Discovery of dual inducible/neuronal nitric oxide synthase (iNOS/nNOS) inhibitor development candidate 4-((2-cyclobutyl-1 H-imidazo[4,5- b ]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-2(1 H)-one (KD7332) Part 2: Identification of a novel, potent, and selective series of benzimidazole- quinolinone iNOS/nNOS dimerization inhibitors that are orally active in pain models

Article abstract of DOI:10.1021/jm100828n

Three isoforms of nitric oxide synthase (NOS), dimeric enzymes that catalyze the formation of nitric oxide (NO) from arginine, have been identified. Inappropriate or excessive NO produced by iNOS and/or nNOS is associated with inflammatory and neuropathic pain. Previously, we described the identification of a series of amide-quinolinone iNOS dimerization inhibitors that although potent, suffered from high clearance and limited exposure in vivo. By conformationally restricting the amide of this progenitor series, we describe the identification of a novel series of benzimidazole-quinolinone dual iNOS/nNOS inhibitors with low clearance and sustained exposure in vivo. Compounds were triaged utilizing an LPS challenge assay coupled with mouse and rhesus pharmacokinetics and led to the identification of 4,7-imidazopyrazine 42 as the lead compound. 42 (KD7332) (J. Med. Chem. 2009, 52, 3047 -3062) was confirmed as an iNOS dimerization inhibitor and was efficacious in the mouse formalin model of nociception and Chung model of neuropathic pain, without showing tolerance after repeat dosing. Further 42 did not affect motor coordination up to doses of 1000 mg/kg, demonstrating a wide therapeutic margin.

Full text of DOI:10.1021/jm100828n

J. Med. Chem. 2010, 53, 7739–7755 7739
DOI: 10.1021/jm100828n
Discovery of Dual Inducible/Neuronal Nitric Oxide Synthase (iNOS/nNOS) Inhibitor Development
Candidate 4-((2-Cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-2(1H)-one
(KD7332) Part 2: Identification of a Novel, Potent, and Selective Series of Benzimidazole-Quinolinone
iNOS/nNOS Dimerization Inhibitors That Are Orally Active in Pain Models
Joseph E. Payne,†,#,[ Celine Bonnefous,†,#,3 Kent T. Symons,‡,1 Phan M. Nguyen,‡ Marciano Sablad,§,O
ꢀ
Natasha Rozenkrants,§,O Yan Zhang,§,O Li Wang, ,z Nahid Yazdani,^,þ Andrew K. Shiau,‡,] Stewart A. Noble,†
Peter Rix, ,3 Tadimeti S. Rao,§,O Christian A. Hassig,‡,b and Nicholas D. Smith*,†,3
†Department of Chemistry, ‡Department of Biology, §Department of Pharmacology, Department of Drug Metabolism and Pharmacokinetics, and
^Department of Analytical Chemistry , Kalypsys, Inc., 10420 Wateridge Circle, San Diego, California 92121. # These authors contributed equally
to this work. 3 Current affiliation: Aragon Pharmaceuticals, San Diego, California 92121. O Current affiliation: Johnson and Johnson PRD,
San Diego, California 92121. [ Current affiliation: Nitto Denko Technical Corporation, Carlsbad, California 92058. z Current affiliation:
Bioquant, San Diego, California 92121. þ Current affiliation: Helicon, San Diego, California 92121. 1 Current affiliation: Dart Neuroscience,
San Diego, California 92121. b Current affiliation: Sanford Burnham Research Center, San Diego, California 92121. ] Current affiliation:
Ludwig Cancer Research Center, University of California, San Diego, California 92037
Received July 3, 2010
Three isoforms of nitric oxide synthase (NOS), dimeric enzymes that catalyze the formation of nitric
oxide (NO) from arginine, have been identified. Inappropriate or excessive NO produced by iNOS and/
or nNOS is associated with inflammatory and neuropathic pain. Previously, we described the
identification of a series of amide-quinolinone iNOS dimerization inhibitors that although potent,
suffered from high clearance and limited exposure in vivo. By conformationally restricting the amide of
this progenitor series, we describe the identification of a novel series of benzimidazole-quinolinone dual
iNOS/nNOS inhibitors with low clearance and sustained exposure in vivo. Compounds were triaged
utilizing an LPS challenge assay coupled with mouse and rhesus pharmacokinetics and led to the
identification of 4,7-imidazopyrazine 42 as the lead compound. 42 (KD7332) (J. Med. Chem. 2009, 52,
3047-3062) was confirmed as an iNOS dimerization inhibitor and was efficacious in the mouse formalin
model of nociception and Chung model of neuropathic pain, without showing tolerance after repeat
dosing. Further 42 did not affect motor coordination up to doses of 1000 mg/kg, demonstrating a wide
therapeutic margin.
Introduction
expressed and is critical in the maintenance of homeostatic
blood pressure, requiring that NOS inhibitor based therapeu-
tics should selectively inhibit the iNOS and/or nNOS isoforms,
and not inhibit eNOS.
Three isoforms of nitric oxide synthase (NOSa), dimeric
enzymes that catalyze the formation of nitric oxide (NO) from
arginine, have been identified.1 Of the three isoforms, iNOS
and nNOS are closely associated with pain. iNOS, which is
synthesized in response to inflammatory and immunologic
stimuli and is up-regulated following peripheral nerve injury,
is implicated in the pathogenesis of numerous diseases includ-
ing asthma, inflammatory bowel disease, arthritis, and neuro-
pathic pain.2,3 While NO derived from constitutively expressed
nNOS has been shown to augment N-methyl-D-aspartate
(NMDA) release and is associated with the development of
chronic pain,4 the third isoform, eNOS, is also constitutively
Drug discovery efforts to date have primarily focused on the
development of iNOS selective inhibitors, particularly substrate
competitive inhibitors based on the structure of arginine, lead-
ing to compounds such as L-NIL (1), a nonselective NOS
inhibitor (Figure 1).5 Also related to the structure of arginine,
GW271450 (2) is an iNOS selective, time-dependent inhibitor
that has demonstrated efficacy in preclinical models of pain.6
However, its close structural similarity to arginine may lead to
interference with physiological processes dependent on arginine
transport and metabolism.7 Small molecule inhibitors that are
structurally distinct from arginine but that still incorporate a
guanidine motif have also been identified. Among these is 3
(AR-C102222),8 a substrate competitive inhibitor which
showed good selectivity for iNOS and was efficacious in a rat
adjuvant-induced arthritis model. Inhibitors of iNOS dimeriza-
tion have also been identified such as 4 (BBS-4),9 a potent and
selective imidazole-based inhibitor that coordinates to the heme
iron in the active site of the enzyme. Although, there have been
far fewer reports of nNOS selective inhibitors, examples do exist
*To whom correspondence should be addressed. Phone: (858) 754-3481.
Fax: (858) 754 3301. E-mail: nsmith@aragonpharm.com. Address: Aragon
Pharmaceuticals, Inc., 4215 Sorrento Valley Boulevard, Suite 215, San Diego,
California 92121.
aAbbreviations: NOS, nitric oxide synthase; NO, nitric oxide; iNOS,
inducible NOS; eNOS, endothelial NOS; nNOS, neuronal NOS; hiNOS,
human iNOS; miNOS, mouse iNOS; LPS, lipopolysaccharide; SDS-
PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis;
NMDA, N-methyl-D-aspartate; DAN, 2,3-diaminonaphthalene; LRMS,
low-resolution mass spectra; HRMS, high-resolution mass spectra.
r
2010 American Chemical Society
Published on Web 10/08/2010
pubs.acs.org/jmc

7740 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Payne et al.
Figure 1. NOS inhibitors.
2(1H)-one (KD7332),12 a potent dual iNOS/nNOS inhi-
bitor with efficacy in multiple animal pain models. The “diaryl
series” represented by 11 will be discussed in separate
publication.
Chemistry
The compounds described in this paper were generally
prepared by the alkylation of an appropriately substituted
benzimidazole core with the bromomethyl quinolinones 57 or
58 (Scheme 1). Synthons 57 and 58 were prepared as follows:
keto-amide 53 formed by the reaction of 2-fluoroaniline 51
with methyl 3-oxobutanoate was selectively brominated at the
R position14 to give 55, which was cyclized under acidic
conditions to afford 8-fluoroquinolinone 57 (R1 = H). The
7,8-difluoroquinolinone 58 was similarly prepared via an
analogous sequence. The 2-substituted benzimidazole cores
78-98 were prepared under standard condensation condi-
tions from the appropriate carboxylic acid and 1,2-diamiono-
benzene and then alkylated with either 57 or 58 using NaH/
DMF to afford target compounds 10 and 12-32.
Compounds 33-36, 42, and 44-50, which contain a nitro-
gen in the six-membered ring of the benzimidazole, were
prepared as shown in Scheme 2. Thus cores 104-110 were
prepared from an appropriately substituted carboxylic acid
and a 1,2-diaminoheterocycle, deprotonated (NaH/DMF),
and reacted with bromide 58.15 However, in contrast to the
benzimdazolecores 78-98 shownin Scheme1, the presence of
additional ring nitrogens in 104-110 meant that there was
more than one potential site of alkylation (and hence product
regioselectivity). In the case of compound 42, which has a
symmetrical benzimidazole core (X1 = X4 = N; X2 = X3 =
CH), regiochemical assignment was confirmed by the distinc-
tive chemical shift of the quinolinone C3 hydrogen16 and by
NOE experiments. The regiochemistry of the related com-
pounds 44-50 were similarly confirmed. The regiochemistry
of compounds 33-36, which contain nonsymmetrical benz-
imidazole cores (X1 or X2 or X3 or X4 = N), were determined
by NOE experiments.
Figure 2. Quinolione iNOS dimerization inhibitor 7.
such as aminobenzothiazole (5)10 and 2-aminopyridine (6),11
both of which showed good selectivity over eNOS (>200-fold)
and, in the case of 5, 50-fold selective over iNOS There have
been no potent dual iNOS/nNOS inhibitors with good selec-
tivity over eNOS reported to date.
Previously, we described the identification of quinolinone-
based iNOS dimerization inhibitor 7 from an ultra high-
throughput screen (Figure 2).12 Optimization of 7 resulted
in quinolinone amide 8, a highly potenthumaniNOSinhibitor
(hiNOS = 0.011 μM), that was >2000-fold selective over
human eNOS. Compound 8 was orally active in a mouse
lipopolysaccharide challenge assay measuring plasma nitrates
(ED50 = 10 mg/kg po) and also in formalin and chronic
constriction injury pain models.12 Although useful as a phar-
macological tool, 8 exhibited high clearance (Clp >100 mL/
min/kg) and a short half-life (0.2 h) in mice, which limited
exposureandcontributed toa shortdurationofaction inthese
models. We thussought toevolve the quinolinoneamideseries
to identify potent iNOS selective or dual iNOS/nNOS inhibi-
tors with selectivity over eNOS, but that also had good
pharmacokinetics and a suitable drug property profile for
development as a neuropathic pain therapeutic.
Toward this end, we looked to remove the amide function-
ality of 8 and to restrict conformational flexibility by exploring
the two new series generated by performing connections “A”
and “B” (Figure 3): the “benzimidazole series” (represented by
10) and the “diaryl series” (represented by 11).13 This paper
describes the evolution of the benzimidazole series of human
iNOS inhibitors to the clinical candidate 42, 4-((2-cyclobutyl-
1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-
Compounds 37-41, wherein the 7-position of the imidazo-
pyridine was substituted, were prepared as shown in Scheme3.
In the case of the 7-methyl derivative (37), commercially
available 1,2-diaminopyridine 112 was converted to 113 and
then to the final product 37 by employing conditions detailed
in Scheme 2. For compounds 39-41, the substituent at the
7-position was installed via displacement of the 7-halopyridine

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7741
Figure 3. Restricting conformations of the amide of 8 leads to new series.
Scheme 1a
a Reagents and conditions: (a) methyl 3-oxobutanoate, pyridine, xylene, reflux, 8.5 h, 38-41% yield; (b) (i) bromine, AcOH, 25 ꢀC, 5 h, (ii) acetone,
25 ꢀC, 18 h, 59-68% yield; (c) H2SO4, 45 ꢀC,18 h, 65-89% yield; (d) PPA, 200 ꢀC, 4 h, 21-88% yield; (e) HCl, 100 ꢀC, 18 h, 24-82% yield; (f) NaH,
DMF, 25 ꢀC, 1.25 h, 5-83% yield.
Scheme 2a
As with compound 42, the regiochemistry of alkylation was
confirmed by observing the characteristic chemical shift of the
quinolinone C3 hydrogen.16
Results and Discussion
Cyclization of amide 9 via connection A (Figure 3) gave
benzimidazole 10 (R = H), which resulted in a >10-fold loss in
potency (human iNOS EC50 = 3.7 μM) and a significant
erosion in selectivity over human eNOS (4-fold selective)
(Table 1). Additionally, in contrast to amide 9, benzimidazole
10 was effectively equipotent against human nNOS (hnNOS =
4.3 μM). Previously, we have shown that a 7,8-difluoroquino-
linone substitution pattern led to an increase in potency and, as
expected, incorporation of this moiety into 10 giving 12 led
to an increase in potency (human iNOS EC50 = 1.8 μM).
Importantly, however, 12 showed a dramatic improvement in
a Reagents and conditions: (a) PPA, 100-120 ꢀC, 4 h, 25-90% yield;
mouse microsomal stability compared to 8 (mouse liver micro-
some t1/2 = 98 min versus 9 min)17 and crucially a correspond-
(b) CDI, ACN, 90 ꢀC, 6 h, 72% yield; (c) NaH, DMF, 25 ꢀC, 1.25 h,
ing improvement in in vivo clearance (Clp = 12 mL/min/kg
versus >100 mL/min/kg) and bioavailability (39%F versus
7%F) in mice (Table 6). Encouraged by the improvement in
mouse pharmacokinetics, we sought to increase the potency
and selectivity of this benzimidazole class of NOS inhibitors.
0.5-25% yield; (d) 250 ꢀC, 5 min, 31-75% yield.
intermediates 115 or 116, followed by alkylation with bromo-
methyl quinolinone 58. The 7-chloropyridine intermediate 115
was in turn prepared from pyridine n-oxide 114 using POCl3.

7742 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Payne et al.
Scheme 3a
a Reagents and conditions: (a) PPA, 100 ꢀC, 4 h, 53-77% yield; (b) mCPBA, acetone, 25 ꢀC, 16 h, 84% yield; (c) POCl3, reflux, 2 h, 75% yield; (d) NaI,
TMS-Cl, ACN, reflux, 18 h, 67% yield; (e) dimethylamine, THF, 110 ꢀC, 18 h, 51% yield; (f) sodium methoxide, MeOH, 150 ꢀC, 3 h, 52% yield; (g) KF,
CuI, TMSi-CF3, 55 ꢀC, 18 h, 36% yield; (h) NaH, DMF, 25 ꢀC, 1.25 h, 10-65% yield.
From a SAR perspective, we assumed that the thiazole group of
amide 9 mapped onto the thiazole group of benzimidazole 10
(Figure 3) and incorporated groups that had previously been
shown to be potent thiazole replacements in the amide series as
C-2 benzimidazole substituents (R2, Table 1). As with the
amide series, the ortho-methyl substituent on the R2 ring of
12 was beneficial for potency, as its removal (13) reduced
potency 5-fold (human iNOS EC50 = 8.6 μM). Imidazole 14
and 3-pyridyl 15, two groups previously shown to be of high
potency in the amide series, were also prepared but unexpect-
when comparing the mouse iNOS in vitro potency and in vivo
activity of 20 (in vitro mouse EC50 = 4.4 μM; LPS ED50
=
6 mg/kg po) with that of 8 (in vitro mouse EC50 = 0.12 μM;
ED50 = 10 mg/kg po), clearly illustrating the importance of
good pharmacokinetics for robust in vivo activity (Table 6).
Despite good mouse PK, 20 was still poorly selective over the
human eNOS isoform (14-fold selective), a critical issue that
needed to be addressed. The SAR of the R2 isopropyl sub-
stituent was found to be quite sensitive to steric bulk, as
truncation to an ethyl group (21; human iNOS EC50 = 4.3 μM)
or expansion to a tertiary butyl group (22; human iNOS
EC50 = 48 μM) led to significant decreases in potency, whereas
the simple n-propyl substituent maintained activity (23; human
iNOS EC50 = 0.73 μM). Cyclization of the isopropyl group
to afford the cyclo-propyl derivative 24 led to a minor loss
in potency (human iNOS EC50 = 1.6 μM), however upon
expansion of the ring size to cyclo-butyl (25; human iNOS
edly resulted in reduced iNOS potency (human iNOS EC50
>
5 μM), suggesting that simple overlap between the two series
could not be assumed. With this in mind, a systematic R2
pyridine scan was performed (16, 17, 18), resulting in the
demonstration that the 4-pyridyl derivative (18) was the more
potent inhibitor (human iNOS EC50 = 2.2 μM). Incorporation
of an ortho-methyl substituent on the R2 ring of 18 gave an
increase in potency (19: human iNOS EC50 = 0.34 μM) similar
to that observed for the 13/12 pairing. However, further
increases in potency (and eNOS selectivity) of the benzimid-
azole series could not be achieved by R2 aromatic substitution,
although compounds typically had long liver microsomal half-
lives, suggesting that the good metabolic stability of 12 was a
general characteristic of the benzimidazole scaffold.
Previously, an isopropyl group was shown to be a thiazole
replacement in amide8 (human EC50 = 0.16μM),12 and so we
also examined aliphatic groups at the 2-position of the
benzimidazole (Table 2). We were pleased to discover that
the isopropyl substituted benzimidazole 20 had submicromo-
lar potency against human iNOS (EC50 = 0.80 μM), was
equipotent against human nNOS (EC50 = 0.65 μM), and was
stable in mouse liver microsomes (t1/2 > 120 min). Further,
when tested in vivo, 20 demonstrated improved mouse phar-
macokinetics (Cl = 5.9 mL/min/kg, %F = 58; Table 6) and
was efficacious in the mouse LPS challenge assay (ED50 = 6
mg/kg po). This result in the LPS challenge assay is noteworthy
EC50 = 0.26 μM) or cyclo-pentyl (26; human iNOS EC50
=
0.75 μM), potency improved again. Further increase in ring size
to the cyclo-hexyl derivative 27 led to a loss in potency (human
iNOS EC50 = 8.9 μM), thus demonstrating subtle R2 size
requirements for optimal potency. Of note was the sensitivity
of human eNOS potency to changes in the cyclo-alkyl ring
size, with cyclo-propyl24 andcyclo-butyl 25 inhibiting human
eNOS at <10 μM, whereas with a larger ring size, 26 and 27
were effectively noninhibitory (human eNOS > 100 μM).
Taken with the human iNOS potency trends, this resulted
in improvements in selectivity against human eNOS for 25
(23 fold) and 26 (>130 fold) compared to 20 (14-fold). Com-
pound 25 also had a long half-life in mouse liver microsomes
(t1/2 > 120 min) and had good exposure following oral dosing
in mouse (Cmax = 5.7 μg/mL, AUC = 10 μg h/mL at 10 mg/
3
kg po), which translated into an ED50 = 4 mg/kg in the LPS
challenge assay. We were able to further exploit the subtle
nature of human iNOS/eNOS SAR by preparing the chain
extended homologues of 20, isobutyl derivative 28, and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7743
Table 1. SAR Examining Heterocycles at the R2 Position of the Benzimidazole
a Cell-based NOS assay using transiently transfected HEK293 cells; NO measured using 2,3-diaminonapthalene (DAN). Each EC50 is determined from three
separate assay runs, each using a 10-point concentration curve with n = 8/point for iNOS and n = 3/point for eNOS and nNOS. Standard deviation shown in
brackets. b Elimination rate under conditions of combined phase I oxidation and phase II glucuronide conjugation (mouse). c Not applicable. See Figure 3.
d <50% inihition @ 100 μM. e <50% inhibition @ 10 μM (100% being maximal inhibition of control compond 4). f Not determined. g n = 1 data.
neopentyl derivative 29. Both 28 and 29 maintained activity
against human iNOS (EC50 = 0.38 μM and EC50 =1.36 μM,
respectively) but as with 26 and 27 were highly selective over
human eNOS (EC50 > 100 μM), giving good selectivity ratios
(28: >250 fold; 29: >70 fold). Preparation of the cyclopro-
pylmethylene 30 further demonstrated the importance of
steric bulk for selectivity over human eNOS. Compared to
the closely related isopropyl analogue 28, cyclo-propyl
on the benzimidazole core employing the cyclo-butyl deriva-
tive 25 as the parent molecule (Table 3).
With the exception of 34 (X6 = N; human iNOS EC50
=
18 μM), systematic placement of nitrogen at each position of
the benzimidazole corewas generally tolerated, and in the case
of 36 (human iNOS EC50 = 0.18 μM), a slight increase in
potency was observed compared to 25. As with the alkyl R2
SAR (Table 2), subtle effects were observed with human
eNOS potency, with 33 inhibiting human eNOS with an
EC50 = 8.8 μM (15-fold selective), while 35 and 36 were
effectively inactive on human eNOS (EC50>100 μM), leading
to good selectivity for these molecules (>200-fold selective).
With its improved human iNOS potency and good selectivity
over human eNOS, imidazopyridine 36 represented an im-
portant SAR development. Preliminary SAR on the benzimi-
dazole series (data not shown) had shown that a methyl
substituent was tolerated at the 7-position of the benzimida-
zole, whereas substitution at the 4-, 5- and 6-positions led to a
significant loss in human iNOS potency. Thus the 7-position
of the benzimidazole was investigated further using imidazo-
pyridine 36 as the core (Table 4).
derivative 30 was equipotent against human iNOS (EC50
=
0.60 μM) but had poor selectivity over human eNOS (human
eNOS EC50 = 3.9 μM; selectivity = 7 fold) compared to
>250-fold selectivity for 28. Similar to 30, the cyclobutyl
derivative 31 had poor human eNOS selectivity, but expan-
sion of the ring size to cyclo-pentyl (32) once again led to more
selective human iNOS inhibitors (human eNOS EC50 > 100 μM)
while maintaining potency against human iNOS (EC50
=
1.77 μM). As with isopropyl derivative 20, the compounds
presented in Table 2 generally had good microsomal half-lives
and were equipotent against human nNOS, making them dual
iNOS/nNOS inhibitors.
Having shown that careful choice of alkyl substituents at
the R2 position of the benzimidazole could be used to mod-
ulate human iNOS potency and selectivity over human eNOS,
wenextturnedtoinvestigatethe effect of nitrogen substitution
As illustrated by compounds 37-41 (Table 4), although a
variety of substituents are tolerated at the 7-position, includ-
ing electron donating (39, 40) and electron withdrawing (41)

7744 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Table 2. SAR Examining Aliphatic Groups at the R2 Position
Payne et al.
a Cell-based NOS assay using transiently transfected HEK293 cells; NO measured using 2,3-diaminonapthalene (DAN). Each EC50 is determined
from three separate assay runs, each using a 10-point concentration curve with n = 8/point for iNOS and n = 3/point for eNOS and nNOS. Standard
deviation shown in brackets. b Elimination rate under conditions of combined phase I oxidation and phase II glucuronide conjugation (mouse). c <50%
inihition @ 100 μM. d <50% inhibition @ 10 μM (100% being maximal inhibition of control compond 4).
groups, hiNOS potency was not improved relative to 36
(although the compounds do maintain excellent selectivity over
eNOS). Further, it was found that these 7-substituted deriva-
tives suffered from limited exposure following oral dosing in
mice and corresponding poor performance in the LPS chal-
EC50 = 0.088 μM) demonstrate that human iNOS potency
typically improves 3-5-fold when compared to the unsubsti-
tuted benzimidazole core (compare to 25, 26, 28: Table 2). An
interesting exception to this trend is the isopropyl derivative 46,
which is nearly identical in its in vitro profile to 20 (Table 2).
Compound 45, which is equipotent to 42 against human iNOS
but has improved selectivity over human eNOS (440 fold), also
had good mouse pharmacokinetics (Cl = 6.1 mL/min/kg; t1/2 =
6.0 h ; F = 39%, Table 6) and performed well in the LPS
challenge assay (ED50 = 0.5 mg/kg po, Table 6).
As described in the Introduction, although a dual human
iNOS and nNOS inhibitor was felt to be optimal for efficacy
in neuropathic pain models, we also sought to identify human
iNOS inhibitors that had selectivity over human nNOS (>10-
fold), both as pharmacological tools but also as potential
clinical back-ups. One such compound that showed a modest
increase in selectivity over human nNOS was 3,3-difluorocy-
lenge assay (e.g., 40, AUC = 0.7 μg h/mL at 10 mg/kg po,
3
Table 6). However, a crucial piece of SAR was discovered by
incorporation of a ring nitrogen at the 7-position of 36, giving
symmetrical 4,7-imidazopyrazine 42. Compound 42 exhibited
improved human iNOS potency (human iNOS EC50
0.091 μM), maintained good selectivity over human eNOS
(180 fold selective), was potent against human nNOS (EC50
=
=
0.30 μM), had good mouse pharmacokinetics (Cl = 4.5 mL/
min/kg, F = 62%, Table 6) and an excellent ED50 in the LPS
challenge model (ED50 = 1 mg/kg po, Table 6). Further, the
symmetrical nature of the imidazopyrazine core of 42 simplified
the regioselectivity issues in the synthesis of the other analogues
illustrated in Table 4.18 Having identified this 4,7-imidazopyr-
azine substitution pattern, we returned to incorporate other
potent R2 alkyl groups on this core (Table 5).
clobutane47, whichwas potent against human iNOS (EC50
=
0.12 μM) and showed a 10-fold selectivity over human nNOS
(human nNOS EC50 = 1.1 μM). Further increases in selec-
tivity over human nNOS were obtained by applying pre-
viously established SAR that showed that compounds that
4,7-Imidazopyrazines 42 (human iNOS EC50 = 0.091 μM),
44 (human iNOS EC50 = 0.28 μM), and 45 (human iNOS

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7745
Table 3. Effect of Nitrogen Substitution on the Benzimidazole Core
EC50 ( μM)a
selectivity
compd
X7
X6
X5
X4
iNOS
eNOS
nNOS
eNOS/iNOS
nNOS/iNOS
liver microsome t1/2 (min)b
25
33
34
35
36
C
N
C
C
C
C
C
N
C
C
C
C
C
N
C
C
C
C
C
N
0.26 (0.09)
0.59 (0.1)
18 (7.8)
5.9 (2.4)
8.8 (2.4)
21 (3.8)
>100c
0.32 (0.12)
0.52 (0.25)
24 (21)
23
15
1.2
0.9
1.3
1.6
7.2
>120
>120
>120
NDe
50
1.2
>220
>560
0.46(0.2)
0.18 (0.01)
0.73d
>100c
1.3 (0.3)
a Cell-based NOS assay using transiently transfected H EK293 cells; NO measured using 2,3-diaminonapthalene (DAN). Each EC50 is determined
from three separate assay runs, each using a 10-point concentration curve with n = 8/point for iNOS and n = 3/point for eNOS and nNOS. Standard
deviation shown in brackets. b Elimination rate under conditions of combined phase I oxidation and phase II glucuronide conjugation (mouse). c <50%
inihition @ 100 μM. d n = 1 data. e Not determined.
Table 4. SAR of Substitution at the 7-Position of the Benzimidazole
EC50 (μM)a
selectivity
compd
X
Y
iNOS
eNOS
nNOS
eNOS/iNOS
nNOS/iNOS
liver microsome t1/2 (min)b
36
37
38
39
40
41
42
C
C
C
C
C
C
N
H
Me
0.18 (0.01)
0.27 (0.04)
0.27 (0.07)
0.21 (0.05)
0.17 (0.04)
0.15 (0.06)
0.091 (0.01)
>100c
>100c
>100c
>100c
>100c
>100c
16.5 (3.2)
1.3 (0.3)
1.6 (0.4)
0.76 (0.3)
1.3 (0.1)
1.0 (1.0)
2.0 (1.7)
0.30 (0.06)
>560
>800
>370
>480
>590
>670
180
7.2
12
50
92
Cl
2.8
6.2
5.9
13
36
OMe
NMe2
CF3
>120
8
86
3.3
>120
a Cell-based NOS assay using transiently transfected HEK293 cells; NO measured using 2,3-diaminonapthalene (DAN). Each EC50 is determined
from three separate assay runs, each using a 10-point concentration curve with n = 8/point for iNOS and n = 3/point for eNOS and nNOS. Standard
deviation shown in brackets. b Elimination rate under conditions of combined phase I oxidation and phase II glucuronide conjugation (mouse). c <50%
inihition @ 100 μM.
incorporated an 8-fluoroquinolinone had greater selectivity
over human nNOS than the 7,8-difluoroquinolinone motif.
We thus prepared the 8-fluoroquinolinone derivatives 48, 49,
and 50, which although they lost 2-4-fold in human iNOS
potency, as predicted had greater selectivity over human
nNOS (14-24-fold). Importantly, 48 (Cl = 5.9 mL/min/kg;
F = 91%, Table 6) and 50 (Cl = 6.2 mL/min/kg; F = 55%,
Table 6) had good mouse PK and were efficacious in the LPS
challenge assay with ED50s = 1.5 mg/kg po and 3 mg/kg po,
respectively (Table 6).
Having identified potent iNOS inhibitors with good selec-
tivity over eNOS, favorable mouse pharmacokinetics and
performance in the LPS challenge assay, we selected 42, 45,
48, and 50 for further profiling in rhesus pharmacokinetics. As
shown in Table 7, 42, 48, and 50 all had low clearance (4-9 mL/
min/kg) and good bioavailability (%F = 60-100) in rhesus
monkey, resulting in sustained exposure (AUC@10 mpk
∼20 μg h/mL). A clear exception to this good rhesus pharma-
3
cokinetics was 45, which compared to 42 wasofhigherclearance
(Cl = 18 mL/min/kg) and lower Cmax (0.21 μg/mL) and AUC
(3.2 μg h/mL). Because of the poorer rhesus pharmacokinetics
3
of 45, compound 42 was selected as the lead compound for
further in vitro characterization and profiling in pharmacology
models (vide infra).
Compound 42 was confirmed to disrupt iNOS dimer-
ization by using low-temperature SDS-PAGE to examine
the quaternary structure of the iNOS enzyme. As illus-
trated in Figure 4, treatment with 42 or 4 (an imidazole-
based dimerization inhibitor) resulted in loss of dimeric
enzyme population, being mechanistically distinct from
a substrate competitive inhibitor such as SEITU. These
data demonstrate that compound 42, like its amide

7746 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Payne et al.
Table 5. Incorporation of Favored R2 Groups onto the 4,7-Imidazopyrazine Core
a Cell-based NOS assay using transiently transfected HEK293 cells; NO measured using 2,3-diaminonapthalene (DAN). Each EC50 is determined
from three separate assay runs, each using a 10-point concentration curve with n = 8/point for iNOS and n = 3/point for eNOS and nNOS. Standard
deviation shown in brackets. b Elimination rate under conditions of combined phase I oxidation and phase II glucuronide conjugation (mouse). c <50%
inihition @ 100 μM.
Table 6. Mouse Pharmacokineticsa and LPS ED50 of Selected Compounds
hiNOS
EC50 ( μM)
miNOS
EC50 ( μM)
Clp
(mL/min/kg)
Vdss
(L/kg)
Cmax
( μg/mL)
AUC
( μg h/mL)
LPS ED50
(mg/kg po)
compd
8c
t1/2 (h)
t1/2 (hr)b
%F
3
0.011 (0.002)
1.8 (0.15)
0.12 (0.001)
10.3 (4.2)
4.4 (1.7)
>100
12
0.2
2.3
2.1
NDd
1.7
NDd
2.1
6
1.6
1.2
1.4
3.8
14
0.45
5.67
19.3
10
0.9
3.1
2.6
1.4
5.2
1.4
2.3
1.8
3.1
2.3
7
39
10
12
20
25
28
40
42
45
48
50
30%e
6
0.80 (0.14)
0.26 (0.09)
0.38 (0.07)
0.17 (0.04)
0.091 (0.01)
0.088 (0.01)
0.19 (0.01)
0.22 (0.05)
5.9
0.7
58
2.1 (0.6)
3.1 (1.3)
1.8 (0.6)
NDd
12
NDd
1.1
5.7
8.7
0.4
15
3.7 b
15 b
8.8
NDd
81
4
13.3
0.7
25
85%e
30%e
1
NDd
4.5
NDd
0.39
6.8
NDd
62
0.43 (0.36)
0.44 (0.1)
1.3 (0.7)
6.1
5.9
11
25
39
91
0.5
1.5
3
1.3
4.0
0.47
0.67
3.3 (1.4)
6.2
15
55
a 3 mg/kg iv; 10 mg/kg po. b Per oral t1/2 c 1 mg/kg iv; 10 mg/kg po. d Not determined. e % Reduction in nitrate levels compared to vehicle control
.
(compound dosed at 30 mg/kg po).
Table 7. Rhesus Pharmacokinetics of Selected Compoundsa
compd
Clp (mL/min/kg)
t1/2 (h)
Vdss (L/kg)
Cmax ( μg/mL)
AUC ( μg h/mL)
t1/2 (h)b
%F
3
42
45
48
50
6.0
18
13
21
3.0
19
3.3
0.21
1.8
3.4
18
3.2
23
21
9.0
15
63
59
4.3
8.6
6.8
6.6
1.2
1.8
10
6.1
93
100
a 3 mg/kg iv; 10 mg/kg po. HCl salt. b Per oral t1/2
.
progenitor compound 9,12 acts as an iNOS dimerization
inhibitor.
Compound 42 was also profiled for off-target activity
(Table 8) and did not exhibit any appreciable inhibitory
activity against the five major human CYP P450 isoforms or
in the hERG patch-clamp assay, with the estimated IC50 value
for all targets exceeding 30 μM (similarly 45, 48, and 50 were
also highly selective). The selectivity profile of 42 was also
profiled against a panel of 50 targets comprised of G-protein
coupled receptors, ion channels, transporters of biogenic
Figure 4. Compound 42 disrupts formation of the iNOS dimer.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7747
Table 8. Cytochrome P450 Inhibition and hERG Values of Selected
Compounds
cytochrome P450 inhibition EC50 (μM)
hERG
EC50
compd CYP1A2 CYP3A4 CYP2D6 CYP2C9 CYP2C19 ( μM)
42
45
48
50
>100a
>100a
>100a
35
>100a
>60a
>60a
23
>100a
>100a
>100a
>100a
>60a
>38a
>60a
>60a
27
>30a
>30a
>30a
NTb
29
>60a
22
a <50% inhibition @ indicated concentration. b NT: not tested.
Figure 6. Oral administration of 42 attenuates tactile allodynia in
the Chung model without loss of efficacy following repeated
administration.
Figure 5. Oral dosing of 42 reduces phase II pain behaviors in the
mouse formalin model.
amines, and enzymes (CEREP Screen) at a test concentration
of 10 μM. Under these conditions, 42 did not show any
measurable interaction at any of the targets examined, indicat-
ing that it is remarkably selective and has a unique profile
relative to known iNOS inhibitors described in the literature.2b
Mouse Models of Pain: Formalin and Spinal Nerve Ligation
(Chung) Models. We investigated the ability of compound 42
to reduce pain behaviors in a mouse formalin model of
nociceptive pain.19 Upon injection of formalin into the hind
paw, a biphasic pain response was induced. The duration of
pain behaviors displayed by the injured paw was then counted,
with phase II being from 25 to 45 min postformalin injection.
The duration of pain behaviors evoked in phase II are believed
to involve the central sensitization of spinal neurons.20 As can
be seen from Figure 5, orally administered 42 gave a dose-
responsive attenuation of formalin-induced nocifensive phase
II behaviors, with an ED50 = 13 mg/kg po.21 This compared
favorably to the corresponding ED50 determined for gabapen-
tin in this model (ED50 = 110 mg/kg po).22
Figure 7. Compound 42 has no measurable impact on motor
coordination up to doses of 1000 mg/kg.
motor coordination using the rotarod assay and employing
gabapentin as a reference compound. As can be seen from
Figure 7, gabapentin adversely impacts motor coordination
in mice with a dose-response relationship that effectively
overlaps its efficacy profile in the formalin assay. In con-
trast, compound 42 demonstrated a superior therapeutic
window with no measurable impact on rotarod perfor-
mance (up to a dose of 1000 mg/kg).
Having shown efficacy in a range of neuropathic pain
models22 with a lack of tolerance and side effects, compound
42, a dual iNOS/nNOS inhibitor with good cross species PK,
was nominated as a development progression candidate.
Conclusion
The efficacy of 42 was also assessed in the mouse Chung
model of neuropathic pain induced by spinal nerve ligation
(Figure 6). In this model, oral dosing of 42 (30 mg/kg)
attenuated tactile allodynia to a similar extent as gabapentin
at 300 mg/kg.22 Further, following four days of repeated
administration (30 mg/kg BID), the antiallodynic effects of
42 did not show tolerance, which is in contrast to the
behavior of morphine, which exhibits rapid tolerance in this
model.22
We also profiled 42 in a capsaicin-induced thermal hyper-
algesia model in rhesus monkeys and demonstrated efficacy
in this model. This data and the profiling of compound 42 in
additional rodent pain and inflammation models will be
presented in a future publication.22
Despite being a potent and selective iNOS inhibitor, the
previously described uHTS-derived amide 8 suffered from
highinvivoclearance and correspondingpoor performance in
rodent pain models. By conformationally restricting amide 8,
we identified a novel series of benzimidazole-quinolinone
dual iNOS/nNOS inhibitors that demonstrated improved in
vivo clearance and sustained exposure following oral dosing.
Although initial derivatives suffered from low iNOS potency
and poor selectivity over eNOS, SAR studies led to the identi-
fication of a 4,7-imidazopyrazine core that greatly improved
iNOS potency (and selectivity over eNOS). Compounds were
triaged using an LPS challenge assay measuring reduction in
plasma nitrates, leading to the identification of a number of
potent compounds (LPS ED50 < 5 mg/kg po). LPS assay
performance coupled with mouse and rhesus PK led to the
selection of 42 for further profiling. 42 was confirmed as an
iNOS dimerization inhibitor and was found to be highly
selective against a panel of off-target receptors, enzymes,
Motor Coordination: Rotarod Assay. A number of the
currently prescribed neuropathic pain medications have the
significant adverse side effect of impairing motor coordina-
tion. We thus examined compound 42 for its impact on

7748 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Payne et al.
and channels. 42 reduced phase II pain behaviors in a mouse
formalin model (ED50 = 13 mg/kg) and was efficacious in a
mouse Chung model at 30 mg/kg po (compared to gabapentin
at 300 mg/kg po), without showing tolerance after repeat
dosing. Further, 42 did not impact motor coordination up to
doses of 1000 mg/kg, resulting in a wide therapeutic window
over beneficial antiallodynic effects. In summary, we have
identified development candidate 42, a potent and selective
dual iNOS/nNOS inhibitor with good cross species PK that is
efficacious in models of neuropathic pain.
resonance not observed, 9.18 (s, 1H), 7.87 (dd, 1H, J = 6.6, 1.2 Hz),
7.72 (dd, 1H, J = 6.4, 1.2 Hz), 7.65 (d, 1H, J = 8.4 Hz),
7.52-7.41 (m, 3H), 7.25 (m, 1H), 5.86 (s, 2H), 5.54 (s, 1H), 2.49
(s, 3H). 13C NMR (400 MHz, DMSO-d6, HCl salt) δ 161.4,
158.5, 157.8, 149.7 (d, J = 245 Hz, F coupling), 145.7, 145.4,
137.1, 134.7, 128.5, 128.4, 126.0, 125.9, 122.6 (d, J = 7.3 Hz),
120.8, 119.4, 117.7, 117.0 (d, J = 16.8 Hz), 115.1, 113.1, 46.4,
17.2. LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for C21H15FN4-
OS 391.1, found 391.6 [M þ H]þ/calcd 389.0873, found 389.0872
[M - H]-.
7,8-Difluoro-4-((2-(4-methylthiazol-5-yl)-1H-benzo[d]imidazol-
1-yl)methyl)quinolin-2(1H)-one (12). Compound 12 was synthe-
sized as described for compound 10 using 4-(bromomethyl)-7,
8-difluoroquinolin-2(1H)-one (58) and 5-(1H-benzo[d]imidazol-
2-yl)-4-methylthiazole (78) as the starting materials (37% yield).
HPLC: tR = 5.16 (>99%); mp > 300 ꢀC. 1H NMR (400 MHz,
DMSO-d6, HCl salt) δ NH resonance not observed, 9.22 (s, 1H),
7.89 (dd, 1H, J = 8.4, 1.6 Hz), 7.73-7.69 (m, 2H), 7.51-7.44 (m,
2H), 7.36 (m, 1H), 5.88 (s, 2H), 5.55 (s, 1H), 2.50 (s, 3H). 13C
NMR (400 MHz, DMSO-d6, HCl salt) δ 161.6, 157.5, 157.0,
151.1 (dd, J = 247, 9.5 Hz, F coupling), 146.1, 145.8, 139.4, 137.7
(dd, J = 247, 15.3 Hz, F coupling), 135.2, 130.3, 125.4, 125.0,
121.6, 118.8, 118.1, 116.6, 115.7, 112.5, 111.3 (d, J = 18.4 Hz),
45.9, 17.2. LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for C21H14-
F2N4OS 409.1, found 409.5 [M þ H]þ/calcd 407.0778, found
407.0779 [M - H]-.
Experimental Section
Chemistry. All reactions were carried out under a nitrogen
atmosphere. All solvents and reagents were obtained from
commercial sources (unless otherwise specified) and used with-
out further purification. All yields reported are not optimized.
Silica gel column chromatography was carried out on either a
Jones Chromatography Flashmaster II or an Isco Combiflash
Companion XL using prepacked silica gel columns. Reversed-
phase semipreparative HPLC purifications were carried out
using a Shimadzu Discovery VP system with a SPD-20A
prominence UV/vis detector (190-700 nm range). The columns
used were either a Waters SunFire C18 (19 mm ꢀ 150 mm) or a
YMC-Pack Pro ODS-A (20 mm ꢀ 150 mm) with a ACN/H2O
solvent mixture stabilized with 0.1% TFA. Proton nuclear
magnetic resonances (1H NMR) and carbon nuclear magnetic
resonances (13C NMR) were recorded on a Varian 400 MHz
Mercury 400VX, and the chemical shifts are reported in parts
per million (δ) from an internal standard of residual DMSO
(2.50 ppm, 39.5 ppm), methanol (3.31 ppm, 49.0 ppm), or
chloroform (7.26 ppm, 77.2 ppm). Proton chemical data are
reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, m = multiplet, b = broad), integration,
coupling constant. Analytical purity was determined by HPLC and
was performed on an Agilent (1100 Series): 1 mg/mL in methanol,
5 μL injection, 254 nm detection, 1 mL/min, 1-95% ACN þ 0.05%
TFA/H2O þ 0.05% TFA gradient, 15 min. Purity of compounds is
>95%. Low-resolution mass spectra (LRMS) were recorded on
either a WatersMicromass ZQ or a Waters Acquity (ultra high
performance LC) system using electrospray positive ionization.
High-resolution mass spectra (HRMS) were obtained on a Waters
LCT Premier time-of-flight mass spectrometer using electrospray
positive or negative ionization. Exact masses are calculated by
MassLynx(c) using the method of calculation based on a published
procedure.23 Melting points were measured on Mettler Toledo FP62
automated melting point apparatus using glass capillary tubes and
are uncorrected.
8-Fluoro-4-((2-(4-methylthiazol-5-yl)-1H-benzo[d]imidazol-1-
yl)methyl)quinolin-2(1H )-one (10). NaH (60%, 94 mg, 2.34
mmol) was added to a DMF (5 mL) solution of 5-(1H-benzo-
[d]imidazol-2-yl)-4-methylthiazole (78, 219 mg, 1.02 mmol) at
25 ꢀC. After 15 min (end of gas evolution), 4-(bromomethyl)-
8-fluoroquinolin-2(1H)-one (57, 200 mg, 0.78 mmol) was added
as a solid to give a brown solution, and the reaction mixture was
stirred at 25 ꢀC for 1 h. MeOH was added to quench any
unreacted NaH, and the mixture was purified via reverse-phase
semipreparative HPLC (ACN:H2O þ 0.1% TFA). The frac-
tions containing the desired compound were combined, and
ACN was removed under reduced pressure. Aqueous sodium
bicarbonate (50 mL) was added to the aqueous layer, and it was
extracted with DCM (3 ꢀ 30 mL). The organics were combined,
dried, and concentrated under reduced pressure. HCl (4 equiv,
4 M in 1,4-dioxane) was then added, and the resulting mixture
was stirred at 25 ꢀC for 1 h, followed by solvent removal under
reduced pressure to afford 69 mg (22%) of 8-fluoro-4-((2-(4-
methylthiazol-5-yl)-1H-benzo[d]imidazol-1-yl)methyl)quinolin-
2(1H)-one (10) as an HCl salt. HPLC: tR = 5.04 (>99%); mp =
289.1 ꢀC. 1H NMR (400 MHz, DMSO-d6, HCl salt) δ NH
7,8-Difluoro-4-((2-(thiazol-5-yl)-1H-benzo[d]imidazol-1-yl)-
methyl)quinolin-2(1H)-one (13). Compound 13 was synthesized
as described for compound 10 using 4-(bromomethyl)-7,8-difluoro-
quinolin-2(1H)-one (58) and 5-(1H-benzo[d]imidazol-2-yl)thiazole
(79) as the starting materials (23% yield). HPLC: tR = 4.89
1
(>99%); mp > 300 ꢀC. H NMR (400 MHz, DMSO-d6, HCl
salt) δ NH resonance not observed, 9.28 (s, 1H), 8.25 (s, 1H),
7.83-7.80 (m, 2H), 7.69 (d, 1H, J = 6.0 Hz), 7.44-7.36 (m, 3H),
6.10 (s, 2H), 5.31 (s, 1H). 13C NMR (400 MHz, DMSO-d6, HCl
salt) δ 160.9, 158.3, 150.4 (dd, J = 248, 9.5 Hz, F coupling), 145.3,
144.6, 144.0, 139.5, 137.7 (dd, J = 247, 15.0 Hz, F coupling),
135.2, 129.8, 125.3, 124.5, 124.1, 121.0, 118.2, 116.8, 115.0, 111.4,
110.5 (d, J = 19.1 Hz), 45.1. LRMS (ESIþ)/HRMS (ESI-) m/z:
calcd for C20H12F2N4OS 395.1, found 395.2 [M þ H]þ/calcd
393.0622, found 393.0619 [M - H]-.
7,8-Difluoro-4-((2-(1-methyl-1H-imidazol-5-yl)-1H-benzo[d ]-
imidazol-1-yl)methyl)quinolin-2(1H)-one (14). Compound 14 was
synthesized as described for compound 10 using 4-(bromomethyl)-
7,8-difluoroquinolin-2(1H)-one (58) and 2-(1-methyl-1H-imida-
zol-5-yl)-1H-benzo[d]imidazole (80) as the starting materials
(10% yield). HPLC: tR = 4.91 (100%); mp > 300 ꢀC. 1H NMR
(400 MHz, DMSO-d6, HCl salt) δ 12.09 (s, 1H), 9.27 (s, 1H),
7.90-7.86 (m, 2H), 7.70 (m, 1H), 7.63 (m, 1H), 7.43-7.36 (m, 3H),
5.94 (s, 2H), 5.22 (s, 1H), 4.01 (s, 3H). 13C NMR (400 MHz,
DMSO-d6, HCl salt) δ 161.0, 151.1 (dd, J = 247, 9.5 Hz, F
coupling), 145.6, 142.0, 140.7, 138.6, 137.7 (dd, J = 247, 15.3 Hz, F
coupling), 134.9, 129.8, 124.5, 123.4, 122.2, 121.5, 120.8, 120.0,
116.8, 115.2, 111.4, 110.3 (d, J = 19.0 Hz), 44.6, 35.3. LRMS
(ESIþ)/HRMS (ESI-) m/z: calcd for C21H15F2N5O 392.1, found
392.5 [M þ H]þ/calcd 390.1167, found 390.1167 [M - H]-.
7,8-Difluoro-4-((2-(4-methylpyridin-3-yl)-1H-benzo[d]imidazol-
1-yl)methyl)quinolin-2(1H)-one (15). Compound 15 was synthe-
sized as described for compound 10 using 4-(bromomethyl)-7,8-
difluoroquinolin-2(1H)-one (58) and 2-(4-methylpyridin-3-yl)-
1H-benzo[d]imidazole (81) as the starting materials (51% yield).
HPLC: tR = 4.94 (100%); mp = 242.5 ꢀC. 1H NMR (400 MHz,
DMSO-d6, HCl salt) δ NH resonance not observed, 8.88 (s, 1H),
8.78 (d, 1H, J = 6.0 Hz), 7.90 (dd, 1H, J = 6.4, 1.6 Hz), 7.83 (d,
1H, J = 5.6 Hz), 7.71 (d, 1H, J = 7.2 Hz), 7.57 (m, 1H),
7.51-7.44 (m, 2H), 7.29 (m, 1H), 5.82 (s, 2H), 5.64 (s, 1H), 2.42
(s, 3H). 13C NMR (400 MHz, DMSO-d6, HCl salt) δ 160.8, 154.3,
150.3 (dd, J = 248, 9.0 Hz, F coupling), 147.4, 146.6, 145.3, 144.8,
138.4, 136.8 (dd, J = 247, 15.4 Hz, F coupling), 133.9, 129.5 (d,
J = 7.3 Hz), 127.5, 125.0, 124.9, 124.5, 120.9, 118.1, 117.8, 115.0,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7749
112.4, 110.3 (d, J = 18.3 Hz), 45.0, 19.8. LRMS (ESIþ)/HRMS
(ESI-) m/z: calcd for C23H16F2N4O 403.1, found 403.5 [M þ H]þ/
calcd 401.1214, found 401.1211 [M - H]-.
HRMS (ESI-) m/z: calcd for C20H17F2N3O 354.1, found 354.6
[M þ H]þ/calcd 352.1262 found 352.1259 [M - H]-.
7,8-Difluoro-4-((2-ethyl-1H-benzo[d]imidazol-1-yl)methyl)-
quinolin-2(1H)-one (21). Compound 21 was synthesized as de-
scribed for compound 10 using 4-(bromomethyl)-7,8-difluoro-
quinolin-2(1H)-one (58) and 2-ethyl-1H-benzo[d]imidazole (97,
commercially available) as the starting materials (62% yield).
HPLC: tR = 3.05 (>98%); mp > 300 ꢀC. 1H NMR (400 MHz,
DMSO-d6, HCl salt) δ 12.19 (s, 1H), 7.91 (d, 1H, J = 8.4 Hz),
7.81 (d, 1H, J = 8.0 Hz), 7.79-7.76 (m, 1H), 7.63-7.55 (m, 2H),
7.47 (m, 1H), 6.11 (s, 2H), 5.60 (s, 1H), 3.20 (q, 2H, J = 7.2 Hz),
1.40 (t, 3H, J = 7.2 Hz). 13C NMR (400 MHz, DMSO-d6, HCl
salt) δ 160.9, 156.3, 150.3 (dd, J = 247, 9.5 Hz, F coupling),
143.9, 136.8 (dd, J = 246, 15.4 Hz, F coupling), 132.0, 131.0,
129.6, 125.9, 125.6, 121.1, 116.9, 115.0, 114.5, 112.7, 110.4 (d,
J = 19.1 Hz), 44.5, 18.8, 10.6. LRMS (ESIþ)/HRMS (ESI-)
m/z: calcd for C19H15F2N3O 340.1, found 340.6 [M þ H]þ/calcd
338.1105 found 338.1109 [M - H]-.
7,8-Difluoro-4-((2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)-
methyl)quinolin-2(1H)-one (16). Compound 16 was synthe-
sized as described for compound 10 using 4-(bromomethyl)-
7,8-difluoroquinolin-2(1H)-one (58) and 2-(pyridin-2-yl)-1-
H-benzo[d]imidazole (82) as the starting materials (8% yield).
HPLC: tR = 5.53 (>94%); mp > 300 ꢀC. 1H NMR (400
MHz, DMSO-d6, HCl salt) δ NH resonance not observed,
8.48 (d, 1H, J = 4.4 Hz), 8.41 (d, 1H, J = 7.6 Hz), 8.01 (td, 1H,
J = 7.8, 2.0 Hz), 7.86 (m, 2H), 7.67 (d, 1H, J = 6.8 Hz), 7.48
(m, 1H), 7.43-7.38 (m, 3H), 6.44 (s, 2H), 5.28 (s, 1H). LRMS
(ESIþ)/HRMS (ESI-) m/z: calcd for C22H14F2N4O 389.1,
found 389.5 [M þ H]þ/calcd 387.1058, found 387.1058
[M - H]-.
7,8-Difluoro-4-((2-(pyridin-3-yl)-1H-benzo[d]imidazol-1-yl)-
methyl)quinolin-2(1H)-one (17). Compound 17 was synthesized
as described for compound 10 using 4-(bromomethyl)-7,8-di-
fluoroquinolin-2(1H)-one (58) and 2-(pyridin-3-yl)-1H-benzo-
[d]imidazole (83) as the starting materials (9% yield). HPLC:
tR = 4.63 (100%); mp > 300 ꢀC. 1H NMR (400 MHz, DMSO-
d6, HCl salt) δ NH resonance not observed, 8.99 (d, 1H, J = 2.4
Hz), 8.81 (dd, 1H, J = 5.2, 1.6 Hz), 8.26 (dt, 1H, J = 8.0, 1.8
Hz), 7.91 (d, 1H, J = 7.2 Hz), 7.72-7.67 (m, 3H), 7.52-7.46 (m,
2H), 7.36 (m, 1H), 5.98 (s, 2H), 5.71 (s, 1H). 13C NMR (400
MHz, DMSO-d6, HCl salt) δ 161.0, 154.3, 150.3 (dd, J = 248,
9.4 Hz, F coupling), 150.3, 149.2, 144.8, 138.7, 136.8 (dd, J =
246, 15.4 Hz, F coupling), 137.2, 134.4, 129.6, 125.1, 124.9,
124.7, 123.4, 120.9, 117.6, 117.5, 115.0, 112.3, 110.4 (d, J = 19.1
Hz), 45.6. LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for C22H14-
F2N4O 389.1, found 389.5 [M þ H]þ/calcd 387.1058, found
387.1059 [M - H]-.
7,8-Difluoro-4-((2-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)-
methyl)quinolin-2(1H)-one (18). Compound 18 was synthesized
as described for compound 10 using 4-(bromomethyl)-7,8-di-
fluoroquinolin-2(1H)-one (58) and 2-(pyridin-4-yl)-1H-benzo-
[d]imidazole (84) as the starting materials (5% yield). HPLC:
tR = 4.40 (>97%). 1H NMR (400 MHz, DMSO-d6, HCl salt) δ
NH resonance not observed, 8.83 (dd, 2H, J = 4.8, 1.6 Hz),
7.94-7.89 (m, 3H), 7.73 (m, 1H), 7.65 (m, 1H), 7.45-7.39 (m,
3H), 5.99 (s, 2H), 5.49 (s, 1H). LRMS (ESIþ)/HRMS (ESI-)
m/z: calcd for C22H14F2N4O 389.1, found 389.2 [M þ H]þ/calcd
387.1058 found 387.1056 [M - H]-.
7,8-Difluoro-4-((2-(3-methylpyridin-4-yl)-1H-benzo[d]imidazol-1-
yl)methyl)quinolin-2(1H)-one (19). Compound 19 was synthesized
as described for compound 10 using 4-(bromomethyl)-7,8-difluor-
oquinolin-2(1H)-one (58) and 2-(3-methylpyridin-4-yl)-1H-benzo-
[d]imidazole (85) as the starting materials (31% yield). HPLC: tR =
5.14 (>99%); mp = 292.4 ꢀC. 1H NMR (400 MHz, DMSO-d6,
HCl salt) δ NH resonance not observed, 8.97 (s, 1H), 8.77 (d, 1H,
J = 6.0 Hz), 7.94-7.90 (m, 2H), 7.68 (m, 1H), 7.58 (m, 1H),
7.48-7.44 (m, 2H), 7.33 (m, 1H), 5.81 (s, 2H), 5.52 (s, 1H), 2.44 (s,
3H). LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for C23H16F2N4O
403.1, found 403.5 [M þ H]þ/calcd 401.1214 found 401.1216
[M - H]-.
4-((2-tert-Butyl-1H-benzo[d]imidazol-1-yl)methyl)-7,8-difluoro-
quinolin-2(1H)-one (22). Compound 22 was synthesized as de-
scribed for compound 10 using 4-(bromomethyl)-7,8-difluoroqui-
nolin-2(1H)-one (58) and 2-tert-butyl-1H-benzo[d]imidazole (98,
commercially available) as the starting materials (12% yield).
HPLC: tR = 4.19 (>97%); mp > 300 ꢀC. 1H NMR (400 MHz,
DMSO-d6, HCl salt) δ 12.05 (s, 1H), 7.94 (m, 1H), 7.92 (d, 2H, J =
8.4 Hz), 7.63-7.55 (m, 2H), 7.51-7.44 (m, 2H), 6.16 (s, 2H), 5.63
(s, 1H), 1.56 (s, 9H). LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for
C21H19F2N3O 368.1, found 368.6 [M þ H]þ/calcd 366.1418 found
366.1418 [M - H]-.
7,8-Difluoro-4-((2-propyl-1H-benzo[d]imidazol-1-yl)methyl)-
quinolin-2(1H)-one (23). Compound 23 was synthesized as de-
scribed for compound 10 using 4-(bromomethyl)-7,8-difluoro-
quinolin-2(1H)-one (58) and 2-propyl-1H-benzo[d]imidazole
(86) as the starting materials (78% yield). HPLC: tR = 4.00
(>97%); mp > 300 ꢀC. 1H NMR (400 MHz, DMSO-d6, HCl
salt) δ 12.19 (s, 1H), 7.89 (d, 1H, J = 8.0 Hz), 7.78 (m, 2H),
7.61-7.43 (m, 3H), 6.12 (s, 2H), 5.53 (s, 1H), 3.17 (t, 2H, J = 7.2
Hz), 1.85 (sextet, 2H, J = 7.6 Hz), 0.98 (t, 3H, J = 7.2 Hz). 13C
NMR (400 MHz, DMSO-d6, HCl salt) δ 160.9, 155.1, 150.3 (dd,
J = 247, 9.5 Hz, F coupling), 144.1, 136.8 (dd, J = 246, 15.4 Hz,
F coupling), 132.0, 131.2, 129.5, 125.9, 125.6, 121.1, 116.8,
115.0, 114.6, 112.7, 110.4 (d, J = 18.3 Hz), 44.6, 26.6, 19.8, 13.4.
LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for C20H17F2N3O
354.1, found 354.6 [M þ H]þ/calcd 352.1262 found 352.1259
[M - H]-.
4-((2-Cyclopropyl-1H-benzo[d]imidazol-1-yl)methyl)-7,8-di-
fluoroquinolin-2(1H)-one (24). Compound 24 was synthesized as
described for compound 10 using 4-(bromomethyl)-7,8-difluoro-
quinolin-2(1H)-one (58) and 2-cyclopropyl-1H-benzo[d]imida-
zole (87) as the starting materials (24% yield). HPLC: tR = 3.76
(>98%); mp = 216 ꢀC (sublimed). 1H NMR (400 MHz, DMSO-
d6, HCl salt) δ 12.05 (s, 1H), 7.80-7.78 (m, 3H), 7.57-7.41 (m,
3H), 6.17 (s, 2H), 5.64 (s, 1H), 2.54 (m, 1H), 1.47 (m, 2H), 1.31 (m,
2H). 13C NMR (400 MHz, DMSO-d6, HCl salt) δ 160.9, 156.6,
150.3 (dd, J = 247, 9.5 Hz, F coupling), 144.0, 136.8 (dd, J = 246,
15.4 Hz, F coupling), 132.2, 130.9, 129.6, 125.9, 125.3, 121.1, 117.0,
115.1, 114.3, 112.2, 110.5 (d, J = 18.3 Hz), 44.7, 10.3 (2 carbons),
6.5. LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for C20H15F2-
N3O 352.1, found 352.6 [M þ H]þ/calcd 350.1105 found 350.1102
[M - H]-.
4-((2-Cyclobutyl-1H-benzo[d]imidazol-1-yl)methyl)-7,8-di-
fluoroquinolin-2(1H)-one (25). Compound 25 was synthesized as
described for compound 10 using 4-(bromomethyl)-7,8-difluoro-
quinolin-2(1H)-one (58) and 2-cyclobutyl-1H-benzo[d]imidazole
(88) as the starting materials (56% yield). HPLC: tR = 4.19
(>99%); mp>300 ꢀC. 1H NMR (400 MHz, DMSO-d6, HCl salt)
δ 12.11 (s, 1H), 7.89 (d, 1H, J = 7.8 Hz), 7.79-7.76 (m, 2H),
7.60-7.42 (m, 3H), 5.99 (s, 2H), 5.53 (s, 1H), 4.15 (quintet, 1H,
J = 8.6 Hz), 2.60 (m, 2H), 2.32 (m, 2H), 2.06 (m, 2H). 13C NMR
(400 MHz, DMSO-d6, HCl salt) δ 161.6, 157.5, 151.1 (dd, J = 247,
7,8-Difluoro-4-((2-isopropyl-1H-benzo[d]imidazol-1-yl)methyl)-
quinolin-2(1H)-one (20). Compound 20 was synthesized as de-
scribed for compound 10 using 4-(bromomethyl)-7,8-difluoroqui-
nolin-2(1H)-one (58) and 2-isopropyl-1H-benzo[d]imidazole (96,
commercially available) as the starting materials (52% yield).
HPLC: tR = 3.36 (>99%); mp = 262.3 ꢀC. 1H NMR (400 MHz,
DMSO-d6, HCl salt) δ 12.12 (s, 1H), 7.89 (d, 1H, J = 8.0 Hz),
7.80-7.76 (m, 2H), 7.59 (td, 1H, J = 7.6, 0.8 Hz), 7.49 (td, 1H,
J = 7.6, 0.8 Hz), 7.45 (m, 1H), 6.14 (s, 2H), 5.52 (s, 1H), 3.62 (m,
1H, J = 6.8 Hz), 1.42 (d, 6H, J = 6.8 Hz). 13C NMR (400 MHz,
DMSO-d6, HCl salt) δ 161.6, 159.9, 151.1 (dd, J = 247, 9.5 Hz, F
coupling), 145.1, 137.6 (dd, J = 248, 14.0 Hz, F coupling), 132.5,
132.0, 130.4, 126.8, 126.4, 122.0, 117.5, 115.7, 115.4, 113.6, 111.1
(d, J = 19.1 Hz), 45.5, 26.0, 21.2 (2 carbons). LRMS (ESIþ)/

7750 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Payne et al.
9.5 Hz, F coupling), 144.9, 137.6 (dd, J= 248, 14.6 Hz, F coupling),
132.8, 132.0, 130.2, 126.6, 126.4, 121.8, 117.6, 115.7, 115.4, 113.3,
111.1 (d, J = 18.3 Hz), 45.3, 30.5, 27.5 (2 carbons), 18.8. LRMS
(ESIþ)/HRMS (ESI-) m/z: calcd for C21H17F2N3O 366.1, found
366.6 [M þ H]þ/calcd 364.1262 found 364.1258 [M - H]-.
4-((2-Cyclopentyl-1H-benzo[d]imidazol-1-yl)methyl)-7,8-di-
fluoroquinolin-2(1H)-one (26). Compound 26 was synthesized
as described for compound 10 using 4-(bromomethyl)-7,8-
difluoroquinolin-2(1H)-one (58) and 2-cyclopentyl-1H-benzo-
[d]imidazole (89) as the starting materials (36% yield). HPLC:
tR = 4.75 (>99%); mp > 300 ꢀC. 1H NMR (400 MHz,
DMSO-d6, HCl salt) δ 12.11 (s, 1H), 7.89 (d, 1H, J = 8.0
Hz), 7.81-7.77 (m, 2H), 7.58 (t, 1H, J = 7.2 Hz) 7.53-7.43 (m,
2H), 6.14 (s, 2H), 5.53 (s, 1H), 3.68 (quintet, 1H, J = 8.6 Hz),
2.09 (m, 2H), 2.00 (m, 2H), 1.85 (m, 2H), 1.65 (m, 2H). 13C
NMR (400 MHz, DMSO-d6, HCl salt) δ 160.9, 158.3, 150.3 (dd,
J = 247, 9.5 Hz, F coupling), 144.4, 136.8 (dd, J = 246, 15.4 Hz, F
coupling), 132.0, 131.4, 129.5, 125.9, 125.5, 121.2, 116.8, 115.0,
114.7, 112.7, 110.4 (d, J = 19.0 Hz), 44.7, 35.2, 31.8 (2 carbons),
25.3 (2 carbons). LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for
C22H19F2N3O 380.1, found 380.6 [M þ H]þ/calcd 378.1418 found
378.1416 [M - H]-.
4-((2-Cyclohexyl-1H-benzo[d]imidazol-1-yl)methyl)-7,8-difluoro-
quinolin-2(1H)-one (27). Compound 27 was synthesized as de-
scribed for compound 10 using 4-(bromomethyl)-7,8-difluoro-
quinolin-2(1H)-one (58) and 2-cyclohexyl-1H-benzo[d]imidazole
(90) as the starting materials (39% yield). HPLC: tR = 5.24
(>97%); mp > 300 ꢀC. 1H NMR (400 MHz, MSO-d6, HCl salt)
δ 12.15 (s, 1H), 7.90 (d, 1H, J = 7.8 Hz), 7.81 (m, 1H), 7.76 (d, 1H,
J = 8.0 Hz), 7.59 (t, 1H, J = 7.6 Hz), 7.54-7.44 (m, 2H), 6.18 (s,
2H), 5.52 (s, 1H), 3.35 (m, 1H), 1.97-1.68 (m, 7H), 1.38-1.24 (m,
3H). 13C NMR (400 MHz, MSO-d6, HCl salt) δ 160.9, 158.0, 150.3
(dd, J = 247, 9.5 Hz, F coupling), 144.5, 136.8 (dd, J = 248, 15.4
Hz, F coupling), 131.7, 131.3, 129.5, 126.0, 125.6, 121.3, 116.7,
115.0, 114.6, 112.9, 110.4 (d, J = 19.0 Hz), 44.7, 33.9, 30.3 (2
carbons), 24.9 (2 carbons), 24.8. LRMS (ESIþ)/HRMS (ESI-)
m/z: calcd for C23H21F2N3O 394.2, found 394.5 [M þ H]þ/calcd
392.1575 found 392.1573 [M - H]-.
4-((2-(Cyclopropylmethyl)-1H-benzo[d]imidazol-1-yl)methyl)-
7,8-difluoroquinolin-2(1H)-one (30). Compound 30 was synthe-
sized as described for compound 10 using 4-(bromomethyl)-7,
8-difluoroquinolin-2(1H)-one (58) and 2-(cyclopropylmethyl)-
1H-benzo[d]imidazole (93) as the starting materials (78% yield).
HPLC: tR = 4.27 (>97%); mp > 300 ꢀC. 1H NMR (400 MHz,
DMSO-d6, HCl salt) δ 12.09 (s, 1H), 7.94 (d, 1H, J = 8.0 Hz),
7.82 (d, 1H, J = 8.0 Hz), 7.78-7.75 (m, 1H), 7.63 (td, 1H, J =
7.6, 0.8 Hz), 7.56 (td, 1H, J = 7.8, 1.2 Hz), 7.48 (m, 1H), 6.13 (s,
2H), 5.52 (s, 1H), 3.16 (d, 2H, J = 7.2 Hz), 1.23 (m, 1H), 0.58 (m,
2H), 0.39 (m, 2H). 13C NMR (400 MHz, DMSO-d6, HCl salt) δ
160.8, 155.1, 150.3 (dd, J = 248, 9.5 Hz, F coupling), 143.9,
136.8 (dd, J = 247, 15.4 Hz, F coupling), 131.9, 130.9, 129.6,
126.1, 125.8, 121.1, 116.9, 115.0, 114.7, 112.8, 110.4 (d, J = 19.0
Hz), 44.7, 29.2, 7.9, 4.8 (2 carbons). LRMS (ESIþ)/HRMS
(ESI-) m/z: calcd for C21H17F2N3O 366.1, found 366.6
[M þ H]þ/calcd 364.1262 found 364.1258 [M - H]-.
4-((2-(Cyclobutylmethyl)-1H-benzo[d]imidazol-1-yl)methyl)-
7,8-difluoroquinolin-2(1H)-one (31). Compound 31 was synthe-
sized as described for compound 10 using 4-(bromomethyl)-7,
8-difluoroquinolin-2(1H)-one (58) and 2-(cyclobutylmethyl)-1H-
benzo[d]imidazole (94) as the starting materials (83% yield).
HPLC: tR = 4.96 (>98%); mp > 300 ꢀC. 1H NMR (400 MHz,
DMSO-d6, HCl salt) δ 12.09 (s, 1H), 7.90 (d, 1H, J = 8.0 Hz),
7.79-7.75 (m, 2H), 7.61 (t, 1H, J = 7.6 Hz), 7.54 (t, 1H, J = 8.4
Hz), 7.48 (m, 1H), 6.13 (s, 2H), 5.44 (s, 1H), 3.34 (d, 2H, J = 7.2
Hz), 2.85 (m, 1H), 2.06 (m, 2H), 1.83 (m, 4H). 13C NMR (400
MHz, DMSO-d6, HCl salt) δ 160.8, 153.9, 150.3 (dd, J = 247, 9.5
Hz, F coupling), 144.1, 136.8 (dd, J = 246, 15.4 Hz, F coupling),
131.8, 130.9, 129.5, 126.1, 125.7, 121.2, 116.7, 115.0, 114.6, 112.8,
110.4 (d, J = 18.3 Hz), 44.6, 32.4, 31.0, 27.3 (2 carbons), 17.7.
LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for C22H19F2N3O 380.1,
found 380.6 [M þ H]þ/calcd 378.1418 found 378.1413 [M - H]-.
4-((2-(Cyclopentylmethyl)-1H-benzo[d]imidazol-1-yl)methyl)-
7,8-difluoroquinolin-2(1H)-one (32). Compound 32 was synthe-
sized as described for compound 10 using 4-(bromomethyl)-7,
8-difluoroquinolin-2(1H)-one (58) and 2-(cyclopentylmethyl)-
1H-benzo[d]imidazole (95) as the starting materials (12% yield).
HPLC: tR = 5.42 (>99%); mp > 300 ꢀC. 1H NMR (400 MHz,
DMSO-d6, HCl salt) δ 12.10 (s, 1H), 7.91 (d, 1H, J = 8.0 Hz),
7.81-7.77 (m, 2H), 7.61 (td, 1H, J = 7.6, 0.8 Hz), 7.54 (td, 1H,
J = 7.8, 0.8 Hz), 7.48 (m, 1H), 6.15 (s, 2H), 5.45 (s, 1H), 3.24 (d,
2H, J = 8.0 Hz), 2.43 (m, 1H), 1.74 (m, 2H), 1.63 (m, 2H), 1.49
(m, 2H), 1.26 (m, 2H). LRMS (ESIþ)/HRMS (ESI-) m/z: calcd
for C23H21F2N3O 394.2, found 394.6 [M þ H]þ/calcd 392.1575
found 392.1573 [M - H]-.
7,8-Difluoro-4-((2-isobutyl-1H-benzo[d]imidazol-1-yl)methyl)-
quinolin-2(1H)-one (28). Compound 28 was synthesized as de-
scribed for compound 10 using 4-(bromomethyl)-7,8-difluoro-
quinolin-2(1H)-one (58) and 2-isobutyl-1H-benzo[d]imidazole
(91) as the starting materials (43% yield). HPLC: tR = 4.55
(>99%); mp > 300 ꢀC. 1H NMR (400 MHz, DMSO-d6, HCl
salt) δ 12.21 (s, 1H), 7.89 (d, 1H, J = 8.0 Hz), 7.80 (m, 1H), 7.75
(d, 1H, J = 8.4 Hz), 7.59 (t, 1H, J = 7.4 Hz), 7.54-7.46 (m, 2H),
6.12 (s, 2H), 5.46 (s, 1H), 3.01 (d, 2H, J = 7.2 Hz), 2.21 (m, 1H),
0.97 (d, 6H, J = 6.8 Hz). 13C NMR (400 MHz, DMSO-d6, HCl
salt) δ 160.8, 154.3, 150.3 (dd, J = 248, 9.2 Hz, F coupling),
144.1, 136.8 (dd, J = 246, 15.4 Hz, F coupling), 131.9, 131.1,
129.6, 126.1, 125.7, 121.3, 116.8, 115.0, 114.6, 112.9, 110.4 (d,
J = 18.4 Hz), 44.7, 33.0, 27.0, 21.9 (2 carbons). LRMS (ESIþ)/
HRMS (ESI-) m/z: calcd for C21H19F2N3O 368.1, found 368.6
[M þ H]þ/calcd 366.1418 found 366.1415 [M - H]-.
4-((2-Cyclobutyl-1H-imidazo[4,5-b]pyridin-1-yl)methyl)-7,8-
difluoroquinolin-2(1H)-one (33). Compound 33 was synthe-
sized as the first eluting regioisomer as described for compound
10 using 4-(bromomethyl)-7,8-difluoroquinolin-2(1H)-one
(58) and 2-cyclobutyl-1H-imidazo[4,5-c]pyridine (104) as the
starting materials (purification by silica gel column chroma-
tography with a step gradient 4 CVs at 2% MeOH/DCM,
followed by 4 CVs at 4% MeOH/DCM) (10% yield). HPLC:
7,8-Difluoro-4-((2-isopentyl-1H-benzo[d]imidazol-1-yl)methyl)-
quinolin-2(1H)-one (29). Compound 29 was synthesized as de-
scribed for compound 10 using 4-(bromomethyl)-7,8-difluoro-
quinolin-2(1H)-one (58) and 2-isopentyl-1H-benzo[d]imidazole
(92) as the starting materials (50% yield). HPLC: tR = 5.20
t
R = 1.56 (>98%). 1H NMR (400 MHz, DMSO-d6, HCl salt)
δ NH resonance not observed, 8.63 (d, 1H, J = 4.4 Hz), 8.41 (d,
1H, J = 4.0 Hz), 7.80 (m, 1H), 7.65 (dd, 1H, J = 8.0, 5.6 Hz),
7.34 (m, 1H), 5.98 (s, 2H), 5.52 (s, 1H), 4.02 (m, 1H), 2.70-1.98
(m, 6H). The regiochemistry of 33 was determined by NMR
spectroscopy measuring nuclear Overhauser effects (NOEs).24
By irradiating the C-9 methylene, NOEs were observed with
the cyclobutyl C-14 methine and the proximal C-13 CH on the
pyridine ring.25 LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for
1
(>99%); mp > 300 ꢀC. H NMR (400 MHz, DMSO-d6, HCl
salt) δ 12.10 (s, 1H), 7.89 (d, 1H, J = 8.0 Hz), 7.81-7.77 (m, 2H),
7.61-7.43 (m, 3H), 6.12 (s, 2H), 5.53 (s, 1H), 3.16 (t, 2H, J = 7.8
Hz), 1.72 (q, 2H, J = 5.7 Hz), 1.63 (m, 1H), 0.89 (d, 6H, J = 6.4
Hz). 13C NMR (400 MHz, DMSO-d6, HCl salt) δ 160.8, 155.4,
150.3 (dd, J = 247, 9.5 Hz, F coupling), 144.0, 136.8 (dd, J = 246,
15.4 Hz, F coupling), 131.9, 131.0, 129.6, 125.9, 125.6, 121.2,
116.9, 115.0, 114.6, 112.7, 110.4 (d, J = 19.1 Hz), 44.6, 34.5, 27.1,
23.0, 21.9 (2 carbons). LRMS (ESIþ)/HRMS (ESI-) m/z: calcd
for C22H21F2N3O 382.2, found 382.5 [M þ H]þ/calcd 380.1575
found 380.1569 [M - H]-.
C
20H16F2N4O 367.1, found 367.3 [M þ H]þ/calcd 365.1214,
found 365.1215 [M - H]-.
4-((2-Cyclobutyl-1H-imidazo[4,5-c]pyridin-1-yl)methyl)-7,8-
difluoroquinolin-2(1H)-one (34). Compound 34 was synthesized
as the first eluting regioisomer as described for compound 10 using
4-(bromomethyl)-7,8-difluoroquinolin-2(1H)-one (58) and 2-cyclo-
butyl-1H-imidazo[4,5-c]pyridine (105) as the starting materials

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7751
(purification via reverse-phase semipreparative HPLC eluting with
ACN:H2O þ 0.1% TFA) (0.5% yield due to the tight separation).
HPLC: tR = 2.17 (>96%). 1H NMR (400 MHz, CD3OD) δ NH
resonance not observed, 9.21 (s, 1H), 8.56 (d, 1H, J = 6.6 Hz), 8.20
(d, 1H, J = 6.6 Hz), 7.79 (m, 1H), 7.34 (m, 1H), 6.00 (s, 2H), 5.42 (s,
1H), 3.91(m, 1H), 2.67-2.03 (m, 6H). The regiochemistry of 34 was
assigned by NMR spectroscopy by measuring nuclear Overhauser
effects (NOEs). By irradiating the C-9 methylene, NOEs were
observed with the cyclobutyl C-14 methine and the proximal C-13
CH on the pyridine ring, so allowing definitive identification of this
signal (δ = 8.20). Further, this C-13 CH is a doublet, suggesting
coupling to the C-12 CH (δ = 8.56) and hence the regiochemistry
shown.24 LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for C20H16F2-
N4O 367.1, found 367.3 [M þ H]þ/calcd 365.1214, found 365.1209
[M - H]-.
4-((2-Cyclobutyl-3H-imidazo[4,5-c]pyridin-3-yl)methyl)-7,8-
difluoroquinolin-2(1H)-one (35). Compound 35 was synthe-
sized as the second eluting regioisomer as described for com-
pound 10 using 4-(bromomethyl)-7,8-difluoroquinolin-2(1H)-
one (58) and 2-cyclobutyl-1H-imidazo[4,5-c]pyridine (105) as
the starting materials (purification by reverse-phase semipre-
parative HPLC eluting with ACN:H2O þ 0.1% TFA) (0.5%
yield due to the tight separation). HPLC: tR = 2.26 (>99%).
1H NMR (400 MHz, CD3OD) δ NH resonance not observed,
9.28 (s, 1H), 8.52 (d, 1H, J = 6.0 Hz), 8.03 (d, 1H, J = 6.4 Hz),
7.80 (m, 1H), 7.34 (m, 1H), 5.96 (s, 2H), 5.39 (s, 1H), 3.92 (m,
1H), 2.65-2.01 (m, 6H). The regiochemistry of 35 was assigned
by NMR spectroscopy measuring nuclear Overhauser effects
(NOEs). By irradiating the C-9 methylene, an NOE were
observed with the cyclobutyl C-14 methine. Although we were
not able to observe the NOE between the C-9 methylene and
the C-13 CH (limited material available), we assigned the
stereochemistry shown based on the NOE results for 34.23
LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for C20H16F2N4O
367.1, found 367.3 [M þ H]þ/calcd 365.1214, found 365.1214
[M - H]-.
4-((2-Cyclobutyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)-7,8-
difluoroquinolin-2(1H)-one (36). Compound 36 was synthe-
sized as the second eluting regioisomer as described for com-
pound 10 using 4-(bromomethyl)-7,8-difluoroquinolin-2(1H)-
one (58) and 2-cyclobutyl-1H-imidazo[4,5-c]pyridine (104) as
the starting materials (purification by silica gel column chro-
matography with a step gradient 4 CVs at 2% MeOH/DCM,
followed by 4 CVs at 4% MeOH/DCM) (23% yield). HPLC:
tR = 5.60 (>99%); mp > 300 ꢀC. 1H NMR (400 MHz,
DMSO-d6, HCl salt) δ 8.47 (d, 1H, J = 4.4 Hz), 8.29 (d, 1H,
J = 8.0 Hz), 7.83 (m, 1H), 7.53 (m, 1H), 7.39 (m, 1H), 5.84 (s,
2H), 5.60 (s, 1H), 4.09 (m, 1H), 2.60-1.88 (m, 6H). The
regiochemistry of 36 was determined by NMR spectroscopy
measuring nuclear Overhauser effects (NOEs). By irradiating
the C-9 methylene, an NOE was only observed with the
cyclobutyl C-14 methine (and not any CH on the pyridine ring
as with 33).23 13C NMR (400 MHz, DMSO-d6) δ 160.9, 158.5,
150.3 (dd, J = 247, 9.5 Hz, F coupling), 145.7, 145.1, 144.6,
136.9 (dd, J = 246, 15.4 Hz, F coupling), 129.5 (d, J = 7.4 Hz),
127.4, 124.6, 120.9, 120.8, 117.4, 115.0, 110.5 (d, J = 18.4 Hz),
42.7, 30.6, 26.6 (2 carbons), 18.0. LRMS (ESIþ)/HRMS (ESI-)
m/z: calcd for C20H16F2N4O 367.1, found 367.3 [M þ H]þ/calcd
365.1214, found 365.1211 [M - H]-.
4-((2-Cyclobutyl-7-methyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl)-7,8-difluoroquinolin-2(1H)-one (37). Compound 37
was synthesized as described for compound 10 using 4-(bromo-
methyl)-7,8-difluoroquinolin-2(1H)-one (58) and 2-cyclobutyl-7-
methyl-3H-imidazo[4,5-b]pyridine (113) as the starting materials
(12% yield). HPLC: tR = 4.43 (>99%); mp > 300 ꢀC. 1H NMR
(400 MHz, DMSO-d6) δ 12.02 (s, 1H), 8.13 (d, 1H, J = 4.8 Hz),
7.85 (m, 1H), 7.38 (m, 1H), 7.16 (d, 1H, J = 5.2 Hz), 5.70 (s, 2H),
5.20 (s, 1H), 3.85 (qt, 1H, J = 8.6 Hz), 2.62 (s, 3H), 2.45-1.82 (m,
6H). 13C NMR (400 MHz, DMSO-d6) δ 160.9, 157.8, 150.3 (dd,
J = 247, 9.5 Hz, F coupling), 145.2, 144.8, 136.9, 136.8 (dd,
J = 246, 15.4 Hz, F coupling), 129.6, 129.5, 127.8, 121.7, 120.7,
117.2, 115.0, 110.5 (d, J = 19.0 Hz), 42.7, 30.5, 26.9 (2 carbons),
18.0, 16.6. LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for
C21H18F2N4O 381.1, found 381.3 [M þ H]þ/calcd 379.1371 found
379.1369 [M - H]-.
4-((7-Chloro-2-cyclobutyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)-
7,8-difluoroquinolin-2(1H)-one (38). Compound 38 was synthesized
as described for compound 10 using 4-(bromomethyl)-7,8-difluor-
oquinolin-2(1H)-one (58) and 7-chloro-2-cyclobutyl-3H-imidazo-
[4,5-b]pyridine (115) as the starting materials (65% yield). HPLC:
tR = 5.75 (100%). 1H NMR (400 MHz, DMSO-d6) δ 12.06 (s, 1H),
8.21 (d, 1H, d = 5.2 Hz), 7.82 (m, 1H), 7.44 (d, 1H, J=5.6Hz),7.37
(m, 1H), 5.73 (s, 2H), 5.22 (s, 1H), 3.86 (qt, 1H, J = 8.6 Hz),
2.42-1.82 (m, 6H). LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for
C20H15ClF2N4O 401.1, found 401.2 [M þ H]þ/calcd 399.0824,
found 399.0819 [M - H]-.
4-((2-Cyclobutyl-7-methoxy-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl)-7,8-difluoroquinolin-2(1H)-one (39). Compound 39 was
synthesized as described for compound 10 using 4-(bromo-
methyl)-7,8-difluoroquinolin-2(1H)-one (58) and 2-cyclobutyl-
7-methoxy-3H-imidazo[4,5-b]pyridine (118) as the starting ma-
1
terials (15% yield). HPLC: tR = 4.59 (100%). H NMR (400
MHz, DMSO-d6; TFA salt) δ NH resonance not observed, 8.05
(d, 1H, J = 4.6 Hz), 7.65 (m, 1H), 7.21 (m, 1H), 6.82 (d, 1H, J =
4.4 Hz), 5.58 (s, 2H), 5.11 (s, 1H), 3.90 (s, 3H), 3.68 (m, 1H),
2.43-1.62 (m, 6H). LRMS (ESIþ)/HRMS (ESIþ) m/z: calcd
for C21H18F2N4O2 397.1, found 397.3 [M þ H]þ/calcd 397.1476,
found 397.1475 [M þ H]þ.
4-((2-Cyclobutyl-7-(dimethylamino)-3H-imidazo[4,5-b]pyridin-3-
yl)methyl)-7,8-difluoroquinolin-2(1H)-one (40). Compound 40 was
synthesized as described for compound 10 using 4-(bromomethyl)-
7,8-difluoroquinolin-2(1H)-one (58) and 2-cyclobutyl-N,N-di-
methyl-3H-imidazo[4,5-b]pyridin-7-amine (117) as the starting
materials (10% yield). HPLC: tR = 5.33 (>97%). 1H NMR
(400 MHz, DMSO-d6; TFA salt) δ 12.09 (s, 1H), 7.95 (d, 1H,
J = 7.2 Hz), 7.75 (m, 1H), 7.41 (m, 1H), 6.59 (d, 1H, J = 6.8 Hz),
5.69 (s, 2H), 5.15 (s, 1H), 3.77 (qt, 1H, J = 8.4 Hz), 3.55 (s, 6H),
2.40-1.86 (m, 6H). LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for
C22H21F2N5O 410.2, found 410.5 [M þ H]þ/calcd 408.1636, found
408.1638 [M - H]-.
4-((2-Cyclobutyl-7-(trifluoromethyl)-3H-imidazo[4,5-b]pyridin-3-
yl)methyl)-7,8-difluoroquinolin-2(1H)-one (41). Compound 41 was
synthesized as described for compound 10 using 4-(bromomethyl)-
7,8-difluoroquinolin-2(1H)-one (58) and 2-cyclobutyl-7-(trifluoro-
methyl)-3H-imidazo[4,5-b]pyridine (119) as the starting materials
1
(55% yield). HPLC: tR = 6.19 (100%). H NMR (400 MHz,
DMSO-d6) δNH resonance not observed, 8.46 (d, 1H, J= 4.8Hz),
7.83 (m, 1H), 7.62 (d, 1H, J = 5.2 Hz), 7.39 (m, 1H), 5.78 (s, 2H),
5.22 (s, 1H), 3.91 (qt, 1H, J = 8.4 Hz), 2.45-1.88 (m, 6H). LRMS
(ESIþ)/HRMS (ESI-) m/z: calcd for C21H15F5N4O 435.2, found
435.2 [M þ H]þ/calcd 433.1088, found 433.1090 [M - H]-.
4-((2-Cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-
difluoroquinolin-2(1H)-one (42). Compound 42 was synthesized
as described for compound 10 using 4-(bromomethyl)-7,8-di-
fluoroquinolin-2(1H)-one (58) and 2-cyclobutyl-1H-imidazo-
[4,5-b]pyrazine (106) as the starting materials (25% yield).
HPLC: tR = 5.29 (>99%); mp = 261 ꢀC. 1H NMR (400 MHz,
DMSO-d6; HClsalt) δ8.53 (d, 1H), 8.33 (d, 1H), 7.83 (m, 1H), 7.40
(m, 1H), 5.78 (s, 2H), 5.36 (s, 1H), 3.95 (m, 1H), 2.5-1.8 (m, 6H).
13C NMR (400 MHz, DMSO-d6, citrate salt) δ 163.0, 160.9, 150.3
(dd, J = 247, 9.5 Hz, F coupling), 148.3, 145.6, 140.8, 139.5, 137.7,
136.9 (dd, J = 246, 15.4 Hz, F coupling), 129.6 (d, J = 8.1 Hz),
120.7, 117.1, 115.1, 110.5 (d, J = 18.3 Hz), 42.0, 31.6, 26.6 (2
carbons), 18.0. LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for
C19H15F2N5O 368.1, found 368.2 [M þ H]þ/calcd 366.1167, found
366.1170 [M - H]-.
The minor regioisomer (43), 4-((2-cyclobutyl-4H-imidazo-
[4,5-b]pyrazin-4-yl)methyl)-7,8-difluoroquinolin-2(1H)-one), was
1
also isolated. H NMR (400 MHz, DMSO-d6) δ NH resonance
not observed, 8.85 (d, 1H, J = 4.4 Hz), 8.76 (d, 1H, J = 4.0 Hz),

7752 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Payne et al.
7.84 (m, 1H), 7.37 (m, 1H), 6.32 (s, 2H), 6.26 (s, 1H), 4.01 (m, 1H),
2.55-1.95 (m, 6H).
4-((2-Cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-8-
fluoroquinolin-2(1H)-one (48). Compound 48 was synthesized
as described for compound 47 using 4-(bromomethyl)-8-
fluoroquinolin-2(1H)-one (51) and 2-cyclobutyl-1H-imidazo-
[4,5-b]pyrazine (106) as the starting materials (75% yield).
4-((2-Cyclopentyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-
difluoroquinolin-2(1H)-one (44). Compound 44 was synthesized
as described for compound 10 using 4-(bromomethyl)-7,8-di-
fluoroquinolin-2(1H)-one (58) and 2-cyclopentyl-1H-imidazo-
[4,5-b]pyrazine (107) as the starting materials (23% yield). HPLC:
tR = 5.39 (>97%); mp = 277.4 ꢀC. 1H NMR (400 MHz, DMSO-
d6) δNH resonance not observed, 8.49 (d, 1H, J = 2.8 Hz), 8.30 (d,
1H, J = 2.8 Hz), 7.84 (m, 1H), 7.39 (m, 1H), 5.86 (s, 2H), 5.33 (s,
1H), 3.46 (qt, 1H, J = 8.0 Hz), 2.01-1.58 (m, 8H). 13C NMR (400
MHz, DMSO-d6) δ 164.6, 160.9, 150.3 (dd, J = 247, 9.6 Hz, F
coupling), 147.6, 145.6, 140.4, 139.7, 137.9, 136.9 (dd, J= 246, 15.4
Hz, F coupling), 129.6 (d, J= 9.0 Hz), 120.8, 117.1, 115.1, 110.5 (d,
J = 19.1 Hz), 42.2, 36.5, 31.9 (2 carbons), 25.5 (2 carbons). LRMS
(ESIþ)/HRMS (ESI-) m/z: calcd for C20H17F2N5O 382.1, found
382.3 [M þ H]þ/calcd 380.1323, found 380.1326 [M - H]-.
7,8-Difluoro-4-((2-isobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)-
methyl)quinolin-2(1H)-one (45). Compound 45 was synthesized as
described for compound 10 using 4-(bromomethyl)-7,8-difluoro-
quinolin-2(1H)-one (58) and 2-isobutyl-1H-imidazo[4,5-b]pyra-
1
HPLC: tR = 4.98 (>98%); mp = 255.6 ꢀC. H NMR (400
MHz, DMSO-d6, HCl salt) δ NH resonance not observed,
8.51 (d, 1H, J = 2.8 Hz), 8.31 (d, 1H, J = 2.8 Hz), 7.77 (d, 1H,
J = 8.4 Hz), 7.51 (dd, 1H, J = 10.8, 8.4 Hz), 7.28 (m, 1H), 5.77
(s, 2H), 5.40 (s, 1H), 3.95 (qt, 1H, J = 8.0 Hz), 2.44-1.88 (m,
6H). 13C NMR (DMSO-d6) δ 162.9, 160.6, 149.0 (d, J = 245
Hz, F coupling), 147.5, 145.5 (d, J = 2.2 Hz), 140.5, 139.7,
138.0, 127.6 (d, J = 13.2 Hz), 121.8 (d, J = 6.6 Hz), 119.9 (d,
J = 3.7 Hz), 118.9 (d, J = 2.9 Hz), 118.3, 116.2 (d, J = 17.6
Hz), 42.2, 31.5, 26.6 (2 carbons), 18.0. LRMS (ESIþ)/HRMS
(ESI-) m/z: calcd for C19H16FN5O 350.1, found 350.3
[M þ H]þ/calcd 348.1261, found 348.1258 [M - H]-.
8-Fluoro-4-((2-isobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-
quinolin-2(1H)-one (49). Compound 49 was synthesized as de-
scribed for compound 10 using 4-(bromomethyl)-8-fluoroquin-
olin-2(1H)-one (51) and 2-isobutyl-1H-imidazo[4,5-b]pyrazine
(108) as the starting materials (25% yield). HPLC: tR = 5.13
(>95%); mp = 250.6 ꢀC. 1H NMR (400 MHz, CD3OD) δ 8.69
(d, 1H, J = 2.8 Hz), 8.57 (d, 1H, J = 2.8 Hz), 7.84 (d, 1H, J = 8.0
Hz), 7.51 (ddd, 1H, J = 10.4, 8.0, 0.8 Hz), 7.39 (m, 1H), 6.07 (s,
2H), 5.85 (s, 1H), 3.10 (d, 2H, J = 7.6 Hz), 2.31 (m, 1H), 1.07 (d,
6H, J = 6.8 Hz). 13C NMR (400 MHz, DMSO-d6) δ 160.6, 160.2,
149.0 (d, J = 245 Hz, F coupling), 147.7, 145.5, 140.3, 139.7,
137.9, 127.6 (d, J = 13.2 Hz), 121.8 (d, J = 7.3 Hz), 120.0 (d, J =
2.9 Hz), 118.9 (d, J = 3.0 Hz), 118.2, 116.2 (d, J = 17.6 Hz), 42.3,
35.4, 26.6, 22.2 (2 carbons). LRMS (ESIþ)/HRMS (ESI-) m/z:
calcd for C19H18FN5O 352.1, found 352.3 [M þ H]þ/calcd
350.1417, found 350.1415 [M - H]-.
4-((2-(3,3-Difluorocyclobutyl)-1H-imidazo[4,5-b]pyrazin-1-yl)-
methyl)-8-fluoroquinolin-2(1H)-one (50). Compound 50 was syn-
thesized as described for compound 47 using 4-(bromomethyl)-8-
fluoroquinolin-2(1H)-one (51) and 2-(3,3-difluorocyclobutyl)-
1H-imidazo[4,5-b]pyrazine (110) as the starting materials (31%
yield). HPLC: tR = 4.38 (>99%). 1H NMR (400 MHz, DMSO-
d6) δ 12.08 (s, 1H), 8.54 (s, 1H), 8.35 (s, 1H), 7.75 (m, 1H), 7.39
(m, 1H), 7.28 (m, 1H), 5.80 (s, 2H), 5.42 (s, 1H), 3.85 (m, 1H),
3.16-3.01 (m, 4H). 13C NMR (400 MHz, DMSO-d6, HCl salt) δ
161.6, 161.4, 149.7 (d, J = 245 Hz, F coupling), 148.6, 146.2, 141.7,
140.6, 138.9, 128.4 (d, J = 13.2 Hz), 122.5 (d, J= 7.3 Hz), 120.8 (d,
J = 3.0 Hz), 120.3 (dd, J = 282, 269 Hz, F coupling), 119.7 (d, J =
3.0 Hz), 119.0, 116.9 (d, J = 17.6 Hz), 43.1, 38.6, 20.9 (dd, J =
16.5, 5.4 Hz, 2 carbons). LRMS (ESIþ)/HRMS (ESI-) m/z: calcd
for C19H14F3N5O 386.1, found 386.2 [M þ H]þ/calcd 384.1072,
found 384.1074 [M - H]-.
zine (108) as the starting materials (20% yield). HPLC: tR
=
5.12 (100%). 1H NMR (400 MHz, DMSO-d6) δ 12.08 (s, 1H), 8.50
(d, 1H, J = 2.4 Hz), 8.31 (d, 1H, J = 2.8 Hz), 7.83 (m, 1H), 7.38
(m, 1H), 5.85 (s, 2H), 5.31 (s, 1H), 2.85 (d, 2H, J = 6.8 Hz), 2.26
(qt, 1H, J = 7.0 Hz), 0.95 (d, 6H, J = 6.8 Hz). 13C NMR (400
MHz, DMSO-d6) δ 160.9, 160.2, 150.4 (dd, J = 247, 9.6 Hz, F
coupling), 148.3, 145.5, 140.4, 139.6, 137.7, 137.0 (dd, J= 247, 14.8
Hz, F coupling), 129.6 (d, J= 9.0 Hz), 120.8, 117.0, 115.2, 110.5 (d,
J = 19.1 Hz), 42.1, 35.5, 26.6, 22.2 (2 carbons). LRMS (ESIþ)/
HRMS (ESI-) m/z: calcd for C19H17F2N5O 370.1, found 370.3
[M þ H]þ/calcd 368.1323, found 368.1318 [M - H]-.
7,8-Difluoro-4-((2-isopropyl-1H-imidazo[4,5-b]pyrazin-1-yl)-
methyl)quinolin-2(1H)-one (46). Compound 46 was synthesized
as described for compound 10 using 4-(bromomethyl)-7,8-di-
fluoroquinolin-2(1H)-one (58) and 2-isopropyl-1H-imidazo-
[4,5-b]pyrazine (109) as the starting materials (18% yield).
1
HPLC: tR = 4.48 (100%). H NMR (400 MHz, DMSO-d6) δ
12.10 (s, 1H), 8.49 (d, 1H, J = 2.8 Hz), 8.30 (d, 1H, J = 2.4 Hz),
7.83 (m, 1H), 7.37 (m, 1H), 5.86 (s, 2H), 5.32 (s, 1H), 3.31 (qt, 1H,
J=6.8Hz),1.31(d,6H,J=6.8Hz).13C NMR (400 MHz, DMSO-
d6) δ 165.4, 160.9, 150.4 (dd, J = 247, 9.6 Hz, F coupling), 148.3,
145.8, 140.4, 139.7, 137.8, 137.0 (dd, J = 246, 14.6 Hz, F coupling),
129.6 (d, J = 9.0 Hz), 120.8, 117.0, 115.2, 110.6 (d, J = 18.3 Hz),
42.1, 26.4, 21.3 (2 carbons). LRMS (ESIþ)/HRMS (ESI-) m/z:
calcd for C18H15F2N5O 356.1, found 356.2 [M þ H]þ/calcd
354.1167, found 354.1161 [M - H]-.
4-((2-(3,3-Difluorocyclobutyl)-1H-imidazo[4,5-b]pyrazin-1-yl)-
methyl)-7,8-difluoroquinolin-2(1H)-one (47). A mixture of 2-(3,3-
difluorocyclobutyl)-1H-imidazo[4,5-b]pyrazine (110, 115 mg,
0.54 mmol) and 4-(bromomethyl)-7,8-difluoroquinolin-2(1H)-
one (58, 50 mg, 0.18 mmol) were combined into a 4 mL vial,
stirred with a spatula for 1 min, and heated in a sand bath at 250 ꢀC
for 5 min. During this time, the powdered mixture congealed,
darkened, and melted. After cooling to 25 ꢀC, the crude solid was
taken up in DMSO and purified via reverse-phase semiprepara-
tive HPLC (ACN:H2O þ 0.1% TFA) to afford 35 mg (48%) of
4-((2-(3,3-difluorocyclobutyl)-1H-imidazo[4,5-b]pyrazin-1-yl)-
methyl)-7,8-difluoroquinolin-2(1H)-one. HPLC: tR = 4.78
(>93%); mp > 300 ꢀC. 1H NMR (400 MHz, DMSO-d6; HCl
salt) δ NH resonance not observed, 8.54 (d, 1H, J = 2.4 Hz),
8.34 (d, 1H, J = 2.8 Hz), 7.80 (m, 1H), 7.38 (m,1H), 5.81 (s,
2H), 5.42 (s, 1H), 3.85 (qt, 1H, J = 8.2 Hz), 3.13-3.00 (m, 4H).
13C NMR (400 MHz, DMSO-d6; HCl salt) δ 160.9, 160.8, 150.3
(dd, J = 247, 9.6 Hz, F coupling), 147.9, 145.3, 140.9, 139.9, 138.2,
136.9 (dd, J = 246, 15.4 Hz, F coupling), 129.6 (d, J = 8.1 Hz),
120.8, 119.6 (dd, J = 281, 268 Hz, F coupling), 117.2, 115.2, 110.4
(d, J = 19.1 Hz), 42.2, 39.6, 20.1 (dd, J = 16.5, 5.5 Hz, 2 carbons).
LRMS (ESIþ)/HRMS (ESI-) m/z: calcd for C19H13F4N5O 404.1,
found 404.2 [M þ H]þ/calcd 402.0978, found 402.0983 [M - H]-.
N-(2-Fluorophenyl)-3-oxobutanamide (53). A solution of
methyl 3-oxobutanoate (54 mL, 0.5 mol) in xylene/pyridine
(200 mL/5 mL) was heated to gentle reflux for 30 min. 2-Fluoro-
aniline (51, 40.0 g, 0.36 mol) was then added dropwise into the
above reaction mixture, and it was refluxed for 8 h, with removal
of distillate through a Dean-Stark tube. The solution was
allowed to cool to 25 ꢀC and was extracted with 20 g of NaOH
in 150 mL of H2O. The aqueous layer was separated and made
weakly acidic with conc HCl. The resulting precipitate was
collected by filtration, washed with H2O, and dried to give 28 g
(38%) of N-(2-fluorophenyl)-3-oxobutanamide (53) as a white
crystalline solid. LRMS m/z: calcd for C10H10FNO2 195.0, found
196.0 [M þ H]þ.
N-(2,3-Difluorophenyl)-3-oxobutanamide (54). Compound 54
was synthesized as described for compound 53 using 3-oxobutano-
ate and 2,3-difluoroaniline (52) as starting materials (41% yield).
1H NMR (400 MHz, CDCl3) δ 9.52 (s, 1H), 7.99 (m, 1H), 7.04 (m,
1H), 6.89 (s, 1H), 3.64 (s, 2H), 2.33 (s, 3H). LRMS (ESIþ) m/z:
calcd for C10H9F2NO2 214.0, found 214.1 [M þ H]þ.
4-Bromo-N-(2-fluorophenyl)-3-oxobutanamide (55). Bromine
(7.9 mL, 1.1 equiv) was added dropwise via an addition funnel to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7753
a solution of N-(2-fluorophenyl)-3-oxobutanamide (53, 27.2 g,
0.14 mol) in AcOH (100 mL). The resulting solution was stirred
at 25 ꢀC for 5 h. Acetone (10 mL) was then added, and the
solution was stirred at 25 ꢀC for 18 h. The mixture was then
concentrated to ∼20% volume and worked up via EtOAc/H2O
extraction. Purification by silica gel column chromatography
(10% to 20% EtOAc/hexanes) afforded 26 g (68%) of 4-bromo-
Recombinant eNOS and nNOS Assay (293 Transient Transfec-
tion Assays). HEK293 cells (ATCC) were seeded into 150 mm ꢀ
25 mm tissue culture plates and grown to approximately 70%
confluence in DMEM containing 10% FBS, 100 U/mL peni-
cillin, and 100 μg/mL streptomycin. Each plate was transfected
with 1 mL of DMEM with Pen/Strep containing 10 μg of
human or murine nNOS or 15 μg of human eNOS expression
plasmid along with Fugene 6 (added in a 3:1 ratio of μL Fugene
to μg plasmid) and incubated for 4 h at 37 ꢀC and 10% CO2.
Cells were trypsinized, resuspended to 5e5 cells/mL, and plated
in 384-well plates, followed by immediate treatment with
compound. SEITU (100 μM final) was added as a positive
control. Cells were incubated at 37 ꢀC with 10% CO2 for 24 h
and then activated by calcium ionophore addition of either
A23187 (83 nM final) for eNOS or ionomycin (30 μM final) for
nNOS. After 18 h of incubation, NO production was measured
as described for recombinant iNOS assay (above).
LPS Assay. A dose of 10 mg/kg of LPS (Sigma) was used in
mouse endotoxemia studies. LPS was dissolved in 0.9% sodium
chloride and was given as an intraperitonel injection at volume
of 10 mL/kg. Male mice (Balb/c) were returned to their home
cages after injection and sacrificed 6 h later. Test compounds
were administered by oral gavage (prepared in 90% PEG/5%
Tween 80/5% PVP þ 90% CMC (0.5% w/v)) immediately
before LPS injection. Blood samples were collected into EDTA
containing tubes and processed for plasma collection for anal-
ysis of nitrate and drug levels. Plasma nitrate (marker of iNOS
activity) levels increase gradually with peak induction achieved
6-8 h postinjection. Prior to starting the reactions, plasma
samples were subjected to filtration through a 10 kDa molecular
weight cutoff filter (Fisher Scientific) at 2500 rpm. Nitrates were
converted to nitrites using nitrate reductase, then diamino-
naphthalene (DAN) was added followed by the addition of
NaOH, which converted the nitrite-DAN adduct into a fluoro-
phore. Measurement of the fluorescence of the nitrite-DAN
adduct using an Aquest plate reader (Molecular Devices) accu-
rately determined NO2 concentration.
Mouse Formalin Pain Model. The formalin solution for
intraplantar injection was first prepared by diluting a 10%
formalin solution to 5% with 0.9% saline. Test compounds
were administered by oral gavage (prepared in 90% PEG/5%
Tween 80/5% PVP þ 90% CMC (0.5% w/v) 30 min prior to
formalin injection. A volume of 20 μL of formalin solution was
injected into the left hind paw of each animal. Immediately after
injection of formalin into the left hind paw, the mouse was
placed in a see-through observation chamber and a timer was
activated. The duration of pain behaviors (hind paw flinches,
licking, and biting) displayed was counted in 5 min intervals.
Phase II was observed from 25 to 45 min postformalin injection.
Side effects observed were also noted. Data are presented as time
spent in nociceptive behaviors during phase II (25-45 min
postformalin). There was a single observer throughout the
study. All results are expressed as mean ( SE (n = 5-6 mice).
Statistical significance is inferred from one-way analysis of
variance (ANOVA) followed by appropriate post hoc analyses
(GraphPad Prizm, V5.0).
Mouse Chung Model. Mice underwent a surgical procedure as
described in Chung.27 Briefly, mice were initially anesthetized
with 5% isoflurane in medical-grade pure oxygen and main-
tained with 2-3% isoflurane during the surgical procedure.
Using aseptic techniques, the left Lumbar (L) L5 and L6 spinal
nerves were exposed and isolated with a surgical hook, distal to
their respective dorsal root ganglion. A tight ligation was made
on each nerve using a 6-0 black braided silk thread. The back
muscles were sutured (6-0 braided black silk) and the skin
laceration was stapled with surgical wound clips. Mice were
allowed to recover from spinal nerve injury for approximately
5-10 days. The development of mechanical (tactile) allodynia
was assessed using a set of calibrated von Frey filaments
(Stoelting Co., IL) with increasing stiffness (0.04, 0.07, 0.16,
1
N-(2-fluorophenyl)-3-oxobutanamide (55). H NMR (400 MHz,
CDCl3) δ 8.77 (s, 1H), 8.23 (t, 1H, J = 8.0 Hz), 7.11 (m, 3H), 4.07
(s, 2H), 3.85 (s, 2H). LRMS m/z: calcd for C10H9BrFNO2 272.9,
found 273.8 [M þ H]þ.
4-Bromo-N-(2,3-difluorophenyl)-3-oxobutanamide (56). Com-
pound 56 was synthesized as described for compound 55 using
N-(2,3-difluorophenyl)-3-oxobutanamide (54) as the starting
1
material (59% yield). H NMR (400 MHz, CDCl3) δ 8.91 (s,
1H), 8.05 (m, 1H), 7.09 (m, 1H), 6.95 (m, 1H), 4.02 (s, 2H), 3.82
(s, 2H). LRMS (ESIþ) m/z: calcd for C10H8BrF2NO2 291.9,
found 292.0 [M þ H]þ.
4-(Bromomethyl)-8-fluoroquinolin-2(1H)-one (57). A mixture
of 4-bromo-N-(2-fluorophenyl)-3-oxobutanamide (55, 26 g, 95
mmol) and concentrated H2SO4 (100 mL) was heated to 45 ꢀC
for 18 h. After cooling to 25 ꢀC, the solution was poured into ice
H2O. The solid was filtered, washed with H2O, 10% aqueous
sodium bicarbonate, and again with H2O to give 15 g (65%) of
4-(bromomethyl)-7,8-difluoroquinolin-2(1H)-one (57) as an off-
white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H), 7.65
(d, 1H, J = 8.0 Hz), 7.44 (t, 1H, J = 8.1 Hz), 7.22 (m, 1H), 6.80 (s,
1H), 4.89 (s, 2H). LRMS m/z: calcd for C10H7BrFNO 254.9, found
256.1 [M þ H]þ.
4-(Bromomethyl)-7,8-difluoroquinolin-2(1H)-one (58). Com-
pound 58 was synthesized as described for compound 57 using
4-bromo-N-(2,3-difluorophenyl)-3-oxobutanamide (56) as the
starting material (89%). 1H NMR (400 MHz, DMSO-d6) δ 7.70
(m, 1H), 7.34 (m, 1H), 6.78 (s, 1H), 4.90 (s, 2H).
5-(1H-Benzo[d ]imidazol-2-yl)-4-methylthiazole (78). Benzene-
1,2-diamine (59, 1 g, 9.3 mmol) and 4-methylthiazole-5-car-
boxylic acid (60, 1.32 g, 9.3 mmol) were suspended in poly-
phosphoric acid (PPA, 20 mL) and heated to 200 ꢀC for 4 h.
The hot mixture was carefully and slowly poured into an ice/H2O
slurry with vigorous stirring. NaOH (3M) was added until a fine
solid crashed out. The solid was filtered, washed with H2O, and
dried for 18 h to afford 1.2 g (60%) of 5-(1H-benzo[d]imidazol-2-
1
yl)-4-methylthiazole (78) as a gray solid. H NMR (400 MHz,
DMSO-d6) δ 12.68 (s, 1H), 9.12 (s, 1H), 7.65 (m, 1H), 7.54 (m,
1H), 7.21 (m, 2H), 2.78 (s, 3H). LRMS (ESIþ) m/z: calcd for
C11H9N3S 216.1, found 216.0 [M þ H]þ.
2-Cyclobutyl-1H-imidazo[4,5-b]pyrazine (106). Compound
106 was synthesized as described for compound 78 using
pyrazine-2,3-diamine (101) and cyclobutanecarboxylic acid
(70) as the starting materials (heated to 100 ꢀC) (90% yield).
1H NMR (400 MHz, DMSO-d6) δ NH resonanace not ob-
served, 8.28 (s, 2H), 3.76 (m, 1H), 2.50-1.80 (m, 6H).
Biological Methods. Recombinant iNOS Assay (293 Transi-
ent Transfection Assay). HEK293 cells (ATCC) were seeded
into 150 mm ꢀ 25 mm tissue culture plates and grown to
approximately 70% confluence in DMEM containing 10%
FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Each plate was transfected with 1 mL DMEM with Pen/Strep
containing 10 μg of human or murine iNOS expression
plasmid and 30 μL of Fugene 6 and incubated for 4 h at
37 ꢀC and 10% CO2. Cells were trypsinized, resuspended to
5e5 cells/mL, and plated in 1536-well plates, followed by
immediate treatment with compound. 1400W (100 μM final)
was added as a positive control. After 18 h of incubation, NO
production was measured by adding 2,3-diaminonaphthalene
(DAN) at 10 μg/mL final concentration (Invitrogen) diluted
in DMEM supplemented with hydrochloric acid (0.1 N final)
and incubated for 20 min at room temperature. Fluorescence
was measured after increasing pH with sodium hydroxide
(0.12 N final).26

7754 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Payne et al.
0.4, 1.0, 1.4, 2.0 g/force) and exerted increasing force when
pushed against the tested paw for a 10 s period. The stimulation
with a von Frey filament was completed within a 10 s period, and
the percent response frequency was calculated using Dixon’s up
and down formula (Chaplan 1994). Control animals with no
surgery or animals that underwent sham-surgery showed a 50%
withdrawal threshold of 1.5-2 g (data not shown). Only those
animals that showed baseline 50% withdrawal thresholds of
∼0.2-0.3 g anytime between 5 and 10 days postsurgery were
considered to have achieved tactile allodynia and were included
in all drug treatment studies.
Mouse Rotarod Assay. Effects of 42 on motor coordination
were assessed in the rotarod assay.28 Male C57Bl/6j mice were
acclimated to testing room for 30 min and to rotarod device for
5 min at 4 rpm, during which animals were repeatedly returned
to the device as the animals fall. Approximately 30 min post-
acclimation to rotarod device, animals were subjected to two
training sessions 30 min apart. Each session involved acclima-
tion to accelerating rotarod (4-40 rpm in 300 s) with latencies to
drop recorded. Only those mice with latencies of >60 s were
included in the assay. Following oral administration of either
gabapentin or 42, latencies to fall on accelerating rotarod were
determined at 1, 2, and 3 h postdose. Animals unable to stay on
the rotarod at 4 rpm were assigned a latency of 0 s; animals
completing a trial were assigned a latency of 300 s.
A. C.; Beaton, H. G.; Boughton-Smith, N.; Cook, T. R.; Cooper, S. L.;
Fraser-Rae, L.; Hallam, K.; Hamley, P.; McInally, T.; Nicholls, D. J.;
Pimm, A. D.; Wallace, A. V. 1,2-Dihydro-4-quinazolinamines: Potent,
Highly Selective Inhibitors of Inducible Nitric Oxide Synthase Which
Show Antiinflammatory Activity in Vivo. J. Med. Chem. 2003, 46 (6),
913–916.
(9) Davey, D. D.; Adler, M.; Arnaiz, D.; Eagen, K.; Erickson, S.;
Guilford, W.; Kenrick, M.; Morrissey, M. M.; Ohlmeyer, M.; Pan,
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and Activity of 2-Imidazol-1-ylpyrimidine Derived Inducible Ni-
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(10) Patman, J.; Bhardwaj, N.; Ramnauth, J.; Annedi, S. C.; Renton, P.;
Maddaford, S. P.; Rakhit, S.; Andrews, J. S. Novel 2-aminobenzo-
thiazoles as selective neuronal nitric oxide synthase inhibitors.
Bioorg. Med. Chem. Lett. 2007, 17, 2540–2544.
(11) Nason, D. M.; Heck, S. D.; Bodenstein, M. S.; Lowe, J. A., III;
Nelson, R. B.; Liston, D. R.; Nolan, C. E.; Lanyon, L. F.; Ward,
K. M.; Volkmann, R. A. Substituted 6-phenyl-pyridin-2-ylamines:
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(12) Bonnefous, C.; Payne, J. E.; Roppe, J.; Zhang, H.; Chen,
X.; Symons, K. T.; Nguyen, P. H.; Sablad, M.; Rozenkrants,
N.; Zhang, Y.; Wang, L.; Severance, D.; Walsh, J. P.; Yazdani,
N.; Shiau, A. K.; Noble, S. A.; Rix, P.; Rao, T. S.; Hassig, C. A.;
Smith, N. D. Discovery of Inducible Nitric Oxide Synthase
(iNOS) Inhibitor Development Candidate KD7332, Part 1:
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3062.
Acknowledgment. We thank John Walsh for high resolu-
tion mass spectra and Xing Cheng for assistance with NMR
spectroscopy.
(13) As disclosed previously, modification of the quinolinone portion of
8 did not lead to an improvement in rodent pharmacokinetics. See
ref 11.
(14) Choi, H. Y.; Chi, D. Y. Nonselective Bromination-Selective
Debromination Strategy: Selective Bromination of Unsymmetrical
Ketones on Singly Activated Carbon against Doubly Activated
Carbon. Org. Lett. 2003, 5 (4), 411–414.
(15) Later in the project, we found higher yields could be obtained by
alkylating the benzimidazole core with 58 by heating the two
starting materials neat under a N2 atmosphere (see preparation
of compound 47).
Supporting Information Available: Experimental details on
the preparation of intermediates 79-119, biological methods,
copies of the 1H NMR, 13C NMR, LC trace, and high resolution
mass spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
(16) The regioselectivity of the desired compound 42 was confirmed by
the characteristic shift of the quinolinone C3 hydrogen (DMSO-d6:
δ = 5.36 (s, 1H)) compared to the undesired regiosiomer 43
(DMSO-d6: δ = 6.26 (s, 1H)). A similar shift was seen for
compound 25 (DMSO-d6: δ = 5.53 (s, 1H)), the structure of which
was confirmed by X-ray Diffraction. Similar chemical shifts of the
quinolinone C3 hydrogen were observed with analogues 33-41,
44-50.
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