Discovery of clinical candidate 1-(4-(3-(4-(1 H-benzo[ d ]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)ethanone (AMG 579), A potent, selective, and efficacious inhibitor of phosphodiesterase 10A (PDE10A)

Article abstract of DOI:10.1021/jm500713j

We report the identification of a PDE10A clinical candidate by optimizing potency and in vivo efficacy of promising keto-benzimidazole leads 1 and 2. Significant increase in biochemical potency was observed when the saturated rings on morpholine 1 and N-a

Full text of DOI:10.1021/jm500713j

Article
pubs.acs.org/jmc
Discovery of Clinical Candidate 1‑(4-(3-(4-(1H‑Benzo[d]imidazole-2-
carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)ethanone (AMG 579), A
Potent, Selective, and Efficacious Inhibitor of Phosphodiesterase 10A
(PDE10A)
Essa Hu,*,† Ning Chen,† Matthew P. Bourbeau,† Paul E. Harrington,† Kaustav Biswas,†
Roxanne K. Kunz,† Kristin L. Andrews,‡ Samer Chmait,‡ Xiaoning Zhao,⊥ Carl Davis,§ Ji Ma,∇
Jianxia Shi,∇ Dianna Lester-Zeiner,# Jean Danao,# Jessica Able,# Madelyn Cueva,# Santosh Talreja,#
Thomas Kornecook,∥ Hang Chen,# Amy Porter,∥ Randall Hungate,† James Treanor,∥
and Jennifer R. Allen†
‡
§
†Department of Medicinal Chemistry, Department of Molecular Structure and Characterization, Department of Pharmacokinetics
∥
and Drug Metabolism, Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California
93012-1799, United States
#
⊥Department of Molecular Structure and Characterization, Department of Neuroscience, ∇Department of Pharmacokinetics and
Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: We report the identification of a PDE10A clinical candidate by optimizing
potency and in vivo efficacy of promising keto-benzimidazole leads 1 and 2. Significant increase
in biochemical potency was observed when the saturated rings on morpholine 1 and N-acetyl
piperazine 2 were changed by a single atom to tetrahydropyran 3 and N-acetyl piperidine 5. A
second single atom modification from pyrazines 3 and 5 to pyridines 4 and 6 improved the
inhibitory activity of 4 but not 6. In the in vivo LC−MS/MS target occupancy (TO) study at
10 mg/kg, 3, 5, and 6 achieved 86−91% occupancy of PDE10A in the brain. Furthermore, both
CNS TO and efficacy in PCP-LMA behavioral model were observed in a dose dependent
manner. With superior in vivo TO, in vivo efficacy and in vivo PK profiles in multiple
preclinical species, compound 5 (AMG 579) was advanced as our PDE10A clinical candidate.
treated with typical and atypical antipsychotics have been linked
to either direct interaction with the dopaminergic receptors or
polypharmacology.33,34 Therefore, we hypothesized targeting
PDE10A would provide a promising approach, using a single
selective molecule, to both treat schizophrenia and circumvent
the adverse events.
We recently reported a detailed structure−activity relationship
(SAR) study that led to the identification of keto-benzimidazoles
1 and 2 as novel and potent inhibitors of PDE10A with mitigated
P-gp efflux (Figure 1).35 Both analogues achieved single-digit
inhibitory activity against PDE10A enzyme in the biochemical
assay (IC50 = 4.5 and 5.1 nM, respectively). Despite their similar
in vitro potencies and structures, their difference in in vivo
efficacies in our rodent LC−MS/MS target occupancy (TO)
assay was pronounced. At 10 mg/kg PO, morpholine 1 only
produced 21.3% TO of PDE10A in the CNS while N-acetyl
piperazine 2 afforded a significantly higher TO of 57.1%. The
realization that a small structural difference between 1 and 2
could have such a dramatic impact on in vivo efficacy prompted
INTRODUCTION
■
Of the 11 isoforms of phosphodiesterase (PDE) identified to
date, PDE10A has recently received much attention in the field of
neuroscience research.1 Because PDE10A has the highest
expression level in the striatum,2−4 modulation of this CNS
target offers an opportunity to regulate signal transmissions in a
region of the brain associated with various neurological
conditions such as schizophrenia and Huntington’s disease.5−13
At the preparation of this manuscript, two PDE10A inhibitors
have been reported to advance into clinical studies for treatment
of schizophrenia.14,15 Other inhibitors have also been described
in the literature.16−26
The function of PDEs is to reduce levels of cyclic nucleotide
monophosphates (cNMP) by cleavage of their phosphodiester
bonds.27 Thus, inhibition of PDE10A enzyme would increase the
levels of intracellular secondary messengers, cyclic adenosine
monophosphate (cAMP) and cyclic guanosine monophosphate
(cGMP).28 It has been hypothesized that this mechanism
effectively replicates the downstream functional effect of current
standard of care (SOC) which targets dopamine receptors (D1
and D2) and NMDA receptors.29−32 Intolerable adverse events
that contributed to compliance issues in schizophrenic patients
Received: May 8, 2014
Published: July 25, 2014
© 2014 American Chemical Society
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3 did change the preferred binding orientation of the saturated
rings relative to the pyrazine rings, however. Even though both
rings were freely rotating, the densities of the co-crystal
structures captured the more favored binding conformation.
For 1, the morpholine ring was nearly coplanar with the pyrazine
ring, presumably due to the stabilizing delocalization of the
nitrogen lone pair into the anti-p-orbitals on the pyrazine ring. In
contrast, the tetrahydropyran ring on 3 preferred a nonplanar
alignment with the pyrazine ring, presumably to minimize the
steric interactions between the two ring systems. We postulated
that the greater torsion angle between the two rings on 3 enabled
the tetrahydropyran to establish more hydrophobic contacts with
the hydrophobic residues in the binding pocket, resulting in an
increase in binding affinity. This computational prediction of the
binding conformation of 3 was later supported by an X-ray co-
crystal structure of pyridyl analogue 4, which showed the
tetrahydropyran in a nonplanar orientation relative to the
pyridine ring analogous to the conformation predicted for 3.
We then discovered a second single atom replacement that
produced a significant effect (Table 1). With a pyridine ring
Figure 1. Promising keto-benzimidazole leads 1 and 2.
us to explore additional changes to seek further optimization. We
sought to further increase the in vitro potency and in vivo efficacy
of our PDE10A inhibitors in order to deliver a clinical candidate
with a low projected human dose and minimal drug burden on
patients.
RESULTS AND DISCUSSION
■
Of all the small structural changes we had explored, the first
breakthrough discovery was a simple nitrogen atom to carbon
atom replacement on the saturated rings. As shown in Figure 2,
Table 1. Structure−Activity Relationships of Keto-
benzimidazoles 1−6
PDE10A
IC
HLM
RLM
50a
b
b
compd
X
Y
Z
(nM)
(μL/min/mg) (μL/min/mg)
Figure 2. Single atom change resulted in 3 with increased potency.
1
3
4
2
5
6
N
N
C
N
N
C
N
C
C
N
C
C
O
4.5
0.8
0.1
5.1
0.1
0.1
55
50
74
119
>399
108
41
O
tetrahydropyran 3 showed PDE10A IC50 = 0.8 nM, a 5-fold
increase in potency from morpholine 1. To analyze the structural
impact of this single atom change, we compared our previously
reported X-ray co-crystal structure of 135 in human PDE10A
catalytic domain with computational model of 336 in human
PDE10A catalytic domain (Figure 3). As expected, the keto-
benzimidazole region on 3 was bound to PDE10A catalytic
domain in a manner identical to the keto-benzimidazole region
on 1. Both pyrazine rings were sandwiched between Phe719 and
Phe686 stabilized by π-stacking interactions. The conserved Gln
716 further anchored the scaffold by forming a hydrogen
bonding interaction with one of the pyrazine nitrogens.
Meanwhile, the phenyl keto-benzimidazole region was locked
into a planar binding conformation by a second hydrogen
bonding interaction between Tyr683 and one of the nitrogens on
the benzimidazole ring. The one atom difference between 1 and
O
100
34
NAc
NAc
NAc
20
29
463
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
instead of pyrazine ring, 4 experienced an 8-fold improvement in
PDE10A inhibitory activity (IC50 = 0.1 nM). Compared to initial
morpholine pyrazine lead 1, N-acetyl piperidine pyridine 4
represented 45-fold improvement in potency. Further profiling
showed each single atom exchange had a different impact on in
Figure 3. Comparison of X-ray co-crystal structures of 1 (left), computational model of 3 (center), and co-crystal structure of 4 (right) in human
PDE10A catalytic domain.
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a
Scheme 1. Synthesis of Keto-benzimidazoles 3
a
Reagents and conditions: (a) cesium carbonate, NMP, 100 °C, 70% yield; (b) 5 mol % PdCl2(P(t-Bu)2(PhNMe2))2, potassium carbonate,
acetonitrile, water 80 °C, 91% yield; (c) 5 mol % Pd(OH)2/C, hydrogen, ethanol, 65% yield; (d) (i) 13, HC(Oi-Pr)3, toluene, reflux, (ii) LDA, THF,
−78 °C, 12, 84% yield.
a
Scheme 2. Synthesis of Keto-benzimidazoles 4, 5, and 6
a
Reagents and conditions: (a) sodium carbonate, Pd(PPh3)2Cl2, ether, water, 80 °C, 89% yield; (b) 9.5 mol % Pd(OH)2, hydrogen, ethanol, 96%
yield; (c) I2, Ph3P, imidazole, THF, 84% yield; (d) (i) zinc dust, TMSCl, 1,2-dibromoethane, DMA, (ii) 3 mol % PdCl2dppf, CuI, DMA, 80 °C, two
step yields 85% (20a) and 70% (20b); (e) Pd(OH)2, hydrogen, toluene, acetic anhydride, 91% (21a), 92% (21b); (f) para-methoxybenzyl chloride,
potassium carbonate, NaI, DMF, 98% yield; (g) (i) HC(OEt)3, benzenesulfonic acid, toluene, (ii) LDA, 66% yield; (h) TFA, dichloromethane, 99%
yield; (i) cesium carbonate, NMP, 120 °C, 48% yield (4), 87% yield (5), 66% yield (6).
vitro clearance. Pyrazine 3 retained human and rat in vitro
metabolic stability comparable to 1. However, rat metabolic
stability of pyridine 4 deteriorated significantly. Interestingly,
compared to morpholine lead 1, the SAR of these same
modifications on N-acetyl piperazine lead 2 was not entirely the
same. Similar to 3, one nitrogen atom to carbon atom change
made N-acetyl piperidine 5 over 50-fold more potent than N-
acetyl piperazine 2 (IC50 = 0.1 vs 5.1 nM, respectively) without
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impacting its rat and human in vitro microsomal clearance (HLM
= 20 μL/min/mg; RLM = 41 μL/min/mg). Unlike 4, further
improvement in potency was not observed when the pyrazine
ring on 5 was replaced with a pyrdine ring (6). As with 4, pyridine
6 also experienced a dramatic decrease in its RLM to 463 μL/
min/mg.
Scheme 1 depicts the synthetic route used to obtain keto-
benzimidazoles 3. The synthesis began with SNAr displacement
of dichloropyrazine 7 with ethyl 4-hydroxybenzoate 8. Suzuki
coupling of ethyl 4-((3-chloropyrazin-2-yl)oxy)benzoate 9 with
2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxa-
borolane 10 using PdCl2(P(t-Bu)2(PhNMe2))2 produced ethyl
4-((3-(3,6-dihydro-2H-pyran-4-yl)pyrazin-2-yl)oxy)benzoate
11 in 91% yield.37 Palladium catalyzed hydrogenation reduced
the olefin and afforded ethyl 4-((3-(tetrahydro-2H-pyran-4-
yl)pyrazin-2-yl)oxy)benzoate 12. A two-step, one-pot protocol
beginning with in situ N−H protection of benzimidazole 13
followed by LDA deprotonation formed (1H-benzo[d]imidazol-
2-yl)(4-((3-(tetrahydro-2H-pyran-4-yl)pyrazin-2-yl)oxy)-
phenyl)methanone 3 in 84% yield.
Table 2. PDE10A in Vivo Target Occupancy and Behavioral
PD Readouts of Keto-benzimidazoles 1−6
a
b
c
d
single dose TO (at dose response TO reversal of PCP-LMA
compd
10 mg/kg) (%)
EC50 (nMtotal
)
(MED) (mg/kg)
1
3
4
21.3
91.0
69.2
117
1.0
2
5
6
57.1
86.3
86.5
711
965
0.3
0.3
a
Dose and methods: LC−MS/MS target occupancy measurements
b
were taken 1 h post PO dosing of compounds. Dose and methods: at
single dose of 10 mg/kg, n = 8. Dose and methods: at doses 0.1, 0.3,
1, 3, 10, and 30 mg/kg n = 4. Reversal of PCP-induced locomotor
c
d
activity was assessed at doses 0.1, 0.3, 1, and 3 mg/kg, n = 10.
Compounds were dosed PO 1 h prior to administration of PCP.
Number of laser beam breaks post PCP dosing was collected at 10 min
intervals and compiled over 2 h period. MED was determined at the
lowest dose which a statistically significant reduction of locomotor
activity occurred.
Syntheses of keto-benzimidazoles 4, 5, and 6 were
accomplished by a convergent route illustrated in Scheme 2.
Two routes were developed to access the three left-hand
fragments. Dihydropyran pyridine 15 was synthesized by Suzuki
coupling of 10 to 3-bromo-2-fluoropyridine 14. Palladium
catalyzed hydrogenation of the olefin afforded 16 in 96% yield.
For the N-acetyl piperidine fragments (pyrazine 21a and pyridine
21b), our synthesis began with iodination of benzyl 4-
hydroxypiperidine-1-carboxylate 17 to form benzyl 4-iodopiper-
idine-1-carboxylate 18 using iodine and triphenylphosphine in
84% yield. In a key transformation, Negishi coupling of 18 to
pyrazine 19 or pyridine 14 via zinc activation38 and palladium
catalysis produced N-Cbz piperidine pyrazine 20a and pyridine
20b. A one-pot Cbz deprotection and acetylation afforded N-
acetyl piperidines 21a and 21b. Preparation of the right-hand
fragment, keto-benzimidazole 23, began with para-methoxyben-
zyl (PMB) protection of ethyl 4-hydroxybenzoate 8 to form
protected phenol ester 22. Ketone formation using a similar one-
pot benzimidazole protection and deprotonation protocol
followed by trifluoroacetic acid promoted removal of the PMB
protecting group resulted in (1H-benzo[d]imidazol-2-yl)(4-
hydroxyphenyl)methanone 23. Final coupling of tetrahydropyr-
an pyridine 16, N-acetyl piperidines 21a or 21b, to keto-phenol
23 was accomplished using cesium carbonate to give keto-
benzimidazoles 4, 5, and 6 in 48%, 87%, and 66% yield,
respectively. The synthetic routes developed allowed for
compounds 3−6 to be prepared on a large scale in order to
enable rodent efficacy assessment as well as rodent and
nonrodent pharmacokinetic and exploratory preclinical toxicol-
ogy studies.
MS/MS technology platform has enabled us to identify
compounds with high in vivo PDE10A specificity and coverage
efficacy. Measurements of CNS PDE10A target occupancy were
conducted 1 h post PO dosing of inhibitors and 10 min post IV
dosing of tracer. TO was determined by comparing the tracer
levels in the striatum, a region of the brain most highly expressed
with PDE10A, versus the thalamus, a control region for
nonspecific binding. At a single dose of 10 mg/kg PO, keto-
benzimidazole 3 achieved 91% TO, a significant improvement
from 21% TO of 1. Even though 4 was biochemically more
potent than 3, 4 exhibited reduced 69.2% TO, presumably due to
its reduced metabolic stability in rat. Meanwhile, N-acetyl
piperidine 5 showed enhanced 86.3% TO at 10 mg/kg compared
to 57.1% TO of 2. Pyridine 6 also afforded comparable TO of
86.5% despite its higher rat in vitro clearance. Compounds 3, 5,
and 6 were advanced into dose response LC−MS/MS TO study
at doses 0.1, 0.3, 1, 3, 10, and 30 mg/kg, and all three compounds
exhibited dose-dependent occupancy of PDE10A receptors in
the brain (Figure 4). On the basis of these data, TO EC50 was
To determine whether these structural changes resulted in
improvements in in vivo efficacies, keto-benzimidazoles 3, 4, 5,
and 6 were advanced into LC−MS/MS target occupancy (TO)
measurement study and phencyclidine induced-locomotor
activity (PCP-LMA) model (Table 2). We recently reported
the development of an in vivo LC−MS/MS TO assay platform
and a selective PDE10A tracer 5-(6,7-dimethoxycinnolin-4-yl)-
N-isopropyl-3-methylpyridin-2-amine (AMG 7980, 24) to
measure PDE10A target occupancy of drug candidates in rat
brain.39 By measuring compound levels in brain regions with
high levels of PDE10A (striatum) and low expression levels of
PDE10A (thalamus) and measuring PDE10A tracer level
changes in the striatum due to compound binding, this LC−
Figure 4. Dose dependent in vivo target occupancy with keto-
benzimidazole 5.
calculated to be 117 nM for 3, 711 nM for 5, and 965 nM for 6
(total plasma). High occupancy PDE10A inhibitors 3, 5, and 6
were then tested in the PCP-LMA model, a widely used
preclinical rodent model to mimic positive symptoms of
schizophrenia (Figure 5).40 Compounds were dosed at 0.1, 0.3,
1, and 3 mg/kg PO 1 h prior to administration of PCP. The
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ethanone 5 passed all our preclinical toxicological studies and
advanced as AMG 579, our PDE10A clinical candidate for
treatment of schizophrenia.
Starting with promising keto-benzimidazole leads 1 and 2, we
discovered single atom replacements from nitrogen atom to
carbon atom produced significant improvements in both
PDE10A enzyme potency and in vivo efficacy in the LC−MS/
MS TO assay. The first single atom modification of the saturated
rings from N-linked to C-linked (1 to 3 and 2 to 5) increased the
biochemical potency by 5-fold and 50-fold, respectively. X-ray
co-crystal structures of 1 and 3 in the human PDE10A enzyme
catalytic domain showed the single atom replacement changed
the preferred orientation of the saturated ring relative to the
pyrazine ring. A second nitrogen atom to carbon atom change
from pyrazine ring to pyridine ring (3 to 4 and 5 to 6) resulted in
8-fold increase of PDE10A activity for 4 but no potency
improvement for 6. Both pyrazines (3 and 5) maintained their in
vitro metabolic stability, while both pyridines (4 and 6) showed
much higher in vitro clearance in rat. It is worth noting that these
modifications had minimal impact on the permeability, efflux
ratio, or PDE selectivity of these keto-benzimidazole analogues
compared to original leads 1 and 2. In vivo LC−MS/MS TO
studies at 10 mg/kg showed 3, 5, and 6 achieved 86−91% TO. All
three compounds were efficacious in the PCP-LMA model, a
widely used model of positive schizophrenic behaviors in
preclinical species. Both occupancy of CNS PDE10A receptors
and efficacy in PCP-LMA model with keto-benzimidazole 5 were
found to occur in a dose dependent manner. Keto-benzimidazole
5 was selected as our clinical candidate upon comparison of in
vivo efficacies and in vivo PK profiles of compounds 3, 5, and 6 in
rat, dog, and nonhuman primate (Figure 6). This molecule was
also highly selective with IC50 > 30 μM against all other PDE
isoforms. Advanced preclinical and clinical data will be disclosed
in separate publications in the near future.
Figure 5. Dose dependent reversal of PCP-induced locomotor activity
model with keto-benzimidazole 5.
magnitude of rat locomotor activity was quantified as number of
beam breaks over a 2 h period. Compared to the untreated group,
all three inhibitors showed statistically significant reduction of
PCP induced behavior in rats over the 2 h period. Minimum
effective doses (MED) for efficacy in PCP-LMA model were
determined to be at 1 mg/kg for 3, 0.3 mg/kg for 5, and 0.3 mg/
kg for 6. In line with CNS target occupancy, all compounds were
effective in reversing PCP-induced locomotor activity in a dose
dependent manner. It is also worth noting that compared to the
vehicle treated group, none of our compounds impacted the
locomotor activity of these animals prior to PCP challenge.
All three compounds (3, 5, and 6) that achieved greater than
80% TO in the single dose LC−MS/MS assay and demonstrated
efficacy in the PCP-LMA behavioral model were also profiled in
rat in vivo PK (Table 3). Both 3 and 5 showed modest in vivo
clearance in rat (0.62 and 0.54 L/h/kg, respectively) compared
to high rat clearance observed with 6 (3.03 L/h/kg). However,
oral bioavailability of 3 was low (22% F) compared to 5 (49% F)
and 6 (63% F). Higher species in vivo PK with 5 and 6 showed
acceptable in vivo clearance in both dog and nonhuman primate.
However, in dog, 5 exhibited superior oral bioavailability of 72%
compared to 45% with 6. With a clearly more robust metabolic
profile across multiple preclinical species and a low projected
human dose of 0.4 mg/kg QD, 1-(4-(3-(4-(1H-benzo[d]-
imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-
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, EMD 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
Table 3. In Vivo PK Data for Keto-benzimidazoles 3, 5, and 6
compd
species
rat
dose (mg/kg)/route
Cl (L/h/kg)
0.62
Vss (L/kg)
2.27
AUC (μM·h)
T1/2 (h)
%F
a
3
2, IV
7.75
7.93
4.26
3.99
b
10, PO
22
a
5
6
rat
2, IV
0.54
0.074
0.34
0.81
0.77
0.83
8.31
19.80
13.70
16.90
3.41
3.16
3.35
6.89
11.0
1.09
2.48
b
10, PO
49
72
71
b
dog
NHP
0.5, IV
b
1, PO
d
0.5, IV
c
0.5, PO
2.40
a
rat
2, IV
3.03
0.052
0.82
31.2
0.50
1.62
2.78
29.50
21.50
9.59
23.1
1.72
7.41
5.07
1.26
b
10, PO
63
45
c
dog
NHP
0.5, IV
b
0.5, PO
d
0.5, IV
1.61
a
b
c
Dosing vehicle: 100% DMSO. Dosing vehicles: Tween 80, 2% HPMC, 97% water, methanesulfonic acid pH 2.0. Dosing vehicles: 30% captisol,
d
70% water, methanesulfonic acid, pH 4.0. Dosing vehicle: 30% hydroxypropyl β-cyclodextrin, 70% water. NHP = nonhuman primate.
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Bu)PPhNMe2)2 (0.07 g, 0.11 mmol), and potassium acetate (0.52 g, 5.3
mmol) was added acetonitrile (7.0 mL), water (0.70 mL), and 10 (0.52
g, 2.5 mmol). The reaction mixture was degassed by bubbling nitrogen
through the solution for 5 min and heated to 80 °C. After 2.5 h, the
reaction was diluted with EtOAc, water, and brine. The aqueous phase
was extracted with EtOAc (1×). The combined organic extracts were
washed with brine (1×), dried over MgSO4, filtered, and concentrated.
Purification by flash column chromatography on silica gel (eluted with
10−50% EtOAc in hexanes) gave 11 (0.60 g, 91% yield) as a pale-yellow
solid. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.40 (t, J = 7.14 Hz, 3 H),
2.76 (dd, J = 4.40, 2.64 Hz, 2 H), 3.97 (t, J = 5.48 Hz, 2 H), 4.36−4.43
(m, 4 H), 7.03 (br s, 1 H), 7.18 (d, J = 8.61 Hz, 2 H), 7.92 (d, J = 2.35 Hz,
1 H), 8.12 (d, J = 8.80 Hz, 2 H), 8.30 (d, J = 2.54 Hz, 1 H). LC−MS m/z
[M + H] 327.2.
Step 3. Ethyl 4-(3-(Tetrahydro-2H-pyran-4-yl)pyrazin-2-yloxy)-
benzoate (12). To a mixture of 11 (3.0 g, 9.2 mmol) and palladium
hydroxide (20% by weight palladium on carbon, 0.32 g, 0.46 mmol)
under argon was added EtOH (18 mL). The argon atmosphere was
replaced by hydrogen from a double balloon, and the reaction mixture
was stirred at room temperature. After 3 h, the solution was filtered and
concentrated. Purification by flash column chromatography on silica gel
(eluted with 10−40% EtOAc in hexanes) gave 12 (2.0 g, 65% yield) as a
colorless oil. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.40 (t, J = 7.14 Hz,
3 H), 1.88 (d, J = 13.30 Hz, 2 H), 2.02−2.13 (m, 2 H), 3.43 (tt, J = 11.70,
3.30 Hz, 1 H), 3.61 (t, J = 11.83 Hz, 2 H), 4.13 (dd, J = 11.25, 3.81 Hz, 2
H), 4.39 (q, J = 7.24 Hz, 2 H), 7.18 (d, J = 8.61 Hz, 2 H), 7.94 (d, J = 2.74
Hz, 1 H), 8.12 (d, J = 8.61 Hz, 2 H), 8.26 (d, J = 2.74 Hz, 1 H). LC−MS
m/z [M + H] 329.2.
Figure 6. Profile of clinical candidate 5.
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 reactor.
1H 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 chromatography (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 acetonitrile; 0.0−3.0 min,
5−95% B; 3.0−3.5 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 electro-
spray 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.
Synthesis of (1H-Benzo[d]imidazol-2-yl)(4-(3-(tetrahydro-2H-
pyran-4-yl)pyrazin-2-yloxy)phenyl)methanone (3). Step 1. Ethyl 4-
(3-Chloropyrazin-2-yloxy)benzoate (9). To a mixture of cesium
carbonate (72.8 g, 223 mmol) and 8 (30.1 g, 181 mmol) was added
NMP (200 mL) and 7 (33.0 mL, 317 mmol). The reaction mixture was
degassed by bubbling nitrogen through the solution for 5 min and
heated to 100 °C. After 15 min, the mixture was diluted with EtOAc.
The organic phase was washed with water (2×), brine (1×), dried over
MgSO4, filtered, and concentrated. The concentrate was diluted with
hexanes (50 mL), and the mixture was allowed to stand at −15 °C for 15
h. The precipitate that formed was collected by filtration, washed with
hexanes, and dried under high vacuum to give 9 (35.3 g, 70% yield) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.33 (t, J = 7.14 Hz,
3 H), 4.33 (q, J = 7.20 Hz, 2 H), 7.41 (d, J = 8.41 Hz, 2 H), 8.05 (d, J =
8.41 Hz, 2 H), 8.22 (d, J = 2.54 Hz, 1 H), 8.29 (d, J = 2.54 Hz, 1 H). LC−
MS m/z [M + H] 279.1.
Step 4. (1H-Benzo[d]imidazol-2-yl)(4-(3-(tetrahydro-2H-pyran-4-
yl)pyrazin-2-yloxy)phenyl)methanone (3). A suspension of 13 (5.9 g,
50 mmol) and triisopropyl orthoformate (63.6 mL, 287 mmol) in
toluene (150 mL) in a round-bottomed flask fitted with a Dean−Stark
trap topped with a reflux condenser was heated to reflux under nitrogen
(bath temperature 140 °C). After 1 h, the reaction was cooled to
ambient temperature and was concentrated in vacuo. The crude
orthoester amide was dried under high vacuum for 6 h and diluted with
tetrahydrofuran (150 mL). Compound 12 (15.7 g, 47.9 mmol) was
azeotroped with dry toluene (3×15 mL), dissolved in tetrahydrofuran
(100 mL), and added via cannula to the reaction flask containing the
benzimidazole orthoester amide. The resulting mixture was then cooled
to −78 °C under nitrogen. Lithium diisopropylamide (27.5 mL of a 2 M
solution in heptane/tetrahydrofuran/ethylbenzene, 55.0 mmol) was
added in a dropwise fashion over 75 min. After 2 h, the reaction was
removed from the acetone−dry ice bath and quenched with via slow
addition of 2 N HCl (25 mL). The solution was stirred at 23 °C for 45
min and neutralized to pH = 7.0 with solid sodium carbonate. The
suspension was diluted with EtOAc (500 mL) and washed with
saturated sodium bicarbonate solution (2 × 150 mL) and brine (150
mL), dried over magnesium sulfate, concentrated in vacuo, and slurried
with ethanol (100 mL). The residue was filtered and dried under
1
vacuum to afford 3 (16 g, 84% yield) as a white solid. H NMR (400
MHz, DMSO-d6) δ ppm 13.52 (1 H, br s), 8.70 (2 H, d, J = 8.8 Hz), 8.41
(1 H, d, J = 2.5 Hz), 8.11 (1 H, d, J = 2.5 Hz), 7.87 (1 H, br s), 7.64 (1 H,
br s), 7.20−7.48 (4 H, m), 3.82−4.13 (2 H, m), 3.52 (2 H, td, J = 11.2,
3.1 Hz), 3.36−3.45 (1 H, m), 1.63−2.12 (4 H, m). LC−MS m/z [M +
H] 401.1. HRMS (ES+) calcd for [C23H21N4O3]+ 401.1614; found
401.1611. 13C NMR (101 MHz, DMSO-d6) δ ppm 30.21, 36.55, 66.87,
121.03, 132.16, 132.96, 138.95, 139.06, 147.94, 149.68, 156.55, 157.80,
182.13.
Synthesis of Common Intermediate Benzyl 4-Iodopiperidine-1-
carboxylate (18) Used in the Syntheses of Keto-benzimidazoles 5
and 6. Benzyl 4-Iodopiperidine-1-carboxylate (18). To a mixture of
imidazole (11.5 g, 168 mmol), triphenylphosphine (43.9 g, 167 mmol),
and 17 (32.9 g, 140 mmol) was added THF (100 mL). The solution was
cooled to 0 °C, and a solution of iodine (42.7 g, 168 mmol) in THF (100
mL) at 0 °C was added dropwise over 20 min. After the addition, the
reaction mixture was warmed to room temperature. After 18 h, the
reaction was quenched with saturated Na2SO3 and diluted with hexanes
(200 mL). The aqueous phase was extracted with hexanes (3×). The
combined organic extracts were washed with brine (1×), dried over
Step 2. Ethyl 4-(3-(3,6-Dihydro-2H-pyran-4-yl)pyrazin-2-yloxy)-
benzoate (11). To a mixture of 9 (0.56 g, 2.0 mmol), PdCl2((t-
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MgSO4, filtered, and concentrated. The concentrate was diluted with
DCM (50 mL) followed by hexanes (200 mL). The solution was
concentrated to half the original volume, and the precipitate that formed
was removed and the filtrate was concentrated. Purification by flash
column chromatography on silica gel (eluted with 0−20% EtOAc in
hexanes) gave 18 (40.4 g, 84% yield) as a colorless oil. 1H NMR (400
MHz, CDCl3-d) δ ppm 2.03 (br s, 4 H), 3.40 (dt, J = 13.60, 5.72 Hz, 2
H), 3.66 (dt, J = 13.45, 5.31 Hz, 2 H), 4.46 (quin, J = 5.80 Hz, 1 H), 5.13
(s, 2 H), 7.31−7.39 (m, 5 H). LC−MS m/z [M + H] 346.1.
Synthesis of Common Intermediate (1H-Benzo[d]imidazol-2-
yl)(4-hydroxyphenyl)methanone (23) Used in Syntheses of Keto-
benzimidazoles 4, 5, and 6. Step 1. Ethyl 4-((4-Methoxybenzyl)oxy)-
benzoate (22). To a stirred solution of 8 (467 g, 2.8 mol) in DMF (5.3
L) were added potassium carbonate powder (1 kg, 7.2 mol), NaI (42 g,
0.28 mol), and PMB-Cl (460 mL, 3.37 mol) at ambient temperature.
The reaction mixture was heated at 50 °C overnight under a nitrogen
atmosphere. The reaction mixture was cooled to ambient temperature.
Ice−water (8 L) was added, and the resulting mixture was stirred for 2 h.
The precipitate was filtered and washed with water (4 × 2 L). The solid
was then slurried in water (2 × 3.5 L) for 30 min, filtered, and dried at 45
°C under vacuum for 3 days to obtain 22 (794 g, 98% yield).
Step 2. (1H-Benzo[d]imidazol-2-yl)(4-((4-methoxybenzyl)oxy)-
phenyl)methanone. A solution of 13 (112.8 g, 0.95 mol), triethyl
orthoformate (317 mL, 1.9 mol), and benzenesulfonic acid (4.1 g, 25.8
mmol) in toluene (1 L) was heated to reflux, and 500 mL was removed
by distillation. Toluene (750 mL) was added again, and 500 mL was
removed by slow distillation. The reaction mixture was cooled and
neutralized with diisopropylamine (13.4 mL, 95.4 mmol). To the
reaction mixture was added THF (1 L) and 22 (300 g, 1.0 mol). The
mixture was cooled to −78 °C before addition of LDA (572 mL, 2 M in
THF/hexane/ethylbenzene, 1.1 mol) dropwise over 4 h. After the
addition, the reaction mixture was stirred at −78 °C for 2 h and then
allowed to gradually warm to ambient temperature. A solution of 2 N
aqueous HCl (3.9 L) was added, and the resulting mixture was stirred
overnight. The mixture was diluted with an additional 2 L of water, and
the layers were separated. The organic layer was washed with 10%
aqueous NaHCO3 solution (2 L) and brine (1.5 L), dried over Na2SO4,
and filtered. The filtrate was concentrated. Purification by flash
chromatography on silica gel (0% to 50% DCM/EtOAc) to afford
product that was further triturated with MTBE to give (1H-
benzo[d]imidazol-2-yl)(4-((4-methoxybenzyl)oxy)phenyl)methanone
(229 g, 66% yield).
0−20% EtOAc in hexanes) gave 15 (6.09 g, 89% yield). 1H NMR (400
MHz, DMSO-d6) δ 8.13 (td, J = 1.69, 4.84 Hz, 1H), 7.89−8.00 (m, 1H),
7.37 (ddd, J = 2.15, 4.99, 7.34 Hz, 1H), 6.21−6.30 (m, 1H), 4.24 (q, J =
2.74 Hz, 2H), 3.82 (t, J = 5.38 Hz, 2H), 2.39−2.47 (m, 2H). LC−MS m/
z [M + H] 180.2.
Step 2. 2-Fluoro-3-(tetrahydro-2H-pyran-4-yl)pyridine (16). To a
mixture of palladium hydroxide (20% by weight palladium on carbon,
0.98 g, 1.4 mmol) and 15 (2.6 g, 15 mmol) was added EtOH (45 mL).
The argon atmosphere was replaced by hydrogen from a double balloon,
and the reaction mixture was stirred at room temperature. After 4 h, the
reaction mixture was filtered and concentrated to give 16 (2.5 g, 96%
yield) as a pale-yellow solid that was used without additional
1
purification. H NMR (400 MHz, CDCl3-d) δ ppm 1.76−1.87 (m, 4
H), 3.01−3.11 (m, 1 H), 3.50−3.62 (m, 2 H), 4.09 (dt, J = 11.35, 3.03
Hz, 2 H), 7.17 (ddd, J = 7.19, 5.14, 1.56 Hz, 1 H), 7.64 (td, J = 8.42, 1.56
Hz, 1 H), 8.07 (d, J = 4.89 Hz, 1 H). LC−MS m/z [M + H] 182.2.
Step 3. (1H-Benzo[d]imidazol-2-yl)(4-(3-(tetrahydro-2H-pyran-4-
yl)pyridin-2-yloxy)phenyl)methanone (4). A mixture of 16 (1 g, 5.5
mmol), 23(2.6 g, 11.0 mmol), and cesium carbonate (3.6 g, 11.0 mmol)
in NMP (11 mL) was heated in a microwave reactor at 180 °C for 1 h
and at 200 °C for 2 h. The mixture was partitioned between water (20
mL) and DCM (30 mL). The layers were separated, and the aqueous
layer was extracted with DCM (3 × 30 mL). The combined organic
layers were dried over magnesium sulfate and concentrated under
reduced pressure. Purification by flash chromatography on silica gel (2−
6% MeOH/DCM). The isolated material was then recrystallized from
ethanol to deliver 4 (1.1 g, 2.7 mmol, 48.0% yield). 1H NMR (300 MHz,
DMSO-d6) δ 13.49 (br s, 1H), 8.68 (d, J = 8.92 Hz, 2H), 8.06 (d, J = 4.58
Hz, 1H), 7.86 (d, J = 7.02 Hz, 1H), 7.31 (d, J = 8.92 Hz, 4H), 7.22 (d, J =
2.78 Hz, 1H), 3.97 (d, J = 11.40 Hz, 2H), 3.39−3.56 (m, 2H), 3.08−3.25
(m, 1H), 1.71−1.88 (m, 4H). LC−MS m/z [M + H] 400.1. HRMS (ES
+) calcd for [C24H22N4O3]+ 400.1661; found 400.1658. 13C NMR (75
MHz, DMSO-d6) δ 182.6, 160.2, 159.7, 148.5, 145.4, 143.7, 137.8,
134.6, 133.5, 131.8, 130.0, 126.1, 123.6, 121.7, 121.0, 120.9, 113.3, 67.8,
35.1, 32.2.
Synthesis of 1-(4-(3-(4-(1H-Benzo[d]imidazole-2-carbonyl)-
phenoxy)pyrazin-2-yl)piperidin-1-yl)ethanone (5). Step 1. Benzyl 4-
(3-Fluoropyrazin-2-yl)piperidine-1-carboxylate (20a). To zinc dust
(0.26 g, 4.0 mmol) at room temperature was added dimethylacetamide
(2.5 mL), and 0.08 mL of a 7:5 v/v mixture of TMSCl/1,2-
dibromoethane was added over 1 min. After 2 min, the solution became
warm and bubbled. The solution was stirred for an additional 15 min,
and 18 (1.3 g, 3.3 mmol) was added dropwise over 5 min. The mixture
was stirred for 30 min and added via cannula to a degassed mixture of
PdCl2dppf (0.01 g, 0.01 mmol), CuI (0.02 g, 0.24 mmol), and 19 (0.51
g, 2.3 mmol) in dimethylacetamide (3 mL) at room temperature. The
reaction mixture was heated to 80 °C for 75 min and diluted with EtOAc
and 1 M NaH2PO4. The aqueous phase was extracted with EtOAc (2×),
and the combined organic extracts were washed with brine (1×), dried
over MgSO4, filtered, and concentrated. Purification by flash column
chromatography on silica gel (eluted with 5−30% EtOAc in hexanes)
Step 3. (1H-Benzo[d]imidazol-2-yl)(4-hydroxyphenyl)methanone
(23). To a slurry of compound (1H-benzo[d]imidazol-2-yl)(4-((4-
methoxybenzyl)oxy)phenyl)methanone (220 g, 0.61 mol) in DCM (2.2
L) at ambient temperature was added TFA (235 mL, 3.1 mol) slowly
using an addition funnel over a period of 2.5 h. The reaction mixture was
stirred at ambient temperature for 18 h. The reaction mixture was
neutralized with saturated aqueous NaHCO3 (4 L) and stirred for an
additional 30 min. The resulting solid was filtered and washed with water
(3 × 1 L) and air-dried. The material was further dried under vacuum at
45 °C to remove water. The material was washed with hexanes:EtOAc
(3:2; 3×500 mL) and dried under vacuum at 45 °C to give 23 (136 g,
1
gave 20a (0.61 g, 85% yield) as an orange oil. H NMR (400 MHz,
CDCl3-d) δ ppm 1.81−1.94 (m, 4 H), 2.96 (br s, 2 H), 3.19 (quin, J =
7.60 Hz, 1 H), 4.35 (br s, 2 H), 5.15 (s, 2 H), 7.29−7.39 (m, 5 H), 8.05
(t, J = 2.15 Hz, 1 H), 8.41 (dd, J = 4.11, 2.54 Hz, 1 H). LC−MS m/z [M
+ H] 316.2.
1
99% yield). H NMR (300 MHz, DMSO-d6) δ 13.39 (s, 1H), 10.62
(s,1H), 8.62 (d, J = 9 Hz., 2H), 7.87 (d, J = 8.1 Hz, 1H), 7.6 (d, J = 5.7
Hz, 1H), 7.32−7.42 (m, 2H), 6.98 (d, J = 9 Hz, 2H). LC−MS m/z [M +
H] 239.1.
Step 2. 1-(4-(3-Fluoropyrazin-2-yl)piperidin-1-yl)ethanone (21a).
To a mixture of palladium hydroxide (20% by weight palladium on
carbon, 19.4 g, 13.8 mmol) and 20a (57.0 g, 181 mmol) was added
PhMe (700 mL) and acetic anhydride (100 mL, 1.1 mol). The argon
atmosphere was replaced by hydrogen from a double balloon. Hydrogen
was bubbled directly into the solution via a needle for 10 min, and the
reaction mixture was stirred at room temperature. After 9 h, the solution
was filtered. The filtrate was diluted with brine and EtOAc, and the pH
was adjusted to 9 with 5 M NaOH. The aqueous phase was extracted
with EtOAc (5×), and the combined organic extracts were washed with
brine (1×), dried over MgSO4, filtered, and concentrated. Purification
by flash column chromatography on silica gel (eluted with 30−80%
EtOAc (10% MeOH)) gave 21a (36.8 g, 91% yield) as a yellow oil. 1H
NMR (400 MHz, CDCl3-d) δ ppm 1.75−1.97 (m, 4 H) 2.14 (s, 3 H)
Synthesis of (1H-Benzo[d]imidazol-2-yl)(4-(3-(tetrahydro-2H-
pyran-4-yl)pyridin-2-yloxy)phenyl)methanone (4). Step 1. 3-(3,6-
Dihydro-2H-pyran-4-yl)-2-fluoropyridine (15). To a 500 mL round-
bottom flask was added 14 (8.7 g, 49 mmol), 10 (8.0 g, 38 mmol), and
disodium carbonate (12.1 g, 114 mmol) in DME (75 mL) and water (25
mL). Argon was bubbled through the resulting solution for 3 min.
Bis(triphenylphosphine)palladium(II) chloride (1.5 g, 2.1 mmol) was
added while still keeping the reaction under argon. The resulting
mixture was heated to 80 °C. After overnight heating, the reaction
mixture was evaporated by rotovap to remove DME. The residue was
diluted with water and extracted with EtOAc (2×). The organic layers
were combined and washed with brine and dried over sodium sulfate.
Purification by flash column chromatography on silica gel (eluted with
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2.74 (td, J = 13.10, 2.74 Hz, 1 H), 3.20−3.29 (m, 2 H), 3.97 (dm, J =
13.30 Hz, 1 H), 4.77 (dm, J = 13.11 Hz, 1 H), 8.07 (t, J = 2.15 Hz, 1 H),
8.42 (dd, J = 4.21, 2.64 Hz, 1 H). LC−MS m/z [M + H] 224.2.
Step 3. 1-(4-(3-(4-(1H-Benzo[d]imidazole-2-carbonyl)phenoxy)-
pyrazin-2-yl)piperidin-1-yl)ethanone (5). To a mixture of cesium
carbonate (7.4 g, 22.6 mmol), 23 (5.3 g, 22.4 mmol), and 21a (2.5 g,
11.2 mmol) was added NMP (22 mL). The mixture was degassed by
bubbling argon through the solution for 5 min and heated to 120 °C.
After 5 h, the reaction was diluted with EtOAc, saturated NH4Cl, and
water. The aqueous phase was extracted with EtOAc (4×). The
combined organic extracts were washed with brine (1×), dried over
MgSO4, filtered, and concentrated. Purification by flash column
chromatography on silica gel (eluted with 50−100% EtOAc in hexanes)
gave 5 (4.3 g, 87% yield) as a white solid. 1H NMR (400 MHz, CDCl3-d)
δ ppm 1.81−2.06 (m, 4 H), 2.16 (s, 3 H), 2.78 (td, J = 12.86, 2.64 Hz, 1
H), 3.23−3.32 (m, 1 H), 3.38−3.51 (m, 1 H), 4.00 (dm, J = 14.28 Hz, 1
H), 4.81 (dm, J = 13.11 Hz, 1 H), 7.31 (d, J = 8.80 Hz, 2 H), 7.39 (td, J =
7.20, 1.17 Hz, 1 H), 7.45 (td, J = 7.20, 0.98 Hz, 1 H), 7.60 (d, J = 8.22 Hz,
1 H), 7.95−7.99 (m, 2 H), 8.28 (d, J = 2.74 Hz, 1 H), 8.89 (d, J = 8.10
Hz, 2 H), 10.50 (br s, 1 H). LC−MS m/z [M + H] 442.3. HRMS (ES+)
calcd for [C25H24N5O3]+ 442.1879; found 442.1877. 13C NMR (101
MHz, CDCl3-d) δ ppm 21.55, 29.86, 30.15, 37.99, 41.75, 46.61, 112.00,
120.98, 122.27, 123.82, 126.50, 132.20, 133.14, 133.58, 138.99, 139.02,
144.02, 147.81, 149.91, 156.94, 158.19, 168.94, 182.19.
J = 9.59, 7.63, 1.76 Hz, 1 H) 8.04−8.13 (m, 1 H). LC−MS m/z [M + H]
223.1.
Step 3. 1-(4-(2-(4-(1H-Benzo[d]imidazole-2-carbonyl)phenoxy)-
pyridin-3-yl)piperidin-1-yl)ethanone (6). To a mixture of cesium
carbonate (2.4 g, 7.3 mmol), 23 (1.7 g, 7.0 mmol), and 21b (0.89 g, 4.0
mmol) was added NMP (8 mL). The reaction mixture was degassed and
heated to 145 °C for 3 h, 150 °C for 12 h, 180 °C for 1 h, and then at 200
°C for 2 h. The reaction mixture was diluted with EtOAc and water. The
pH was adjusted to 7 using concentrated hydrogen chloride solution.
The aqueous phase was extracted with EtOAc (3×), and the combined
organic extracts were washed with brine (1×), dried over magnesium
sulfate, filtered, and concentrated. Purification by flash column
chromatography on silica gel (30−100% of 10% MeOH/EtOAc and
hexanes) gave the product. The residual NMP was removed by
dissolving the product in EtOAc (200 mL). The organic phase was
washed with water (3×) and brine (1×), dried over magnesium sulfate,
filtered, concentrated, and dried under high vacuum to give 6 (1.2 g, 2.7
1
mmol, 68% yield) as a white solid. H NMR (300 MHz, CDCl3-d) δ
ppm 1.62−1.79 (m, 2 H) 1.93−2.10 (m, 2 H) 2.15 (s, 3 H) 2.69 (td, J =
12.93, 2.63 Hz, 1 H) 3.16−3.31 (m, 2 H) 3.97 (d, J = 13.74 Hz, 1 H)
4.80−4.91 (m, 1 H) 7.10 (dd, J = 7.53, 4.90 Hz, 1 H) 7.24−7.26 (m, 1
H) 7.27−7.29 (m, 1 H) 7.35−7.50 (m, 2 H) 7.56−7.64 (m, 2 H) 7.97 (d,
J = 8.33 Hz, 1 H) 8.10 (dd, J = 4.82, 1.75 Hz, 1 H) 8.83−8.91 (m, 2 H)
10.38 (br s, 1 H). LC−MS m/z [M + H] 441.2. HRMS (ES+) calcd for
[C26H25N4O3]+ 441.1927; found 441.1924. 13C NMR (101 MHz,
CDCl3-d) δ ppm 21.57, 31.03, 32.32, 35.97, 42.13, 47.02, 112.01,
120.13−120.32, 122.20, 123.69, 126.32, 129.38, 131.31, 133.19, 133.59,
136.56, 144.00, 145.50, 147.98, 159.89, 169.01, 182.26.
Synthesis of 1-(4-(2-(4-(1H-Benzo[d]imidazole-2-carbonyl)-
phenoxy)pyridin-3-yl)piperidin-1-yl)ethanone (6). Step 1. Benzyl 4-
(2-Fluoropyridin-3-yl)piperidine-1-carboxylate (20b). To an oven-
dried 25 mL round-bottomed flask was charged dry dimethylacetamide
(5 mL) and zinc dust (2.1 g, 32 mmol). The reaction mixture was stirred
at room temperature, while a mixture of chlorotrimethylsilane (0.33 mL,
2.6 mmol) and 1,2-dibromoethane (0.22 mL, 2.6 mmol) was added
slowly. The resulting slurry was aged for 15 min. A solution of 18 (8.9 g,
25.8 mmol) in dimethylacetamide (13 mL) was then added slowly to the
above mixture. After stirring for 30 min, the resulting milky solution was
cooled to room temperature (solution A). To an oven-dried flask were
charged 14 (3.3 g, 18.6 mmol), dichloro[1,1′-bis(diphenylphosphino)-
ferrocene]palladium(II) dichloromethane adduct (0.46 g, 0.56 mmol),
copper(I) iodide (0.21 g, 1.1 mmol), and dimethylacetamide (25 mL).
The resulting mixture was degassed with alternating vacuum/nitrogen
purges. Then solution A from above was filtered through a fitted funnel
into the reaction mixture. The resulting mixture was degassed one more
time and then heated to 80 °C with stirring for 16 h. After cooling to
room temperature, the reaction mixture was partitioned between EtOAc
and 1 N ammonium chloride solution. The aqueous layer was back
extracted with EtOAc (2×), and the combined organic layers were
washed with 1 N ammonium chloride solution and brine, dried over
sodium sulfate, and concentrated. The crude material as purified by
chromatography using a Redi-Sep prepacked silica gel column (120 g),
eluting with a gradient of 0−40% EtOAc in hexane, to provide 20b (4.1
Previously Reported Biological Assays. Detailed descriptions of
our PDE10A biochemical assay, permeability and transcellular transport
assay, rat and human liver microsomal assay, and LC−MS/MS target
occupancy assay have been reported in refs 35 and 39.
Ex vivo Target Occupancy Assays. Animals. Adult male
Sprague−Dawley rats weighing 180−225 g (Harlan, San Diego) were
cared for in accordance to the Guide for the Care and Use of Laboratory
Animals, 8th edition. Animals were group-housed at an Association for
Assessment and Accreditation of Laboratory Animal Committee,
internationally accredited facility in nonsterile ventilated microisolator
housing on corn cob bedding. All research protocols were approved by
the Amgen, Thousand Oaks Institutional Animal Care and Use
Committee (IACUC). Animals had ad libitum access to pelleted feed
(Harlan Teklad 2020X, Indianapolis, IN) and water (on-site generated
reverse osmosis) via automatic watering system. Animals were
maintained on a 12−12 h light−dark cycle in rooms at (70 5 °F, 50
20% RH) and had access to enrichment opportunities (nesting
materials and plastic domes). All animals were sourced from approved
vendors who meet or exceed animal health specifications for the
exclusion of specific pathogens (i.e., mouse parvovirus, Helicobacter).
Rats were allowed at least 3 days of acclimation prior to any procedures.
Ex Vivo RO Assay with PO Administration. PDE10A inhibitors were
dissolved in 2% hydroxypropylmethylcellulose (HPMC), 1% Tween-80,
pH 2.2 with methanesulfonic acid. Four rats per group were dosed orally
with either vehicle or 3 mg/kg PDE10A inhibitors and then returned to
their home cage to allow for absorption of the compounds. After 4 h, rats
were sacrificed by CO2 inhalation.
1
g, 13.0 mmol, 70% yield) as light-yellow oil. H NMR (400 MHz,
CDCl3-d) δ ppm 1.58−1.72 (m, 2 H) 1.87 (d, J = 12.32 Hz, 2 H) 2.82−
3.03 (m, 3 H) 4.36 (m, 2 H) 5.16 (s, 2 H) 7.15 (ddd, J = 7.19, 5.13, 1.56
Hz, 1 H) 7.29−7.42 (m, 5 H) 7.59 (ddd, J = 9.59, 7.63, 1.76 Hz, 1 H)
8.07 (dt, J = 4.74, 1.44 Hz, 1 H). LC−MS m/z [M + H] 315.2.
Step 2. 1-(4-(2-Fluoropyridin-3-yl)piperidin-1-yl)ethanone (21b).
To a solution of 20b (2.9 g, 9.1 mmol) in tetrahydrofuran (46 mL) was
added glacial acetic acid (1.1 mL, 18.3 mmol), acetic anhydride (4.3 mL,
46 mmol), and 10% palladium on carbon (0.97 g, 0.91 mmol). The
reaction mixture was hydrogenated under 1 atm of hydrogen for 4 h.
The reaction mixture was filtered through a pad of Celite and washed
with tetrahydrofuran (3×). The filtrate was concentrated in vacuo. To
the residue were added EtOAc and 1N NaOH. The aqueous layer was
back extracted with EtOAc (3×), and the combined organic layers were
washed with brine, dried over sodium sulfate, and concentrated to give
21b (1.9 g, 8.4 mmol, 92% yield) as a white solid. 1H NMR (400 MHz,
CDCl3-d) δ ppm 1.57−1.72 (m, 2 H) 1.85−2.01 (m, 2 H) 2.14 (s, 3 H)
2.65 (td, J = 12.96, 2.64 Hz, 1 H) 3.03 (tt, J = 12.23, 3.52 Hz, 1 H) 3.21
(td, J = 13.11, 2.54 Hz, 1 H) 3.95 (dt, J = 13.69, 1.96 Hz, 1 H) 4.83 (dt, J
= 13.35, 2.13 Hz, 1 H) 7.16 (ddd, J = 7.14, 5.09, 1.66 Hz, 1 H) 7.60 (ddd,
Sample Analysis and Target occupancy Determination. Blood was
obtained by heart puncture, and plasma was frozen and stored at −80 °C
for exposure analysis. Brains were removed and immediately frozen in
chilled methylbutane and stored at −80 °C until cutting. Three coronal
brain slices per brain containing the striatum were cut at 20 μm using a
cryostat and placed onto microscope slides, air-dried, and stored at −20
°C. For radioligand binding experiments, slides were thawed at room
temperature and then incubated with 1 nM 3H-24 in binding buffer (150
mM phosphate-buffered saline containing 2 mM MgCl2 and 100 mM
DTT, pH 7.4) for 1 min at 4 °C. To assess nonspecific binding, slides
containing adjacent brain sections were incubated in the same solution
with addition of 10 mM of test compound. Afterward, slides were
washed 3 times in ice-cold binding buffer, dipped into distilled water to
remove buffer salts, and dried under a stream of cold air. Emission of
beta particles from the sections was counted for 8 h in a Beta Imager
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Article
2000 (Biospace, Paris, France) and digitized and analyzed using M3
Vision software (Biospace, Paris, France). Total binding radioactivity in
the striatum was measured as cpm/mm2 in hand-drawn regions of
interest and averaged across the three sections per brain. Nonspecific
binding was subtracted to obtain specific binding values and percent
occupancy was calculated by setting vehicle specific binding as 0%
occupancy.
chromatography−tandem mass spectroscopy; LDA, lithium
diisopropylamide; NaI, sodium iodide; NHP, nonhuman
primate; NMP, N-methyl-2-pyrrolidone; PCP-LMA, phencycli-
dine induced locomotor activity; PDE, phosphodiesterase; QD,
once a day dosing; RLM, rat liver microsome; SAR, structure−
activity relationship; SOC, standard of care; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; TMSCl, trimethylsilyl chloride; TO,
target occupancy
Behavioral Model. 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 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 12−12 h light−dark cycle, with food and water available ad
libitum. Animals were allowed a minimum 3 days of adaptation to the
laboratory conditions prior to being utilized in the experiments.
Reversal of PCP-Induced Hyperlocomotor Activity. To measure
locomotor activity, group-housed animals were taken from the animal
colony room, weighed and numbered, and placed individually into a new
cage with bedding. The cage was then placed in a 4 × 8 photobeam
frame connected to a computer for activity monitoring (Home Cage
Photo-Beam Activity System, San Diego Instruments, San Diego, CA).
The number of photobeam breaks is correlated with locomotor activity,
and total photobeam breaks were measured and recorded every 5 min.
Assessment of locomotor activity occurred over 3 phases: In phase 1,
animals were acclimated to the novel testing environment in the absence
of any drug treatment for 60 min. To begin phase 2, animals were
administered the test article (5, n = 10; risperidone, n = 9; or vehicle, n =
9), returned to the test cage, and activity was recorded for another 60
min period. Finally, in phase 3, animals were injected subcutaneously
with PCP (8 mg/kg), and locomotor activity was recorded for an
additional 120 min.
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Statistical Analysis. Behavioral results are expressed as the mean
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ASSOCIATED CONTENT
* Supporting Information
■
S
Details of computational model of 3, methods for determination
of X-ray co-crystal structure of 4 with PDE10A, and summary of
data collection are provided. Methods and data for co-crystal
structure of 1 were previously reported in ref 35. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Accession Codes
The PDB ID codes for coordinates of 1 and 4 with PDE10A are
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AUTHOR INFORMATION
Corresponding Author
*Phone: 805-313-5300. E-mail: ehu@amgen.com.
Notes
■
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
cAMP, cyclic adenosine monophosphate; cGMP, cyclic
guanosine monophosphate; cNMP, cyclic nucleotide mono-
phosphate; DMA, dimethylacetamide; DMF, dimethylforma-
mide; DMSO, dimethyl sulfoxide; HLM, human liver micro-
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