Design, optimization, and biological evaluation of novel keto-benzimidazoles as potent and selective inhibitors of phosphodiesterase 10A (PDE10A)

Article abstract of DOI:10.1021/jm401234w

Our development of PDE10A inhibitors began with an HTS screening hit (1) that exhibited both high p-glycoprotein (P-gp) efflux ratios in rat and human and poor metabolic stability. On the basis of cocrystal structure of 1 in human PDE10A enzyme, we designed a novel keto-benzimidazole 26 with comparable PDE10A potency devoid of efflux liabilities. On target in vivo coverage of PDE10A in rat brain was assessed using our previously reported LC-MS/MS receptor occupancy (RO) technology. Compound 26 achieved 55% RO of PDE10A at 30 mg/kg po and covered PDE10A receptors in rat brain in a dose-dependent manner. Cocrystal structure of 26 in PDE10A confirmed the binding mode of the novel scaffold. Further optimization resulted in the identification of keto-benzimidazole 34, which showed an increased in vivo efficacy of 57% RO in rats at 10 mg/kg po and an improved in vivo rat clearance and oral bioavailability.

Full text of DOI:10.1021/jm401234w

Article
pubs.acs.org/jmc
Design, Optimization, and Biological Evaluation of Novel Keto-
Benzimidazoles as Potent and Selective Inhibitors of
Phosphodiesterase 10A (PDE10A)
Essa Hu,*,† Roxanne K. Kunz,† Ning Chen,† Shannon Rumfelt,† Aaron Siegmund,† Kristin Andrews,‡
Samer Chmait,‡ Sharon Zhao,‡ Carl Davis,§ Hang Chen,⊥ Dianna Lester-Zeiner,⊥ Ji Ma,∥
Christopher Biorn,⊥ Jianxia Shi,∥ Amy Porter,⊥ James Treanor,# and Jennifer R. Allen†
†Department of Medicinal Chemistry, ‡Department of Molecular Structure, §Department of Pharmacokinetics and Drug Metabolism,
∥Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States
#
⊥Department of Neuroscience and Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard,
South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: Our development of PDE10A inhibitors began
with an HTS screening hit (1) that exhibited both high p-
glycoprotein (P-gp) efflux ratios in rat and human and poor
metabolic stability. On the basis of cocrystal structure of 1 in
human PDE10A enzyme, we designed a novel keto-
benzimidazole 26 with comparable PDE10A potency devoid
of efflux liabilities. On target in vivo coverage of PDE10A in rat
brain was assessed using our previously reported LC-MS/MS
receptor occupancy (RO) technology. Compound 26 achieved
55% RO of PDE10A at 30 mg/kg po and covered PDE10A receptors in rat brain in a dose-dependent manner. Cocrystal
structure of 26 in PDE10A confirmed the binding mode of the novel scaffold. Further optimization resulted in the identification
of keto-benzimidazole 34, which showed an increased in vivo efficacy of 57% RO in rats at 10 mg/kg po and an improved in vivo
rat clearance and oral bioavailability.
have the additional benefit of addressing multiple symptoms of
schizophrenia unlike the current SOC. On the basis of these
compelling biological findings, we sought to design potent,
efficacious, and selective compounds to inhibit PDE10A in the
striatum.
Traditionally, measuring drug levels in the brain homogenate
or cerebrospinal fluid (CSF) has been used to assess target
coverage in the central nervous system (CNS). However,
because these levels could not distinguish specific from
nonspecific binding in the brain, this approach could not
provide definitive proof of an on-target effect. We recently
described the development of a PDE10A tracer, AMG-7980, to
enable measurement of PDE10A receptor occupancy (RO) in a
rat brain using in vivo LC-MS/MS technology.15 In this report,
we used this technology to evaluate the effectiveness of target
coverage of drug candidates and successfully identified several
potent in vivo inhibitors of PDE10A in the brain.
INTRODUCTION
■
A significant unmet medical need still exists in the treatment of
schizophrenia. The current standard of care (SOC) does not
address all three major symptoms of schizophrenia (positive,
negative, and cognitive symptoms). While a subpopulation of
patients remains unresponsive to SOC, others struggle with
intolerable side effects.1−3 As the research community searches
for new approaches and mechanisms to treat schizophrenia,4−9
inhibition of phosphodiesterase 10A (PDE10A) has emerged as
a potential novel solution to address this unmet medical need.
Unlike most atypical antipsychotics which directly target
dopamine receptors, PDE10A regulates the levels of second
messenger cyclic adenosine monophosphate (cAMP) and cyclic
guanosine monophosphate (cGMP) in the signaling pathway
downstream of the dopamine receptors.10 The PDE10A
enzyme is also highly expressed in the medium spiney neuron
of the striatum, the section of the brain most associated with
schizophrenia.11,12 These attributes have led to the hypothesis
that PDE10A modulators could offer an efficacious treatment of
schizophrenia devoid of the undesired side effects of current
SOC because they would not directly interact with the
dopaminergic receptors. In addition, PDE10A is also reported
to be downstream of the glutamine pathway, which has been
postulated to be responsible for the cognitive deficits of
schizophrenic patients.13,14 Thus, inhibition of PDE10A could
An internal HTS screening campaign revealed compound 1
as a potent hit (IC50 = 9.7 nM) with acceptable average
permeability in LLCK-PK1 cells of 18.1 μcm/s (Figure 1).
However, the compound also exhibited several undesirable
attributes: (1) sub-μM off-target kinase activities against Aurora
Received: August 8, 2013
Published: October 8, 2013
© 2013 American Chemical Society
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remaining chlorine atom on pyrazine 15 with morpholine
produced pyrazine aminopyridine 16.
Synthesis of keto-benzthiazole 22 began with alkylation of
methyl 4-hydroxybenzoate 17 with benzyl bromide to produce
methyl 4-(benzyloxy)benzoate 18 (Scheme 4). Nucleophilic
addition of lithium benzothiolate afforded keto-benzthiazole 19.
Deprotection with boron trifluoride revealed phenol 20.
Chloropyrazine keto-benzthiazole 21 was prepared via SNAr
displacement of phenol 20 with dichloropyrazine 2a. Heating
with morpholine resulted in formation of benzo[d]thiazol-2-
yl(4-((3-morpholinopyrazin-2-yl)oxy)phenyl)methanone 22.
Preparation of keto-benzimidazoles 26 and 27 started with
SNAr reaction of dichloropyrazine 2a and ethyl 4-hydrox-
ybenzoate 23 (Scheme 5). A one-pot, two-step reaction formed
(1H-benzo[d]imidazol-2-yl)(4-((3-chloropyrazin-2-yl)oxy)-
phenyl)methanone 25. The reaction sequence began with acid
catalyzed cyclization of benzene-1,2-diamine to form 1H-
benzo[d]imidazole. LDA was then added to the reaction
mixture to enable nucleophilic addition of 1H-benzo[d]-
imidazole to ethyl 4-((3-chloropyrazin-2-yl)oxy)benzoate 24
to produce 25. Synthesis of (1H-benzo[d]imidazol-2-yl)(4-((3-
morpholinopyrazin-2-yl)oxy)phenyl)methanone 26 was accom-
plished by SNAr displacement of chloropyrazine 25 with
morpholine. Alkylation of imidazole amine with iodomethane
produced (1-methyl-1H-benzo[d]imidazol-2-yl)(4-((3-mor-
pholinopyrazin-2-yl)oxy)phenyl)methanone 27. Keto-benzimi-
dazoles 28−37 were synthesized in similar SNAr reactions by
combining various cyclic amines with chloropyrazine keto-
benzimidazole intermediate 25 (Scheme 6).
Figure 1. Initial HTS screening hit 1.
1 and Aurora 2 kinases, high efflux ratio (ER) of 24.8 in human
and 13.1 in rat, and modest to poor in vitro liver microsomal
clearance in human (HLM = 127 μL/min/mg) and rat (RLM =
297 μL/min/mg). Because we were targeting the PDE10A
receptors in the brain, high efflux ratios could significantly
impair a drug’s ability to penetrate the blood−brain barrier
(BBB).16 Furthermore, excessive systemic exposure needed to
compensate for the poor CNS penetration would pose an
unnecessary drug load burden on the patients. Thus, we
decided to address the efflux issue first.
CHEMISTRY
■
As shown in Scheme 1, syntheses of aminobenzimidazoles 5
and 7 began with a SNAr displacement of 4-aminophenol (3)
with fluoropyridine (2a) or dichloropyrazine (2b). Amino-
benzimidazoles (5, 6) were formed by cyclization with di-2-
pyridyl-thionocarbonate or thiophosgene and benzene-1,2-
diamine. SNAr displacement of chloropyrazine benzimidazole
6 with morpholine afforded 7.
Synthesis of N-(4-((3-morpholinopyrazin-2-yl)oxy)phenyl)-
benzo[d]thiazol-2-amine (10) began with coupling of morpho-
line with dichloropyrazine (2a) to form 4-(3-chloropyrazin-2-
yl)morpholine (8) (Scheme 2). SNAr displacement of pyrazine
chloride (8) with 4-aminophenol (3) produced 4-((3-
morpholinopyrazin-2-yl)oxy)aniline (9). Palladium catalyzed
heteroaryl coupling with 2-bromobenzo[d]thiazole gave desired
analog 10.
As shown in Scheme 3, synthesis of N-(4-((3-morpholino-
pyrazin-2-yl)oxy)phenyl)pyridin-2-amine 16 began with Buch-
wald coupling of 1-bromo-4-methoxybenzene (11) to pyridin-
2-amine (12) to afford the diaryl-aniline core (13). Cleavage of
the methyl ether by boron tribromide revealed phenol 14.
Sequential SNAr displacement of dichloropyrazine 2b with
phenol 14, followed by a second SNAr reaction to replace the
RESULTS AND DISCUSSION
■
A cocrystal structure of screening hit 1 in human PDE10A
enzyme’s catalytic domain was first obtained to help elucidate
critical binding interactions (Figure 2). The internal pyridine
ring appeared to be held in place by π-stacking interactions with
Ph719 and Ph686. The nitrogen atom on the internal pyridine
ring was further anchored by hydrogen bonding interactions
with Gln716. A second hydrogen bonding interaction between
one of the nitrogens on the benzimidazole ring and Tyr683
induced a coplanar orientation of benzimidazole ring relative to
the middle phenyl ring. A water molecule was resolved
coordinating the benzimidazole N−H bond, while the N−H
bond on the amino linker appeared to be oriented toward the
solvent exposed region. Thus, neither N−H bonds seemed to
a
Scheme 1. Synthesis of Amino Phenyl Ether Analogs 5 and 7
a
Reagents and conditions: (a) cesium carbonate, DMSO, 80 °C, 16 h, 94% yield for 4a; (b) method A, (i) di-2-pyridyl-thionocarbonate, DCM,
room temperature, 16 h, 18% yield for 5; method B, (i) thiophosgene, sodium carbonate, room temperature CHCl3, (ii) DCC, benzene-1,2-diamine,
THF, 75 °C, 78% yield for 6; (c) morpholine, DMSO, 80 °C, 16 h, 34% yield.
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a
Scheme 2. Synthesis of Amino Phenyl Ether Analog 10
a
Reagents and conditions: (a) morpholine, DMSO, 120 °C, 16 h, 40% yield; (b) cesium carbonate, DMSO, 80 °C, 16 h; (c) Pd2(dba)3, S-phos,
sodium tert-butoxide, 2-bromobenzo[d]thiazole, toluene, 80 °C, 5% yield.
a
Scheme 3. Synthesis of Aminopyridine Analog 16
a
Reagents and conditions: (a) Pd2(dba)3, di-tert-butyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine, sodium tert-butoxide, toluene, 100 °C, 3 h; (b)
BBr3, DCM, −78 °C to room temperature, 3 h, 70% yield; (c) cesium carbonate, DMF, 100 °C, 16 h, 72% yield; (d) morpholine, DMSO, 70 °C, 16
h, 80% yield.
a
Scheme 4. Synthesis of Keto-benzimidazole Analog 22
a
Reagents and conditions: (a) potassium carbonate, benzyl bromide, DMF, room temperature, 8 h, 89% yield; (b) benzothiazole, LDA, −70 °C, 2 h,
46% yield; (c) boron trifluoride diethyl etherate, dimethyl sulfide, DCM, room temperature 72 h, 72% yield; (d) cesium carbonate, DMSO, 90 °C, 6
h, 52% yield; (e) morpholine, DMSO, 100 °C, 16 h, 57% yield.
have a critical function in the binding to the catalytic domain of
PDE10A.
PDE10A potency improved by 9-fold (IC50 = 1.1 nM) and the
off-target Aurora 1 and 2 kinase activities of screening hit 1
were completely eliminated. Thus, we turned our attention to
the rest of the scaffold to address the efflux issue while keeping
the morpholine functionality in hopes of retaining its beneficial
effects.
We recently reported that replacement of the N−H proton
on a benzimidazole containing scaffold significantly lowered its
efflux ratio.17 Because our cocrystal structure of 1 in PDE10A
enzyme seemed to suggest the benzimidazole N−H on 1 was
not critical for binding, we decided to apply this strategy and
replaced benzimidazole with benzthiazole (10) and pyridine
(16). Indeed, compound 10 produced acceptable efflux ratios
of 2.4 in human and 2.1 rat. This modification also retained
comparable potency against PDE10A (IC50 = 4.3 nM).
However, its in vitro rat clearance remained high. Efflux ratios
of pyridine 16 also improved significantly to 3.7 in human and
2.6 in rat. Nevertheless, the pyridyl replacement also decreased
To eliminate the high efflux ratio, we adopted a systematic
approach to investigate the structure−activity relationship of
this new scaffold while retaining the key interactions observed
in the PDE10A cocrystal structure (Table 1). The truncated
substructure 5 was synthesized to assess the impact of the
methyl-amino-pyridine functionality on screening hit 1.
Interestingly, without the methyl-amino-pyridine functionality
and the two fluorine atoms on 1, pyridine ether 5 only lost its
PDE10A inhibitory activity by 5-fold while its efflux ratio
improved significantly to 4.9 in rat and 4.7 in human.
Furthermore, the pyridine ring could also be replaced with a
pyrimidine ring with little impact on potency (data not shown).
Substitution of the pyridine ring on 1 with morpholine
produced 7, which resulted in a decrease of the efflux ratios
to 11.1 in human but an increase of efflux ratio to 32.8 in rat.
This modification, however, brought two improvements: its
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a
Scheme 5. Synthesis of Keto-benzimidazoles 26 and 27
a
Reagents and conditions: (a) cesium carbonate, DMSO, 80 °C, 16 h, 79% yield; (b) (i) benzene-1,2-diamine, triethoxymethane, benzenesulfonic
acid, toluene, reflux, (ii) LDA, −78 °C→ 23 °C, 41% yield; (c) morpholine, DMSO, 80 °C, 16 h, 59% yield; (d) cesium carbonate, iodomethane,
DMF, room temperature, overnight, 75% yield.
a
μL/min/mg) in both species. N-Methylated benzimidazole 27
resulted in a 3-fold loss in potency, increased efflux ratio, and in
vitro clearance compared to 26. It is worth noting that there
appeared to be a trend correlating cLogP with low efflux ratio.
Of the five compounds with cLogP values less than 4
(compounds 7, 16, 22, 26, and 27), four exhibited efflux ratios
of 4 or less in both species.
Scheme 6. Synthesis of Keto-benzimidazoles 28−37
To confirm our design hypothesis, a second cocrystal
structure of keto-benzimidazole 26 in human PDE10A enzyme
was obtained (Figure 3). The cocrystal structure confirmed that
the new keto-benzimidazole scaffold maintained the coplanarity
of middle phenyl ring and benzimidazole ring. Essential
hydrogen bonding interactions (pyrimidine nitrogen with
Gln716, benzimidazole nitrogen with Tyr683) and π-stacking
interactions (pyrimidine with Ph719 and Phe686) previously
observed in cocrystal structure of 1 in PDE10A enzyme (Figure
2) were also retained.
a
Reagents and conditions: (a) cyclic amine, DMSO, 80 °C, 16 h.
To assess whether our SAR efforts were on track, compound
26 was advanced into the LC-MS/MS RO assay recently
reported by our group to assess its CNS target coverage in
vivo.1 Using our reported PDE10A tracer AMG-7980, we were
gratified to see that keto-benzimidazole 26 produced 21% RO
at 10 mg/kg and 55% RO at 30 mg/kg. Compared to high in
vivo rat clearance of screening hit 1 (3.90 L/h/kg), compound
26 achieved modest clearance of 0.53 L/h/kg and good T1/2
(3.19 h), albeit a low oral bioavailability (10%F). Dose
dependent occupancy of CNS PDE10A receptors by 26 was
also evaluated using LC-MS/MS RO dose response assay at 0.3,
1, 3, 10, and 30 mg/kg doses. Poor oral bioavailability of 26 was
circumvented by dosing the compound ip instead. Occupancy
measurements were taken 30 min post dosing. As shown in
Figure 4, keto-benzimidazole 26 exhibited an increase in
PDE10A occupancy in the rat brain in vivo in a dose dependent
manner. This in vivo validation of the keto-benzimidazole
scaffold provided the critical data we needed to continue to
optimize lead compound 26.
To address the poor PKDM profile of 26, we initiated an
investigation of structure−activity relationship of the morpho-
line region. To further expedite our discovery timeline, our LC-
MS/MS RO technology platform was set up to handle multiple
compounds a week with short turnaround time. This enabled
us to advance compounds that exhibited acceptable in vitro
PDE10A inhibitory activity and in vitro metabolic stability
directly into the LC-MS/MS RO studies instead of the
Figure 2. Cocrystal structure of 1 in human PDE10A enzyme.
the PDE10A inhibitory activity of 16 by more than 30-fold
compared to 7.
Because the cocrystal structure of 1 in PDE10A suggested
that the N−H bond on the amine linker was not essential, we
replaced the amine linker on 7 and 10 with a carbonyl group
and synthesized 22 and 26 to maintain the planarity of the
phenyl and the benzimidazole ring observed in the cocrystal
structure. In several aspects, ketone replacement was
detrimental to the benzthiazole analog. Compared to amino-
benzthiazole 10, keto-benzthiazole 22 was more than 30-fold
less potent with higher in vitro microsomal clearance in human
and rat. Keto-benzimidazole 26, on the other hand, showed just
slightly reduced PDE10A potency compared to its amino-
benzimidazole analog 7. Unlike all the previous analogs, keto-
benzimidazole 26 was the first compound to exhibit both low
efflux ratio (human ER = 0.9; rat ER = 1.0) and low in vitro
microsomal clearance (HLM = 55 μL/min/mg; RLM = 178
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Table 1. Structural Modifications to Reduce Efflux Ratio of Screening Hit 1
a
Each IC50 value reported is an average of at least two independent experiments with 22 point dose response curve in duplicates at each
b
concentration. Efflux ratio reported is based on the ratio of the basolateral to apical permeability over apical to basolateral permeability of a
c
compound. MDR1−Madin−Darby canine kidney (MDCK) cells were used to measure the compound’s apparent permeability in each direction. In
vitro microsomal stability studies were conducted in the presence of NADPH at 37 °C for 30 min at a final compound concentration of 1 μM of
compound.
Figure 4. Dose dependent increase in PDE10A receptor occupancy in
CNS observed for keto-benzimidazole 26.
Figure 3. Cocrystal structure of 26 in human PDE10A enzyme.
significantly reduced the number of in vivo rat PK studies we
needed to support the project advancement.
A variety of N-linked rings were synthesized to examine the
effects of electronics and ring sizes on the in vitro metabolic
stability of the keto-benzimidazole scaffold (Table 2).
traditional order of rat PK before in vivo PD studies. Using LC-
MS/MS RO technology to filter out compounds also
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with an alcohol (32) did not impact PDE10A activity but
slightly increased RLM. Interestingly, insertion of a carbonyl
group α to the distal piperidine nitrogen (33) increased its
Table 2. Modification of the Morpholine Ring
inhibitory activity against PDE10A to single digit nM (IC50
=
1.7 nM) compared to its N-methyl-piperazine analog 30. The
polar functional group also restored in vitro clearance in both
species (HLM = 20 μL/min/mg; RLM = 122 μL/min/mg).
This improvement in binding affinity was likely due to the
carbonyl group making a hydrogen bonding interaction with
one of the water molecules occupying the solvated metal
binding pocket in the PDE10A catalytic domain.18 To further
exploit this finding, we proceeded to install various oxygen
containing polar groups on the N-linked six-membered ring. N-
Acetyl piperazine 34 did not experience the potency increase
observed with 33 even though its HLM and RLM profiles
improved slightly. Compared to the secondary alcohol on
piperidinol 32, tertiary alcohol (35) was similarly potent but
exhibited significantly higher in vitro clearance in rat. Changing
the electronic property of the alcohol by replacing one of the
gem-dimethyl groups with a trifluoromethyl group (36) resulted
in 8-fold loss in activity against PDE10A.
On the basis of their respective in vitro PDE10A inhibitory
potency and in vitro metabolic stability in both human and rat,
compounds 32, 33, and 34 were advanced into the LC-MS/MS
RO assays (Table 3). Piperidinol 32 was more efficacious in
Table 3. Receptor Occupancy Measurements of Keto-
benzimidazoles 26, 32, 33, and 34 Using ex Vivo and LC-
MS/MS Methods
a
compd no.
LC-MS/MS receptor occupancy (%)
21.3
26
b
55.3
32
33
34
39.4
0
57.1
a
Receptor occupancy measurements were taken 1 h post po dosing of
b
compounds at 10 mg/kg. 1 h post po dosing of compound at 30 mg/
kg.
vivo than 26, exhibiting 39% RO when dosed at 10 mg/kg po.
Piperazinone 33 was not able to cover the PDE10A receptors in
the rat brain (RO = 0% at 10 mg/kg). Acetyl piperidine 34,
which had comparable PDE10A potency as 26 but reduced in
vitro clearance in rat, produced an improved in vivo occupancy
of 57% RO at 10 mg/kg po in the LC-MS/MS RO assay.
Compared to high in vivo rat clearance of screening hit 1
(3.90 L/h/kg) and the poor oral bioavailability of compound
26 (10%F), keto-benzimidazoles 32 and 34 exhibited improved
in vivo rat PK profiles (Table 4). Compound 32 afforded
improved oral bioavailability of 52% even though it was inferior
to 26 in terms of in vivo clearance (1.10 L/h/kg) and T1/2
(1.96 h). Meanwhile, compound 34 showed the best overall
improvement with a low in vivo clearance of 0.074 L/h/kg,
good T1/2 of 3.9 h, and acceptable oral bioavailability of 56%.
a
Each IC50 value reported is an average of at least two independent
experiments with 22 point dose response curve in duplicates at each
concentration. In vitro microsomal stability studies were conducted
in the presence of NADPH at 37 °C for 30 min at final compound
concentration of 1 μM of compound.
b
Compared to morpholine 26, piperidine 28, with one atom
difference, showed comparable potency but increased in vitro
microsomal clearance in human and rat. Increasing the ring size
(29) did not impact PDE10A potency but significantly
increased RLM. N-Methyl piperazine 30 exhibited a modest
4-fold decrease in activity while its in vitro metabolic stability
increased slightly in both human and rat. 4-Methoxy piperidine
(31) showed comparable potency to morpholine 26 but higher
in vitro clearance in both species. Replacing the methyl ether
CONCLUSION
■
HTS screening hit 1 was identified as a potent inhibitor of
PDE10A (IC50 = 9.7 nM). The molecule displayed high efflux
ratios in both human and rat, as well as high in vitro and in vivo
clearance in rat and off-target kinase activities. Cocrystal
structure of 1 in human PDE10A catalytic domain revealed two
key interactions: binding of pyridine nitrogen to the conserved
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a
Table 4. In Vivo Rat PK of Compounds 1, 26, 32, and 34
b
compd no.
route
dose (mg/kg)
Cl (L/h/kg)
Vss (L/kg)
AUC (μM·h)
T1/2 (h)
%F
1
iv
1
2
3.90
0.53
2.52
1.35
0.58
9.32
0.81
2.63
3.19
20.4
4.21
1.96
1.65
3.90
26
iv
po
ip
iv
5
4.35
10
10
2
1.40
1.10
27.6
1.28
12.10
4.50
32
34
po
iv
10
2
11.70
62.10
79.2
52
56
0.074
0.165
po
5
a
b
Study in fed male Sprague−Dawley rats. Vehicles used: 100% DMSO for iv; 1% Tween, 2% HPMC with methanesulfonic acid, pH 2.2, for po and
ip.
3.5 min, 95% B; 3.5−3.51 min, 5% B. Flow from UV detector was split
(50:50) to the MS detector, which was configured with APIES as
ionizable source. All high resolution mass spectrometry (HRMS) data
were acquired on a Synapt G2 HDMS instrument (Waters
Corporation, Manchester, UK) operated in positive electrospray
ionization mode. The sample was diluted to 10 μM in 50% acetonitrile
(v/v), 0.1% formic acid (v/v), and infused into the mass spectrometer
at a flow rate of 5 μL/min through an electrospray ionization source
operated with a capillary voltage of 3 kV. The sample cone was
operated at 30 V. The time-of-flight analyzer was operated at a
resolution (fwhm) of 30000 at m/z 785 and was calibrated over the m/
z range 50−1200 using a 1 μM sodium iodide (NaI) solution (50% v/
v acetonitrile solution). To obtain accurate mass measurements, an
internal lock-mass correction was applied using leucine enkephalin
(m/z 556.2771). Collision induced fragmentation (CID) was
performed using an injection voltage of 28 V. Instrument control
was performed through the software suite MassLynx, version 4.1.
4-(Pyridin-2-yloxy)benzenamine (4a). To a solution of 3 (545 mg,
5.0 mmol) in DMSO (8 mL) was added cesium carbonate (1.95 g, 6.0
mmol) and 2a (0.43 mL, 5.0 mmol). The resulting mixture was heated
in an 80 °C oil bath for 16 h. After cooling to RT, the reaction mixture
was diluted with DCM and washed with water and brine several times
to remove DMSO. The organic layer was dried (Na2SO4) and
concentrated to give 4a (875 mg, 94% yield) as a tan solid. LC-MS m/
z [M + H] 187.2.
4-(3-Chloropyrazin-2-yloxy)benzenamine (4b). A mixture of 2b
(470 mg, 3.16 mmol) with 3 (344 mg, 3.16 mmol) and cesium
carbonate (1233 mg, 3.79 mmol) in DMSO and was heated to 70 °C
for 3 h. The mixture was cooled to room temperature then transferred
to a separatory funnel rinsing with ethyl acetate and water. The layers
were separated, and the aqueous layer was back extracted with ethyl
acetate (3×). The combined organic layers were rinsed with brine,
dried over Na2SO4, filtered, and concentrated to produce 4b as a solid
(620 mg, 88% yield), which was carried forward without further
purification. LC-MS m/z [M + H] 222.0.
Gln716 and binding of imidazole nitrogen to Tyr683. While
maintaining these two essential hydrogen bonding interactions,
we systematically examined the remainder of the scaffold and
identified keto-benzimidazole 26 as a promising new scaffold.
The key bonding interactions and binding conformation of this
new keto-benzimidazole scaffold was confirmed with a second
cocrystal structure of 26 in human PDE10A. For proof of
concept, keto-benzimidazole 26 with nM PDE10A potency and
modest in vitro rat microsomal clearance was advanced into an
in vivo LC-MS/MS RO studies and showed a promising 55%
RO at 30 mg/kg by po dosing. Additional LC-MS/MS RO
dose response study further demonstrated that 26 was able to
cover the PDE10A receptors in a dose dependent manner.
Modifications of the morpholine region of the scaffold to
optimize metabolic stability led to identification of compound
34. Compared to lead compound 26, keto-benzimidazole 34
possessed a superior in vivo efficacy, achieving 57% RO at 10
mg/kg po in the LC-MS/MS RO assay. In addition, 34
exhibited improved in vivo rat clearance and oral bioavailability.
Both compounds 26 and 34 were highly selective for PDE10A,
exhibiting 7−30 μM inhibitory activities against the remaining
PDE isoforms (PDE1, 2, 3, 4, 5, 6, 7, 8, 9, and 11). Our work to
further increase the in vivo efficacy of these keto-benzimidazole
PDE10A inhibitors will be reported in a future communication.
EXPERIMENTAL METHODS
■
Chemistry, Materials, and General Methods. Unless otherwise
noted, all reagents and solvents were obtained from commercial
suppliers such as Aldrich, Sigma, Fluka, Acros, EDM Sciences, etc., and
used without further purification. Dry organic solvents (dichloro-
methane, acetonitrile, DMF, etc.) were purchased from Aldrich
packaged under nitrogen in Sure/Seal bottles. All reactions involving
air or moisture sensitive reagents were performed under a nitrogen or
argon atmosphere. Silica gel chromatography was performed using
prepacked silica gel cartridges (Biotage or RediSep). Microwave
assisted reactions were performed in Biotage Initiator Sixty microwave
N-(4-(Pyridin-2-yloxy)phenyl)-1H-benzo[d]imidazol-2-amine (5).
In a round-bottomed flask were added 4a (186 mg, 1.0 mmol), di-
2-pyridyl thionocarbonate (244 mg, 1.05 mmol), and DCM (5 mL).
The reaction mixture was stirred at RT for 16 h. The reaction mixture
was partitioned between DCM and water. The aqueous layer was
extracted with DCM, and the combined organics were washed with
brine, dried (Na2SO4), and concentrated. The crude material was
dissolved in THF (20 mL), to which was added 1,2-phenylenediamine
(162 mg, 1.5 mmol) and PS-carbodiimide resin (Argonaut, 0.88
mmol/g, 3.2 g, 2.85 mmol). The reaction was heated at 70 °C for 4 h,
cooled to RT, filtered, and concentrated. The crude product was
dissolved in DCM and chromatographed through a Redi-Sep
prepacked silica gel column (40 g), eluting with a gradient of 10−
100% EtOAc in hexane, to provide 5 (55 mg, 18% yield) as off-white
1
reactor. H NMR spectra were recorded on a Bruker DRX 300 MHz,
Bruker DRX 400 MHz, Bruker AV 400 MHz, Varian 300 MHz, or a
Varian 400 MHz spectrometer at ambient temperature. Chemical
shifts are reported in parts per million (ppm, δ units). Data are
reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling
constants, and number of protons. Reactions were monitored using
Agilent 1100 series LC/MSD SL high performance liquid chromatog-
raphy (HPLC) systems with UV detection at 254 nm and a low
resonance electrospray mode (ESI). All final compounds were purified
to >95% purity, as determined by high performance liquid
chromatography (HPLC). HPLC methods used the following: Agilent
1100 spectrometer, Zorbax SB-C18 column (50 mm × 3.0 mm, 3.5
μm) at 40 °C with a 1.5 mL/min flow rate; solvent A of 0.1% TFA in
water, solvent B of 0.1% TFA in MeCN; 0.0−3.0 min, 5−95% B; 3.0−
1
solid. H NMR (400 MHz, CDCl3) δ ppm 6.71 (s, 1 H), 6.96 (d, J =
8.53 Hz, 1 H), 7.00−7.06 (m, 1 H), 7.08−7.16 (m, 4 H), 7.28−7.42
(m, 4 H), 7.72 (t, J = 7.78 Hz, 1 H), 8.19 (d, J = 4.52 Hz, 1 H). LC-
MS m/z [M + H] 303.2.
N-(4-(3-Chloropyrazin-2-yloxy)phenyl)-1H-benzo[d]imidazol-2-
amine (6). To a mixture of 4b (620 mg, 2.80 mmol) and sodium
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carbonate (652 mg, 6.15 mmol) in chloroform was added
thiophosgene (0.24 mL, 3.08 mmol). The solution turned cloudy
and was stirred overnight at room temperature. Reaction mixture was
filtered, and the filtrate was concentrated by rotovap to give a brown
solid.
dried over sodium sulfate and concentrated. Purification by column
chromatography (15% ethyl acetate:hexane) produced 13. H NMR
(DMSO-d6): δ ppm 8.076 (d, 1H), 7.543 (d, 2H), 7.409 (m, 1H),
6.865 (d, 2H), 6.727 (d, 1H), 6.665 (m, 1H), 3.708 (s, 3H).
1
4-(Pyridin-2-ylamino)phenol (14). A solution of 13 (3.0 g, 14.9
mmol) in DCM (50 mL) was cooled to −78 °C before dropwise
addition of BBr3 (4.5 mL, 44.1 mmol, 3.0 equiv) under nitrogen
atmosphere over 30 min. The temperature was maintained at −78 °C
for 1 h before allowing to warm to room temperature over 2 h.
Reaction was cooled to 0 °C and quenched with saturated sodium
bicarbonate solution. The resulting mixture was stirred for 30 min and
extracted by ethyl acetate (200 × 3 mL). The combined organic
extracts were dried over sodium sulfate and concentrate to give 14
A mixture of 2-chloro-3-(4-isothiocyanatophenoxy)pyrazine (190
mg, 0.72 mmol), benzene-1,2-diamine (93.5 mg, 0.87 mmol), and
N,N′-dicyclohexylcarbodiimide (223 mg, 1.08 mmol) in THF was
heated at 75 °C for 1.5 h. The reaction mixture was cooled to room
temperature, diluted with DCM, and absorbed onto silica gel for
purification. Gradient elution with 20−100% EtOAc/hexanes afforded
6 as a solid (191 mg, 78% yield). LC-MS m/z [M + H] 338.0.
N-(4-(3-Morpholinopyrazin-2-yloxy)phenyl)-1H-benzo[d]-
imidazol-2-amine (7). A mixture of 6 (191 mg, 0.57 mmol) and
morpholine (0.074 mL, 0.85 mmol) in DMSO (2 mL) was heated to
80 °C overnight. The hot solution was then poured onto ice water,
which caused a brown solid to precipitate. The solid was collected by
1
(1.95 g, 70.0% yield), which was used directly for next step. H NMR
(DMSO-d6): δ ppm 9.114 (s, 1H), 8.205 (d, 2H), 8.171 (d, 1H), 7.755
(d, 2H), 7.592 (t, 1H), 7.154 (d, 2H), 6.840 (d, 1H), 6.767(t, 1H).
N-(4-(3-Chloropyrazin-2-yloxy)phenyl)pyridin-2-amine (15). A
solution of 14 (10 g, 51 mmol) and 2b (7.75 g, 81 mmol, 1.4
equiv) in DMF (100 mL) was added cesium carbonate (34.5 g, 106
mmol, 2 equiv) and heated to 100 °C overnight. Reaction mixture was
concentrated under vacuum and precipitated with water. The mixture
was stirred for 1 h and filtered to afford a brown solid. The solid was
1
filtration, washed, and dried to give 7 (75 mg, 34% yield). H NMR
(300 MHz, DMSO-d6) δ ppm 3.47−3.59 (m, 4 H), 3.69−3.83 (m, 4
H), 6.89−7.06 (m, 2 H), 7.12 (m, J = 8.92 Hz, 2 H), 7.22−7.39 (m, 2
H), 7.55 (d, J = 2.63 Hz, 1 H), 7.79 (m, J = 8.92 Hz, 2 H), 7.90 (d, J =
2.78 Hz, 1 H), 9.43 (s, 1 H), 10.92 (s, 1 H). LC-MS m/z [M + H]
389.1.
1
then washed with ether to afford 15 (11.5 g, 72% yield). H NMR
4-(3-Chloropyrazin-2-yl)morpholine (8). A solution of 2a (2.03 g,
13.63 mmol), morpholine (1.22 mL, 14.00 mmol), and toluene (10
mL) was stirred at 120 °C for 16 h. The reaction was poured into 10%
Na2CO3 solution (20 mL) and extracted with EtOAc (2 × 30 mL).
The combined organics were washed with brine and concentrated in
vacuo. The crude product was adsorbed onto a plug of silica gel and
chromatographed through a Redi-Sep prepacked silica gel column (40
g), eluting with 0% to 30% EtOAc in hexane, to provide 8 (1.11 g,
40.8% yield).
4-(3-Morpholinopyrazin-2-yloxy)benzenamine (9). To a 25 mL
round-bottomed flask was added 8 (1.094 g, 5.5 mmol), 3 (0.60 g, 5.5
mmol), and cesium carbonate (2.25 g, 6.6 mmol) in DMSO at 80 °C.
Reaction mixture was allowed to cool to room temperature, diluted
with water, and extracted with CH2Cl2. The organic extract was
washed with water and saturated NaCl solution and dried with
MgSO4. The concentrated residue was taken up in DCM and loaded
onto SCX cartridges. The cartridges were rinsed with DCM 3×,
MeOH 3×, and 2.0 M ammonia in MeOH collecting fractions during
each rinse. The ammonia fractions were combined and concentrated
to give 9. LC-MS m/z [M + H] 273.2.
(DMSO-d6): δ ppm 9.116 (s. 1H), 8.197 (d, 2H), 8.165 (d, 1H), 7.750
(d, 2H), 7.589 (t, 1H), 7.147 (d, 2H), 6.835 (d, 1H), 6.760 (t, 1H).
N-(4-((3-(4-Morpholinyl)-2-pyrazinyl)oxy)phenyl)-2-pyridinamine
(16). A suspension of N-(4-((3-chloro-2-pyrazinyl)oxy)phenyl)-2-
pyridinamine (285 mg, 0.95 mmol) and morpholine (0.42 mL, 4.77
mmol) in DMSO (2 mL) was heated at 70 °C for 72 h. Reaction was
then partitioned between 9:1 CHCl3/IPA (30 mL) and 1 M NaOH
(30 mL). The separated organic layer was dried over MgSO4,
concentrated under reduced pressure, then purified by silica gel
chromatography, eluting products with a 0−4% gradient of MeOH/
CH2Cl2 over 20 column volumes to afford 16 (267 mg, 80% yield,
97.7% purity) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ
ppm 3.48−3.57 (m, 4 H), 3.71−3.81 (m, 4 H), 6.73 (dd, J = 6.65, 5.28
Hz, 1 H), 6.81 (d, J = 8.41 Hz, 1 H), 7.02−7.10 (m, 2 H), 7.50−7.60
(m, 2 H), 7.66−7.72 (m, 2 H), 7.89 (d, J = 2.74 Hz, 1 H), 8.13 (dd, J =
5.09, 1.37 Hz, 1 H), 9.04 (s, 1 H). LC-MS m/z [M + H] 350.1. HRMS
(ES+) calcd for [C19H19N5O2]+, 349.154; found, 350.162.
4-Benzyloxy-benzoic Acid Methyl Ester (18). In a 2 L two neck
round-bottom flask, 17 (100 g, 657 mmol) was dissolved in 500 mL of
DMF and potassium carbonate (180 g, 1300 mmol) and cooled to 0
°C. Benzyl bromide (77 mL, 657 mmol) was added dropwise for 30
min. After stirring for 8 h at room temperature, reaction mixture was
diluted with 500 mL of water. The precipitated solid was filtered and
washed with water to afford 18 (150 g, 89% yield). 1H NMR (CDCl3,
300 MHz): δ ppm 3.8 (s, 3H), 5.2 (s, 1H), 6.9 (d, 1H), 7.32 (m, 5H),
7.98 (m, 2H).
Benzothiazol-2-yl-(4-benzyloxy-phenyl)-methanone (19). In a
100 mL round-bottom flask, a mixture of 18 (0.8 g, 3.1 mmol) and
benzothiazole (312 mg, 3.1 mmol) in dry THF was cooled to −70 °C.
LDA (15.2 mL 1.6 M, 18.6 mmol) was added slowly for 5 min.
Reaction mixture was stirred for an additional 2 h at −70 °C. Reaction
mixture was quenched with 1N HCl solution and extracted with ethyl
acetate, washed with brine solution, and dried over anhydrous sodium
sulfate. Volatile solvents were evaporated under vacuum. The crude
product was recrystallized from hexane ethyl acetate mixture to afford
19 (500 mg, 46% yield). 1H NMR (CDCl3, 300 MHz): δ ppm 5.2 (s,
2H), 7.1 (m, 2H), 7.34 (m, 5H), 7.5−7.6 (m, 2H), 8.0 (m, 1H), 8.22
(m, 1H), 8.62 (m, 2H).
Benzothiazol-2-yl-(4-hydroxy-phenyl)-methanone (20). In a 1 L
round-bottom flask 19 (30 g, 86.9 mmol) was stirred in a solution of
boron trifluoride diethyl etherate (101.2 g, 312 mmol) and
dimethylsulfide (112 g, 809 mmol) in dry CH2Cl2 (300 mL) at
room temperature for 72 h. The mixture was then quenched with
water and diluted with CH2Cl2. The organic phase was washed with
brine and dried over anhydrous Na2SO4. Crude product was purified
by silica gel column chromatography using hexane and ethyl acetate to
afford 20 (16 g, 72% yield). 1H NMR (CDCl3, 300 MHz): δ ppm 6.95
N-(4-(3-Morpholinopyrazin-2-yloxy)phenyl)benzo[d]thiazol-2-
amine (10). A microwave reaction vessel was charged with 9 (0.109 g,
0.40 mmol), 2-bromobenzo[d]thiazole (0.074 g, 0.35 mmol),
Pd2(dba)3 (0.0277 g, 0.024 mmol), 2-dicyclohexylphosphino-2′,6′-
dimethoxy-1,1′-biphenyl (0.0198 g, 0.048 mmol), and sodium t-
butoxide (0.0974 g, 1.2 mmol). The reaction mixture was stirred and
heated in a Discover model microwave reactor (CEM, Matthews, NC)
at 150 °C for 20 min (125 W, Powermax feature on, ramp time 10
min). Solvent was concentrated in vacuo. The crude product was
purified by reverse-phase preparative HPLC using a Phenomenex
Gemini column, 5 μm, 150 mm × 30 mm, 0.1% TFA in CH3CN/
H2O, gradient 10−100% over 15 min to afford 10 (8.6 mg, 5.3%
1
yield). H NMR (300 MHz, CDCl3) δ ppm 3.59−3.69 (m, 4 H),
3.84−3.94 (m, 4 H), 7.11−7.22 (m, 3 H), 7.35 (td, J = 7.75, 1.17 Hz, 1
H), 7.54−7.57 (m, 2 H), 7.57−7.59 (m, 1 H), 7.59−7.67 (m, 2 H),
7.88 (d, J = 2.78 Hz, 1 H). LC-MS m/z [M + H] 406.0.
N-(4-Methoxyphenyl)pyridin-2-amine (13). A solution of 11 (5.0
g, 26.7 mmol), 12 (3.58 g, 37.4 mmol, 1.4 equiv), and sodium tert-
butoxide (7.68 g, 80 mmol, 3.0 equiv) in 50 mL of toluene was
degassed by nitrogen purging for 30 min before addition of Pd(dba)2
(0.541 g, 0.66 mmol, 0.025 equiv) and di-tert-butyl(2′,4′,6′-
triisopropylbiphenyl-2-yl)phosphine (0.566 g, 1.3 mmol, 0.05 equiv).
The reaction mixture was degassed for an additional 15 min before
heating to 100 °C for 3 h. The mixture was cooled to room
temperature and diluted with water and extracted with ethyl acetate
(200 × 3 mL). The combined organic layers were filtered through
Celite. The Celite cake was washed with ethyl acetate. The filtrate was
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(s, 2H), 7.5−7.62 (m, 2H), 8.01 (m, 1H), 8.22 (m, 1H), 8.58 (m, 2H).
LC-MS m/z [M + H] 256.
with copious amounts of MeOH. The solids were dried under vacuum
overnight to afford 27 (155 mg, 75% yield). H NMR (300 MHz,
1
Benzothiazol-2-yl-[4-(3-chloro-pyrazin-2-yloxy)-phenyl]-metha-
none (21). In 250 mL round-bottom flask 20 (6 g, 23.5 mmol) was
dissolved in DMSO (60 mL). Compound 2b (3.48 g, 23.5 mmol) and
cesium carbonate (15 g, 47 mmol) were added, and the resulting
mixture was stirred at 90 °C for 6 h. Reaction mixture was diluted with
cold water, and the precipitate was collected by filtration. The crude
compound was purified by silica gel column chromatography to afford
21 (4.5g, 52% yield). H NMR (CDCl3, 300 MHz): δ ppm 7.35 (m,
2H), 7.5−7.62 (m, 2H), 8.01 (m, 2H), 8.13(d, 1H), 8.73 (m, 2H). LC-
MS m/z [M + H] 368.
DMSO-d6) δ ppm 3.55 (d, J = 4.82 Hz, 4 H), 3.75 (br s, 4 H), 4.12 (s,
3 H), 7.38 (d, J = 8.77 Hz, 3 H), 7.43−7.54 (m, 1 H), 7.61−7.67 (m, 1
H), 7.75−7.82 (m, 1 H), 7.82−7.89 (m, 1 H), 7.97−8.06 (m, 1 H),
8.30−8.42 (m, 2 H). LC-MS m/z [M + H] 416.1.
(1H-Benzo[d]imidazol-2-yl)(4-(3-(piperidin-1-yl)pyrazin-2-yloxy)-
phenyl)methanone (28). In a 50 mL round-bottom flask was placed
25 (0.1915 g, 0.546 mmol) in DMSO. Piperidine (0.270 mL, 2.73
mmol) was added, and the temperature was brought to 90 °C to stir
overnight. The reaction mixture was diluted with water and extracted
with CH2Cl2. The organic extract was washed with water and saturated
NaCl and dried with MgSO4. The crude product was adsorbed onto a
plug of silica gel and chromatographed through a Biotage prepacked
silica gel column, eluting with a gradient of 0.5−4% MeOH in CH2Cl2,
to provide 28 (3.49 mg, 16.0% yield). 1H NMR (300 MHz, CDCl3) δ
ppm 1.69 (br s, 6 H), 3.57 (d, J = 5.41 Hz, 4 H), 7.30 (d, J = 8.77 Hz,
2 H), 7.35−7.50 (m, 2 H), 7.54 (d, J = 2.63 Hz, 1 H), 7.60 (d, J = 7.89
Hz, 1 H), 7.94 (d, J = 2.78 Hz, 1 H), 7.98 (d, J = 8.18 Hz, 1 H), 8.87
(d, J = 8.92 Hz, 2 H). LC-MS m/z [M + H] 400.1.
1
Benzo[d]thiazol-2-yl(4-(3-morpholinopyrazin-2-yloxy)phenyl)-
methanone (22). To a solution of 21 (185 mg, 0.5 mmol) in DMSO
(2 mL) was added morpholine (0.22 mL, 2.5 mmol). The reaction
mixture was heated at 100 °C for 2 h. The reaction mixture was
partitioned between EtOAc and brine. The precipitate formed was
collected by filtration and washed with EtOAc and water. The solid
1
was dried to provide 22 (120 mg, 57% yield) as light-yellow solid. H
NMR (400 MHz, DMSO-d6) δ ppm 3.49−3.60 (m, 4 H), 3.69−3.79
(m, 4 H), 7.44 (d, J = 8.80 Hz, 2 H), 7.60−7.75 (m, 3 H), 8.04 (d, J =
2.74 Hz, 1 H), 8.26−8.37 (m, 2 H), 8.55 (d, J = 8.80 Hz, 2 H). LC-MS
m/z [M + H] 419.0.
(4-(3-(1,4-Oxazepan-4-yl)pyrazin-2-yloxy)phenyl)(1H-benzo[d]-
imidazol-2-yl)methanone (29). A solution of 25 (0.100 g, 0.285
mmol) and homomorpholine hydrochloride (0.118 g, 0.855 mmol) in
DMSO (2 mL) in a sealed tube was heated to 70 °C for 5 h. The
reaction mixture was quenched with water (75 mL) and extracted with
DCM (2 × 50 mL). The organic layer was washed with water (50 mL)
and brine (50 mL), dried over magnesium sulfate, concentrated, and
dried to give a crude product. The crude material was purified by flash
column chromatography (ISCO 12 g, 0−30% EtOAc in hexane) to
Ethyl 4-(3-Chloropyrazin-2-yloxy)benzoate (24). To a solution of
23 (11.2 g, 67.1 mmol) and 2a (10.00 g, 67.1 mmol) in DMSO was
added cesium carbonate (26.2 g, 80.5 mmol). The mixture was heated
at 70 °C for 6 h. The mixture was cooled to room temperature and
diluted with water and DCM, and the layers were separated and the
aqueous was extracted with DCM (2×). The combined organics were
washed with brine, dried over Na2SO4, filtered, and concentrated. The
crude material was purified by Biotage, using 15−20% ethyl acetate/
hexanes gradient to give 24 as a white solid (14.9g, 79% yield). LC-MS
m/z [M] 278.9.
(1H-Benzo[d]imidazol-2-yl)(4-(3-chloropyrazin-2-yloxy)phenyl)-
methanone (25). A solution of benzene-1,2-diamine (750 mg, 6.93
mmol), triethyl orthoformate (2.85 mL, 18.66 mmol), and
benzenesulfonic acid (37 mg, 0.24 mmol) in toluene was heated to
reflux for 4 h and then slowly distilled to remove half of the solvent.
The mixture was then cooled to room temperature and neutralized
with 0.1 mL of diisopropyl amine, followed by addition of a solution of
24 (2.13 g, 7.63 mmol) in THF (7 mL). The mixture was cooled to
−78 °C, and 1.2 equiv of LDA (4.62 mL, 8.32 mmol) was added. After
aging at −78 °C for 1.5 h, the mixture was warmed to room
temperature after 1.5 h and then 2 N HCl solution was added. The
resulting mixture was agitated for 30 min. The pH of mixture was
adjusted to pH 9 with 1N NaOH solution. Ethyl acetate was added
and the layers were separated. The aqueous layer was back extracted
with ethyl acetate (3×), and the combined organics were washed with
brine, dried over Na2SO4, filtered, and concentrated to give 24 (987
mg, 41% yield). LC-MS m/z [M + H] 351.0.
1
give 29 (8.34 mg, 70.4% yield). H NMR (400 MHz, DMSO-d6) δ
ppm 1.95 (br s, 2 H), 3.65−3.94 (m, 8 H), 7.39 (d, J = 7.43 Hz, 4 H),
7.45−7.50 (m, 1 H), 7.61−7.89 (m, 2 H), 7.89−7.98 (m, 1 H), 8.56−
8.76 (m, 2 H), 13.48 (br s, 1 H). LC-MS m/z [M + H] 416.0.
(1H-Benzo[d]imidazol-2-yl)(4-(3-(4-methylpiperazin-1-yl)pyrazin-
2-yloxy)phenyl)methanone (30). In a 50 mL RBF was placed 25
(100.5 mg, 0.287 mmol) in DMSO. Methylpiperazine (151 mg, 1.43
mmol) was added, and the temperature was brought to 90 °C to stir
overnight. The reaction mixture was diluted with water (15 mL) and
extracted with CH2Cl2 (3 × 10 mL). The organic extract was washed
with water (2 × 10 mL) and saturated NaCl (2× 10 mL) and dried
with MgSO4. The crude product was adsorbed onto a plug of silica gel
and chromatographed through a Biotage prepacked silica gel column,
eluting with a gradient of 0.5−8% MeOH in CH2Cl2, to provide 30
1
(3.69 mg, 31.1% yield). H NMR (300 MHz, CDCl3) δ ppm 1.43−
1.74 (m, 2 H), 2.19 (d, J = 10.08 Hz, 2 H), 2.64 (s, 3 H), 2.99−3.21
(m, 2 H), 3.90 (d, J = 13.45 Hz, 2 H), 6.12 (d, J = 7.60 Hz, 1 H), 6.61
(dd, J = 6.14, 2.34 Hz, 1 H), 6.69 (d, J = 2.34 Hz, 1 H), 7.71−7.90 (m,
7 H), 8.02 (d, J = 6.14 Hz, 1 H). LC-MS m/z [M + H] 415.1.
(1H-Benzo[d]imidazol-2-yl)(4-(3-(4-methoxypiperidin-1-yl)-
pyrazin-2-yloxy)phenyl)methanone (31). A suspension of 25 (75.0
mg, 0.21 mmol), 4-methoxypiperidine (73.9 mg, 0.64 mmol), and
triethylamine (29.7 μL, 0.21 mmol) in DMSO (0.50 mL) in a round-
bottom flask was heated to 90 °C for 1.5 h. Water (10 mL) was added
to quench the reaction. Precipitates were collected by filtration and
washed with water. The cake was dissolved in DCM (50 mL) and
washed with water (30 mL) and brine (30 mL). The organic layer was
dried over magnesium sulfate, concentrated, and dried in vacuo to give
(1H-benzo[d]imidazol-2-yl)(4-(3-(4-methoxypiperidin-1-yl)pyrazin-2-
yloxy)phenyl)methanone (57.2 mg, 62.3% yield). 1H NMR (400
MHz, DMSO-d6) δ ppm 13.51 (s, 1H), 8.68 (d, J = 8.6 Hz, 2H), 8.00
(d, J = 2.5 Hz, 1H), 7.87 (br s, 1H), 7.67 (br s, 1H), 7.60 (d, J = 2.5
Hz, 1H), 7.29−7.47 (m, 4H), 3.86−8.97 (m, 2H), 3.38−3.48 (m, 1H),
3.27 (s, 3H), 3.18−3.27 (m, 2H), 1.89−2.02 (m, 2H), 1.47−1.60 (m,
2H). LC-MS m/z [M + H] 430.1.
(1H-Benzo[d]imidazol-2-yl)(4-(3-morpholinopyrazin-2-yloxy)-
phenyl)methanone (26). A solution of 25 (23.0 g, 65.6 mmol) and
morpholine (17.2 mL, 197 mmol) was heated to 90 °C in DMSO.
After complete consumption of the starting material, the hot solution
was dripped into ice water, which caused a yellow solid to precipitate.
The solid was slurried in boiling EtOH, and enough tetrahydrofuran
was added to completely dissolve the solids. The solution was
transferred to the freezer for crystallization. The solid was collected by
1
filtration and air-dried to give 26 (15.5 g, 59% yield). H NMR (300
MHz, CDCl3) δ ppm 3.57−3.70 (m, 4 H), 3.79−3.95 (m, 4 H), 7.22−
7.34 (m, 3 H), 7.35−7.49 (m, 2 H), 7.59 (d, J = 2.63 Hz, 1 H), 7.78
(br s, 2 H), 7.95 (d, J = 2.78 Hz, 1 H), 8.77−8.95 (m, 2 H). LC-MS
m/z [M + H] 402.0. HRMS (ES+) calcd for [C22H20N5O3]+,
402.1566; found, 402.1564.
(1-Methyl-1H-benzo[d]imidazol-2-yl)(4-(3-morpholinopyrazin-2-
yloxy)phenyl)methanone (27). To a suspension of 26 (0.2 g, 0.5
mmol) in DMF was added cesium carbonate (0.2 g, 0.7 mmol) and
iodomethane (0.04 mL, 0.6 mmol). The resulting mixture was stirred
at room temperature overnight. The compound was precipitated from
ACN/MeOH, and the resulting orange solid was filtered and rinsed
(1H-Benzo[d]imidazol-2-yl)(4-(3-(4-hydroxypiperidin-1-yl)-
pyrazin-2-yloxy)phenyl)methanone (32). In a 50 mL round-bottom
flask was placed 25 (154.7 mg, 0.44 mmol) in DMSO. Piperidin-4-ol
(333.5 mg, 2.21 mmol) was added, and the temperature was brought
to 90 °C to stir overnight. The reaction mixture was diluted with water
(15 mL) and extracted with CH2Cl2 (3 × 10 mL). The organic extract
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was washed with water (2 × 10 mL) and saturated NaCl (2 × 10 mL)
and dried with MgSO4. The crude product was adsorbed onto a plug
of silica gel and chromatographed through a Biotage prepacked silica
gel column, eluting with a gradient of 0.5−8% MeOH in CH2Cl2, to
provide 32 (75.1 mg, 41% yield). 1H NMR (300 MHz, CDCl3) δ ppm
1.56−1.81 (m, 3 H), 1.90−2.12 (m, 2 H), 3.25 (ddd, J = 13.15, 9.94,
2.92 Hz, 2 H), 3.95 (tt, J = 8.64, 4.15 Hz, 1 H), 4.07 (dt, J = 13.30,
4.09 Hz, 2 H), 7.21−7.33 (m, 2 H), 7.41 (br s, 2 H), 7.55 (d, J = 2.78
Hz, 1 H), 7.93 (d, J = 2.78 Hz, 1 H), 8.84 (d, J = 8.92 Hz, 2 H). LC-
MS m/z [M + H] 416.1. HRMS (ES+) calcd for [C23H22N5O3]+,
416.1723; found, 416.1718.
was adsorbed onto a plug of silica gel and chromatographed through a
Biotage prepacked silica gel column, eluting with a gradient of 0.5−4%
MeOH in CH2Cl2, to 36 (18.1 mg, 11.7% yield). 1H NMR (300 MHz,
CDCl3) δ ppm 1.35 (d, J = 0.73 Hz, 3 H), 1.45−1.70 (m, 3 H), 1.81−
2.09 (m, 4 H), 2.81−3.01 (m, 2 H), 4.23−4.53 (m, 2 H), 7.31 (d, J =
8.92 Hz, 2 H), 7.43 (dd, J = 6.21, 3.14 Hz, 2 H), 7.58 (d, J = 2.78 Hz, 1
H), 7.95 (d, J = 2.78 Hz, 1 H), 8.85 (d, J = 8.92 Hz, 2 H). LC-MS m/z
[M + H] 512.0.
Pharmacology. PDE10A Biochemical Assay. Functional inhib-
ition of human recombinant PDE10A was measured as described in
the IMAP TR-FRET (time-resolved fluorescence energy transfer)
assay kit protocol (Molecular Devices, Sunnyvale, CA. cat. nos. R8160,
R8176, or R8159). Compound was serially diluted in 100% DMSO
from a 10 mM stock and further diluted in Complete reaction buffer
(10 mM Tris-HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3, 0.01% Tween
20, and 1 mM freshly added DTT) to a 4× concentration.
Recombinant PDE10A enzyme and cAMP substrate were diluted in
Complete reaction buffer. The optimized binding buffer was 70%
binding buffer A and 30% binding buffer B. Binding reagent (1:800)
and terbium donor (1: 400) were added to the binding buffer. All
incubations were carried out at room temperature. Testing or control
compounds (5 μL of each 4× concentration per well, 12-point dose−
response curve ranging from 5.1 pM to 10 μM, tested in
quadruplicate) were incubated with 5 μL of recombinant human
PDE10A (0.06 units per well) in a 384-well microplate. After 30 min,
10 μL of cAMP substrate was added to each well for a final substrate
concentration of 100 nM. After 1 h, 60 μL of binding buffer was added
to each well. The plate was then incubated from 3 h to overnight
before reading on an EnVision plate reader. The IMAP binding
reagent binds to the nucleotide monophosphate generated from cyclic
nucleotides (cAMP/cGMP) through phosphodiesterases and enables
measurement of substrate turnover.
4-(3-(4-(1H-Benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-
yl)-1-methylpiperazin-2-one (33). A mixture of 25 (250 mg, 0.71
mmol), cesium carbonate (697 mg, 2.14 mmol), and 1-methylpiper-
azin-2-one (244 mg, 2.14 mmol) was combined in DMSO and heated
to 100 °C overnight. The mixture was cooled to room temperature
and diluted with H2O and DCM. The layers were separated, and the
aqueous layer was extracted with DCM (2×). The combined organics
were washed with saturated NaCl solution, dried over Na2SO4, filtered,
and concentrated. The crude residue was purified by silica gel
1
chromatography to give 33 (34 mg, 11% yield). H NMR (300 MHz,
CDCl3) δ ppm 3.05 (s, 3 H), 3.45−3.57 (m, 3 H), 3.94 (t, J = 5.26 Hz,
2 H), 4.36 (s, 2 H), 7.20−7.36 (m, 4 H), 7.38−7.48 (m, 2 H), 7.60 (d,
J = 2.48 Hz, 1 H), 7.77 (br s, 2 H), 7.93 (d, J = 2.34 Hz, 1 H), 8.85 (d,
J = 8.62 Hz, 2 H). LC-MS m/z [M + H] 429.0. HRMS (ES+) calcd for
[C23H21N6O3]+, 429.1675; found, 429.1674.
1-(4-(3-(4-(1H-Benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-
2-yl)piperazin-1-yl)ethanone (34). In a 50 mL round-bottom flask
was placed 25 (99.7 mg, 0.284 mmol) in DMSO. 1-(Piperazin-1-
yl)ethanone (187 mg, 1.42 mmol) was added, and the temperature
was brought to 90 °C to stir overnight. Reaction mixture was diluted
with water (15 mL) and extracted with CH2Cl2 (3 × 10 mL). The
organic extract was washed with water (2 × 10 mL) and saturated
NaCl solution (2 × 10 mL), dried with MgSO4, filtered, and
concentrated. The crude product was adsorbed onto a plug of silica gel
and chromatographed through a Biotage prepacked silica gel column,
eluting with a gradient of 0.5−4% MeOH in CH2Cl2, to provide 1-(4-
(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)-
piperazin-1-yl)ethanone (28.1 mg, 22.3% yield). 1H NMR (300 MHz,
CDCl3) δ ppm 2.17 (s, 3 H), 3.59−3.69 (m, 6 H), 3.74−3.83 (m, 2
H), 7.31 (d, J = 8.92 Hz, 2 H), 7.43 (dd, J = 6.07, 3.00 Hz, 2 H), 7.63
(d, J = 2.63 Hz, 1 H), 7.96 (d, J = 2.78 Hz, 1 H), 8.88 (d, J = 8.92 Hz,
2 H). LC-MS m/z [M + H] 443.1. HRMS (ES+) calcd for
[C24H23N6O3]+, 443.1832; found, 443.1828.
Permeability and Transcellular Transport. Materials. Digoxin and
3
mannitol were purchased from Sigma-Aldrich (St. Louis, MO). H-
Digoxin and 14C-mannitol were purchased from PerkinElmer Life and
Analytical Sciences (Boston, MA). Transport buffer was prepared
using Hank’s Balanced Salt Solution (HBSS) supplemented with 10
mM Hepes, pH 7.4, and 0.1% BSA (HHBSS, Invitrogen, Grand Island,
NY, BSA, Bovine Serum Albumin, Calbiochem, La Jolla, CA).
Cell Lines and Cultures. Cultures were incubated at 37 °C in a
humidified (95% relative humidity) atmosphere of 5% CO2/95% air.
The parental cell line LLC-PK1 (porcine renal epithelial cells) was
purchased from American Type Culture Collection (ATCC, Manassas,
VA). Human MDR1 and Sprague−Dawley rat mdra1 transfectants in
LLC-PK1 were generated at Amgen (Thousand Oaks, CA). Cells were
cultured in Medium 199 supplemented with 2 mM L-glutamine,
penicillin (50 units/mL), streptomycin (50 μg/mL), and 10% (v/v)
fetal bovine serum (all from Invitrogen).19
(1H-Benzo[d]imidazol-2-yl)(4-(3-(4-(2-hydroxypropan-2-yl)-
piperidin-1-yl)pyrazin-2-yloxy)phenyl)methanone (35). In a 50 mL
round-bottom flask was placed 25 (181.4 mg, 0.517 mmol) in DMSO.
2-(Piperidin-4-yl)propan-2-ol (0.51 g, 2.59 mmol) was added, and the
temperature was brought to 90 °C to stir overnight. The reaction
mixture was diluted with water (15 mL) and extracted with CH2Cl2 (3
× 10 mL). The organic extract was washed with water (2 × 10 mL)
and saturated NaCl solution (2 × 10 mL), dried with MgSO4, filtered,
and concentrated. The crude product was adsorbed onto a plug of
silica gel and chromatographed through a Biotage prepacked silica gel
column, eluting with a gradient of 0.5−4% MeOH in CH2Cl2, to
Permeability and Transcellular Transport of Test Compounds.
LLC-PK1, MDR1-LLC-PK1, and mdr1a-LLC-PK1 cell monolayers
were seeded onto porous (1.0 μm) polycarbonate 96-well transwell
membrane filters (Millipore Corp., Billerica, MA) and cultured for six
days with one media replacement on day four prior to transwell
experiments. Cells were washed once with warmed HHBS prior to
transwell experiments. Experiments were initiated by replacing the
buffer in each compartment with 0.15 mL of HBSS containing 0.1%
BSA with and without 5 μM of test compound in triplicate wells. The
plates were incubated for 2 h at 37 °C in an EVO incubator with
shaking. Aliquots (100 μL) from both donor and receiver chambers
were transferred to 96-well plates or scintillation vials. Protein was
precipitated by addition of 200 μL of acetonitrile containing 0.1%
formic acid and prazosin (25 ng/mL) as internal standard. After
vortexing and centrifugation at 3000 rpm for 20 min, 150 μL
supernatant samples were transferred to a new plate containing 50 μL
of water for LC-MS/MS analysis. Transcellular transport of 3H-digoxin
was used as a positive control for Pgp. Paracellular permeability of 14C-
mannitol was used to measure the integrity of the monolayer. Sample
radioactivity was measured using a liquid scintillation counter (Packard
Tri-Carb 2910TR, PerkinElmer).
1
provide 35 (0.0918 g, 38.8% yield). H NMR (300 MHz, CDCl3) δ
ppm 1.21 (s, 6 H), 1.32−1.67 (m, 3 H), 1.88 (d, J = 10.52 Hz, 2 H),
2.87 (t, J = 12.06 Hz, 2 H), 4.38 (d, J = 13.15 Hz, 2 H), 7.24−7.33 (m,
3 H), 7.42 (d, J = 10.67 Hz, 2 H), 7.54 (d, J = 2.63 Hz, 1 H), 7.59 (d, J
= 6.87 Hz, 1 H), 7.93 (d, J = 2.63 Hz, 1 H), 7.97 (br s, 1 H), 8.85 (d, J
= 8.92 Hz, 2 H). LC-MS m/z [M + H] 458.0.
(S)-(1H-Benzo[d]imidazol-2-yl)(4-(3-(4-(1,1,1-trifluoro-2-hydroxy-
propan-2-yl)piperidin-1-yl)pyrazin-2-yloxy)phenyl)methanone (36).
In a 50 mL round-bottom flask was placed 25 (106.3 mg, 0.303 mmol)
in DMSO. (S)-1,1,1-Trifluoro-2-(piperidin-4-yl)propan-2-ol (198.3 g,
1.06 mmol) was added, and the temperature was brought to 90 °C to
stir overnight. The reaction mixture was diluted with water (15 mL)
and extracted with CH2Cl2 (3 × 10 mL). The organic extract was
washed with water (2 × 10 mL) and saturated NaCl solution (2 × 10
mL), dried with MgSO4, filtered, and concentrated. The crude product
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The apparent permeability coefficient (Papp) of all tested agents was
estimated from the slope of cumulative amount (dQ) of the agent vs
time (dt), and the equation:
analysis: one site − specific binding
equation: Y = Bmax × X/(Kd + X)
P
= (dQ /dt)/(A × C0)
app
Rat and Human Liver Microsomal Assays. Liver microsomal
stability was measured at 37 °C in phosphate buffer (66.7 mM, pH
7.4). Test compounds (1 μM) were incubated with pooled human or
rat liver microsomes at 0.25 mg/mL of protein, with or without
NADPH (1 mM). After 30 min, the reaction was stopped by the
addition of acetonitrile containing 0.5% formic acid and internal
standard. The quenched samples were centrifuged at 1650g for 20 min.
The supernatants were analyzed directly for unchanged test compound
using liquid chromatography and tandem mass spectrometric
detection (LC-MS/MS). Intrinsic clearance was calculated based on
substrate disappearance rate, assuming first-order elimination of
compound over the 30 min incubation.
where dQ/dt is the penetration rate of the agent (μm/s), A is the
surface area of the cell layer on the Transwell (0.11 cm2), and C0 is the
initial concentration of the test compound (μM).
LC-MS/MS RO Assays. Animals. All experiments were conducted
under approved research protocols by Amgen’s Animal Care and Use
Committee (IACUC) and in accordance with National Institutes of
Health Guide for Care and Use of Laboratory Animals guidelines in
facilities accredited by the Association for the Assessment and
Accreditation of Laboratory Animal Care (AALAC). Adult male
Sprague−Dawley (SD) rats (250−280 g) were purchased from Harlan
(Harlan, Indianapolis). Rats were group housed on a filtered, forced air
isolation rack and maintained on sterile wood chip bedding in a quiet
room on a 12 h light-dark cycle, with food and water available ad
libitum. Animals were allowed for minimum 3 days of adaptation to
the laboratory conditions prior to being utilized in the experiments.
LC-MS RO Assay with po Administration. Sprague−Dawley rats
were pretreated with vehicle or compound via po dosing. Then 50 min
after dosing, our PDE10A tracer AMG798015 was administered by
bolus IV injection via lateral tail vein, then 10 min after tracer
injection, animals under isoflurane anesthesia were euthanized by
decapitation; samples of blood and brain samples were collected for
analysis.
ASSOCIATED CONTENT
* Supporting Information
■
S
Method for determination of cocrystal structures of 1 and 26
with PDE10A and summary of data collection. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
The PDB ID codes for coordinates of 1 and 26 with PDE10A
are 4MUW and 4MVH, respectively.
LC-MS RO Assay with ip Administration. Sprague−Dawley rats
were pretreated with vehicle or compound via ip dosing. Then 20 min
after dosing vehicle or compound, our PDE10A tracer AMG798015
was administered by bolus IV injection via lateral tail vein, and 10 min
after tracer injection, animals under isoflurane anesthesia were
euthanized by decapitation; samples of blood and brain samples
were collected for analysis.
AUTHOR INFORMATION
Corresponding Author
*Phone: 805-313-5300. E-mail: ehu@amgen.com.
Notes
■
The authors declare no competing financial interest.
Sample Analysis and Receptor Occupancy Determination of 26,
32, 33, and 34. In both ip and po studies, striatum was used as the
target tissue due to high endogenous expression of PDE10A protein,
and thalamus was chosen as the reference tissue due to lower
expression of PDE10A.13
Brain samples were weighed and HPLC-grade water was added
(20% weight/volume), followed by homogenization using a Covaris
E110 acoustic homogenizer (model E210, Covaris, Inc., Woburn,
MA). Covaris settings: 4 °C, duty cycle 20%, intensity 8, cycles per
Burst 500, treatment time 6 × 10 s. Homogenized samples were stored
at −20 °C. Extraction and analysis of compound and AMG7980 tracer
concentrations by API 4000 LC-MS/MS (Applied Biosystems,
Carlsbad, CA).
ABBREVIATIONS USED
■
ACN, acetonitrile; BBB, blood−brain barrier; cAMP, cyclic
adenosine monophosphate; cGMP, cyclic guanosine mono-
phosphate; ER, efflux ratio; EtOAc, ethyl acetate; HLM, human
liver microsome; IPA, isopropyl alcohol; LC-MS/MS, liquid
chromatography−tandem mass spectrometry; MeOH, methyl
alcohol; PDE, phosphodiesterase; RLM, rat liver microsome;
RO, receptor occupancy; SOC, standard of care
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