additional general
experimental details, see Ref. 21).
Use of the Advanced Photon Source at Argonne National Labora-
tory was supported by the US Department of Energy, Office of Sci-
ence, Office of Basic Energy Sciences, under Contract No. DE-AC02-
06CH11357. Use of the IMCA-CAT beamline 17-ID at the Advanced
Photon Source was supported by the companies of the Industrial
Macromolecular Crystallography Association.
Even with this subsequent exploration of the S20 subsite, how-
ever, sulfonamide 3x (in vitro profile in Table 4) still represented
the best lead in this series and was evaluated in vivo in 6-week-
old, pre-plaque CRND8 mice.21 This potent compound reduced
plasma Ab40 levels to background with single subcutaneous22
doses at or above 30 mg/kg (Fig. 3). Interestingly, these single
doses produced an effect that was not only robust at the 3 h time
point, but also persistent. The effect lasted 6 h for the 30 mg/kg
dose, at least 24 h for the 100 mg/kg dose, and the 300 mg/kg dose
still showed more than 90% reduction of plasma Ab40 at 12 h
(Fig. 4). Unfortunately, there was no effect on Ab40 levels in the cor-
tex of the mice at any of these doses or time points. Like many
hydroxyethylamine-based peptidomimetics.16b,23,24 sulfonamide
3x is a substrate for P-gp (Caco-2 efflux ratio >100). Despite this
high ratio, subcutaneous dosing generated modest levels of com-
pound in the brain (Fig. 3). With mouse plasma protein binding
of approximately 95%, however, the fraction of compound un-
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,
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100
80
60
40
20
0
-8%
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-67%
-94%
vs.
0 1
3
6
12
Time (h)
24
30 mg/kg
100 mg/kg
300 mg/kg
6. McConlogue, L.; Buttini, M.; Anderson, J. P.; Brigham, E. F.; Chen, K. S.;
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Figure 4. Time course of changes in plasma Ab40 following
a single dose of
piperazinone sulfonamide 3x in 6-week-old pre-plaque CRND8 mice (three to eight
animals per dose, compound administered subcutaneously, Ab40 levels for each
dose were measured at time points shown. Changes in Ab40 levels are normalized
relative to vehicle (100%) and are expressed as mean standard error. For
additional general experimental details, see Ref. 21).
8. As a representative example, coordinates for the X-ray structure of compound
1
complexed with BACE1 have been deposited in the Protein Data Bank
(www.rcsb.org), and can be accessed under PDB ID 2QP8.

2842
J. Cumming et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2837–2842
9. We employed both the de novo design method Allegrow (v. 020802, Boston De
Novo, Boston, MA) as well as minimization using the Polak–Ribiere conjugated
gradient algorithm until convergence of 0.001, CFF91 force field, Insight 2000/
CDISCOVER (Accelrys Inc., San Diego, CA).
10. Prepared from commercially available Boc-3,5-difluorophenylalanine: Reetz,
M. T.; Drewes, M. W.; Schwickardi, R. Org. Synth. 1999, 76, 110.
11. (a) Reetz, M. T.; Drewes, M. W.; Schmitz, A. Angew. Chem., Int. Ed. Engl. 1987, 26,
1141; For an extensive review of this chemistry, see: (b) Reetz, M. T. Chem. Rev.
1999, 99, 1121.
12. Diastereomeric ratios at the stereocenter formed on the piperazinone ring
typically ranged from 5:4 to 3:2, always in favor of the desired isomer. The
minor component was easily separable by silica gel chromatography to cleanly
afford adduct 6.
13. Maillaird, M.; Hom, R.; Gailunas, A.; Jagodzinska, B.; Fang, L. Y.; John, V.; Freskos,
J. N.; Pulley, S. R.; Beck, J. P.; Tenbrink, R. E. PCT Int. Appl. WO 2002002512.
14. Inhibition of BACE1 in vitro was determined using an APP-derived peptide
containing the Swedish mutant: (a) Kennedy, M. E.; Wang, W.; Song, L.; Lee, J.;
Zhang, L.; Wong, G.; Wang, L.; Parker, E. Anal. Biochem. 2003, 319, 49; Cellular
IC50 values for inhibition of Ab40 production were determined by incubating
HEK293 cells, stably transfected with the human APP cDNA containing both
Swedish and London FAD mutations, with increasing concentrations of BACE
inhibitors. Ab40 levels were measured in the cell culture media using an Ab1–40
specific ELISA assay: (b) Zhang, L.; Song, L.; Terracina, G.; Liu, Y.; Pramanik, B.;
Parker, E. Biochemistry 2001, 40, 5049.
15. Coordinates for the X-ray structures of compounds 3a, 12, and 13 complexed
with BACE1 have been deposited in the Protein Data Bank (www.rcsb.org), and
can be accessed under PDB ID 3LPI, 3LPJ, and 3LNK, respectively.
16. (a) Varghese, J.; Maillard, M.; Jagodzinska, B.; Beck, J. P.; Gailunas, A.; Fang, L.;
Sealy, J.; Tenbrink, R.; Freskos, J.; Mickelson, J.; Samala, L.; Hom, R. PCT Int.
Appl. WO 2003040096.; (b) Iserloh, U.; Pan, J.; Stamford, A. W.; Kennedy, M. E.;
Zhang, Q.; Zhang, L.; Parker, E. M.; McHugh, N. A.; Favreau, L.; Strickland, C.;
Voigt, J. Bioorg. Med. Chem. Lett. 2008, 18, 418; (c) Thompson, L. A.; Boy, K., M.;
Shi, J.; Macor, J. E. PCT Int. Appl. WO 2006099352.
17. (a) Barrow, J. C.; Coburn, C. A.; Nantermet, P. G.; Selnick, H. G.; Stachel, S. J.;
Stanton, M. G.; Stauffer, S. R.; Zhuang, L.; Davis, J. R. PCT Int. Appl. WO
2005065195.; (b) Eickmeier, C.; Fuchs, K.; Peters, S.; Dorner-Ciossek, C.; Heine,
N.; Handschuh, S.; Klinder, K.; Kostka, M. PCT Int. Appl. WO 2006103038.; (c)
Chirapu, S. R.; Pachaiyappan, B.; Nural, H. F.; Cheng, X.; Yuan, H.; Lankin, D. C.;
Abdul-Hay, S. O.; Thatcher, G. R. J.; Shen, Y.; Kozikowski, A. P.; Petukhov, P. A.
Bioorg. Med. Chem. Lett. 2009, 19, 264.
18. (a) Beswick, P.; Charrier, N.; Clarke, B.; Demont, E.; Dingwall, C.; Dunsdon, R.;
Faller, A.; Gleave, R.; Hawkins, J.; Hussain, I.; Johnson, C. N.; MacPherson, D.;
Maile, G.; Matico, R.; Milner, P.; Mosley, J.; Naylor, A.; O‘Brien, A.; Redshaw, S.;
Riddell, D.; Rowland, P.; Skidmore, J.; Soleil, V.; Smith, K. J.; Stanway, S.; Stemp,
G.; Stuart, A.; Sweitze, S.; Theobald, P.; Vesey, D.; Walter, D. S.; Ward, J.;
Wayne, G. Bioorg. Med. Chem. Lett. 2008, 18, 1022; (b) Clarke, B.; Demont, E.;
Dingwall, C.; Dunsdon, R.; Faller, A.; Hawkins, J.; Hussain, I.; MacPherson, D.;
Maile, G.; Matico, R.; Milner, P.; Mosley, J.; Naylor, A.; O‘Brien, A.; Redshaw, S.;
Riddell, D.; Rowland, P.; Soleil, V.; Smith, K. J.; Stanway, S.; Stemp, G.; Sweitzer,
S.; Theobald, P.; Vesey, D.; Walter, D. S.; Ward, J.; Wayne, G. Bioorg. Med. Chem.
Lett. 2008, 18, 1017; (c) Hussain, I.; Hawkins, J.; Harisson, D.; Hille, C.; Wayne,
G.; Cutler, L.; Buck, T.; Walter, D.; Demont, E.; Howes, C.; Naylor, A.; Jeffrey, P.;
Gonzalez, M. I.; Dingwall, C.; Michel, A.; Redshaw, S.; Davis, J. B. J. Neurochem.
2007, 100, 802; (d) Demont, E. H.; Faller, A.; MacPherson, D. T.; Milner, P. H.;
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WO 2004050619.
19. (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. J. Med. Chem. 2004, 47, 6117; (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. J. Med. Chem. 2004,
47, 6447.
20. Coordinates for the X-ray structure of compound 3x complexed with BACE1
have been deposited in the Protein Data Bank (www.rcsb.org), and can be
accessed under PDB ID 3LPK.
21. CRND8-APP mice are models for early onset (familial) AD that express human
APP containing both Swedish and London mutations that enhance the rate of
APP cleavage by BACE1 and favor production of Ab42 over Ab40 in the
c-
secretase cleavage step: Hyde, L. A.; Kazdoba, T. M.; Grilli, M.; Lozza, G.; Brussa,
R.; Zhang, Q.; Wong, G. T.; McCool, M. F.; Zhang, L.; Parker, E. M.; Higgins, G. A.
Behav. Brain Res. 2005, 160, 344.
22. Subceutaneous dosing was chosen due to the poor oral bioavailability of lead
compound 3x: rapid rat AUC0–6h (10 mg/kg, PO) = 0 nM h.
23. (a) Meredith, J. E., Jr.; Thompson, L. A.; Toyn, J. H.; Marcin, L.; Barten, D. M.;
Marcinkeviciene, J.; Kopcho, L.; Kim, Y.; Lin, A.; Guss, V.; Burton, C.; Iben, L.;
Polson, C.; Cantone, J.; Ford, M.; Drexler, D.; Fiedler, T.; Lentz, K. A.; Grace, J. E.,
Jr.; Kolb, J.; Corsa, J.; Pierdomenico, M.; Jones, K.; Olson, R. E.; Macor, J. E.;
Albright, C. F. J. Pharmacol. Exp. Ther. 2008, 326, 502; (b) Stanton, M. G.; Stauffer,
S. R.; Gregro, A. R.; Steinbeiser, M.; Nantermet, P.; Sankaranarayanan, S.; Price,
E. A.; Wu, G.; Crouthamel, M.; Ellis, J.; Lai, M.; Espeseth, A. S.; Shi, X.; Jin, L.;
Colussi, D.; Pietrak, B.; Huang, Q.; Xu, M.; Simon, A. J.; Graham, S. L.; Vacca, J. P.;
Selnick, H. J. Med. Chem. 2007, 50, 3431.
24. PK/PD data for BACE1 inhibition in a rhesus monkey model were recently
reported for
a fully de-peptidized transition state isostere with low
susceptibility to P-gp transport: Sankaranarayanan, S.; Holahan, M. A.;
Colussi, D.; Crouthamel, M.-C.; Devanarayan, V.; Ellis, J.; Espeseth, A.; Gates,
A. T.; Graham, S. L.; Gregro, A. R.; Hazuda, D.; Hochman, J. H.; Holloway, K.; Jin,
L.; Kahana, J.; Lai, M.-t.; Lineberger, J.; McGaughey, G.; Moore, K. P.; Nantermet,
P.; Pietrak, B.; Price, E. A.; Rajapakse, H.; Stauffer, S.; Steinbeiser, M. A.;
Seabrook, G.; Selnick, H. G.; Shi, X.-P.; Stanton, M. G.; Swestock, J.; Tugusheva,
K.; Tyler, K. X.; Vacca, J. P.; Wong, J.; Wu, G.; Xu, M.; Cook, J. J.; Simon, A. J. J.
Pharmacol. Exp. Ther. 2009, 328, 131.
25. (a) Zhu, Z.; Sun, Z.-Y.; Ye, Y.; Voigt, J.; Strickland, C.; Smith, E. M.; Cumming, J.;
Wang, L.; Wong, J.; Wang, Y.-S.; Wyss, D. F.; Chen, X.; Kuvelkar, R.; Kennedy, M.
E.; Favreau, L.; Parker, E.; McKittrick, B. A.; Stamford, A.; Czarniecki, M.;
Greenlee, W.; Hunter, J. C. J. Med. Chem. 2010, 53, 951; (b) Wang, Y.-S.;
Strickland, C.; Voigt, J.; Kennedy, M. E.; Beyer, B. M.; Senior, M. M.; Smith, E. M.;
Nechuta, T.; Madison, V.; Czarniecki, M.; McKittrick, B. A.; Stamford, A.; Parker,
E.; Hunter, J.; Greenlee, W.; Wyss, D. F. J. Med. Chem. 2010, 53, 942.