Discovery of inducible nitric oxide synthase (iNOS) inhibitor development candidate KD7332, part 1: Identification of a novel, potent, and selective series of quinolinone iNOS dimerization inhibitors that are orally active in rodent pain models

Article abstract of DOI:10.1021/jm900173b

There are three isoforms of dimeric nitric oxide synthases (NOS) that convert arginine to citrulline and nitric oxide. Inducible NOS is implicated in numerous inflammatory diseases and, more recently, in neuropathic pain states. The majority of existing NOS inhibitors are either based on the structure of arginine or are substrate competitive. We describe the identification from an ultra high-throughput screen of a novel series of quinolinone small molecule, nonarginine iNOS dimerization inhibitors. SAR studies on the screening hit, coupled with an in vivo lipopolysaccharide (LPS) challenge assay measuring plasma nitrates and drug levels, rapidly led to the identification of compounds 12 and 42 - potent inhibitors of the human and mouse iNOS enzyme that were highly selective over endothelial NOS (eNOS). Following oral dosing, compounds 12 and 42 gave a statistical reduction in pain behaviors in the mouse formalin model, while 12 also statistically reduced neuropathic pain behaviors in the chronic constriction injury (Bennett) model.

Full text of DOI:10.1021/jm900173b

J. Med. Chem. 2009, 52, 3047–3062
3047
Discovery of Inducible Nitric Oxide Synthase (iNOS) Inhibitor Development Candidate
KD7332, Part 1: Identification of a Novel, Potent, and Selective Series of Quinolinone iNOS
Dimerization Inhibitors that are Orally Active in Rodent Pain Models
Ce´line Bonnefous,† Joseph E. Payne,† Jeffrey Roppe,† Hui Zhuang,† Xiaohong Chen,† Kent T. Symons,‡ Phan M. Nguyen,‡
Marciano Sablad,§ Natasha Rozenkrants,§ Yan Zhang,§ Li Wang,| Daniel Severance,† John P. Walsh, Nahid Yazdani,
Andrew K. Shiau,‡ Stewart A. Noble,† Peter Rix,| Tadimeti S. Rao,§ Christian A. Hassig,‡ and Nicholas D. Smith*,†
Department of Chemistry, Department of Biology, Department of Pharmacology, Department of Drug Metabolism and Pharmacokinetics,
Department of Analytical Chemistry, Kalypsys Inc., 10420 Wateridge Circle, San Diego, CA 92121
ReceiVed February 10, 2009
There are three isoforms of dimeric nitric oxide synthases (NOS) that convert arginine to citrulline and
nitric oxide. Inducible NOS is implicated in numerous inflammatory diseases and, more recently, in
neuropathic pain states. The majority of existing NOS inhibitors are either based on the structure of arginine
or are substrate competitive. We describe the identification from an ultra high-throughput screen of a novel
series of quinolinone small molecule, nonarginine iNOS dimerization inhibitors. SAR studies on the screening
hit, coupled with an in vivo lipopolysaccharide (LPS) challenge assay measuring plasma nitrates and drug
levels, rapidly led to the identification of compounds 12 and 42spotent inhibitors of the human and mouse
iNOS enzyme that were highly selective over endothelial NOS (eNOS). Following oral dosing, compounds
12 and 42 gave a statistical reduction in pain behaviors in the mouse formalin model, while 12 also statistically
reduced neuropathic pain behaviors in the chronic constriction injury (Bennett) model.
Introduction
structural similarity to arginine confers risk of potential interfer-
Nitric oxide synthases (NOSa) are dimeric enzymes that
catalyze the formation of nitric oxide (NO) and citrulline from
arginine and molecular oxygen using heme, biopterin, and
NADPH as cofactors. Three isoforms of NOS have been
identified: two of these, endothelial NOS (eNOS) and neuronal
NOS (nNOS), are constitutively expressed and play important
roles in blood pressure regulation and neurotransmission
respectively. The third isoform, inducible NOS, is synthesized
in response to exposure to inflammatory and immunologic
stimuli and has been implicated in the pathogenesis of numerous
diseases including septic shock, asthma, inflammatory bowel
disease, osteoarthritis, and rheumatoid arthritis.1 More recently,
there is increasing evidence of the involvement of iNOS in the
development and maintenance of neuropathic pain states.2
There has been substantial activity in the pharmaceutical
industry to identify selective iNOS inhibitors, with selectivity
over eNOS being particularly important as inhibition can result
in an elevation of blood pressure. Much of the initial work
centered on substrate competitive inhibitors structurally related
to arginine. L-NNA (1) and L-NIL (2) exemplify this structural
class and exhibit poor selectivity for iNOS over eNOS.3
GW271450 (3), a structurally related compound exhibits time-
dependent inhibition, improved selectivity for iNOS over eNOS
and is efficacious in preclinical models of pain.4 Given that
ence with physiological processes dependent on arginine
transport and metabolism,5 we prioritized the identification and
characterization of novel nonsubstrate competitive NOS inhibitors.
Small molecule iNOS inhibitors (Figure 1) that are structurally
distinct from arginine have precedent and often incorporate a
guanidine mimetic. For example, compound 4, a substrate
competitive inhibitor, displayed high iNOS potency and good
efficacy in a rat LPS assay measuring nitrate release, however,
selectivity over eNOS was poor (40-fold).6 Another substrate
competitive inhibitor AR-C102222 (5), demonstrated good
selectivity for iNOS over eNOS and was efficacious in a rat
adjuvant-induced arthritis model and other models of neuro-
pathic and surgical pain.7
More recently, BBS-4 (6), an imidazole-based inhibitor that
is able to disrupt the formation of the active NOS dimer by
direct coordination of the imidazole to the heme iron in the
active site of the enzyme, was reported.8 Compound 6 is a potent
and selective iNOS inhibitor, however, compounds that actively
coordinate to heme iron have been shown to be potent inhibitors
of cytochrome P450s,9 resulting in the potential for drug-drug
interactions. NOS dimerization inhibitors that lack cytochrome
P450 cross reactivity are expected to exhibit a unique in vivo
profile distinct from substrate competitive inhibitors and have
utility in neuropathic pain. We thus conducted a cell-based ultra
high-throughput screen on a small molecule library of ap-
proximately 880000 compounds looking for nonarginine, non-
imidazole-based dimerization inhibitors of iNOS. From this
screen, four series of human iNOS inhibitors were identified,
including a set of quinolinones represented by 7 (Figure 2).
Quinolinone 7 had an EC50 of 1.3 µM against human iNOS
(hiNOS) and had good selectivity over human eNOS (heNOS
> 70-fold), however it had poor inhibitory activity against the
mouse iNOS isoform (miNOS EC50 ) 46 µM)sa key issue as
activity against miNOS would be required for future pharma-
cological evaluation of the series in rodents. This paper describes
* To whom correspondence should be addressed. Phone: (858)754-3481.
Fax: (858)754-3301. E-mail: nsmith@kalypsys.com.
†
Department of Chemistry.
Department of Biology.
Department of Pharmacology.
Department of Drug Metabolism and Pharmacokinetics.
‡
§
|
Department of Analytical Chemistry.
a NOS, nitric oxide synthase; NO, nitric oxide; iNOS, inducible NOS;
eNOS, endothelial NOS; nNOS, neuronal NOS; uHTS, ultra high-throughput
screening; LPS, lipopolysaccharide; SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis; CCI, chronic constriction injury; DAN,
diaminonaphthalene.
10.1021/jm900173b CCC: $40.75
2009 American Chemical Society
Published on Web 04/17/2009

3048 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Bonnefous et al.
Figure 1. iNOS inhibitors.
Scheme 2a
Figure 2. Ultra high-throughput screening hit quinolinone 7.
Scheme 1a
a Reagents and conditions: (a) DMSO, 50 °C, 1 h. (b) (i) Carboxylic
acid, oxalyl chloride, DMF, DCM, 25 °C, 2 h; (ii) DIEA, NMP, 25 °C,
14-18 h; (iii) Propylamine, 25 °C, 30 min.
to give 50. Cyclization of 50 under acidic conditions gave
8-fluoroquinolinone (52) (R1 ) F), which was reacted with
aniline followed by acylation with either furan-2-carbonyl
chloride or 4-methylthiazole-5-carbonyl chloride to give 8 or
10, respectively. Of note is the use of two equivalents of
acylating reagent, which first alkylates the quinolinone, followed
by the aniline nitrogen; addition of propylamine then removes
the quinolinone acyl group to give the desired product. Similarly,
starting from commercially available quinolinone 51 (R1 ) H),
compounds 7 and 9 were prepared.
Scheme 2 summarizes the preparation of compounds 11-22
and 24-26. Thus substituted anilines were alkylated with
8-fluoroquinolinone (52) to give intermediates 55-61, which
were subsequently acylated with 4-methylthiazole-5-carbonyl
chloride to give compounds 11-17. Additionally, 3-chlorophe-
nylaniline (56) was acylated with a selection of aryl and alkyl
acid chlorides to give 18-22 and 24-26.
a Reagents and conditions: (a) Methyl 3-oxobutanoate, pyridine, xylene,
reflux, 30 min. (b) (i) Bromine, AcOH, 25 °C, 5 h; (ii) acetone, 25 °C,
18 h. (c) H2SO4, 45 °C, 18 h. (d) Aniline, DMSO, 50 °C, 1 h. (e) (i)
Carboxylic acid, oxalyl chloride, DMF, DCM, 25 °C, 2 h; (ii) DIEA, NMP,
25 °C, 14 h; (iii) propylamine, 25 °C, 30 min.
the characterization and structure-activity relationship of this
quinolinone series of iNOS dimerization inhibitors and the
evolution of uHTS hit 7 to potent inhibitors of human and mouse
iNOS that are orally active in mouse LPS challenge, mouse
formalin, and mouse chronic constriction injury models.
Chemistry. The synthetic route used to make uHTS hit 7
and compounds 8-10 is shown in Scheme 1. Thus keto-amide
49 formed by the reaction of fluoroaniline 48 with methyl
3-oxobutanoate, was selectively brominated at the R position10

iNOS Inhibitor DeVelopment Candidate KD7332
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3049
Scheme 3a
was acylated to give compound 33. Oxidation of 33 with
m-CPBA gave isoquinoline N-oxide (34).
Scheme 8 illustrates the synthesis of compounds 35-41,
43, and 44, which further explores the impact of substitution
on the quinolinone nucleus. Thus keto-amides 98-102
formed by the reaction of anilines 92-96 with methyl
3-oxobutanoate were selectively brominated at the R-position
to give 50 and 103-107. Using 1-chloromethyl-4-fluoro-1,4-
diazoniabicyclo[2.2.2]octane bis-(tetrafluoroborate), fluorine
was introduced at the 2-position of 50 (R4 ) 8-F) to give
108, which was then cyclized in hot sulfuric acid to give
quinolinone 114 (R4 ) 8-F, R5 ) F). Similarly, 103-107
were converted to quinolinones 51 and 109-113. These
quinolinone intermediates were then processed as described
in Scheme 1 to give compounds 35-38, 40, and 44.
When the synthesis of 5-fluoroquinolinone derivative 39 was
initially attempted using 3-fluoroaniline as a starting material,
it was found that cyclization of intermediate 104 occurred at
the 4-position to give undesired quinolinone 110 rather than at
the 2-position to give 123 (Scheme 9). Thus to access compound
39, the 4-position of intermediate 104 was blocked with bromine
leading via intermediate quinolinone 112 (R4 ) 5-F, 8-Br, R5
) H) to 122 (R4 ) 5-F, 8-Br, R5 ) H). Removal of bromine
from 122 by hydrogenation then gave desired 39. Compounds
41 and 43 were prepared by acylation of 120 (R4 ) 7,8-diF, R5
) H) with the respective carbonyl chloride, while imidazole
42 was prepared as shown in Scheme 3 but using 113 (Scheme
8, R4 ) 7,8-diF, R5 ) H) in place of 52 as the starting
quinolinone.
a Reagents and conditions: (a) TBDMS-Cl, DMF, 25 °C, 4 h. (b) NaI,
acetone, 25 °C, 2 h. (c) HATU, Et3N, DMF, 25 °C, 18 h. (d) NaH, DMF,
25 °C, 3.5 h.
Imidazole-amide (23) required an alternative route from that
shown in Scheme 2 for its preparation that utilized a protected
quinolinone alkylating agent (Scheme 3). Thus 8-fluoroquino-
linone (52) was protected with a tertiary-butyl dimethylsilyl
group and converted to the corresponding iodide 63. Alkylation
of amide 66 with iodide 63 gave 23 (deprotection of the silyl
protecting group occurred under the reaction conditions).
Finally, the 7,8-difluorocoumarin (45) was prepared as shown
Scheme 4 but using 1-(3,4-difluoro-2-hydroxyphenyl)ethanone16
as the starting material, while the isoquinoline (46) and
isoquinoline N-oxide (47) were prepared as shown in Scheme
7 but using 2,3-difluoro-6-iodobenzoic acid as the starting
material.
Compounds 27-34 that explore the SAR of the quinolinone
portion of 12 required a number of diverse synthetic routes for
their preparation (Schemes 4-7). Thus pyridone 27 was
prepared in three steps as shown in Scheme 4: 3-chloroaniline
was alkylated with 67,11 followed by acylation with 4-meth-
ylthiazole-5-carbonyl chloride to give 69. Hydrolysis of the
2-chloropyridine of 69 in acetic acid then gave pyridone 27.
Tetrahydroquinolinone (28) was prepared in five steps starting
from ester 70. Thus reduction of ester 70 to alcohol 71 with
LAH was followed by conversion to the benzyllic bromide (72)
with PBr3, which in an analogous fashion to 27 was converted
to tetrahydroquinolinone (28). Coumarin 29 was prepared from
hydroxyketone 75 in four steps as shown in Scheme 4. Reaction
of 75 with phosphoranylidene ester (76)12 gave coumarin 77,
which was converted to the benzylbromide 78 using NBS/AIBN.
Alkylation of 3-chloroaniline with 78 followed by acylation with
4-methylthiazole-5-carbonyl chloride gave 29.
Results and Discussion
The compounds prepared in Schemes 1-8 were tested for
inhibitory activity against iNOS, eNOS, and nNOS using a cell-
based NOS assay using transiently transfected HEK293 cells.
Nitric oxide was measured using 2,3-diaminonapthalene with
each EC50 determined from three separate assay runs (each using
a 10-point concentration curve). In this phenotypic cell-based
assay, compounds are examined for their ability to inhibit the
production of nitric oxide from the three indicated NOS
isoforms. Initial SAR work was focused on changing the
substituent R1 at positions around the quinolinone core and the
amide group R2 of 7. Selected SAR illustrating single-point
changes relative to 7 is shown in Table 1. Incorporation of an
8-fluoro group on the quinolinone 8 led to a slight improvement
in potency (hiNOS EC50 ) 1.1 µM), while changing the
2-furylamide to a 5-thiazolyl amide (9) improved human iNOS
potency to 0.50 µM. Combining these two changes into a single
molecule (10) led to an additive improvement in hiNOS potency
(hiNOS EC50 ) 0.20 µM) while still maintaining excellent
selectivity over heNOS (>500 fold).
Access to methoxyquinoline (30) and N-methyl-quinolinone
(31) is depicted in Scheme 5. Thus alkylation of 12 with NaH/
dimethylsulfate gave a mixture of regioisomers which were
separated to give 30 and 31. The identity of 30 and 31 was
confirmed by the independent synthesis of 30.13
Compound 32 was prepared from 8-fluoroquinoline (80)14
in three steps as shown in Scheme 6. Quinoline benzyl bromide
81 resulting from the bromination of 80 with NBS/AIBN was
reacted with 3-chloroaniline to give 82; acylation of 82 then
gave 32.
Isoquinoline (33) and its N-oxide 34 were prepared as shown
in Scheme 7. Thus palladium catalyzed cyclization15 of ortho-
iodoalkene (85) gave isoquinolinone (86), which was depro-
tected and converted to 1-chloroisoquinoline (88) using POCl3.
Intermediate 88 was converted to benzylbromide (89) using
NBS/AIBN, which was used to alkylate 3-chloroaniline to afford
90. Palladium catalyzed hydrogenation led to selective reduction
of the chlorine of the 1-chloroisoquinoline to give 91, which
While maintaining the 8-fluoroquinolinone moiety of 10, the
SAR of the phenylamide portion of the molecule was examined
as shown in Table 2. It quickly became apparent that lipophillic
groups at the 3-position were favored as demonstrated by a R1
substituent scan. Thus 2-chloro derivative 11 lost potency
compared to 10, whereas the 3-chloro 12 showed a significant
increase in potency (hiNOS EC50 ) 0.011 µM) while the
4-chloro derivative 13 was equipotent to 10. When examining

3050 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Bonnefous et al.
Scheme 4a
a Reagents and conditions: (a) 3-Chloroaniline, Na2CO3 or K2CO3, DMF or DMSO, 2 h. (b) 4-Methylthiazole-5-carbonyl chloride, DMF or NMP, 25 °C.
(c) AcOH, 140 °C. (d) LiAlH4, THF, 0 °C, 30 min. (e) PBr3, DCM 0-25 °C, 30 min. (f) 76, toluene, 120 °C, 18 h. (g) NBS, AIBN, CCl4, reflux, 18 h.
Scheme 5a
Because of the breakthrough improvements in human and
mouse iNOS potency, 12 was characterized further. As il-
lustrated in Figure 3, single crystal X-ray diffraction confirmed
the structure of 12 and showed a trans- relationship between
the thiazole and quinolinone about the amide bond.
The quaternary structure of the iNOS enzyme in the presence
of 12 was investigated using low-temperature SDS-PAGE,
followed by immunoblotting with antibodies recognizing the
iNOS protein.18 Under these conditions, monomeric and dimeric
a Reagents and conditions: (a) NaH, dimethylsulfate, DCM, 25 °C, 18 h.
iNOS can be resolved on the basis of having distinct mobility
within the gel matrix. Enzyme isolated from cells treated with
vehicle (DMSO) or substrate competitive inhibitor SEITU (50
Scheme 6a
µM)19 retained a population of dimeric iNOS (Figure 4). In
contrast, compound 12 reduced the level of iNOS dimer in a
concentration-dependent manner, indicating that 12 is an iNOS
dimerization inhibitor (seven-point dose response; 1 µM to 0.2
nM). A similar reduction of dimer population was seen with
imidazole-based dimerization inhibitor BBS-4 at 1 µM (6). This
data suggests that 12 (and the compounds described in this
paper) binds iNOS in a monomeric or loose dimeric state and
interferes with the formation of a stable, enzymatically active
dimer.
a Reagents and conditions: (a) NBS, AIBN, CCl4, reflux, 18 h. (b)
3-Chloroaniline, K2CO3, DMF, 60 °C, 2 h. (c) 4-Methylthiazole-5-carbonyl
chloride, NMP, 25 °C, 18 h.
the nature of the group at the 3-position, small lipophilic groups
such as 3-fluoro (hiNOS EC50 ) 0.062 µM), 3-cyano (hiNOS
EC50 ) 0.056 µM), or 3-methyl (hiNOS EC50 ) 0.104 µM)
maintained activity relative to 12, whereas more polar groups
such as 3-methoxy (hiNOS EC50 ) 0.99 µM) or 3-pyridyl
(hiNOS EC50 ) 1.8 µM)17 lost significant activity.
Not only did 3-chloro derivative 12 show a greatly improved
human iNOS potency, it also maintained good selectivity over
human eNOS (heNOS EC50 ) 26 µM, 2300 fold selective) and
nNOS (hnNOS EC50 ) 2.3 µM, 210 fold selective). Crucially,
compared to uHTS hit 7 (miNOS ) 46 µM), 12 was potent
against the mouse iNOS enzyme (miNOS ) 0.12 µM), allowing
for proof of concept mouse pharmacology experiments (vide
infra).
Having identified lead molecule 12, we next examined the
SAR of the amide region of the molecule (R2; Table 2). It was
found that the ortho-methyl group on the thiazole of 12 was
important for potency, thus des-methyl thiazole amide 18
(hiNOS EC50 ) 0.053 µM) lost potency relative to 12 (hiNOS
EC50 ) 0.011 µM), a trend that was seen for a number of other
R2 heterocyclic groups.20 A pyridyl scan while fixing an ortho-
methyl also demonstrated that the position of the heteroatom
in the R2 group was important. Thus 20 and 21 maintained
activity (hiNOS EC50 < 0.1 µM), whereas 19 (hiNOS EC50
)
0.35 µM) and 22 (hiNOS EC50 ) 0.42 µM) lost significant
activity relative to 12. Incorporating these two lessons into
alternative R2 five-membered ring heterocycles led to the

iNOS Inhibitor DeVelopment Candidate KD7332
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3051
Scheme 7a
a Reagents and conditions: (a) (i) SOCl2, 80 °C, 1.5 h; (ii) Et3N, THF, 5-25 °C, 2 h. (b) Dicyclohexylamine, PPh3, Pd(OAc)2, DMA, 100 °C, 18 h. (c)
H2SO4, 150 °C, 2.5 h. (d) POCl3, 80 °C, 2 h. (e) NBS, AIBN, CCl4, reflux, 18 h. (f) 3-Chloroaniline, K2CO3, DMF, 60 °C, 2 h. (g) H2, Pd/C, MeOH, 25 °C,
4 h. (h) 4-Methylthiazole-5-carbonyl chloride, NMP, 25 °C, 18 h. (i) m-CPBA, DCM, 25 °C, 1 h.
Scheme 8a
a Reagents and conditions: (a) Methyl 3-oxobutanoate, pyridine, xylene, reflux, 30 min. (b) (i) Bromine, AcOH, 25 °C, 5 h; (ii) acetone, 25 °C, 18 h. (c)
1-Chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis-(tetrafluoroborate) ACN, 60 °C, 2 h. (d) H2SO4, 45 °C, 18 h. (e) 3-Chloroaniline, DMSO, 50
°C, 1 h. (f) (i) Carboxylic acid, oxalyl chloride, DMF, DCM, 25 °C, 2 h; (ii) DIEA, NMP, 25 °C, 14 h; (iii) propylamine, 25 °C, 30 min. (g) H2, Pd/C,
MeOH, 25 °C, 18 h.
discovery of imidazole 23 (hiNOS EC50 ) 0.009 µM) and
isoxazole 24 (hiNOS EC50 ) 0.016 µM), two potent human
iNOS inhibitors that still maintained good selectivity over
heNOS (>400 fold). Of note is the higher selectivity for human

3052 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Table 1. Selected SAR of uHTS Hit 7
Bonnefous 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 <50% inhibition @ 100 µM. c <50% inhibition
@ 10 µM (100% being maximal inhibition of control compond 6).
Figure 3. Single crystal X-ray diffraction of compound 12.
Table 2. SAR of Amide (R2) and Aryl (R1) of Compound 10
Figure 4. Compound 12 destabilizes murine iNOS dimer in cells.
iNOS over human nNOS of compound 12 (210-fold selective)
relative to compounds 19-24 (10-45-fold selective). While
excellent hiNOS potency was obtained with five-membered
heteroaromatic R2 groups such as 12, aliphatic cyclopentane (25)
(hiNOS EC50 ) 0.77 µM) lost activity relative to 12. Interest-
ingly however, when the cyclopentane was truncated to iso-
propyl amide (26), an improvement in potency was observed
(hiNOS EC50 ) 0.16 µM).
Having identified 12 through optimization of the amide and
aryl ring regions of the original screening hit, we returned to
examine the SAR of the quinolinone ring further using 12 as a
template (Tables 3 and 4). As shown in Table 3, truncation of
the quinolinone to give 27 led to a complete loss of potency,
whereas saturated system 28 maintained some level of potency
(hiNOS EC50 ) 1.14 µM).21 Recently, Sanofi-Aventis reported
coumarin-based iNOS inhibitors,22 however, upon preparation,
coumarin 29 (hiNOS EC50 ) 2.0 µM) was 200-fold less potent
than the corresponding quinolinone 12. Further SAR examining
the embedded amide of the quinolinone of 12 indicated the
importance of this region of the molecule for potency against
the iNOS enzyme. Thus methylation of either the oxygen (30)
or nitrogen (31) or the removal of the amide as in quinoline 32
led to a complete loss of hiNOS potency. Interestingly however,
isoquinoline 33 had submicromolar potency against the enzyme
(hiNOS EC50 ) 0.76 µM), and conversion to the N-oxide 34
gave a further 10-fold increase in potency (hiNOS EC50 ) 0.087
µM). Isoquinoline N-oxide (34) represents a potent quinolinone
replacement but one without the hydrogen bond donor capabili-
ties of the quinolinone.
a Cell-based NOS assay using transiently transfected HEK293 cells; NO
measured using 2,3- diaminonapthalene (DAN). Each EC50 is determnd 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 <50% inihition @ 100 µM. c <50% inhibition
@ 10 µM (100% being maximal inhibition of control compond 6). d Not
determined. e n ) 1 data.
Examination of the SAR around the quinolinone ring of 12
illustrated that the size and position of the halogen greatly
effected hiNOS potency (Table 4). For example, removing the
8-fluoro of 12, giving 35 or changing it to an 8-chloro (36), led
to a decrease in potency of 10-100 fold. In moving the position

iNOS Inhibitor DeVelopment Candidate KD7332
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3053
Table 3. SAR Examining Quinolinone Portion of Compound 12
Table 5. Incorporation of a Difluoro Motif into Coumarin 29,
Isoquinoline 33, and Isoquinoline N-oxide 34
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 Not determined. c n ) 1 data. d n ) 2 data.
e <50% inhibition @ 10 µM (100% being maximal inhibition of control
compond 6).
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 <50% inhibition @ 100 µM.
quinolinone 35, we prepared 7,8-difluoroquinolinone 40 and
were pleased to see a further increase in potency (hiNOS EC50
) 0.002 µM) compared to 12. Additionally, when this 7,8-
disubstituted quinolinone motif was combined with other potent
R2 groups identified previously, giving pyridine 41, imidazole
42, and isoxazole 43, a corresponding increases in potency was
observed, leading to a series of very potent hiNOS inhibitors
(hiNOS EC50s < 0.010 µM). These 7,8-difluoroquinolinone
derivatives still maintained good selectivity over heNOS
(100-1500-fold), although it was noted that in general selectiv-
ity was eroded compared to the corresponding monofluoro
derivatives (400-2300-fold). Further, the 7,8-difluoroquinoli-
none derivatives showed moderately lower selectivity for human
iNOS over human nNOS, i.e., compound 40 (60-fold selective)
versus 12 (210-fold) and 42 (23-fold selective) versus 23 (40-
fold). We also prepared the 3,8-difluoroquinolinone derivative
44, however, with an EC50 ) 0.026 µM, this did not show an
improvement in potency compared to 12.
Table 4. Identification of a Potency-Enhancing 7,8-Difluoroquinolinone
Motif
Having identified a 7,8-difluoro substitution pattern on the
quinolinone moiety as being optimal for potency, we installed
this same fluorine substitution pattern on the coumarin and
isoquinolines described in Table 3. As shown in Table 5, 7,8-
difluorocoumarin (45), -isoquinoline (46), and -isoquinoline
N-oxide (47) all show a 3-5-fold increase in hiNOS potency
relative to the corresponding monofluoro derivative (29, 33, and
34; Table 3). Once again, isoquinoline N-oxide 47 is a potent
quinolinone replacement, demonstrating excellent hiNOS po-
tency (hiNOS EC50 ) 0.019 µM) and selectivity over human
eNOS (1800-fold) and nNOS (170-fold).
In Vivo Characterization: Mouse LPS Assay. As described
in Tables 1-5, SAR studies on uHTS hit 7 led to the
identification of a large number of potent human iNOS
inhibitors. As the SAR evolved during the course of the project,
we utilized a LPS challenge assay23 to confirm that the increases
seen in in vitro iNOS potency translated to increases in in vivo
activity, as determined by measuring a biomarker (NO) reflective
of activity in mechanism. To further leverage this LPS challenge
assay, we obtained drug levels and so looked to establish a
PK-PD relationship, enabling us to effectively triage com-
pounds. In this LPS assay, test articles (30 mpk) or vehicle
control were administered orally at t ) 0 h, followed im-
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 <50% inhibition @ 100 µM. c <50%
inhibition @ 10 µM (100% being maximal inhibition of control compound
6). d n ) 1 data.
of the fluorine atom around the ring, it was found that the
7-fluoro derivative 37 (hiNOS EC50 ) 0.053 µM) maintained
good levels of potency, while the 6- and 5-fluoro derivatives
led to a decrease in potency relative to 12. As the 7-fluoro
derivative 37 increased potency relative to the unsubstituted

3054 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Bonnefous et al.
Table 6. Reduction of Plasma Nitrate Levels by Selected Compounds Following LPS Challengea
compd
hiNOS EC50 (µM)
miNOS EC50 (µM)
% nitrates rdn @ 30 mg/kg po (%)b
drug plasma @ 6 h (ng/mL)
LPS ED50 (mg/kg) po
42
41
12
23
43
40
44
34
47
26
0.003 (0.002)
0.011 (0.003)
0.011 (0.002)
0.009 (0.003)
0.003 (0.001)
0.002 (0.001)
0.026 (0.01)
0.087 (0.02)
0.019 (0.02)
0.16 (0.02)
0.014 (0.001)
0.057 (0.02)
0.12 (0.01)
0.034 (0.03)
0.022 (0.001)
0.032 (0.01)
0.13 (0.12)
0.66 (0.25)
0.074 (0.004)
2.7 (0.6)
95
95
90
67
67
62
29
20
10
6
18
63
77
5
16
10
7
13
BLQc
8
NDd
13
BLQc
39
NDd
NDd
NDd
NDd
20
21
a Compounds administered at t ) 0 h (30 mg/kg po) immediately followed by LPS (10 mg/kg ip). Plasma nitrate and drug levels measured at t ) 6 h.
b % Reduction compared to vehicle control. c Below limits of quantification. d Not determined.
mediately by injection of LPS (10 mpk ip). At t ) 6 h, a time
corresponding to peak nitrate induction, nitrate and drug levels
were measured in plasma, with the reduction in nitrate levels
compared to vehicle indicating the level of iNOS inhibition.
Clearly, potency against the mouse iNOS enzyme was crucial
for the assessment of compounds in rodent pharmacology
experiments such as the LPS challenge assay. Importantly, as
shown for a selection of compounds in Table 6, mouse iNOS
potency did increase proportionally as human iNOS potency
was optimized. It should be noted though that the compounds
are 5-10-fold less potent against the mouse iNOS enzyme than
the human iNOS enzymesa trend that was observed for all the
compounds in this series.
As is evident on inspection of Table 6, an in vitro potency/
PK/PD correlation is generally observed. Compounds 42, 41,
and 12 exhibit near complete reduction in plasma nitrate levels
(>90%) consistent with high miNOS potency and favorable
exposure, as assessed by circulating drug levels (>20ng/mL)
at 6 h. In contrast, compounds 23, 43, and 30, despite having
good potency against mouse iNOS (miNOS EC50 < 0.035 µM),
achieve lower levels of plasma nitrate reduction (∼65%) due
to less favorable oral exposure (in each case drug levels at 6 h
postdose are <10 ng/mL). Finally, as predicted, compounds with
low potency against mouse iNOS, with moderate drug levels
at 6 h (34, 26) perform poorly in reducing circulating nitrate
levels (<30% reduction). For compounds 42, 41, 12, 23, and
40, a full dose response in the LPS challenge assay was carried
out, with 42 and 12 attaining ED50s following oral dosing of 5
and 10 mg/kg, respectively. This assay was used to triage >35
compounds and identified 42 and 12 as compounds for further
profiling.
Mouse pharmacokinetics was carried out on 12 and 42 (Table
7) and showed that the compounds were orally bioavailable with
Cmaxs (10 mg/kg po) of 1.4 and 2.2 µM, respectively. These
plasma levels are 12× and 150× the respective mouse iNOS
EC50s, meaning that 12 and 42 were good tools for carrying
out proof-of-concept studies in rodent models of pain (vide
infra).
Despite achieving good plasma concentrations after oral
dosing, both molecules suffered from high clearance (Clp >100
mL/min/kg), suggesting these molecules would not be capable
of sustained duration of action in pharmacology models. As part
of an effort to understand the mechanism of clearance,
compound 12 was incubated under conditions of either NADPH-
dependent oxidation (NADPH only), UGT-dependent glucu-
ronide conjugation (UGT only), or combined NADPH and UGT
activities (NADPH+UGT) (Figure 5). The results clearly
demonstrate that oxidation and glucuronide conjugation both
contribute to the in vitro clearance of 12. Further SAR studies
on the quinolinone series utilizing this assay led to the
identification of iNOS inhibitors with high metabolic stability
and good rodent and nonhuman primate pharmacokinetics; these
results will be discussed in a future publication.
Inhibition values against the five major cytochrome P450
isoforms for 12 and 42 as well as other representative
compounds from the series are shown in Table 8. In general,
the series showed minimal inhibition of CYP’s 1A2, 2D6, 2C9,
and 2C19 (EC50’s >10 µM), with only CYP3A4 inhibition being
in the 1-10 µM range. Compounds 12 and 42 were also profiled
in a hERG patch clamp assay and determined to have EC50
values of 12 and 8 µM, respectively.
Mouse Models of Pain: Formalin and Chronic
Constriction Injury Models. We investigated the ability of
compounds 12 and 42 to reduce pain behaviors in a mouse
formalin model of nociceptive pain.24 In this model, upon
injection of formalin into the hind paw, a biphasic pain response
is induced. The duration of pain behaviors displayed by the
injured paw is then counted, with phase II being from 25-45
min postformalin injection. The duration of pain behaviors
evoked in phase II are believed to involve the central sensitiza-
tion of spinal neurons.25 From Figure 6, it can be seen that
compounds 12 and 42 (100 mg/kg po, 30 min predose to
formalin challenge) both statistically reduced phase II pain
behaviors in the mouse formalin model (∼40% and ∼65%
reduction, respectively) relative to vehicle treated animals,
consistent with these compounds reducing or blocking the
development of central sensitization.26 The attenuation of these
formalin induced pain behaviors by compound 12 was not
sensitive to the opiate antagonist naloxone, thereby suggesting
a lack of involvement of an opiate mechanism in this response.
Compound 12 was also examined in the chronic constriction
injury (CCI) model of peripheral neuropathy in which the sciatic
nerve is loosely ligated, leading to spontaneous pain behaviors
such as guarding or licking of the injured paw.27 Additionally,
allodynia and hyperalgesia due to mechanical and thermal
stimuli may be observed.
As shown in Figure 7, compound 12 (100 mg/kg po, 30 min
prior to testing) gave a statistically significant reduction (∼45%)
in the duration of pain behaviors induced by application of
acetone to the injured paw (cold allodynia modality). This result
indicates that 12 is orally active in a rodent model of peripheral
neuropathy.
Conclusion
From an ultrahigh throughput screen we identified a novel
series of quinolinone iNOS dimerization inhibitors exemplified
by compound 7 (hiNOS EC50 ) 1.3 µM; miNOS EC50 ) 46
µM). SAR studies on compound 7 led to a number of potent
human iNOS inhibitors (hiNOS EC50 < 0.01 µM) that main-
tained good selectivity over eNOS and also demonstrated a
corresponding increase in mouse iNOS potency (miNOS EC50

iNOS Inhibitor DeVelopment Candidate KD7332
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3055
tors that display excellent selectivity over eNOS and that are
orally active in rodent pain models. Future publications will
address the high clearance of the quinolinone series and the
subsequent evolution of the series to a clinical development
candidate.
Experimental Section
Chemistry. All reactions were carried out under a nitrogen
atmosphere. All solvents and reagents were obtained from com-
mercial sources (unless otherwise specified) and used without 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
HPLC (for purity determination) were performed on an Agilent
(1100 Series): 1 mg/mL in methanol, 5 µL injection, 254 nm
detection, 1 mL/min, 1-95% ACN + 0.05% TFA /H2O + 0.05%
TFA gradient, 15 min. Compound purity was determined as g95%
purity by this method. 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.28 Melting points were measured on
Mettler Toledo FP62 automated melting point apparatus using glass
capillary tubes and are uncorrected.
Figure 5. Elimination rate of compound 12 under conditions of
combined phase I oxidation and phase II glucuronide conjugation and
each separately.
Figure 6. Compounds 12 and 42 reduce phase II pain behaviors in a
mouse formalin model.
N-(2-Fluorophenyl)-3-oxobutanamide (49). A solution of
methyl 3-oxobutanoate (54 mL, 0.5 mol) in xylene/pyridine (200
mL/5 mL) was heated to gentle reflux for 0.5 h. 2-Fluoroaniline
(48, 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 concentrated HCl. The resulting precipitate was collected by
filtration, washed with H2O, and dried to give 28 g (38%) of N-(2-
fluorophenyl)-3-oxobutanamide (49) as a white crystalline solid.
LRMS m/z: calcd for C10H10FNO2 195.0, found 196.0 [M + H]+.
4-Bromo-N-(2-fluorophenyl)-3-oxobutanamide (50). Bromine
(7.9 mL, 1.1 equiv) was added dropwise via an addition funnel to
a solution of N-(2-fluorophenyl)-3-oxobutanamide (49, 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. Purifica-
tion by silica gel column chromatography (10% to 20% EtOAc/
hexanes) afforded 26 g (68% yield) of 4-bromo-N-(2-fluorophenyl)-
3-oxobutanamide (50). 1H 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 + H]+.
4-(Bromomethyl)-8-fluoroquinolin-2(1H)-one (52). A mixture
of 4-bromo-N-(2-fluorophenyl)-3-oxobutanamide (50, 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 water.
The solid was filtered, washed with water, 10% aqueous sodium
Figure 7. Compound 12 reduces pain behaviors in the chronic
constriction injury model.
< 0.05 µM). These compounds were efficiently triaged utilizing
a mouse LPS challenge assay measuring plasma nitrates and
drug levels and led to the identification of 12 (human iNOS
EC50 ) 0.011 µM; mouse LPS ED50 ) 10 mg/kg po) and 42
(human iNOS EC50 ) 0.003 µM; mouse LPS ED50 ) 5 mg/kg
po) as lead molecules. In contrast to the majority of previously
reported iNOS inhibitors, compound 12 is structurally unrelated
to arginine and was confirmed to function as an iNOS enzyme
dimerization inhibitor. Further, 12 and 42 were found to have
minimal cross-reactivity with cytochrome P450 enzymes and
the hERG channel. Despite having high clearance in vivo,
compounds 12 and 42 achieved good per oral Cmaxs and were
tested in mouse models of neuropathic pain. Following oral
dosing, compounds 12 and 42 gave a statistical reduction in
pain behaviors in the mouse formalin model (central sensitiza-
tion model), while 12 also statistically reduced pain behaviors
in the chronic constriction injury model (peripheral neuropathy
model). In summary, we have identified a new class of highly
potent, small molecule, nonarginine iNOS dimerization inhibi-

3056 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Bonnefous et al.
bicarbonate, and again with water to give 15 g (65%) of 4-(bro-
115.7, 49.7, 16.6. LRMS (ESI+)/HRMS (ESI-) m/z: calcd for
C21H17N3O2S 376.1, found 376.5 [M + H]+/374.0963 found
374.0959 [M - H]-.
momethyl)-7,8-difluoroquinolin-2(1H)-one (52) as an off-white
1
solid. H 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 + H]+.
N-((8-Fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-4-methyl-
N-phenylthiazole-5-carboxamide (10). Compound 10 was synthe-
sized as described for 7 using 8-fluoro-4-((phenylamino)meth-
yl)quinolin-2(1H)-one (54) and 4-methylthiazole-5-carboxylic acid
4-((Phenylamino)methyl)quinolin-2(1H)-one (53). A mixture
of 4-(bromomethyl) quinolin-2(1H)-one (51, commercially avail-
able, 200 mg, 0.84 mmol) and aniline (90 mg, 0.97 mmol) in DMSO
(15 mL) was stirred at 50 °C for 1 h. The reaction mixture was
cooled to 25 °C and poured into ice water (100 mL). The resulting
precipitate was collected via vacuum filtration. The filter cake was
washed with 0.1 N HCl (3 × 10 mL) and water (50 mL) to afford
100 mg (48%) of 4-((phenylamino)methyl)quinolin-2(1H)-one (53)
as a yellow solid. LRMS m/z: calcd for C16H14N2O 250.1, found
251.0 [M + H]+.
1
as starting materials; HPLC: tR ) 6.13(>92%); mp > 300 °C. H
NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 8.89 (s, 1H), 7.68
(d, 1H, J ) 8.0 Hz), 7.44 (m, 1H), 7.29-7.16 (m, 4H), 7.14 (m,
2H), 6.38 (s, 1H), 5.33 (s, 2H), 2.49 (s, 3H). LRMS (ESI+)/HRMS
(ESI-) m/z: calcd for C21H16FN3O2S 394.1, found 394.6 [M + H]+/
392.0869 found 392.0870 [M - H]-.
N-(2-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-4-methylthiazole-5-carboxamide (11). Compound 11 was
synthesized as described for compound 7 using 4-((2-chlorophe-
nylamino)methyl)-8-fluoroquinolin-2(1H)-one (55) and 4-meth-
ylthiazole-5-carboxylic acid as starting materials; HPLC: tR ) 6.46
(>97%); mp ) 273.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.79
(s, 1H), 8.86 (s, 1H), 7.71 (d, 1H, J ) 8.0 Hz), 7.47-7.41 (m,
2H), 7.35-7.28 (m, 3H), 7.20 (m, 1H), 6.36 (s, 1H), 5.50 (d, 1H,
J ) 15.6 Hz), 4.92 (d, 1H, J ) 15.6 Hz), 2.49 (s, 3H). LRMS
(ESI+)/HRMS (ESI+) m/z: calcd for C21H15ClFN3O2S 428.1, found
428.5 [M + H]+/428.0636 found 428.0641 [M + H]+.
8-Fluoro-4-((phenylamino)methyl)quinolin-2(1H)-one (54).
Compound 54 was synthesized as described for 53 using 4-(bro-
momethyl)-8-fluoroquinolin-2(1H)-one (52) and aniline as starting
1
materials. H NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H), 7.62
(d, 1H, J ) 8.0 Hz), 7.37 (ddd, 1H, J ) 10.8, 7.6, 0.8 Hz), 7.13
(m, 1H), 7.01 (m, 2H), 6.51 (m, 3H), 6.40 (s, 1H), 6.25 (m, 1H),
4.46 (s, 2H). LRMS m/z: calcd for C16H13FN2O 268.1, found 269.2
[M + H]+.
N-((2-oxo-1,2-Dihydroquinolin-4-yl)methyl)-N-phenylfuran-2-
carboxamide (7). Oxalyl chloride (86 µL, 1.0 mmol) was added to
a solution of furan-2-carboxylic acid (94 mg, 0.84 mmol) and
catalytic DMF (7 µL, 0.084 mmol) in DCM (10 mL) at 25 °C. The
resulting mixture was stirred for 2 h, after which time it was
concentrated to dryness under reduced pressure and redissolved in
NMP (2 mL). The resulting solution was added at 25 °C to a
separate mixture of 4-((phenylamino)methyl) quinolin-2(1H)-one
(53, 100 mg, 0.4 mmol) and DIEA (350 µL, 2.0 mmol) in NMP (3
mL). After 14 h, the reaction mixture was treated with neat
propylamine (100 µL, 1.2 mmol), stirred for an additional 30 min
at 25 °C, and purified via reversed-phase semipreparative HPLC
(ACN:H2O + 0.1% TFA) to afford 40 mg (30%) of N-((2-oxo-
1,2-dihydroquinolin-4-yl)methyl)-N-phenylfuran-2-carboxamide (7)
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-4-methylthiazole-5-carboxamide (12). Compound 12 was
synthesized as described for compound 7 using 4-((3-chlorophe-
nylamino)methyl)-8-fluoroquinolin-2(1H)-one (56) and 4-meth-
ylthiazole-5-carboxylic as starting materials; HPLC: tR ) 6.56
1
(100%); mp ) 201.9 °C. H NMR (DMSO-d6) δ 11.75 (s, 1H),
8.92 (s, 1H), 7.65 (d, 1H, J ) 8.4 Hz), 7.47-7.41 (m, 2H),
7.30-7.19 (m, 3H), 7.07 (dt, 1H, J ) 8.0, 1.3 Hz), 6.41 (s, 1H),
5.34 (s, 2H), 2.42 (s, 3H). 13C NMR (400 MHz, DMSO-d6) δ 163.5,
161.6, 156.6, 156.2, 149.7 (d, J ) 245 Hz, F coupling), 146.1,
143.3, 134.0, 131.5, 128.6, 128.4 (d, J ) 13.9 Hz), 128.1, 127.4,
124.7, 122.4 (d, J ) 7.3 Hz), 122.1, 120.6 (d, J ) 2.9 Hz), 120.3
(d, J ) 2.9 Hz), 116.6 (d, J ) 16.9 Hz), 50.4, 17.4. LRMS (ESI+)/
HRMS (ESI-) m/z: calcd for C21H15ClFN3O2S 428.1, found 428.5
[M + H]+/426.0480 found 426.0486 [M - H]-.
N-(4-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-4-methylthiazole-5-carboxamide (13). Compound 13 was
synthesized as described for compound 7 using 4-((4-chlorophe-
nylamino)methyl)-8-fluoroquinolin-2(1H)-one (57) and 4-meth-
ylthiazole-5-carboxylic acid as starting materials; HPLC: tR ) 6.22
(100%); mp ) 146.4 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.77
(s, 1H), 8.92 (s, 1H), 7.66 (d, 1H, J ) 8.0 Hz), 7.44 (dd, 1H, J )
11.2, 8.8 Hz), 7.35 (m, 2H), 7.22 (m, 3H), 6.38 (s, 1H), 5.32 (s,
2H), 2.42 (s, 3H). 13C NMR (400 MHz, DMSO-d6) δ 162.7, 160.8,
155.8, 155.5, 149.0 (d, J ) 245 Hz, F coupling), 145.4, 140.0,
132.2, 129.5 (2C), 129.3 (2C), 127.7 (d, J ) 13.2 Hz), 123.9, 121.7
(d, J ) 7.3 Hz), 121.6, 119.8 (d, J ) 3.7 Hz), 119.6 (d, J ) 2.9
Hz), 115.8 (d, J ) 16.9 Hz), 49.7, 16.7. LRMS (ESI+)/HRMS
(ESI-) m/z: calcd for C21H15ClFN3O2S 428.1, found 428.2 [M +
H]+/426.0480 found 426.0478 [M - H]-.
1
as a white solid’ HPLC: tR ) 6.30 (>99%); mp ) 249.4 °C. H
NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H), 7.82 (s, 1H, J ) 7.6
Hz), 7.68 (s, 1H), 7.49 (td, 2H, J ) 8.4, 1.2 Hz), 7.38-7.29 (m,
4H), 7.21-7.18 (m, 2H), 6.40 (m, 1H), 6.29 (s, 1H), 5.88 (d, 1H,
J ) 3.7 Hz), 5.26 (s, 2H). 13C NMR (400 MHz, DMSO-d6) δ 161.9,
159.1, 146.9, 146.5, 146.2, 142.4, 139.5, 131.3, 130.2, 128.7, 128.3,
124.7, 122.6, 120.9, 118.3, 117.4, 116.4, 112.1, 50.8. LRMS
(ESI+)/HRMS (ESI-) m/z: calcd for C21H16N2O3 345.1, found
345.5 [M + H]+/343.1083 found 343.1085 [M - H]-.
N-((8-Fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-N-phenyl-
furan-2-carboxamide (8). Compound 8 was synthesized as de-
scribed for 7 using 8-fluoro-4-((phenylamino)methyl)quinolin-
2(1H)-one (54) and furan-2-carboxylic acid as starting materials;
1
HPLC: tR ) 6.36 (>94%); mp ) 247.3 °C. H NMR (400 MHz,
DMSO-d6) δ 11.74 (s, 1H), 7.69 (m, 1H), 7.66 (d, 1H, J ) 8.4
Hz), 7.49-7.33 (m, 4H), 7.22-7.18 (m, 3H), 6.39 (m, 1H), 6.36
(s, 1H), 5.87 (d, 1H, J ) 3.6 Hz), 5.26 (s, 2H). 13C NMR (400
MHz, DMSO-d6) δ 160.8, 158.4, 149.0 (d, J ) 244 Hz, F coupling),
146.1, 145.6 (d, J ) 2.2 Hz), 145.5, 141.6, 129.5 (2C), 128.0, 127.6,
127.5 (2C), 121.7 (d, J ) 7.3 Hz), 121.5, 119.8 (d, J ) 3.6 Hz),
119.7 (d, J ) 2.9 Hz), 116.7, 115.7 (d, J ) 16.8 Hz), 111.4, 50.1.
LRMS (ESI+)/HRMS (ESI-) m/z: calcd for C21H15FN2O3 363.1,
found 363.5 [M + H]+/361.0989 found 361.0985 [M - H]-.
4-Methyl-N-((2-oxo-1,2-dihydroquinolin-4-yl)methyl)-N-phe-
nylthiazole-5-carboxamide (9). Compound 9 was synthesized as
described for 7 using 4-((phenylamino)methyl)quinolin-2(1H)-one
(53) and 4-methylthiazole-5-carboxylic acid as starting material;
N-((8-Fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-N-(3-fluo-
rophenyl)-4-methylthiazole-5-carboxamide (14). Compound 14 was
synthesized as described for compound 7 using 4-((3-fluorophe-
nylamino)methyl)-8-fluoroquinolin-2(1H)-one (58) and 4-meth-
ylthiazole-5-carboxylic acid as starting materials; HPLC: tR ) 5.95
(>98%); mp ) 227.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.78
(s, 1H), 8.92 (s, 1H), 7.66 (d, 1H, J ) 8.4 Hz), 7.44 (m, 1H),
7.31-7.19 (m, 3H), 7.11 (m, 1H), 6.94 (d, 1H, J ) 8.4 Hz), 6.42
(s, 1H), 5.34 (s, 2H), 2.42 (s, 3H). 13C NMR (400 MHz, DMSO-
d6) δ 162.8, 161.9 (d, J ) 244 Hz, F coupling), 160.8, 155.8, 155.4,
149.0 (d, J ) 244 Hz, F coupling), 145.3, 142.7 (d, J ) 10.3 Hz),
130.8 (d, J ) 9.5 Hz), 127.6 (d, J ) 14.0 Hz), 124.0, 121.7 (d, J
) 6.6 Hz), 121.4, 119.8, 119.6, 115.8 (d, J ) 16.9 Hz), 114.9,
114.8, 114.7, 49.7, 16.7. LRMS (ESI+)/HRMS (ESI-) m/z: calcd
for C21H15F2N3O2S 412.1, found 412.2 [M + H]+/410.0775 found
410.0771 [M - H]-.
1
HPLC: tR ) 6.20 (>95%); mp ) 281.6 °C. H NMR (400 MHz,
DMSO-d6) δ 11.75 (s, 1H), 8.89 (s, 1H), 7.84 (d, 1H, J ) 8.0 Hz),
7.51 (td, 2H, J ) 8.4, 1.2 Hz), 7.31-7.20 (m, 4H), 7.15 (m, 2H),
6.31 (s, 1H), 5.33 (s, 2H), 2.48 (s, 3H). 13C NMR (400 MHz,
DMSO-d6) δ 162.8, 161.1, 155.4, 155.1, 145.7, 141.2, 138.8, 130.6,
129.2 (2C), 127.8, 127.6 (2C), 124.3, 123.9, 121.8, 120.2, 117.5,

iNOS Inhibitor DeVelopment Candidate KD7332
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3057
N-(3-Cyanophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)-
methyl)-4-methylthiazole-5-carboxamide (15). Compound 15 was
synthesized as described for compound 7 using 8-fluoro-4-((3-
cyanophenylamino)methyl)quinolin-2(1H)-one (59) and 4-me-
thylthiazole-5-carboxylic acid as starting materials; HPLC: tR
7.44 (m, 1H), 7.37 (m, 1H), 7.33 (m, 1H), 7.25 (m, 1H), 7.18 (m,
1H), 6.99 (d, 1H, J ) 8.0 Hz), 6.58 (s, 1H), 5.49 (s, 2H), 2.52 (s,
3H). LRMS (ESI+)/HRMS (ESI-) m/z: calcd for C23H17ClFN3O2
422.1, found 422.3 [M + H]+/420.0915 found 420.0915 [M - H]-.
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-4-methylnicotinamide (21). Compound 21 was synthe-
sized as described for compound 7 using 8-fluoro-4-((3-chlorophe-
nylamino)methyl)quinolin-2(1H)-one (56) and 4-methylnicotinic
acid as starting materials; HPLC: tR ) 5.21 (>99%). 1H NMR (400
MHz, DMSO-d6, TFA salt) δ 10.52 (s, 1H), 8.44 (d, 1H, J ) 5.2
Hz), 8.34 (s, 1H), 8.27 (d, 1H, J ) 4.8 Hz), 7.85 (d, 1H, J ) 7.6
Hz), 7.42 (t, 1H, J ) 8.0 Hz), 7.31-7.28 (m, 2H), 7.22-7.15 (m,
2H), 7.08 (m, 1H), 6.53 (s, 1H), 5.51 (s, 2H), 2.42 (s, 3H). LRMS
(ESI+)/HRMS (ESI-) m/z: calcd for C23H17ClFN3O2 422.1, found
422.3 [M + H]+/420.0915 found 420.0919 [M - H]-.
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-3-methylpicolinamide (22). Compound 22 was synthe-
sized as described for compound 7 using 8-fluoro-4-((3-chlorophe-
nylamino)methyl)quinolin-2(1H)-one (56) and 3-methylpicolinic
acid as starting materials; HPLC: tR ) 6.23 (>93%). 1H NMR (400
MHz, DMSO-d6, TFA salt) δ 11.77 (s, 1H), 8.20 (s, 1H), 7.70 (d,
1H, J ) 8.0 Hz), 7.59 (d, 1H, J ) 6.8 Hz), 7.46 (t, 1H, 8.0 Hz),
7.26-7.18 (m, 3H), 7.13 (m, 2H), 6.91 (s, 1H), 6.54 (s, 1H), 5.39
(s, 2H), 2.47 (s, 3H). 13C NMR (400 MHz, DMSO-d6, TFA salt) δ
168.0, 160.9, 152.9, 149.0 (d, J ) 244 Hz, F coupling), 145.9,
145.5, 145.4, 142.1, 138.3, 132.7, 130.5, 130.2, 127.0, 126.5, 125.6,
124.0, 121.7 (d, J ) 6.6 Hz), 121.3, 119.9, 119.6, 115.9 (d, J )
16.8 Hz), 48.4, 17.2. LRMS (ESI+)/HRMS (ESI-) m/z: calcd for
C23H17ClFN3O2 422.1, found 422.1 [M + H]+/420.0915 found
420.0919 [M - H]-.
1
) 5.84 (>98%); mp ) 273.2 °C. H NMR (400 MHz, DMSO-
d6) δ 11.76 (s, 1H), 8.93 (s, 1H), 7.98 (s, 1H), 7.71 (m, 1H),
7.64 (d, 1H, J ) 8.4 Hz), 7.47-7.42 (m, 3H), 7.22 (m, 1H),
6.45 (s, 1H), 5.37 (s, 2H), 2.47 (s, 3H). LRMS (ESI+)/HRMS
(ESI-) m/z: calcd for C22H15FN4O2S 419.1, found 419.0 [M +
H]+/417.0822 found 417.0821 [M - H]-.
N-(3-Methylphenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-4-methylthiazole-5-carboxamide (16). Compound 16 was
synthesized as described for compound 7 using 8-fluoro-4-((m-
tolylamino)methyl)quinolin-2(1H)-one (60) and 4-methylthiazole-
5-carboxylic acid as starting materials; HPLC: tR ) 6.15 (100%).
1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 8.89 (s, 1H), 7.67
(d, 1H, J ) 8.0 Hz), 7.44 (dd, 1H, J ) 10.8, 8.4 Hz), 7.23-7.12
(m, 2H), 7.06 (m, 2H), 6.88 (d, 1H, J ) 7.6 Hz), 6.38 (s, 1H), 5.30
(s, 2H), 2.43 (s, 3H), 2.19 (s, 3H). LRMS (ESI+)/HRMS (ESI-)
m/z: calcd for C22H18FN3O2S 408.1, found 408.3 [M + H]+/
406.1026 found 406.1027 [M - H]-.
N-(3-Methoxyphenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-
4-yl)methyl)-4-methylthiazole-5-carboxamide (17). Compound 17
was synthesized as described for compound 7 using 8-fluoro-4-
((3-methoxyphenylamino)methyl)quinolin-2(1H)-one (61) and 4-me-
thylthiazole-5-carboxylic acid as starting materials; HPLC: tR
)
5.98 (100%). 1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 8.90
(s, 1H), 7.68 (d, 1H, J ) 8.0 Hz), 7.44 (ddd, 1H, J ) 10.0, 7.2, 0.8
Hz), 7.24-7.13 (m, 2H), 6.80 (m, 2H), 6.63 (d, 1H, J ) 7.6 Hz),
6.40 (s, 1H), 5.32 (s, 2H), 3.64 (s, 3H), 2.43 (s, 3H). 13C NMR
(400 MHz, DMSO-d6) δ 162.8, 160.9, 159.6, 155.6, 155.2, 149.0
(d, J ) 245 Hz, F coupling), 145.6, 142.1, 130.0, 127.6 (d, J )
13.9 Hz), 124.2, 121.6 (d, J ) 13.9 Hz), 121.4, 120.0, 119.9 (d, J
) 3.0 Hz), 119.7 (d, J ) 3.0 Hz), 115.8 (d, J ) 17.6 Hz), 113.5,
113.3, 55.2, 49.7, 16.7. LRMS (ESI+)/HRMS (ESI-) m/z: calcd
for C22H18FN3O3S 424.1, found 424.3 [M + H]+/422.0975 found
422.0974 [M - H]-.
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-1-methyl-1H-imidazole-5-carboxamide (23). Sodium hy-
dride (19 mg, 0.50 mmol) was added to a stirred solution of N-(3-
chlorophenyl)-1-methyl-1H-imidazole-5-carboxamide (66, 100 mg,
0.42 mmol) in DMF (6 mL) at 25 °C. After 1 h, 2-(tert-
butyldimethylsilyloxy)-8-fluoro-4-(iodomethyl)quinoline (63, 208
mg, 0.50 mmol) was added in one portion. The resulting mixture
was stirred for 2.5 h, diluted with 1:1 hexanes:EtOAc (100 mL),
washed with H2O (50 mL), 5% aqueous sodium bicarbonate (50
mL), and brine, and then dried over MgSO4, filtered, and concen-
trated under reduced pressure. The crude residue was purified by
reversed-phase semipreparative HPLC (ACN:H2O + 0.1%TFA) to
afford N-(3-chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-
4-yl)methyl)-1-methyl-1H-imidazole-4-carboxamide (23, 29 mg,
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)thiazole-5-carboxamide (18). Compound 18 was synthe-
sized as described for compound 7 using 8-fluoro-4-((3-chlorophe-
nylamino)methyl)quinolin-2(1H)-one (56) and thiazole-5-carboxylic
acid as starting materials; HPLC: tR ) 6.14 (>97%); mp > 300
°C. 1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 9.10 (s, 1H),
7.66 (s, 1H), 7.62 (d, 1H, J ) 8.0 Hz), 7.58 (t, 1H, J ) 2.0 Hz),
7.48-7.37 (m, 3H), 7.24-7.16 (m, 2H), 6.47 (s, 1H), 5.30 (s, 2H).
LRMS (ESI+)/HRMS (ESI-) m/z: calcd for C20H13ClFN3O2S
414.0, found 414.2 [M + H]+/412.0323 found 412.0323 [M - H]-.
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-2-methylnicotinamide (19). Compound 19 was synthe-
sized as described for compound 7 using 8-fluoro-4-((3-chlorophe-
nylamino)methyl)quinolin-2(1H)-one (56) and 2-methylnicotinic
acid as starting materials; HPLC: tR ) 5.09 (>98%); mp ) 216.2
1
17%) as a white solid. HPLC: tR ) 4.88 (>97%). H NMR (400
MHz, DMSO-d6) δ 11.74 (s, 1H), 7.70 (s, 1H), 7.63 (d, 1H, J )
8.4 Hz), 7.52 (s, 1H), 7.44 (m, 1H), 7.34 (m, 2H), 7.26-7.18 (m,
2H), 6.47 (s, 1H), 6.17 (s, 1H), 5.28 (s, 2H), 3.83 (s, 3H). LRMS
(ESI+)/HRMS (ESI-) m/z: calcd for C21H16ClFN4O2 411.1, found
411.2 [M + H]+/409.0868 found 409.0863 [M - H]-.
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-3,5-dimethylisoxazole-4-carboxamide (24). Compound
24 was synthesized as described for compound 7 using 8-fluoro-
4-((3-chlorophenylamino)methyl)quinolin-2(1H)-one (56) and 3,5-
dimethylisoxazole-4-carboxylic acid as starting materials; HPLC:
1
°C. H NMR (400 MHz, DMSO-d6, TFA salt) δ 11.76 (s, 1H),
8.30 (d, 1H, J ) 3.6 Hz), 7.72 (d, 1H, J ) 7.2 Hz), 7.60 (d, 1H,
J ) 7.6 Hz), 7.45 (m, 1H), 7.39 (s, 1H), 7.26-7.21 (m, 1H),
7.18-7.11 (m, 2H), 7.06 (m, 1H), 6.92 (m, 1H), 6.46 (s, 1H), 5.38
(s, 2H), 2.43 (s, 3H). 13C NMR (400 MHz, DMSO-d6, TFA salt) δ
168.5, 160.8, 153.7, 149.0 (d, J ) 245 Hz, F coupling), 149.3,
145.2, 141.9, 135.1, 133.0, 130.9, 130.4, 127.7, 127.5, 127.4, 126.6,
122.4, 121.7 (d, J ) 6.6 Hz), 120.3, 120.0, 119.6, 115.8 (d, J )
16.8 Hz), 48.6, 22.3. LRMS (ESI+)/HRMS (ESI-) m/z: calcd for
C23H17ClFN3O2 422.1, found 422.3 [M + H]+/420.0915 found
420.0919 [M - H]-.
1
tR ) 6.30 (100%); mp ) 240.2 °C. H NMR (400 MHz, DMSO-
d6) δ 11.75 (s, 1H), 7.65 (d, 1H, J ) 8.4 Hz), 7.49 (m, 1H), 7.44
(m, 1H), 7.27-7.22 (m, 3H), 7.05 (m, 1H), 6.37 (s, 1H), 5.38 (s,
2H), 2.11 (s, 3H), 2.08 (s, 3H). 13C NMR (DMSO-d6) δ 167.9,
162.4, 160.8, 157.9, 149.0 (d, J ) 244 Hz, F coupling), 145.2,
142.1, 133.1, 130.5, 127.6 (d, J ) 13.2 Hz), 127.1, 126.4, 125.8,
121.6 (d, J ) 6.6 Hz), 121.4, 119.8 (d, J ) 3.7 Hz), 119.5 (d, J )
3.0 Hz), 115.8 (d, J ) 17.6 Hz), 112.5, 48.9, 11.7, 10.2. LRMS
(ESI+)/HRMS (ESI-) m/z: calcd for C22H17ClFN3O3 426.1, found
426.3 [M + H]+/424.0864 found 424.0865 [M - H]-.
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-3-methylisonicotinamide (20). Compound 20 was syn-
thesized as described for compound 7 using 8-fluoro-4-((3-
chlorophenylamino)methyl)quinolin-2(1H)-one (56) and 3-methylison-
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)cyclopentanecarboxamide (25). Compound 25 was syn-
thesized as described for compound 7 using 8-fluoro-4-((3-
chlorophenylamino)methyl)quinolin-2(1H)-one (56) and cyclopentane-
carbonyl chloride (acid chloride commercially available) as starting
1
icotinic acid as starting materials; HPLC: tR ) 5.07 (>97%). H
NMR (400 MHz, MeOD, TFA salt) δ 8.63 (s, 1H), 8.50 (d, 1H, J
) 5.6 Hz), 7.81 (d, 1H, J ) 8.4 Hz), 7.76 (d, 1H, J ) 5.6 Hz),
1
materials; HPLC: tR ) 6.94 (>95%); mp ) 188.4 °C. H NMR

3058 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Bonnefous et al.
(400 MHz, DMSO-d6) δ 11.72 (s, 1H), 7.54 (d, 1H, J ) 8.4 Hz),
7.43-7.38 (m, 4H), 7.19-7.11 (m, 2H), 6.28 (s, 1H), 5.09 (s, 2H),
2.59 (m, 1H), 1.70-1.36 (m, 8H). 13C NMR (400 MHz, DMSO-
d6) δ 175.3, 160.9, 149.0 (d, J ) 244 Hz, F coupling), 145.8, 142.9,
133.5, 131.0, 128.0, 127.6, 127.5, 127.1, 121.6, 121.5, 119.8 (d, J
) 3.7 Hz), 119.7, 115.7 (d, J ) 16.9 Hz), 49.3, 41.4, 30.5 (2C),
25.7 (2C). LRMS (ESI+)/HRMS (ESI-) m/z: calcd for
C22H20ClFN2O2 399.1, found 399.3 [M + H]+/397.1119 found
397.1122 [M - H]-.
(2 mL). Dimethylsulfate (6.6 µL, 0.07 mmol) was then added, and
the resulting mixture was stirred at 25 °C for 18 h. The solvent
was removed, and the residue was purified via reversed-phase
semipreparative HPLC (ACN:H2O+0.1% TFA) to afford 12 mg
of N-(3-chlorophenyl)-N-((8-fluoro-1-methyl-2-oxo-1,2-dihydro-
quinolin-4-yl)methyl)-4-methylthiazole-5-carboxamide (31) as the
first eluting isomer; HPLC: tR ) 6.74 (100%); 1H NMR (400 MHz,
DMSO-d6) δ 8.94 (s, 1H), 7.74 (d, 1H, J ) 8.4 Hz), 7.54 (t, 1H,
J ) 8.2 Hz), 7.49 (m, 1H), 7.35-7.26 (m, 3H), 7.10 (dt, 1H, J )
7.5, 2.0 Hz), 6.55 (s, 1H), 5.36 (s, 2H), 3.73 (d, 3H, J ) 9.2 Hz),
2.43 (s, 3H); LRMS (ESI+)/HRMS (ESI+) m/z: calcd for
C22H17ClFN3O2S 442.1, found 442.3 [M + H]+/442.0792 found
442.0789 [M + H]+; and 10 mg of N-(3-chlorophenyl)-N-((8-fluoro-
2-methoxyquinolin-4-yl)methyl)-4-methylthiazole-5-carboxamide
(30) as the second eluting isomer; HPLC: tR ) 7.38 (>99%); mp
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)isobutyramide (26). Compound 26 was synthesized as
described for compound 7 using 8-fluoro-4-((3-chlorophenylami-
no)methyl)quinolin-2(1H)-one (56) and isobutyryl chloride (acid
chloride commercially available) as starting materials; HPLC: tR
1
) 6.45 (100%). H NMR (400 MHz, DMSO-d6) δ 11.72 (s, 1H),
1
) 150.5 °C; H NMR (400 MHz, DMSO-d6) δ 8.92 (s, 1H), 7.87
7.53 (d, 1H, J ) 7.6 Hz), 7.44-7.37 (m, 4H), 7.19-7.14 (m, 2H),
6.28 (s, 1H), 5.08 (s, 2H), 2.48 (m, 1H), 0.96 (d, 6H, J ) 6.8 Hz).
LRMS (ESI+)/HRMS (ESI-) m/z: calcd for C20H18ClFN2O2 373.1,
found 373.3 [M + H]+/371.0963 found 371.0957 [M - H]-.
N-(3-Chlorophenyl)-4-methyl-N-((2-oxo-1,2-dihydropyridin-4-
yl)methyl)thiazole-5-carboxamide (27). N-(3-chlorophenyl)-N-((2-
chloropyridin-4-yl)methyl)-4-methylthiazole-5-carboxamide (69,
400 mg, 0.91 mmol) in AcOH (40 mL) was heated to 140 °C for
2 days. The solvent was removed by evaporation under reduced
pressure and the residue was purified by silica gel column
chromatography (50:1 DCM/MeOH) to afford 200 mg (60%) of
N-(3-chlorophenyl)-4-methyl-N-((2-oxo-1,2-dihydropyridin-4-yl)-
methyl)thiazole-5-carboxamide (27) as a yellow solid. HPLC: tR
) 5.28 (>98%); mp ) 102.1 °C. 1H NMR (400 MHz, DMSO-d6)
δ 11.47 (s, 1H), 8.92 (s, 1H), 7.40 (m, 1H), 7.32 (m, 3H), 7.10 (dt,
1H, J ) 6.0, 2.4 Hz), 6.12 (s, 1H), 6.10 (s, 1H), 4.89 (s, 2H), 2.39
(s, 3H). 13C NMR (400 MHz, DMSO-d6) δ 162.8, 162.2, 155.6,
155.4, 150.5, 142.8, 135.4, 133.2, 130.7, 127.6, 127.3, 126.6, 124.1,
117.4, 104.3, 51.7, 16.7. LRMS (ESI+)/HRMS (ESI-) m/z: calcd
for C17H14ClN3O2S 360.0, found 360.2 [M + H]+/358.0417 found
358.0417 [M - H]-.
(d, 1H, J ) 8.0 Hz), 7.56-7.43 (m, 3H), 7.28 (m, 1H), 7.22 (t,
1H, J ) 8.0 Hz), 7.05 (dt, 1H, J ) 7.6, 1.6 Hz), 6.95 (s, 1H), 5.55
(s, 2H), 3.95 (s, 3H), 2.43 (s, 3H); 13C NMR (400 MHz, DMSO-
d6) δ 162.8, 161.7, 156.3 (d, J ) 251 Hz, F coupling), 155.8, 155.4,
145.4, 142.5, 135.7 (d, J ) 11.7 Hz), 133.3, 130.6, 127.8, 127.4,
126.7, 124.9, 124.1 (d, J ) 7.3 Hz), 124.0, 119.3 (d, J ) 4.4 Hz),
114.5 (d, J ) 19.0 Hz), 112.5, 53.3, 49.6, 16.7; LRMS (ESI+)/
HRMS (ESI-) m/z: calcd for C22H17ClFN3O2S 442.1, found 442.3
[M + H]+/440.0636 found 440.0640 [M - H]-.
N-(3-Chlorophenyl)-N-((8-fluoroquinolin-4-yl)methyl)-4-meth-
ylthiazole-5-carboxamide (32). Compound 32 was synthesized as
described for compound 7 using 3-chloro-N-((8-fluoroquinolin-4-
yl)methyl)benzenamine (82) and 4-methylthiazole-5-carboxylic acid
as starting materials (propylamine step not necessary); HPLC: tR
) 6.18 (>98%); mp ) 175.3 °C. 1H NMR (400 MHz, DMSO-d6)
δ 8.93 (s, 1H), 8.86 (d, 1H, J ) 4.4 Hz), 8.00 (dd, 1H, J ) 8.0, 1.6
Hz), 7.68-7.58 (m, 2H), 7.54 (d, 1H, J ) 4.4 Hz), 7.50 (t, 1H, J
) 2.0 Hz), 7.27 (m, 1H), 7.22 (t, 1H, J ) 8.0 Hz), 7.10 (d, 1H, J
) 7.6 Hz), 5.63 (s, 2H), 2.44 (s, 3H). 13C NMR (400 MHz, DMSO-
d6) δ 162.8, 157.6 (d, J ) 254 Hz, F coupling), 155.8, 155.4, 150.4,
142.6, 142.0 (d, J ) 2.9 Hz), 137.6 (d, J ) 11.7 Hz), 133.2, 130.6,
127.8, 127.7, 127.6 (d, J ) 2.5 Hz), 126.9 (d, J ) 4.4 Hz), 126.8,
123.9, 121.1, 119.5 (d, J ) 5.2 Hz), 113.6 (d, J ) 18.3 Hz), 49.8,
16.7. LRMS (ESI+)/HRMS (ESI-) m/z: calcd for C21H15ClFN3OS
412.1, found 412.2 [M + H]+/410.0530 found 410.0533 [M - H]-.
N-(3-Chlorophenyl)-N-((8-fluoroisoquinolin-4-yl)methyl)-4-me-
thylthiazole-5-carboxamide (33). Compound 33 was synthesized
as described for compound 7 using 3-chloro-N-((8-fluoroisoquino-
lin-4-yl)methyl)benzenamine (91) and 4-methylthiazole-5-carboxy-
lic acid as starting materials (propylamine step not necessary);
HPLC: tR ) 5.59 (>98%); mp ) 100 °C (decomposition). 1H NMR
(400 MHz, DMSO-d6, HCl salt) δ 9.62 (s, 1H), 8.90 (s, 1H), 8.51
(s, 1H), 8.15-8.07 (m, 2H), 7.72 (ddd, 1H, J ) 10.8, 7.6, 1.2 Hz),
7.47 (t, 1H, J ) 2.0 Hz), 7.25 (m, 1H), 7.16 (t, 1H, J ) 8.0 Hz),
6.91 (d, 1H, J ) 8.0 Hz), 5.66 (s, 2H), 2.43 (s, 3H). 13C NMR
(400 MHz, DMSO-d6, HCl salt) δ 162.9, 159.3 (d, J ) 258 Hz, F
coupling), 155.8, 155.6, 143.1 (d, J ) 5.1 Hz), 141.9, 136.6, 136.3
(d, J ) 9.6 Hz), 135.8, 133.3, 130.7, 129.8, 128.2, 127.2, 123.8,
119.9 (d, J ) 4.4 Hz), 118.0 (d, J ) 15.4 Hz), 113.9 (d, J ) 17.6
Hz), 54.9, 47.3, 16.8. LRMS (ESI+)/HRMS (ESI+) m/z: calcd for
C21H15ClFN3OS 412.1, found 412.2 [M + H]+/412.0686 found
412.0682 [M + H]+.
N-(3-Chlorophenyl)-4-methyl-N-((2-oxo-1,2,5,6,7,8-hexahydro-
quinolin-4-yl)methyl)thiazole-5-carboxamide (28). Compound 28
was synthesized as described for compound 27 using N-((2-chloro-
5,6,7,8-tetrahydroquinolin-4-yl)methyl)-N-(3-chlorophenyl)-4-me-
thylthiazole-5-carboxamide (74) as the starting material; HPLC: tR
1
) 6.16 (100%); mp ) 223.4 °C. H NMR (400 MHz, DMSO-d6)
δ 11.30 (s, 1H), 8.93 (s, 1H), 7.43 (s, 1H), 7.31 (m, 2H), 7.15 (m,
1H), 5.98 (s, 1H), 4.86 (s, 2H), 3.33 (m, 3H), 2.48-2.34 (m, 4H),
1.63 (m, 4H). 13C NMR (DMSO-d6) δ 162.6, 161.6, 155.7, 155.3,
149.6, 143.2, 142.7, 133.3, 130.7, 127.5, 127.0, 126.4, 124.1, 113.6,
110.3, 50.3, 26.5, 22.1, 21.9, 20.9, 16.6. LRMS (ESI+)/HRMS
(ESI-) m/z: calcd for C21H20ClN3O2S 414.1, found 414.3 [M +
H]+/412.0887 found 412.0889 [M - H]-.
N-(3-Chlorophenyl)-N-((8-fluoro-2-oxo-2H-chromen-4-yl)methyl)-
5-methylthiazole-4-carboxamide (29). Compound 29 was synthesized
as described for compound 7 using 4-((3-chlorophenylamino)m-
ethyl)-8-fluoro-2H-chromen-2-one (79) and 4-methylthiazole-5-
carboxylic acid as starting materials (propylamine step not neces-
1
sary); HPLC: tR ) 6.83 (>99%); mp ) 186.5 °C. H NMR (400
MHz, DMSO-d6) δ 8.95 (s, 1H), 7.70 (d, 1H, J ) 8.4 Hz), 7.64
(m, 2H), 7.40 (m, 1H), 7.34 (m, 2H), 7.26 (d, 1H, J ) 7.2 Hz),
6.50 (s, 1H), 5.37 (s, 2H), 2.44 (s, 3H). 13C NMR (400 MHz,
DMSO-d6) δ 162.9, 158.1, 156.2, 155.6, 150.3, 148.7 (d, J ) 247
Hz, F coupling), 142.6, 141.2 (d, J ) 11.0 Hz), 133.4, 130.7, 127.9,
127.5, 126.7, 124.4 (d, J ) 6.6 Hz), 123.7, 120.2, 119.5, 118.4 (d,
J ) 16.2 Hz), 113.9, 49.6, 16.8. LRMS (ESI+)/HRMS (ESI-)
m/z: calcd for C21H14ClFN2O3S 429.0, found 429.1 [M + H]+/
427.0320 found 427.0321 [M - H]-.
4-((N-(3-Chlorophenyl)-4-methylthiazole-5-carboxamido)methyl)-
8-fluoroisoquinoline 2-Oxide (34). A mixture of N-(3-chlorophenyl)-
N-((8-fluoroisoquinolin-4-yl)methyl)-4-methylthiazole-5-carboxa-
mide (33, 124 mg, 302 mmol) and m-CPBA (156 mg, 905 mmol)
in DCM (10 mL) was stirred at 25 °C for 1 h. The solvent was
removed under reduced pressure, and the residue was purified by
silica gel column chromatography (0 to 10% MeOH/DCM) to afford
80 mg (62%) of 4-((N-(3-chlorophenyl)-4-methylthiazole-5-car-
boxamido)methyl)-8-fluoroisoquinoline 2-oxide (34) as an off-white
N-(3-Chlorophenyl)-N-((8-fluoro-2-methoxyquinolin-4-yl)methyl)-
4-methylthiazole-5-carboxamide (30) and N-(3-chlorophenyl)-N-
((8-fluoro-1-methyl-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-4-me-
thylthiazole-5-carboxamide (31). Sodium hydride (60% in mineral
oil, 5 mg, 0.12 mmol) was added to a suspension of N-(3-
chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-
4-methylthiazole-5-carboxamide (12, 25 mg, 0.06 mmol) in DCM
1
solid; HPLC: tR ) 6.05 (>99%); mp ) 171.4 °C. H NMR (400
MHz, DMSO-d6) δ 8.91 (s, 1H), 8.78 (s, 1H), 8.10 (s, 1H), 7.94
(d, 1H. J ) 8.4 Hz), 7.70 (m, 1H), 7.55 (m, 1H), 7.44 (s, 1H), 7.32
(m, 1H), 7.21 (t, 1H, J ) 8.0 Hz), 6.92 (d, 1H, J ) 8.0 Hz), 5.54

iNOS Inhibitor DeVelopment Candidate KD7332
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3059
(s, 2H), 2.44 (s, 3H). 13C NMR (400 MHz, DMSO-d6) δ 162.8,
156.7, 155.5, 155.4 (d, J ) 252 Hz, F coupling), 141.7, 138.0,
133.3, 131.1, 130.6, 129.2 (d, J ) 8.1 Hz), 128.6 (d, J ) 4.4 Hz),
128.1, 127.9, 127.7, 127.1, 123.8, 119.9 (d, J ) 17.9 Hz), 119.5
(d, J ) 3.6 Hz), 113.7 (d, J ) 18.3 Hz), 46.7, 16.7. LRMS (ESI+)/
HRMS (ESI-) m/z: calcd for C21H15ClFN3O2S 428.1, found 428.2
[M + H]+/426.0480 found 426.0486 [M - H]-.
N-(3-Chlorophenyl)-4-methyl-N-((2-oxo-1,2-dihydroquinolin-4-
yl)methyl)thiazole-5-carboxamide (35). Compound 35 was synthe-
sized as described for compound 7 using 4-((3-chlorophenylami-
no)methyl)quinolin-2(1H)-one (115) and 4-methylthiazole-5-
carboxylic acid as starting materials; HPLC: tR ) 6.50 (>98%).
1H NMR (400 MHz, DMSO-d6) δ 11.73 (s, 1H), 8.93 (s, 1H), 7.82
(d, 1H, J ) 7.6 Hz), 7.50 (m, 1H), 7.43 (s, 1H), 7.32-7.22 (m,
4H), 7.05 (m, 1H), 6.34 (s, 1H), 5.35 (s, 2H), 2.42 (s, 3H). LRMS
(ESI+)/HRMS (ESI-) m/z: calcd for C21H16ClN3O2S 410.1, found
410.4 [M + H]+/408.0574 found 408.0568 [M - H]-.
N-((8-Chloro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-N-(3-chlo-
rophenyl)-4-methylthiazole-5-carboxamide (36). Compound 36 was
synthesized as described for compound 7 using 8-chloro-4-((3-
chlorophenylamino)methyl)quinolin-2(1H)-one (116) and 4-meth-
ylthiazole-5-carboxylic acid as starting materials; HPLC: tR ) 6.57
(>97%); mp ) 211.4 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.98
(s, 1H), 8.92 (s, 1H), 7.83 (d, 1H, J ) 7.6 Hz), 7.69 (d, 1H, J )
7.6 Hz), 7.47 (s, 1H), 7.32-7.23 (m, 3H), 7.08 (dd, 1H, J ) 7.6,
1.6 Hz), 6.45 (s, 1H), 5.36 (s, 2H), 2.42 (s, 3H). 13C NMR (400
MHz, DMSO-d6) δ 162.8, 161.0, 155.8, 155.5, 145.7, 142.5, 135.2,
133.3, 130.9, 130.7, 127.8, 127.4, 126.7, 123.9, 123.3, 122.4, 121.1,
119.3, 119.0, 49.7, 16.7. LRMS (ESI+)/HRMS (ESI-) m/z: calcd
for C21H15Cl2N3O2S 444.0, found 444.2 [M + H]+/442.0184 found
442.0186 [M - H]-.
N-(3-Chlorophenyl)-N-((7-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-4-methylthiazole-5-carboxamide (37). Compound 37 was
synthesized as described for compound 7 using 4-((3-chlorophe-
nylamino)methyl)-7-fluoroquinolin-2(1H)-one (117) and 4-meth-
ylthiazole-5-carboxylic acid as starting materials; HPLC: tR ) 6.79
(100%); mp ) 258.8 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.83
(s, 1H), 8.94 (s, 1H), 7.90 (m, 1H), 7.46 (s, 1H), 7.30 (m, 2H),
7.13-7.06 (m, 3H), 6.36 (s, 1H), 5.35 (s, 2H), 2.44(s, 3H). 13C
NMR (400 MHz, DMSO-d6) δ 163.0 (d, J ) 246 Hz, F coupling),
162.8, 161.3, 155.8, 155.4, 145.3, 142.5, 140.5 (d, J ) 11.7 Hz),
133.3, 130.7, 127.8, 127.4, 126.7, 126.6, 123.9, 119.3, 114.5, 109.9
(d, J ) 23.4 Hz), 101.5 (d, J ) 24.9 Hz), 49.6, 16.7. LRMS (ESI+)/
HRMS (ESI-) m/z: calcd for C21H15ClFN3O2S 428.1, found 428.5
[M + H]+/426.0480 found 426.0482 [M - H]-.
NMR (DMSO-d6) δ 11.99 (s, 1H), 8.97 (s, 1H), 7.54-7.51 (m,
2H), 7.33 (m, 2H), 7.25 (m, 1H), 7.16 (d, 1H, J ) 8.4 Hz), 7.04
(d, 1H), 6.44 (s, 1H), 5.35 (s, 2H), 2.44 (s, 3H). LRMS (ESI+)/
HRMS (ESI-) m/z: calcd for C21H15ClFN3O2S 428.1, found 428.5
[M + H]+/426.0480 found 426.0487 [M - H]-.
N-(3-Chlorophenyl)-N-((7,8-difluoro-2-oxo-1,2-dihydroquinolin-
4-yl)methyl)-4-methylthiazole-5-carboxamide (40). Compound 40
was synthesized as described for compound 7 using 4-((3-
chlorophenylamino)methyl)-7,8-difluoroquinolin-2(1H)-one (120)
and 4-methylthiazole-5-carboxylic acid as starting materials; HPLC:
tR ) 6.73 (>96%); mp ) 174.2 °C. 1H NMR (DMSO-d6) δ 12.03
(s, 1H), 8.95 (s, 1H), 7.70 (m, 1H), 7.49 (s, 1H), 7.37-7.28 (m,
3H), 7.09 (d, 1H, J ) 8.2 Hz), 6.39 (s, 1H), 5.35 (s, 2H), 2.43 (s,
3H). 13C NMR (400 MHz, DMSO-d6) δ 162.8, 161.0, 155.9, 155.5,
150.0 (dd, J ) 247, 9.2 Hz, F coupling), 145.2, 142.5, 136.9 (dd,
J ) 240, 9.2 Hz, F coupling), 133.3, 130.7, 129.6 (d, J ) 6.6 Hz),
127.9, 127.4, 126.7, 123.9, 120.4, 120.2, 115.7, 110.3 (d, J ) 19.0
Hz), 49.6, 16.7. LRMS (ESI+)/HRMS (ESI-) m/z: calcd for
C21H14ClF2N3O2S 446.0, found 446.5 [M + H]+/444.0385 found
444.0381 [M - H]-.
N-(3-Chlorophenyl)-N-((7,8-difluoro-2-oxo-1,2-dihydroquinolin-
4-yl)methyl)-4-methylnicotinamide (41). Compound 41 was syn-
thesized as described for compound 7 using 4-((3-chlorophenyl-
amino)methyl)-7,8-difluoroquinolin-2(1H)-one(120)and4-methylnicotinic
acid as starting materials; HPLC: tR ) 5.29 (>99%); mp ) 187.7
°C. 1H NMR (400 MHz, DMSO-d6, TFA salt) δ NH resonance not
observed, 8.48 (s, 1H), 8.36 (d, 1H, J ) 4.8 Hz), 7.73 (m, 1H),
7.47 (s, 1H), 7.33 (m, 2H), 7.20-7.12 (m, 2H), 6.95 (m, 1H), 6.44
(s, 1H), 5.37 (s, 2H), 2.35 (s, 3H). 13C NMR (400 MHz, DMSO-
d6, TFA salt) δ 166.8, 161.0, 150.1 (dd, J ) 247, 9.5 Hz, F
coupling), 147.5, 146.7, 145.7, 144.9, 141.6, 137.0 (dd, J ) 240,
9.5 Hz, F coupling), 133.2, 132.5, 130.6, 129.5 (d, J ) 6.6 Hz),
127.8, 127.5, 126.7, 126.0, 121.3, 120.6, 115.8, 110.4 (d, J ) 18.3
Hz), 48.7, 18.8. LRMS (ESI+)/HRMS (ESI-) m/z: calcd for
C23H16ClF2N3O2 440.1, found 440.3 [M + H]+/438.0821 found
438.0820 [M - H]-.
N-(3-Chlorophenyl)-N-((7,8-difluoro-2-oxo-1,2-dihydroquinolin-
4-yl)methyl)-1-methyl-1H-imidazole-5-carboxamide (42). Com-
pound 42 was synthesized as described for compound 23 using
4-(bromomethyl)-7,8-difluoroquinolin-2(1H)-one (113) as the start-
ing material; HPLC: tR ) 5.86 (>97%). 1H NMR (400 MHz,
DMSO-d6, TFA salt) δ NH resonance not observed, 8.54 (s, 1H),
7.63 (m, 2H), 7.39-7.27 (m, 4H), 6.67 (s, 1H), 6.49 (s, 1H), 5.29
(s, 2H), 3.93 (s, 3H). 13C NMR (400 MHz, DMSO-d6, TFA salt) δ
161.2, 160.5, 150.1 (dd, J ) 247, 9.5 Hz, F coupling), 145.6, 143.8,
141.6, 137.0 (dd, J ) 240, 15.4 Hz, F coupling), 134.2, 133.4,
130.8, 127.7, 127.5, 126.6, 124.9, 120.6, 118.8, 115.8, 115.7, 110.4
(d, J ) 19.0 Hz), 49.6, 33.7. LRMS (ESI+)/HRMS (ESI-) m/z:
calcd for C21H15ClF2N4O2 429.1, found 428.9 [M + H]+/427.0774
found 427.0773 [M - H]-.
N-(3-Chlorophenyl)-N-((7,8-difluoro-2-oxo-1,2-dihydroquinolin-
4-yl)methyl)-3,5-dimethylisoxazole-4-carboxamide (43). Compound
43 was synthesized as described for compound 7 using 4-((3-
chlorophenylamino)methyl)-7,8-difluoroquinolin-2(1H)-one (120)
and 3,5-dimethylisoxazole-4-carboxylic acid as starting materials;
HPLC: tR ) 6.42 (>95%). 1H NMR (400 MHz, DMSO-d6) δ 12.00
(s, 1H), 7.68 (m, 1H), 7.50 (s, 1H), 7.40-7.22 (m, 3H), 7.04 (m,
1H), 6.32 (s, 1H), 5.37 (s, 2H), 2.11 (s, 3H), 2.07 (s, 3H). LRMS
(ESI+)/HRMS (ESI-) m/z: calcd for C22H16ClF2N3O3 444.1, found
444.3 [M + H]+/442.0770 found 442.0767 [M - H]-.
N-(3-Chlorophenyl)-N-((6-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-4-methylthiazole-5-carboxamide (38). Compound 38 was
synthesized as described for compound 7 using 4-((3-chlorophe-
nylamino)methyl)-6-fluoroquinolin-2(1H)-one (118) and 4-meth-
ylthiazole-5-carboxylic acid as starting materials. HPLC: tR ) 6.73
(>99%); mp ) 256.6 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.85
(s, 1H), 8.95 (s, 1H), 7.68 (dd, 1H, J ) 10.0, 2.8 Hz), 7.47-7.41
(m, 2H), 7.34-7.30 (m, 3H), 7.26 (t, 1H, J ) 8.0 Hz), 7.04 (m,
1H), 6.41 (s, 1H), 5.38 (s, 1H), 2.40 (s, 3H). 13C NMR (400 MHz,
DMSO-d6) δ 162.8, 160.8, 157.0 (d, J ) 237 Hz, F coupling), 155.8,
155.5, 144.9, 142.4, 135.5, 133.3, 130.7, 127.8, 127.3, 126.7, 123.9,
121.5, 118.7 (d, J ) 24 Hz), 118.1 (d, J ) 8.8 Hz), 117.4 (d, J )
8.8 Hz), 109.5 (d, J ) 24 Hz), 49.4, 16.6. LRMS (ESI+)/HRMS
(ESI-) m/z: calcd for C21H15ClFN3O2S 428.1, found 428.5 [M +
H]+/426.0480 found 426.0472 [M - H]-.
N-(3-Chlorophenyl)-N-((5-fluoro-2-oxo-1,2-dihydroquinolin-4-
yl)methyl)-4-methylthiazole-5-carboxamide (39). A mixture of
N-((8-bromo-5-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-N-(3-
chlorophenyl)-4-methylthiazole-5-carboxamide (122, 80 mg, 0.36
mmol) and Pd/C (cat.) in MeOH (5 mL) was hydrogenated for 18 h
(with a balloon of hydrogen). The Pd/C was removed by filtration
through celite, and the solvent was removed under reduced pressure.
The residue was purified by semipreparative HPLC (ACN/water
+ 0.1% TFA) to afford 5.5 mg (3%) of N-(3-chlorophenyl)-N-((5-
fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-4-methylthiazole-5-
N-(3-Chlorophenyl)-N-((3,8-difluoro-2-oxo-1,2-dihydroquinolin-
4-yl)methyl)-4-methylthiazole-5-carboxamide (44). Compound 44
was synthesized as described for compound 7 using 4-((3-
chlorophenylamino)methyl)-3,8-difluoroquinolin-2(1H)-one (121)
and 4-methylthiazole-5-carboxylic acid as starting materials. HPLC:
1
tR ) 6.19 (>99%). H NMR (400 MHz, DMSO-d6) δ 12.38 (s,
1H), 8.88 (s, 1H), 7.80 (d, 1H, J ) 8.0 Hz), 7.47 (t, 1H, J ) 10.8
Hz), 7.38-7.30 (m, 3H), 7.19 (t, 1H, J ) 8.0 Hz), 6.77 (d, 1H, J
) 7.6 Hz), 5.44 (s, 2H), 2.42 (s, 3H). LRMS (ESI+)/HRMS (ESI-)
m/z: calcd for C21H14ClF2N3O2S 446.0, found 446.2 [M + H]+/
444.0385 found 444.0380 [M - H]-.
1
carboxamide (39) as a white solid; HPLC: tR ) 6.85 (>97%). H

3060 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Table 7. Mouse Pharmacokinetics of Compounds 12 and 42
Bonnefous et al.
compd
Cmax (µM)
Tmax (h)
t1/2(h)c
AUC (µg·h/mL)
Clp (mL/min/kg)
Vdss (L/kg)
12a
1.4
2.2
0.5
0.25
0.9
1.1
0.45
0.52
>100
>100
1.6
1.1
42b
a 1 mg/kg iv; 10 mg/kg po. b 3 mg/kg iv; 10 mg/kg po. c po t1/2
.
Table 8. Cytochrome P450 Inhibition Values of Selected Compounds
cytochrome P450 inhibition EC50 (µM)
2H), 2.42 (s, 3H). LRMS (ESI+)/HRMS (ESI-) m/z: calcd for
C21H14ClF2N3O2S 446.0, found 446.2 [M + H]+/444.0385 found
444.0382 [M - H]-.
compd
CYP1A2
CYP3A4
CYP2D6
CYP2C9
CYP2C19
Biological Methods. Recombinant iNOS Assay (293
Transient 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 of 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 5 × 105 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 (In-
vitrogen) 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).30
12
42
41
40
26
47
>100a
95
7.0
1.4
3.4
4.9
7.6
1.8
>100a
21
33
13
11
30
19
42
93
25
>100a
52
>100a
>100a
>100a
>100a
NDb
22
>100a
NDb
55
>100a
a <50% inhibition @100 µM. b Not determined.
Scheme 9a
Recombinant eNOS and nNOS Assay (293 Transient
Transfection 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
penicillin, and 100 µg/mL streptomycin. Each plate was transfected
with 1 mL DMEM with Pen/Strep containing 10 µg of human or
murine nNOS or 15 ug 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 5 × 105 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 + 90% CMC (0.5% w/v)) immediately before LPS
injection. Blood samples were collected into EDTA containing tubes
and processed for plasma collection for analysis 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, and then diaminonaphthalene (DAN) was added, fol-
lowed by the addition of NaOH, which converts the nitrite-DAN
adduct into a fluorophore. Measurement of the fluorescence of the
nitrite-DAN adduct using an Aquest plate reader (Molecular
Devices) accurately determines NO2 concentration.
a Reagents and conditions: (a) H2 SO4, 45 °C, 1 8 h.
N-(3-Chlorophenyl)-N-((7,8-difluoro-2-oxo-2H-chromen-4-yl)-
methyl)-4-methylthiazole-5-carboxamide (45). Compound 45 was
synthesized as described for compound 29 using 1-(3,4-difluoro-
2-hydroxyphenyl)ethanone29 as the starting material; HPLC: tR )
1
7.02 (100%); mp ) 170 °C. H NMR (400 MHz, DMSO-d6) δ
9.95 (s, 1H), 7.74 (m, 1H), 7.62 (m, 1H), 7.54 (m, 1H), 7.36-7.25
(m, 3H), 6.46 (s, 1H), 5.35 (s, 2H), 2.44 (s, 3H). 13C NMR (400
MHz, DMSO-d6) δ 163.0, 157.9, 156.3, 155.6, 151.3 (dd, J ) 250,
9.5 Hz, F coupling), 150.2, 142.7, 142.5, 137.5 (dd, J ) 249, 15.4
Hz, F coupling), 133.4, 130.8, 127.9, 127.5, 126.8, 123.6, 120.3,
115.7 (d, J ) 3.0 Hz), 112.7 (d, J ) 13.2 Hz), 112.6 (d, J ) 5.1
Hz), 49.5, 16.8. LRMS (ESI+)/HRMS (ESI-) m/z: calcd for
C21H13ClF2N2O3S 447.0, found 447.2 [M + H]+/445.0225 found
445.0220 [M - H]-.
N-(3-Chlorophenyl)-N-((7,8-difluoroisoquinolin-4-yl)methyl)-4-
methylthiazole-5-carboxamide (46). Compound 46 was synthesized
as described for compound 33 using 2,3-difluoro-6-iodobenzoic acid
as the starting material; HPLC: tR ) 6.20 (>95%). 1H NMR (400
MHz, DMSO-d6) δ 9.57 (s, 1H), 8.91 (s, 1H), 8.45 (s, 1H), 8.19
(m, 2H), 7.42 (t, 1H, J ) 2.4 Hz), 7.25 (d, 1H, J ) 8.0 Hz), 7.16
(t, 1H, J ) 8.0 Hz), 6.87 (d, 1H, J ) 8.0 Hz), 5.64 (s, 2H), 2.42
(s, 3H). 13C NMR (400 MHz, DMSO-d6) δ 162.7, 155.7, 155.5,
146.5 (dd, J ) 248, 10.2 Hz, F coupling), 144.9 (dd, J ) 255, 10.0
Hz, F coupling), 144.2, 141.8, 140.0, 133.3, 131.9, 130.6, 128.1
(d, J ) 2.2 Hz), 127.6, 127.2, 124.4, 124.2, 123.9, 121.3, 119.2 (d,
J ) 10.9 Hz), 47.2, 16.7. LRMS (ESI+)/HRMS (ESI+) m/z: calcd
for C21H14ClF2N3OS 430.0, found 430.2 [M + H]+/430.0592 found
430.0585 [M + H]+.
4-((N-(3-Chlorophenyl)-4-methylthiazole-5-carboxamido)methyl)-
7,8-difluoroisoquinoline 2-Oxide (47). Compound 47 was synthe-
sized as described for compound 34 using N-(3-chlorophenyl)-N-
((7,8-difluoroisoquinolin-4-yl)methyl)-4-methylthiazole-5-
carboxamide (46) as the starting material; HPLC: tR ) 6.20 (>99%).
1H NMR (400 MHz, DMSO-d6) δ 8.89 (s, 1H), 8.82 (s, 1H), 8.06
(s, 1H), 7.97 (m, 1H), 7.88-7.81 (m, 1H), 7.45 (m, 1H), 7.29 (m,
1H), 7.21 (t, 1H, J ) 8.0 Hz), 6.91 (d, 1H, J ) 8.0 Hz), 5.51 (s,
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 +
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

iNOS Inhibitor DeVelopment Candidate KD7332
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3061
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.
(hind paw flinches, licking, and biting) displayed was counted in 5
min intervals. Phase II was observed from 25-45 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 (ANO-
VA), followed by appropriate post hoc analyses (GraphPad Prizm,
V5.0).
Mouse Chronic Constriction Injury (CCI) Model. Mice
underwent a surgical procedure previously described by Bennett and
Xie. Briefly, mice were initially anesthetized with 5% isoflurane in
medical grade pure oxygen and maintained at 2-3% isoflurane during
the surgical procedure. Using aseptic techniques, the left sciatic nerve
at the level of the midthigh was exposed and loosely ligated with four
ligatures (6-0 chromic gut, Ethicon). Each ligation was approximately
1 mm apart. The thigh muscles were sutured shut with 6-0 braided
black silk, and the skin was closed with surgical wound clips.
Nociceptive behavioral tests: Mice were allowed to recover from
peripheral nerve injury for approximately 1-2 weeks. The development
of cold allodynia was measured by subjecting the animals injured
hindpaw to acetone.31 Briefly, each animal was placed in an acrylic
chamber that rested on an elevated wire mesh platform. Each animal
was placed in the chamber and allowed to acclimate for 1 h. Using a
1 cm3 syringe and 23 gauge needle, 100 µL of acetone was applied to
the plantar surface of the injured hindpaw and measurements of pain
behaviors (hind foot flinching, licking, and biting) were observed and
recorded for 1 min after acetone application. Animals that did not
display a baseline of more than 8 s of pain behaviors were omitted
from the experiment.
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(13) Compound 30 was independently prepared from 8-fluoroquinolinone
52 in four steps: Treatment with POCl3 gave 2-chloroquinoline 123.
3-Chloroaniline was alkylated with 123, followed by reaction with
sodium methoxide to give the 2-methoxyquinoline. Acylation with
4-methylthiazole-5-carbonyl chloride then gave 30.
Acknowledgment. We thank Xing Cheng for assistance with
NMR spectroscopy and Christine Kao for assistance in NOS
SAR-driving assays.
1
Supporting Information Available: Copies of the H NMR,
13C NMR, LC trace, and high resolution mass spectrum. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(14) Li, H.; McMillen, W. T.; Heap, C. R.; McCann, D. J.; Yan, L.;
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