Synthesis and biological evaluation of N-difluoromethyl-1,2-dihydropyrid-2-one acetic acid regioisomers: Dual Inhibitors of cyclooxygenases and 5-lipoxygenase

Article abstract of DOI:10.1016/j.bmcl.2010.02.040

A new group of acetic acid (7a-c, R¹ = H), and propionic acid (7d-f, R¹ = Me), regioisomers wherein a N-difluoromethyl-1,2-dihydropyrid-2-one moiety is attached via its C-3, C-4, and C-5 position was synthesized. This group of compounds exhibited a more potent inhibition, and hence selectivity, for the cyclooxygenase-2 (COX-2) relative to the COX-1 isozyme. Attachment of the N-difluoromethyl-1,2-dihydropyrid-2-one ring system to an acetic acid, or propionic acid, moiety confers potent 5-LOX inhibitory activity, that is, absent in traditional arylacetic acid NSAIDs. 2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)acetic acid (7c) exhibited the best combination of dual COX-2 and 5-LOX inhibitory activities. Molecular modeling (docking) studies showed that the highly electronegative CHF₂ substituent present in 7c, that showed a modest selectivity for the COX-2 isozyme, is oriented within the secondary pocket (Val523) present in COX-2 similar to the sulfonamide (SO₂NH₂) COX-2 pharmacophore present in celecoxib, and that the N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore is oriented close to the region containing the LOX enzyme catalytic iron (His361, His366, and His545). Accordingly, the N-difluoromethyl-1,2-dihyrdopyrid-2-one moiety possesses properties suitable for the design of dual COX-2/5-LOX inhibitory drugs.

Full text of DOI:10.1016/j.bmcl.2010.02.040

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2168–2173
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of N-difluoromethyl-1,2-dihydropyrid-
2-one acetic acid regioisomers: Dual Inhibitors of cyclooxygenases
and 5-lipoxygenase
Gang Yu a,b, P. N. Praveen Rao c, Morshed A. Chowdhury a, Khaled R. A. Abdellatif a, Ying Dong a,
Dipankar Das a, Carlos A. Velázquez a, Mavanur R. Suresh a, Edward E. Knaus a,
*
a Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
b Jiangsu Academy Agricultural Sciences, Nanjing, Jiangsu 210014, China
c School of Pharmacy, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
a r t i c l e i n f o
a b s t r a c t
A new group of acetic acid (7a–c, R1 = H), and propionic acid (7d–f, R1 = Me), regioisomers wherein a
N-difluoromethyl-1,2-dihydropyrid-2-one moiety is attached via its C-3, C-4, and C-5 position was syn-
thesized. This group of compounds exhibited a more potent inhibition, and hence selectivity, for the
cyclooxygenase-2 (COX-2) relative to the COX-1 isozyme. Attachment of the N-difluoromethyl-1,
2-dihydropyrid-2-one ring system to an acetic acid, or propionic acid, moiety confers potent 5-LOX inhib-
itory activity, that is, absent in traditional arylacetic acid NSAIDs. 2-(1-Difluoromethyl-2-oxo-1,2-dihy-
dropyridin-5-yl)acetic acid (7c) exhibited the best combination of dual COX-2 and 5-LOX inhibitory
activities. Molecular modeling (docking) studies showed that the highly electronegative CHF2 substituent
present in 7c, that showed a modest selectivity for the COX-2 isozyme, is oriented within the secondary
pocket (Val523) present in COX-2 similar to the sulfonamide (SO2NH2) COX-2 pharmacophore present in
celecoxib, and that the N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore is oriented close to the
region containing the LOX enzyme catalytic iron (His361, His366, and His545). Accordingly, the N-difluo-
romethyl-1,2-dihyrdopyrid-2-one moiety possesses properties suitable for the design of dual COX-2/5-
LOX inhibitory drugs.
Article history:
Received 15 January 2010
Revised 8 February 2010
Accepted 8 February 2010
Available online 13 February 2010
Keywords:
NSAIDs
Acetic acids
Regioisomers
N-Difluoromethyl-1,2-dihydropyrid-2-one
pharmacophore
Cyclooxygenase isozyme and
5-lipoxygenase inhibition
Molecular modeling
Ó 2010 Elsevier Ltd. All rights reserved.
The eicosanoid family of inflammatory mediators arise from the
biotransformation of arachidonic acid (AA) via the cyclooxygenase
(COX) and lipoxygenase (LOX) pathways.1 In this regard, the pros-
tanoids (prostaglandins, prostacyclin, and thromboxanes) and leu-
kotrienes (LTs) are produced via their respective COX and LOX
pathways. Traditional non-steroidal anti-inflammatory drugs
(NSAIDs) exert their anti-inflammatory effect by inhibition of the
constitutive COX-1 and inducible COX-2 isozymes.2 Adverse side
effects, in particular gastrointestinal (GI) irritation, ulcerogenicity
and renal toxicity attributed to inhibition of the cytoprotective
COX-1 isozyme, are frequent deterrents to the chronic use of NSA-
IDs.3 Alternatively, LTs produced via the 5-LOX enzyme catalyzed
pathway are known to play a role in the pathogenesis of inflamma-
tory and allergic disorders.1 The related isozyme 15-LOX is linked
to cardiovascular complications since it is known to participate
in oxidative modification of low-density lipoproteins (LDL) leading
to the development of atherosclerosis.4 In addition, the 5-LOX
pathway is up-regulated during COX blockade that may be due
to a shift in the metabolism of AA to the uninhibited LOX pathway.
Accordingly, increased levels of LTs may induce undesirable ad-
verse effects such as bronchial constriction. Therefore, dual inhib-
itors of COXs and LOXs represent an attractive safer clinical
alternative to COX inhibitors in view of their potentially greater
anti-inflammatory efficacy due to a synergistic block of both the
COX and LOX metabolic pathways in the AA cascade.5
In an earlier study, we reported that replacement of the tolyl
ring in celecoxib (1) by a N-difluoromethyl-1,2-dihydropyrid-2-
one 5-LOX pharmacophore6 furnished a novel class of dual COX/
5-LOX inhibitors (2) which exhibited effective AI activity.7 It was
subsequently discovered that phenylacetic acid regioisomers (3)
possessing
a N-difluoromethyl-1,2-dihydropyrid-2-one moiety
attached to its C-2, C-3, or C-4 position also exhibited dual COX/
5-LOX inhibitory activities (see structures in Fig. 1).8 It was antic-
ipated that replacement of the aryl ring in a classical arylacetic acid
NSAID template by a N-difluoromethyl-1,2-dihydropyrid-2-one 5-
LOX pharmacophore would similarly provide dual 5-LOX/COX
inhibitors. Accordingly, we now describe the synthesis of a novel
class of acetic acid regioisomers having a N-difluoromethyl-1,2-
dihydropyrid-2-one moiety (7a–f), their in vitro evaluation as
* Corresponding author. Tel.: +1 780 492 5993; fax: +1 780 492 1217.
E-mail address: eknaus@pharmacy.ualberta.ca (E.E. Knaus).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.040

G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2168–2173
2169
CF3
CF3
N
N
N
N
R1
2
CO2H
N
O
N
F2CH
Me
O
F2HC
3
SO2R1
2, R1 = NH2, Me
SO2NH2
Celecoxib (1)
4
3
Figure 1. Some representative examples of the selective COX-2 inhibitor celecoxib (1), and dual COX/5-LOX inhibitors having a N-difluoromethyl-1,2-dihydropyrid-2-one
pharmacophore (2–3).
COX-1/COX-2, 5-LOX inhibitors, and some molecular modeling
studies.
acetonitrile
with
2,2-difluoro-2-(fluorosulfonyl)acetic
acid
(FSO2CF2CO2H)7,14 in the presence of NaHCO3 under an argon
atmosphere at reflux temperature afforded the respective N-difluo-
romethyl-1,2-dihydropyrid-2-one product (6a–f) in 39–65% yields.
Acid hydrolysis of the cyano substituent present in the acetonitrile
compounds (6a, 6c–d, 6f) using 30% HCl in 1,4-dioxane at 80 °C
afforded the respective carboxylic acid target products 7a, 7c–d,
or 7f in 57–79% yield. Alkaline hydrolysis of the methyl ester
moiety present in compounds 6b and 6e using aqueous 2 N NaOH
in MeOH at reflux furnished the respective target acetic acid (7b,
75%), and propionic acid (7e, 74%), product.15
A group of acetic acid (7a–c, R1 = H), and propionic acid (7d–f,
R1 = Me), regioisomers in which a N-difluoromethyl-1,2-dihydro-
pyrid-2-one moiety was attached via its C-3, C-4, and C-5 position
was synthesized as illustrated in Scheme 1. Reaction of the
chloromethylpyridines (4a, 4c) with NaCN furnished the respective
cyanomethylpyridines (5a, 85%; 5c, 70%).9,10 2-Chloro-4-methyl-
pyridine (4b) was elaborated to methyl 2-chloroisonicotinate (5b,
53% yield) upon reaction with lithium diisopropylamide (LDA)
and methyl chloroformate.11 The subsequent methylation of 5a–c
using iodomethane in the presence of LDA at low temperature
under argon afforded the respective products (5d–f) in 75–83%
yields.12,13 Reaction of the 2-chloropyridyl compounds 5a–f in
Traditional arylacetic acid NSAIDs share a number of similar
structural features that include a carboxyl group separated by
one-carbon atom from a flat aromatic nucleus, and one or more
R2
R1
R1
4
4
c
3
5
a
3
5
N
4
Cl
N
5
Cl
5a: 3-isomer, R1 = H, R2 = CN
5d: 3-isomer, R1 = Me, R2 = CN
5b: 4-isomer, R1 = H, R2 = CO2Me
5e: 4-isomer, R1 = Me, R2 = CO2Me
5c: 5-isomer, R1 = H, R2 = CN
5f: 5-isomer, R1 = Me, R2 = CN
4a: R1 = 3-CH2Cl
b
b
b
4b: R1 = 4-Me
4c: R1 = 5-CH2Cl
R1
R1
R2
4
4
CO2H
3
5
3
5
d
N
O
N
O
CHF2
CHF2
7
6
7a: 3-isomer, R1 = H
7b: 4-isomer, R1 = H
7c: 5-isomer, R1 = H
7d: 3-isomer, R1 = Me
7e: 4-isomer, R1 = Me
7f: 5-isomer, R1 = Me
6a: 3-isomer, R1 = H, R2 = CN
6b: 4-isomer, R1 = H, R2 = CO2Me
6c: 5-isomer, R1 = H, R2 = CN
6d: 3-isomer, R1 = Me, R2 = CN
6e: 4-isomer, R1 = Me, R2 = CO2Me
6f: 5-isomer, R1 = Me, R2 = CN
Scheme 1. Reagents and conditions: (a) NaCN, EtOH/H2O, reflux, 12 h for 5a, 5c; LDA, ClCO2Me, THF, À78 °C, 1 h; then 0 °C, 30 min for 5b; (b) LDA, MeI, THF, À78 °C, 30 min;
then 0 °C, 30 min; (c) FSO2CF2CO2H, NaHCO3, MeCN, argon atmosphere, reflux, 6 h; (d) 30% HCl, 1,4-dioxane, reflux, 8 h for 7a, 7c–d, 7f; aqueous 2 N NaOH, MeOH, reflux, 3 h
for 7b, 7e.

2170
G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2168–2173
large lipophilic groups attached to the aromatic nucleus, that is,
two, three, or four carbon atoms removed from the point of attach-
ment of the acetic acid side chain.16 Similarly, the lipophilic CHF2
substituent present in the 1-difluoromethyl-2-oxo-1,2-dihydro-
pyridyl derivatives of acetic acid (7a–c) and propionic acid (7d–f)
is separated from the point of attachment of the acetic acid side
chain by either 2 or 3 carbon atoms and 1 N atom that are embed-
ded in the nearly planar 2-oxo-1,2-dihydropyridyl ring. In this
regard, the rational for the design of compounds (7a–f) was based
on the belief that replacement of the aromatic ring with an at-
tached lipophilic substituent in an arylacetic acid NSAID by a lipo-
philic N-difluoromethyl-2-oxo-1,2-dihydropyridyl moiety may
furnish a new class of dual COX-2/5-LOX inhibitors. This expecta-
tion is based on a drug design concept that the CONCHF2 fragment
of the N-difluoromethyl-1,2-dihydropyrid-2-one ring present in
7a–f can be viewed as a cyclic hydroxamic acid mimetic. If this
concept is true, there are two plausible mechanisms by which
N-difluoromethyl-1,2-dihydropyrid-2-ones 7a–f could inhibit the
5-LOX enzyme. For example, 7a–f, like acyclic hydroxamic acids,
may chelate iron present in 5-LOX to induce inhibitory activity.
There is a substantial localized negative potential around the two
fluorine atoms of a CHF2 group.17 However, aliphatic fluorine
atoms, in spite of this high electron-density, seldom act as a hydro-
gen-bond acceptor, that is, most likely due to their high electroneg-
ativity and low polarizability.18,19 Therefore, it is also plausible that
the CHF2 group could interact with a positively charged region on
the enzyme that may provide enhanced affinity and competitive
reversible inhibition of the COX and/or 5-LOX enzymes.20
one regioisomers 7b–f exhibited a more potent inhibition, and
hence selectivity, for the COX-2 isozyme (COX-1 IC50 = inactive to
16.5 lM range; COX-2 IC50 = 1.3–12.9 lM range). In comparison,
the acetic acid regioisomer 7a was a nearly equiactive inhibitor
of COX-1 and COX-2. The point of attachment of the acetic acid
(7a–c, R1 = H) substituent to the N-difluoromethyl-1,2-dihydropy-
rid-2-one ring system was a greater determinant of COX-2 inhibi-
tory activity relative to that observed for the propionic acids (7d–f,
R1 = Me). All compounds 7a–f were less potent inhibitors of COX-1
and COX-2 than the reference drug ibuprofen. The most potent
COX-2 inhibitor 7c (IC50 = 1.3
ibuprofen (IC50 = 1.1 M).
lM) was marginally less potent than
l
In vitro 5-LOX inhibition studies showed that attachment of the
N-difluoromethyl-1,2-dhydropyrid-2-one ring system to an acetic
acid moiety confers 5-LOX inhibitory activity, that is, absent in tra-
ditional arylacetic acid NSAIDs. The group of compounds 7a–f
exhibited more potent 5-LOX inhibitory activity (IC50 = 0.25–
3.7 lM range) than the reference drug caffeic acid (IC50 = 4.0 lM).
The point of attachment of the acetic acid (R1 = H) substituent was
not a determinant of activity for the nearly equipotent 3-, 4-, and
5-regioisomers 7a–c (IC50 = 0.44–0.77 lM range). In contrast, the
point of attachment of the propionic acid (R1 = Me) substituent
modulated 5-LOX potency where the 4-regioisomer 7e
(IC50 = 0.25
3- and 5-regioisomers 7d and 7f showing IC50 values of 3.7 and
3.0
M, respectively. The R1 substituent was a determinant of 5-
lM) was greater than 10-fold more potent than the
l
LOX inhibitory potency for the C-3 and C-5 regioisomers (7a and
7c, R1 = H > 7d and 7f, R1 = Me), but not the C-4 regioisomers (7b,
R1 = H approximately equipotent to 7e, R1 = Me).
In vitro COX-1 and COX-2 enzyme inhibition studies (see data in
Table 1) showed that the N-difluoromethyl-1,2-dihydropyrid-2-
Some physicochemical comparisons indicate that the acetic acids
7a–c (R1 = H) are less lipophilic (calculated log P = 0.08–0.21 range)
and smaller in size (calculated volume of 154 Å3) than the corre-
sponding propionic acids 7d–f (R1 = Me; calculated log P = 0.65–
0.82 range; calculated volume of 171 Å3; see data in Table 1).
The binding interactions of an acetic acid derivative possessing
a novel N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore
was investigated by enzyme–ligand molecular modeling (docking)
studies. The binding interaction of 2-(1-difluoromethyl-2-oxo-1,2-
Table 1
In vitro COX-1, COX-2, and 5-LOX enzyme inhibition data for 1-difluoromethyl-2-oxo-
1,2-dihydropyridyl derivatives of acetic acid (7a–c) and propionic acid (7d–f)
R1
4
CO2H
3
5
N
O
CHF2
7: a,d = 3-isomer; b,e = 4-isomer; c,f = 5-isomer
Compound
R1
COX-1
COX-2
5-LOX
Log Pc Volumed
(Å3)
a
a
b
IC50
(lM) IC50 (lM) IC50 (lM)
7a
7b
7c
7d
7e
7f
H
H
H
6.0
Inactive
7.9
8.0
12.9
1.3
5.9
5.5
6.4
1.1e
—
0.77
0.44
0.46
3.7
0.25
3.0
0.21
0.08
0.08
0.82
0.65
0.65
3.68
154.2
154.9
154.7
171.0
170.9
171.0
211.8
Me 16.5
Me 10.7
Me 9.8
2.9
Ibuprofen
—
4.0
Caffeic acidf
—
a
The in vitro test compound concentration required to produce 50% inhibition of
ovine COX-1 or human recombinant COX-2. The result (IC50 M) is the mean of two
,
l
determinations acquired using the enzyme immuno assay kit (Catalog No. 560131,
Cayman Chemicals Inc., Ann Arbor, MI, USA) and the deviation from the mean is
<10% of the mean value.
b
The in vitro test compound concentration required to produce 50% inhibition of
potato 5-LOX (Cayman Chemicals Inc. Catalog No. 60401). The result (IC50, lM) is
the mean of two determinations acquired using a LOX assay kit (Catalog No.
760700, Cayman Chemicals Inc., Ann Arbor, MI, USA) and the deviation from the
mean is <10% of the mean value.
c
The log P value was calculated using the ChemDraw Ultra program, Version 6.0,
CambridgeSoft company.
d
The volume of the molecule, after minimization using the MM3 force field, was
Figure 2. Docking 2-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)acetic acid
calculated using the Alchemy 2000 program, Tripos Inc.
(7c) (ball-and-stick) in the active site of mammalian COX-2 (Eintermolecular
=
e
Data acquired using ovine COX-2 (Catalog No. 560101, Cayman Chemicals Inc.).
À23.08 kcal/mol). Red dotted line represents hydrogen bonding. Hydrogen atoms
f
Caffeic acid: 3,4-dihydroxycinnamic acid.
are not shown for clarity.

G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2168–2173
2171
dihydropyridin-5-yl)acetic acid (7c, COX-2 IC50 = 1.30
l
M) within
located about 3.94 Å from the NH of His361. The distance between
the CHF2 and the NH2 (guanidine) group of Arg403, that is, closer to
the mouth of the 15-LOX binding site is about 10.48 Å. The 1,2-
dihydropyrid-2-one ring is surrounded by His361, His362,
Leu408, Phe415, and Leu597 (distance <5 Å). The C-5 acetic acid
(CH2COOH) side chain is oriented toward the hydrophobic base re-
gion of the 15-LOX active site in the vicinity of Leu408, Ile414,
Phe415, Met419, Ile593, and Leu597 (distance <5 Å). The COOH
substituent is oriented in a polar region comprised of Glu357 and
Gln548 (distance <5 Å). The C@O (CH2COOH) forms a favorable
hydrogen bonding interaction with the NH2 of Gln548 (dis-
tance = 1.92 Å). This docking study supports our hypothesis that
the CONCHF2 fragment present in the N-difluoromethyl-1,2-dihyd-
ropyrid-2-one ring can be viewed as a cyclic hydroxamic acid
mimetic.
In conclusion, a new class of C-3, C-4, and C-5 regioisomeric 1-
difluoromethyl-2-oxo-1,2-dihydropyridyl derivatives of acetic acid
(7a–c) and propionic acid (7d–f) was synthesized23 for evaluation
as dual COX-1/COX-224 and 5-LOX25 enzyme inhibitors. Biological
data acquired indicated that (1-difluoromethyl-2-oxo-1,2-dihydro-
pyridin-5-yl)acetic acid (7c) exhibits the best combination of
in vitro COX-2 and 5-LOX inhibitory potency. Molecular modeling
(docking) studies26 showed that the highly electronegative CHF2
substituent present in 7c is oriented within the secondary pocket
(Val523) present in COX-2 similar to the sulfonamide (SO2NH2)
COX-2 pharmacophore present in celecoxib (1) to confer a modest
selectivity for the COX-2 isozyme, and that the N-difluoromethyl-
1,2-dihydropyrid-2-one pharmacophore is oriented close to the re-
gion containing the enzyme catalytic iron (His361, His366, and
His545). Accordingly, the N-difluoromethyl-1,2-dihyrdopyrid-2-
one moiety possesses properties suitable for the design of dual
COX-2/5-LOX inhibitory AI drugs.
the COX-2 binding site is illustrated in Figure 2. The enzy-
meÀligand complex observed shows that the 1,2-dihydropyrid-2-
one ring is oriented such that the CONCHF2 fragment is positioned
in a region close to both the entrance of the COX-2 active site
(Arg120 and Tyr355) and the COX-2 secondary pocket (His90,
Arg513, and Val523). The C@O oxygen atom (CONCHF2), that
undergoes a favorable hydrogen bonding interaction with the OH
of Tyr355 (distance = 1.87 Å), is located about 2.57 Å from
Arg120. The highly electronegative CHF2 substituent is oriented
within the COX-2 secondary pocket (Val523) in a polar region com-
prised of His90 and Arg513. The CHF2 fluorine atoms are located
about 4.54 and 3.82 Å from the NH of His90 and the NH2 ofArg513,
respectively. The C-5 acetic acid (CH2COOH) side chain is oriented
in a region consisting of Val349, Leu352, Tyr385, Ala527, and
Ser530 (distance <5 Å). The observation that the C@O (CH2COOH)
is oriented closer to the apex of the COX-2 active site (Tyr385
and Ser530) where it is positioned about 4.26 and 4.54 Å from
the OHs of Ser530 and Tyr385, respectively, is consistent with pre-
viously reported crystal structure data for the NSAID diclofenac
[ortho-(2,6-dichloroanilino)phenylacetic acid] bound to the COX-
2 enzyme.21
The closely related mammalian lipoxygenase enzymes (5-, 12-,
and 15-LOX) share 35–80% sequence identity. X-ray data for mam-
malian 15-LOX, but not 5-LOX, has been reported.22 Accordingly,
15-LOX X-ray data was used as a model to investigate the binding
interactions of 2-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-5-
yl)acetic acid (7c, 5-LOX IC50 = 0.46 lM) within a LOX binding site
(Fig. 3). This docking study indicated that the LOX N-difluoro-
methyl-1,2-dihydropyrid-2-one pharmacophore is oriented close
to the region containing the enzyme catalytic iron (His361,
His366, and His545). The CONCHF2 fragment is located in the
vicinity of a polar region where the distance between the fluorine
atoms (CHF2) and the NHs of His361 and His366 are about 4.41 and
4.23 Å, respectively. In addition, the C@O (dihydropyrid-2-one) is
Acknowledgments
We are grateful to the Canadian Institutes of Health Research
(CIHR) (MOP-14712) for financial support of this research. Gang
Yu was a recipient of a Jiangsu Government Abroad Study Scholar-
ship, China.
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Brunetti, M.; Altamura, S.; De Francesco, R.; Matassa, V. G. Bioorg. Med. Chem.
Lett. 2002, 12, 701. and references cited therein.
Figure 3. Docking 2-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)acetic acid
(7c) (ball-and-stick) in the active site of mammalian 15-LOX (Eintermolecular
=
18. Dunitz, J. D.; Taylor, R. Chem. Eur. J. 1997, 3, 89.
À33.52 kcal/mol). Red dotted line represents hydrogen bonding. Hydrogen atoms
19. Howard, J. A. K.; Hoy, V. J.; O’Hagan, D.; Smith, G. T. Tetrahedron 1996, 52,
12613.
are not shown for clarity.

2172
G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2168–2173
20. Chavatte, P.; Yous, S.; Marot, C.; Baurin, N.; Lesieur, D. J. Med. Chem. 2001, 44,
3223.
21. Rowlinson, S. W.; Kiefer, J. R.; Prusakiewicz, J. J.; Pawlitz, J. L.; Kozak, K. R.;
Kalgutkar, A. S.; Stallings, W. C.; Kurumbail, R. G.; Marnett, L. J. J. Biol. Chem.
2003, 278, 45763.
1.85 mL, 18.0 mmol), and then NaHCO3 (0.56 g, 6.6 mmol), was added to a
solution of each 2-chloropyridine compound 5a–f (6.0 mmol) in dry acetonitrile
(30 mL) with stirring. The reaction was allowed to proceed at reflux under an
argon atmosphere for 6 h. After cooling to 25 °C, the solvent was removed in
vacuo, a saturated solution of aqueous NaHCO3 (25 mL) was added to the
residue, and the mixture was extracted with CH2Cl2 (3 Â 50 mL). The combined
organic extracts were washed with 10 N HCl (2 Â 15 mL) to remove any starting
material 5 that was still present. The CH2Cl2 extract was washed with water
(50 mL) and then brine (25 mL), the organic fraction was dried (MgSO4), and the
solvent was removed in vacuo to afford the respective title product 6. Some
physical and spectral data for 6a–f are listed below.
22. Gillmor, S. A.; Villasenor, A.; Fletterick, R.; Sigal, E.; Browner, M. F. Nat. Struct.
Biol. 1997, 4, 1003.
23. Experimental procedures and spectral data for compounds 5, 7–9. General:
Melting points were determined on a Thomas-Hoover capillary apparatus and
are uncorrected. Infrared (IR) spectra were recorded as films on NaCl plates
using a Nicolet 550 Series II Magna FT-IR spectrometer. 1H NMR spectra were
measured on a Bruker AM-300 spectrometer in CDCl3 or DMSO-d6 with TMS as
the internal standard, where J (coupling constant) values are estimated in Hz.
Spin multiples are given as s (singlet), d (doublet), t (triplet), q (quartet), m
(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-3-yl)acetonitrile (6a): Product 6a
was obtained as a pale yellow solid (0.47 g, 43%); mp 76–78 °C; IR (film):
2924, 2252, 1672 cmÀ1 1H NMR (CDCl3) d 3.62 (s, 2H, CH2), 6.40 (dd, J = 6.7,
;
(multiplet), and br (broad). Microanalyses were performed for C, H,
N
6.7 Hz, 1H, H-5), 7.51 (dd, J = 6.7, 1.8 Hz, 1H, H-4), 7.65 (dd, J = 6.7, 1.8 Hz, 1H, H-
6), 7.70 (t, J = 59.8 Hz, 1H, CHF2); 13C NMR (CDCl3) d 19.1, 106.5 (t, J = 234 Hz),
106.6, 116.4, 123.6, 129.1, 138.6, 159.7.
(MicroAnalytical Service Laboratory, Department of Chemistry, University of
Alberta) and were within 0.4% of theoretical values. Silica gel column
chromatography was performed using Merck Silica Gel 60 ASTM (70–230
mesh). 2-Chloro-3-chloromethylpyridine (4a) was prepared starting from
commercially available 2-chloronicotinic acid.27 All other reagents,
purchased from the Aldrich Chemical Company (Milwaukee, WI), were used
without further purification.
2-Chloro-3-cyanomethylpyridine (5a): Sodium cyanide (1.3 g, 26.5 mmol) was
added to a solution of 4a (4.0 g, 24.7 mmol) in EtOH (45 mL) and water (5 mL)
at 25 °C with stirring. The reaction was allowed to proceed at reflux for 12 h,
the reaction mixture was cooled to 25 °C, and the solvent was removed in
vacuo. The residue was partitioned between EtOAc and water, the EtOAc
fraction was washed with water and then brine, the EtOAc fraction was dried
(MgSO4), filtered and the solvent was removed in vacuo. The residue was
purified by flash chromatography (hexane/EtOAc) to afford 5a (3.2 g, 85%) as a
pale brown solid, mp 84–85 °C (lit.9b mp 85–86 °C); 1H NMR (CDCl3) d 3.88 (s,
2H, CH2CN), 7.34 (dd, J = 8.5, 4.9 Hz, 1H, H-5), 7.90 (dd, J = 8.5, 1.8 Hz, 1H, H-4),
8.41 (dd, J = 4.9, 1.8 Hz, H-6).
Methyl (1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)acetate (6b): Product 6b
was obtained as a yellow solid (0.69 g, 53%); mp 74–76 °C; IR (film): 2959, 1733,
1683 cmÀ1 1H NMR (CDCl3) d 3.48 (s, 2H, CH2), 3.74 (s, 3H, OMe), 6.28 (dd,
;
J = 7.9, 1.8 Hz, 1H, H-5), 6.45 (d, J = 1.8 Hz, 1H, H-3), 7.49 (d, J = 7.9 Hz, 1H, H-6),
7.66 (t, J = 59.8 Hz, 1H, CHF2); 13C NMR (CDCl3) d 40.6, 56.8, 107.3 (t, J = 234 Hz),
108.8, 121.2, 129.3, 148.3, 161.3, 169.4.
(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)acetonitrile (6c): Product 6c
was obtained as a pale yellow solid (0.66 g, 60%); mp 86–87 °C; IR (film):
2971, 2245, 1699 cmÀ1 1H NMR (CDCl3) d 3.54 (s, 2H, CH2), 6.63 (d, J = 9.8 Hz,
;
1H, H-3), 7.34 (dd, J = 9.8, 2.4 Hz, 1H, H-4), 7.47 (d, J = 2.4 Hz, 1H, H-6), 7.67 (t,
J = 59.8 Hz, 1H, CHF2); 1C NMR (CDCl3) d 20.3, 107.3 (t, J = 253 Hz), 109.7, 116.1,
122.8, 128.0, 140.8, 159.9.
2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-3-yl)propionitrile (6d): Product
6d was obtained as
a
yellow oil (0.46 g, 39%); IR (film): 2999, 2245,
1.59 (d, J = 7.3 Hz, 3H, CHMe), 4.15 (q,
1671 cmÀ1 1H NMR (CDCl3)
;
d
J = 7.3 Hz, 1H, CHMe), 6.34 (t, J = 7.3 Hz, 1H, H-5), 7.49 (dd, J = 7.3, 1.8 Hz, 1H,
H-4), 7.65 (dd, J = 7.3, 1.8 Hz, 1H, H-6), 7.70 (t, J = 60.4 Hz, 1H, CHF2); 13C NMR
(CDCl3) d 18.4, 26.3, 106.7, 107.5 (t, J = 253 Hz), 120.4, 129.2, 129.8, 137.7, 159.3.
Methyl 2-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)propanoate (6e):
Product 6e was obtained as a pale yellow oil (0.73 g, 53%); IR (film): 2950,
2-Chloro-4-methoxycarbonylmethylpyridine (5b): n-Butyllithium (17.6 mL of
2.5 M in hexane, 44.0 mmol) was added to a solution of diisopropylamine
(6.22 mL, 44.0 mmol) in dry THF (50 mL) at À78 °C under an atmosphere of
argon, and the mixture was stirred for 30 min. A solution of 2-chloro-4-
methylpyridine (4b) (5.11 g, 40.0 mmol) in dry THF (20 mL) was added drop
wise during 20 min, and the mixture was stirred for 1 h at À78 °C. A solution of
methyl chloroformate (3.69 mL, 48.0 mmol) in dry THF (10 mL) was added
drop wise during 20 min, and the reaction was allowed to proceed with stirring
for 1 h at À78 °C and then at 0 °C for 30 min. The mixture was quenched with
aqueous saturated NaHCO3 solution, the organic fraction was extracted with
ether (3 Â 80 mL), the combined organic extracts were washed with brine, and
the organic fraction was dried (MgSO4). Removal of the solvent in vacuo gave a
residue that was purified by silica gel column chromatography using hexane–
ethyl acetate (4:1, v/v) as eluent to afford 5b (3.92 g, 53%) as a pale yellow oil,
1734, 1683 cmÀ1 1H NMR (CDCl3) d 1.45 (d, J = 6.7 Hz, 3H, CHMe), 3.55 (q,
;
J = 6.7 Hz, 1H, CHMe), 3.71 (s, 3H, OMe), 6.27 (dd, J = 7.3, 1.8 Hz, 1H, H-5), 6.44 (d,
J = 1.8 Hz, 1H, H-3), 7.40 (d, J = 7.3 Hz, 1H, H-6), 7.65 (t, J = 60.4 Hz, 1H, CHF2); 13
C
NMR (CDCl3) d 16.8, 45.0, 52.5, 107.2, 107.3 (t, J = 252 Hz), 119.3, 129.1, 154.3,
160.9, 172.5.
2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)propionitrile (6f): Product 6f
was obtained as a yellow gum (0.77 g, 65%); IR (film): 2992, 2241, 1693 cmÀ1 1H
;
NMR (CDCl3) d1.62 (d, J = 7.3 Hz 3H, CHMe), 3.72 (q, J = 7.3 Hz 1H, CHMe), 6.63 (d,
J = 9.7 Hz, 1H, H-3), 7.38 (dd, J = 9.7, 2.4 Hz, 1H, H-4), 7.45 (d, J = 2.4 Hz, 1H, H-6),
7.66 (t, J = 59.8 Hz, 1H, CHF2); 13C NMR (CDCl3) d 19.8, 27.9, 107.3 (t, J = 253 Hz),
116.3, 119.6, 123.0, 126.9, 139.7, 160.0.
IR (film): 2948, 1733, 1593, 1227 cmÀ1 1H NMR (CDCl3) d 3.63 (s, 2H, CH2),
;
3.67 (s, 3H, OMe), 7.17 (dd, J = 4.9, 1.8 Hz, 1H, H-5), 7.29 (d, J = 1.8 Hz, 1H, H-3),
8.35 (d, J = 4.9 Hz, H-6).
General procedure for the synthesis of 1-difluoromethyl-2-oxo-1,2-dihydropyridyl
derivatives of acetic acid (7a, 7c) and propionic acid (7d, 7f): Hydrochloric acid
(15 mL of 10 N) was added to a solution of 6a, 6c, 6d, or 6f (3.0 mmol) in 1,4-
dioxane (10 mL). The mixture was heated at 80 °C for 8 h, cooled to 25 °C, and the
solvent was removed in vacuo. Cold water (30 mL) was added to the residue, the
mixture was adjusted to pH 5 using 5% HCl, and this mixture was extracted with
EtOAc (3 Â 30 mL). The combined EtOAc extracts were washed with water and
then brine, the organic fraction was dried (MgSO4), and the solvent was removed
in vacuo. The residue was purified by silica gel column chromatography
(hexanes–EtOAc = 2:1, v/v) to afford the respective product 7a, 7c, 7d, or 7f.
Physical and spectral data are listed below.
General procedure for synthesis of 1-difluoromethyl-2-oxo-1,2-dihydropyridyl
derivatives of the acetic acid (7b) and propionic acid (7e): A solution of aqueous
NaOH (10 mL of 2 N) was added to a solution of either 6b or 6e (3.0 mmol) in
MeOH (10 mL), and the mixture was stirred at gentle reflux for 3 h prior to
cooling to 25 °C. After nearly complete removal of the MeOH in vacuo, water
(30 mL) was added, the mixture was adjusted to pH 3 using aqueous 5% HCl
solution, and the mixture was extracted with EtOAc (3 Â 30 mL). The combined
EtOAc extracts were washed with water and then brine, the organic fraction
was dried (MgSO4), and the solvent was removed in vacuo. The residue was
purified by silica gel column chromatography (hexanes–EtOAc, 2:1, v/v) to
furnish the respective product 7b or 7e. Physical and spectral data for 7b and
7e are listed below.
2-Chloro-5-cyanomethylpyridine (5c): The title compound was prepared by
reaction of 4c (2.0 g, 12.35 mmol) with NaCN (0.67 g, 13.59 mmol) according to
a reported procedure12 as colorless plates (1.31 g, 70%), mp 52–54 °C (lit.9b mp
51–52 °C); 1H NMR (CDCl3) d 3.77 (s, 2H, CH2CN), 7.40 (d, J = 8.5 Hz, 1H, H-3),
7.70 (dd, J = 8.5, 2.4 Hz, 1H, H-4), 8.41 (d, J = 2.4 Hz, H-6).
General procedure for the synthesis of the propionitriles (5d, 5f) and methyl
propanoate (5e): n-Butyllithium (4.40 mL of 2.5 M in hexane, 11.0 mmol) was
added to a solution of diisopropylamine (1.55 mL, 11.0 mmol) in dry THF
(20 mL) at À78 °C under an atmosphere of argon, and the mixture was stirred
for 30 min. A solution of either 5a, 5b, or 5c (10.0 mmol) in dry THF (10 mL)
was added drop wise during 20 min, and the solution was stirred at À78° for
30 min prior to warming to 0 °C. Methyl iodide (0.75 mL, 12.0 mmol) was
added and the reaction was allowed to proceed with stirring for 30 min. The
reaction mixture was washed with aqueous saturated NH4Cl (15 mL) and the
THF layer was extracted with EtOAc (2 Â 50 mL). The combined organic extracts
were washed with brine and dried over MgSO4. Removal of the solvent in vacuo
afforded a residue that was purified by silica gel column chromatography using
hexane–ethyl acetate (4:1, v/v) as eluent to furnish the respective product 5d,
5e, or 5f. The physical and spectral data for 5d–f are listed below.
2-(2-Chloropyridin-3-yl)propionitrile (5d): Product 5d was obtained as a pale
yellow oil (1.24 g, 75%); IR (film): 2986, 2242 cmÀ1 1H NMR (CDCl3) d 1.67 (d,
;
J = 6.7 Hz, 3H, CHMe), 4.32 (q, J = 6.7 Hz, 1H, CHMe), 7.36 (dd, J = 7.9, 4.9 Hz, 1H,
H-5), 7.95 (dd, J = 7.9, 1.8 Hz, 1H, H-4), 8.41 (dd, J = 4.9, 1.8 Hz, H-6). 13C NMR
(CDCl3) d 18.3, 54.2, 122.3, 123.9, 147.7, 151.9, 153.4, 160.3 Hz.
(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-3-yl)acetic acid (7a): Product 7a
was obtained as a pale brown solid (0.48 g, 79%), mp 98–100 °C; IR (film):
3100 (br), 2919, 1734, 1667 cmÀ1 1H NMR (CDCl3) d 3.61 (s, 2H, CH2), 6.39 (dd,
;
Methyl 2-(2-chloropyridin-4-yl)propanoate (5e): Product 5e was obtained as a
J = 6.7, 6.7 Hz, 1H, H-5), 7.45 (dd, J = 6.7, 1.2 Hz, 1H, H-4), 7.58 (dd, J = 6.7,
1.2 Hz, 1H, H-6), 7.73 (t, J = 60.4 Hz, 1H, CHF2), 9.37 (br s, 1H, COOH); 13C NMR
(CDCl3) d 36.5, 107.4, 107.5 (t, J = 253 Hz), 126.7, 128.7 (t, J = 3 Hz), 140.1,
161.5, 174.1. Anal. Calcd for C8H7F2NO3: C, 47.30; H, 3.47; N, 6.89. Found: C,
47.70; H, 3.71; N, 6.87.
pale yellow oil (1.65 g, 83%); IR (film): 2950, 1735, 1592, 1246 cmÀ1 1H NMR
;
(CDCl3) d 1.50 (d, J = 6.7 Hz, 3H, CHMe), 3.68 (q, J = 6.7 Hz, 1H, CHMe), 3.69 (s,
3H, OMe), 7.16 (dd, J = 4.9, 1.2 Hz, 1H, H-5), 7.27 (d, J = 1.2 Hz, 1H, l H-3), 8.33 (d,
J = 4.9 Hz, H-6); 13C NMR (CDCl3) d 17.8, 44.6, 52.5, 121.6, 123.4, 149.8, 151.9,
152.4, 173.0 Hz.
2-(2-Chloropyridin-5-yl)propionitrile (5f): Product 5f was obtained as a pale
yellow solid (1.27 g, 77%); mp 69–70 °C (lit12 mp 67–69 °C); 1H NMR (CDCl3) d
1.68 (d, J = 7.3 Hz, 3H, CHMe), 3.96 (q, J = 7.3 Hz, 1H, CHMe), 7.40 (d, J = 8.5 Hz,
1H, H-3), 7.95 (dd, J = 8.5, 2.4 Hz, 1H, H-4), 8.41 (d, J = 2.4 Hz, H-6).
General procedure for synthesis of the 1-difluoromethyl-2-oxo-1,2-dihydropyridine
analogs (6a–f): 2,2-Difluoro-2-(fluorosulfonyl)acetic acid (FSO2CF2COOH,
(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)acetic acid (7b): Product 7b
was obtained as a yellow solid (0.45 g, 75%); mp 112–114 °C; IR (film): 3100
(br), 2926, 1723, 1677 cmÀ1 1H NMR (CDCl3 + DMSO) d 3.41 (s, 2H, CH2), 6.28
;
(dd, J = 7.3, 1.2 Hz, 1H, H-5), 6.42 (d, J = 1.2 Hz, 1H, H-3), 7.36 (d, J = 7.3 Hz, 1H,
H-6), 7.66 (t, J = 59.8 Hz, 1H, CHF2), 8.62 (br s, 1H, COOH); 1C NMR (CDCl3) d
40.6, 107.3 (t, J = 234 Hz), 108.8, 121.2, 129.3, 148.3, 161.3, 169.4. Anal. Calcd
for C8H7F2NO3: C, 47.30; H, 3.47; N, 6.89. Found: C, 47.59; H, 3.20; N, 6.67.

G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2168–2173
2173
(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)acetic acid (7c): Product 7c
was obtained as an off-white solid (0.44 g, 73%); mp 128–130 °C; IR (film):
Arbor, MI, USA) (IC50 values,
l
M) were determined using an enzyme immuno
assay (EIA) kit (Catalog No. 760700, Cayman Chemical, Ann Arbor, MI, USA)
3069 (br), 2903, 1734, 1677 cmÀ1 1H NMR (CDCl3) d 3.46 (s, 2H, CH2), 6.60 (d,
;
according to our previously reported method.29
J = 9.2 Hz, 1H, H-3), 7.38 (dd, J = 9.2, 2.4 Hz, 1H, H-4), 7.40 (d, J = 2.4 Hz, 1H, H-
6), 7.82 (t, J = 60.4 Hz, 1H, CHF2), 9.91 (br s, 1H, COOH); 13C NMR (CDCl3) d 36.4,
107.0 (t, J = 253 Hz), 113.7, 120.8, 127.5, 143.3, 160.0, 171.8. Anal. Calcd for
C8H7F2NO3: C, 47.30; H, 3.47; N, 6.89. Found: C, 47.53; H, 3.59; N, 6.64.
2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-3-yl)propanoic acid (7d): Product
7d was obtained as a pale yellow solid (0.39 g, 64%); mp 102–104 °C; IR (film):
26. Molecular modeling (docking) studies: Docking experiments were performed
using Discovery Studio Client v2.5.0.9164 (2005-09), Accelrys Software Inc.
running on a HP xw4600 workstation (Processor x86 family 6 model 23
stepping 10 GenuineIntel 2999 ꢀ Mhz). The coordinates for the X-ray crystal
structure of the enzyme COX-2 and 15-LOX were obtained from the RCSB
Protein Data Bank and hydrogens were added. The ligand molecules were
constructed using the Build Fragment tool and energy minimized for 1000
3098 (br), 2940, 1718, 1667 cmÀ1 1H NMR (CDCl3) d 1.51 (d, J = 7.3 Hz, 3H,
;
CHMe), 3.94 (q, J = 7.3 Hz, 1H, CHMe), 6.43 (dd, J = 6.7, 6.7 Hz, 1H, H-5), 7.38
(dd, J = 6.7, 1.2 Hz, 1H, H-4), 7.40 (dd, J = 6.7, 1.2 Hz, 1H, H-6), 7.74 (t,
J = 60.4 Hz, 1H, CHF2), 9.73 (br s, 1H, COOH); 13C NMR (CDCl3) d 15.5, 39.8,
107.6 (t, J = 253 Hz), 107.8, 128.1, 132.3, 137.9, 161.2, 177.5. Anal. Calcd for
C9H9F2NO3: C, 49.78; H, 4.18; N, 6.45. Found: C, 49.53; H, 4.14; N, 6.35.
2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)propanoic acid (7e): Product
7e was obtained as an off-white solid (0.48 g, 74%); mp 64–66 °C; IR (film):
iterations reaching
a
convergence of 0.01 kcal/mol Å. The docking
experiment on COX-2 was carried out by superimposing the energy
minimized ligand on SC-558 in the PDB file 1cx2 after which SC-558 was
deleted. The coordinates for 15-LOX was obtained from PDB file 1lox and
the energy minimized ligand was superimposed on the inhibitor RS75091
after which RS75091 was deleted. In all these experiments the resulting
ligandÀenzyme complex was subjected to docking using the Libdock
command in the receptor–ligand interactions protocol of Discovery Studio
after defining subsets of the enzyme within 10 Å sphere radius of the ligand.
The force field, Chemistry at HARvard Macromolecular Mechanics
(CHARMM) was employed for all docking purposes. The ligandÀenzyme
3100 (br), 2986, 1682, 1667 cmÀ1 1H NMR (CDCl3) d 1.49 (d, J = 7.3 Hz, 3H,
;
CHMe), 3.60 (q, J = 7.3 Hz, 1H, CHMe), 6.34 (dd, J = 7.3, 1.8 Hz, 1H, H-5), 6.54 (d,
J = 1.8 Hz, 1H, H-3), 7.44 (d, J = 7.3 Hz, 1H, H-6), 7.67 (t, J = 60.4 Hz, 1H, CHF2),
8.65 (br s, 1H, COOH); 13C NMR (CDCl3) d 16.7, 44.5, 107.3 (t, J = 253 Hz), 107.8,
119.3, 129.2, 154.3, 161.3, 176.1. Anal. Calcd for C9H9F2NO3: C, 49.78; H, 4.18;
N, 6.45. Found: C, 49.55; H, 4.20; N, 6.64.
assembly was then subjected to
a molecular dynamics (MD) simulation
using Simulation protocol at a constant temperature of 300 K with a 100
step equilibration for over 1000 iterations and a time step of 1 fs using a
distance dependent dielectric constant 4r. The optimal binding orientation of
the ligandÀenzyme assembly obtained after docking was further minimized
for 1000 iterations using the conjugate gradient method until a convergence
of 0.001 kcal/mol Å was reached after which Eintermolecular (kcal/mol) of the
ligandÀenzyme assembly was evaluated.
2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)propanoic acid (7f): Product
7f was obtained as a white solid (0.37 g, 57%); mp 90–92 °C; IR (film): 3074
(br), 2986, 1734, 1688 cmÀ1 1H NMR (CDCl3) d 1.49 (d, J = 7.3 Hz, 3H, CHMe),
;
3.54 (q, J = 7.3 Hz, 1H, CHMe), 6.62 (d, J = 9.7 Hz, 1H, H-3), 7.37 (d, J = 2.4 Hz,
1H, H-6), 7.49 (dd, J = 9.7, 2.4 Hz, 1H, H-4), 7.68 (t, J = 60.4 Hz, 1H, CHF2), 9.95
(br s, 1H, COOH); 13C NMR (CDCl3) d 17.2, 41.6, 107.4 (t, J = 253 Hz), 119.4,
121.7, 127.1, 141.8, 160.9, 178.0. Anal. Calcd for C9H9F2NO3: C, 49.78; H, 4.18;
N, 6.45. Found: C, 49.46; H, 4.22; N, 6.55.
27. (a) Read, M. W.; Ray, P. S. J. Heterocycl. Chem. 1995, 32, 1595; (b) Zhuang, L. H.;
Wai, J. S.; Embrey, M. W.; Fisher, T. E.; Egbertson, M. S.; Payne, L. S.; Guare, J. P.,
Jr.; Vacca, J. P.; Hazuda, D. J.; Felock, P. J.; Wolfe, A. L.; Stillmock, K. A.; Witmer,
M. V.; Moyer, G.; Schleif, W. A.; Gabryelski, L. J.; Leonard, Y. M.; Lynch, J. J., Jr.;
Michelson, S. R.; Young, S. D. J. Med. Chem. 2003, 46, 453.
24. Cyclooxygenase inhibition assays: The ability of the test compounds listed in
Table 1 to inhibit ovine COX-1 and human recombinant COX-2 (IC50 value, lM)
were determined using an enzyme immuno assay (EIA) kit (Catalog No.
560131, Cayman Chemical, Ann Arbor, MI, USA) according to our previously
reported method.28
28. Rao, P. N. P.; Amini, M.; Li, H.; Habeeb, A.; Knaus, E. E. J. Med. Chem. 2003, 46,
4872.
25. 5-Lipoxygenase inhibition assay: The ability of the test compounds listed in
Table 1 to inhibit potato 5-LOX (Catalog No. 60401, Cayman Chemical, Ann
29. Chowdhury, M. A.; Abdellatif, K. R. A.; Dong, Y.; Das, D.; Suresh, M. R.; Knaus, E.
E. Bioorg. Med. Chem. Lett. 2008, 18, 6138.