Design, synthesis, and pharmacological evaluation of a novel series of pyridopyrazine-1,6-dione γ-secretase modulators

Article abstract of DOI:10.1021/jm401782h

Herein we describe the design and synthesis of a novel series of γ-secretase modulators (GSMs) that incorporates a pyridopiperazine-1,6- dione ring system. To align improved potency with favorable ADME and in vitro safety, we applied prospective physicochemical property-driven design coupled with parallel medicinal chemistry techniques to arrive at a novel series containing a conformationally restricted core. Lead compound 51 exhibited good in vitro potency and ADME, which translated into a favorable in vivo pharmacokinetic profile. Furthermore, robust reduction of brain Aβ42 was observed in guinea pig at 30 mg/kg dosed orally. Through chemical biology efforts involving the design and synthesis of a clickable photoreactive probe, we demonstrated specific labeling of the presenilin N-terminal fragment (PS1-NTF) within the γ-secretase complex, thus gaining insight into the binding site of this series of GSMs.

Full text of DOI:10.1021/jm401782h

Article
pubs.acs.org/jmc
Design, Synthesis, and Pharmacological Evaluation of a Novel Series
of Pyridopyrazine-1,6-dione γ‑Secretase Modulators
Martin Pettersson,*,† Douglas S. Johnson,† Chakrapani Subramanyam,‡ Kelly R. Bales,†
Christopher W. am Ende,‡ Benjamin A. Fish,‡,§ Michael E. Green,† Gregory W. Kauffman,†
Patrick B. Mullins,‡ Thayalan Navaratnam,‡,# Subas M. Sakya,‡,∥ Cory M. Stiff,‡ Tuan P. Tran,‡
Longfei Xie,‡ Liming Zhang,‡,⊥ Leslie R. Pustilnik,‡ Beth C. Vetelino,‡ Kathleen M. Wood,†
Nikolay Pozdnyakov,† Patrick R. Verhoest,† and Christopher J. O’Donnell‡
†Pfizer Worldwide Research & Development, 610 Main Street, Cambridge, Massachusetts 02139, United States
‡Pfizer Worldwide Research & Development, Eastern Point Road, Groton, Connecticut 06340, United States
S
* Supporting Information
ABSTRACT: Herein we describe the design and synthesis of
a novel series of γ-secretase modulators (GSMs) that
incorporates a pyridopiperazine-1,6-dione ring system. To
align improved potency with favorable ADME and in vitro
safety, we applied prospective physicochemical property-driven
design coupled with parallel medicinal chemistry techniques to
arrive at a novel series containing a conformationally restricted
core. Lead compound 51 exhibited good in vitro potency and
ADME, which translated into a favorable in vivo pharmaco-
kinetic profile. Furthermore, robust reduction of brain Aβ42
was observed in guinea pig at 30 mg/kg dosed orally. Through
chemical biology efforts involving the design and synthesis of a
clickable photoreactive probe, we demonstrated specific labeling of the presenilin N-terminal fragment (PS1-NTF) within the γ-
secretase complex, thus gaining insight into the binding site of this series of GSMs.
afford amyloid peptides ranging from 37 to 43 amino acids in
length. Thus, suppression of the de novo synthesis of
neurotoxic Aβ42 peptides via inhibition of BACE and/or
modulation of γ-secretase represents a promising therapeutic
avenue for the treatment of AD.4 Consequently, this approach
is being aggressively pursued throughout the pharmaceutical
industry as well as in academic laboratories.
Development of γ-secretase inhibitors (GSIs) has provided
agents that successfully reduced Aβ in cerebrospinal fluid
(CSF) in humans;5 however, a phase III study with GSI
LY450139 (semagacestat) was recently halted due to adverse
events including decline in cognition and increased incidence of
skin cancer.6 Likewise, development of BMS-708,163 (avagace-
stat) was terminated after completion of phase II trials in which
gastrointestinal and skin-related adverse events were observed.7
Part of the explanation for these failures may be that, in
addition to APP, γ-secretase cleaves multiple other substrates;
inhibition of such processing may result in detrimental side
effects. For example, S3 cleavage of the Notch 1 receptor by γ-
secretase within the membrane domain releases the notch
intracellular domain (NICD). This translocates to the nucleus,
where it activates transcription of target genes that play an
INTRODUCTION
■
Alzheimer’s disease (AD), first described in 1906 by German
neuropathologist and psychiatrist Alois Alzheimer, is a
progressive, neurodegenerative disorder that leads to cognitive
impairment, loss of motor function, and ultimately death.
Current therapies such as acetylcholinesterase inhibitors and
NMDA receptor antagonists temporarily reduce the symptoms
of AD but do not affect progression of the underlying disease.
Currently, 1 in 8 people in America over the age of 65, or 5.2
million people, are affected by AD, and this number is expected
to more than double by 2040 as the population ages.1 AD
represents a significant burden to the afflicted patients,
caregivers, and the healthcare system, and effective treatments
to reduce or halt the progression of this devastating disease
present a major unmet medical need.
Genetic and histopathological evidence points to the
aberrant production and accumulation of amyloid β (Aβ)
peptides as a causative process in AD.2 Oligomers of Aβ42 have
been shown to be neurotoxic in vitro, and these hydrophobic
species have a high propensity for aggregation to generate
insoluble amyloid plaques, a hallmark of AD pathology.3
Formation of Aβ42 is initiated by cleavage of the amyloid
precursor protein (APP) by the β-aspartyl secretase cleaving
enzyme (BACE). The resultant membrane-bound C-terminal
fragment undergoes a subsequent cleavage by γ-secretase to
Received: November 17, 2013
Published: January 15, 2014
© 2014 American Chemical Society
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important role in cell differentiation including neuronal
development.8 Inhibition of Notch processing by GSIs is
thought to be responsible, at least in part, for the gastro-
intestinal adverse events and increased risk of skin cancer that
have been observed in the clinic. Furthermore, the accumu-
lation of APP β-C-terminal fragment (β-CTF) that results from
γ-secretase inhibition has been implicated in neurotoxicity.9
In contrast to GSIs, γ-secretase modulators (GSMs)
selectively alter the cleavage site of APP to reduce the
formation of the neurotoxic peptides Aβ42 and in many cases
Aβ40 as well, while increasing formation of Aβ37 and/or Aβ38,
which are less prone to aggregation. Importantly, processing of
other substrates including Notch 1 is not inhibited by GSMs.10
Furthermore, GSMs do not cause an accumulation of γ-
secretase substrates such as the APP β-CTF. Focus has
therefore shifted away from GSIs in favor of the development
of GSMs, which may offer an improved safety profile.11 Early
efforts focused on lipophilic acid derivatives such as (R)-
flurbiprofen (1, Figure 1), which originated from nonsteroidal
N-terminal fragment of presenilin (PS1-NTF) at distinct
binding sites.13b,c,16 Importantly, GSIs did not compete with
photolabeling of presenilin using either acid or imidazole
probes, indicating that these GSMs do not share the binding
site(s) of GSIs. As an aspartyl protease located within the
membrane, γ-secretase presents a formidable challenge for the
development of potent modulators with aligned absorption,
distribution, metabolism, and excretion (ADME) attributes
along with favorable physicochemical properties. This is evident
when examining the patent literature, where on average, GSMs
have significantly higher lipophilicities and molecular weights as
compared to marketed CNS drugs.11a,b
RESULTS AND DISCUSSION
■
We have previously described our efforts in the heteroaryl series
using 3 as a starting point (Scheme 1):17,18 Aβ42 IC50 = 143
Scheme 1. Evolution of the Amide Series from Heteroaryl
GSM 3
nM, clogP = 4.8,19 lipophilic efficiency (LipE) = 2.3,20 and a
central nervous system multiple parameter optimization score
(CNS MPO) = 3.7/6.0.21 Our primary design objectives were
to remove the cinnamide olefin and to improve overall
physicochemical properties. In particular we sought to reduce
lipophilicity, as a high clogP value has been linked to increased
promiscuity and a higher risk of adverse findings in exploratory
toxicology studies.22 Furthermore, targeting less lipophilic,
more polar chemical space may increase the probability of
achieving good alignment of potency with critical ADME
parameters such as clearance, permeability, and brain
penetration.21,23 These efforts resulted in the discovery of the
Figure 1. Representative acid GSMs 1, 2, and heteroaryl GSM 3.
anti-inflammatory agents (NSAIDS).10b Although 1 entered
clinical development, it was unsuccessful in slowing cognitive
decline in a phase III trial.12 This may be attributed to its low in
vitro potency (ca. 200 μM) in combination with inadequate
exposure at the target site due to poor brain penetration.
Further efforts resulted in significant improvements in Aβ-
lowering activity to afford acids such as 2 (GSM-1, Aβ42 IC50
value of 120−348 nM).13 However, examination of the patent
literature suggests that a majority of the researchers in the field
have shifted their efforts away from the NSAID-derived acids in
favor of nonacid or heteroaryl-type series of GSMs, as
exemplified by 3 (E2012).11a,b This chemotype was initially
discovered at Neurogenetics and was further developed by
scientists at Eisai, who advanced 3 into phase I clinical trials.14
Further development of this compound was halted in favor of a
second generation analogue with an improved potency and
safety profile.
dihydrobenzofuran amide series exemplified by 4 (Aβ42 IC50 =
521 nM, clogP = 3.96, LipE = 3.55, CNS MPO = 4.8) and 5
(Aβ42 IC50 = 188 nM, clogP = 3.96, LipE = 4.12, CNS MPO =
4.8).17 Both compounds demonstrated robust reduction of
brain Aβ42 at 100 mg/kg in guinea pig, and while 5 is less
potent than 3, it achieved an improvement in LipE of
approximately two log units. However, our efforts to further
improve potency through additional optimization of the right-
hand dihydrobenzofuran amide moiety, while maintaining
favorable alignment of ADME and physicochemical properties,
were unsuccessful. Furthermore, the in vitro safety profile of
this series emerged as a significant concern: although 4 and 5
did not pose a DDI risk, with IC50 values for inhibition of CYP
2D6 and 3A4 greater than 30 μM, CYP inhibition increasingly
became a problem with more potent GSMs in this series. This
finding is consistent with observations in related heteroaryl
series reported in the literature.18a
γ-Secretase is an intramembrane-cleaving aspartyl protease
that is composed of four subunits: presenilin, Pen-2, Aph-1, and
nicastrin.15 Although the exact mechanism of APP processing
by γ-secretase is unknown, we and others have developed
photoaffinity probes from both the acid and arylimidazole series
of GSMs and demonstrated that these compounds bind to the
Given the challenges of aligning potency, clearance, brain
penetration, and in vitro safety (DDI) in the amide series, we
opted to focus our efforts on modifying the arylimidazole A-B
moiety through design of a novel, conformationally restricted
core with increased polarity. By introducing conformational
constraint, we anticipated the opportunity to (a) lock the
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molecule into the putative active conformation and therefore
increase potency, and (b) reduce rotatable bonds and the
number of hydrogen bond donors, which should improve
permeability and brain penetration. As shown in Scheme 2,
masking imine 10 as the carbamate phosphonate 11 prior to
cyclization.25 Thus, refluxing 11 in dichloromethane in the
presence of TiCl4 afforded isoquinoline 12 in good yield as a
single regioisomer. Next, oxidation with m-CPBA furnished
isoquinoline N-oxide 13, which underwent a rearrangement to
form isoquinolone 14 upon refluxing in acetic anhydride; this
was followed by hydrolytic removal of the acetate group with
sodium hydroxide. Following N-alkylation to introduce the side
chain, the 4-methylimidazole moiety was installed via a
nucleophilic aromatic substitution. This reaction was compli-
cated by the presence of the methoxy substituent ortho to the
aryl fluoride. Nevertheless, heating a mixture of fluoroquino-
lone, 4-methylimidazole, and potassium carbonate in DMSO to
135 °C afforded the target compound 15, along with the
corresponding regioisomeric imidazole adduct (not shown) in a
2.5:1 ratio, in 53% yield; this mixture was readily separable by
preparative HPLC.
Scheme 2. Design of Constrained Amide GSMs
Synthesis of the 5,6-disubstituted isoquinolones 25a and 25b,
examples of regioisomer 8, commenced via condensation of
aldehyde 16 with malonic acid, followed by decarboxylation to
furnish 18 (Scheme 4). The carboxylic acid was then converted
to the acyl azide 20 in preparation for the key tandem Curtius
rearrangement/Friedel−Crafts acylation. In contrast to the
transformation using diphenylphosphoryl azide (DPPA), the
acyl azide was prepared in excellent overall yield by using
thionyl choride followed by addition of sodium azide. With acyl
azide 20 in hand, the Curtius rearrangement/intramolecular
Friedel−Crafts cascade afforded the 5,6-disubstituted isoquino-
lone intermediate 22 in 51% yield over three steps. The
synthesis was then completed by alkylation with 1-(2-
bromoethyl)-3-chlorobenzene followed by CuI-mediated cou-
pling with 4-methylimidazole. Interestingly, the latter trans-
formation resulted in complete demethylation of the methoxy
group when the reaction mixture was heated at 130 °C:
hydroxy-substituted quinolone 25a was isolated in 74% yield as
a 4:3 mixture of imidazole regioisomers, which were readily
separated by preparative-HPLC. The demethylation could be
suppressed by reducing the temperature to 100 °C, affording
25b in 20% yield after separation of regioisomers by
preparative-HPLC. The synthesis of 3-trifluoromethylbenzyl
analogue 27 was completed in an analogous manner. Finally,
the double bond of the isoquinolone in 27 was reduced via H-
Cube flow hydrogenation to afford the saturated analogue 28.
constraining the amide moiety of 6 by tying back the nitrogen
onto the aryl ring forms a bicyclic core. Given the presence of
the methoxy group, which is important for potency, this
structural modification gives rise to two possible regioisomers,
exemplified by 7 and 8. Preparation of both of these novel cores
would therefore provide valuable insights into the GSM
pharmacophore, as it would lock the methoxy group and
lactam carbonyl on either the same or opposite sides of the
bicyclic ring system. The arylimidazole region of the non-
NSAID derived GSMs is highly conserved, and only limited
exploration of the aryl core had been exemplified in the GSM
patent literature at the time.11a,b
The synthesis of 15, an example of regioisomer 7, involved
the rearrangement of an isoquinoline N-oxide as a key step
(Scheme 3). Imine 10, obtained from the condensation of
aldehyde 9 with 2,2-dimethoxyethanamine, could not be
directly cyclized to isoquinoline 12. This transformation,
known as the Pomeranz−Fritsch reaction,24 typically works
best with aromatic systems bearing multiple electron-donating
substituents. One of the observed side reactions in this case
involved hydrolysis of 10 back to aldehyde 9 under the acidic
reaction conditions. This problem was circumvented by first
a
Scheme 3. Synthesis of 15
a
Reagents and conditions: (a) 2,2-dimethoxyethanamine, toluene, reflux; (b) EtOCOCl, THF, −10 °C to rt; then P(OMe)3; (c) TiCl4,
dichloromethane, reflux, 57% over three steps; (d) m-CPBA, dichloromethane, rt, 46%; (e) Ac2O, reflux; (f) sodium hydroxide, 100 °C, 84% over
two steps; (g) NaH, 1-(2-bromoethyl)-3-chlorobenzene, DMA, rt, 62%; (h) 4-methylimidazole, K2CO3, DMSO, 135 °C, 7%.
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a
Scheme 4. Synthesis of 25b, 27, and 28
a
Reagents and conditions: (a) malonic acid, piperidine, pyridine, 120 °C, 50% (18), 19% (19); (b) SOCl2, rt, then 90 °C; (c) NaN3, H2O, acetone, 0
°C; (d) 1,2-dichlorobenzene, 140 °C, 1 h; then catalytic I2, 185 °C, 1.5 h, 51% (22, three steps), 16% (23, three steps); (e) NaH, 1-(2-bromoethyl)-
3-chlorobenzene, DMA, rt 45%; (f) NaH, 3-CF3-benzyl bromide, DMA, rt, 97%; (g) 4-methylimidazole, CuI, Cs2CO3, DMF, 100 °C, 20%; (h) 4-
methylimidazole, K2CO3, DMF, 120 °C, 27%. (i) H2, Pd/C, H-Cube, 97%.
Next, we determined Aβ42-lowering activity in a whole-cell
assay using Chinese hamster ovary (CHO) cells that over-
express wild type human APP (hereafter expressed as Aβ42
IC50). Key compounds were spot-checked for inhibition of
Notch signaling in HEK 293 NdeltaE cells.26 A majority of the
analogues were also tested in several in vitro ADME assays,
including stability in human liver microsomes.27 The potential
for P-glycoprotein (P-gp)-mediated efflux was examined by
measuring the efflux ratio (MDR Er) in a Madin−Darby canine
kidney (MDCK) cell line that had been transfected with the
human MDR1 gene encoding P-gp.28 Interestingly, constrain-
ing the amide according to path A (Scheme 2) to form 15
(Table 1) resulted in complete loss of activity, whereas
cyclization according to path B to afford the isomeric lactam
25b preserved Aβ42-lowering activity (Aβ42 IC50 = 3.4 μM).
This finding provided valuable insight into the GSM
pharmacophore, as it suggested that the two polar functional
groups need to be situated on opposing sides of the scaffold to
enable the key interactions with the binding site.
Next, we introduced a m-CF3-benzyl substituent on the
amide nitrogen, as we had observed in our amide series that N-
benzyl groups generally have comparable potency to N-
phenethyl groups but offer improved metabolic stability.17
During the SNAr reaction to introduce the 4-methylimidazole
moiety, the corresponding desmethyl (phenol) derivative 27
was isolated as the major product. Phenol 27 was found to be
3-fold more potent (Aβ42 IC50 = 920 nM) than 25b. We
previously observed in the amide series (e.g., 4 and 5) that
introduction of hydrogen bond donors, such as hydroxyl
groups, generally results in increased P-gp liability as measured
by an increased MDR efflux ratio.17 However, polar functional
groups tend to have reduced MDR Er liability when introduced
onto the aryl core (B-ring) as opposed to the lipophilic right-
hand section of the molecule (C- and D-rings).17 Given these
prior observations, we were intrigued to note that phenol 27
maintained a low MDR efflux ratio (MDR Er = 1.1). This
provided the potential to incorporate polarity into the molecule
and to bring the series into better physicochemical property
space without impairing penetration into the brain.
We next sought to reduce the planarity of the bicyclic core by
introducing sp3 centers into the system. Toward this end, 27
was subjected to hydrogenation, affording the saturated lactam
28. This compound represented an important advancement for
the program, as it successfully aligned moderate in vitro
potency with an improved ADME profile: the Aβ42 IC50 of 28
compared favorably to that of the monocyclic parent amide
2917 (0.40 μM vs 0.66 μM, respectively), while also maintaining
a low MDR efflux ratio (MDR Er = 1.9) and good passive
permeability (RRCK Papp,A→B = 10.4 × 10−6 cm/s).29
Furthermore, 28 achieved excellent stability in human liver
microsomes (HLM CLint,app < 8.0 mL/min/kg). The presence
of a phenol, however, was cause for some concern, as phenols
are known to undergo secondary metabolism through
glucuronidation.30 Consequently, 28 was evaluated for
metabolic stability in human hepatocytes and was found to
have only moderate stability (HHEP CLint,app = 90.5 mL/min/
kg; T1/2 = 19.3 min). This indirectly suggested that the phenol
may indeed be a metabolic liability. Therefore, the next phase
of the design process focused on identifying alternative
heterocyclic templates that would maintain or increase the
polarity while avoiding the liability associated with the phenol.
Evaluation of the heterocyclic cores that had been prepared
thus far suggested that the bicyclic lactam of 28 (as its
generalized template 31) could be combined with the pyridyl
amide core of 5 (as its generalized template 30) (Scheme 5).
Introduction of a two-carbon linker to connect the amide
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Table 1. In Vitro Pharmacology and Disposition Data for Constrained Amide GSMs
a
Aβ42 IC50 values were obtained in a whole cell assay using CHO APPwt cells. Aβ42 IC50 values shown here are the geometric mean of at least three
experiments. MDR efflux ratio in a MDR1/MDCK assay utilizing MDCK cells transfected with the gene that encodes human P-glycoprotein.28
b
c
Human liver microsome-derived intrinsic clearance.27
with a phenol. We therefore decided to invest in the
development of an efficient synthetic route to this novel
GSM core.
Scheme 5. Design Strategy Leading to a Novel
Pyridopyrazine-1,6-dione Series
There are only a limited number of published synthetic
approaches to bicyclic heterocycles related to pyridone 32,
none of which were directly applicable to incorporation of the
requisite substitution pattern.31 We therefore developed a facile
synthesis that features a novel one-pot, HATU32-mediated
amide coupling/cyclization reaction. Details of the develop-
ment, scope, and limitations of this transformation have
recently been described.33 The known pyridyl ester 3317,34
was heated in the presence of HBr and AcOH to afford the
desired acid 34 as the HBr salt (Scheme 6). Alternatively, ester
33 could be converted to the HCl salt 35 in quantitative yield
by heating in HCl/1,4-dioxane. Continuing from pyridone
acids 34 or 35, we had originally envisioned a stepwise
approach whereby an amide coupling reaction would be carried
out with amino alcohol 38 followed by conversion of the
hydroxyl group to a suitable leaving group and finally
intramolecular alkylation. To our surprise, subjecting a mixture
of 34 or 35 and amino alcohol 38 (derived from 1-
nitrogen of 30 to the pyridyl nitrogen would require conversion
of the methoxypyridine into a pyridone, as shown in 32. The
resultant pyridopyrazine-1,6-dione structural motif maintains a
polar functional group in the 5-position, allowing for increased
potency and improved properties, but the carbonyl of the
pyridone should not carry the metabolic liabilities associated
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a
Scheme 6. Synthesis Employed for Preparation of 44 and 45
a
Reagents and conditions: (a) HBr, HOAc, 150 °C, 79%; (b) HCl, 1,4-dioxane, 150 °C, quant; (c) NaBH(OAc)3, DIPEA, THF; (d) HCl, MeOH,
(e) HATU, DIPEA, dichloromethane, rt, 30−80%.
a
Scheme 7. Synthesis Employed for Initial Preparation of 46−53
a
Reagents and conditions: (a) HATU, K2CO3, DMF, 100 °C, 56%; (b) K2CO3, DMSO, 100 °C, 30−60%.
phenylethylamine) to 1 equiv of HATU in the presence of N,N-
diisopropylethylamine (DIPEA) did not produce the targeted
alcohol intermediate 39 as the major product (Scheme 6);
instead, cyclized product 32 was isolated, as a mixture with 39
and unreacted acid 34 (3:2:5 ratio, respectively).33 The
cyclization step was found to be promoted by the HATU
coupling reagent, and the one-pot conversion to 32 could be
enhanced by running the reaction in the presence of 2.5 equiv
of HATU. Further optimization resulted in a useful protocol
that allowed access to a diverse set of pyridopyrazine-1,6-dione
derivatives.33 The utility of this transformation was further
expanded by successfully incorporating parallel medicinal
chemistry techniques and utilizing in situ monomer synthesis.
We initially prepared the requisite amino alcohols 38 via
reductive amination of ethanolamine with substituted benzal-
dehydes; however, the range of monomers that could be
prepared using this approach was rather limited. Therefore, an
improved sequence was developed that took advantage of the
large diversity of our amine reagent collection. As shown in
Scheme 6, reductive amination of various amines 37 with 2-
(tert-butyldimethylsilyloxy)acetaldehyde (36) gave TBS-pro-
tected ethanolamine derivatives in good yield. These
intermediates were then deprotected by exposure to methanolic
HCl. The resulting HCl salts were obtained cleanly and could
be used without further purification in the ensuing coupling
reaction. An alternative to this approach involved mono-
alkylation of amines 37 with 2-bromoethanol. All three in situ
monomer syntheses have been successfully applied to the
preparation of pyridopyrazine-1,6-diones in parallel medicinal
chemistry format.
cyclization reaction with bis(2-chloroethyl)amine (40) to
deliver chloroethyl derivative 41 in 42% yield (Scheme 7).
With ready access to this valuable intermediate, we then carried
out alkylations with a diverse set of phenols in parallel format.
In particular, by running the alkylation reactions in DMSO as
solvent, only a filtration was necessary upon completion of the
reaction, whereupon the mixture could be directly purified via
prep-HPLC; this protocol was ideal for rapid exploration of
SAR on small scale. For the purpose of scale-up of key
compounds, the preferred method involved the original
coupling/cyclization reaction (vide supra) using amino alcohols
prepared by alkylation of phenols with dibromoethane,
followed by reaction with ethanolamine to provide the requisite
compounds 38 (see Experimental Section).
With a facile approach to the novel pyridopyrazine dione
GSM template in hand, we set out to explore SAR in this series.
Compound 44, bearing a 3-CF3-benzyl N-substituent, was
prepared first, as a direct comparison to phenol 28. Although
44 was less potent than 28 (Aβ42 IC50 = 1.6 μM vs 400 nM), it
resided in superior physicochemical space as indicated by a
CNS MPO of 5.7/6.0, versus 4.6/6.0 for phenol 28, and
improvement in LipE from 2.3 to 3.5. Furthermore, 44
maintained excellent stability in human liver microsomes. Most
importantly, the increase in polarity of 44 did not lead to an
increase in P-gp efflux liability (MDR Er = 1.5). The favorable
ADME and physicochemical property profile of 44, coupled
with the fact that we had developed a facile, parallel-enabled
synthesis, prompted further investigation of this series to
determine the extent to which potency could be optimized.
Consequently, GSM 45, which incorporated an extended
linker, was rapidly identified. This compound demonstrated
that reasonable potency (Aβ42 IC50 = 410 nM) could indeed
be attained within this series while maintaining excellent
physicochemical properties (clogP = 2.52; CNS MPO = 5.6/
6.0) and microsomal stability in HLMs (HLM CLint,app = 8.1
We soon identified the phenoxyethyl moiety as an amide
substituent warranting further investigation (vide infra) and
elected to deconstruct this enabled monomer to facilitate SAR
exploration of the aryl substitution pattern. Toward this end,
we found that pyridone acid 35 underwent a one-pot coupling/
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Table 2. In Vitro Pharmacology and Disposition Data for Pyridopyrazine-1,6-dione GSMs
a
Aβ42 IC50 values were obtained in a whole cell assay using CHO APPwt cells. Aβ42 IC50 values are the geometric mean of at least three
experiments. MDR efflux ratio using a MDR1/MDCK assay utilizing MDCK cells transfected with the gene that encodes human P-glycoprotein.28
b
c
d
Human liver microsome-derived intrinsic clearance.27 Rat liver microsome-derived intrinsic clearance.
mL/min/kg). Furthermore, in contrast to phenol 28, GSM 45
was also found to have very good stability in human
hepatocytes (HHEP CLint,app < 2.8 mL/min/kg), thus achieving
one of the key design objectives that had prompted the
development of this novel, conformationally constrained core.
A disadvantage to the extended linker of 45 was that it
exhibited high turnover in rat liver microsomes (RLM CLint,app
= 482 mL/min/kg) despite excellent human microsomal
stability (HLM CLint,app = 8.1 mL/min/kg). This was
undesirable because many of our in vivo efficacy and safety
studies are conducted in rodents. On the other hand,
compound 46, which incorporates a phenoxyethyl moiety as
the amide N-substituent, was found to have improved rat
microsomal stability (RLM CLint,app = 97.0 mL/min/kg) along
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with slightly improved potency (Aβ42 IC50 = 311 nM). The
presence of the phenol ether linkage in 46 rendered this
substituent an enabled monomer, providing a valuable synthetic
disconnection to facilitate further SAR efforts. This allowed us
to use our extensive phenol monomer set to rapidly explore
various substitution patterns using phenol alkylation chemistry
in parallel (vide supra). As a result, we quickly established that
an o-CF3 group was key to optimal potency. For example, the
corresponding o-chloro-substituted analogue 47 was >4-fold
less active. Likewise, moving the CF3 group to the meta- (48)
or para-position (49) resulted in a loss of Aβ-lowering activity.
Compound 46 had the best overall in vitro profile thus far;
however, it only exhibited 23% oral bioavailability in rat. We
reasoned that this may in part be due to inadequate rat
microsomal stability, leading to extensive first-pass metabolism.
Furthermore, it was conceivable that the terminal aryl ring
might undergo cytochrome P450 (CYP)-mediated hydroxyla-
tion in the para-position, as a result of activation by the
electron-donating alkoxy linker. To test this hypothesis, GSM
50 was prepared, in which the metabolically labile para-position
was blocked by a fluoro substituent. Not only was this
compound more potent (Aβ42 IC50 = 252 nM), but the rat
microsomal clearance was reduced from 97.0 to 44.1 mL/min/
kg. Moreover, this modification may have contributed to a
significant improvement in rat oral bioavailability: compound
50 had 100% oral bioavailability, which stands in sharp contrast
to the 23% found for desfluoro analogue 46. In addition to low
clearance (HLM CLint,app = 8.8 mL/min/kg), GSM 50 was
found to have excellent permeability (RRCK Papp,A→B = 17.7 ×
10−6 cm/s and MDRA→B = 13.1 × 10−6 cm/s) as well as a low
efflux ratio (MDR Er = 1.5). In terms of physicochemical
properties, 50 is considerably less lipophilic than 3 (cLogP =
2.75 vs 4.8), which contributes to an improvement CNS MPO
and LipE relative to 3. GSMs 50 and 3 have CNS MPO scores
of 4.6 and 3.7, and LipE values of 3.6 and 2.3, respectively.
The design of the pyridopyrazine-1,6-dione series and the
subsequent discovery of GSM 50 allowed us to significantly
improve upon our previous amide series by successfully aligning
potency, clearance, permeability, and MDR efflux ratio within
excellent CNS property space. However, further gains in
potency were necessary to approach an acceptable human dose
projection. Toward this end we identified 51, wherein the p-
fluoro substituent of 50 has been replaced with a chloro
substituent. GSM 51 maintained an in vitro ADME profile
similar to that of 50, but was more potent (Aβ42 IC50 = 101
nM). This compared favorably to the reference compound 3
(Aβ42 IC50 = 143 nM), which had served as the original
starting point for our program. Compound 51 maintained good
permeability (RRCK Papp,A→B = 10.5 × 10−6 cm/s and
MDRA→B = 5.8 × 10−6 cm/s) and low efflux ratio (MDR Er
= 1.8). Although 51 was more lipophilic (cLogP = 3.32) than
50, it remained within good CNS physicochemical property
space, as indicated by a CNS MPO score of 4.6. Additional
chemistry efforts suggested that potency could be improved
further in this series. For example, naphthyl analogue 52 had an
Aβ42 IC50 of 15 nM; however, this compound had insufficient
stability in rat liver microsomal stability to warrant further
studies. Ultimately, GSMs 50 and 51 were selected for further
profiling in vitro and in vivo.
Table 3. Physicochemical Properties of GSMs 50 and 51
log
a
CNS
solubility, pH 6.5
b
c
ClogP
D
TPSA MW
MPO
(μM)
50
51
2.75
3.32
2.98
3.12
69
69
450
466
5.35
4.64
177
95.6
a
b
Shake flask log D.35 Central nervous system multiple parameter
optimization score.21 Kinetic solubility was measured at Analiza,
Inc.36
c
attributed to increased sp3/sp2 ratio. An additional advantage of
this series was an improvement in the cytochrome P450
inhibition profile, which had plagued the earlier phenyl-
imidazole series;18a GSMs 50 and 51 had IC50 values greater
than 30 μM against the major CYP isoforms, although they
exhibited moderate inhibition of CYP 2C8 and CYP 2C9
(Table 4). Finally, in accordance with the typical profile of γ-
secretase modulators, both lead compounds 50 and 51 were
inactive against Notch (IC50 > 50 μM). This was the case for all
compounds in this series that were spot-checked for inhibition
of Notch signaling.
The lead compounds exhibited good pharmacokinetic
parameters in rat, including high oral bioavailability (%F),
low to moderate clearance (CL), and reasonable half-life (T1/2
)
(Table 5). When dosed orally at 5 mg/kg, 50 and 51 reached
plasma concentrations of 964 ng/mL and 4140 ng/mL,
respectively, at the 1 h time point. The lead compounds also
exhibited acceptable brain penetration, as evidenced by
unbound brain (Cb,u) to unbound plasma (Cp,u) concentration
ratios of 0.43 and 0.32 for 50 and 51, respectively. The extent
to which GSM 51 was able to reduce Aβ40/42 in vivo was
evaluated in a guinea pig efficacy model. The compound was
administered orally in doses of 10, 30, and 90 mg/kg, and levels
of brain Aβ42, Aβ40, Aβ total, and Aβ38 were measured 4 h
postdose along with compound exposure. As shown in Figure
2, de novo synthesis of brain Aβ42 was reduced in a dose-
dependent manner with reductions of 19%, 44%, and 68% at
the 10, 30, and 90 mg/kg doses, respectively. Dose-dependent
suppression of Aβ40 was also observed, but the effect of 51 on
Aβ40 was slightly less than for Aβ42. While there was a trend
toward reduction of Aβ total at the higher doses, this change
was not statistically significant. Furthermore, at the highest
dose, an increase in the formation of Aβ38 was observed, which
is consistent with the profile of a γ-secretase modulator. The Aβ
profile of a related compound in guinea pig brain was also
examined by immunoprecipitation followed by MALDI-TOF
MS (IP/MS) analysis of Aβ peptides. Aβ37 was found to be
increased more than Aβ38 (data not shown), which is
consistent with what has been reported for other arylimidazole
GSMs.14b,37
To determine the target of this novel class of GSMs within
the γ-secretase complex, we synthesized a clickable photo-
affinity probe containing a benzophenone photoreactive group
to effect covalent capture of the binding partner(s) upon UV
irradiation and an alkyne handle to enable subsequent click
chemistry-mediated conjugation of biotin (see Supporting
Information).16,38 The resulting photoprobe, GSM-827-BPyne
(53), maintained good potency, exhibiting an Aβ42 IC50 of 135
nM (Figure 3). With a promising photoprobe in hand, we
performed photoaffinity labeling studies in HeLa cell
membranes, which are known to have high γ-secretase
activity.13c,16b,c The photoprobe 53 was incubated with HeLa
cell membranes in the presence or absence of GSM or GSI
Table 3 summarizes the overall physicochemical profiles of
50 and 51, which compare favorably to those of the majority of
GSMs in the patent literature.11a These compounds achieved
adequate aqueous kinetic solubility, which in part may be
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Table 4. CYP Inhibition by GSMs 50 and 51
CYP 1A2 IC50 (μM)
CYP 2C19 IC50 (μM)
CYP 2C8 IC50 (μM)
CYP 2C9 IC50 (μM)
CYP 2D6 IC50 (μM)
CYP 3A4 IC50 (μM)
50
51
>30.0
>30.0
>30.0
>30.0
19.0
12.3
>30.0
15.4
>30.0
>30.0
>30.0
>30.0
Table 5. Pharmacokinetic Profile of GSMs 50 and 51 in Rat
a
a
a
b
c
c
c
d
CL (mL/min/kg)
T1/2 (h)
Vdss (L/kg)
1.91
%F
plasma concentration (ng/mL)
brain concentration (ng/mL)
B/P
Cb,u/Cp,u
50
51
14.3
1.41
2.09
6.88
100
88
964
718
0.74
0.24
0.43
0.32
0.69
4140
1000
a
b
c
Determined from 1 mg/kg intravenous dose. Calculated using the exposure from 1 mg/kg intravenous dose and 5 mg/kg oral dose. 5 mg/kg oral
d
dose, 1 h time point. Plasma free fractions in rat for 50 and 51 were 5.5% and 0.63%, respectively, and rat brain free fractions for 50 and 51 were
3.2% and 0.84%, respectively.
Figure 2. Dose−response of 51 in guinea pig efficacy model reported as percent of vehicle.
Table 6. Plasma, Brain, and CSF Exposure of GSM 51 in the
Guinea Pig Efficacy Model, at 4 h Postdose
a
b
dose (po, mg/kg)
plasma (μM)
brain (μM)
CSF (nM)
10
30
90
0.367 0.182
0.819 0.056
7.30 2.76
2.91 1.39
9.16 4.91
52.4 17.6
12.7 7.2
35.7 5.9
239 57
a
b
Free fraction of 51 in guinea pig plasma (Fu,p) was 7%. Free fraction
of 51 in guinea pig brain (Fu,b) was 0.8%.
competitor compounds followed by UV irradiation to initiate
photo-cross-linking to nearby proteins. The labeled proteins
were tagged with biotin via click chemistry with biotin azide
and enriched by affinity chromatography with streptavidin. The
eluted proteins were separated by SDS-PAGE, and Western
blot analysis demonstrated that presenilin N-terminal fragment
(PS1-NTF) was labeled by 53 (Figure 3). The labeling was
deemed specific, because it was competed by pyridopyrazine-
1,6-dione GSMs 52 and 54 (lanes 2 and 3). Despite the
structural similarity to E2012-BPyne,16b our data indicate that
53 binds differently to PS1-NTF because the labeling is not
competed by 3 (lane 5). Furthermore, unlike E2012-BPyne,
labeling by 53 in the presence of GSI L458 did not result in a
dramatic increase in the labeling of PS1-NTF (lane 4).16b
Figure 3. GSM-827-BPyne (53) specifically labels PS1-NTF in HeLa
membranes. Photoaffinity labeling of 53 (5 μM, lanes 1−5) in HeLa
membranes in the absence (lane 1) or presence of 50 μM GSM 52
(lane 2), 50 μM GSM 5439 (lane 3), 1 μM GSI L458 (lane 4) or 50
μM 3 (lane 5), followed by click chemistry with biotin azide,
streptavidin pull-down, and Western blot analysis with PS1-NTF
antibody.
core as a replacement for the pyridyl amide moiety of 5, to
improve physicochemical properties. We also sought to
improve the alignment of potency and ADME parameters
such as clearance, permeability, and MDR efflux ratio. These
objectives were achieved through incorporation of conforma-
CONCLUSION
■
In summary, starting from our previously described amide
series (i.e., 4 and 5), we have designed a novel series of GSMs.
Our primary design objective was to identify a new heterocyclic
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5% B increasing to 5% A, 95% B. All final test compounds were found
to have purity >95% unless otherwise indicated.
tional constraint, which ultimately led to the discovery of a
novel GSM series containing a pyridopyrazine-1,6-dione
bicyclic core. Within this series we have highlighted compounds
that exhibit a 7-fold improvement in Aβ-lowering activity in
vitro as compared to reference compound 3 and possess
significantly improved physicochemical properties. Lead
compound 51 has good CNS-like physicochemical properties,
as evidenced by improved CNS MPO and LipE relative to 3
(GSMs 51 and 3 have CNS MPO scores of 4.6 and 3.7, and
LipE values of 3.9 and 2.3, respectively). This in turn translated
into low clearance in vitro and in vivo, and excellent
permeability and oral bioavailability. Robust in vivo efficacy
was observed in a guinea pig model at 30 mg/kg, and reduction
of brain Aβ42 was achieved in a dose-dependent manner. Of
note is that this new GSM series successfully addresses the
problems of CYP-inhibition and off-target activity that were
observed with previous arylimidazole series. Our efforts also led
to important insights into the GSM pharmacophore. By
breaking the symmetry around the core and by locking
rotamers through introduction of conformational constraint, we
were able to establish directionality for the key polar
interactions between heteroaryl-type γ-secretase modulators
and the target binding site. Finally, chemical biology efforts
involving design and synthesis of photoaffinity probe 53
demonstrated that the pyridinone class of GSMs binds to the
N-terminal fragment of presenillin (PS1-NTF) within the of γ-
secretase complex at a site that is distinct from that targeted by
γ-secretase inhibitors. Additional efforts to further improve
potency while maintaining the favorable physicochemical
properties, ADME, and safety profiles of 50 and 51 will be
disclosed in due course.
N-(4-Fluoro-3-methoxybenzylidene)-2,2-dimethoxyethanamine
(10). A solution of 4-fluoro-3-methoxybenzaldehyde (9) (7.00 g, 45.4
mmol) and 2,2-dimethoxyethanamine (5.88 mL, 54.5 mmol) in
toluene (60 mL) was heated to reflux under a Dean−Stark trap for 1 h.
The reaction mixture was allowed to cool to room temperature and
concentrated under reduced pressure to provide the title compound
1
(10.9 g, quantitative). LCMS m/z 242.2 (M + 1). H NMR (400
MHz, CDCl3) δ 3.42 (s, 6H), 3.77 (dd, J = 5.3, 1.2 Hz, 2H), 3.94 (s,
3H), 4.68 (t, J = 5.3 Hz, 1H), 7.09 (dd, J = 10.7, 8.4 Hz, 1H), 7.16
(ddd, J = 8.2, 4.7, 1.9 Hz, 1H), 7.50 (dd, J = 8.4, 1.9 Hz, 1H), 8.21 (t, J
= 1.4 Hz, 1H).
6-Fluoro-7-methoxyisoquinoline (12). Ethyl chloroformate (4.04
mL, 40.2 mmol) was added to a solution of N-(4-fluoro-3-
methoxybenzylidene)-2,2-dimethoxyethanamine (10) (9.70 g, 40.2
mmol) in tetrahydrofuran (30 mL) at −10 °C. The reaction was
stirred at −10 °C for 5 min and then warmed to room temperature,
whereupon trimethyl phosphite (6.05 mL, 48.2 mmol) was added. The
reaction mixture was stirred at room temperature for 18 h and then
concentrated under reduced pressure to provide ethyl 2,2-
dimethoxyethyl[(dimethoxyphosphoryl)(4-fluoro-3-methoxyphenyl)-
methyl]carbamate (11) (17.0 g, quantitative), which was used in the
subsequent step without purification. Intermediate 11 (2.41 g, 5.70
mmol) was treated with a 1.0 M solution of titanium tetrachloride in
dichloromethane (34.2 mL, 34.2 mmol). The mixture was heated to
reflux for 24 h, whereupon it was allowed to cool to room temperature
and was poured into an aqueous solution of sodium hydroxide (1 M,
150 mL) and ethyl acetate (50 mL). The resulting white precipitate
was removed by filtration, and the filtrate was extracted with ethyl
acetate (2 × 40 mL). The combined organic layers were dried
(Na2SO4), filtered, and concentrated under reduced pressure. The
resulting residue was purified by chromatography on silica gel
(gradient: 0% to 10% methanol in ethyl acetate) to provide the title
compound (573 mg, 57%). LCMS m/z 178.2 (M + 1). 1H NMR (400
MHz, CDCl3) δ 4.03 (s, 3H), 7.31 (d, J = 8.2 Hz, 1H), 7.43 (d, J =
11.3 Hz, 1H), 7.53 (d, J = 5.7 Hz, 1H), 8.44 (d, J = 5.9 Hz, 1H), 9.12
(s, 1H).
EXPERIMENTAL SECTION
■
General Methods. Solvents and reagents were of reagent grade
and were used as supplied by the manufacturer. All reactions were run
under a N2 atmosphere. Organic extracts were routinely dried over
anhydrous sodium sulfate or magnesium sulfate. Concentration refers
to rotary evaporation under reduced pressure. Chromatography refers
to flash chromatography using disposable RediSepRf 4 to 120 g silica
columns or Biotage disposable columns on a CombiFlash Companion
or Biotage horizon automatic purification system. Microwave reactions
were carried out in a microwave reactor manufactured by Smithcreator
6-Fluoro-7-methoxyisoquinoline 2-oxide (13). m-Chloroperoxy-
benzoic acid (1.57 g, 6.46 mmol) was added to a solution of 6-fluoro-
7-methoxyisoquinoline (12) (573 mg, 3.23 mmol) in dichloromethane
(20 mL). The mixture was stirred at room temperature for 3 h,
whereupon it was poured into a mixture of aqueous sodium hydroxide
solution (1 M, 50 mL) and 20% aqueous sodium thiosulfate solution
(50 mL). The layers were separated, and the aqueous layer was
extracted with dichloromethane (2 × 25 mL). The combined organic
layers were dried (Na2SO4), filtered, and concentrated under reduced
pressure. The resulting residue was purified by chromatography on
silica gel (gradient: 0% to 20% methanol in ethyl acetate) to provide
1
of Personal Chemistry. H NMR spectra were recorded on Varian
INOVA spectrometers (300, 400, or 500 MHz) (Varian Inc., Palo
Alto, CA) at room temperature. Chemical shifts are expressed in parts
per million δ relative to residual solvent as an internal reference. Peak
multiplicities are expressed as follows: singlet (s), doublet (d), triplet
(t), quartet (q), multiplet (m), and broad singlet (br s). HPLC purity
analysis of final test compounds was carried out using one of the
following three methods unless otherwise indicated. Method A:
Compounds 46−49, 52, and 53: liquid chromatography/photodiode
array detector/evaporative light scattering detection coupled to single
quadrupole mass spectrometry (LC/UV/ELSD/MS), Waters Atlantis
dC18 4.6 × 50 mm, 5 μm; mobile phase A: 0.05% TFA in water (v/v);
mobile phase B: 0.05% TFA in acetonitrile (v/v); gradient begins at
95.0% A, 5% B increasing to 5% A, 95% B over 4.0 min, hold at 5% A,
95% B until 5.0 min. Flow rate: 2 mL/min. Method B: Compounds 50
and 51: UPLC/UV/MS using Waters CSH C18 2.1 × 50 mm with 1.7
μm particles; UV purity detected at 215 nm; mass spectrometer ESI
positive negative switching acquiring from m/z 160 to 1000. Mobile
phase A = 0.1% formic acid in water (v/v); mobile phase B = 0.1%
formic acid in acetonitrile (v/v); gradient begins at 95% A, 5% B
increasing to 100% B over 1.2 min, hold at 100% B until 1.5 min. Flow
rate: 1.0 mL/min. Method C: Compounds 15, 25b, 27, 28, and 44:
UPLC/UV/MS. Mobile phase A: 0.05% TFA in water (v/v); mobile
phase B: 0.05% TFA in acetonitrile (v/v); gradient begins at 95.0% A,
1
the title compound (288 mg, 46%). LCMS m/z 193.9 (M + 1). H
NMR (400 MHz, CD3OD) δ 4.02 (s, 3H), 7.50 (d, J = 8.2 Hz, 1H),
7.65 (d, J = 10.9 Hz, 1H), 7.85 (d, J = 7.0 Hz, 1H), 8.10 (dd, J = 7.0,
1.4 Hz, 1H), 8.88 (s, 1H).
6-Fluoro-7-methoxyisoquinolin-1(2H)-one (14). 6-Fluoro-7-me-
thoxyisoquinoline 2-oxide (13) (288 mg, 1.49 mmol) was dissolved
in acetic anhydride (5.0 mL, 40 mmol) and heated to reflux for 3 h.
The mixture was allowed to cool to room temperature and then was
concentrated under reduced pressure. The resulting residue was
treated with an aqueous solution of sodium hydroxide (2 M, 10 mL)
and heated to 100 °C for 1 h. The mixture was cooled to room
temperature and acidified to pH 6 with a 5% aqueous solution of citric
acid. The resulting solid was isolated by filtration to provide the title
compound (242 mg, 84%). LCMS m/z 193.9 (M + 1). 1H NMR (400
MHz, CDCl3) δ 4.03 (s, 3H), 6.49 (d, J = 7.2 Hz, 1H), 7.11 (d, J = 7.2
Hz, 1H), 7.25 (d, J = 11.5 Hz, 1H), 7.92 (d, J = 8.6 Hz, 1H), 10.77 (br
s, 1H).
2-(3-Chlorophenethyl)-6-fluoro-7-methoxyisoquinolin-1(2H)-
one. Sodium hydride (60.2 mg, 60% dispersion in mineral oil, 1.50
mmol) was added to a solution of 6-fluoro-7-methoxyisoquinolin-
1(2H)-one (14) (242 mg, 1.25 mmol) in N,N-dimethylacetamide (10
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mL). The mixture was stirred at room temperature for 1 h and then
treated with 1-(2-bromoethyl)-3-chlorobenzene (0.22 mL, 1.50
mmol). After being stirred for 18 h, the reaction was judged
incomplete by TLC. Additional sodium hydride (30 mg, 60%
dispersion in mineral oil, 0.75 mmol) and 1-(2-bromoethyl)-3-
chlorobenzene (0.200 mL, 1.36 mmol) were added to the mixture.
The reaction was stirred at room temperature for 24 h, whereupon it
was poured into water (40 mL) and extracted with dichloromethane
(3 × 25 mL). The combined organic layers were dried (Na2SO4),
filtered, and concentrated under reduced pressure. The resulting
residue was purified by chromatography on silica gel (gradient: 0% to
50% ethyl acetate in heptane) to provide the title compound (259 mg,
(383 mg, 5.9 mmol) in water (1.2 mL) was added, the cold bath was
removed, and the reaction was stirred at room temperature for 2 h.
The reaction mixture was poured into diethyl ether (30 mL) and water
(30 mL), and the layers were separated. The aqueous layer was
extracted with ether (2 × 30 mL), and the combined organic layers
were dried (Na2SO4), filtered, and concentrated under reduced
pressure to provide the title compound (996 mg, 99%). 1H NMR (400
MHz, CDCl3) δ 4.00 (d, J = 2.3 Hz, 3H), 6.51 (d, J = 16.0 Hz, 1H),
6.99−7.07 (m, 1H), 7.15 (ddd, J = 11.5, 8.2, 1.6 Hz, 1H), 7.31 (d, J =
7.8 Hz, 1H), 8.01 (d, J = 16.0 Hz, 1H).
6-Bromo-5-methoxyisoquinolin-1(2H)-one (22). Thionyl chloride
(2.5 mL, 34 mmol) was added to a round-bottom flask charged with
(E)-3-(3-bromo-2-methoxyphenyl)acrylic acid (18) (0.88 g, 3.4
mmol). The reaction mixture was stirred for 16 h at room temperature
followed by heating at 90 °C for 2 h. The reaction was then allowed to
cool to room temperature, and the volatiles were removed under
reduced pressure. After azeotroping with toluene, the resulting residue
(0.95 g) was dissolved in acetone (3.2 mL) and cooled to 0 °C.
Sodium azide (0.34 g, 5.3 mmol) in water (1.2 mL) was added, and
the reaction mixture was stirred at room temperature for 2 h. Diethyl
ether (30 mL) and water (30 mL) were added, and the layers were
separated. The aqueous layer was extracted with diethyl ether (2 × 30
mL), the combined organic layers were dried (Na2SO4) and filtered,
and the solvent was removed under reduced pressure. The resulting
residue (0.89 g) was dissolved in 1,2-dichlorobenzene (20 mL) and
subsequently heated to 140 °C. After 1 h, when gas evolution had
subsided, catalytic iodine was added to the reaction mixture and the
temperature was increased to 185 °C for an additional 1.5 h. The
reaction mixture was allowed to cool to room temperature, the solvent
was partially removed, and the material was isolated through
crystallization to afford the desired product (0.44 g, 51%). LCMS
m/z 255.9 (M + 1). 1H NMR (400 MHz, CDCl3) δ 3.95 (s, 3H), 6.83
(d, J = 7.2 Hz, 1H), 7.22 (d, J = 7.6 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H),
8.06 (d, J = 8.6 Hz, 1H), 11.2 (br s, 1H).
1
62%). LCMS m/z 332.2 (M + 1). H NMR (400 MHz, CDCl3) δ
3.05−3.11 (m, 2H), 4.02 (s, 3H), 4.17−4.23 (m, 2H), 6.32 (d, J = 7.4
Hz, 1H), 6.77 (d, J = 7.4 Hz, 1H), 7.05−7.10 (m, 1H), 7.18 (d, J =
11.3 Hz, 1H), 7.20−7.25 (m, 3H), 7.96 (d, J = 8.6 Hz, 1H).
2-(3-Chlorophenethyl)-7-methoxy-6-(4-methyl-1H-imidazol-1-
yl)isoquinolin-1(2H)-one (15). 4-Methylimidazole (126 mg, 1.54
mmol) and potassium carbonate (425 mg, 3.08 mmol) were added
to a solution of 2-(3-chlorophenethyl)-6-fluoro-7-methoxyisoquinolin-
1(2H)-one (255 mg, 0.769 mmol) in dimethyl sulfoxide (5 mL). The
mixture was heated to 135 °C for 14 h, whereupon it was cooled to
room temperature and poured into water (20 mL). The aqueous
mixture was extracted with dichloromethane (3 × 30 mL), and the
combined organic layers were dried (Na2SO4), filtered, and
concentrated under reduced pressure. The resulting residue was
purified by silica gel chromatography (gradient: 0% to 6% [2 M
ammonia in methanol] in ethyl acetate) to provide a 2.5:1 mixture of
imidazole regioisomers (160 mg, 53%). This mixture was further
purified by HPLC (Phenomenex Luna (2) C18, 21.2 × 150 mm, 5 μm;
0.1% formic acid modifier, 95% water/5% acetonitrile, linear gradient
to 5% water/95% acetonitrile over 10.0 min, then 100% acetonitrile for
1.0 min; flow rate: 28 mL/min) to provide the title compound (20 mg,
1
7%). LCMS m/z 394.2 (M + 1). H NMR (400 MHz, CDCl3) δ
2.29−2.34 (m, 3H), 3.08 (t, J = 7.3 Hz, 2H), 4.01 (s, 3H), 4.18−4.25
(m, 2H), 6.36 (d, J = 7.4 Hz, 1H), 6.78 (d, J = 7.2 Hz, 1H), 7.03 (t, J =
1.0 Hz, 1H), 7.04−7.10 (m, 1H), 7.19−7.24 (m, 3H), 7.39 (s, 1H),
7.83 (d, J = 1.2 Hz, 1H), 8.01 (s, 1H). HPLC purity >95%.
6-Fluoro-5-methoxyisoquinolin-1(2H)-one (23). A solution of (E)-
3-(3-fluoro-2-methoxyphenyl)acryloyl azide (21) (996 mg, 4.50
mmol) in 1,2-dichlorobenzene (30 mL) was heated to 140 °C for 1
h until gas formation subsided. Catalytic iodine was added, and the
temperature was increased to 185 °C. The reaction mixture was heated
to 185 °C for 4 days, whereupon it was allowed to cool to room
temperature. The reaction was concentrated under reduced pressure,
and the residue was purified via chromatography on silica gel
(gradient: 0% to 10% ethyl acetate in heptane, then 0% to 10% [10%
methanol in ethyl acetate] in heptane). The resulting residue (469 mg)
was purified further by prep-HPLC (Gemini 50 × 100 mm C18;
mobile phase A: 0.1% formic acid in water; mobile phase B:
acetonitrile; gradient: 25% to 75% B; flow rate: 75 mL/min) to afford
the title compound (143 mg, 16%) as a solid. LCMS m/z 194.3 (M +
1). 1H NMR (400 MHz, CD3OD) δ 4.06 (d, J = 1.9 Hz, 3H), 6.87 (d,
J = 7.4 Hz, 1H), 7.14 (d, J = 6.6 Hz, 1H), 7.24 (dd, J = 11.1, 9.0 Hz,
1H), 8.08−8.19 (m, 1H), 10.28 (br s, 1H).
(E)-3-(3-Bromo-2-methoxyphenyl)acrylic Acid (18). To a solution
of 3-bromo-2-methoxybenzaldehyde (16) (7.42 g, 34.5 mmol) in
pyridine (25.1 mL) was added malonic acid (5.50 g, 51.8 mmol)
followed by piperidine (1.06 mL, 10.4 mmol). The reaction mixture
was heated to 120 °C for 6 h, whereupon it was allowed to cool to
room temperature and acidified to pH < 3 using concentrated
hydrochloric acid. The resulting solid was collected by filtration, and
the residue was purified via chromatography on silica gel (5% [2 M
acetic acid in ethyl acetate] in heptane) to provide the title compound
1
(4.43 g, 50%). LCMS m/z 254.1 (M − 1). H NMR (400 MHz,
CD3OD) δ 3.83 (s, 3H), 6.56 (d, J = 16.0 Hz, 1H), 7.09 (t, J = 8.0 Hz,
1H), 7.67 (dd, J = 1.6, 7.8 Hz, 1H), 7.64 (dd, J = 1.6, 8.0 Hz, 1H), 7.91
(d, J = 16.2 Hz, 1H).
(E)-3-(3-Fluoro-2-methoxyphenyl)acrylic Acid (19). A solution of
3-fluoro-2-methoxybenzaldehyde (17) (16.1 g, 104 mmol) and
malonic acid (16.6 g, 157 mmol) in pyridine (51 mL) and piperidine
(3.2 mL) was heated to 120 °C for 6 h. The reaction was allowed to
cool to room temperature, and concentrated aqueous hydrochloric
acid was added to reach pH < 3. The resulting precipitate was
collected via filtration, washed with water, and recrystallized from ethyl
acetate to provide the title compound as a solid (3.84 g, 19%). LCMS
6-Bromo-2-(3-chlorophenethyl)-5-methoxyisoquinolin-1(2H)-one
(24). Sodium hydride (60% dispersion in mineral oil, 0.61 g, 15.3
mmol) was added to a solution of 6-bromo-5-methoxyisoquinolin-
1(2H)-one (22) (0.44 g, 1.7 mmol) in dimethylacetamide (20.0 mL),
and the reaction mixture was stirred at room temperature for 1 h. 1-(2-
Bromoethyl)-3-chlorobenzene (2.25 mL, 15.3 mmol) was added, and
stirring was continued for 48 h. Additional sodium hydride (0.70 g,
17.6 mmol) and 1-(2-bromoethyl)-3-chlorobenzene (2.00 mL, 13.6
mmol) were added, and the reaction mixture was stirred for an
additional 24 h, whereupon it was poured into water (40 mL) and
ethyl acetate (30 mL). The layers were separated, and the aqueous
layer was extracted twice with diethyl ether. The combined organic
layers were dried (Na2SO4), filtered, and concentrated under reduced
pressure. The resulting residue was purified via chromatography on
silica gel (gradient: 0% to 50% ethyl acetate in heptane) to provide the
1
m/z 195.3 (M-1). H NMR (400 MHz, CD3OD) δ 3.95 (d, J = 1.9
Hz, 3H), 6.54 (d, J = 16.0 Hz, 1H), 7.09 (ddd, J = 8.2, 8.0, 5.1 Hz,
1H), 7.18 (ddd, J = 11.5, 8.2, 1.6 Hz, 1H), 7.44 (br d, J = 7.8 Hz, 1H),
7.92 (d, J = 16.2 Hz, 1H).
(E)-3-(3-Fluoro-2-methoxyphenyl)acryloyl Azide (21). A mixture
of (E)-3-(3-fluoro-2-methoxyphenyl)acrylic acid (19) (889 mg, 4.53
mmol) in thionyl chloride (3.3 mL, 45.3 mmol) was stirred at room
temperature for 16 h and then heated to reflux for 2 h. The volatiles
were evaporated under reduced pressure, and the crude material was
azeotroped with toluene. The resulting residue (973 mg) was dissolved
in acetone (3.2 mL) and cooled to 0 °C. A solution of sodium azide
1
desired product (0.30 g, 45%). LCMS m/z 393.8 (M + 1). H NMR
(400 MHz, CDCl3) δ 3.07 (m, 2H), 3.92 (s, 3H), 4.18 (m, 2H), 6.63
(d, J = 7.6 Hz, 1H), 6.84 (d, J = 7.4 Hz, 1H), 7.08−7.06 (m, 1H),
7.22−7.20 (m, 3H), 7.63 (d, J = 8.6 Hz, 1H), 8.08 (d, J = 8.6, 1H).
1056
dx.doi.org/10.1021/jm401782h | J. Med. Chem. 2014, 57, 1046−1062

Journal of Medicinal Chemistry
Article
2-(3-Chlorophenethyl)-5-methoxy-6-(4-methyl-1H-imidazol-1-
yl)isoquinolin-1(2H)-one (25b). Cesium carbonate (0.16 g, 0.48
mmol) and 4-methylimidazole (30 mg, 0.36 mmol) were added to a
solution of 6-bromo-2-(3-chlorophenethyl)-5-methoxyisoquinolin-
1(2H)-one (24) (0.10 g, 0.24 mmol) in N,N-dimethylformamide
(2.0 mL). Nitrogen was gently bubbled through the reaction mixture
for 5 min, and copper(I) iodide (20 mg, 0.12 mmol) was added.
Nitrogen was bubbled through the reaction mixture for an additional
30 min, and the reaction was then heated to 100 °C. After stirring the
reaction at 100 °C for 3 days, it was allowed to cool to room
temperature and filtered through Celite. The filtrate was concentrated
under reduced pressure, and the residue was purified via reversed
phase prep-HPLC (gradient: 5% to 15% acetonitrile in water
containing 0.1% formic acid) to provide the desired product (20
mg, 20%). LCMS m/z 394.5 (M + 1). 1H NMR (400 MHz, CDCl3) δ
2.33 (s, 3H), 3.09 (m, 2H), 3.55 (s, 3H), 4.21 (m. 2H), 6.70 (d, J = 7.4
Hz, 1H), 6.88 (J = 7.6 Hz, 1H), 7.07−7.24 (m, 7H), 7.40 (d, J = 8.6
Hz, 1H). HPLC purity: 93%.
5-(4-Methyl-1H-imidazol-1-yl)-6-oxo-1,6-dihydropyridine-2-car-
boxylic Acid, Hydrobromide Salt (34). A solution of methyl 6-
methoxy-5-(4-methyl-1H-imidazol-1-yl)pyridine-2-carboxylate34 (33)
(3.82 g, 15.9 mmol) in acetic acid (30 mL) and aqueous hydrobromic
acid (48%, 30 mL) was heated to reflux for 4 h. The reaction was
allowed to cool to room temperature and then cooled further to 0 °C
using an ice−water bath. The resulting precipitate was collected via
filtration and washed with ice−water (30 mL). Recrystallization from
ethanol (20 mL) provided the title compound as a light yellow solid
1
(3.79 g, 79%). LCMS m/z 220.1 (M + 1). H NMR (400 MHz,
DMSO-d6) δ 2.34 (br s, 3H), 7.09 (d, J = 7.4 Hz, 1H), 7.88−7.91 (m,
1H), 8.07 (d, J = 7.6 Hz, 1H), 9.58−9.60 (m, 1H), 12.6 (br s, 1H).
5-(4-Methyl-1H-imidazol-1-yl)-6-oxo-1,6-dihydropyridine-2-car-
boxylic Acid, Hydrochloride Salt (35). A solution of methyl 6-
methoxy-5-(4-methyl-1H-imidazol-1-yl)pyridine-2-carboxylate34 (33)
(34.3 g, 139 mmol) in aqueous hydrochloric acid (37%, 230 mL)
and 1,4-dioxane (230 mL) was heated to reflux for 18 h, whereupon it
was allowed to cool to room temperature. The reaction was filtered,
and the solids were washed with 1,4-dioxane (2 × 100 mL). The solids
were mixed with methanol (500 mL), and the volatiles were removed
under reduced pressure. The residue was stirred with methanol (100
mL) for 15 min, and 1,4-dioxane (250 mL) was added. The resulting
mixture was stirred for 15 min, whereupon the solids were collected by
filtration and washed with 1,4-dioxane to provide the title compound
6-Fluoro-5-methoxy-2-[3-(trifluoromethyl)benzyl]isoquinolin-
1(2H)-one (26). Sodium hydride (60% dispersion in mineral oil, 44.4
mg, 1.11 mmol) was added to a solution of 6-fluoro-5-
methoxyisoquinolin-1(2H)-one (23) (143 mg, 0.74 mmol) in N,N-
dimethylformamide (5 mL). The mixture was stirred at room
temperature for 1 h, whereupon 3-(trifluoromethyl)benzyl bromide
(212 mg, 0.89 mmoL) was added. The reaction mixture was stirred at
room temperature overnight, whereupon it was poured into ethyl
acetate (10 mL) and water (30 mL). The layers were separated, and
the aqueous layer was extracted with ethyl acetate (2 × 10 mL). The
combined organic layers were dried (Na2SO4), filtered, and
concentrated under reduced pressure. The resulting residue was
purified by chromatography on silica gel (gradient: 0% to 40% ethyl
acetate in heptane) to afford the title compound as a solid (253 mg,
1
as a beige solid (35.4 g, 99%). LCMS m/z 220.1 (M + 1). H NMR
(300 MHz, DMSO-d6) δ 2.33 (d, J = 0.9 Hz, 3H), 7.09 (d, J = 7.5 Hz,
1H), 7.84−7.87 (m, 1H), 8.04 (d, J = 7.5 Hz, 1H), 9.50 (d, J = 1.6 Hz,
1H).
2-(2-Chloroethyl)-7-(4-methyl-1H-imidazol-1-yl)-3,4-dihydro-2H-
pyrido[1,2-a]pyrazine-1,6-dione (41). Potassium carbonate (195.4 g,
1414 mmol) was added to a mixture of 5-(4-methyl-1H-imidazol-1-yl)-
6-oxo-1,6-dihydropyridine-2-carboxylic acid, hydrochloride salt (35)
(34.5 g, 135 mmol) and bis(2-chloroethyl)amine hydrochloride (40)
(37.8 g, 212 mmol) in N,N-dimethylformamide (670 mL), and the
reaction was stirred for 10 min. HATU (83.5 g, 219 mmol) was added,
and stirring was continued for an additional 3 h and 40 min. The
reaction mixture was poured into water (4 L), stirred for 30 min, and
then extracted with dichloromethane (3 × 1 L). The combined organic
layers were washed with saturated aqueous sodium chloride solution
(3 × 3 L), dried (MgSO4), filtered, and concentrated under reduced
pressure. The residue was stirred in ethyl acetate (approximately 50
mL) for 30 min, whereupon the solids were collected by filtration and
washed with ethyl acetate and pentane to provide the title compound
1
97%). LCMS m/z 352.5 (M + 1). H NMR (400 MHz, CD3OD) δ
4.01 (s, 3H), 5.20 (s, 2H), 6.80 (d, J = 7.6 Hz, 1H), 7.08 (d, J = 7.6
Hz, 1H), 7.14−7.26 (m, 1H), 7.37−7.57 (m, 4H), 8.11 (dd, J = 9.0,
4.9 Hz, 1H).
5-Hydroxy-6-(4-methyl-1H-imidazol-1-yl)-2-[3-(trifluoromethyl)-
benzyl]isoquinolin-1(2H)-one (27). A mixture of 6-fluoro-5-methoxy-
2-[3-(trifluoromethyl)benzyl]isoquinolin-1(2H)-one (26) (253 mg,
0.72 mmol), 4-methylimidazole (118 mg, 1.44 mmol), and potassium
carbonate (398 mg, 2.88 mmol) in N,N-dimethylformamide (5 mL)
was stirred at 100 °C for 3 days and then at 120 °C for 12 h. The
reaction was allowed to cool to room temperature and filtered, the
solids were washed with acetonitrile, and the combined filtrate and
washings were concentrated under reduced pressure. The resulting
residue was purified by prep-HPLC (column: Gemini 50 × 100 mm
C18 (acidic or basic, >150 mg), injection volume: 900 μL × 7; solvent
A: 0.1% formic acid in water; solvent B: acetonitrile; gradient 30% to
80% B; collection time: 3−10 min; flow rate: 75 mL/min; wavelength
220 nm) to afford the desired product as a mixture containing the
imidazole regioisomer side product (142 mg). The regioisomers were
further separated by SFC prep-HPLC (column: Chiralcel OD-H (4.6
mm x 25 cm); mobile phase 25% methanol in carbon dioxide; flow
rate: 2.5 mL/min) to afford the title compound as a solid (80 mg,
1
as a yellow solid (23.0 g, 56%). LCMS m/z 307.1 (M + 1). H NMR
(300 MHz, CDCl3) δ 2.29 (br s, 3H), 3.80−3.93 (m, 6H), 4.38−4.44
(m, 2H), 7.13−7.16 (m, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.45 (d, J = 7.6
Hz, 1H), 8.24 (d, J = 1.0 Hz, 1H).
2-[3-(Trifluoromethyl)benzyl]-7-(4-methyl-1H-imidazol-1-yl)-3,4-
dihydro-2H-pyrido[1,2-a]pyrazine-1,6-dione (44). To a solution of 5-
(4-methyl-1H-imidazol-1-yl)-6-oxo-1,6-dihydropyridine-2-carboxylic
acid, hydrochloride salt (35) (68 mg, 0.20 mmol) in N,N-
dimethylformamide (6.0 mL) was added HATU (140 mg, 0.36
mmol), N,N-diisopropylethylamine (0.30 mL, 2.0 mmol), and finally
[2-(3-(trifluoromethyl)benzylamino]ethanol (50 mg, 0.23 mmol). The
reaction mixture was stirred at room temperature for 14 h, whereupon
additional HATU (102 mg, 0.26 mmol) was added. Stirring was
continued for 4 h, and the reaction mixture was poured into saturated
aqueous sodium bicarbonate solution (50 mL) and washed with ethyl
acetate (2 × 50 mL). The combined organic layers were washed with
saturated aqueous sodium chloride solution (2 × 50 mL), dried
(NaSO4), filtered, and concentrated under reduced pressure. The
resulting residue was purified via chromatography on silica gel
(gradient: 10% to 100% ethyl acetate in heptane) to afford the title
compound as a tan solid (20 mg, 23%). LCMS m/z 403.1 (M + 1). 1H
NMR (400 MHz, CDCl3) δ 2.25 (d, J = 1.0 Hz, 3H), 3.59 (dd, J = 6.5,
5.2 Hz, 2H), 4.29 (dd, J = 6.5, 5.2 Hz, 2H), 4.80 (s, 2H), 7.10 (t, J =
1.2 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.47−
7.51 (m, 2H), 7.52−7.60 (m, 2H), 8.19 (d, J = 1.2 Hz, 1H). HPLC
purity >95%.
1
27%). LCMS m/z 400.5 (M + 1). H NMR (400 MHz, CD3OD) δ
2.30 (s, 3H), 5.35 (s, 2H), 7.10 (dd, J = 7.6, 0.8 Hz, 1H), 7.23 (t, J =
1.2 Hz, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.50−7.60 (m, 2H), 7.60−7.66
(m, 2H), 7.68 (s, 1H), 7.95 (d, J = 8.6 Hz, 1H), 8.05 (d, J = 1.4 Hz,
1H). HPLC purity >95%.
5-Hydroxy-6-(4-methyl-1H-imidazol-1-yl)-2-[3-(trifluoromethyl)-
benzyl]-3,4-dihydroisoquinolin-1(2H)-one (28). 5-Hydroxy-6-(4-
methyl-1H-imidazol-1-yl)-2-[3-(trifluoromethyl)benzyl]isoquinolin-
1(2H)-one (27) (30 mg, 0.075 mmol) in 20 mL of methanol was
subjected to H-Cube hydrogenation (10% Pd/C, 50 bar, 60 °C, 0.4
mL/min). The solvent was evaporated under reduced pressure to
afford the title compound as an oil (29 mg, 97%). LCMS m/z 402.6
(M + 1). 1H NMR (400 MHz, CD3OD) δ 2.07 (s, 3H), 3.08 (t, J = 6.7
Hz, 2H), 3.52 (t, J = 6.7 Hz, 2H), 4.85 (s, 2H), 6.79 (s, 1H), 7.08 (d, J
= 8.2 Hz, 1H), 7.36−7.42 (m, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.51−
7.64 (m, 3H), 7.69 (d, J = 8.2 Hz, 1H). HPLC purity >95%.
1057
dx.doi.org/10.1021/jm401782h | J. Med. Chem. 2014, 57, 1046−1062

Journal of Medicinal Chemistry
Article
2-(tert-Butyldimethylsilyloxy)-N-{[1-(4-chlorophenyl)cyclopropyl]-
methyl}ethanamine. A solution of [1-(4-chlorophenyl)cyclopropyl]-
methanamine (10.0 mg, 0.055 mmol) and (tert-butyldimethylsilyloxy)-
acetaldehyde (9.3 mg, 0.053 mmol) in dichloroethane (17.0 mL) was
stirred for 5 min at room temperature, whereupon sodium
triacetoxyborohydride (15.0 mg, 0.071 mmol) was added. The mixture
was stirred at room temperature for 18 h and then poured into a
saturated aqueous solution of sodium bicarbonate (50 mL) and
washed with dichloromethane (2 × 20 mL). The combined organic
layers were washed with brine (2 × 50 mL), dried (Na2SO4), filtered,
and concentrated under reduced pressure to afford the title compound
(20 mg, LCMS m/z 340.2 (M + 1) which was used directly in the next
without further purification.
2-{[1-(4-Chlorophenyl)cyclopropyl]methylamino}ethanol. A sol-
ution of 0.4 M HCl in methanol at 0 °C (0.5 mL, 0.2 mmol) was
added to a solution of 2-(tert-butyldimethylsilyloxy)-N-{[1-(4-
chlorophenyl)cyclopropyl]methyl}ethanamine (20 mg, 0.059 mmol)
in methanol (0.5 mL). The reaction was stirred at room temperature
for 64 h, whereupon it was poured into a saturated solution of aqueous
sodium bicarbonate (50 mL) and washed with ethyl acetate (50 mL).
The layers were separated, and the organic layer was washed with
brine (2 × 50 mL), dried (Na2SO4), filtered, and concentrated under
reduced pressure. The residue was purified by chromatography on
silica gel (gradient: 10% to 100% ethyl acetate in heptane) to afford
the title compound as a clear colorless oil (13 mg, quant.). LCMS m/z
226.2 (M + 1).
2-{[1-(4-Chlorophenyl)cyclopropyl]methyl}-7-(4-methyl-1H-imi-
dazol-1-yl)-3,4-dihydro-2H-pyrido[1,2-a]pyrazine-1,6-dione, Tri-
fluoroacetate Salt (45). To a solution of 5-(4-methyl-1H-imidazol-
1-yl)-6-oxo-1,6-dihydropyridine-2-carboxylic acid, hydrobromide salt
(34) (22 mg 0.06 mmol) and 2-{[1-(4-chlorophenyl)cyclopropyl]-
methylamino}ethanol (13 mg, 0.06 mmol) in N,N-dimethylformamide
(1.5 mL) were added HATU (50 mg, 0.13 mmol) and N,N-
diisopropylethylamine (0.30 mL, 2.0 mmol). The reaction mixture was
stirred at room temperature for 17 h, whereupon it was partitioned
between dichloromethane and water. The aqueous layer was extracted
with dichloromethane, and the combined organic layers were dried
(Na2SO4), filtered through silica gel, and concentrated under reduced
pressure. The residue was dissolved in dimethyl sulfoxide (1 mL) and
purified by reversed-phase HPLC Column: Waters Sunfire C18 19 ×
100, 5 μm; mobile phase A: 0.05% TFA in water (v/v); mobile phase
B: 0.05% TFA in acetonitrile (v/v); gradient: 85.0% water/15.0%
acetonitrile linear to 0% water/100% acetonitrile in 8.5 min, hold at
0% water/100% acetonitrile to 10.0 min. Flow rate: 25 mL/min. Purity
was determined using liquid chromatography/photodiode array
detector/evaporative light scattering detection coupled to single
quadrupole mass spectrometry (LC/UV/ELSD/MS) using these
standard conditions: Waters Atlantis dC18 columns, 6 × 50 mm, 5
μm, at 2 mL/min flow rate; mobile phase A 0.05% trifluoroacetic acid
in water (v/v); mobile phase B 0.05% trifluoroacetic acid in
acetonitrile (v/v); 95% water/5% acetonitrile to 5% water/95%
acetonitrile linear gradient over 4 min, and holding at final gradient for
an additional 1 min. Retention time: 2.4 min. LCMS m/z 409.2 (M +
1). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 1.2 Hz, 1H), 7.42 (d, J
= 7.8 Hz, 1H), 7.31−7.22 (m, 4H), 7.17 (d, J = 7.8 Hz, 1H), 7.10 (s,
1H), 4.08−4.02 (m, 2H), 3.74 (s, 2H), 3.38−3.32 (m, 2H), 2.26 (d, J
= 0.8 Hz, 3H), 1.04−0.99 (m, 2H), 0.95−0.91 (m, 2H). HPLC purity
>95%.
× 100, 5 μm; mobile phase A: 0.05% trifluoroacetic acid in water (v/
v); mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v/v).
Flow rate: 25 mL/min.
7-(4-Methyl-1H-imidazol-1-yl)-2-{2-[2-(trifluoromethyl)phenoxy]-
ethyl}-3,4-dihydro-1H-pyrido[1,2-a]pyrazine-1,6(2H)-dione (46).
1
LCMS m/z 433.1 (M + 1). H NMR (600 MHz, DMSO-d6) δ 9.25
(s, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.76 (s, 1H), 7.67−7.57 (m, 2H),
7.31 (d, J = 8.3 Hz, 1H), 7.19−7.14 (m, 1H), 7.11 (t, J = 7.5 Hz, 1H),
4.35 (t, J = 5.0 Hz, 2H), 4.28−4.24 (m, 2H), 3.93 (t, J = 5.3 Hz, 2H),
3.86−3.82 (m, 2H), 2.30 (s, 3H). HPLC purity >95%.
2-[2-(2-Chlorophenoxy)ethyl]-7-(4-methyl-1H-imidazol-1-yl)-3,4-
dihydro-1H-pyrido[1,2-a]pyrazine-1,6(2H)-dione (47). LCMS m/z
1
399.1 (M + 1). H NMR (600 MHz, DMSO-d6) δ 9.24 (s, 1H), 8.01
(d, J = 7.5 Hz, 1H), 7.75 (s, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.33−7.28
(m, 1H), 7.20−7.15 (m, 2H), 6.97 (t, J = 7.7 Hz, 1H), 4.30−4.28 (m,
4H), 3.94−3.92 (m, 4H), 2.30 (s, 3H). HPLC purity >95%.
7-(4-Methyl-1H-imidazol-1-yl)-2-{2-[3-(trifluoromethyl)phenoxy]-
ethyl}-3,4-dihydro-1H-pyrido[1,2-a]pyrazine-1,6(2H)-dione (48).
1
LCMS m/z 433.1 (M + 1). H NMR (600 MHz, DMSO-d6) δ 9.21
(br s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.74 (s, 1H), 7.56−7.49 (m, 1H),
7.32−7.26 (m, 2H), 7.16 (d, J = 7.5 Hz, 1H), 4.30 (t, J = 5.5 Hz, 2H),
4.28−4.23 (m, 2H), 3.91 (t, J = 5.3 Hz, 2H), 3.89−3.86 (m, 2H), 2.30
(s, 3H). HPLC purity >95%.
7-(4-Methyl-1H-imidazol-1-yl)-2-{2-[4-(trifluoromethyl)phenoxy]-
ethyl}-3,4-dihydro-1H-pyrido[1,2-a]pyrazine-1,6(2H)-dione (49).
LCMS m/z 433.1 (M + 1). 1H NMR (600 MHz, DMSO-d6): δ
9.43 (s, 1H), 8.05 (dd, J = 7.7, 2.0 Hz, 1H), 7.82 (s, 1H), 7.66 (d, J =
7.0 Hz, 2H), 7.12−7.20 (m, 3H), 4.24−4.34 (m, 4H), 3.85−3.94 (m,
4H), 2.33 (s, 3H). HPLC purity >95%.
2-[2-(7-Fluoronaphthalen-1-yloxy)ethyl]-7-(4-methyl-1H-imida-
zol-1-yl)-3,4-dihydro-1H-pyrido[1,2-a]pyrazine-1,6(2H)-dione (52).
1
LCMS m/z 433.3 (M + 1). H NMR (400 MHz, CDCl3) δ 8.20 (s,
1H), 7.79 (dd, J = 5.5, 9.0 Hz, 1H), 7.69 (dd, J = 2.5, 10.4 Hz, 1H),
7.46−7.38 (m, 2H), 7.32 (t, J = 7.9 Hz, 1H), 7.29−7.22 (m, 2H), 7.10
(s, 1H), 6.83 (d, J = 7.6 Hz, 1H), 4.42 (t, J = 4.8 Hz, 2H), 4.40−4.34
(m, 2H), 4.11 (t, J = 4.8 Hz, 2H), 4.00−3.94 (m, 2H), 2.26 (s, 3H).
HPLC purity >95%.
1-(2-Bromoethoxy)-4-fluoro-2-(trifluoromethyl)benzene. A mix-
ture of 4-fluoro-2-(trifluoromethyl)phenol (1.05 g, 5.83 mmol), 2-
bromoethanol (0.62 mL, 8.7 mmol), and triphenylphosphine (2.29 g,
8.73 mmol) in tetrahydrofuran (20 mL) was stirred for 5 min.
Diisopropyl azodicarboxylate (94%, 1.84 mL, 8.71 mmol) was added
dropwise over 20 min, and the reaction was stirred at room
temperature for 16 h. Water (50 mL) was added, and the mixture
was extracted with dichloromethane (2 × 75 mL). The combined
organic layers were washed with saturated aqueous sodium chloride
solution (50 mL), dried (MgSO4), filtered, and concentrated under
reduced pressure. Purification via chromatography on silica gel
(gradient: 0% to 10% ethyl acetate in heptane) afforded the title
compound as a white solid (600 mg, 36%). GCMS m/z 286. 1H NMR
(400 MHz, CDCl3) δ 3.66 (dd, J = 6.6, 6.4 Hz, 2H), 4.35 (dd, J = 6.5,
6.4 Hz, 2H), 6.97 (br dd, J = 9.0, 4.1 Hz, 1H), 7.18−7.24 (m, 1H),
7.32 (br dd, J = 8.3, 3.2 Hz, 1H).
2-({2-[4-Fluoro-2-(trifluoromethyl)phenoxy]ethyl}amino)ethanol.
A mixture of 1-(2-bromoethoxy)-4-fluoro-2-(trifluoromethyl)benzene
(600 mg, 2.09 mmol) and 2-aminoethanol (2.20 mL, 52.4 mmol) was
heated to 80 °C for 1.5 h. The reaction was allowed to cool to room
temperature, diluted with ethyl acetate (75 mL), and washed with
aqueous sodium hydroxide solution (1 N, 4 × 50 mL). The organic
layer was dried (MgSO4), filtered, and concentrated under reduced
pressure to provide the title compound as a white solid (550 mg,
Parallel Medicinal Chemistry Protocol for Preparation of 2-[2-
(Aryloxy)ethyl]- and 2-[2-(heteroaryloxy)ethyl]-7-(4-methyl-1H-imi-
dazol-1-yl)-3,4-dihydro-2H-pyrido[1,2-a]pyrazine-1,6-diones 46−49
and 52. 2-(2-Chloroethyl)-7-(4-methyl-1H-imidazol-1-yl)-3,4-dihy-
dro-2H-pyrido[1,2-a]pyrazine-1,6-dione (41) (20−35 mg) and a
suitably substituted phenol (1−1.5 equiv) were combined in dimethyl
sulfoxide (1.0 mL). After addition of potassium carbonate (3.5 equiv),
the reaction mixture was heated at 100 °C until the reaction was
judged to be complete via LCMS analysis (generally 1 to 3 h). The
mixture was allowed to cool to room temperature and filtered, and the
filtrate was purified using reversed phase HPLC with an appropriate
gradient using the following system: Column: Waters Sunfire C18 19
1
99%). LCMS m/z 268.1 (M + 1). H NMR (400 MHz, CD3OD) δ
2.80 (br dd, J = 5.5, 5.4 Hz, 2H), 3.04 (dd, J = 5.4, 5.2 Hz, 2H), 3.68
(br dd, J = 5.6, 5.4 Hz, 2H), 4.20 (dd, J = 5.3, 5.3 Hz, 2H), 7.22 (br dd,
J = 8.8, 4.2 Hz, 1H), 7.30−7.37 (m, 2H).
2-{2-[4-Fluoro-2-(trifluoromethyl)phenoxy]ethyl}-7-(4-methyl-
1H-imidazol-1-yl)-3,4-dihydro-2H-pyrido[1,2-a]pyrazine-1,6-dione
(50). 5-(4-Methyl-1H-imidazol-1-yl)-6-oxo-1,6-dihydropyridine-2-car-
boxylic acid, hydrobromide salt (34) (201 mg, 0.729 mmol) and 2-
({2-[4-fluoro-2-(trifluoromethyl)phenoxy]ethyl}amino)ethanol (214
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mg, 0.801 mmol) were combined in dichloromethane (15 mL) and
N,N-diisopropylethylamine (0.508 mL, 2.92 mmol), and the mixture
was stirred for 5 min at room temperature. HATU (97%, 857 mg, 2.19
mmol) was added in one portion, and the reaction was stirred for an
additional 16 h. Water (50 mL) was added, and the mixture was
extracted with dichloromethane (3 × 50 mL). The combined organic
layers were washed with saturated aqueous sodium bicarbonate
solution (50 mL), water (50 mL), and saturated aqueous sodium
chloride solution (50 mL), dried (MgSO4), filtered, and concentrated
under reduced pressure. Purification was carried out twice by
chromatography on silica gel (gradient 1: 0% to 20% methanol in
dichloromethane; gradient 2: 0% to 70% [10% 2 N ammonia in
methanol/90% ethyl acetate] in ethyl acetate) to afford the title
compound as a white solid (268 mg, 82%). LCMS m/z 451.0 (M + 1).
1H NMR (400 MHz, CD3OD) δ 2.24 (d, J = 0.9 Hz, 3H), 3.90−3.95
(m, 2H), 4.01 (dd, J = 5.1, 5.1 Hz, 2H), 4.32−4.37 (m, 4H), 7.21−
7.26 (m, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.30−7.32 (m, 1H), 7.32−7.39
(m, 2H), 7.76 (d, J = 7.8 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H). 13C NMR
(125 MHz, CDCl3) δ 158.0, 157.3, 155.7, 155.3, 152.2, 138.3, 136.2,
134.3, 130.4, 127.5, 119.9, 119.7, 114.7, 113.8, 113.7, 108.4, 67.9, 47.9,
46.5, 40.1, 13.4. HPLC purity >95%.
1-(2-Bromoethoxy)-4-chloro-2-(trifluoromethyl)benzene. Cesium
carbonate (16.60 g, 50.95 mmol) was added to a solution of 4-chloro-
2-(trifluoromethyl)phenol (5.00 g, 25.4 mmol) and 1,2-dibromo-
ethane (23.90 g, 127.2 mmol) in acetonitrile (70 mL), and the reaction
was heated to 70 °C for 16 h. The mixture was allowed to cool to
room temperature, diluted with ethyl acetate, and washed with water.
The organic layer was dried (MgSO4), filtered, and concentrated
under reduced pressure. Purification via chromatography on silica gel
(gradient: 10% to 30% ethyl acetate in heptanes) afforded the title
compound as a clear viscous oil (7.05 g, 91%). GCMS m/z 302 (M+).
1H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 2.5 Hz, 1H), 7.46 (dd, J =
carbonate (456 mg, 1.40 mmol) were added to a solution of (3-
hydroxyphenyl)[4-(pent-4-ynyloxy)phenyl]methanone (196 mg, 0.70
mmol) in acetonitrile (15 mL). The reaction mixture was heated to 70
°C and was stirred overnight, whereupon it was allowed to cool to
room temperature. The reaction mixture was then poured into
dichloromethane (25 mL), washed with water (2 × 20 mL), dried
(MgSO4), filtered, and concentrated under reduced pressure. The
resulting residue was purified by chromatography on silica gel
(gradient: 10% to 30% ethyl acetate in heptane) to afford the title
1
compound (271 mg, 54%). LCMS m/z 387.2 (M + 1). H NMR
(CDCl3, 400 MHz) δ 7.79 (d, J = 8.4 Hz, 2H), 7.29−7.37 (m, 3H),
7.10 (d, J = 8 Hz, 1H), 6.94 (d, J = 8.4 Hz, 2H), 4.32 (t, J = 6.2 Hz,
2H), 4.14 (t, J = 6.2 Hz, 2H), 3.63 (t, J = 6.2 Hz, 2H), 2.40 (td, J = 6.9,
6.9 Hz, 2H), 2.01 (m, 2H), 1.95 (m, 1H).
{3-[2-(2-Hydroxyethylamino)ethoxy]phenyl}[4-(pent-4-ynyloxy)-
phenyl]methanone. A solution of [3-(2-bromoethoxy)phenyl][4-
(pent-4-ynyloxy)phenyl]methanone (147 mg, 0.38 mmol) in ethanol-
amine (0.23 mL, 3.8 mmol) was heated to 80 °C for 3 h. The reaction
was allowed to cool to room temperature and was diluted with
dichloromethane (15 mL). The mixture was washed with 0.5 N
hydrochloric acid (2 × 15 mL), water (15 mL), and brine (10 mL).
The organic layer was dried (MgSO4), filtered, and concentrated
under reduced pressure to provide the title compound (87 mg, 62%)
1
as a white solid. LCMS m/z 368.3 (M + 1). H NMR (CDCl3, 400
MHz) δ 7.79 (d, J = 8.8 Hz, 2H), 7.34 (m, 2H), 7.28 (m, 1H), 7.08 (d,
J = 8.4 Hz, 1H), 6.93 (d, J = 7.2 Hz, 2H), 4.11 (m, 4H), 3.64 (m, 2H),
3.02 (m, 2H), 2.83 (m, 2H), 2.41 (m, 2H), 2.00 (m, 3H).
7-(4-Methyl-1H-imidazol-1-yl)-2-(2-{3-[4-(pent-4-yn-1-yloxy)-
benzoyl]phenoxy}ethyl)-3,4-dihydro-2H-pyrido[1,2-a]pyrazine-1,6-
dione (53). HATU (212 mg, 0.558 mmol) was added to a solution of
5-(4-methyl-1H-imidazol-1-yl)-6-oxo-1,6-dihydropyridine-2-carboxylic
acid, hydrobromide salt (34) (60 mg, 0.22 mmol), {3-[2-(2-
hydroxyethylamino)ethoxy]phenyl}[4-(pent-4-ynyloxy)phenyl]-
methanone (87 mg, 0.24 mmol), and N,N-diisopropylethylamine (0.16
mL, 0.87 mmol) in dichloromethane (2 mL), and the reaction was
stirred for 19 h at room temperature. The mixture was diluted with
dichloromethane (15 mL) and washed with a saturated aqueous
solution of sodium bicarbonate (15 mL) and with water (15 mL). The
organic layer was dried (MgSO4), filtered, and concentrated under
reduced pressure. The resulting residue was purified by chromatog-
raphy on silica gel (gradient: 0% to 40% methanol in ethyl acetate) to
provide the title compound (76 mg, 63%) as a solid. LCMS m/z 551.2
(M + 1). 1H NMR (CDCl3) δ 1.99−2.09 (m, 3H), 2.29 (s, 3H), 2.42−
2.48 (m, 2H), 3.91−3.96 (m, 2H), 3.98−4.03 (m, 2H), 4.15−4.20 (m,
2H), 4.30−4.34 (m, 2H), 4.36−4.42 (m, 2H), 6.95−7.00 (m, 2H),
7.07−7.12 (m, 1H), 7.14 (br s, 1H), 7.26−7.47 (m, 5 H, assumed;
partially obscured by solvent peak), 7.79−7.83 (m, 2H), 8.23 (br s,
1H). HPLC purity >95%.
2.5, 8.8 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 4.36 (t, J = 6.6 Hz, 2H),
3.66 (t, J = 6.6 Hz, 2H).
2-({2-[4-Chloro-2-(trifluoromethyl)phenoxy]ethyl}amino)ethanol.
A solution of 1-(2-bromoethoxy)-4-chloro-2-(trifluoromethyl)benzene
(7.05 g, 23.2 mmol) in 2-aminoethanol (14.20 g, 232.5 mmol) was
heated to 80 °C for 4 h. The reaction mixture was allowed to cool to
room temperature, diluted with dichloromethane, and washed with
aqueous sodium hydroxide solution (0.5 N) and water. The organic
layer was dried (MgSO4), filtered, and concentrated under reduced
pressure to provide the title compound as a white solid (6.31 g, 96%).
LCMS m/z 284.1 (M + 1). 1H NMR (400 MHz, CDCl3) δ 7.55 (d, J
= 2.7 Hz, 1H), 7.45 (dd, J = 2.6, 8.9 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H),
4.15 (t, J = 5.0 Hz, 2H), 3.69−3.63 (m, 2H), 3.07 (t, J = 5.0 Hz, 2H),
2.89−2.84 (m, 2H).
2-{2-[4-Chloro-2-(trifluoromethyl)phenoxy]ethyl}-7-(4-methyl-
1H-imidazol-1-yl)-3,4-dihydro-2H-pyrido[1,2-a]pyrazine-1,6-dione
(51). HATU (15.7 g, 40.5 mmol) was added to a solution of 5-(4-
methyl-1H-imidazol-1-yl)-6-oxo-1,6-dihydropyridine-2-carboxylic acid,
hydrobromide salt (34) (4.85 g, 16.2 mmol), 2-({2-[4-chloro-2-
(trifluoromethyl)phenoxy]ethyl}amino)ethanol (5.04 g, 17.2 mmol),
and N,N-diisopropylethylamine (8.62 mL, 2.92 mmol) in dichloro-
methane (100 mL). The reaction was stirred at room temperature for
16 h, whereupon it was diluted with dichloromethane and washed with
a saturated aqueous solution of sodium bicarbonate and with water.
The organic layer was dried (MgSO4), filtered, and concentrated
under reduced pressure. Purification was carried out twice by
chromatography on silica gel (gradient: 0% to 30% methanol in
dichloromethane) to afford the title compound as a white solid (4.01
g, 53%). LCMS m/z 467.1 (M + 1). 1H NMR (400 MHz, CD3OD) δ
2.24 (d, J = 0.9 Hz, 3H), 3.90−3.94 (m, 2H), 4.01 (dd, J = 5.1, 5.0 Hz,
2H), 4.32−4.39 (m, 4H), 7.21−7.27 (m, 2H), 7.30−7.32 (m, 1H),
7.56−7.60 (m, 2H), 7.76 (d, J = 7.7 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H).
13C NMR (125 MHz, CDCl3) δ 158.1, 155.7, 154.6, 138.2, 136.1,
ASSOCIATED CONTENT
* Supporting Information
■
S
The following experimental information is provided: cell-based
Aβ production assay, determination of Aβ lowering activity in
vivo, and photoaffinity labeling of HeLa membranes with
clickable GSMs followed by Western blot analysis. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (617) 395-0705. E-mail: Martin.Pettersson@pfizer.
com.
■
Present Addresses
§255 W. MLK Jr. Blvd., Unit 817, Charlotte, NC 28202.
⊥Clinical Pharmacology, Takeda Pharmaceuticals, 1 Takeda
Parkway, Deerfield, IL 60015.
133.3, 130.3, 127.5, 127.4, 126.1, 123.9, 121.8, 114.7, 113.7, 108.4,
67.7, 47.9, 46.5, 40.1, 13.3. HRMS: calcd for C21H19ClF3N4O3 (M +
H)+ = 467.1098, found =467.1091. HPLC purity >95%.
[3-(2-Bromoethoxy)phenyl][4-(pent-4-ynyloxy)phenyl]-
methanone. Dibromoethane (0.60 mL, 7.0 mmol) and cesium
∥BioDuro Shanghai, BioDuro Co., Ltd., 233 North Fute Road,
Weigaoqiao Free Trade Zone, Shanghai 200131, China.
1059
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Journal of Medicinal Chemistry
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#Toronto Research Chemicals, 2 Brisbane Road, Toronto,
Ontario M3J 2J8, Canada.
Notes
avagacestat in a phase 2 study of mild to moderate Alzheimer disease.
Arch. Neurol. 2012, 69, 1430−1440.
(8) Haapasalo, A.; Kovacs, D. M. The many substrates of presenilin/
γ-secretase. J. Alzheimer’s Dis. 2011, 25, 3−28.
The authors declare no competing financial interest.
(9) 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.
ACKNOWLEDGMENTS
■
We thank Stacey Becker, Emily Miller, Michael Marconi, Emily
Sylvain, and Karin Wallace for their contributions to the in vivo
studies. We also thank Katherine Brighty for editing the
manuscript, and the Pfizer ADME technology group for
generating the in vitro pharmacokinetic data to support the
SAR efforts.
(10) (a) Weggen, S.; Eriksen, J. L.; Das, P.; Sagi, S. A.; Wang, R.;
Pietrzik, C. U.; Findlay, K. A.; Smith, T. E.; Murphy, M. P.; Bulter, T.;
Kang, D. E.; Marquez-Sterling, N.; Golde, T. E.; Koo, E. H. A subset of
NSAIDs lower amyloidogenic Aβ42 independently of cyclooxygenase
activity. Nature 2001, 414, 212−216. (b) Eriksen, J. L.; Sagi, S. A.;
Smith, T. E.; Weggen, S.; Das, P.; McLendon, D. C.; Ozols, V. V.;
Jessing, K. W.; Zavitz, K. H.; Koo, E. H.; Golde, T. E. NSAIDs and
enantiomers of flurbiprofen target γ-secretase and lower Aβ42 in vivo.
J. Clin. Invest. 2003, 112, 440−449. (c) Weggen, S.; Eriksen, J. L.; Sagi,
S. A.; Pietrzik, C. U.; Golde, T. E.; Koo, E. H. Aβ42-Lowering
nonsteroidal anti-inflammatory drugs preserve intramembrane cleav-
age of the amyloid precursor protein (APP) and ErbB-4 receptor and
signaling through the APP intracellular domain. J. Biol. Chem. 2003,
278, 30748−30754. (d) Weggen, S.; Eriksen, J. L.; Sagi, S. A.; Pietrzik,
C. U.; Ozols, V.; Fauq, A.; Golde, T. E.; Koo, E. H. Evidence that
nonsteroidal anti-inflammatory drugs decrease amyloid β42 produc-
tion by direct modulation of γ-secretase activity. J. Biol. Chem. 2003,
278, 31831−31837.
ABBREVIATIONS USED
■
Aβ, amyloid-β peptide; AD, Alzheimer’s disease; ADME,
absorption, distribution, metabolism, and excretion; APP,
amyloid precursor protein; AB, apical to basolateral; BA,
basolateral to apical; CLint,app, apparent intrinsic clearance; CNS
MPO, central nervous system multiparameter optimization;
CSF, cerebrospinal fluid; CYP, cytochrome P450; GSM, γ-
secretase modulator; HHEP, human hepatocytes; HLM, human
liver microsomes; LipE, lipophilic efficiency; MDCK, Madin−
Darby canine kidney; MDR1, multidrug resistance protein (P-
glycoprotein, P-gp); PK, pharmacokinetic; PD, pharmacody-
namic; PS, presenilin; RLM, rat liver microsomes; SAR,
structure−activity relationship; TPSA, topological polar surface
area
(11) (a) Pettersson, M.; Stepan, A. F.; Kauffman, G. W.; Johnson, D.
S. Novel γ-secretase modulators for the treatment of Alzheimer’s
disease: a review focusing on patents from 2010 to 2012. Expert Opin.
Ther. Pat. 2013, 23, 1349−1366. (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, 205−226. (c) 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.
(12) Green, R. C.; Schneider, L. S.; Amato, D. A.; Beelen, A. P.;
Wilcock, G.; Swabb, E. A.; Zavitz, K. H. Effect of tarenflurbil on
cognitive decline and activities of daily living in patients with mild
Alzheimer disease: A randomized controlled trial. JAMA, J. Am. Med.
Assoc. 2009, 302, 2557−2564.
REFERENCES
■
(1) Thies, W.; Bleiler, L. 2013 Alzheimer’s disease facts and figures.
Alzheimer’s Dementia 2013, 9, 208-245.
(2) Bertram, L.; Tanzi, R. E. The genetics of Alzheimer’s disease.
Prog. Mol. Biol. Transl. Sci. 2012, 107, 79−100.
(3) (a) Walsh, D. M.; Selkoe, D. J. Aβ oligomers - a decade of
discovery. J. Neurochem. 2007, 101, 1172−1184. (b) Karran, E.;
Mercken, M.; De Strooper, B. The amyloid cascade hypothesis for
Alzheimer’s disease: an appraisal for the development of therapeutics.
Nat. Rev. Drug Discovery 2011, 10, 698−712.
́
(13) (a) Page, R. M.; Baumann, K.; Tomioka, M.; Perez-Revuelta, B.
(4) De Strooper, B.; Vassar, R.; Golde, T. The secretases: enzymes
with therapeutic potential in Alzheimer disease. Nat. Rev. Neurol. 2010,
6, 99−107.
I.; Fukumori, A.; Jacobsen, H.; Flohr, A.; Luebbers, T.; Ozmen, L.;
Steiner, H.; Haass, C. Generation of Aβ38 and Aβ42 is independently
and differentially affected by familial Alzheimer disease-associated
presenilin mutations and γ-secretase modulation. J. Biol. Chem. 2008,
283, 677−683. (b) Ohki, Y.; Higo, T.; Uemura, K.; Shimada, N.;
Osawa, S.; Berezovska, O.; Yokoshima, S.; Fukuyama, T.; Tomita, T.;
Iwatsubo, T. Phenylpiperidine-type γ-secretase modulators target the
transmembrane domain 1 of presenilin 1. EMBO J. 2011, 30, 4815−
4824. (c) Crump, C. J.; Fish, B. A.; Castro, S. V.; Chau, D.-M.; Gertsik,
N.; Ahn, K.; Stiff, C.; Pozdnyakov, N.; Bales, K. R.; Johnson, D. S.; Li,
Y.-M. Piperidine acetic acid based γ-secretase modulators directly bind
to presenilin-1. ACS Chem. Neurosci. 2011, 2, 705−710.
(5) (a) Bateman, R. J.; Siemers, E. R.; Mawuenyega, K. G.; Wen, G.;
Browning, K. R.; Sigurdson, W. C.; Yarasheski, K. E.; Friedrich, S. W.;
DeMattos, R. B.; May, P. C.; Paul, S. M.; Holtzman, D. M. A γ-
secretase inhibitor decreases amyloid-β production in the central
nervous system. Ann. Neurol. 2009, 66, 48−54. (b) Tong, G.;
Castaneda, L.; Wang, J.-S.; Sverdlov, O.; Huang, S.-P.; Slemmon, R.;
Gu, H.; Wong, O.; Li, H.; Berman, R. M.; Smith, C.; Albright, C.;
Dockens, R. C. Effects of single doses of avagacestat (BMS-708163)
on cerebrospinal fluid Aβ levels in healthy young men. Clin. Drug
Invest. 2012, 32, 761−769.
(14) (a) Hashimoto, T.; Ishibashi, A.; Hagiwara, H.; Murata, Y.;
Takenaka, O.; Miyagawa, T. E2012: A novel gamma-secretase
modulator - pharmacology. Alzheimer’s Dementia 2010, 6 (Suppl),
S242. (b) Portelius, E.; Van Broeck, B.; Andreasson, U.; Gustavsson,
M. K.; Mercken, M.; Zetterberg, H.; Borghys, H.; Blennow, K. Acute
effect on the Aβ isoform pattern in CSF in response to γ-secretase
modulator and inhibitor treatment in dogs. J. Alzheimer’s Dis. 2010, 21,
1005−1012.
(6) (a) Imbimbo, B. P.; Panza, F.; Frisardi, V.; Solfrizzi, V.;
D’Onofrio, G.; Logroscino, G.; Seripa, D.; Pilotto, A. Therapeutic
intervention for Alzheimer’s disease with γ-secretase inhibitors: Still a
viable option? Expert Opin. Invest. Drugs 2011, 20, 325−341.
(b) Sambamurti, K.; Greig, N. H.; Utsuki, T.; Barnwell, E. L.;
Sharma, E.; Mazell, C.; Bhat, N. R.; Kindy, M. S.; Lahiri, D. K.;
Pappolla, M. A. Targets for AD treatment: conflicting messages from
γ-secretase inhibitors. J. Neurochem. 2011, 117, 359−374. (c) Schor, N.
F. What the halted phase III γ-secretase inhibitor trial may (or may
not) be telling us. Ann. Neurol. 2011, 69, 237−239.
(7) Coric, V.; van Dyck, C. H.; Salloway, S.; Andreasen, N.; Brody,
M.; Richter, R. W.; Soininen, H.; Thein, S.; Shiovitz, T.; Pilcher, G.;
Colby, S.; Rollin, L.; Dockens, R.; Pachai, C.; Portelius, E.;
Andreasson, U.; Blennow, K.; Soares, H.; Albright, C.; Feldman, H.
H.; Berman, R. M. Safety and tolerability of the γ-secretase inhibitor
(15) De Strooper, B.; Iwatsubo, T.; Wolfe, M. S. Presenilins and γ-
secretase: structure, function, and role in Alzheimer disease. Cold
Spring Harbor Perspect. Med. 2012, 2, a006304.
(16) (a) Crump, C. J.; Johnson, D. S.; Li, Y.-M. Development and
mechanism of γ-secretase modulators for Alzheimer’s disease.
Biochemistry 2013, 52, 3197−3216. (b) Pozdnyakov, N.; Murrey, H.
E.; Crump, C. J.; Pettersson, M.; Ballard, T. E.; am Ende, C. W.; Ahn,
1060
dx.doi.org/10.1021/jm401782h | J. Med. Chem. 2014, 57, 1046−1062

Journal of Medicinal Chemistry
Article
K.; Li, Y.-M.; Bales, K. R.; Johnson, D. S. γ-Secretase modulator
(GSM) photoaffinity probes reveal distinct allosteric binding sites on
presenilin. J. Biol. Chem. 2013, 288, 9710−9720. (c) Crump, C. J.; am
Ende, C. W.; Ballard, T. E.; Pozdnyakov, N.; Pettersson, M.; Chau, D.-
M.; Bales, K. R.; Li, Y.-M.; Johnson, D. S. Development of clickable
active site-directed photoaffinity probes for γ-secretase. Bioorg. Med.
Chem. Lett. 2012, 22, 2997−3000. (d) Crump, C. J.; Johnson, D. S.; Li,
Y.-M. Target of γ-secretase modulators, presenilin marks the spot.
EMBO J. 2011, 30, 4696−4698. (e) Ebke, A.; Luebbers, T.; Fukumori,
A.; Shirotani, K.; Haass, C.; Baumann, K.; Steiner, H. Novel γ-secretase
enzyme modulators directly target presenilin protein. J. Biol. Chem.
2011, 286, 37181−37186. (f) Jumpertz, T.; Rennhack, A.; Ness, J.;
Baches, S.; Pietrzik, C. U.; Bulic, B.; Weggen, S. Presenilin is the
molecular target of acidic γ-secretase modulators in living cells. PLoS
ONE 2012, 7, e30484.
(17) 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. P.;
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.
(22) Hughes, J. D.; Blagg, J.; Price, D. A.; Bailey, S.; DeCrescenzo, G.
A.; Devraj, R. V.; Ellsworth, E.; Fobian, Y. M.; Gibbs, M. E.; Gilles, R.
W.; Greene, N.; Huang, E.; Krieger-Burke, T.; Loesel, J.; Wager, T.;
Whiteley, L.; Zhang, Y. Physiochemical drug properties associated with
in vivo toxicological outcomes. Bioorg. Med. Chem. Lett. 2008, 18,
4872−4875.
(23) Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M.
D.; Verhoest, P. R.; Villalobos, A.; Will, Y. Defining desirable central
nervous system drug space through the alignment of molecular
properties, in vitro ADME, and safety attributes. ACS Chem. Neurosci.
2010, 1, 420−434.
(24) Chrzanowska, M.; Rozwadowska, M. D. Asymmetric synthesis
of isoquinoline alkaloids. Chem. Rev. 2004, 104, 3341−3370.
(25) Hendrickson, J. B.; Rodríguez, C. An efficient synthesis of
substituted isoquinolines. J. Org. Chem. 1983, 48, 3344−3346.
(26) Stepan, A. F.; Karki, K.; McDonald, W. S.; Dorff, P. H.; Dutra, J.
K.; Dirico, K. J.; Won, A.; Subramanyam, C.; Efremov, I. V.;
O’Donnell, C. J.; Nolan, C. E.; Becker, S. L.; Pustilnik, L. R.; Sneed, B.;
Sun, H.; Lu, Y.; Robshaw, A. E.; Riddell, D.; O’Sullivan, T. J.; Sibley,
E.; Capetta, S.; Atchison, K.; Hallgren, A. J.; Miller, E.; Wood, A.;
Obach, R. S. Metabolism-directed design of oxetane-containing
arylsulfonamide derivatives as γ-secretase inhibitors. J. Med. Chem.
2011, 54, 7772−7783.
(18) For some alternative, recent examples, see (a) Oehlrich, D.;
Rombouts, F. J.; Berthelot, D.; Bischoff, F. P.; De Cleyn, M. A.;
Jaroskova, L.; Macdonald, G.; Mercken, M.; Surkyn, M.; Trabanco, A.
A.; Tresadern, G.; Van Brandt, S.; Velter, A. I.; Wu, T.; Gijsen, H. J.
Design and synthesis of bicyclic heterocycles as potent gamma-
secretase modulators. Bioorg. Med. Chem. Lett. 2013, 23, 4794−4800.
(b) Chen, J. J.; Qian, W.; Biswas, K.; Yuan, C.; Amegadzie, A.; Liu, Q.;
Nixey, T.; Zhu, J.; Ncube, M.; Rzasa, R. M.; Chavez, F., Jr.; Chen, N.;
Demorin, F.; Rumfelt, S.; Tegley, C. M.; Allen, J. R.; Hitchcock, S.;
Hungate, R.; Bartberger, M. D.; Zalameda, L.; Liu, Y.; McCarter, J. D.;
Zhang, J.; Zhu, L.; Babu-Khan, S.; Luo, Y.; Bradley, J.; Wen, P. H.;
Reid, D. L.; Koegler, F.; Dean, C., Jr.; Hickman, D.; Correll, T. L.;
Williamson, T.; Wood, S. Discovery of 2-methylpyridine-based biaryl
amides as gamma-secretase modulators for the treatment of
Alzheimer’s disease. Bioorg. Med. Chem. Lett. 2013, 23, 6447−6454.
(c) Bischoff, F.; Berthelot, D.; De Cleyn, M.; Macdonald, G.; Minne,
G.; Oehlrich, D.; Pieters, S.; Surkyn, M.; Trabanco, A. A.; Tresadern,
G.; Van Brandt, S.; Velter, I.; Zaja, M.; Borghys, H.; Masungi, C.;
Mercken, M.; Gijsen, H. J. Design and synthesis of a novel series of
bicyclic heterocycles as potent gamma-secretase modulators. J. Med.
Chem. 2012, 55, 9089−9106. (d) Gijsen, H. J.; Mercken, M. γ-
Secretase modulators: Can we combine potency with safety? Int. J.
Alzheimer’s Dis. 2012, 2012, 295207. (e) Sun, Z. Y.; Asberom, T.; Bara,
T.; Bennett, C.; Burnett, D.; Chu, I.; Clader, J.; Cohen-Williams, M.;
Cole, D.; Czarniecki, M.; Durkin, J.; Gallo, G.; Greenlee, W.; Josien,
H.; Huang, X.; Hyde, L.; Jones, N.; Kazakevich, I.; Li, H.; Liu, X.; Lee,
J.; Maccoss, M.; Mandal, M. B.; McCracken, T.; Nomeir, A.; Mazzola,
R.; Palani, A.; Parker, E. M.; Pissarnitski, D. A.; Qin, J.; Song, L.;
Terracina, G.; Vicarel, M.; Voigt, J.; Xu, R.; Zhang, L.; Zhang, Q.;
Zhao, Z.; Zhu, X.; Zhu, Z. Cyclic hydroxyamidines as amide isosteres:
Discovery of oxadiazolines and oxadiazines as potent and highly
efficacious γ-secretase modulators in vivo. J. Med. Chem. 2012, 55,
489−502. Also see ref 37. For comprehensive reviews, see ref 11.
(19) Values for clogP were calculated using the BIOBYTE (www.
biobyte.com) program clogP, version 4.3.
(27) Assay method adapted from published protocols: (a) Riley, R. J.;
McGinnity, D. F.; Austin, R. P. A unified model for predicting human
hepatic, metabolic clearance from in vitro intrinsic clearance data in
hepatocytes and microsomes. Drug Metab. Dispos. 2005, 33, 1304−
1311. (b) Obach, R. S. Prediction of human clearance of twenty-nine
drugs from hepatic microsomal intrinsic clearance data: An
examination of in vitro half-life approach and nonspecific binding to
microsomes. Drug Metab. Dispos. 1999, 27, 1350−1359.
(28) (a) Feng, B.; Mills, J. B.; Davidson, R. E.; Mireles, R. J.;
Janiszewski, J. S.; Troutman, M. D.; de Morais, S. M. In vitro P-
glycoprotein assays to predict the in vivo interactions of P-glycoprotein
with drugs in the central nervous system. Drug Metab. Dispos. 2008,
36, 268−275. (b) A compound with a MDR efflux ratio (MDR Er)
less than 2.5 is considered to have minimal P-gp efflux liability.
(29) Callegari, E.; Malhotra, B.; Bungay, P. J.; Webster, R.; Fenner, K.
S.; Kempshall, S.; LaPerle, J. L.; Michel, M. C.; Kay, G. G. A
comprehensive non-clinical evaluation of the CNS penetration
potential of antimuscarinic agents for the treatment of overactive
bladder. Br. J. Clin. Pharmacol. 2011, 72, 235−246.
(30) Parkinson, A.; Ogilvie, B. W. Biotransformation of Xenobiotics.
In Casarett & Doull’s Toxicology, The Basic Science of Poisons, 7th ed.;
Klaassen, C. D., Ed.; The McGraw-Hill Co., Inc.: New York, 2008;
Chapter 6, pp 161−304. Also available online: http://ilmufarmasis.
files.wordpress.com/2011/07/ casarett- and-doulls-toxicology-the-
basic-science-of-poisons7th-ed.pdf (accessed Oct 23, 2013).
(31) (a) Wai, J. S.; Kim, B.; Fisher, T. E.; Zhuang, L.; Embrey, M. W.;
Williams, P. D.; Staas, D. D.; Culberson, C.; Lyle, T. A.; Vacca, J. P.;
Hazuda, D. J.; Felock, P. J.; Schleif, W. A.; Gabryelski, L. J.; Jin, L.;
Chen, I.-W.; Ellis, J. D.; Mallai, R.; Young, S. D. Dihydroxypyridopyr-
azine-1,6-dione HIV-1 integrase inhibitors. Bioorg. Med. Chem. Lett.
2007, 17, 5595−5599. (b) Kawahara, N.; Nakajima, T.; Itoh, T.;
Ogura, H. A Synthesis of pyrido[1,2-a]quinoxalines and pyrido[1,2-
a]pyrazines. Heterocycles 1983, 20, 1721−1725.
(32) HATU: O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluro-
nium hexafluorophosphate.
(20) (a) Leeson, P. D.; Springthorpe, B. The influence of drug-like
concepts on decision-making in medicinal chemistry. Nat. Rev. Drug
Discovery 2007, 6, 881−890. (b) Ryckmans, T.; Edwards, M. P.;
Horne, V. A.; Correia, A. M.; Owen, D. R.; Thompson, L. R.; Tran, I.;
Tutt, M. F.; Young, T. Rapid assessment of a novel series of selective
CB2 agonists using parallel synthesis protocols: A lipophilic efficiency
(LipE) analysis. Bioorg. Med. Chem. Lett. 2009, 19, 4406−4409.
(21) Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A. Moving
beyond rules: The development of a central nervous system
multiparameter optimization (CNS MPO) approach to enable
alignment of druglike properties. ACS Chem. Neurosci. 2010, 1,
435−449.
(33) Tran, T. P.; Mullins, P. B.; am Ende, C. W.; Pettersson, M.
Synthesis of pyridopyrazine-1,6-diones from 6-hydroxypicolinic acids
via a one-pot coupling/cyclization reaction. Org. Lett. 2013, 15, 642−
645.
(34) am Ende, C. W.; Johnson, D. S.; O’Donnell, C. J.; Pettersson,
M. Y.; Subramanyam, C. Preparation of heteroaryl imidazoles and
heteroaryl triazoles as γ-secretase modulators. WO2011048525A1,
2011.
(35) Assay method adapted from published protocol: Hay, T.; Jones,
R.; Beaumont, K.; Kemp, M. Modulation of the partition coefficient
between octanol and buffer at pH 7.4 and pKa to achieve the optimum
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Journal of Medicinal Chemistry
Article
balance of blood clearance and volume of distribution for a series of
tetrahydropyran histamine type 3 receptor antagonists. Drug Metab.
Dispos. 2009, 37, 1864−1870.
(36) Solubility was measured by Analiza, Inc., Cleveland, OH.
(http://analiza.com/physchem/logd.html#qf.
(37) Borgegard, T.; Jureus, A.; Olsson, F.; Rosqvist, S.; Sabirsh, A.;
Rotticci, D.; Paulsen, K.; Klintenberg, R.; Yan, H.; Waldman, M.;
Stromberg, K.; Nord, J.; Johansson, J.; Regner, A.; Parpal, S.;
Malinowsky, D.; Radesater, A. C.; Li, T.; Singh, R.; Eriksson, H.;
Lundkvist, J. First and second generation gamma-secretase modulators
(gsms) modulate amyloid-beta (abeta) peptide production through
different mechanisms. J. Biol. Chem. 2012, 287, 11810−11819.
(38) Lapinsky, D. J. Tandem photoaffinity labeling-bioorthogonal
conjugation in medicinal chemistry. Bioorg. Med. Chem. 2012, 20,
6237−6247.
(39) GSM 54 refers to a proprietary GSM containing the same
pyridone imidazole core as GSM 52, but a structurally distinct right-
hand fragment attached to the lactam nitrogen.
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