Design and Synthesis of a Novel Series of Bicyclic Heterocycles As Potent γ-Secretase Modulators

Article abstract of DOI:10.1021/jm201710f

The design and the synthesis of several chemical subclasses of imidazole containing γ-secretase modulators (GSMs) is described. Conformational restriction of pyridone 4 into bicyclic pyridone isosteres has led to compounds with high in vitro and in vivo p

Full text of DOI:10.1021/jm201710f

Article
pubs.acs.org/jmc
Design and Synthesis of a Novel Series of Bicyclic Heterocycles As
Potent γ-Secretase Modulators
Francois Bischoff, Didier Berthelot, Michel De Cleyn, Gregor Macdonald, Garrett Minne, Daniel Oehlrich,
Serge Pieters, Michel Surkyn, Andres A. Trabanco, Gary Tresadern, Sven Van Brandt, Ingrid Velter,
́
Mirko Zaja, Herman Borghys, Chantal Masungi, Marc Mercken, and Harrie J. M. Gijsen*
Janssen Research & Development, Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium
S
* Supporting Information
ABSTRACT: The design and the synthesis of several
chemical subclasses of imidazole containing γ-secretase
modulators (GSMs) is described. Conformational restriction
of pyridone 4 into bicyclic pyridone isosteres has led to
compounds with high in vitro and in vivo potency. This has
resulted in the identification of benzimidazole 44a as a GSM
with low nanomolar potency in vitro. In mouse, rat, and dog,
this compound displayed the typical γ-secretase modulatory
profile by lowering Aβ42 and Aβ40 levels combined with an especially pronounced increase in Aβ38 and Aβ37 levels while
leaving the total levels of amyloid peptides unchanged.
semagacestat, was interrupted during a phase III clinical trial as
it was associated with worsening of clinical measures of
cognition and an increase in the risk of skin cancer. Multiple
reasons have been proposed to explain the lack of efficacy and
observed side effects, including the rebound effect on Aβ42 at
low levels of semagacestat, poor selectivity versus Notch
cleavage, and accumulation of neurotoxic C99.4b,5 In contrast to
GSIs, γ-secretase modulators (GSMs) are defined as molecules
that change the relative proportion of the Aβ isoforms while
maintaining the rate at which APP is processed.6 Targeted
modulation of γ-secretase activity in such a way that the release
of the longer amyloidogenic Aβ-peptides, especially Aβ42, is
disfavored with regard to the shorter and more soluble Aβ-
peptides, especially Aβ37 and Aβ38, has been and still is an
important strategy to tackle AD.7,8 Mechanistically, GSMs have
been postulated to act as activators of the γ-secretase complex,
enhancing the stepwise cleavage of APP toward the shorter Aβ-
peptides.9 In a recent study to further discriminate between
modulation and inhibition, a GSM successfully improved
cognitive deficits in APP-transgenic mice whereas a GSI caused
cognitive impairment likely due to accumulation of C99.5
Two major classes of compounds are known to modulate the
γ-secretase activity.10 The first of these are carboxylic acids
derived from nonsteroidal anti-inflammatory drugs
(NSAIDs).11 Initial examples of this class, such as tarenflurbil
(1), were of low potency and displayed poor brain penetration,
resulting in the failure of 1 in phase III clinical trials (Figure 1).
Subsequent, more potent analogues suffered from very high
INTRODUCTION
■
Alzheimer’s disease (AD) is a chronic and progressive
neurodegenerative disorder characterized by the deposition of
extraneuronal amyloid plaques and intraneuronal neurofibrillary
tangles of hyperphosphorylated tau protein in the limbic and
cortical regions of the brain.1 These pathological depositions
were first discovered in 1906 by Alois Alzheimer during the
autopsy of patients with loss of memory, cognition, and
behavioral stability.2 Nowadays, AD is the leading form of
dementia and the third leading cause of death after
cardiovascular disease and cancer.
AD does not have a simple etiology, but a substantial body of
evidence supports the amyloid hypothesis which supposes that
the accumulation of the amyloid-β peptide (Aβ) caused by
altered production or clearance is the causative event in AD.3
Aβ-peptides are derived from the amyloid precursor protein
(APP) by two sequential proteolytic cleavages. The first
cleavage of APP is catalyzed by the aspartyl protease BACE1
(β-secretase), yielding a soluble N-terminal fragment (sAPP)
which is excreted from the cell, and a short membrane-bound
C-terminal fragment (C99). The latter is a substrate for γ-
secretase, and it is cleaved at variable positions of the
transmembrane domain to release a 37−43 amino acid long
Aβ-peptide. The two most common forms of Aβ produced in
this way are Aβ40 and Aβ42, containing 40 and 42 amino acid
residues respectively. It is the longer and more hydrophobic
Aβ42 that forms the nucleus of the amyloid plaques and is
thought to cause the neurotoxicity associated with AD.
The search for γ-secretase inhibitors (GSIs) that lower the
production of all Aβ isoforms has been a first strategy to
prevent the formation of amyloid plaques. Several GSIs have
moved to the clinic, and their progress has been reviewed
recently.4 The development of the most advanced compound,
Special Issue: Alzheimer's Disease
Received: December 20, 2011
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Examples of γ-secretase modulators. In vitro data were obtained from in-house experiments. The IC50 values are a mean of at least three
determinations.
Scheme 1
lipophilicity, limiting the potential use of these compounds as
GSMs. The second class of GSMs is characterized by non-
NSAID-scaffolds without a carboxylic acid group. These were
initially reported by Neurogenetics in 2004, claiming imidazole
containing compounds of general structure 2 as Aβ-modulating
agents with potencies below 1 μM.12 Subsequent work by Eisai
has led to E-2012 (3), the first compound from this series
entering the clinic (Figure 1).13 However, further development
of E-2012 was halted due to lenticular toxicity in preclinical
trials.6 Compound 3 was used as a starting point for the design
of our own non-NSAID γ-secretase modulators for the
treatment of AD.14
The cinnamide group in 3 has an E orientation of the double
bond. Our first hit was obtained by speculating that an NH
linker as in compound 4 would preserve a similar geometry due
to favorable electrostatic interaction between the NH and the
carbonyl oxygen. Also, a steric clash between the carbonyl and
the methoxyphenyl ring of 4 would disfavor alternative
conformations. Computational modeling of 4 suggested that
the orientation as sketched was the most energetically favorable
conformation.15 This type of aminopyridones has also been
described by Schering/Merck, including 4.14a Despite the
conformational similarity between 3 and 4, subsequent testing
of both compounds in vitro showed a loss in potency of 4
versus 3 (550 vs 81 nM, respectively). Upon oral admin-
istration in mice at 30 mg/kg, no significant change in Aβ brain
levels was observed after 4 h for 4, while dosing of 3 resulted in
a 43% reduction of Aβ42 levels. To improve the in vitro
potency and in vivo activity of 4, we set out to further
conformationally restrict the aminopyridone scaffold in 4. In
this paper, we will discuss our findings in this area and the
development of a number of subseries with high in vitro and in
vivo potency.16
CHEMISTRY
■
All the final compounds were synthesized via a Buchwald
coupling between the anilino- or bromo-derivatives I and the
corresponding halo- or anilino- heterocylic intermediates II,
respectively (Scheme 1).
Synthesis of Intermediates I. In a first approach, the
aniline precursors (I, R′′ = NH2) were prepared in two steps,
starting from commercially available 4-halo-nitroarenes, as
exemplified for anilines 8(a,c,f) in Scheme 2. Nucleophilic
aromatic substitution of a 4-halide in 5 with 4-methylimidazole
gave a mixture of regioisomers, from which 7 was isolated in
good yield. Subsequent catalytic hydrogenation of the nitro
functionality in 7 provided the desired anilines 8(a,c,f) in good
yields. 8a could then be submitted to Sandmeyer reaction
conditions to provide the bromo precursor 11 (I, R′′ = Br). In
another approach, the selective substitution of the 5-bromide in
2,5-dibromopyridine with 4-methylimidazole followed by a
copper oxide amination with ammonium hydroxide afforded
aniline 8e.17 Alternatively, compounds 8b and 8d were
prepared in one step, via an Ullmann type coupling of the 4-
methylimidazole with 4-halo-anilines 9b and 9d, respectively.18
Separation of the two regioisomers provided the major, and
desired, regioisomers 8b and 8d in good yields. A different
approach was used for synthesis of the pyridylmethoxy
derivative 14. Regioselective nucleophilic addition of sodium
methoxide to commercially available 3-amino-2,6-dibromopyr-
idine provided the methoxypyridylamine 12.19 The amine
moiety in 12 was converted to the desired 4-methylimidazole
ring in 13 in a sequence of reactions that included formylation
with acetic formic anhydride,20 followed by alkylation with 1-
chloropropanone and finally ring closure of the resulting
formyloxopropyl amine with ammonium acetate. Bromo-
intermediate 13 could easily be converted to amino-derivative
B
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2. General Synthetic Route Towards Intermediates I
a
Reagents and conditions: (a) 4-methylimidazole, K2CO3, DMSO, 150 °C, 6 h; (b) H2, Pd(C), MeOH, 50 °C, 12 h; (c) NaNO2, H2SO4, 10 °C, 30
min, then CuBr, HBr; 10 °C to rt, 1 h; (d) 4-methylimidazole, CuI, 1,10-phenanthroline, Cs2CO3, DMSO, 130 °C, 2 d; (e) NaOMe, 1,4-dioxane, rt,
8 h; (f) AcOH, HCO2H, THF, 60 °C, 12 h; (g) 1-chloropropanone, KI, Cs2CO3, DMF, rt, 12 h; (h) NH4OAc, AcOH, 100 °C, 1 h; (i) Cu2O,
NH4OH, ethylene glycol, 100 °C, 16 h.
14 via copper oxide catalyzed amination with ammonium
hydroxide.17
Scheme 3. Synthesis of Imidazopyridines and Benzimidazole
Building Blocks
a
Synthesis of Intermediates II. Imidazopyridines. The
imidazopyridine nucleus 17 was prepared by condensation of 2-
amino-3-bromopyridine 15 with α-haloketones 16 (Scheme 3).
Benzimidazoles. The initial synthesis of benzimidazole
analogues involved acylation of 1,2-diamino-3-nitrobenzene
(18) to provide the corresponding anilides (Scheme 3). These
compounds were then condensed to a benzimidazole ring
system 19 at 150 °C in strong acidic conditions under
microwave irradiation. Selective alkylation at N1 of 19 with the
corresponding alkylbromides, followed by reduction of the
nitro moiety, gave the desired 1,2-substituted 4-amino-
benzimidazoles 20(a,b,c,e).
Alternatively, benzimidazoles 23(a,d,f,h) were prepared from
2-bromo-6-fluoro-nitrobenzene (21). Aromatic nucleophilic
substitution on 21 with methylamine followed by reduction
of the nitro functionality provided aniline 22, which was
cyclized with acyl derivatives 25 to provide 23 in good yields.
Indazoles. Condensation of 3-bromo-2-nitrotoluene 24 with
dimethylformamide dimethyl acetal (DMF-DMA) in the
presence of pyrrolidine, followed by oxidation with NaIO4,
gave 3-bromo-2-nitrobenzaldehyde 26 in a modest 20% yield
over two steps (Scheme 4). Alternatively, oxidation of the
commercially available 3-chloro-2-nitrobenzyl alcohol 25 gave
a
Reagents and conditions: (a) EtOH, reflux, 4 h; (b) RCOCl, Et3N,
CH2Cl2, rt, 2 h; (c) AcOH, HCl, MW 150 °C, 30 min; (d) LiHMDS,
R′Br, THF, 0−150 °C, 1−4 h; (e) Fe, AcOH, 60 °C, 1 h; (f) MeNH2,
EtOH, 0 °C−rt, 15 h; (g) Et3N, CH2Cl2, rt, 15 h then AcOH, HCl,
100 °C, 2 h.
C
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 4. Synthesis of Indazole and Benzoxazole Building Blocks
a
Reagents and conditions: (a) DMF−DMA, pyrrolidine, 115 °C, 22 h; (b) NaIO4, DMF/H2O, 3 h, rt; (c) PCC, CH2Cl2, rt, 2 h; (d) R(CH2)nNH2,
sodium triacetoxyborohydride, HOAc, 1,2-dichloroethane, rt, 3 h; (e) SnCl2·2H2O, EtOH, 40 °C, 16 h; (f) LDA, THF, −78 to 0 °C, 15 min, MeI,
−78 °C to rt, 16 h; (g) (1) NaNO2, 6N aq HCl soln, H2O, 30 min, 60 °C, (2) t-butylmercaptan, EtOH, 1 h, 0 °C; (h) KOtBu, DMSO, 2 h, rt; (i)
R2SO4, toluene, 16 h, 110 °C or RBr, N-methyl-dicyclohexylamine, DMF, 16 h, 30 °C; (j) RCOCl, THF, rt, 15 h; (k) CuI, 1,10-phenantroline,
Cs2CO3, DME, 90 °C, 15 h.
the 3-chloro analogue 27, which was also used to synthesize the
final products. Reductive amination of benzaldehydes 26 and
27 with the corresponding anilines or benzylamines, followed
by reduction of the nitro group with concomitant cyclization to
the indazole nucleus, provided 28.21 The 3-position of 28a and
28c was also methylated using lithium diisopropyl amide
(LDA) and methyl iodide in THF to provide bromo indazoles
29a and 29c, respectively.
This synthetic route proceeded smoothly when the R
substituent at the 2-position of indazoles was an aromatic
ring. When R was an alkyl substituent, the major product of the
cyclization step (e) in Scheme 4 was a reduced diamino-
derivative such as 30, which was not isolated. Therefore an
alternative route for the preparation of the 2-alkyl indazoles was
applied, starting from 2-bromo-6-methylaniline 31. Diazotation
of 31 followed by condensation with t-butylmercaptan afforded
diazene 32. Subsequently, 32 underwent cyclization to indazole
33 by treatment with potassium t-butylate.22 N-Alkylation of
33, either with a dialkylsulfate, or with an alkylhalide, gave a
mixture of the two regioisomers 34 and 35, which could be
separated via chromatography.
modification of the assay as described.24 The cell/compound
mixtures were incubated overnight at 37 °C, 5% CO2. Aβ42
and Aβtotal concentrations were quantified in the cell
supernatant using sandwich immunoassays via the Alphalisa
technology (Perkin-Elmer). To obtain the reported IC50 values,
the data are calculated as percentage of the maximum amount
of Aβ42 or Aβtotal measured in the absence of the test
compound. The IC50 represents the concentration of a
compound that is required for reducing the Aβ peptide levels
by 50%. In this assay, a GSM will lead to a lowering of Aβ42
while leaving Aβtotal levels unchanged. Other mechanisms of
lowering the Aβ levels, such as γ- or β-secretase inhibition or
cytotoxicity, would result in lowering of all Aβ peptides levels,
including Aβtotal. None of the described compounds showed a
significant lowering of the Aβtotal levels up to the highest
concentration tested (10 μM), confirming that they are not γ-
or β-secretase inhibitors.
In Vivo Mouse Screening Assay. To determine the effect
on Aβ42 and Aβtotal levels upon acute administration of a
compound in vivo, nontransgenic CD1 mice were used.
Compounds were formulated as suspensions in 20% of Captisol
(a sulfobutyl ether of β-cyclodextrin) in water and administered
as a single oral dose of 30 mg/kg. After four hours, the animals
were sacrificed and their brains dissected in two. One half was
used for quantification of Aβ levels using sandwich immuno-
assays. The other half was analyzed for compound levels and
compared to compound levels in blood plasma collected at the
same time point. Each quoted value is the mean of at least six
animals. Changes in Aβ peptide levels are expressed as a
percentage relative to untreated animals. Reduction of Aβ
peptide levels of greater than 20% are considered significant. In
all runs, compound 3 was dosed as the positive control. In
Benzoxazoles. The synthesis of benzoxazole analogues is
shown in Scheme 4. Acylation of dibromoaniline 36 with the
corresponding acyl chlorides provided anilides 37. In turn,
these compounds were submitted to cyclization via Ullmann-
type coupling conditions in the presence of a catalytic Cu(I)−
phenanthroline system23 to give bromobenzoxazoles 38 in
good to excellent yields.
BIOLOGY
■
Cellular in Vitro Assay. All compounds were tested using
SKNBE2 cells carrying the APP 695−wild type according to
D
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 5. Rationale for the Synthesis of Imidazopyridine 40
a
Mouse in vivo at 30 mpk po after 4 h (n = 6) expressed as a percentage of Aβ42 lowering in brain compared with untreated animals.
agreement with the cellular in vitro data (vide supra), none of
the tested compounds showed a significant lowering of the
Aβtotal levels.
Further biological experiments are explained in the next
section and detailed in the Experimental Section or Supporting
Information.
in vivo in mice, displayed a moderate 20−40% lowering of
Aβ42 brain levels, with total levels of Aβ peptides remaining
unchanged. No clear correlation could be observed between in
vitro potency and in vivo activity, even upon consideration of
Table 1. In Vitro/in Vivo Activities, Brain/Plasma Levels,
and Solubility for Imidazopyridines
RESULTS AND DISCUSSION
■
Imidazopyridines. To regain the loss in potency, when
going from cinnamide 3 to aminopyridone 4 (Figure 1), we set
up to investigate conformational restriction of 4. Assuming that
the proposed intramolecular hydrogen bond in 4 would indeed
mimic the conformation of the cinnamide 3, the N-pyridone
benzyl substituent was targeted, being the most flexible part of
the molecule. A bioisosteric replacement of a pyridone by an
imidazopyridine heterocycle has been described as a successful
strategy for other targets25 and would preserve the intra-
molecular hydrogen bond arrangement present in 4. The 4-
fluorophenyl ring present in both 3 and 4 can be oriented in
two ways on the imidazopyridine ring, and both regioisomers
were prepared (39 and 40, Scheme 5). Only regioisomer 40, in
which the aromatic ring was attached to the 2-position, showed
an improvement of in vitro activity.26 Although with an IC50 of
182 nM still lower in potency than cinnamide 3, this now
translated in vivo into a 35% reduction of Aβ42 levels in mouse
brain 4 h after an oral dose of 30 mg/kg.
Given this result, the influence of various substituents at the
2-position of the imidazopyridine ring in 40 was evaluated. The
most relevant data are presented in Table 1. In general, an
aromatic substituent at the 2-position of imidazopyridines
brought good in vitro potency, with a range of ring
substitutions being allowed. For instance, replacing the para-
fluorine atom on the aromatic ring in 40a by an electron
donating meta-methoxy group (40b) slightly increased the
potency. A reasonable in vitro activity was also achieved with an
unsubstituted phenyl ring (40c). The introduction of a weak
basic center, such as a 3-pyridyl ring in 40f, led to a loss of
primary activity. When the hydrogen atom at the 3-position of
the imidazopyridines was replaced by a bulkier group, such as a
methyl group, potency was improved further, see 40c versus
40d. Most of the compounds in Table 1, which were screened
inhib
IC50
plasma
Aβ42
(nM)
brain levels
levels
vivo
a
b
b
c
compd
R′
R
Aβ42
(μM)
(μM)
(%)
40a
40b
H
4-F-Ph
182
138
11.7 0.7
3.0 0.3
17.2 0.7
11.1 0.7
35
20
H
3-MeO-
Ph
40c
40d
40e
H
Ph
339
78
16.2 1.1
5.4 0.4
1.2 0.3
26.3 1.6
16.0 2.0
4.9 0.8
15
34
44
Me Ph
Me 2-CF3-
Ph
26
40f
H
3-
pyridyl
1216
100
nd
nd
nd
44
40g
40h
H
4-F-Ph-
CH2
6.2 0.3
17.5 0.7
H
H
H
Me
442
194
213
13.6 1.5
4.9 0.3
5.6 0.4
15.5 1.6
10 0.2
33
13
7
d
40i
n-Bu
THP
40j
11.3 0.7
a
IC50 represents the concentration of a compound that is required for
reducing the Aβ42 level by 50%. The IC50 values are a mean of at least
three determinations. The confidence intervals for the IC50 values are
reported in the Supporting Information. Plasma and brain samples
were analyzed for the tested compound using a qualified research LC-
MS/MS method. Data are expressed as the geometric mean values of
the standard error of the mean (SEM). nd: not
determined. Mouse in vivo at 30 mpk p.o. after 4 h (n = 6) expressed
b
at least 4 runs
c
as a percentage of Aβ42 lowering in brain compared with untreated
animals. nd: not determined. Mouse in vivo at 20 mpk p.o. after 4 h
(n = 4) expressed as a percentage of Aβ42 lowering in brain compared
d
with vehicle treated animals.
E
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
compound brain levels. An effort was made to correlate in vivo
activity with the estimated free brain concentration, using the
fraction unbound in brain (fu,brain).27 For most in vivo active
compounds, fu,brain was less than 0.1% and therefore could not
be accurately determined. Nevertheless, we observed that many
compounds demonstrated robust vivo activity while the
estimated free brain concentration was substantially lower
than the in vitro IC50 for Aβ42 lowering. Notably, 40e was one
of the most potent compounds in vivo with 44% lowering of
Aβ42 levels after 4 h, despite relatively low brain levels and
fu,brain < 0.03%. In terms of ADME parameters, most of the 2-
aryl substituted analogues had poor kinetic solubility at pH =
7.4 (<2 μM) and were cytochrome P450 (CyP) inhibitors and
hERG channel binders (data not shown). Replacement of the
2-aryl group on the imidazopyridine nucleus by various alkyl
groups (40h−40j) resulted in active compounds and did
increase the solubility but overall did not improve the ADME
profile. Compound 40g, bearing a benzyl substituent, was one
of the most potent analogues in vivo, lowering the Aβ42 levels
with 44% in mice at 30 mg/kg per os (po). Upon oral dosing,
most analogues of the imidazopyridine series displayed high
compound plasma levels despite their low solubility. On the
other hand, the most soluble compound within this series, the
tetrahydropyranyl (THP) derivative 40j (kinetic solubility at
pH 7.4 = 75 μM), had no significant effect on Aβ42 levels in
vivo despite moderate in vitro activity, good brain levels, and
fu,brain = 1.9%, highlighting again the disconnect between free
brain concentrations and vivo activity.
Table 2. In Vitro/in Vivo Activities, Brain/Plasma Levels,
and Solubility for Benzimidazoles
inhib
IC50
plasma
Aβ42
Aβ42
brain levels
levels
vivo
a
b
b
c
compd
R′
R
(nM)
(μM)
(μM)
(%)
41a
41b
41c
41d
H
4-F-Ph
203
17
nd
nd
nd
43
nd
45
Me 4-F-Ph
iPr 4-F-Ph
Me 3-
OMePh
7.0 0.6
nd
17.3 1.3
nd
25
43
5.5 0.5
19 1.0
41e
41f
Me 2,4-F-Ph
69
82
10.3 0.8
4.8 0.4
27 1.6
54
23
Me 4-F-Ph-
CH2
15.7 1.8
41g
41h
Me Me
Me iBu
1660
107
nd
nd
nd
45
10.8 1.3
14.1 1.3
a
IC50 represents the concentration of a compound that is required for
reducing the Aβ42 level by 50%. The IC50 values are a mean of at least
three determinations. The confidence intervals for the IC50 values are
b
reported in the Supporting Information Plasma and brain samples
were analyzed for the tested compound using a qualified research LC-
MS/MS method. Data are expressed as the geometric mean values of
at least four runs the standard error of the mean (SEM). nd: not
c
determined. Mouse in vivo at 30 mpk po after 4 h (n = 6, except for
Having identified activity with the imidazopyridines 40,
alternative bicyclic systems were synthesized which also
contained a hydrogen bond acceptor in the same position
relative to the NH linker (Figure 2). These examples included
41(d,f), where n = 4) expressed as a percentage of Aβ42 lowering in
brain compared with untreated animals. nd: not determined.
Indazoles. Also compounds from the indazole (Table 3)
series turned out to be highly potent. The most active in vitro
compounds were substituted on the 2-position of the indazole
nucleus with an aromatic ring either directly connected (42a−
c) or via a methylene spacer (42d). Again, additional
substitution on the 3-position, for instance with a methyl
group, resulted in an improvement of the primary activity (42c
versus 42b). In the case of an alkyl substituent on the 2-
position, good in vitro activity was retained with a n-butyl
group (42f), whereas a smaller substituent such as a methyl
group (42e) led to a poorly active compound. Several
compounds demonstrated in vivo activity, with up to 41%
inhibition of Aβ42 lowering in mouse for compound 42c.
Benzoxazoles. Benzoxazole derivatives were clearly less
active than their imidazopyridine or benzimidazole analogues
(Table 4). For instance 4-F-Ph derivative 43a was almost 2
orders of magnitude less active than the corresponding
imidazopyridine 40a. In this series, the most active compounds
were substituted either with an alkyl substituent on the 2-
position of the benzoxazole nucleus (43d) or an alkylaryl
substituent (43c). However, both compounds were not potent
enough to significantly reduce Aβ42 levels in vivo.
The high potency of compounds across the imidazopyridine,
benzimidazole, and indazole series underlines the benefit of
conformational restriction of the original pyridone core in 4.
Additionally, the cyclization promotes a beneficial orientation
of the aromatic substituent at the 2-position and allows
augmentation of the hydrogen bonding capability of the sp2
nitrogen at the 1-position. The same heterocyclic nitrogen can
form an intramolecular interaction to the NH aniline linker and
stabilize an E-like conformation similar to 3 and 4.
Figure 2. Isosteres of imidazopyridines.
benzimidazole, benzoxazole, and indazole heterocycles. A
preference was given to compounds bearing a relatively large
substituent R at the 2-position of the heterocyclic nucleus in
order to preserve the preferred orientation established within
the imidazopyridine series 40.
Benzimidazoles. In general, the benzimidazole series
retained the potency of the imidazopyridines and showed a
similar SAR profile as for the imidazopyridines (Table 2). The
positive influence on potency of an additional substituent R′ at
the 3-position of the benzimidazole nucleus became even more
apparent in this series, with 41a being at least 10-fold less
potent than 41b and 41c. For compound 41b, the high in vitro
potency translated into a good in vivo efficacy, this compound
lowered the levels of Aβ42 peptides in mouse by 43%. As in the
imidazopyridine series, a drop in potency was observed when
small aliphatic substituents were introduced at the 2-position of
the benzimidazole ring (41g). Larger alkyl substituents, such as
in 41h, retained good in vitro potency, and despite a lower
metabolic stability (data not shown), this compound displayed
high brain levels which translated to a 45% reduction of Aβ42
levels.
F
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
ring. This is achieved by the presence of a small alkyl
substituent at the 3-position of the imidazopyridines,
benzimidazoles, and indazoles (steric clash indicated in Figure
3). The inherent lack of such a substituent in the benzoxazoles
Table 3. In Vitro/in Vivo Activities, Brain/Plasma Levels,
and Solubility for Indazoles
IC50
plasma
inhib
(nM)
brain levels
levels
Aβ42 vivo
a
b
b
c
compd
R′
R
Aβ42
(μM)
(μM)
(%)
42a
Me 2,4-diF-
Ph
133
nd
nd
nd
42b
42c
42d
H
3-
131
18
6.9 0.3
12.8 0.6
6
OMe-
Ph
Me 3-
4.7 1.0
6.8 0.6
14.4 0.6
11.6 1.0
41
31
Figure 3. Factors influencing the potency in the bicyclic heterocycles
series.
OMe-
Ph
H
4-F-Ph-
CH2
69
can explain the overall lower potency of this series. Non-
planarity is also achieved by using ortho-substituted aromatic
rings or replacing the aromatic ring with a benzylic or alkyl
group. Indeed, in the benzoxazole series all these variations led
to an increase in potency (43a versus 43b, 43c, and 43d,
respectively).
42e
42f
H
H
Me
2252
363
nd
nd
nd
nd
nd
nd
n-Bu
a
IC50 represents the concentration of a compound that is required for
reducing the Aβ42 level by 50%. The IC50 values are a mean of at least
three determinations. The confidence intervals for the IC50 values are
reported in the Supporting Information. Plasma and brain samples
b
The carbonyl of the cinnamide 3 and pyridone 4 as well as
the heterocyclic sp2 nitrogen in the series presented here all
maintain a hydrogen bond acceptor in the same position. It was
expected that this is an important feature for the activity of
these molecules. The hydrogen bonding capability of the
heterocyclic nitrogen (N1, Figure 3) was evaluated by
calculating the minimum electrostatic potential, Vmin, for this
atom. This parameter has been shown to be a useful predictor
for hydrogen bond strength.28 A weak correlation (r2 0.38) was
seen between the Vmin descriptor and activity, with more
negative values of Vmin and therefore stronger H-bond acceptor
capability correlating with potency, see Supporting Information
for more details. When comparing analogous molecules, the
imidazopyridine and benzimidazoles had the strongest hydro-
gen bond acceptor potential, while the benzoxazole tended to
be the weakest. The hydrogen bond acceptor capabilities of the
heterocyclic acceptor were modulated by the introduction of a
methyl substituent at the 3-position, as in methyl substituted
41b, Vmin −25.7 kcal/mol, and unsubstituted 41a, Vmin −20.3
kcal/mol. The effects of the methyl are likely 2-fold: electron
donating and, as discussed before, disturbing the planarity
between the benzimidazole and the 4-F-phenyl- substituent and
thereby reducing electron delocalization. Although there was
weak overall correlation, a single descriptor is unlikely to
capture all properties important for activity and the correlation
was seen to break down as more structural diversity at the 2-
position was included. Nevertheless, the calculations illustrate
an important consideration in the design of the target
molecules.
were analyzed for the tested compound using a qualified research LC-
MS/MS method. Data are expressed as the geometric mean values of
at least four runs the standard error of the mean (SEM). nd: not
determined. Mouse in vivo at 30 mpk po after 4 h (n = 6) expressed
as a percentage of Aβ42 lowering in brain compared with vehicle
c
treated animals. nd: not determined.
Table 4. In Vitro/in Vivo Activities, Brain/Plasma Levels,
and Solubility for Benzoxazoles
plasma
IC50 (nM) brain levels
levels
inhib Aβ42
c
a
b
b
compd
R
Aβ42
(μM)
(μM)
vivo (%)
43a
43b
4-F-Ph
2-
OMePh
5289
2113
11.6 1.1
nd
6.4 0.3
nd
3
nd
43c
4-F-Ph-
CH2−
696
6.8 0.7
9.4 1.0
15
43d
iBu
603
10 2.4
4.7 0.9
18
a
IC50 represents the concentration of a compound that is required for
reducing the Aβ42 level by 50%. The IC50 values are a mean of at least
three determinations. The confidence intervals for the IC50 values are
b
reported in the Supporting Information. Plasma and brain samples
were analyzed for the tested compound using a qualified research LC-
MS/MS method. Data are expressed as the geometric mean values of
In general, the designed series of compounds showed
moderate to excellent in vitro activity, which in some cases
translated in very good lowering of Aβ42 peptides in vivo. In
contrast to γ-secretase inhibitors, no relevant reduction in
Aβtotal levels was observed for any of the tested compounds
and no inhibition of Notch processing up to the highest
concentration tested (10 μM, data not shown). In all chemical
classes, the high potency was accompanied by high lipophilicity
(in general, cLogP >5) and a TPSA below 75 Å, explaining the
c
at least four runs the standard error of the mean (SEM). Mouse in
vivo at 30 mpk po after 4 h (n = 6) expressed as a percentage of Aβ42
lowering in brain compared with vehicle treated animals. nd: not
determined.
The most potent compounds within the various series are
those where the aromatic substituent at the 2-position is
pushed out of the plane of the central bicyclic heteroaromatic
G
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 4. Aza benzimidazole analogues. The IC50 values are a mean of at least three determinations.
Table 5. In Vitro/in Vivo Activities, Brain/Plasma Levels, Solubility, and hERG Binding of Methoxyphenyl Variations
a
IC50 represents the concentration of a compound that is required for reducing the Aβ42 level by 50%. The IC50 values are a mean of at least three
b
determinations. The confidence intervals for the IC50 values are reported in the Supporting Information. Plasma and brain samples were analyzed
for the tested compound using a qualified research LC-MS/MS method. Data are expressed as the geometric mean values of at least six runs the
c
standard error of the mean (SEM). nd: not determined. Mouse in vivo at 30 mpk po after 4 h (n = 6, except for 44a where n = 4) expressed as a
d
percentage of Aβ42 lowering in brain compared with untreated animals. Inhibition of binding of tritiated dofetilide to the human ether-a-go-go
related gene (hERG) expressed in HEK-293 cells.
good brain penetration.29 However, the majority of compounds
also suffered from hERG binding with IC50s below 1 μM, CyP
inhibition and low solubility, particularly at pH = 7.4 (data not
shown). These unfavorable ADME properties can likely be
attributed to the combination of high lipophilicity and low
TPSA.30 Despite the low solubility, most of the compounds had
good oral bioavailability, with high levels in plasma and brain.
We speculate that this is likely related to the weakly basic
character of the imidazole moiety (pKa 5−6), helping to
solubilize the compounds in the acidic environment of the
stomach, for subsequent absorption in the small intestine.31
Among the most potent in vivo γ-secretase modulators were
benzimidazoles 41b and 41e (Table 2), which prompted us to
further explore the benzimidazole series. To improve the drug-
likeness, analogues were designed in which the benzimidazole
nucleus (see Figure 4) was kept constant while variations were
made on the methoxyphenyl ring as illustrated in compounds
44a−44f, Table 5.
H
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
In the majority of these modifications, the in vitro potency
remained below 100 nM, which in vivo translated in the
reduction of Aβ42 levels. Gratifyingly, some of the in vitro
ADME parameters were also improved. For example, where
compound 41b inhibited the hERG channel with an IC50 of 2
μM, several of the analogues had an IC50 above 5 μM.
Compound 44a showed the best in vivo lowering of Aβ42
peptides (62%) in mouse brain 4 h after a dose of 30 mg/kg po,
most likely related to the improved brain/plasma ratio
compared to 41b and other 44 analogues. Additional nitrogen
analogues 44g and 44h were also prepared (Figure 4).
Compound 44g, with the N-atom transferred to the
benzimidazole and ortho to the aniline linker, maintained the
in vitro activity but showed a lower reduction of the Aβ42
peptides. Interestingly, with ortho N-atoms present in both
adjacent rings to the amino linker as in 44h, the in vitro activity
of 44h was reduced 10-fold compared to 44a or 44g. This may
be due to an electronically induced unfavorable torsion angle
between the two heteroaromatic rings in 44h.
Compound 44a remained the best analogue of the
benzimidazole series and also one of the most potent GSMs
reported to date. The compound was extensively evaluated for
its in vitro ADME properties and in vivo potency in several
species. The most relevant physicochemical and in vitro data
are tabulated in Table 6. The metabolic stability of 44a was
shown to be good across multiple species with a low turnover
in human, mouse, and rat microsomes and a moderate turnover
in dog microsomes.32 In human liver microsomes, no reactive
metabolites were observed upon trapping experiments with
glutathione or cyanide (data not shown). 44a was a moderate
to weak inhibitor of cytochromes P450, with IC50 values
varying from 2.9 to more than 30 μM for the different isoforms
in human liver microsomes. No genotoxicity alerts were found
in the Ames II33 or GreenScreen HC34 tests. In accordance
with the high lipophilicity of 44a, over 98% plasma protein
binding was observed across species. The weak affinity of 44a
to bind to the hERG channel (IC50 8.5 μM) translated into
only a weak effect on the potassium membrane current
recorded with an automated patch-clamp system (30%
inhibition at 3 μM versus 17% for vehicle). Compound 44a
was poorly soluble across pH range (<0.001 mg/mL at pH 4
and pH 7.4, Table 6), but it did solubilize in a strongly acidic
environment (1.2−2.3 mg/mL in 0.01 N HCl).31 To have an
estimate of the solubility of 44a in the gastro-intestinal tract
(GI tract), the solubility was determined also in the fasted-state
and fed-state simulated intestinal fluid buffers (FaSSIF and
FeSSIF respectively, Table 6).35 In the FeSSIF test, 44a showed
an improved, albeit low solubility of 20 μg/mL (Table 6). The
fact that 44a had a moderate melting point of 139 °C (see
Experimental Section) would also suggest a better solubility
across the GI tract than that determined with the pH 4 and pH
7.4 buffers.36 44a displayed a moderate permeability in MDR1-
expressing LLC-PK1 cells (LLC-MDR1, 8 × 10−6 cm/s, Table
6),37 with no indication of P-glycoprotein mediated efflux (0.5
efflux ratio).
Table 6. In Vitro Drug-Like Profile of 44a
assay
44a
LogP
5.25
a
Metabolic Stability (% Conversion)
human
mouse
rat
6
0
16
37
dog
b
CyP450 Inhibition, IC50 (μM)
3A4 (midazolam)
8.7
3A4 (testosterone)
2D6 (dextrometorphan)
2C9 (tolbutamide)
2C19 (S-mephenytoin)
1A2 (phenacetin)
2.9
3.6
21.9
3.5
>30
Genotoxicity
Ames II31
GreenScreen HC32
negative
negative
Plasma Protein Binding (% free)
human
mouse
rat
0.13
0.04
0.04
0.03
30
dog
hERG patch clamp (%inh, at 3 μM)
Thermodynamic Solubility
buffer pH 4.0
buffer pH 7.4
0.01N HCl
<0.001 mg/mL
<0.001 mg/mL
1.17−2.33 mg/mL
0.003 mg/mL
FaSSIF
FeSSIF
0.020 mg/mL
c
Permeability (LLC-MDR1 Cells)
Papp (A−B) (10−6 cm/s)
8
4
Papp (B−A) (10−6 cm/s)
a
Metabolism after incubation of 44a at 1 μM with liver microsomes
for 15 min.30 Inhibition of the metabolism of isoform specific
b
substrates (given in parentheses) in human liver microsomes. The IC50
values are given as a mean of three determinations. A−B = apical to
basolateral; B−A = basolateral to apical.
c
dependent reduction in Aβ42 was observed, with a concomitant
increase in Aβ38 levels. As expected for a GSM, no significant
change in Aβ total level was observed, although at 8 h a
tendency of reduction was observed. After 24 h, Aβ levels were
normalized, except for the Aβ42 level at the 30 mg/kg dose,
where still some reduction appeared to be present (77 3%
versus vehicle). In this experiment, an additional analysis was
performed at 48 h, at which time-point also Aβ42 levels had
returned to normal (101 7% versus vehicle, plasma and brain
levels of 44a below quantifiable limit (BQL), data not shown in
Figure 5).
Standard pharmacokinetic (PK) studies in multiple species
revealed a good and rapid absorption in mouse and dog and a
good and slow absorption in rat. Bioavailability for all species
was high in rat (95%), mouse (100%), and dog (64%). Detailed
PK data can be found in the Supporting Information. Although
these PK data were generated with 44a formulated as a solution
In addition, 44a was screened on 51 receptors and
transporters and 37 enzymes in a selectivity screen performed
at CEREP.38 Selectivity overall was good, and the only
interactions greater than 50% at 10 μM were with NK3
(71%) and with the Na+ channel (site 2, 60%).
For the in vivo evaluation, 44a was first tested further in
mouse at multiple time points at a dose of 10 and 30 mg/kg po
and including measurement of Aβ38 (Figure 5). A dose
I
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. Changes in mouse brain Aβ-levels after a single 10 and 30 mg/kg dose of 44a. Expressed as mean % change versus T = 0 h, n = 4.
(1 mg/mL in 20% aqueous SBE-β-cyclodextrin solution at pH
4), a comparable exposure was observed when dosed as a
methocell suspension. Subsequent in vivo experiments were
therefore carried out using methocell suspensions. We recently
reported on the use of a canine model to evaluate efficacy and
safety of γ-secretase inhibitors (GSIs) and GSMs.39 In Figure 6,
and Aβ38 were increased, with an especially strong increase in
Aβ37 of 4-fold in brain and 8-fold in CSF (Figure 7). Apart
from this difference in change for the shorter Aβ species, in rat
no significant difference or time delay was observed for the Aβ
changes in brain versus CSF.
CONCLUSIONS
■
The present studies described the successful application of
conformational restriction of aminopyridone 4 into pyridone
isosteres such as imidazopyridines, benzimidazoles, benzox-
azoles, and indazoles, resulting in new series of GSMs with
good in vitro and in vivo activity. The potency was enhanced
further by inducing nonplanarity between the central bicyclic
heterocycle and the pendant 2-substituent. (Figure 3). Despite
the low solubility across the series, good oral bioavailability
combined with acceptable brain penetration resulted in
considerable reductions in Aβ42 levels in mouse brain without
significant changes in Aβtotal levels. Replacement of the
methoxyphenyl ring with a methoxypyridyl ring has led to 44a
with an improved drug-like profile and enhanced in vivo
activity. Benzimidazole analogue 44a had an IC50 of 16 nM in
lowering Aβ42 in a cellular assay and displayed good in vivo
activity across species such as mouse, rat, and dog. In all three
species, a typical γ-secretase modulatory profile was observed
with a decrease in the longer Aβ-isoforms Aβ40 and Aβ42 and
an increase in the shorter isoforms Aβ37 and Aβ38. Unlike
carboxylic acid-derived GSMs recently described40 with no
effect on Aβ40 levels, 44a is efficient in lowering Aβ40,
suggesting a different mode of interaction with the γ-secretase
complex. The especially strong increase in the short Aβ-
peptides Aβ37 and Aβ38 after treatment with 44a is
noteworthy. Currently, little is known about the toxicity or
relevance to plaque formation of these more soluble isoforms
Aβ37 and Aβ38. Studies with compounds such as 44a may
provide further insight in the role of these isoforms.8 As many
of the GSMs described to date, the series detailed in this paper,
including 44a, is suffering from suboptimal physicochemical
properties: low solubility, high lipophilicity, and high
aromaticity.30 For 44a, this has translated into signs of liver
toxicity after dosing in dog at 20 mg/kg.39 Further optimization
of the drug-like properties of this series is ongoing. For this
work, 44a will be serving as a benchmark for potency, as well as
in studies toward required changes of Aβ-levels needed for
disease modification of Alzheimer’s disease.
Figure 6. Changes in dog CSF Aβ-levels after a single 20 mg/kg dose
of 44a. Expressed as mean % change versus T = 0 h, n = 4.
the effect of 44a on Aβ38, 40, and 42 in beagle dog
cerebrospinal fluid (CSF) is shown after a single oral dose of
20 mg/kg, as determined via MesoScale Discovery analysis. At
8 h, an increase of 84% in Aβ38 and a decrease of 36% and 48%
of the longer Aβ-isoforms Aβ40 and Aβ42, respectively, were
observed. After 24 h, the Aβ levels were normalized again.
Although the compound levels were substantially higher at 4 h
than at 8 h, at 4 h only a marginal reduction in Aβ40 and Aβ42
was seen. This can be explained by a delay in Aβ-level changes
in CSF compared to brain, where the actual production of Aβ
peptides is taking place.
To bridge the data obtained in mouse brain with those in dog
CSF, in a subsequent experiment rats were treated with 10 mg/
kg of 44a in order to investigate its efficacy to modulate the
release of Aβ-peptides in brain and CSF within the same
species. In addition, the MesoScale analysis was expanded to
include the measurement of Aβ37 levels. In agreement with the
experiments in mouse and dog, a decrease in Aβ40 and Aβ42
was seen in both brain and CSF. The shorter isoforms Aβ37
J
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 7. Changes in rat brain and CSF Aβ and compound levels of 44a after a single oral dose (10 mg/kg). Expressed as mean % change versus T =
0 h, n = 5.
A, a 0.25% NH4HCO3 solution in water; phase B, CH3OH; phase C,
EXPERIMENTAL SECTION
■
CH3CN).
Chemistry. Melting points were determined with a DSC823e
In general, final compounds obtained as solids were triturated with
(Mettler-Toledo) and were measured with a temperature gradient of
diisopropylether (DIPE). Alternatively, some compounds were
1
30 °C/min. The reported values are peak values. H NMR spectra
precipitated and isolated as HCl salt by dissolving them in
were recorded with Bruker Avance DPX 400 and 360 spectrometers,
diethylether, followed by the addition of a 1N HCl solution in
and chemical shifts (δ) are expressed in parts per million (ppm) with
TMS as internal standard. Elemental analyses were performed with a
Carlo-Erba EA1110 analyzer. Mass spectral data were obtained via
GC-MS analysis, using an Agilent 6890 series GC combined with a
5973N mass selected detector. Combustion analytical results were
within 0.4% of the theoretical values except when noted otherwise. For
all tested and final compounds where no analytical purity is
mentioned, compounds were confirmed >95% pure via HPLC
methods. The analytical LC measurement was performed using an
Acquity UPLC (Waters) system comprising a binary pump, a sample
organizer, a column heater (set at 55 °C), a diode array detector
(DAD), and a bridged ethylsiloxane/silica hybrid (BEH) C18 column
(1.7 μm, 2.1 mm × 50 mm; Waters Acquity) with a flow rate of 0.8
mL/min. Two mobile phases (mobile phase A consisting of 0.1%
formic acid in H2O/methanol 95/5; mobile phase B consisting of
methanol) were used to run a gradient condition from 95% A and 5%
B to 5% A and 95% B in 1.3 min and held for 0.2 min. An injection
volume of 0.5 μL was used. Cone voltage was 10 V for positive
ionization mode and 20 V for negative ionization mode. Flow from the
column was split to a mass spectrometer. The MS detector was
configured with an electrospray ionization source. Mass spectra were
acquired by scanning from 100 to 1000 in 0.18 s using a dwell time of
0.02 s. The capillary needle voltage was 3.5 kV, and the source
temperature was maintained at 140 °C. Nitrogen was used as the
nebulizer gas. Sensitive reactions were performed under nitrogen.
Commercial solvents were used without any pretreatment. Preparative
HPLC purifications were carried out using a Shandon Hyperprep
column: C18 base deactivated silica (BDS), 8 μm, 250 g, I.D. 5 cm,
and eluting with a gradient of three subsequent mobile phases (phase
diethylether.
Compounds 313 and 414a were synthesized according to literature
procedures.
1-(2-Methoxy-4-nitro-phenyl)-4-methyl-1H-imidazole (7a). A
mixture of 1-chloro-2-methoxy-4-nitrobenzene (150 g, 0.8 mol), 4-
methyl-1H-imidazole (131.3 g, 1.6 mol), and K2CO3 (110.5 g, 0.8
mol) in DMSO (1.5 L) was reacted in an autoclave under a N2
atmosphere for 6 h at 150 °C. The reaction mixture was poured into
ice−water (6 L). The solid was filtered and washed with water, and
then it was dissolved in CH2Cl2 and further washed with water. The
residue was partitioned between CH2Cl2 and water. The organic layer
was dried over MgSO4, filtered, and then the filtrate was concentrated.
The residue was purified by chromatography over silica gel (eluent:
CH2Cl2/methanol 100/0 to 97/3). The desired fractions were
collected and the solvent was evaporated. The residue was triturated
in DIPE, filtered off and dried to give 48.5 g (26%) of 7a; mp 136.9
°C. 1H NMR (360 MHz, DMSO-d6) δ (ppm) 2.17 (s, 3 H), 3.99 (s, 3
H), 7.31 (s, 1 H), 7.70 (d, J = 8.4 Hz, 1 H), 7.95 (dd, J = 8.8, 2.5 Hz, 1
H), 7.97−8.01 (m, 2 H). MS (ESI) m/z 234 [M + H]+.
3-Methoxy-4-(4-methyl-imidazol-1-yl)-phenylamine (8a). To
a solution of 7a (48.5 g, 0.21 mol) in MeOH (1 L) was added Pd/C
(10%, 5.0 g), and the resulting suspension was stirred at 50 °C under
an atmospheric pressure of H2 overnight, until 3 equiv of H2 were
absorbed. The catalyst was filtered over dicalite. The filtrate was
concentrated, and the resulting residue was suspended in DIPE and
stirred for 1 h. The solid was filtered and dried to yield 39.9 g (94%) of
8a; mp 128.3 °C. 1H NMR (360 MHz, DMSO-d6) δ (ppm) 2.10 (s, 3
H), 3.67 (s, 3 H), 5.37 (br s, 2 H), 6.17 (dd, J = 8.0, 2.2 Hz, 1 H), 6.34
(d, J = 2.2 Hz, 1 H), 6.88 (s, 1 H), 6.91 (d, J = 8.4 Hz, 1 H), 7.49 (d, J
= 1.4 Hz, 1 H). MS (ESI) m/z 204 [M + H]+.
K
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4-Methoxy-5-(4-methyl-imidazol-1-yl)-pyridin-2-ylamine
(8b). A solution of tert-butyl (5-iodo-4-methoxy-pyridin-2-yl)-
carbamate (1.2 g, 3.4 mmol), K2CO3 (947 mg, 6.9 mmol), 4-
methyl-1H-imidazole (394 mg, 4.8 mmol), CuI (653 mg, 3.4 mmol),
and 1,10-phenanthroline (618 mg, 3.4 mmol) in DMF (15 mL) was
degassed and stirred for 6 days at 120 °C. The reaction mixture was
cooled to room temperature and filtered through dicalite. The filtrate
was concentrated, and the residue was dissolved in EtOAc and washed
with a saturated aqueous NaHCO3 solution and brine. The organic
layer was dried over MgSO4, filtered, and concentrated. The crude
residue was purified via reversed phase HPLC to provide 120 mg
3-Fluoro-4-(4-methyl-imidazol-1-yl)-phenylamine (8f). Step
1. Synthesis of 1-(2-Fluoro-4-nitro-phenyl)-4-methyl-1H-imidazole
(7f). K2CO3 (15.3 g, 111 mmol) and 4-methyl-1H-imidazole (9.1 g,
111 mmol) were added to a solution of 3,4-difluoro-nitrobenzene (15
g, 92 mmol) in DMF (200 mL). The reaction mixture was stirred at
125 °C for 4 h. The reaction mixture was cooled down to room
temperature, diluted with water, and extracted with EtOAc. The
organic layer was dried over MgSO4 and concentrated. The residue
was purified via reversed phase. The desired fractions were collected,
and the solvent was evaporated and coevaporated with methanol,
yielding 13 g (64%) of 7f. 1H NMR (360 MHz, CDCl3) δ (ppm) 2.32
(s, 3 H), 7.01−7.17 (m, 1 H), 7.51−7.66 (m, 1 H), 7.88 (s, 1 H),
8.08−8.28 (m, 2 H). MS (ESI) m/z 222 [M + H]+.
1
(17%) of 8b. H NMR (360 MHz, CDCl3) δ (ppm) 2.28 (s, 3 H),
3.82 (s, 3 H), 4.65 (br s, 2 H), 6.09 (s, 1 H), 6.79 (br s, 1 H), 7.52 (br
s, 1 H), 7.89 (br s, 1 H). MS (ESI) m/z 205 [M + H]+.
Step 2. Synthesis of 8f. Intermediate 7f (7.5 g, 34 mmol) and 1 mL
of 4% thiophene solution in DIPE were added to a solution of 1 g of
palladium on carbon (10% Pd) in methanol (150 mL). The reaction
mixture was stirred at 25 °C under hydrogen atmosphere until 3 equiv
of H2 were absorbed. The catalyst was removed by filtration over
dicalite. The filtrate was concentrated, and the residue was triturated in
heptane, filtered, and dried in vacuo to 6.4 g (100% yield) of 7f, which
was used as such in the next step. MS (ESI) m/z 192 [M + H]+.
1-(4-Bromo-2-methoxy-phenyl)-4-methyl-1H-imidazole
(11). A solution of 8a (20.0 g, 98.4 mmol) in AcOH (200 mL) was
added at 10 °C to a stirred solution of NaNO2 (7.47 g, 108 mmol) in
concd H2SO4 (160 mL) at such a rate that the temperature of the
reaction was maintained below 10 °C. After addition was completed,
the resulting mixture was stirred at room temperature for 30 min
before being added dropwise to a stirred solution of CuBr (28.2 g, 197
mmol) in 48% HBr (200 mL). The ensuing reaction was stirred for 1 h
and then diluted with ice−water (1 L). The resulting white precipitate
was filtered, washed with water, and then suspended in a biphasic
mixture of CH2Cl2 and saturated aqueous Na2CO3 solution (1:1). The
resulting slurry was filtered over dicalite. The layers were separated,
and the organic layer was washed with a diluted ammonia solution.
The organic phase was dried over MgSO4, filtered, and concentrated
to give a first batch of 11 (8.1 g) as a brown solid. The aqueous filtrate
was brought to pH = 10 by addition of solid Na2CO3 and then
extracted 3 times with CH2Cl2. The combined organic extracts were
washed with diluted ammonia until the disappearance of the blue color
of the water phase. The organic phase was dried over MgSO4 and
filtered. The first batch of 11 was added to the filtrate, which was
concentrated to give 24.0 g of the title compound (91.3%) as a brown
5-Methoxy-6-(4-methyl-imidazol-1-yl)-pyridin-3-ylamine
(8c). Na2CO3 (528 mg, 5 mmol) and 4-methyl-1H-imidazole (409 mg,
5 mmol) were added to a solution of 2-chloro-3-methoxy-5-nitro-
pyridine (940 mg, 5 mmol) in DMF (10 mL). The reaction mixture
was stirred at 100 °C for 16 h, and then it was cooled to room
temperature and the solvent was evaporated under reduced pressure.
The residue was dissolved in EtOAc and was washed with water. The
water layer was extracted with EtOAc, and the combined organic layers
were dried over MgSO4, filtered, and concentrated. The crude residue
(7c) was added to a suspension of iron powder (835 mg, 15 mmol) in
acetic acid (10 mL). The reaction mixture was stirred at 60 °C for 1 h,
after which the solvent was evaporated. The residue was dissolved in
EtOAc and washed with a saturated aqueous solution of NaHCO3 and
brine. The organic layer was dried over MgSO4, filtered, and
concentrated to provide 245 mg (21%) of 8c, which was used as
such in the next reaction. MS (ESI) m/z 205 [M + H]+.
5-(4-Methyl-imidazol-1-yl)-pyridin-2-ylamine (8d). CuI (8.26
g, 43.4 mmol) was added to a solution of Cs2CO3 (9.4 g, 28.9 mmol),
4-methyl-1H-imidazole (5.9 g, 72.3 mmol), and 5-bromo-pyridin-2-
ylamine (5 g, 29 mmol) in DMSO (100 mL). The reaction mixture
was stirred at 130 °C for 2 days, after which it was cooled to room
temperature, triturated in acetonitrile, and filtered. The filtrate was
concentrated, and the residue was dissolved in CH2Cl2. The organic
layer was washed with water, dried over MgSO4, filtered, and
concentrated. The residue was purified by flash column chromatog-
raphy over silica gel (eluent: CH2Cl2/(7N NH3 in MeOH), 100/0 to
96/4) to provide 160 mg of 8d. The water layer was concentrated until
a precipitate started to form. The solid was filtered, yielding an
additional 628 mg of 8d (15% overall yield) as a single regioisomer. 1H
NMR (360 MHz, CDCl3) δ (ppm) 2.29 (s, 3 H), 4.68 (br s, 2 H),
6.58 (d, J = 8.8 Hz, 1 H), 6.87 (s, 1 H), 7.42 (dd, J = 8.8, 2.56 Hz, 1
H), 7.60 (s, 1 H), 8.11 (d, J = 2.9 Hz, 1 H). MS (ESI) m/z 175 [M +
H]+ .
6-(4-Methyl-imidazol-1-yl)-pyridin-3-ylamine (8e). Step 1.
Synthesis of 5-Bromo-2-(4-methyl-imidazol-1-yl)-pyridine. K2CO3
(8.8 g, 63 mmol) and 4-methyl-1H-imidazole (3.5 g, 42 mmol) were
added to a solution of 2,5-dibromo-pyridine (5 g, 21 mmol) in N-
methyl-2-pyrrolidone (50 mL). The reaction mixture was stirred at 85
°C for 16 h, and then it was cooled to room temperature and
partitioned between CH2Cl2 and water. The two layers were separated,
and the organic layer was dried over MgSO4 and concentrated under
reduced pressure (85 °C at 10 mmHg) to provide 3.3 g (62%) of the
title compound as a mixture of regioisomers (85/10). MS (ESI) m/z
238 [M + H]+.
Step 2. Synthesis of 8e. A 250 mL stainless steel autoclave was
charged with a solution of the intermediate from step 1 (2.8 g, 12
mmol) in THF (10 mL). Ammonia (90 mL) and Cu2O (90 mg, 0.6
mmol) were added. The autoclave was closed, and the reaction
mixture was stirred and heated at 150 °C for 18 h. The mixture was
allowed to cool to room temperature and extracted three times with
CH2Cl2. The combined organic layers were dried over MgSO4,
filtered, and concentrated. The residue was purified via reversed phase
HPLC to provide 250 mg (12%) of 8e. 1H NMR (360 MHz, CDCl3)
δ (ppm) 2.29 (d, J = 1.1 Hz, 3 H), 3.76 (br s, 2 H), 7.11 (d, J = 1.1 Hz,
1 H), 7.11 (s, 1 H), 7.22−7.24 (m, 1 H), 7.93 (dd, J = 2.2, 1.5 Hz, 1
H), 8.07 (d, J = 1.5 Hz, 1 H). MS m/z 175 [M + H]+.
1
solid; mp 69.4−70.7 °C. H NMR (360 MHz, CDCl3) δ (ppm) 2.27
(s, 3 H), 3.83 (s, 3 H), 6.84−6.89 (m, 1 H), 7.10 (dd, J = 7.3, 1.1 Hz, 1
H), 7.13 (d, J = 1.8 Hz, 1 H), 7.15 (s, 1 H), 7.63 (d, J = 1.4 Hz, 1 H).
MS (ESI) m/z 267 [M + H]+.
6-Bromo-2-methoxy-pyridin-3-ylamine (12). NaOCH3 (176 g,
3.3 mol) was added portionwise to a solution of 3-amino-2,6-
dibromopyridine (100 g, 0.4 mol) in 1,4-dioxane (1 L), and the
resulting mixture was refluxed for 3 h. The reaction mixture was then
allowed to cool to room temperature, after which it was poured into an
aqueous saturated NH4Cl solution (2 L). After stirring for 30 min,
diethyl ether (2 L) was added and the bilayer was stirred for 30 min.
The layers were separated, and the aqueous layer was diluted with
water (1.5 L) and then extracted with diethyl ether (6 × 500 mL). The
combined organic layers were washed brine (2 × 500 mL), dried over
MgSO4, and concentrated. The residue was purified by silica gel
chromatography (eluent: CH2Cl2) to provide 67 g (78%) of 12, as a
brown−orange solid. GC-MS m/z 202.
6-Bromo-2-methoxy-3-(4-methyl-imidazol-1-yl)-pyridine
(13). Step 1. Synthesis of N-(6-Bromo-2-methoxy-pyridin-3-yl)-
formamide. A mixture of formic acid (12.8 mL, 340 mmol) and acetic
acid anhydride (8.54 mL, 91 mmol) was stirred at room temperature
for 40 min. Subsequently, a solution of 3-amino-6-bromo-2-methoxy-
pyridine (12) (5.0 g, 24.6 mmol) in THF (30 mL) was added
dropwise, and the resulting reaction mixture was stirred overnight at
60 °C. The mixture was cooled and poured into ice−water. The
resulting solid was filtered, washed with water, and dried to give 5.2 g
(76%) of the title compound, which was used as such in the next step.
MS (ESI) m/z 231 [M + H]+
L
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Step 2. Synthesis of N-(6-Bromo-2-methoxy-pyridin-3-yl)-N-(2-
oxo-propyl)-formamide. 1-Chloro-propan-2-one (4.34 g, 46.9 mmol)
was added dropwise to a mixture of N-(6-bromo-2-methoxy-pyridin-3-
yl)-formamide (5.2 g, 18.8 mmol), KI (0.34 g, 2.0 mmol), and Cs2CO3
(21.4 g, 66 mmol) in DMF (50 mL). The reaction mixture was stirred
at room temperature overnight, and then it was poured into ice−water
and extracted with EtOAc. The combined organic layers were dried
over MgSO4, filtered, and concentrated. The residue was triturated
with DIPE, filtered, and dried to give 4.43 g (82%) of the title
compound. 1H NMR (360 MHz, CDCl3) δ (ppm) 2.18 (s, 3 H), 4.00
(s, 3 H), 4.48 (s, 2 H), 7.14 (d, J = 8.0 Hz, 1 H), 7.49 (d, J = 8.0 Hz, 1
H), 8.23 (s, 1 H). GC-MS m/z 286.
DIPE, and then dried to provide 1.00 g (69%) of 19a as HCl salt. 1H
NMR (360 MHz, DMSO-d6) δ (ppm) 6.82 (br. s, 2 H), 7.36−7.58
(m, 3 H), 8.16 (t, J = 8.0 Hz, 2 H), 8.37−8.56 (m, 2 H). MS (ESI) m/
z 258 [M + H]+.
2-(4-Fluoro-phenyl)-1-methyl-1H-benzimidazol-4-ylamine
(20b, R = 4-F-Ph, R′ = Me). Step 1. Synthesis of 2-(4-Fluoro-
phenyl)-1-methyl-4-nitro-1H-benzimidazole. A solution of lithium
hexamethyldisilazide (1 M in THF, 2.96 mL, 2.96 mmol) was added
dropwise at 0 °C to a solution of 19 (290 mg, 0.99 mmol) in THF (10
mL) under a flow of nitrogen, and the resulting mixture was stirred at
this temperature for 30 min. Then CH3I (92 μL, 1.48 mmol) was
added and the resulting mixture was stirred at 55 °C for 12 h. The
reaction was quenched with brine. The organic layer was separated,
dried over MgSO4, filtered, and concentrated. The resulting residue
was triturated with a DIPE/2-propanol mixture. The precipitate was
filtered and dried to provide 160 mg (60%) of the title compound. 1H
NMR (400 MHz, DMSO-d6) δ (ppm) 3.97 (s, 3 H), 7.41−7.55 (m, 3
H), 7.93−8.02 (m, 2 H), 8.12 (ddd, J = 12.9, 8.1, 0.8 Hz, 2 H). MS
(ESI) m/z 272 [M + H]+.
Step 3. Synthesis of 13. N-(6-Bromo-2-methoxy-pyridin-3-yl)-N-
(2-oxo-propyl)-formamide (4.4 g, 15.3 mmol) was added to a mixture
of NH4OAc (5.41 g, 70.2 mmol) in AcOH (50 mL). The reaction
mixture was heated at reflux for 1 h, and then it was cooled to room
temperature and poured into ice−water. The mixture was brought to
pH 9 via the addition of an aqueous NaOH solution (50% w/v), and
then it was extracted with EtOAc. The organic layer was separated,
dried over MgSO4, filtered, and concentrated to give 3.0 g (73%) of 13
Step 2. Synthesis of 20b. Iron (812 mg, 14.5 mmol) was added to a
solution of 2-(4-fluoro-phenyl)-1-methyl-4-nitro-1H-benzimidazole
(750 mg, 2.76 mmol) in AcOH (50 mL), and the resulting mixture
was mechanically stirred at 60 °C for 1 h. After this time, the solvent
was removed under reduced pressure. The resulting residue was
dissolved in CH2Cl2 and washed with a saturated aqueous solution of
NaHCO3 and then brine. The organic layer was separated, active
charcoal and MgSO4 were added, and the resulting mixture stirred at
room temperature for 20 min. Filtration over dicalite, followed by
1
as a solid, which was used as such in the next step. H NMR (360
MHz, CDCl3) δ (ppm) 2.29 (s, 3 H), 4.03 (s, 3 H), 6.92 (t, J = 1.1 Hz,
1 H), 7.17 (d, J = 8.0 Hz, 1 H), 7.40 (d, J = 7.7 Hz, 1 H), 7.73 (d, J =
1.4 Hz, 1 H). MS (ESI) m/z 268 [M + H]+.
6-Methoxy-5-(4-methyl-imidazol-1-yl)-pyridin-2-ylamine
(14). Cu2O (166 mg, 1.16 mmol) and ammonia (24 mL) were added
to a solution of 13 (3.12 g, 11.64 mmol) in ethylene glycol (25 mL).
The mixture was stirred at 100 °C during 16 h. It was then cooled,
diluted with water, and extracted with EtOAc. The organic layer was
dried over Na2SO4 and concentrated. The residue was triturated with
1
concentration of the filtrate, yielded 250 mg (37%) of 20b. H NMR
(360 MHz, DMSO-d6) δ (ppm) 3.77 (s, 3 H), 6.41 (d, J = 7.3 Hz, 1
H), 6.73 (d, J = 8.0 Hz, 1 H), 6.98 (t, J = 7.9 Hz, 1 H), 7.37−7.43 (m,
2 H), 7.84−7.90 (m, 2 H). MS (ESI) m/z 242 [M + H]+.
1
DIPE to yield 1.51 g (63%) of 14 as a light-brown solid. H NMR
(360 MHz, CDCl3) δ (ppm) 2.28 (s, 3 H), 3.89 (s, 3 H), 4.45 (br s, 2
H), 6.10 (d, J = 8.0 Hz, 1 H), 6.80 (s, 1 H), 7.29 (d, J = 8.0 Hz, 1 H),
7.55 (s, 1 H). MS (ESI) m/z 205 [M + H]+.
3-Bromo-N1-methyl-benzene-1,2-diamine (22). A solution of
methylamine in ethanol (8M, 100 mL, 0.8 mol) was added at 0−5 °C
to 1-bromo-3-fluoro-2-nitro-benzene (19.8 g, 90 mmol). The mixture
was stirred overnight at room temperature, and then the solvent was
evaporated and the residue was partitioned between water and
CH2Cl2. The organic layer was dried over MgSO4, filtered, and
concentrated to provide 20 g (96%) of crude (3-bromo-2-nitro-
phenyl)-methyl-amine, which was dissolved in HOAc (150 mL). Iron
powder (15 g, 269 mmol) was added, and the resulting suspension was
stirred and heated at 60 °C for 1 h. The reaction mixture was
concentrated in vacuo, and the residue was partitioned between
CH2Cl2 and a saturated aqueous NaHCO3 solution. The organic layer
was dried over MgSO4, filtered, and concentrated to give 14 g (80%)
8-Bromo-2-(4-fluoro-phenyl)-imidazo[1,2-a]pyridine (17a). A
solution of 2-amino-3-bromopyridine (50.0 g, 0.29 mol) and 4-
fluorophenacyl bromide (75.3 g, 0.35 mol) in ethanol (300 mL) was
stirred at reflux temperature for 17 h. After this time, the reaction
mixture was cooled to room temperature. The resulting precipitate was
filtered, and the filtrate was partially concentrated to result in a second
crop of precipitate. The combined precipitates were triturated with
acetonitrile and ethanol. The resulting solid was suspended in a
saturated aqueous solution of NaHCO3 (500 mL) and stirred for 30
min. Then, the mixture was extracted with CH2Cl2. The organic layer
was separated, dried over Na2SO4, and concentrated. The residue was
recrystallized from EtOAc, to provide 46.5 g (55%) of 17a as a white
solid; mp 165.3 °C. 1H NMR (360 MHz, DMSO-d6) δ (ppm) 6.83 (t,
J = 6.9 Hz, 1 H), 7.29 (m, 2 H), 7.60 (d, J = 7.3 Hz, 1 H) 8.02 (m, J =
5.8 Hz, 2 H) 8.53 (s, 1 H) 8.56 (d, J = 7.0 Hz, 1 H). MS (ESI) m/z
291 [M + H]+.
1
of 22, which was used as such in the next step. H NMR (360 MHz,
CDCl3) δ (ppm) 2.87 (s, 3 H), 3.73 (br s, 3 H), 6.60 (dd, J = 7.8, 1.3
Hz, 1 H), 6.71 (t, J = 8.1 Hz, 1 H), 6.95 (dd, J = 8.1, 1.1 Hz, 1 H). GC-
MS m/z 200.
4-Bromo-2-(4-fluoro-phenyl)-1-methyl-1H-benzimidazole
(23a, R = 4-F-Ph, R′ = Me). 4-Fluoro-benzoylchloride (5.5 g, 34.7
mmol) was added at room temperature to a solution of 22 (10.0 g,
39.8 mmol) and triethylamine (8.05 g, 79.6 mmol) in CH2Cl2 (250
mL), and the resulting mixture was stirred for 15 h. The reaction
mixture was quenched with water, and the two layers were separated.
The organic layer was dried over MgSO4, filtered, and concentrated.
The residue was dissolved in HOAc (100 mL), and concd aq HCl (3
mL), and the resulting solution was stirred for 2 h at 100 °C. The
solvent was removed by evaporation, and the residue was dissolved in
CH2Cl2 and washed with a saturated aqueous NaHCO3 solution. The
organic layer was dried over MgSO4, filtered, and concentrated. The
residue was triturated with DIPE/2-propanol and then filtered to give
6.8 g (64%) of 23a. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 3.88 (s,
3 H), 7.24 (t, J = 7.7 Hz, 1 H), 7.40−7.47 (m, 2 H), 7.49 (dd, J = 7.7,
0.8 Hz, 1 H), 7.66 (dd, J = 8.1, 0.8 Hz, 1 H), 7.90−7.97 (m, 2 H). MS
(ESI) m/z 305 [M + H]+ .
2-(4-Fluoro-phenyl)-4-nitro-1H-benzimidazole (19a, R = 4-F-
Ph, R′ = H). Step 1 Synthesis of N-(2-amino-3-nitro-phenyl)-4-
fluoro-benzamide. A solution of 1,2-diamino-3-nitrobenzene (1.53 g,
10 mmol), triethylamine (2.78 mL, 20 mmol), and 4-fluorobenzoyl
chloride (1.58 g, 10 mmol) in a mixture of CH2Cl2/acetonitrile (2/1,
75 mL) was stirred at room temperature for 2 h. After this time, the
reaction mixture was diluted with CH2Cl2 (20 mL) and washed with
water (20 mL). The two layers were separated, and the organic layer
was dried over MgSO4, filtered, and concentrated. The resulting
residue triturated with DIPE to give 2.3 g (83%) of the title
1
compound; mp 181.5 °C. H NMR (360 MHz, DMSO-d6) δ (ppm)
6.70 (dd, J = 8.6, 7.5 Hz, 1 H), 7.21 (s, 2 H), 7.32−7.42 (m, 2 H), 7.48
(dd, J = 7.3, 1.1 Hz, 1 H), 7.99 (dd, J = 8.8, 1.4 Hz, 1 H), 8.05−8.16
(m, 2 H), 9.89 (s, 1 H). MS (ESI) m/z 551 [2M + H]+.
Step 2. Synthesis of 19a. A solution of N-(2-amino-3-nitro-
phenyl)-4-fluoro-benzamide (1.35 g, 5.0 mmol) and concd aq HCl
(0.5 mL, 6.0 mmol) in acetic acid (15.0 mL) was submitted to
microwave irradiation at 150 °C, for 30 min. The reaction mixture was
cooled to room temperature, and the product precipitated upon
cooling. The precipitate was filtered, washed with acetic acid and
3-Bromo-2-nitro-benzaldehyde (26). A mixture of 3-bromo-2-
nitrotoluene (10 g, 46.3 mmol), N,N-dimethylformamide dimethyl
acetal (16.5 g, 139 mmol), and pyrrolidine (3.3 g, 46.3 mmol) was
heated at 115 °C for 22 h. The reaction mixture was cooled to 0 °C,
M
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and a cooled solution of NaIO4 (29.7 g, 139 mmol) in DMF (75 mL)
and water (100 mL) was added dropwise. After the addition, the
reaction mixture was allowed to warm to room temperature. The
suspension was filtered on dicalite and washed with EtOAc (400 mL).
The filtrate was washed with water (2 × 150 mL), and the organic
layer was concentrated. The residue was purified by flash
chromatography over silica gel (eluent: heptane/CH2Cl2 1/1 to 0/
1). The product fractions were collected, and the solvent was
evaporated to give 2.72 g (20%) of 26a, which was used as such in the
was partitioned between water and CH2Cl2. The organic layer was
dried over MgSO4, filtered, and concentrated. The residue was purified
by flash column chromatography over silica gel (eluent: heptane/
1
CH2Cl2, 30/70) to give 110 mg (69%) of 38d. H NMR (360 MHz,
CDCl3) δ (ppm) 1.04 (d, J = 6.6 Hz, 6 H), 2.23−2.42 (m, 1 H), 2.85
(d, J = 7.3 Hz, 2 H), 7.18 (t, J = 8.1 Hz, 1 H), 7.44 (d, J = 8.1 Hz, 1
H), 7.48 (d, J = 8.1 Hz, 1 H). MS ESI m/z 254 [M + H]+.
[2-(4-Fluoro-phenyl)-imidazo[1,2-a]pyridin-8-yl]-[3-me-
thoxy-4-(4-methyl-imidazol-1-yl)-phenyl]-amine (40a). Sodium
tert-butoxide (0.77 g, 8 mmol), BINAP (93 mg, 0.15 mmol),
Pd(OAc)2 (22 mg, 0.1 mmol), and 8a (0.72 g, 3 mmol) were added
to a solution of 17a (0.58 g, 2 mmol) in toluene (15 mL), and the
mixture was purged with N2 for 5 min. The reaction mixture was
stirred and heated at 100 °C overnight under a N2 atmosphere. The
reaction mixture was cooled to room temperature, water was added,
and the mixture was extracted with CH2Cl2. The organic layer was
dried over MgSO4, filtered, and concentrated. The residue was purified
via reversed phase HPLC. The desired fraction were collected, and the
solvent was evaporated. The residue was crystallized from CH3CN/
DIPE. The solid was collected and dried to give 0.42 g (51%) of 40a;
mp 209.2 °C. 1H NMR (360 MHz, DMSO-d6) δ (ppm) 2.15 (s, 3 H),
3.80 (s, 3 H), 6.80 (t, J = 6.9 Hz, 1 H), 6.98−7.08 (m, 3 H), 7.22−7.34
(m, 4 H), 7.68 (d, J = 1.4 Hz, 1 H), 8.01−8.10 (m, 3 H), 8.38 (s, 1 H),
8.46 (s, 1 H). MS (ESI) m/z 414 [M + H]+. HRMS calcd for
C24H21FN5O (M + H)+, 414.1730, found, 414.1739.
[2-(4-Fluoro-phenyl)-1-methyl-1H-benzoimidazol-4-yl]-[3-
methoxy-4-(4-methyl-imidazol-1-yl)-phenyl]-amine (41 b).
Compound 41b was prepared starting from 11 and 20b in a manner
identical to the preparation of 40a. The crude residue was purified via
column chromatography on silica gel [eluent: CH2Cl2/(7N NH3 in
MeOH), 100/0 to 97/3)] to give 41b in 63% yield; mp 130.9 °C. 1H
NMR (360 MHz, DMSO-d6) δ (ppm) 2.14 (s, 3 H), 3.74 (s, 3 H),
3.86 (s, 3 H), 6.93 (dd, J = 8.5, 2.2 Hz, 1 H), 7.01 (s, 1 H), 7.12−7.23
(m, 5 H), 7.43 (t, J = 8.8 Hz, 2 H), 7.63 (d, J = 1.3 Hz, 1 H), 7.92 (dd,
J = 8.6, 5.5 Hz, 2 H), 8.46 (s, 1 H). MS (ESI) m/z 428 [M + H]+.
Anal. (C25H22FN5O) C, H, N. HRMS calcd for C25H23FN5O (M +
H)+, 428.1886; found, 428.1882.
1
next step. H NMR (360 MHz, CDCl3) δ ppm 7.59 (t, J = 7.9 Hz, 1
H), 7.92−7.96 (m, 2 H), 9.90 (s, 1 H).
7-Bromo-2-(3-methoxy-phenyl)-2H-indazole (28b, R = 3-
CH3O-Ph, R′ = H). Step 1. Synthesis of (3-Bromo-2-nitro-benzyl)-(3-
methoxy-phenyl)-amine. To a solution of 3-bromo-2-nitro-benzalde-
hyde (5.5 g, 23.9 mmol), 3-methoxy-aniline (2.94 g, 23.9 mmol), and
HOAc (7.18 g, 119 mmol) in 1,2-dichloroethane (60 mL) was added
sodium triacetoxyborohydride (7.6 g, 35.8 mmol). The reaction
mixture was stirred for 3 h at room temperature. The reaction mixture
was diluted with CH2Cl2 and washed with an aqueous 10% K2CO3
solution and brine. The organic layer was dried over MgSO4, filtered,
and concentrated. The residue was purified via reversed phase HPLC.
The desired fraction was collected and the solvent was evaporated,
1
yielding 5.68 g (70%) of the title compound. H NMR (360 MHz,
CDCl3) δ (ppm) 3.75 (s, 3 H), 4.18 (br s, 1 H), 4.38 (s, 2 H), 6.11 (t,
J = 2.4 Hz, 1 H), 6.19 (ddd, J = 8.1, 2.4, 0.9 Hz, 1 H), 6.27−6.36 (m, 1
H), 7.08 (t, J = 8.1 Hz, 1 H), 7.31 (t, J = 8.1 Hz, 1 H), 7.48−7.64 (m, 2
H). MS (ESI) m/z 337 [M + H]+.
Step 2. Synthesis of 28b. To a solution of (3-bromo-2-nitro-
benzyl)-(3-methoxy-phenyl)-amine (5.68 g, 16.8 mmol) in EtOH (100
mL) was added tin chloride dihydrate (7.6 g, 34 mmol). The reaction
mixture was stirred at 40−60 °C overnight. The solvent was
evaporated, and CH2Cl2 was added together with a small amount of
water. The mixture was filtered on dicalite, and the filtrate was washed
with water. The water layer was extracted with CH2Cl2, and the
organic layer was dried over MgSO4 and concentrated. The residue
was purified by flash chromatography on silica gel (eluent: heptane/
CH2Cl2 40/60 to 0/100) to yield 3.63 g (71%) of 27b. 1H NMR (360
MHz, CDCl3) δ (ppm) 3.92 (s, 3 H), 6.92−7.04 (m, 2 H), 7.39−7.50
(m, 2 H), 7.51−7.59 (m, 2 H), 7.67 (d, J = 8.4 Hz, 1 H), 8.49 (s, 1 H).
MS (ESI) m/z 303 [M + H]+.
[2-(3-Methoxy-phenyl)-3-methyl-2H-indazol-7-yl]-[3-me-
thoxy-4-(4-methyl-imidazol-1-yl)-phenyl]-amine (42c). Cs2CO3
(0.47 g, 1.44 mmol), X-Phos (50 mg, 0.105 mmol), Pd2(dba)3 (44 mg,
0.048 mmol), and 8a (97 mg, 0.48 mmol) were added to a solution of
28c (152 mg, 0.48 mmol) in tert-butanol (10 mL), and the mixture
was purged with N2 for 5 min. The reaction mixture was stirred and
heated at 110 °C for 20 h under a N2 atmosphere. The reaction
mixture was cooled to room temperature, and water was added. The
mixture was extracted with CH2Cl2. The organic layer was dried over
MgSO4, filtered, and concentrated. The residue was purified by
column chromatography on silica gel (eluent: CH2Cl2/MeOH, 100/0
7-Bromo-2-(3-methoxy-phenyl)-3-methyl-2H-indazole (29c,
R = 3-CH3O-Ph, R′ = Me). A 2 M LDA-solution in THF (7.4 mL,
14.8 mmol) was added dropwise to a solution of 28b (3 g, 9.9 mmol)
in THF (50 mL) at −78 °C. The reaction mixture was allowed to
warm up to 0 °C, stirred at 0 °C for 5 min, and then cooled again to
−78 °C. CH3I (0.92 mL, 14.8 mmol) was added, and the reaction
mixture was allowed to warm up to room temperature. After stirring
for 16 h, water was added and the water layer was extracted with
diethyl ether. The organic layer was dried over MgSO4, filtered, and
concentrated. The residue was purified by flash chromatography on
silica gel (eluent: heptane/CH2Cl2 50/50 to 0/100) to yield 2.2 g
1
to 95/5) to yield 0.13 g (62%) of 42c; mp 184.4 °C. H NMR (360
MHz, DMSO-d6) δ (ppm) 2.14 (s, 3 H), 2.64 (s, 3 H), 3.75 (s, 3 H),
3.84 (s, 3 H), 6.92−7.00 (m, 2 H), 7.01 (s, 1 H), 7.09−7.16 (m, 3 H),
7.19 6 (d, J = 8.5 Hz, 1 H), 7.23−7.31 (m, 3 H), 7.52 (t, J = 8.4 Hz, 1
H), 7.63 (d, J = 1.1 Hz, 1 H), 8.33 (s, 1 H). MS (ESI) m/z 440 [M +
H]+. HRMS calcd for C26H26N5O2 (M + H)+, 440.2086; found,
440.2083.
1
(70%) of 29c. H NMR (360 MHz, DMSO-d6) δ ppm 2.64 (s, 3 H),
3.85 (s, 3 H), 6.98 (dd, J = 8.4, 7.3 Hz, 1 H), 7.10−7.19 (m, 1 H),
7.20−7.29 (m, 2 H), 7.48−7.56 (m, 1 H), 7.58 (d, J = 7.3 Hz, 1 H),
7.81 (d, J = 8.4 Hz, 1 H). MS (ESI) m/z 317 [M + H]+.
N-(2,6-Dibromo-phenyl)-3-methyl-butyramide (37d, R = i-
Bu). Isovaleryl chloride (2.9 mL, 23.9 mmol) was added at room
temperature to a solution of 2,6-dibromoaniline (2.00 g, 7.98 mmol) in
THF (30 mL). The reaction mixture was stirred at room temperature
overnight, and then it was diluted with EtOAc and the organic phase
was washed with a saturated aqueous NaHCO3 solution, dried over
MgSO4, filtered, and concentrated. The residue was triturated with
heptane, filtered, extensively washed with heptanes, and then dried to
yield 2.19 g (82%) of 37d. 1H NMR (360 MHz, CDCl3) δ (ppm) 1.09
(d, J = 6.2 Hz, 6 H), 2.16−2.41 (m, 3 H), 6.97 (br s, 1 H), 7.03 (t, J =
8.1 Hz, 1 H), 7.59 (d, J = 8.1 Hz, 2 H). MS (ESI) m/z 334 [M + H]+ .
4-Bromo-2-isobutyl-benzooxazole (38d, R = i-Bu). A mixture
of 37d (0.21 g, 0.63 mmol), CuI (12 mg, 0.063 mmol), Cs2CO3 (0.31
g, 0.94 mmol), and 1,10-phenantroline (23 mg, 0.125 mmol) in DME
(3 mL) was stirred at 90 °C in a sealed tube overnight. The mixture
(2-Isobutyl-benzooxazol-4-yl)-[3-methoxy-4-(4-methyl-imi-
dazol-1-yl)-phenyl]-amine (43d). Compound 43d was prepared
starting from 8a and 38a in a manner identical to the preparation of
42c, yielding 71% of 43d. 1H NMR (360 MHz, CDCl3) δ (ppm) 1.05
(d, J = 6.7 Hz, 6 H), 2.23−2.36 (m, 1 H), 2.30 (s, 3 H), 2.81 (d, J = 7.2
Hz, 2 H), 3.81 (s, 3 H), 6.72 (s, 1 H), 6.86−6.91 (m, 3 H), 7.03−7.09
(m, 1 H), 7.16 (d, J = 9.1 Hz, 1 H), 7.19−7.24 (m, 2 H), 7.62 (d, J =
1.3 Hz, 1 H). MS(ESI) m/z 377 [M + H]+. HRMS calcd for
C22H25N4O2 (M + H)+, 377.1977; found, 377.1970.
[2-(4-Fluoro-phenyl)-1-methyl-1H-benzoimidazol-4-yl]-[6-
methoxy-5-(4-methyl-imidazol-1-yl)-pyridin-2-yl]-amine (44a).
To a solution of 23a (6.0 g, 19.6 mmol) in tert-butanol (250 mL) were
added aniline 14 (4.0 g, 19.66 mmol), Pd2(dba)3 (1.44 g, 1.57 mmol),
X-Phos (2.25 g, 4.72 mmol), and Cs2CO3 (19.23 g, 59.0 mmol) under
a N2 atmosphere. The reaction mixture was heated at 100 °C for 3 h.
N
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
The solvent was evaporated. Then water was added and the mixture
was extracted twice with CH2Cl2. The combined organic layers were
dried over MgSO4, filtered, and concentrated. The residue was purified
by flash column chromatography [eluent: CH2Cl2/(7N NH3 in
MeOH) 100/0 to 96/4]. The product fractions were collected and
concentrated. The residue was triturated with DIPE and dried, yielding
NH3 in MeOH), 100/0 to 98/2). The resulting residue was purified
further via reversed phase HPLC to provide 95 mg (23%); mp 116 °C.
1H NMR (400 MHz, CDCl3) δ (ppm) 2.31 (s, 3 H), 3.85 (s, 3 H),
6.92 (s, 1 H), 6.99−7.03 (m, 1 H), 7.06 (dd, J = 8.7, 2.5 Hz, 1 H), 7.15
(s, 1 H), 7.18−7.30 (m, 6 H), 7.65 (s, 1 H), 7.74 (dd, J = 8.5, 5.3 Hz, 2
H). MS (ESI) m/z 416 [M + H]+. HRMS calcd for C24H20F2N5 (M +
H)+, 416.1687; found, 416.1680.
1
5.34 g (63.4%) of 44a; mp 139.3 °C. H NMR (360 MHz, CDCl3) δ
(ppm) 2.31 (s, 3 H), 3.86 (s, 3 H), 4.08 (s, 3 H), 6.60 (d, J = 8.4 Hz, 1
H), 6.84−6.91 (m, 1 H), 7.04 (dd, J = 8.0, 0.7 Hz, 1 H), 7.22−7.27
(m, 1 H), 7.28−7.32 (m, 1 H), 7.34 (d, J = 1.0 Hz, 1 H), 7.42 (d, J =
8.0 Hz, 1 H), 7.62−7.66 d, J = 1.0 Hz, 1 H), 7.72−7.80 (m, 2 H), 7.93
(s, 1 H), 8.19 (d, J = 8.0 Hz, 1 H). ESI(MS) m/z 429 [M + H]+. Anal.
(C24H21FN6O) C, H, N. HRMS calcd for C24H22FN6O (M + H)+,
429.1839; found, 429.1854.
Biology. Cell-Based in Vitro Assay Method. Screening was carried
out using SKNBE2 human neuroblastoma cells carrying the hAPP
695−wild type, grown in Dulbecco’s Modified Eagle’s Medium/
Nutrient mixture F-12 (DMEM/NUT-mix F-12) (HAM) provided by
Invitrogen (cat. no. 10371−029) containing 5% Serum/Fe supple-
mented with 1% nonessential amino acids, L-glutamine 2 mM, Hepes
15 mM, penicillin 50 U/mL (units/ml), and streptomycin 50 μg/mL.
Cells were grown to near confluency.
The screening was performed using a modification of the assay as
described.24 Briefly, cells were plated in a 384-well plate at 104 cells/
well in Ultraculture (Lonza, BE12−725F) supplemented with 1%
glutamine (Invitrogen, 25030−024), 1% nonessential amino acid
(NEAA), penicillin 50 U/mL, and streptomycin 50 μg/mL in the
presence of test compound at different test concentrations. The cell/
compound mixture was incubated overnight at 37 °C, 5% CO2. The
next day, the media were assayed for Aβ42 and Aβtotal by two
sandwich immunoassays, using the AphaLisa technology (Perkin-
Elmer) according to the manufacturer’s instructions. To quantify the
amount of Aβ42 in the cell supernatant, monoclonal antibody specific
to the C-terminus of Aβ42 (JRF/cAβ42/26) was coupled to the
receptor beads and biotinylated antibody specific to the N-terminus of
Aβ (JRF/AβN/25) was used to react with the donor beads. To
quantify the amount of Aβtotal in the cell supernatant, monoclonal
antibody specific to the N-terminus of Aβ (JRF/AβN/25) was coupled
to the receptor beads and biotinylated antibody specific to the mid
region of Aβ (biotinylated 4G8) was used to react with the donor
beads.
Mouse in Vivo Assay Method. Compounds were formulated in
20% of Captisol (a sulfobutyl ether of β-cyclodextrin) in water. The
compounds were administered as a single oral dose to overnight fasted,
male CD1 Swiss Specific Pathogen Free (SPF) mice (Charles River,
Germany). After the desired time of treatment (4 h in the screening
model), the animals were sacrificed and Aβ levels were analyzed. Blood
was collected by decapitation and exsanguinations in EDTA-treated
collection tubes. Blood was centrifuged at 1900g for 10 min (min) at 4
°C and the plasma recovered and flash frozen for later analysis. The
brain was removed from the cranium and hindbrain. The cerebellum
was removed, and the left and right hemisphere were separated. The
left hemisphere was stored at −18 °C for quantitative analysis of test
compound levels. The right hemisphere was rinsed with phosphate-
buffered saline (PBS) buffer and immediately frozen on dry ice and
stored at −80 °C until homogenization for biochemical assays.
Mouse brains were resuspended in 8 volumes of 0.4% diethylamine
(DEA)/50 mM NaCl containing protease inhibitors (Roche-
11873580001 or 04693159001) per gram of tissue, e.g., for 0.158 g
brain, add 1.264 mL of 0.4% DEA. All samples were homogenized in
the FastPrep-24 system (MP Biomedicals) using lysing matrix D
(MPBio no. 6913−100) at 6 m/s for 20 s. Homogenates were
centrifuged at 221300g for 50 min. The resulting high speed
supernatants were then transferred to fresh Eppendorf tubes. Nine
parts of supernatant were neutralized with 1 part 0.5 M Tris-HCl pH
6.8 and used to quantify Aß-peptide levels. To quantify the amount of
Aβ-peptides in the soluble fraction of the brain homogenates, Enzyme-
linked immunosorbent assays (ELISA) were used. Briefly, the
standards (a dilution of synthetic Aβ42, Bachem, and Aβ38,
ANASPEC) were prepared in a 1.5 mL Eppendorf tube in
Ultraculture, with final concentrations ranging from 10000 to 0.3
pg/mL. The samples and standards were coincubated with HRPO-
labeled N-terminal antibodies for Aβ42 and Aβ38 detection and with
the biotinylated mid-domain antibody 4G8 for Aβtotal detection.
Then 50 μL of conjugate/sample or conjugate/standards mixtures
were added to the antibody-coated plate. The capture antibodies
selectively recognize the C-terminal end of Aβ42 and Aβ38 and the N-
[2-(4-Fluoro-phenyl)-1-methyl-1H-benzoimidazol-4-yl]-[4-
methoxy-5-(4-methyl-imidazol-1-yl)-pyridin-2-yl]-amine (44b).
Compound 44b was prepared starting from 8b and 23a in a manner
identical to the preparation of 42c. The crude residue was purified by
flash column chromatography over silica gel (eluent: CH2Cl2/(7N
1
NH3 in MeOH), 100/0 to 96/4), yielding 100 mg (40%) of 44b. H
NMR (360 MHz, DMSO-d6) δ (ppm) 2.15 (s, 3 H), 3.83 (s, 3 H),
3.86 (s, 3 H), 7.05 (s, 1 H), 7.13−7.27 (m, 3 H), 7.45 (t, J = 9.0 Hz, 2
H), 7.67 (s, 1 H), 6 7.95 (dd, J = 8.8, 5.5 Hz, 2 H), 8.08 (s, 1 H), 8.32
(d, J = 7.3 Hz, 1 H), 9.05 (s, 1 H). MS (ESI) m/z 429 [M + H]+.
HRMS calcd for C24H22FN6O (M + H)+, 429.1839; found, 429.1840.
[2-(4-Fluoro-phenyl)-1-methyl-1H-benzoimidazol-4-yl]-[5-
methoxy-6-(4-methyl-imidazol-1-yl)-pyridin-3-yl]-amine (44c).
Sodium tert-butoxide (288 mg, 3.0 mmol), BINAP (94 mg, 0.15
mmol), Pd(OAc)2 (23 mg, 0.1 mmol), and 23a (305 mg, 1 mmol)
were added to a solution of 8c (245 mg, 1.2 mmol) in toluene (15
mL), and the mixture was purged with N2 for 5 min. The reaction
mixture was stirred and heated at 150 °C for 5 h under microwave
irradiation, and then it was cooled to room temperature. Water was
added, and the mixture was extracted CH2Cl2. The organic layer was
separated, dried (MgSO4), filtered, and concentrated. The residue was
purified via reversed phase HPLC, to provide 79 mg (18%) of 44c. 1H
NMR (360 MHz, DMSO-d6) δ (ppm) 2.16 (s, 3 H), 3.84 (s, 3 H),
3.86 (s, 3 H), 7.14−7.27 (m, 3 H), 7.37 (s, 1 H), 7.43 (t, J = 9.0 Hz, 2
H), 7.53 (d, J = 2.2 Hz, 1 H), 7.92 (dd, J = 8.8, 5.5 Hz, 2 H), 8.03 (d, J
= 2.2 Hz, 1 H), 8.04 (br s, 1 H), 8.69 (s, 1 H). MS (ESI) m/z 429 [M
+ H]+. HRMS calcd for C24H22FN6O (M + H)+, 429.1839; found,
429.1853.
[2-(4-Fluoro-phenyl)-1-methyl-1H-benzoimidazol-4-yl]-[5-
(4-methyl-imidazol-1-yl)-pyridin-2-yl]-amine (44d). Compound
44d was prepared starting from 8d and 23a in a manner identical to
the preparation of 42c. The residue was purified by flash column
chromatography over silica gel (eluent: CH2Cl2/(7N NH3 in MeOH),
100/0 to 98/2) to provide 50 mg (16%) of 44d. 1H NMR (360 MHz,
CDCl3) δ (ppm) 2.31 (s, 3 H), 3.86 (s, 3 H), 6.93 (s, 1 H), 6.99−7.06
(m, 2 H), 7.26 (t, 2 H), 7.34 (t, J = 8.2 Hz, 1 H), 7.53 (dd, J = 8.8, 2.7
Hz, 1 H), 7.66 (s, 1 H), 7.76 (dd, J = 8.5, 5.3 Hz, 2 H), 7.92 (s, 1 H),
8.24 (d, J = 8.0 Hz, 1 H), 8.36 (d, J = 2.7 Hz, 1 H). MS (ESI) m/z 399
[M + H]+. HRMS calcd for C23H20FN6 (M + H)+, 399.1733; found,
399.1724.
[2-(4-Fluoro-phenyl)-1-methyl-1H-benzoimidazol-4-yl]-[6-
(4-methyl-imidazol-1-yl)-pyridin-3-yl]-amine (44e). Compound
44e was prepared starting from 8e and 23a in a manner identical to
the preparation of 42c. The crude residue was purified by flash column
chromatography over silica gel (eluent: CH2Cl2/(7N NH3 in MeOH),
100/0 to 98/2). The product fractions were collected, yielding 95 mg
(23%) of 44e. 1H NMR (360 MHz, CDCl3) δ (ppm) 2.31 (d, J = 0.7
Hz, 3 H), 3.86 (s, 3 H), 6.95−7.04 (m, 2 H), 7.11 (d, J =7.3 Hz, 1 H),
7.17−7. 32 (m, 5 H), 7.69−7.80 (m, 3 H), 8.16 (d, J = 1.1 Hz, 1 H),
8.45 (d, J = 2.6 Hz, 1 H). MS (ESI) m/z 399 [M + H]+. HRMS calcd
for C23H20FN6 (M + H)+, 399.1733; found, 399.1767.
[3-Fluoro-4-(4-methyl-imidazol-1-yl)-phenyl]-[2-(4-fluoro-
phenyl)-1-methyl-1H-benzoimidazol-4-yl]-amine (44f). Com-
pound 44f was prepared starting from 8f and 23a in a manner
identical to the preparation of 42c. The crude residue was purified by
flash column chromatography over silica gel (eluent: CH2Cl2/(7N
O
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
terminal end for Aβ-total detection. Antibody JRF/cAβ42/26 was used
for Aβ42 detection, antibody J&JPRD/Aβ38/5 for Aβ38 detection,
and antibody JRF/rAβ/2 for Aβtotal detection. The plate was allowed
to incubate overnight at 4 °C in order to allow formation of the
antibody−amyloid complex. Following this incubation and subsequent
wash steps, the ELISA for Aβ42 and Aβ38 quantification was finished
by addition of Quanta Blu fluorogenic peroxidase substrate according
to the manufacturer’s instructions (Pierce Corp., Rockford, Il). A
reading was performed after 10−15 min (excitation 320 nm/emission
420 nm). For Aβtotal detection, a streptavidine-peroxidase conjugate
was added, followed 60 min later by an addional wash step and
addition of Quanta Blu fluorogenic peroxidase substrate according to
the manufacturer’s instructions (Pierce Corp., Rockford, Il). A reading
was performed after 10−15 min (excitation 320 nm/emission 420
nm).
Dog and Rat in Vivo Assays. For details on the in vivo
experimental procedures for the dog experiment; see ref 39. The
experiments in rat were carried out using male Sprague−Dawley rats of
180−220 g, which were fasted overnight before dosing. 44a was dosed
via gastric intubation of a 5 mg/mL suspension in 0.5%
methocellulose. After the indicated time points (2, 4, 7, and 25 h),
the rats were anesthesized in isoflurane, and 50 μL of CSF was
sampled from the cisterna magna and frozen on dry ice. Then the
animals were sacrificed by decapitation, and blood and brain were
collected. Further analysis of compound levels and Aβ levels were
performed in analogy to the mouse in vivo assay. Aβ levels in dog and
rat derived samples were measured using MesoScale Discovery
technology-based sandwich immune-assays. The antibodies against
Aβ38 and Aβ42 were the same as described above. For Aβ40, the JRF/
cAβ40/28 antibody was used; for Aβ37, the antibody JRD/Aβ37/3
was used.
weight; nd, not determined; NSAIDs, nonsteroidal anti-
inflammatory drugs; po, per os
REFERENCES
■
(1) (a) Jakob-Roetne, R.; Jacobsen, H. Alzheimer’s disease: from
pathology to therapeutic approaches. Angew. Chem., Int. Ed. 2009, 48,
3030−3059. (b) Berchtold, N. C.; Cotman, C. W. Evolution in the
conceptualization of dementia and Alzheimer’s disease: Greco-Roman
period to the 1960s. Neurobiol. Aging 1998, 19, 173−189.
(c) Tiraboschi, P.; Hansen, L. A.; Thal, L. J.; Corey-Bloom, J. The
importance of neurotic plaques and tangles to the development and
evolution of AD. Neurology 2004, 62, 1984−1989.
(2) Small, D. H.; Cappai, R. Alois Alzheimer and Alzheimer’s disease:
a centennial perspective. J. Neurochem. 2006, 99, 708−710.
(3) (a) Lahiri, D. K.; Ghosh, C.; Ge, Y. W. A proximal gene promoter
region for the β-amyloid precursor protein provides a link between
development, apotosis and Alzheimer’s disease. Ann. N. Y. Acad. Sci.
2003, 1010, 643−647. (b) Walsh, D. M.; Selkoe, D. J. Deciphering the
molecular basis of memory failure in Alzheimers disease. Neuron 2004,
44, 181−193. (c) Tolia, A.; De Strooper, B. Review structure and
function of γ-secretase. Semin. Cell Dev. Biol. 2009, 20, 211−218.
(d) Takami, M.; Nagashima, Y.; Sano, Y.; Ishihara, S.; Morishima-
Kawashima, M.; Funtamoto, S.; Yasuo, I. γ-Secretase: successive
tripeptide and tetrapeptide release from the transmembrane domain of
β-carboxyl terminal fragment. J. Neurosci. 2009, 29, 13042−13052.
(4) (a) Kreft, A. F.; Martone, R.; Porte, A. Recent advances in the
identification of γ-secretase inhibitors to clinically test the Aβ oligomer
hypothesis of Alzheimer’s disease. J. Med. Chem. 2009, 52, 6169−6188.
(b) D’Onofrio, G.; Panza, F.; Frisardi, V.; Solfrizzi, V.; Imbimbo, B. P.;
Paroni, G.; Cascavilla, L.; Seripa, D.; Pilotto, A. Advances in the
identification of γ-secretase inhibitors for the treatment of Alzheimer’s
disease. Expert Opin. Drug Discovery 2012, 7, 19−37.
(5) Mitani, Y.; Yarimizu, J.; Saita, K.; Uchino, H.; Akashiba, H.;
Shitaka, Y.; Ni, K.; Matsuoka, N. Differential effects between γ-
secretase inhibitors and modulators on cognitive function in amyloid
precursor protein-transgenic and nontransgenic mice. J. Neurosci.
2012, 32, 2037−2050.
(6) 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.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis and analytical data of all intermediates and final
compounds not described in the Experimental Section. Details
of the PK studies in mouse, rat, and dog for 44a. Description of
molecular modeling. Confidence intervals for the cellular Aβ42
IC50 values. This material is available free of charge via the
Internet at http://pubs.acs.org.
(7) (a) Hardy, J. A.; Higgins, G. A. Alzheimer’s disease: the amyloid
cascade hypothesis. Science 1992, 256, 184−5. (b) Bergmans, B. A.; De
Strooper, B. γ-Secretases: from cell biology to therapeutic strategies.
Neurology 2010, 9, 215−225. (c) Panza, F.; Solfrizzi, V.; Frisardi, V.;
Capurso, C.; D’Introno, A.; Colacicco, A. M.; Vendemiale, G.;
Capurso, A.; Imbibo, B. P. Disease-modifying approach to the
treatment of Alzheimer’s Disease. Drugs Aging 2009, 26, 537−555.
(8) Karran, E.; Mercken, M.; De Strooper, B. The amyloid cascade
hypothesis for Alzheimer’s disease: an appraisal for the development of
therapeutics. Nature Rev. Drug Discovery 2011, 10, 698−712.
AUTHOR INFORMATION
Corresponding Author
*Phone: +32-14-606830. Fax: +32-14-605344. E-mail:
hgijsen@its.jnj.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
́ ́
(9) Chavez-Gutierrez, L.; Bammens, L.; Benilova, I.; Vandersteen, A.;
■
Benurwar, M.; Borgers, M.; Lismont, S.; Zhou, L.; Van
Cleynenbreugel, S.; Esselmann, H.; Wiltfang, J.; Serneels, L.; Karran,
E.; Gijsen, H.; Schymkowitz, J.; Rousseau, F.; Broersen, K.; De
Strooper, B. The mechanism of γ-secretase dysfunction in familial
Alzheimer disease. EMBO J. 2012, 31, 2261−2274.
(10) For comprehensive reviews on work on γ-secretase modulators
see: ref 6 (a) (b) 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, 205−225. (c) Zettl, H.; Weggen, S.; Schneider, P.;
Schneider, G. Exploring the chemical space of γ-secretase modulators.
We thank our co-workers at Cellzome, the members of the
Janssen R&D Biology, ADME, and Screening Departments,
and the members of the purification and analysis team.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; Aβ, amyloid-β peptide; ADME,
absorption, distribution, metabolism and excretion; APP,
amyloid precursor protein; BACE1, β-secretase; C99, C-
terminal fragment obtained after cleavage of APP by BACE1;
Captisol, a sulfobutyl ether of β-cyclodextrin; CyP, cytochrome
Trends Pharmacol. Sci. 2010, 31, 402−410. (d) Baumann, S.; Hotteke,
̈
P
450; FaSSIF, fasted state simulated intestinal fluid; FeSSIF, fed
N.; Narlawar, R.; Schmidt, B. γ-Secretase as a target for AD. In
Medicinal Chemistry of Alzheimer’s Disease; Gil, A. M., Eds.; Transworld
Research Network: Trivandrum, India, 2008; pp 193−224; (e) Gar-
ofalo, A. W. Patents targeting γ-secretase inhibition and modulation for
the treatment of Alzheimer’s disease: 2004−2008. Expert Opin. Ther.
Pat. 2008, 18, 693−703. (f) Tomita, T. Secretase inhibitors and
state simulated intestinal fluid; fu,brain, fraction of compound
unbound in brain; GI, gastro-intestinal; GSM, γ-secretase
modulator; GSI, γ-secretase inhibitor; hERG, potassium
channel encoded by the human ether-a-
mpk, milligram of compound per kilogram of animal body
̀
go-go related gene;
P
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
modulators for Alzheimer’s disease treatment. Expert Rev. Neurother.
2009, 9, 661−679. (g) Pissarnitski, D. Advances in γ-secretase
modulation. Curr. Opin. Drug Discovery 2007, 10, 392−402.
(h) Peretto, I.; La Porta, E. γ-Secretase modulation and its promise
for Alzheimer’s disease: a medicinal chemistry perspective. Curr. Top.
Med. Chem. 2008, 8, 38−46.
(11) (a) Abdul-Hay, S. O.; Edirisinghe, P.; Thatcher, G. R. J.
Selective modulation of amyloid-β peptide degradation by flurbiprofen,
fenofibrate, and related compounds regulates Aβ levels. J. Neurochem.
2009, 111, 683−695. (b) Van Broeck, B.; Chen, J.-M.; Treton, G.;
Desmidt, M.; Hopf, C.; Ramsden, N.; Karran, E.; Mercken, M.;
Rowley, A. Chronic treatment with a novel γ-secretase modulator, JNJ-
40418677, inhibits amyloid plaque formation in a mouse model of
Alzheimer’s disease. Br. J. Pharmacol. 2011, 163, 375−389.
(12) (a) Cheng, S.; Comer, D. D.; Mao, L.; Balow, G. P.; Pleynet, D.
Aryl compounds and uses in modulating amyloid β. Patent WO
2004110350, 2004; (b) Kounnas, M. Z.; Danks, A. M.; C., Soan, T. 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.
(13) Kimura, T.; Kawano, K.; Doi, E.; Kitazawa, N.; Shin, K.;
Miyagawa, T.; Kaneko, T.; Ito, K.; Takaishi, M.; Sasaki, T.; Hagiwara,
H. Preparation of cinnamide, 3-benzylidenepiperidin-2-one, phenyl-
propynamide compounds as amyloid β production inhibitors. Patent
WO 2005115990, 2005.
(16) (a) Gijsen, H. J. M.; Bischoff, F. P.; Zhuang, W.; Van Brandt, S.
F. A.; Surkyn, M.; Zaja, M.; Berthelot, D. J.-C.; De Cleyn, M. A. J.;
Macdonald, G. J.; Oehlrich, D. Substituted benzoxazole, benzimida-
zole, oxazolopyridine and imidazopyridine derivatives as γ-secretase
modulators and their preparation and use for the treatment of diseases.
Patent WO 2010094647, 2010; (b) Gijsen, H. J. M.; Velter, A. I.;
Macdonald, G. J.; Bischoff, F. P.; Wu, T.; Van Brandt, S. F. A.; Surkyn,
M.; Zaja, M.; Pieters, S. M. A.; Berthelot, D. J.-C.; De Cleyn, M. A. J.;
Oehlrich, D. Novel substituted bicyclic heterocyclic compounds as
gamma secretase modulators and their preparation and use in the
treatment of diseases. Patent WO 2010089292, 2010; (c) Gijsen, H. J.
M.; Macdonald, G. J.; Bischoff, F. P.; Tresadern, G. J.; Trabanco-
Suarez, A. A.; Van Brandt, S. F. A.; Berthelot, D. J.-C. Preparation of
substituted bicyclic imidazole derivatives as gamma secretase
modulators. Patent WO 2010070008, 2010; (d) Bischoff, F. P.;
Berthelot, D. J.-C.; De Cleyn, M. A. J.; Macdonald, G. J; Oehlrich, D.;
Surkyn, M.; Trabanco-Suarez, A. A.; Tresadern, G. J.; Van Brandt, S. F.
A.; Velter, A. I.; Borghys, H.; Masungi, C.; Mercken, M.; Gijsen. H. J.
M. Discovery of JNJ-42601572, a γ-Secretase Modulator with Potent,
Central Activity in Mouse and Dog. 242nd ACS National Meeting,
Denver, CO, August 28−September 1, 2011, Abstract MEDI-3.
(17) Lang, F.; Zewge, D.; Houpis, I. N.; Volante, R. P. Amination of
aryl halides using copper catalysis. Tetrahedron Lett. 2001, 42, 3251−
3254.
(18) Venuti, M. C.; Stephenson, R. A.; Alvarez, R.; Bruno, J. J.;
Strosberg, A. M. Inhibitors of cyclic AMP phosphodiesterase. 3.
Synthesis and biological evaluation of pyrido and imidazolyl analogues
of 1,2,3,5-tetrahydro-2-oxoimidazo[2,1-b]quinazoline. J. Med. Chem.
1988, 31, 2136−2145.
(14) Similar strategies have been used by others: (a) Huang, X.;
Aslanian, R.; Zhou, W.; Zhu, X.; Qin, J.; Greenlee, W.; Zhu, Z.; Zhang,
L.; Hyde, L.; Chu, I.; Cohen-Williams, M.; Palani, A. The Discovery of
Pyridone and Pyridazone Heterocycles as γ-Secretase Modulators. ACS
Med. Chem. Lett. 2010, 1, 184−187. (b) Qin., J.; Dhondi, P.; Huang,
X.; Mandal, M.; Zhao, Z.; Pissarnitski, D.; Zhou, W.; Aslanian, R.; Zhu,
Z.; Greenlee, W.; Clader, J.; Zhang, L.; Cohen-Williams, M.; Jones, N.;
Hyde, L.; Palani, A. Discovery of fused 5,6-bicyclic heterocycles as γ-
secretase modulators. Bioorg. Med. Chem. Lett. 2011, 21, 664−669.
(c) Fischer, C.; Shah, S.; Hughes, B. L.; Nikov, G. N.; Crispino, J. L.;
Middleton, R. E.; Szewczak, A. A.; Munoz, B.; Shearman, M. S.
Quinazolinones as γ-secretase modulators. Bioorg. Med. Chem. Lett.
2011, 21, 773−776. (d) Qin., J.; Zhou, W; Huang, X.; Dhondi, P.;
Palani, A; Aslanian, R.; Zhu, Z.; Greenlee, W.; Cohen-Williams, M.;
Jones, N.; Hyde, L.; Zhang, L. Discovery of a potent pyrazolopyridine
series of γ-secretase modulators. ACS Med. Chem. Lett. 2011, 2, 471−
476. (e) Wan, Z.; Hall, A.; Sang, Y; Xiang, J.-N.; Yang, E.; Smith, B;
Yang, G.; Yu, H.; Wang, J.; Lau, L. F.; Li, T.; Zhao, W.; Su, X.; Zhang,
X.; Zhou, Y.; Jin, Y.; Tong, Z.; Cheng, Z.; Hussain, I.; Elliott, J. D.;
Matsuoka, Y. Pyridine-derived γ-secretase modulators. Bioorg. Med.
Chem. Lett. 2011, 21, 4832−4835. (f) Pettersson, M.; Johnson, D. S.;
Subramanyam, C.; Bales, K. R.; am Ende, C. W.; Fish, B. A.; Green, M.
E.; Kauffman, G. W.; Lira, R.; Mullins, P. B.; Navaratnam, T.; Sakya, S.
M.; Stiff, C. M.; Tran, T. M.; Vetelino, B. C.; Xie, L.; Zhang, L.;
Pustilnik, L. R.; Wood, K. M.; O’Donnell, C. J. Design and synthesis of
dihydrobenzofuran amides as orally bioavailable, centrally active γ-
secretase modulators. Bioorg. Med. Chem. Lett. 2012, 22, 2906−2911.
(g) Fischer, C.; Zultanski, S. L.; Zhou, H.; Methot, J. L.; Shah, S.;
Nuthall., H.; Hughes, B. L.; Smotrov, N.; Hill, A.; Szewczak, A. A.;
Moxham, C. M.; Bays, N.; Middleton, R. E.; Munoz, B.; Shearman, M.
S. Triazoloamides as potent γ-secretase modulators with reduced
hERG liability. Bioorg. Med. Chem. Lett. 2012, 22, 3140−3146.
(15) Conformational analysis was performed on molecule 4 with the
Molecular Operating Environment (MOE) software (Molecular
Operating Environment 2010.10 (MOE); Chemical Computing Group
Inc.: Montreal Quebec Canada, 2010, http://www.chemcomp.com)
using the MMFF94x force field and the LowModeMD approach with
default settings. Unfavourable conformers with alternative conforma-
tions of the methoxyphenyl relative to the pyridone were at least 6.3
kcal/mol higher in energy. This suggested the preferred orientation
would be significantly more likely to occur.
(19) Parlow, J. J.; South, M. S. Synthesis of 2-pyridones as tissue
factor VIIa inhibitors. Tetrahedron 2003, 59, 7695−7701.
(20) Kimura, T.; Kawano, K.; Doi, E.; Kitazawa, N.; Takaishi, M.; Ito,
K.; Kaneko, T.; Sasaki, T.; Sato, N.; Miyagawa, T.; Hagiwara, H.
Preparation of morpholine type cinnamides as amyloid-β production
inhibitors. U.S. Patent US 20070117798, 2007.
(21) Shi, D.-Q.; Dou, G.-L.; Ni, S.-N.; Shi, J.-W.; Li, X.-Y.; Wang, X.-
S.; Wu, H.; Ji, S.-J. A novel and efficient synthesis of 2-aryl-2H-
indazoles via SnCl2-mediated cyclization of 2-nitrobenzylamines.
Synlett 2007, 2509−2512.
(22) Dell’Erba, C.; Novi, M.; Petrillo, G.; Tavani, C. A novel
approach to 1H-indazoles via arylazidosulfides. Tetrahedron 1994, 50,
3529−3536.
(23) Evindar, G.; Batey, R. A. Parallel synthesis of a library of
benzoxazoles and benzothiazoles using ligand-accelerated copper-
catalyzed cyclizations of ortho-halobenzanilides. J. Org. Chem. 2006, 71,
1802−1808.
(24) Citron, M.; Westaway, D.; Xia, W.; Carlson, G.; Diehl, T.;
Levesque, G.; Johnson-Wood, K.; Lee, M.; Seubert, P.; Davis, A.;
Kholodenko, D.; Motter, R.; Sherrington, R.; Perry, B.; Yao, H.;
Strome, R.; Lieberburg, I.; Rommens, J.; Kim, S.; Schenk, D.; Fraser,
P., St.; George Hyslop, P.; Selkoe, D. J. Mutant presenilins of
Alzheimer’s disease increase production of 42-residue amyloid β-
protein in both transfected cells and transgenic mice. Nature Med.
1997, 3, 67−72.
(25) Tresadern, G.; Cid, J. M.; Macdonald, G. J.; Vega, J. A.; de
Lucas, A. I.; Garcia, A.; Matesanz, E.; Linares, M. L.; Oehlrich, D.;
Lavreysen, H.; Biesmans, I.; Trabanco, A. A. Scaffold hopping from
pyridones to imidazo[1,2-a]pyridines. New positive allosteric modu-
lators of metabotropic glutamate 2 receptor. Bioorg. Med. Chem. Lett.
2010, 20, 175−179.
(26) An overlay of the 3-D structures of compounds 3 and 40 is
provided in the Supporting Information.
(27) Read, K. D.; Braggio, S. Assessing brain free fraction in early
drug discovery. Expert Opin. Drug Metab. Toxicol. 2010, 6, 337−344.
(28) Murray, J. S.; Ranganathan, S.; Politzer, P. Correlations between
the solvent hydrogen bond acceptor parameter β and the calculated
molecular electrostatic potential. J. Org. Chem. 1991, 56, 3734−3739.
(29) Ghose, A. K.; Torsten, H.; Hudkins, R. L.; Dorsey, B. D.;
Mallamo, J. P. Knowledge-based, central nervous system (CNS) lead
Q
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
selection and lead optimization for CNS drug discovery. Chem.
Neurosci. 2012, 3, 50−68 and the references therein..
(30) For a recent review regarding the correlation between in vitro
potency, ADMET, and physicochemical parameters, see Hann, M. M.
Molecular obesity, potency and other addictions in drug discovery.
Med. Chem. Commun. 2011, 2, 349−355 and the references therein..
(31) For a review on factors that influence the oral absorption of
drugs, see Hurst, S.; Loi, C.-M.; Brodfuehrer, J.; El-Kattan, A. Impact
of physiological, physicochemical and biopharmaceutical; factors in
absorption and metabolism mechanisms on the drug oral bioavail-
ability of rats and humans. Expert Opin. Drug. Metab. Toxicol. 2007, 3,
469−489.
(32) For assay conditions, see: Kantharaj, E.; Tuytelaars, A.; Proost,
P. E. A.; Ongel, Z.; van Assouw, H. P.; Gilissen, R. A. H. J.
Simultaneous measurement of drug metabolic stability and identi-
fication of metabolites using ion-trap mass spectrometry. Rapid
Commun. Mass Spectrom. 2003, 17, 2661−2668.
(33) Gee, P.; Sommers, C. H.; Melick, A. S.; Gidrol, X. M.; Todd, M.
D.; Burris, R. B.; Nelson, M. E.; Klemm, R. C.; Zeiger, E. Comparison
of responses of base-specific Salmonella tester strains with the
traditional strains for identifying mutagens: The results of a validation
study. Mutat. Res. 1998, 412, 115−130.
(34) Hastwell, P. W.; Chai, L.-L.; Roberts, K. J.; Webster, T. W.;
Harvey, J. S.; Rees, R. W.; Walmsley, R. M. High-specificity and high-
sensitivity genotoxicity assessment in a human cell line: Validation of
the GreenScreen HC GADD45a-GFP genotoxicity assay. Mutat. Res.,
Genet. Toxicol. Environ. Mutagen. 2006, 607, 160−175.
(35) Dressman, J. B.; Reppas, C. In vitro−in vivo correlations for
lipophilic, poorly water soluble drugs. Eur. J. Pharm. Sci. 2000, S73−
S78.
(36) Yamaoka, Y.; Roberts, R. D.; Stella, V. J. Low melting phenytoin
prodrugs as alternative oral delivery modes for phenytoin: a model for
other high-melting sparingly water-soluble drugs. J. Pharmacol. Sci.
1983, 72, 400−405.
(37) Salama, N. N.; Yang, Z.; Bui, T.; Ho, R. J. Y. MDR1 haplotypes
significantly minimize intracellular uptake and transcellular P-gp
substrate transport in recombinant LLC-PK1 cells. J. Pharm. Sci.
2006, 95, 2293−2308.
(38) The CEREP selectivity screen was performed on the following
receptors and transporters: A1, A2A, A3, alpha 1, alpha 2, beta 1, AT1,
BZD, B2, CCK1, CCKA, D1, D2S, ETA, GABA, GAL2, CXCR2 (IL-
8B), CCR1, H1, H2, MC4, MT1 (ML1A), M1, M2, M3, NK2, NK3,
Y1, Y2, NTS1 (NT1), delta 2 (DOP), kappa (KOP), mu (MOP),
NOP (ORL1), 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT3, 5-HT5a,
5-HT6, 5-HT7, sst, VPAC1 (VIP1), V1a, Ca2+ channel (L, verapamil
site) (phenylalkylamine), KV channel, SKCa channel, Na+ channel
(site 2), Cl− channel (GABA-gated) norepinephrine transporter, and
dopamine transporter; and on the following enzymes: PLA2, COX1,
COX2, 12-lipoxygenase, PDE2A, PDE3A, PDE4D, PDE5, PDE6,
ACE, ACE-2, BACE-1, ECE-1, elastase, caspase-3, caspase-8, cathepsin
D, cathepsin L, HIV-1 protease, neutral endopeptidase, MMP-1,
MMP-3, MMP-9, tryptase, TACE, phosphatase 1B (PTP1B),
phosphatase CDC25A, phosphatase MKP1, acetylcholinesterase,
MAO-A, MAO-B, HDAC3, HDAC4, HDAC6, HDAC11, sirtuin 1,
sirtuin 2.
(39) For a comprehensive study of this modulator’s behavior in a dog
model, see: Borghys, H; Tuefferd, M; Van Broeck, B.; Clessens, E.;
Dillen, L.; Cools, W.; Vinken, P.; Straetemans, R.; de Ridder, F.;
Gijsen, H.; Mercken, M. A canine model to evaluate efficacy and safety
of γ-secretase inhibitors and modulators. J. Alzheimer’s Dis. 2012, 28,
809−822.
(40) Peng, H.; Talreja, T.; Xin, Z.; Cuervo, J. H.; Kumaravel, G.;
Humora, M. J.; Xu, L.; Rohde, E.; Gan, L.; Jung, M.; Shackett, M. N.;
Chollate, S.; Dunah, A. W.; Snodgrass-Belt, P. A.; Arnold, H. M.;
Taveras, A. G.; Rhodes, K. J.; Scannevin, R. H. Discovery of BIIB042, a
potent, selective, and orally bioavailable γ-secretase modulator. ACS
Med. Chem. Lett. 2011, 2, 786−791.
R
dx.doi.org/10.1021/jm201710f | J. Med. Chem. XXXX, XXX, XXX−XXX