Cyclic hydroxyamidines as amide isosteres: Discovery of oxadiazolines and oxadiazines as potent and highly efficacious γ-secretase modulators in vivo

Article abstract of DOI:10.1021/jm201407j

Cyclic hydroxyamidines were designed and validated as isosteric replacements of the amide functionality. Compounds with these structural motifs were found to be metabolically stable and to possess highly desirable pharmacokinetic profiles. These designs were applied in the identification of γ-secretase modulators leading to highly efficacious agents for reduction of central nervous system Aβ₄₂ in various animal models.

Full text of DOI:10.1021/jm201407j

Article
pubs.acs.org/jmc
Cyclic Hydroxyamidines as Amide Isosteres: Discovery of
Oxadiazolines and Oxadiazines as Potent and Highly Efficacious
γ-Secretase Modulators in Vivo†
Zhong-Yue Sun,*,‡ Theodros Asberom,‡ Thomas Bara,‡ Chad Bennett,‡ Duane Burnett,‡ Inhou Chu,#
John Clader,‡ Mary Cohen-Williams,§ David Cole,‡ Michael Czarniecki,‡ James Durkin,‡
Gioconda Gallo,‡ William Greenlee,‡ Hubert Josien,‡ Xianhai Huang,‡ Lynn Hyde,§ Nicholas Jones,§
Irina Kazakevich,⊥ Hongmei Li,‡ Xiaoxiang Liu,‡ Julie Lee,§ Malcolm MacCoss,‡ Mihir B. Mandal,‡
Troy McCracken,‡ Amin Nomeir,# Robert Mazzola,‡ Anandan Palani,‡ Eric M. Parker,§
Dmitri A. Pissarnitski,‡ Jun Qin,‡ Lixin Song,§ Giuseppe Terracina,§ Monica Vicarel,‡ Johannes Voigt,∥
Ruo Xu,‡ Lili Zhang,§ Qi Zhang,§ Zhiqiang Zhao,‡ Xiaohong Zhu,‡ and Zhaoning Zhu*,‡
§
∥
⊥
‡Department of Medicinal Chemistry, Department of Neuroscience, Department of Structural Chemistry, Department of
#
Pharmaceutical Sciences, and Department of Drug Metabolism and Pharmacokinetics, Schering Plough Research Institute,
2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United States
ABSTRACT: Cyclic hydroxyamidines were designed and
validated as isosteric replacements of the amide functionality.
Compounds with these structural motifs were found to be
metabolically stable and to possess highly desirable pharma-
cokinetic profiles. These designs were applied in the identi-
fication of γ-secretase modulators leading to highly efficacious
agents for reduction of central nervous system Aβ42 in various
animal models.
is the Notch cleavage,5 producing a cell surface receptor in-
INTRODUCTION
Alzheimer’s disease (AD) is an ultimately fatal neurodegener-
■
volved in regulating cell differentiation and proliferation.6 GSI’s
selectivity issues against Notch and other biological pathways
ative disease that is the most common cause of age-related
became a significant concern when their clinical trials identified
dementia. More than 35 million people suffer from AD
higher incidence of skin cancer in the treatment group than in
worldwide, with an estimated annual cost of over $600 billion,
the placebo group among other known mechanism-based toxi-
and the AD population may increase to more than 115 million
by 2050 according to a report from Alzheimers Disease Inter-
cological findings.7
national.1 A great collective effort is being made across aca-
In 2001, a class of small molecules was found that alter the
product distribution among the known Aβ species without
demic and industrial laboratories to develop therapies to halt or
even reverse AD. There are two distinct pathological hallmarks
in the AD brains: extracellular amyloid plaques and intracellular
neurofibrillary tangles. Amyloid plaques are composed of insoluble
overall inhibition of the γ-secretase enzyme function including
APP and Notch processing. GSMs selectively reduce certain Aβ
species while enhancing the production of others.8,9 A sub-
class of these γ-secretase modulators (GSMs) exemplified by
deposits of amyloid-β (Aβ), particularly Aβ42, a 42 amino acid long
some nonsteroidal anti-inflammatory drugs (NSAIDs) such as
peptide produced from its precursor protein (APP) through
sequential cleavages by BACE12 and γ-secretase.3 Aβ42 and its
oligomers were found to be highly toxic in cells and were
ibuprofen, indomethacin, and sulindac sulfide was found to
elevate the level of the shorter Aβ species (Aβ37, Aβ38, and
proposed4 as the root cause for AD’s pathology, leading to
Aβ39) while inhibiting longer and more toxic Aβ40 and Aβ42.
This type of GSM presents a unique approach to address the
neurofibrillary tangle formation and eventual cell death.
central tenet of the amyloid hypothesis (i.e., Aβ42 inhibition)
while effecting a gentler perturbation of Aβ natural metabolism,
potentially leading to less mechanism-based toxicity involving
Aβ and γ-secretase inhibition.10 Therefore, it is highly attractive
to identify GSMs that can selectively reduce Aβ42 and elevate
the Aβ40/Aβ42 ratio and can be evaluated as anti-AD agents for
Aβ42, despite being the predominant component of the
amyloid plaque, is produced in minor proportion (5−10%) of
the total Aβ species composed of Aβ37 to Aβ42. They differ at the
C-terminus because of poor selectivity at the γ-secretase
cleavage step. Several classes of γ-secretase inhibitors (GSIs)
have been reported to effectively eliminate production of all Aβ
peptides in vitro and in vivo. However, γ-secretase has a wide
variety of substrates in addition to APP that are involved in
diverse biological processes. One of the most notable processes
Received: October 18, 2011
Published: November 18, 2011
© 2011 American Chemical Society
489
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
their safety and efficacy profiles in comparison to other
approaches targeting the Aβ process. Flurizan (Figure 1), the
hands, E-2012 showed good in vitro potency (HEK 293Swe‑Lon
cell Aβ42 IC50 = 64 nM) and a desirable GSM profile (Aβtotal
/
Aβ42 IC50 ratio of 226). It also exhibits robust rat in vivo
efficacy in reducing cerebrospinal fluid (CSF) Aβ42 by 55% and
cortical Aβ42 by 44% after a single oral dose of 30 mg/kg.
MODULATOR DESIGN
■
In an effort to design novel GSMs, we decided to focus on the
non-NSAID GSM class represented by compound 1 and try to
further optimize its overall profile and address its lenticular
toxicity issue. A survey of related patent literature indicated that
the imidazolylphenyl group is a highly conserved pharmaco-
phore across multiple subclasses of modulators from a number
of research laboratories. In contrast, the middle part of the
molecule, represented by the δ-lactam in 1, can be highly
variant, leaving room for exploration of a diverse range of
structures. Realizing that compound 1 is a GSM with a highly
optimized pharmacokinetic and pharmacodynamic profile, in
our modulator design, we aimed to learn from its electronic
topology while exploring significantly different chemical
structures. One striking SAR point represented by the potent
E-2012 analogue compound III (Aβ42 IC50 = 56 nM, com-
pound 639 in ref 13) indicates high tolerance in the enzyme
environment around the quarternalized benzylic position for
large structural changes on this portion of the molecule. More
importantly, the steric bulkiness at this site precluded the right-
hand lone electron pair of the lactam carbonyl group from
interacting with any potential H-bonding donor of the enzyme.
The combination of potentially high tolerance for structural
change and an idling right-hand lone electron pair on the
carbonyl offers an attractive opportunity to build a ring from
the position of the carbonyl oxygen to the benzylic position. An
imino group would be required to offer an extra valence to form
the ring while still preserving the left-hand lone pair electron
Figure 1. NSAID and non-NSAID γ-secretase modulators.
most advanced GSM developed by Myriad Genetics, failed to
demonstrate statistically significant efficacy in AD patients in a
phase III clinical trial. This result was not surprising given the
compound’s weak in vitro activity against γ-secretase Aβ42 cleavage
and its poor central nervous system (CNS) penetration,11
highlighting the need for a more potent and brain penetrant GSM.
In 2004, a new class of non-NSAID class of GSMs was
reported by scientists from Torrey Pines Therapeutics12 with
impressive potency in reduction of Aβ42 in cells. Compound I
(Figure 1) was also shown to reduce both plasma and brain
Aβ42 and Aβ40 in Tg 2576 mice. Furthermore, chronic
treatment of Tg 2576 mice with I lowered plaque density
and amyloid load in both hippocampus and cortex.9 Following
this lead, a biarylcinnamide class of GSM was reported later
that preserved the imidazolylphenyl group with the central
aminothiazole of I replaced by an α,β-unsaturated amide (e.g.,
1, II, and III in Figure 2).13,14 Compound 1 entered phase I
clinical studies in 2006, but the clinical development was
temporarily halted because of the finding of rat lenticular
toxicity after administration of high doses for 13 weeks.8e In our
Figure 2. Design of 1,2,4-oxadiazoline as an amide isostere.
490
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Monocyclic Oxadiazoline 4
a
Reagents and conditions: (a) microwave 150 °C, 30 min, THF; (b) NH2OH·HCl, KOAc, MeOH; (c) NCS/Py, DCM; (d) Et3N, DCM.
a
Scheme 2. Synthesis of 5-Alkyl-5-aryl Bicyclic Oxadiazolines
a
Reagents and conditions: (a) NBS, DMF; (b) Et3N, DMF; (c) t-BuOK, THF; (d) t-BuOK, THF; (e) NaBH4, THF/MeOH.
density as a replacement of the carbonyl group. However, an
imino group seldom is a viable replacement for the carbonyl
because of its high basicity and reactivity; a neutral version of
the imino group therefore should be a far better choice.
Hydroxylated imines such as oximes, hydroxyamidines,15 and
hydroxyguanidines16 are known to be much less basic than their
imine counterparts and in most cases far more stable. There-
fore, a cyclized oxadiazoline or oxadiazine appeared to be a
reasonable isosteric replacement of the lactam carbonyl in 1.
Comparing the core conformations of compound 1 (gray)
and oxadiazoline 2a (red), we found that they are completely
superimposible (Figure 2). Molecular designs such as 2a have
minimum perturbation of electronic topology compared to 1
while offering a different molecular framework for us to further
optimize its profile. However, oxadiazolines have not been
explored as viable pharmacophores, and their metabolic and
toxicological profiles were unknown. The monocyclic oxadiazo-
lines seem unstable under acidic conditions,17 but some fused
tricyclic oxadiazolines are stable in hot 20% HCl in MeOH.18
On the other hand, a ring expanded analogue of oxadiazoline,
oxadiazine, has been explored in the context of p38 MAP kinase
inhibitors, one of which was evaluated in phase II human clinic
trials for cardiovascular disease.19 In order to expeditiously
validate the proposal of isosteric replacement of the lactam
carbonyl group with cyclic hydroxyamidines, we first focused
on monocyclic oxadiazolines because of their straightforward
syntheses. Despite their known stability issues, the monocyclic
oxadiazolines (e.g., 4 in Scheme 1) should be a suitable probe
for the validity of the GSM molecular design using the in vitro
cellular assay, before more resource was applied to this direc-
tion. If successful, we planned to move to bicyclic oxadiazolines
(e.g., 2a in Figure 2) and oxadiazines (e.g., 2b in Figure 2) to
address any druggability issues.
CHEMISTRY
■
The monocyclic 1,2,4-oxadiazoline 4 was synthesized in three
steps as outlined in Scheme 1. The imidazolylbenzaldehyde 613
reacted with (formylmethylene)triphenylphosphorane to form
imidazolylcinnamaldehyde 7 which was subsequently converted
to oxime 8. Nitrile oxide 9, generated in situ by treatment of
8 with N-chlorosuccinimide and pyridine,20 reacted with the
ketimine 10 through a [3 + 2] dipolar cycloaddition to form
compound 4.
The bicyclic oxadiazoline compounds 2a, 3, and 12−19 were
prepared as described in Scheme 2 from the key intermediate
oxadiazolines (31a−j), which were synthesized from the [3 + 2]
491
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
a
Scheme 3. Synthesis of 3-(4-Fluorophenyl)-3-hydroxymethyl or 3-Aminomethyl Analogues
a
Reagents and conditions: (a) MeMgBr, Et3N, THF; (b) (S)-2-methyl-CBS-oxazaborolidine in toluene, BH3·Me2S, THF; (c) NaBH4, THF/MeOH;
(d) MeI, NaH, DMF; (e) Swern oxidation. (f) For 36 and 37: NaBH(OAc)3, HOAc, the corresponding amine, DCE. For 38: the reductive
amination with 2-aminoethylbenzoate followed by hydrolysis with LiOH. (g) Acetic anhydride, Et3N, DCM; (h) MeSO2Cl, Et3N, DCM.
a
Scheme 4. Synthesis of the Ketimine 30b
a
Reagents and conditions: (a) TMSCl, Et3N, DCM; (b) TMSOTf, DCM, reflux.
dipolar cycloaddition of the corresponding 3-bromopropylke-
timines 30a−j and a nitrile oxide generated from bromooxime
29 in situ.21 A potassium tert-butoxide mediated cyclization of
31a−j led to formation of the bicyclic oxadiazoline compounds
32a−j, which were finally coupled with the imidazolylbenzal-
dehyde 6 using Horner−Emmons conditions to give 2a, 3, and
12−19, respectively (Scheme 2).
The ester group in the bicyclic oxadiazolines 3 and 12−19 is
highly versatile in order to access alcohols and other functional
groups (Schemes 2 and 3). The success of the [3 + 2]
cycloaddition was highly dependent on the quality of the
corresponding imines, for which three synthetic methods had
to be developed based on different starting amines and
ketones. Nonfunctionalized ketimine 10 was generated from
3-methoxypropylamine and 4-fluoroacetophenone using 3 Å
molecular sieves as the dehydrating agent. Ketimine 30a con-
taining a 3-bromopropyl group was obtained with condensation
conditions involving TiCl4 and Et3N.22 Formation of ketimines
(30b−j) containing both ester and 3-bromopropyl groups was
found to be most efficient using TMS triflate catalyzed imine
synthesis from the TMS protected amine (42)23 and the corre-
sponding arylketoesters (Scheme 4)24 that were obtained from
the corresponding aryl bromides (43, 44) and diethyl oxalate
through the Grignard reaction as shown in Scheme 5.
Compound 3a is an enantiomer obtained by resolving the
racemate 3. The absolute structure of 3a was determined as the
492
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
a
only slowly decomposing in aqueous 0.1 N HCl at room tem-
perature over 3 h, leading to the corresponding acetophenone.
A cyclized version of the oxadiazoline such as compound 2a
would further stabilize the heterocyclic ring18 and may lead to a
suitable “druggable motif”.27 Indeed, the active enantiomer of
racemic 2a(α) is far more stable under the same conditions,
with deterioration of enantiomeric excess from 99.6% to 55.7%
overnight as the only observable change. The compound has a
pKa of 6.7, a value higher than the reported pKa for 1-phenyl-
imidazole (pKa = 5.1),28 indicating that the oxadiazoline core is
more basic than the N-phenylimidazole group. Compound
2a(α) possesses an excellent pharmacokinetic profile in all
animal species examined (Table 4) and has a low drug−drug
interaction liability as evidenced by lack of inhibition of human
cytochrome P450s, such as 3A4, 2D6, and 2C9, and lack of
induction of human hPXR. It has a hERG ion channel IC50 of
740 nM (Table 3) in a voltage clamp cellular assay and thus has
some risk of QTc prolongation in humans. More interestingly,
compound 2a(α) demonstrates a pharmacological profile indis-
tinguishable from E-2012 in vitro, with an IC50 of 58 nM for
reduction of Aβ42 and an Aβtotal/Aβ42 IC50 ratio of 237 in an
HEK293 cell assay (Table 1). When administered to rats,
compound 2a(α) dose dependently reduces CSF levels of Aβ42
by 51% and 30% 3 h after single oral doses of 10 and 3 mg/kg,
respectively, with corresponding brain exposure of 5 and
0.8 μM.
Scheme 5. Synthesis of the Ketoesters 45 and 46
a
Reagents and conditions: (a) i-PrMgCl·LiCl, THF.
S-configuration through its hydroxyethyl derivatives 11a and
11b, using the NMR spectra of their corresponding Mosher’s
esters.25
As illustrated in Scheme 6, oxadiazine 53 was synthesized via
lactam 51 which was prepared from 48 and 49 using a similar
method described in ref 13. The enantiomer of amino alcohol
48 was synthesized by the Sharpless aminohydroxylation26
from styrene 47. By use of a Mitsunobu reaction/deprotection
sequence, lactam 51 was then converted into hydroxylamine
52, followed by cyclization using P2O5 in refluxing EtOH
overnight to yield oxadiazine 53 in 90% enantiomeric excess.
RESULTS AND DISCUSSION
■
Racemic oxadiazoline 4, designed based on compound II
(Figure 2) for the initial GSM design validation, has IC50 of 187
nM for Aβ42 and IC50 of 2 μM for Aβ40 in the cellular assays.
Compared to the IC50 of compound II of 60 nM for Aβ42
reduction, these data confirm the modulator profile and the
validity of oxadiazoline as an isosteric replacement of the lactam
carbonyl group. Compound 4 is stable under neutral conditions,
The highly desirable in vitro and in vivo profile of 2a(α)
highlighted the urgency to resolve the hERG issue. A com-
prehensive medicinal chemistry effort was therefore launched to
further optimize this series. Among many directions, we
examined substitution effects on both 3-phenyl and 3-methyl
a
Scheme 6. Synthesis of Oxadiazine 53
a
Reagents and conditions: (a) CbzNH2, NaOH/tert-butyl hypochlorite, (DHQ)2PHAL, K2OsO2(OH)4, PrOH/water; (b) H2/Pd(OH)2; (c) EDCI,
HOBT, DIEA, DMF; (d), NaOMe, MeOH; (e) N-hydroxyphthalimide, PPh3, DIAD, THF; (f) NH2NH2, EtOH; (g) P2O5, EtOH, reflux.
493
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
Table 1. In Vitro Activity of 3-Aryl-3-alkyl Substituted Bicyclic Oxadiazoline Analogues
a
IC50, nM
compd
Ar
4-F-Ph
R
C3 config
Aβ42
Aβtotal
IC50(Aβtotal)/IC50(Aβ42)
b
2a(α)
5a
Me
58
29
13702
14130
>20000
3210
237
488
>3
4-F-Ph
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
S
5b
4-F-Ph
R
b
7643
96
20a
21a
22a
23a
24a
25a
26a
27a
Ph
33
b
b
b
b
b
b
b
3-F-Ph
26
7164
280
189
>171
316
1023
192
>189
2-F-Ph
53
10052
>20000
6750
4-CN-Ph
3,5-di-F-Ph
3,4,5-tri-F-Ph
5-F-Py-2-yl
5-F-Py-3-yl
117
21
15
15810
19716
>20000
103
106
a
b
Determined in HEKSwe‑Lon 293 cells. Active enantiomer.
groups on the oxadiazoline ring as shown in Table 1 with an
emphasis on reduction of hydrophobicity of these compounds
(see also ref 14 for examples of similar approaches around
E-2012). Hydroxylation of the 3-methyl group is tolerated with
an eudismic ratio between the two enantiomers (5a and 5b)
higher than 260-fold. The S-configuration (5a) is the preferred
stereochemistry, which agrees with what was reported for 1.
Additional fluorine substitution modestly improves potency
with the 3,4,5-trifluorophenyl (25a) being the most potent
(Aβ42 IC50 of 15 nM). Polar group incorporation into the 3-aryl
group (23a, 26a, and 27a) had a negative impact on GSM
potency.
Compound 5b, with a pKa of 5.8, was found to be more
stable compared to 2a(α), as it showed no sign of decom-
position or deterioration of enantiomeric excess after
7 days at room temperature in 0.1 N HCl/MeOH (1:1). The
excellent stability of 5b can be attributed to the increased
barrier to decomposition imposed by the inductive effect of
3-hydroxymethyl and potential stabilization from an intra-
molecular hydrogen bond between the hydroxyl group and the
oxadiazoline oxygen.
Encouraged by the tolerance to the C3-methyl modification,
we further explored the scope of the SAR at this position.
Replacement of the C3-methyl group with either configuration
of 1-hydroxyethyl is tolerated though no improvement was seen
(11a, 11b). Charged groups (36, 37) seem to reduce modu-
lator potency while the less basic hydroxyethylamino group has
the least impact (38). Small nonbasic nitrogen derivatives such
as amide (39) and sulfonamide (40) are modestly tolerated
(Table 2).
With the full validation of five-membered cyclic hydroxy-
amidines as lactam carbonyl isosteric replacement, we further
explored other variations of this concept by first examining
oxadiazines with a choice of trifluorophenyl group as exempli-
fied by compound 25a. Compound 53 displays two pKa values
of 7.4 and 2.7, indicating that the oxadiazine ring is modestly
more basic than oxadiazolines. This weak basicity gives 53 a
highly desirable aqueous solubility of greater than 100 mM at
pH 3.5 as an HCl salt, despite the fact that its measured log D
(5.29 at pH 7.4) suggested a molecule with a rather hydrophobic
Table 2. In Vitro Activity of (S)-3-(4-Fluorophenyl)-3-alkyl
Substituted Bicyclic Oxadiazoline Analogues
a
IC50, nM
IC50(Aβtotal)/-
IC50(Aβ42 )
compd
R
Aβ42
Aβtotal
11a
11b
35
CH(OH)Me (R)
CH(OH)Me (S)
CH2OMe
46
89
16237
>20000
17466
350
>225
144
122
191
196
80
36
CH2NHMe
>20000
18833
>105
96
37
CH2NMe2
38
CH2NHCH2CH2OH
CH2N(Me)COMe
CH2N(Me)SO2Me
>20000
>20000
19308
>250
>101
269
39
199
72
40
a
Determined in HEKSwe‑Lon 293 cells.
nature. Its nominal basicity notwithstanding, compound 53 is
a very potent GSM in vitro, indicating an excellent isosteric
replacement of the carbonyl group (Table 3).
Table 3. In Vitro Profiles of 2a(α), 5a, and 53
2a(α)
5a
53
1
a
a
Aβ42 IC50 (nM)
Aβ40 IC50 (nM)
58
29
33
64
267
170
123
2300
a
Aβtotal IC50 (nM)
14000
14130
>50000
3300
302
>20000
>50000
1000
246
14000
a
Notch IC50 (nM)
b
hERG IC50 (nM)
740
346
0.6
1200
579
1.2
c
Pa‑b (nm/s)
c
Pb‑a/Pa‑b
1.4
2
a
b
determined in HEK293Swe‑Lon cells. determined using a voltage-
c
clamp method. Pa‑b is the permeability when applied to the apical
side, while Pb‑a is the permeability when applied to the basolateral side
in a Caco-2 membrane assay (nm/s). The ratio of these two values
is a measure of Pgp efflux, while the Pa‑b value is a measure of
permeability.
494
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
The GSM characteristics of 5a and 53 were further con-
firmed by the changes they induce in the Aβ species produced
by HEK293Swe‑Lon cells in vitro as assessed by mass spectrom-
etry. Incubation of HEK293Swe‑Lon with 10 μM 53 selectively
reduced Aβ42, Aβ40, and Aβ39 while profoundly increasing
production of Aβ37 and Aβ38 (Figure 3). A similar modulator
profile was also observed for 5a.
side effects or biomarker changes following repeated admin-
istration of the highest tolerable doses of 5a and 53 to rats.
Neither compound produced goblet cell meta/hyperplasia in
the ileum, nor did they decrease thymus size or dramatically
affect body weight, all of which are common side effects of
γ-secretase inhibitors that reduce Notch cleavage. In addition,
HES-1 and adipsin expression, biomarkers for Notch activity,
were unaffected in the jejunum of treated rats.
Compound 53 (3 mg/kg, orally) reduces cortical Aβ42 to
63% of the level seen after vehicle administration, while 5a
(10 mg/kg, orally) reduces cortical Aβ42 levels to 37% (Table 5).
Compounds 5a and 53 were also found to dose-dependently
reduce Aβ42 preferentially over Aβtotal and Aβ40 upon acute
administration to rat (Figure 4). Similar results were observed
after sub-chronic (6 day) dosing in rat (data not shown). Four
hours after a single dose of 30 mg/kg to cynomolgous monkeys
(n = 3), 53 reduces Aβ42 in the cortex and CSF to 44% and
70% of the values seen after vehicle administration (Figure 5).
The corresponding plasma exposure of 53 4 h after admini-
stration to the monkeys was ∼6 μM, while the average brain/
plasma ratio in the monkeys was determined to be 1.4.
SUMMARY AND CONCLUSION
■
Cyclic hydroxyamidines were designed as novel isosteric
replacements of amide with a consideration of hydrogen
bonding characteristics and lack of strong basicity. Both five-
membered oxadiazolines and six-membered oxadiazines were
found to be ideal replacements for a lactam group. Importantly,
these novel druggable motifs were found to possess highly
desirable pharmacokinetic and toxicological profiles, offering ex-
cellent opportunities for exploration of amide bond replace-
ment, a frequently encountered but potentially difficult task in
drug discovery. Most valuable of all, both scaffolds were found
to be modestly basic, enabling salt formation for rapid dissolu-
tion with an appropriate formulation while maintaining amide
characteristics at physiological pH. The successful and expe-
ditious validation of these novel amide isosteres led to GSMs
that are highly efficacious in vitro and in vivo in a number of
animal models. Further exploration of these compounds as
potential drugs to fight Alzheimer’s disease is underway and will
be subjects of additional publications.
Figure 3. Mass spectrometric analysis of Aβ peptides generated from
HEK293Swe‑Lon cells after treatment with vehicle (DMSO) or 10 μM 53.
Compounds 5a and 53 are highly permeable in a Caco-2
membrane permeability assay and do not appear to be
substrates for PGP efflux (Table 3).29 These two compounds
possess excellent pharmacokinetic profiles typical of their struc-
tural classes in dog, rat, and monkey (Table 4). Both com-
pounds displayed a better overall hERG profile than compound
2a(α) because both reduced potency for blocking the hERG
channel and decreased exposure at the effective doses (Table 3).
Neither compound affected QTc intervals in dogs at sufficient
exposure multiples over exposures required for pharmacological
effect (data not shown).
Compounds 5a and 53 did not inhibit APP processing
(Table 3) in vitro as indicated by a lack of substrate accumu-
lation of C89 and C100, the cleavage products of preceding
α-secretase and BACE1 cleavages. Nor did they affect Notch
processing in HEK293 cells expressing the human Notch1
protein at concentrations up to 50 μM. The shift in specificity
of APP cleavage (Figure 3) rather than a reduction in overall
γ-secretase activity and the resulting lack of changes in substrate
accumulation and Notch processing confirm that both com-
pounds are potent GSMs. There was no evidence of Notch-related
EXPERIMENTAL SECTION
■
Cell Based γ-Secretase Assay. Human embryonic kidney
(HEK) 293 cells stably transfected with a human APP cDNA with
both the Swedish and London familial AD mutations in the pcDNA3.1
vector (Invitrogen, HEK293Swe‑Lon cells) were treated with GSM
a
Table 4. PK Parameters in Rat, Dog, and Monkey
a
b
b
c
d
d
d
compd
species
AUC (μM·h)
Cmax (μM)
BA (%)
T1/2 (h)
Vdss (L/kg)
CL (mL min−1 kg−1
)
2a(α)
rat
19
27
30
61
26
30
265
27
11
4.9
5.3
5.6
7.5
4.0
8.8
19.8
7.9
78
69
57
98
38
48
100
69
34
2.5
1.7
2
1.6
2.2
1
6.8
23
7.3
6.1
5
dog
monkey
rat
5a
53
3.4
1.4
3
1.6
0.4
1.4
0.4
0.6
0.8
dog
monkey
rat
6
3.5
0.8
0.9
1.4
9
dog
monkey
4.9
11
a
b
10 mg/kg for po; 3 mg/kg for iv in rat, dog, and monkey. AUC (area under the curve) and Cmax (maximal drug concentration) obtained with po
c
d
administration. BA: bioavalability. T1/2 (half-life), Vdss (steady state volume of distribution), and CL (in vivo clearance) obtained with iv
administration.
495
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
Table 5. In Vivo Profile of 5a and 53 in Rat
a
a
a
CSF Aβ (%)
Aβ40
cortex Aβ (%)
plasma Aβ (%)
Aβ40
compd
po (mg/kg)
plasma (μM)
B/P ratio
Aβ42
Aβtotal
Aβ42
Aβ40
Aβ42
Aβtotal
b
b
b
5a
10
3
3.6
3
0.15
0.49
66
65
104
120
88
37
77
84
41
44
77
86
c
53
74
63
32
50
a
b
Expressed as % vehicle in CSF, cortex, and plasma at 3 h after dosing. p < 0.05 vs vehicle-treated rats (ANOVA on raw values followed by
c
Dunnett’s post hoc t test comparing each treatment group to vehicle). Results for 53 are average of data from several studies; see Figure 4 for data
and variance.
Figure 4. Aβtotal, Aβ40, and Aβ42 in plasma (left), CSF (middle), and cortex (right) from rats treated acutely with various single oral doses of 53
(1−30 mg/kg). Data are the mean standard error of the mean averaged from several studies, expressed as % vehicle.
MSD-based sandwich immunoassay with Notch antibodies (Cell
Signaling).
CSF and Brain Aβ Assays. CSF Aβ42 was measured using
AlphaLISA Aβ1−42 and Aβ1−40 kits (Perkin-Elmer) according to the
manufacturer’s instructions. CSF total Aβ was measured using
MesoScale Discovery technology with biotinylated antibody 4G8
(Signet) and S-tag labeled antibody W02. Aβ from rat and monkey
brain was enriched by solid phase extraction using an Oasis HLB LP
96-well plate (Waters Corp.) and analyzed using AlphaLISA Aβ1−42
and Aβ1−40 kits.
Drug Treatment in Animals. Male CD rats (∼100 g, Crl:CD-
(SD); Charles River Laboratories, Kingston, NY) were group housed
and acclimated to the vivarium for 5−7 days prior to use in a study.
Compounds were formulated in 20% hydroxypropyl-β-cyclodextrin
and administered orally with a dosing volume of 5 mL/kg. Three
hours after drug administration, rats were euthanized with excess CO2.
Immediately following euthanasia, CSF was collected from the cisterna
magna and quickly frozen on dry ice. Only visibly clear CSF samples
were analyzed. Blood was collected from the vena cava, and plasma
was isolated using heparin as an anticoagulant. Finally, the brain was
removed from the skull and immediately frozen on dry ice. All tissues
were stored at −70 °C until Aβ quantification.
Adult male purpose bred cynomolgous monkeys (Macaca
fascicularis), weighing between 6 and 8 kg, were used. Monkeys
were fasted overnight prior to orogastric gavage tube administration of
compound (formulated in 0.4% MC with 1−2 equiv of tartrate acid as
an excipient) in a 1 mL/kg dosing volume. Three hours after drug
administration, monkeys were given an intramuscular injection of
ketamine HCl, a blood sample was collected from the femoral vein,
and the animals were euthanized by an intravenous dose of sodium
pentobarbital. CSF was collected from the cisterna magna immediately
following euthanasia, and then the brain was removed and frozen on
dry ice.
Figure 5. Aβtotal, Aβ40, and Aβ42 in plasma, CSF, and cortex from
monkeys treated with 53 orally at 30 mg/kg at 4 h. Data are expressed
as a percent of the predose value from the same animals (plasma) or as
a percent of the control (vehicle)-treated monkeys (CSF and cortex).
compounds for 5 h. Aβ in conditioned medium was measured using
MesoScale Discovery (MSD) technology-based sandwich immuno-
assays. Aβ42 was measured using a pair of labeled antibodies TAG-G2-
11 and biotin-4G8. Aβ40 was measured using the antibody pair of
TAG-G2-10 and biotin-4G8; total Aβ was measured using TAG-W02
and biotin-4G8. In some cases, a cell based γ-secretase assay was also
carried out in HEK293 cells stably coexpressing a C99 expression
construct and PS1ΔE9, a familial Alzheimer's disease mutant form
presenilin.30 Aβ in conditioned medium was measured using the same
methods described above.
Mass Spectrometric Analysis of Aβ Profiles. The Aβ profile in
conditioned medium was analyzed using surface enhanced laser
desorption/ionization (SELDI) mass spectrometry as described pre-
viously.31 The γ-secretase mixture of 50 μL was applied on a PS20
protein chip array (BioRad) that had been precoated with 1 μg of
antibody W02. The array was incubated overnight at 4 °C. Nonspecific
protein binding to the array was removed by washing three times in
phosphate buffered saline (PBS) with 0.2% Tween 20 followed by
three times in PBS. The array was rinsed briefly with water and air-
dried before being analyzed on BioRad.
Chemistry. 1H NMR spectra were measured on a Varian Oxford
400 or Bruker 500 UltraShield spectrometer. Chemical shifts δ are
reported relative to CDCl3 at 7.26 ppm as an internal standard. LCMS
spectra were obtained on a Shimadzu VP-Sciex API 150EX system.
HPLC spectra were obtained on a Shimadzu VP system. Normal
phase column chromatography was performed on prepacked silica
gel columns using ISCO CombiFlash system. Reverse phase column
chromatography was performed on Phenomenex Luna 10 μm C18
Notch Assay. HEK293 cells stably expressing a human Notch
protein with a truncated extracellular EGF repeat domain (NΔE)32
were treated with GSI or GSM compounds. Production of the Notch
intracellular domain (NICD) in cell lysates was measured using a
496
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
columns using Alltech 627 HPLC pump or Varian SD-1 HPLC
system. Chiral columns were from Chiral Technologies. The purity of
final compounds was analyzed on two independent reverse phase
HPLC systems with different gradient. LC−electrospray mass spectrom-
etry with a C-18 column using a gradient of 5−95% MeCN in water as
the mobile phase was used to determine the molecular mass and
retention time. The purity of the samples was assessed using a mass
detector and a UV detector at 254 nm. An additional analytical reverse
phase HPLC system was used to assess the purity of final compounds
using a UV detector monitored at both 219 and 254 nm and an ELSD
detector. All compounds have purity greater than 95%.
(E)-3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-imida-
zol-1-yl)benzylidene)-3-methyl-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridine (2a) and Its Enantiomers (2a(α) and
2a(β)). 2a, 2a(α), and 2a(β) were prepared from 30a similar to the
method for 3, 3a, and 3b. Compound 2a was resolved on an OD
column, eluting with 50% isopropanol in hexanes containing 0.1%
Et2NH to give 2a(α) and 2a(β). 1H NMR (400 MHz, CDCl3): δ 7.81
(1H, s), 7.58−7.55 (2H, m), 7.51 (1H, s), 7.26−7.24 (1H, d), 7.10−
7.06 (2H, t), 7.02−7.00 (1H, d), 6.98 (1H, s), 6.94 (1H, s), 3.85 (3H, s),
3.17−3.12 (1H, m), 2.87−2.78 (1H, m), 2.78−2.69 (1H, m), 2.69−
2.60 (1H, m), 2.32 (3H, s), 1.97−1.78 (2H, m), 1.89 (3H, s). LCMS
for 2a(α): m/z 433 [M + 1]+, 100% purity. LCMS for 2a(β): m/z 433
[M + 1]+, 99.9% purity.
in anhydrous THF (116 mL) in an ice bath, followed by slow addition
of anhydrous MeOH. The reaction mixture was stirred at room
temperature for 2 h, quenched with a mixture of ice and brine, and
extracted with EtOAc. The combined organic phase was washed with
brine, dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The crude was purified on a silica gel column, eluting with
MeCN containing 5% Et3N/EtOAc with a gradient from 0% to 100%
1
to give 5a (8.1 g, 77.7% yield). H NMR (400 MHz, CDCl3): δ 7.72
(1H, s), 7.52−7.48 (2H, m), 7.45 (1H, s), 7.26−7.23 (1H, d), 7.16−
7.08 (2H, t), 7.03−6.96 (2H, m), 6.92 (1H, s), 4.23−4.19 (1H, d),
4.09−4.05 (1H, d), 3.84 (3H, m), 3.36−3.32(1H, m), 2.99−2.75
(1H, m), 2.75−2.68 (2H, m), 2.60 (1H, br s), 2.29 (3H, s), 2.04−1.92
(1H, m), 1.92−1.80 (1H, m). LCMS m/z 449 [M + 1]+, 99.0% purity.
Compound 5b was prepared from 3b by the same method. HPLC:
96.8% purity.
Method b. Compound 3 (3 g, crude) was reduced according to
method a to give 5 (2.8 g, crude). This product (500 mg) was resolved
on an AD column, eluting with 65% isopropanol in hexanes containing
0.1% Et2NH to give 5a (183 mg, 36.6%) and 5b.
(E)-3-(3-Methoxy-4-(4-methyl-1H-imidazol-1-yl)phenyl)-
acrylaldehyde (7). A solution of 3-methoxy-4-(4-methyl-1H-imida-
zol-1-yl)benzaldehyde (6) (1.00 g, 4.6 mmol) and (formylmethylene)-
triphenylphosphorane (1.83 g, 6.0 mmol) in THF was heated at
150 °C in a microwave reactor for 30 min. Solvent was evaporated
under reduced pressure, and the residue was purified on a silica gel
column, eluting with EtOAc/hexanes in a gradient from 0% to 100%
to give 7 (1.05 g), which was used without further purification in the
preparation of 8.
3-(3-Methoxy-4-(4-methyl-1H-imidazol-1-yl)phenyl)-
acrylaldehyde Oxime (8). Hydroxylamine hydrochloride (2.35 g,
33.8 mmol) was added portionwise to a solution of 7 (6.3 g,
26.0 mmol) and potassium acetate in MeOH (300 mL) in an ice bath.
The reaction mixture was stirred at 0 °C for 90 min and then at room
temperature for 30 min. Solvent was evaporated under reduced pres-
sure. The residue was partitioned in EtOAc and brine, and aqueous
NaHCO3 was added to adjust the pH to 8. The aqueous phase was
extracted with EtOAc, and the combined organic phase was washed
with brine and dried over anhydrous Na2SO4. The crude was briefly
purified on a C18 column, eluting with MeCN/water containing 0.02%
concentrated HCl. The collected fraction was concentrated under reduced
pressure to remove MeCN, neutralized with aqueous NaHCO3, and
extracted with EtOAc to give 8 (2.48 g). LCMS m/z 258 [M + 1]+. It was
used without further purification in the preparation of 4.
(R)-1-((S,E)-3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-
1H-imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)ethanol (11a) and (S)-1-((S,E)-3-
(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-imidazol-1-yl)-
benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]oxadiazolo[4,3-a]-
pyridin-3-yl)ethanol (11b). Under N2 protection, a 1 M solution of
(S)-2-methyl-CBS-oxazaborolidine (4.1 mL, 4.1 mmol) in toluene was
added to a solution of the crude 33 (2.2 g, 4.9 mmol) in anhydrous
THF (15 mL) at room temperature, followed by slow addition of 2 M
solution of BH3·Me2S (3.7 mL, 7.3 mmol) in THF. The reaction
solution was stirred for 2 h, quenched with a mixture of brine and ice,
and extracted with EtOAc. The combined organic phase was washed
with brine and dried over anhydrous Na2SO4. The crude was purified
on a silica gel column, eluting with EtOAc/hexanes from 0% to 100%.
The desired fraction was concentrated under reduced pressure. The
resulting residue (1.3 g) was dissolved in THF (27 mL), treated with
concentrated hydrochloride (0.45 mL, 5.5 mmol) at room tempera-
ture for 1 h, neutralized with aqueous NaHCO3, concentrated under
reduced pressure, diluted with brine, and extracted with EtOAc. The
combined organic phase was washed with brine and dried over
anhydrous Na2SO4. The crude was purified on a C18 column, elut-
ing with MeCN/water containing 0.1% TFA. The desired frac-
tions were neutralized with aqueous NaHCO3, concentrated under
reduced pressure, and extracted with EtOAc to give 11a (0.56 g, 25.3%
yield) and 11b (0.12 g, 5.4% yield). Analytical data for 11a: 1H NMR
(400 MHz, CDCl3) δ 7.94 (1H, s), 7.50−7.46 (2H, m), 7.42 (1H, s),
7.23−7.21 (1H, d), 7.11−7.07 (2H, t), 6.97−6.91 (3H, m), 4.53−4.49
(1H, q), 3.82 (3H, s), 3.42−3.37 (1H, m), 2.99−2.95 (1H, m),
(E)-Ethyl 3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridine-3-carboxylate (3), and Its S-enan-
tiomer (3a) and R-Enantiomer (3b). Under N2 protection, a solu-
tion of potassium t-BuOK (22.3 g, 199.5 mmol) in anhydrous THF
(200 mL) was added slowly to a solution of 32b (81.3 g, 190.0 mmol)
and 3-methoxy-4-(4-methyl-1H-imidazol-1-yl)benzaldehyde (6)
(39.0 g, 180.5 mmol) in anhydrous THF (1900 mL) at −78 °C.
The reaction mixture was stirred at −78 °C for 45 min, allowed to
warm to −20 °C over a period of 30 min, diluted with brine, and
extracted with EtOAc. The combined organic phase was washed with
brine, dried over anhydrous Na2SO4, and concentrated under reduced
pressure to give 3 (91 g, crude). This product (88 g) was resolved in an AS
column, eluting with 55% isopropanol in hexanes containing 0.1% Et2NH
to give the S-enantiomer 3a (30.6 g, 34.8% yield) and the R-enantiomer
3b. Analytical data for 3a: 1H NMR (400 MHz, CDCl3) δ 7.70 (1H, s),
7.53−7.47 (3H, m), 7.24−7.22 (1H, d), 7.13−7.09 (2H, t), 7.00−6.98
(1H, d), 6.96 (1H, s), 6.91 (1H, s), 4.40−4.28 (2H, m), 3.83 (1H, s),
3.58−3.55 (1H, m), 2.88−2.85 (1H, m), 2.76−2.63 (2H, m), 2.28 (3, s),
1.99−1.91 (1H, m), 1.73−1.69 (1H, m), 1.35−1.31 (3H, t). LCMS m/z
491 [M + 1]+, 100% ee. 3b: LCMS m/z 491 [M + 1]+, 99% ee.
(E)-5-(4-Fluorophenyl)-3-(3-methoxy-4-(4-methyl-1H-imida-
zol-1-yl)styryl)-4-(3-methoxypropyl)-5-methyl-4,5-dihydro-
1,2,4-oxadiazole (4). A mixture of 4-fluoroacetophenone (1.36 g,
9.9 mmol), 3-methoxypropylamine (1.76 g, 19.8 mmol), 3 Å molecular
sieves, and 18 mL of DCM in a sealed tube was heated at 50 °C for 3 h
and then was at room temperature for several days. The filtrate was
concentrated under reduced pressure to give 10 (1.03 g). A mixture
of 8 (100 mg, 0.39 mmol), NCS (51.9 mg, 0.39 mmol), and pyridine
(7.8 mg, 0.1 mmol) in DCM (1.2 mL) was stirred at room tem-
perature for 10 min, followed by additions of 10 (98 mg, 0.39 mmol)
and Et3N (0.8 mL). The reaction mixture was stirred overnight,
diluted with DCM, washed with water, and dried over anhydrous
Na2SO4. The crude was purified on a silica gel column MeOH con-
taining 2% Et3N/DCM with a gradient from 0% to 5%, followed by
purification on a C18 column, eluting with MeCN/water containing
1
0.1% formic acid to give 4 (27 mg). H NMR (400 MHz, CDCl3):
δ 7.80 (1H, br s), 7.59−7.54 (2H, m), 7.48−7.44 (1H, d), 7.27−7.25
(1H, d), 7.18−7.16 (1H, d) 7.12 (1H, s), 7.08−7.04 (2H, t), 6.94
(1H, br s), 6.58−6.54 (1H, d). LCMS m/z 465 [M + 1]+, 97.7% purity.
(S,E)-(3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-imi-
dazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)methanol (5a) and (R,E)-(3-(4-
Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-imidazol-1-yl)-
benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]oxadiazolo[4,3-a]-
pyridin-3-yl)methanol (5b). Method a. Sodium borohydride
(1.3 g, 34. mmol) was added to a solution of 3a (11.4 g, 23.2 mmol)
497
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
2.79−2.71 (1H, m), 2.54−2.48 (1H, m), 2.30 (3H, s), 1.95−1.76 (2H, m),
1.15−1.13 (3H, d); LCMS m/z 463 [M + H]+, 97.8% purity. 11b:
1H NMR (400 MHz, CDCl3) δ 8.03 (1H, s), 7.63−7.59 (2H, m), 7.44
(1H, s), 7.24−7.22 (1H, d), 7.10−7.06 (2H, t), 6.98−6.92 (3H, m),
4.58−4.53 (1H, q), 3.83 (3H, s), 3.27−3.22 (1H, m), 2.88−2.84 (1H,
m), 2.75−2.71 (1H, m), 2.55−2.49 (1H, m), 2.31 (3H, s), 1.86−1.79
(2H, m), 1.37−1.35 (3H, d). LCMS m/z 463 [M + H]+, 99.0% purity.
(E)-(8-(3-Methoxy-4-(4-methyl-1H-imidazol-1-yl)-
benzylidene)-3-phenyl-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)methanol (20) and Its Enan-
tiomer (20a). 20 and 20a were prepared from 30c, similar to the
procedure for 5a in method b. The racemate 20 was resolved on an OJ
procedure for 5a in method b from 30i which was made from 45
according to the procedure for 30b. The racemate 26 was resolved on
an OJ column, eluting with 40% isopropanol in hexanes to give 26a.
1H NMR (400 MHz, CDCl3) δ 8.41 (1H, m), 7.84−7.81 (1H, m),
7.72 (1H, s), 7.53−7.48 (1H, m), 7.46 (1H, s), 7.26−7.23 (1H, d),
7.00−6.97 (1H, d), 6.96 (1H, s), 6.92 (1H, s), 4.45−4.42 (1H, d),
4.05−4.02 (1H, d), 3.84 (3H, s), 3.54−3.48 (1H, m), 3.19−3.14 (1H, m),
2.73−2.63 (2H, m), 2.29 (3H, s), 1.97−1.89 (1H, m), 1.83−1.75
(1H, m). LCMS m/z 450 [M + H]+, 91.4% purity.
(E)-(3-(5-Fluoropyridin-3-yl)-8-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)methanol (27) and Its Enan-
tiomer (27a). 27 and 27a were prepared according to the similar
procedure for 5a in method b from 30j which was made from 46
according to the procedure for 30b. Analytical data for 27a: 1H NMR
(400 MHz, CDCl3) δ 8.55−8.51 (2H, m), 7.72 (1H, s), 7.68−7.65
(1H, m), 7.46 (1H, s), 7.26−7.24 (1H, d), 7.00−6.93 (3H, m), 4.25−
4.22 (1H, d), 4.10−4.07 (1H, d), 3.84 (3H, s), 3.46−3.40 (1H, m),
3.09−3.04 (1H, m), 2.75−2.70 (2H, m), 2.29 (3H, s), 2.05−1.85 (2H, m).
LCMS m/z 450 [M + H]+, 95% purity.
3-Bromo-N-(1-(4-fluorophenyl)ethylidene)propan-1-amine
(30a). Under N2 protection, Et3N (40 mL, 29.0 mmol) was added
slowly to a solution of 4-fluoroacetophenone (5 g, 36.2 mmol) and
3-bromopropan-1-amine hydrobromide (41) (10.3 g, 47.1 mmol) in
anhydrous DMF (35 mL) and DCM (10 mL) while vigorously
stirring. A solution of TiCl4 (6.2 g, 32.6 mmol) in DCM (29 mL)
was added dropwise at 0 °C. The reaction suspension was vigorously
stirred at room temperature overnight. The reaction mixture was
diluted with ether and filtered to remove solid. The filtrate was washed
with ice cold brine 4 times and dried over anhydrous Na2SO4 to give
30a (6.8 g, 73.0% yield). The product was used without further
purification in the preparation of 1.
1
column, eluting with 65% isopropanol in hexanes to give 20a. H
NMR (400 MHz, CDCl3): δ 7.71 (1H, s), 7.52−7.49 (2H, m), 7.46−
7.39 (4H, m), 7.26−7.23 (1H, d), 7.00−6.97 (1H, d), 6.96 (1H, s),
6.92 (1H, s), 4.25−4.22 (1H, d), 4.10−4.07 (1H, d), 3.84 (3H, s),
3.39−3.33 (1H, m), 3.05−3.00 (1H, m,), 2.73−2.70 (2H, m), 2.29
(3H, s), 2.00−1.84 (2H, m). LCMS m/z 431 [M + 1]+, 100% purity.
(E)-(3-(3-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-imida-
zol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]oxadiazolo-
[4,3-a]pyridin-3-yl)methanol (21) and Its Enantiomers
(21a). 21 and 21a were prepared from 30d, similar to the procedure
1
for 5a in method b. Analytical data for 21a: H NMR (400 MHz,
CDCl3) δ 7.73 (1H, s), 7.46 (1H, s), 7.43−7.38 (1H, m), 7.26−7.24
(3H, m), 7.12−7.07 (1H, m), 7.01−6.98 (1H, d), 6.97 (1H, s), 6.93
(1H,s), 4.22−4.19 (1H, d), 4.07−4.03 (1H, d), 3.85 (3H, s), 3.40−
3.34 (1H, m), 3.06−3.01 (1H, m), 2.74−2.71 (2H, m), 2.30 (3H, s),
2.01−1.84 (2H, m). LCMS m/z 449 [M + 1]+, 96.0% purity.
(E)-(3-(2-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-imida-
zol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]oxadiazolo-
[4,3-a]pyridin-3-yl)methanol (22) and Its Enantiomers
(22a). 22 and 22a were prepared from 30e, similar to the procedure
1
for 5a in method b. Analytical data for 22a: H NMR (400 MHz,
Ethyl 2-(3-Bromopropylimino)-2-(4-fluorophenyl)acetate
(30b). Under N2 protection, the reaction solution of 42 (306.8 g,
1.1 mol), ethyl 2-(4-fluorophenyl)-2-oxoacetate (209 g, 1.1 mol), and
trimethylsilyl trifluoromethanesulfonate (11.8 g, 0.05 mol) in DCM
(1500 mL) was refluxed for 4 days, cooled to room temperature,
washed with cold aqueous NaHCO3 and brine, and dried over
anhydrous Na2SO4 to give 30b (332 g, 98.6% yield). 1H NMR
(400 MHz, CDCl3): δ 7.71−7.67 (2H, m), 7.24−7.05 (2H, m), 4.45−
4.39 (2H, q), 3.66−3.62 (2H, t), 3.55−3.52 (2H, t), 2.28−2.2.25
(2H, m), 1.41−1.37 (3H, t).
CDCl3) δ 7.75−7.71 (2H, m), 7.44 (1H, s), 7.41−7.35 (1H, m),
7.25−7.21 (2H, m), 7.12−7.07 (1H, m), 6.99−6.92 (3H, m), 4.44−
4.41 (1H, d), 4.06−4.03 (1H, d), 3.84 (3H, s), 3.52−3.47 (1H, m),
3.15−3.10 (1H, m), 2.83−2.75 (1H, m), 2.63−2.55 (1H, m) 2.29 (3H, s),
1.97−1.88 (2H, m). LCMS m/z 449 [M + 1]+, 97.0% purity.
(E)-4-(3-(Hydroxymethyl)-8-(3-methoxy-4-(4-methyl-1H-imi-
dazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)benzonitrile (23) and Its Enan-
tiomer (23a). 23 and 23a were prepared from 30f, similar to the
procedure for 5a in method b. Analytical data for 23a: 1H NMR (400
MHz, CDCl3) δ 7.74−7.72 (3H, m), 7.65−7.63 (2H, d), 7.45 (1H, s),
7.26−7.24 (1H, d), 7.01−6.98 (1H, d), 6.96 (1H, s), 6.93 (1H, s),
4.24−4.21 (1H, d), 4.07−4.04 (1H, d), 3.85 (3H, s), 3.43−3.37 (1H, m),
3.05−2.99 (1H, m), 2.75−2.71 (2H, m), 2.30 (3H, s), 2.04−1.84
(2H, m). LCMS m/z 456 [M + 1]+, 95.3% purity.
(E)-(3-(3,5-Difluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)methanol (24) and Its Enan-
tiomer (24a). 24 and 24a were prepared from 30g, similar to the
procedure for 5a in method b. The racemate 24 was resolved on an
OD-H column by SFC using IPA as a cosolvent to give 24a. 1H NMR
(400 MHz, CDCl3) δ 7.72 (1H, s), 7.45 (1H, s), 7.26−7.25 (1H, d),
7.06−7.04 (2H, m), 7.00−6.98 (1H, d), 6.96 (1H, s), 6.93 (1H, d),
6.87−6.82 (1H, m), 4.18−4.15 (1H, d),), 4.04−4.01 (1H, d), 3.85
(3H, s), 3.43−3.37 (1H, m), 3.10−3.03 (1H, m), 2.74−2.71 (2H, m),
2.30 (3H, s), 2.00−1.85 (2H, m). LCMS m/z 467 [M + 1]+, 100% purity.
(E)-(8-(3-Methoxy-4-(4-methyl-1H-imidazol-1-yl)-
benzylidene)-3-(3,4,5-trifluorophenyl)-5,6,7,8-tetrahydro-3H-
[1,2,4]oxadiazolo[4,3-a]pyridin-3-yl)methanol (25) and Its
Enantiomer (25a). 25 and 25a were prepared from 30h, similar to
the procedure for 5a in method b. Analytical data for 25a: 1H NMR (500
MHz, CDCl3) δ 7.76 (1H, s), 7.46 (1H, s), 7.30−7.20 (3H, m), 7.01−6.96
(3H, m), 4.18−4.16 (1H, d), 4.10−4.08 (1H, d), 3.86 (3H, s), 3.46−3.40
(1H, m), 3.10−3.04 (1H, m), 2.80−2.60 (2H, m), 2.31 (3H, s), 2.00−1.95
(1H, m), 1.95−1.88 (1H, m). LCMS m/z 485 [M + 1]+, 96.0% purity.
(E)-(3-(5-Fluoropyridin-2-yl)-8-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)methanol (26) and Its Enan-
tiomer (26a). 26 and 26a were prepared according to the similar
Ethyl 4-(3-Bromopropyl)-3-((diethoxyphosphoryl)methyl)-5-
(4-fluorophenyl)-4,5-dihydro-1,2,4-oxadiazole-5-carboxylate
(31b). Under N2 protection, NBS (101.3 g, 569 mmol) in DMF
(240 mL) was added dropwise to a solution of diethyl 2-(hydroxy-
imino)ethylphosphonate (28) (111 g, 569 mmol) in DMF (470 mL)
at −30 °C. The cooling bath was removed, and the reaction mixture
was stirred for about 1 h to give 29 in DMF solution. This solution
was slowly mixed with 30b (178.8 g, 569 mmol) in DMF (400 mL) at
−10 °C, followed by dropwise addition of Et3N (39.5 mL, 285 mmol)
in DMF (250 mL) in an ice bath. The resulting reaction mixture was
stirred at room temperature overnight. Additional 39.5 mL of Et3N in
DMF (250 mL) was added dropwise in an ice bath, and the reaction
mixture was stirred at room temperature for 4 h. Then an additional
1 equiv of 29 was made as described above and added dropwise to the
reaction mixture in an ice bath, followed by slow addition of Et3N
(79 mL) in two portions using the procedure described above. The reac-
tion mixture was diluted with 70% EtOAc in hexanes and half-saturated
NaCl in water. The aqueous phase was extracted with 70% EtOAc in
hexanes. The combined organic phase was washed with a mixture of
NaHCO3 and NaCl in water, with half-saturated NaCl in water, and dried
over anhydrous Na2SO4. The crude was purified on a silica gel column,
eluting with EtOAc/hexanes in a gradient from 0% to 70% to give 31b
1
(122.6 g, 42.6% yield). H NMR (400 MHz, CDCl3) δ 7.49−7.46 (2H,
m), 7.12−7.07 (2H, m), 4.33−4.27 (2H, m), 4.21−4.16 (4H, m), 3.54−
3.46 (1H, m), 3.42−3.34 (1H, m) 3.22−3.18 (2H, m), 2.99−2.93(2H, m),
1.81−1.70 (1H, m), 1.62−1.51 (1H, m), 1.36−1.26 (9H, m).
Ethyl 8-(Diethoxyphosphoryl)-3-(4-fluorophenyl)-5,6,7,8-
tetrahydro-3H-[1,2,4]oxadiazolo[4,3-a]pyridine-3-carboxylate
(32b). Under N2 protection, t-BuOK (29.7 g, 265.0 mmol) in
498
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
anhydrous THF (265 mL) was added dropwise to a solution of 31b
(122.6 g, 240.9 mmol) in anhydrous THF (2400 mL) at −70 °C. The
reaction mixture was allowed to warm to 12 °C over a period of 2 h,
diluted with brine, and extracted with EtOAc. The combined organic
phase was washed with brine and dried over anhydrous Na2SO4. The
crude was purified on a silica gel column, eluting with EtOAc/hexanes
in a gradient from 0% to 100% to give 32b (82.6 g, 80.1% yield), a
mixture of diastereomers A and B. The analytical diastereomer samples
were obtained by purification on silica gel column again. 1H NMR
(400 MHz, CDCl3), diastereomer A: δ 7.45−7.41 (2H, m), 7.08−7.04
(2H, m), 4.30−4.12 (6H, m), 3.33−3.28 (1H, m), 3.13−3.05 (1H, tt),
2.79−2.74 (1H, m), 2.13−1.75 (4H, m), 1.34−1.26 (9H, m). Diastereo-
mer B: δ 7.45−7.41 (2H, m), 7.10−7.05 (2H, m), 4.33−4.14 (6H, m),
3.42−3.37 (1H, m), 3.15−3.06 (1H, tt), 2.62−2.56 (1H, m), 2.18−2.08
(2H, m), 1.92−1.78 (1H, m), 1.68−1.57 (1H, m), 1.36−1.27 (9H, m).
(S,E)-1-(3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)ethanone (33). A solution of 3 M
MeMgBr in ether (3.4 mL, 10.3 mmol) and Et3N (4.3 mL, 31 mmol)
in THF (6.8 mL) was added dropwise to a stirred solution of 3a (2.5 g,
5.2 mmol) in 15.6 mL of THF at −60 °C under nitrogen atmosphere.
The reaction mixture was allowed to warm to 10 °C over a period of
5 h, stirred for an additional 1 h at room temperature, quenched with a
mixture of brine and ice, diluted with EtOAc, filtered through a Celite
pad, and extracted with EtOAc. The combined organic phase was
washed with brine and dried over anhydrous Na2SO4. The crude was
purified on a silica gel column, eluting with EtOAc/hexanes from 0% to
75% to give a crude of 33 (1.7 g) containing 50% of 3a. This crude was
used without further purification in the preparation of 11a and 11b. The
analytical sample was purified on a C18 column, eluting with MeCN/
water containing 0.1% formic acid. 1H NMR (400 MHz, CDCl3): δ 7.67
(1H, s), 7.47−7.43 (3H, m), 7.21−7.19 (1H, d), 7.12−7.08 (2H, t),
6.96−6.94 (1H, d), 6.92 (1H, s), 6.88 (1H, s), 3.80 (3H, s), 3.58−3.53
(1H, m), 2.83−2.77 (1H, m), 2.70−2.59 (2H, m), 2.25−2.24 (6H,
ss),1.92−1.87 (1H, m), 1.72−1.66 (1H, m).
added to a stirred reaction mixture of 34 (200 mg, 0.45 mmol), 2 M
solution methylamine in THF (0.5 mL, 1 mmol), and acetic acid (0.051 mL,
0.9 mmol) in DCE (12 mL) at 0 °C under nitrogen atmosphere.
The reaction mixture was stirred at room temperature overnight,
quenched with aqueous NaHCO3, and extracted with EtOAc. The
organic phase was dried over Na2SO4 and concentrated under reduced
pressure. The crude was purified on a C18 column with MeCN/water
containing 0.1% formic acid. The desired fraction was neutralized with
aqueous NaHCO3, concentrated under reduced pressure, and extracted
1
with EtOAc to give 36 (87.9 mg, 42.5% yield). H NMR (400 MHz,
CDCl3): δ 9.15 (1H, s), 7.44−7.36 (3H, m), 7.13−7.09 (2H, t), 7.05
(1H, s), 6.94 (1H, s), 6.79 (1H, s), 6.73−6.71 (1H, d), 3.99−3.95 (1H,
d), 3.82 (3H, s), 3.72−3.69 (1H, d), 3.44−3.38 (1H, m), 2.93 (3H, s),
2.76−2.67 (2H, m), 2.44 (3H, s), 2.39−2.32 (1H, m), 1.99−1.87
(1H, m), 1.73−1.61 (1H, m). LCMS m/z 462 [M + H]+, 99.9% purity.
(S,E)-1-(3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)-N,N-dimethylmethanamine
(37). 37 was made from 34 and dimethylamine according to the
procedure for 36. 1H NMR (400 MHz, CDCl3): δ 7.69 (1H, s), 7.54−
7.50 (2H, m), 7.46 (1H, s), 7.22−7.20 (1H, d), 7.09−7.05 (3H, t),
6.97−6.95 (1H, d), 6.94 (1H, s), 6.91 (1H, s), 3.82 (3H, s), 3.23−3.18
(1H, m), 3.09−3.05 (1H, d), 2.95−2.91 (1H, d), 2.85−2.79 (1H, m),
2.76−2.70 (1H, m), 2.60−2.52 (1H, m), 2.50 (6H, s), 2.28 (3H, s),
1.90−1.79 (2H, m). LCMS m/z 476 [M + H]+, 98.1% purity.
(S,E)-2-((3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)methylamino)ethanol (38). 38
was made from 34 and 2-aminoethyl benzoate according to the
procedure for 36, followed by hydrolysis of the benzoate with lithium
1
hydroxide. H NMR (400 MHz, CDCl3): δ 9.03 (1H, s), 7.45−7.41
(2H, m), 7.35−7.33 (1H, d), 7.15−7.09 (3H, m), 7.05 (1H, s), 6.88
(1H, s), 6.81−6.79 (1H, d), 4.20−4.17 (1H, d), 4.02−3.97 (2H, m),
3.85 (3H, s), 3.66−3.63 (1H, d), 3.46−3.43 (2H, m), 3.39−3.35 (1H, m),
2.76−2.70 (2H, m), 2.45 (3H, s), 2.37−2.29 (1H, m), 1.94−1.86 (1H, m),
1.75−1.69 (1H, m). LCMS m/z 492 [M + H]+, 99.4% purity.
(S,E)-N-((3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)methyl)-N-methylacetamide
(39). Acetic anhydride (5.3 mg, 0.052 mmol) in DCM (0.1 mL)
was added dropwise to a stirred reaction mixture of 36 (20 mg,
0.043 mmol) and Et3N (0.02 mL) in anhydrous DCM (0.4 mL) at
0 °C under nitrogen atmosphere. The reaction mixture was stirred for
30 min, allowed to warm to room temperature, quenched with water,
concentrated to remove DCM, and purified on a C18 column, eluting
with MeCN/water containing 0.1% formic acid. The desired fraction
was neutralized with aqueous NaHCO3, concentrated under reduced
(S,E)-3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-imi-
dazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridine-3-carbaldehyde (34). Under N2 pro-
tection, DMSO (0.16 mL, 2.2 mmol) was added dropwise to a
solution of oxalyl dichloride (0.11 mL, 1.3 mmol) in anhydrous DCM
(11.1 mL) at −78 °C, followed by dropwise addition of 5a (500 mg,
1.1 mmol) in anhydrous DCM (1.5 mL). After the mixture was stirred
at −78 °C for 1 h, Et3N (0.62 mL, 4.5 mmol) was added dropwise.
The reaction mixture was stirred for 30 min, allowed to warm to room
temperature, diluted with cold brine, and extracted with DCM. The
combined organic phase was washed with brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure to give 34 (507
mg), which was used without further purification in the preparations of
36, 37, and 38. MS m/z 447.
1
pressure, and extracted with EtOAc to give 39 (9.4 mg, 43.1%). H
NMR (400 MHz, CDCl3): δ 7.69 (1H, s), 7.58−7.55 (2H, m), 7.44
(1H, s), 7.23−7.21 (1H, d), 7.11−7.07 (2H, t), 6.98−6.94 (2H, m),
6.90 (1H, s), 4.86−4.82 (1H, d), 3.82 (3H, s), 3.53−3.49 (1H, m),
3.47−3.43 (1H, d), 3.22 (3H, s), 2.79−2.72 (1H, m), 2.63−2.57 (1H, m),
2.45−2.37 (1H, m), 2.28 (3H, s), 2.09 (3H, s), 1.80−1.70 (2H, m).
LCMS m/z 504 [M + H]+, 100% purity.
(S,E)-N-((3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)methyl)-N-methylmethanesulfo-
namide (40). 40 was made from methanesulfonyl chloride according
to the procedure for 39 in 46.6% yield. 1H NMR (400 MHz, CDCl3):
δ 7.94 (1H, s), 7.54−7.51 (2H, m), 7.45 (1H, s), 7.24−7.22 (1H, d),
7.13−7.09 (2H, t), 6.99−6.96 (2H, m), 6.93 (1H, s), 4.28−4.24 (1H,
d), 3.84 (3H, s), 3.47−3.43 (1H, d), 3.43−3.39 (1H, m), 3.11 (3H, s),
2.84 (3H, s), 2.82−2.78 (1H, m), 2.73−2.66 (1H, m), 2.45−2.39 (1H, m),
2.32 (3H, s), 1.90−1.80 (2H, m). LCMS m/z 540 [M + H]+, 96.0%
purity.
(S,E)-3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-imi-
dazol-1-yl)benzylidene)-3-(methoxymethyl)-5,6,7,8-tetrahy-
dro-3H-[1,2,4]oxadiazolo[4,3-a]pyridine (35). Sodium hydride
(2 mg, 60% in mineral oil) and methyl iodide (16.6 mg, 0.12 mmol))
were added to a stirred solution of 5a (7.5 mg, 0.017 mmol) in an-
hydrous DMF (0.2 mL) at room temperature under nitrogen atmo-
sphere. The reaction mixture was stirred for 15 min, quenched with
iced brine, and extracted with EtOAc. The organic phase was dried
over anhydrous Na2SO4 and concentrated. The crude was purified on
a C18 column with MeCN/water containing 0.1% formic acid. The
desired fraction was neutralized with aqueous NaHCO3, concentrated
under reduced pressure to remove MeCN, and extracted with EtOAc
1
to give 35. H NMR (400 MHz, CDCl3): δ 8.05 (1H, s), 7.55−7.51
(2H, m), 7.46 (1H, s), 7.26−7.24 (1H, d), 7.11−7.06 (2H, t), 7.02−
6.93 (3H, m), 4.04−4.01 (1H, d), 3.96−3.93 (1H, d), 3.84 (3H, s),
3.50 (3H, s), 3.29−3.26 (1H, m), 2.97−2.94 (1H, m), 2.71−2.68 (2H, m),
2.33 (3H, s), 1.95−1.85 (1H, m), 1.85−1.75 (1H, m). LCMS m/z
463 [M + H]+, 98.5% purity.
N-(3-Bromopropyl)-N,N-bis(trimethylsilyl)amine (42). Chloro-
trimethylsilane (273 g, 2.5 mol) was added to a suspension of 41 in
anhydrous DCM (3000 mL) in an ice bath, followed by slow addition
of Et3N (525 mL, 3.8 mol). The reaction mixture was stirred at room
temperature overnight and filtered to remove white solid. The filtrate
was concentrated under reduced pressure, diluted with ether, filtered,
(S,E)-1-(3-(4-Fluorophenyl)-8-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)-5,6,7,8-tetrahydro-3H-[1,2,4]-
oxadiazolo[4,3-a]pyridin-3-yl)-N-methylmethanamine
(36). Sodium triacetoxy borohydride (252 mg, 1.2 mmol) was slowly
499
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
and concentrated again to give 42 (306.8 g, 95.1% yield). The product
was used without further purification in the preparation of 30b.
Ethyl 2-(5-Fluoropyridin-2-yl)-2-oxoacetate (45). Isopropyl-
magnesium chloride−lithium chloride complex (1.3 M in THF)
(131 mL, 170.5 mmol) was added slowly to a solution of 2-bromo-
5-fluoropyridine (43) (25 g, 142.1 mmol) in 200 mL of THF over
20 min at 0 °C. The resulting mixture was allowed to warm to room
temperature over 1 h and stirred at room temperature for 30 min. The
prepared organometallic solution was added to a solution of diethyl
oxalate (22.7 g, 135 mmol) in 100 mL of THF at −78 °C slowly via
cannulation. The reaction mixture was allowed to warm to room
temperature overnight, quenched with saturated NH4Cl, and extracted
with ether. The combined organic layers were dried over anhydrous
Na2SO4, filtered, and concentrated to dryness. The residue was puri-
fied on a silica gel column, eluting with EtOAc/hexanes in a gradient
from 0% to 100% to give 45 (13 g, 47% yield). H NMR (400 MHz,
CDCl3): δ 8.57 (1H, m), 8.19−8.15 (1H, m), 7.62−7.57 (1H, m),
4.50−4.45 (2H, q), 1.42−1.38 (3H, t).
Ethyl 2-(5-Fluoropyridin-3-yl)-2-oxoacetate (46). 46 was pre-
pared from 3-bromo-5-fluoropyridine (44) according to the procedure
for 45. The product was used without further purification in the
preparation of 30j.
(S)-2-Amino-2-(3,4,5-trifluorophenyl)ethanol (48). Sodium
hydroxide (8.2 g, 205 mmol) in water (300 mL) was added to a
solution of CbzNH2 (31.5 g, 208 mmol) in PrOH (300 mL). The
mixture was stirred for 3 min, followed by dropwise addition of tert-
butyl hypochlorite (23.5 mL, 205 mmol). After this mixture was stirred
for another 6 min, a solution of (DHQ)2PHAL (600 mg, 0.75 mmol)
and 1,2,3-trifluoro-5-vinylbenzene 47 (100 mmol) in PrOH (150 mL)
was added, followed by addition of K2OsO2(OH)4 (380 mg, 1.0 mmol).
The resulting mixture turned green immediately. Stirring was continued
for 2 h, upon which the solution turned light yellow. The reaction was
quenched by stirring the mixture with an excess of Na2S2O3 for 2 h. The
PrOH layer was then separated and concentrated in vacuum. The solid
residue was dissolved in EtOAc, which was passed through a silica gel
pad, and concentrated. The residue was treated with 15% EtOAc/
hexanes to remove solid CbzNH2. The filtrate was reconcentrated and
crystallized in EtOAc/hexanes to give an undesired regioisomer (3 g).
The filtrate was concentrated again to provide a solid mixture (18 g)
with the desired Cbz-protected regioisomer as the major component
(>70%). This mixture was directly subjected to hydrogenation catalyzed
with Pd(OH)2/C (9 g, 50% water, 20% Pd on dry basis) in MeOH
(250 mL) for 1 h. The reaction mixture was filtered through Celite and
concentrated to give 48 (8.6 g, 45.0% yield), which was used without
further purification in the preparation of 51.
(S,E)-1-(2-Hydroxy-1-(3,4,5-trifluorophenyl)ethyl)-3-(3-me-
thoxy-4-(4-methyl-1H-imidazol-1-yl)benzylidene)piperidin-2-
one (51). 51 was prepared from 48 and (E)-5-chloro-2-(3-methoxy-
4-(4-methyl-1H-imidazol-1-yl)benzylidene)pentanoic acid TFA salt
(49) using a similar method described in the ref 13. 1H NMR
(400 MHz, CDCl3): δ 7.82 (1H, s), 7.70 (1H, s), 7.24−7.22 (1H, d),
7.00−6.91 (4H, m), 6.92 (1H, s), 5.78−5.76 (1H, m), 4.2−4.05 (2H, m),
3.84 (3H, s), 3.42−3.35 (1H, m), 3.20−3.12 (1H, m), 2.88−2.76
(2H, m), 2.28 (3H, s), 1.90−1.70 (2H, m). LCMS m/z 472 [M + H]+,
96.8% purity.
(400 MHz, CDCl3): δ 7.86 (1H, br s), 7.71 (1H, s), 7.25−7.23 (1H, m),
7.05−7.02 (2H, m), 6.95−6.92 (3H, m), 6.33−6.28 (1H, br t), 5.70
(2H, br s), 4.13−4.10 (2H, m), 3.84 (3H, s), 3.32−3.25 (1H, m),
3.07−3.02 (1H, m), 2.92−2.75 (2 H, m), 2.28 (3H, s), 1.92−1.85 (1H, m),
1.78−1.70 (1H, m).
(S,E)-9-(3-Methoxy-4-(4-methyl-1H-imidazol-1-yl)-
benzylidene)-4-(3,4,5-trifluorophenyl)-3,4,6,7,8,9-
hexahydropyrido[2,1-c][1,2,4]oxadiazine (53). A solution of 52
(2.7 g, 5.5 mmol) in EtOH (30 mL) was added to a mixture of P2O5
(7.5 g, 52.8 mmol) in EtOH (35 mL). The resulting mixture was then
stirred at 80 °C overnight, cooled to room temperature, and diluted
with DCM and water. Aqueous K2CO3 was added to adjust the pH to
∼9−10. The organic layer was then collected and dried over an-
hydrous Na2SO4. After the DCM was evaporated, the residue was
purified on a silica gel column, eluting with MeOH/DCM containing
1
1
0.1% Et3N to afford 53 (2.2 g, 84.6% yield). H NMR (400 MHz,
CDCl3): δ 7.69 (1H, s), 7.51 (1H, br s), 7.24−7.21 (1H, m), 7.00−
6.95 (4H, m), 6.91 (1H, s), 4.26−4.24 (1H, m), 4.04−4.02 (2H, m),
3.83 (3H, s), 3.18−3.03 (2H, m), 2.86−2.79 (1H, m), 2.75−2.68 (1H,
m), 2.28 (3H, s), 1.93−1.75 (2H, m). LCMS m/z 469 [M + H]+,
96.6% purity, 90% ee.
AUTHOR INFORMATION
Corresponding Author
■
*For Z.-Y.S.: phone, +1 917 657 6061; e-mail, ZYSUN66011@yahoo.
com. For Z.Z.: phone, +1 732 822 7384; e-mail johnnyzzhu@
msn.com.
ACKNOWLEDGMENTS
■
We thank Mary Humphries, Matthew Nienart, and Kathryn
Galvin for conducting the in life portion of the cynomolgous
study; Alexei Buevich, Rebecca Osterman, Mary Senior, and
Tze-Ming Chan for NMR structural analysis; Steve Sorota for
all hERG cellular data; and Jessy Wong, Xian Liang, Yan Jin,
Teresa Andreani, Yang Cao, and Michael Starks for scale-up
and chiral separation.
DEDICATION
■
†This paper is dedicated to Professor Ronald Breslow on the
occasion of his 80th birthday.
ABBREVIATIONS USED
■
GSM, γ-secretase modulator; AD, Alzheimer’s disease; CNS,
central nervous system; GSI, γ-secretase inhibitor; APP,
amyloid precursor protein; NSAID, nonsteroidal anti-inflam-
matory drug; Aβ, amyloid β; BACE, β-site amyloid cleaving
enzyme; CSF, cerebral spinal fluid; HEK, human embryonic
kidney cell; PBS, phosphate buffered saline
REFERENCES
■
(1) Wimo, A.; Prince, M. World Alzheimer Report 2010: The Global
Economic Impact of Dementia; Alzheimer’s Disease International
(ADI): London, U.K., September 2010.
(S,E)-1-(2-(Aminooxy)-1-(3,4,5-trifluorophenyl)ethyl)-3-(3-
methoxy-4-(4-methyl-1H-imidazol-1-yl)benzylidene)piperidin-
2-one (52). Under N2 protection, DIAD (3.0 g, 15 mmol) was added
dropwise to a mixture of 51 (4.7 g, 10 mmol), PPh3 (3.9 g, 15 mmol),
and N-hydroxylphthalimide (2.4 g, 15 mmol) in anhydrous THF
(100 mL) at −10 °C. The mixture was allowed to warm to room
temperature and stirred for 30 min. After evaporation of THF, the
residue was dissolved in EtOAc, washed with aqueous NaHCO3, dried
over anhydrous Na2SO4, and concentrated. The residue was then
treated with NH2NH2 monohydrate (1.0 g, 20 mmol) in EtOH
(100 mL) overnight. After evaporation of EtOH, the residue was
dissolved in DCM, washed consecutively with water and brine, and
dried over anhydrous Na2SO4. The DCM was then removed and the
residue was purified on a silica gel column, eluting with MeOH/
(2) (a) Yan, R.; Bienkowski, M. J.; Shuck, M. E.; Miao, H.; Tory, M. C.;
Pauley, A. M.; Brashler, J. R.; Stratman, N. C.; Mathews, W. R.;
Buhl, A. E.; Carter, D. B.; Tomasselli, A. G.; Parodi, L. A.; Heinrikson,
R. L.; Gurney, M. E. Membrane-anchored aspartyl protease with
Alzheimer’s disease β-secretase specificity. Nature 1999, 402, 533−
537. (b) Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz,
E. A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.;
Luo, Y.; Fisher, S; Fuller, J.; Edenson, S.; Lile, J.; Jarosinski, M. A.;
Biere, A. L.; Curran, E.; Burgess, T.; Louis, J.-C.; Collins, F.; Treanor,
J.; Rogers, G.; Citron, M. β-Secretase cleavage of Alzheimer’s amyloid
precursor protein by the transmembrane aspartic protease BACE.
Science 1999, 286, 735−741. (c) Hussain, I.; Powell, D.; Howlett, D. R.;
Tew, D. G.; Meek, T. D.; Chapman, C.; Gloger, I. S.; Murphy, K. E.;
1
DCM containing 0.1% Et3N to afford 52 (3.5 g, 72.2%). H NMR
500
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
Southan, C. D.; Ryan, D. M.; Smith, T. S.; Simmons, D. L.; Walsh, F. S.;
Dingwall, C.; Christie, G. Identification of a novel aspartic protease
(Asp 2) as beta-secretase. Mol. Cell. Neurosci. 1999, 14, 419−427.
(d) Sinha, S.; Anderson, J. P.; Barbour, R.; Basi, G. S.; Caccavello, R.;
Davis, D.; Doan, M.; Dovey, H. F.; Frigon, N.; Hong, J.; Jacobson-
Croak, K.; Jewett, N.; Keim, P.; Knops, J.; Lieberburg, I.; Power, M.;
Tan, H.; Tatsuno, G.; Tung, J.; Schenk, D.; Seubert, P.; Suomensaari,
S. M.; Wang, S.; Walker, D.; Zhao, J.; McConlogue, L.; Varghese, J.
Purification and cloning of amyloid precursor protein β-secretase from
human brain. Nature 1999, 402, 537−540.
disease: disappointments and hopes. Curr. Top. Med. Chem. 2011, 11,
1555−1570. (h) Schmidt, B.; Baumman, S.; Braun, H. A.; Larbig, G.
Inhibitors and modulators of β- and γ-secretase. Curr. Top. Med. Chem.
2006, 6, 377−392.
(9) Kounnas, M. Z.; Danks, A. M.; Cheng, S.; Tyree, C.; Ackerman,
E.; Zhang, X.; Ahn, K.; Nguyen, P.; Comer, D.; Mao, L.; Yu, C.;
Pleynet, D.; Digregorio, P. J.; Velicelebi, G.; Stauderman, K. A.;
Comer, W. T.; Mobley, W. C.; Li, Y.-M; Sisodia, S. S.; Tanzi, R. E.;
Wagner, S. L. Modulation of γ-secretase reduces β-amyloid deposition
in a transgenic mouse model of Alzheimer’s disease. Neuron 2010, 67,
769−780.
(10) (a) Kim, J.; Onstead, L.; Randle, S.; Price, R.; Smithson, L.;
Zwizinski, C.; Dickson, D. W.; Golde, T.; McGowan, E. Aβ40 inhibits
amyloid deposition in vivo. J. Neurosci. 2007, 27, 627−633. (b) Chen,
Y.; Dong, C. Aβ40 promotes neuronal cell fate in neural progenitor
cells. Cell Death Differ. 2009, 16, 386−394. (c) Abramov, E.; Dolev, I.;
Fogel, H.; Ciccotosto, G. D.; Ruff, E.; Slutsky, I. Amyloid-β as a
positive endogenous regulator of release probability at hippocampal
synapses. Nat. Neurosci. 2009, 12, 1567−1576. (d) Wang, R.; Wang,
B.; He, W.; Zheng, H. Wild-type presenilin 1 protects against
Alzheimer disease mutation-induced amyloid pathology. J. Biol. Chem.
2006, 281, 15330−15336.
(11) (a) Imbimbo, B. P. Why did tarenflurbil fail in Alzheimer’s
disease? J. Alzheimer’s Dis. 2009, 17, 757−760. (b) Eriksen, J. L.; Sagi,
S. A.; Smith, T. E.; Weggen, S.; Das, P.; McLendon, D. C.; Ozols,
V. V.; Jessing, K. W.; Zavitz, K. H.; Koo, E. H.; Golde, T. E. NSAIDs
and enantiomers of flurbiprofen target γ-secretase and lower Aβ42 in
vivo. J. Clin. Invest. 2003, 112, 440−449.
(12) Cheng, S.; Comer, D. D.; Mao, L.; Balow, G. P.; Pleynet, D. Aryl
Compounds and Uses Thereof in Modulating Amyloid β. WO
2004110350, 2004.
(13) Kimura, T.; Kawano, K.; Doi, E.; Kitazawa, N.; Shin, K.;
Miyagawa, T.; Kaneko, T.; Ito, K.; Takaishi, M.; Sasaki, T.; Hagiwara,
H. Cinnamide Compound. US 20060004013, 2006.
(14) Kimura, T.; Kawano, K.; Doi, E.; Kitazawa, N.; Takaishi, M.; Ito,
K.; Kaneko, T.; Sasaki, T.; Sato, N.; Miyagawa, T.; Hagiwara, H.
Morpholine Type Cinnamide Compound. US20070117798, 2007
́
(3) Guardia-Laguarta, C.; Pera, M.; Lleo, A. γ-Secretase as a ther-
apeutic target in Alzheimer’s disease. Curr. Drug Targets 2010, 11,
506−517.
(4) (a) Hardy, J. A.; Higgins, G. A. Alzheimer’s disease: the amyloid
cascade hypothesis. Science 1992, 256, 184−185. (b) Hardy, J.; Selkoe,
D. J. The amyloid hypothesis of Alzheimer’s disease: progress and
problem on the road to therapeutics. Science 2002, 297, 353−356.
(c) Walsh, D. M.; Selkoe, D. J. Aβ oligomers: a decade of discovery.
J. Neurochem. 2007, 101, 1172−1184.
(5) Artavanis-Tsakonas, S.; Rand, M. D.; Lake, R. J. Notch signaling:
cell fate control and signal integration in development. Science 1999,
284, 770−776.
(6) Hull, M.; Berger, M.; Heneka, M. Disease-modifying therapies in
̈
Alzheimer’s disease. Drug 2006, 66, 2075−2093.
(7) (a) Siemers, E. R.; Quinn, J. F.; Kaye, J.; Farlow, M. R.;
Porsteinsson, A.; Tariot, P.; Zoulnouni, P.; Galvin, J. E.; Holtzman,
D. M.; Knopman, D. S.; Satterwhite, J.; Gonzales, C.; Dean, R. A.; May,
P. C. Effects of a γ-secretase inhibitor in a randomized study of patients
with Alzheimer disease. Neurology 2006, 66, 602−604. (b) Fleisher,
A. S.; Raman, R.; Siemers, E. R.; Becerra, L.; Clark, C. M.; Dean, R. A.;
Farlow, M. R.; Galvin, J. E.; Peskind, E. R.; Quinn, J. F.; Sherzai, A.;
Sowell, B. B.; Aisen, P. S.; Thal, L. J. Phase 2 safety trial targeting
amyloid β production with a γ-secretase inhibitor in Alzheimer disease.
Arch. Neurol. 2008, 65, 1031−1038. (c) Siemers, E.; Skinner, M.;
Dean, R. A.; Gonzales, C.; Satterwhite, J.; Farlow, M.; Ness, D.; May,
P. C. Safety, tolerability, and changes in amyloid β concentrations after
administration of a γ-secretase inhibitor in volunteers. Clin.
Neuropharmacol. 2007, 28, 126−132. (d) Bateman, R. J.; Siemers,
E. R.; Mawuenyega, K. G.; Wen, G.; Browning, K. R.; Sigurdson, W. C.;
Yarasheski, K. E.; Friedrich, S. W.; DeMattos, R. B.; May, P. C.; Paul,
S. M.; Holtzman, D. M. A γ-secretase inhibitor decreases amyloid-β
production in the central nervous system. Ann. Neurol. 2009, 66, 48−
64. (e) Carlson, C.; Estergard, W.; Oh, J.; Suhy, J.; Jack, C. R. Jr.;
Siemers, E.; Barakos, J. Prevalence of asymptomatic vasogenic edema
in pretreatment Alzheimer’s disease study cohorts from phase 3
trials of semagacestat and solanezumab. Alzheimer’s Dementia 2011, 7,
396−401.
(15) Gustafsson, D.; Nystrom, J.-E.; Carlsson, S.; Bredberg, U.;
̈
Eriksson, U.; Gyzander, E.; Elg, M.; Antonsson, T.; Hoffmann, K.-J.;
Ungell, A.-L.; Sorensen, H.; Någård, S.; Abrahamsson, A.; Bylund, R.
̈
The direct thrombin inhibitor melagatran and its oral prodrug H
376/95: intestinal absorption properties, biochemical and phar-
macodynamic effects. Thromb. Res. 2001, 101, 171−181.
(16) Tomczuk, B.; Lu, T.; Soll, R. M.; Fedde, C.; Wang, A.; Murphy,
L.; Crysler, C.; Dasgupta, M.; Eisennagel, S.; Spurlino, J.; Bone, R.
Oxyguanidines: application to non-peptidic phenyl-based thrombin
inhibitors. Bioorg. Med. Chem. Lett. 2003, 13, 1495−1498.
(8) (a) Weggen, S.; Eriksen, J.; Das, P.; Saqi, S. A.; Wang, R.; Pietrzik,
C. U.; Findlay, K. A.; Smith, T. E.; Murphy, M. P.; Bulter, T.; Kang,
D. E.; Marquez-Sterlingk, N.; Golde, T. E.; Koo, E. H. A subset of
NSAIDs lower amyloidogenic Aβ42 independently of cyclooxygenase
activity. Nature 2001, 414, 212−216. (b) Kukar, T.; Murphy, M. P.;
Eriksen, J. L.; Sagi, S. A.; Weggen, S.; Smith, T. E.; Ladd, T.; Khan, M.
A.; Kache, R.; Beard, J.; Dodson, M.; Merit, S.; Ozols, V. V.;
Anastasiadis, P. Z.; Das, P.; Fauq, A.; Koo, E. H.; Golde, T. E. Diverse
compounds mimic Alzheimer disease-causing mutations by
augmenting Aβ42 production. Nat. Med. 2005, 11, 545−550.
(c) Pettersson, M.; Kauffman, G. W.; am Ende, C. W.; Patel, N. C.;
Stiff, C.; Tran, T. P.; Johnson, D. S. Novel γ-secretase modulators: a
review of patents from 2008 to 2010. Expert Opin. Ther. Pat. 2011, 21,
205−226. (d) Czirr, E.; Weggen, S. γ-Secretase modulation with Aβ42-
lowering nonsteroidal anti-inflammatory drugs and derived
compounds. Neurodegener. Dis. 2006, 3, 298−304. (e) Oehlrich, D.;
Berthelot, D. J.-C; Gijsen, H. J. M. γ-Secretase modulators as potential
disease modifying anti-Alzheimer’s drugs. J. Med. Chem. 2011, 54,
669−698. (f) Olson, R. E.; Marcin, L. R. Secretase inhibitors and
modulators for the treatment of Alzheimer’s disease. Annu. Rep. Med.
Chem. 2007, 42, 27−47. (g) Imbimbo, B. P.; Giardina, G. A. M.
γ-Secretase inhibitors and modulators for the treatment of Alzheimer’s
(17) Gandolfi, R.; Gamba, A.; Grunanger, P. 1,3-Dipolar cyclo-
̈
addition of nitrile oxides to 8-azaheptafulvenes. Heterocycles 1995, 40,
619−638.
(18) Malamdou-Xenikaki, E.; Coutouli-Argyropoulou, E. Preparation
and catalytic hydrogenation of 1,2,4-oxadiazolo[4,5-a]indolines.
Tetrahedron 1990, 46, 7865−7872.
(19) Kabakov, A. E.; Budagova, K. R.; Malyutina, Y. V.; Latchman, D. S.;
Csermely, P. Pharmacological attenuation of apoptosis in re-oxygenated
endothelial cells. Cell. Mol. Life Sci. 2005, 61, 3076−3086.
(20) Azizian, J.; Madani, M.; Souzangarzadeh, S. One-pot synthesis of
novel derivatives of spiro-[6H-indolo[2,1-b]quinazoline-6,3′-
[1,2,4]oxadiazoline]. Synth. Commun. 2005, 35, 765−768.
(21) Tsuge, O.; Kanemasa, S.; Suga, H.; Nakagawa, N. Synthesis of
2-(1-phosphorylalkyl)acetonitrile oxide and its cycloaddition to olefins.
Synthetic applications to 3,5-disubstituted 2-isoxazolines. Bull. Chem.
Soc. Jpn. 1987, 60, 2463−2473.
(22) Selva, M.; Tundo, P.; Marques, C. A. A simple one-port
synthesis of functionalized ketimines from ketones and amine
hydrochloride salts. Synth. Commun. 1995, 25, 369−378.
(23) Lee, J.-D.; Lee, Y.-J.; Jeong, H.-J.; Lee, J. S.; Lee, C.-H.; Ko, J.;
Kang, S. O. Practical synthesis of aminoethyl-o-carboranes.
Organometallics 2003, 22, 445−449.
501
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502

Journal of Medicinal Chemistry
Article
(24) Morimoto, T.; Sekiya, M. A convenient synthetic method for
Schiff bases. The trimethyl trifluoromethanesulfonate-catalyzed
reaction of N,N-bis(trimethylsilyl)amines with aldehydes and
ketones. Chem. Lett. 1985, 1371−1372.
(25) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. High-field FT
NMR application of Mosher’s method. The absolute configurations of
marine terpenoids. J. Am. Chem. Soc. 1991, 113, 4092−4096.
(26) Reddy, K. L.; Sharpless, K. B. From styrenes to enantiopure
R-arylglycines in two steps. J. Am. Chem. Soc. 1998, 120, 1207−1217.
(27) We propose to use the term “druggable motif” to describe some
of the well understood pharmacophores that have proven records for
achieving good pharmacokinetic, pharmacodynamic, and toxicological
profiles. Deliberate attention to suitable “druggable motifs” would help
medicinal chemists achieve a balanced approach in evaluating SAR
directions. Detailed discussions will be the subject of another publication.
(28) Pozharskii, A. F.; Sitkina, L. M.; Simonov, A. M.; Chegolya,
T. N. Nature of the interaction of phenyl and imidazole rings in N-aryl-
imidazoles. IV. Derivatives of N-phenylimidazole with substituents on the
benzene ring. Khim. Geterotsikl. Soedin. 1970, 6, 209−213.
(29) (a) Rubas, W.; Jezyk, N.; Grass, G. M. Comparison of the
permeability characteristics of a human colonic epithelial (Caco-2) cell
line to colon of rabbit, monkey, and dog intestine and human drug
absorption. Pharm. Res. 1993, 10, 113−118. (b) Makhey, V. D.; Guo,
A.; Norris, D. A.; Hu, P.; Yan, J.; Sinko, P. J. Characterization of the
regional intestinal kinetics of drug efflux in rat and human intestine
and in Caco-2 cells. Pharm. Res. 1998, 15, 1160−1167.
(30) Ida, N.; Hartmann, T.; Pantel, J.; Schroder, J.; Zerfass, R.; Forstl,
H.; Sandbrink, R.; Masters, C. L.; Beyreuther, K. Analysis of heterogeneous
Aβ42 peptide in human cerebrospinal fluid and blood by a newly deve-
loped sensitive Western blot assay. J. Biol. Chem. 1996, 271, 22908−
22914.
(31) Lee, J.; Song, L.; Terracina, G.; Bara, T.; Josien, H.; Asberom,
T.; Sasikumar, T. K.; Burnett, D. A.; Clader, J.; Parker, E. M.; Zhang,
L. Identification of presenilin 1-selective γ-secretase inhibitors with
reconstituted γ-secretase complexes. Biochemistry 2011, 50, 4973−4980.
(32) Wong, G. T.; Manfra, D.; Poulet, F. M.; Zhang, Q.; Josien, H.;
Bara, T.; Engstrom, L.; Pinzon-Ortiz, M.; Fine, J. S.; Lee, H.-J. J.;
Zhang, L.; Higgins, G. A.; Parker, E. M. Chronic treatment with the
γ-secretase inhibitor LY-411,575 inhibits β-amyloid peptide produc-
tion and alters lymphopoiesis and intestinal cell differentiation. J. Biol.
Chem. 2004, 279, 12876−12882.
NOTE ADDED AFTER ASAP PUBLICATION
■
After this paper was published online December 8, 2011, a
correction was made to the straddle heading structure of Table 5.
The corrected version was published December 14, 2011.
502
dx.doi.org/10.1021/jm201407j | J. Med. Chem. 2012, 55, 489−502