Phenylacetic acid regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore: Evaluation as dual inhibitors of cyclooxygenases and 5-lipoxygenase with anti-inflammatory activity

Article abstract of DOI:10.1016/j.bmcl.2009.12.073

A novel class of phenylacetic acid regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore attached to its C-2, C-3 or C-4 position was designed for evaluation as anti-inflammatory (AI) agents. A number of compounds exhibited a com

Full text of DOI:10.1016/j.bmcl.2009.12.073

Bioorganic & Medicinal Chemistry Letters 20 (2010) 896–902
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phenylacetic acid regioisomers possessing a N-difluoromethyl-1,2-dihydropy-
rid-2-one pharmacophore: Evaluation as dual inhibitors of cyclooxygenases
and 5-lipoxygenase with anti-inflammatory activity
Gang Yu a,b, Morshed A. Chowdhury a, Khaled R. A. Abdellatif a, Ying Dong a, P. N. Praveen Rao c,
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, Alta, 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
Article history:
A novel class of phenylacetic acid regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one
pharmacophore attached to its C-2, C-3 or C-4 position was designed for evaluation as anti-inflammatory
(AI) agents. A number of compounds exhibited a combination of potent in vitro cyclooxygenase-2 (COX-2)
and 5-lipoxygenase (5-LOX) inhibitory activities. 2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-4-
yl)phenylacetic acid (9a) exerted the most potent AI activity among this group of compounds. Molecular
modeling studies showed that the N-difluoromethyl-1,2-dihydropyridin-2-one moiety present in 9a inserts
into the secondary pocket present in COX-2 to confer COX-2 selectivity, and that the N-difluoromethyl-1,2-
dihydropyrid-2-one group (9a) binds close to the region of the 15-LOX enzyme containing catalytic iron
(His361, His366). Accordingly, the N-difluoromethyl-1,2-dihyrdopyrid-2-one moiety possesses properties
that make it an attractive pharmacophore suitable for the design of dual COX-2/5-LOX inhibitory AI drugs.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 26 November 2009
Revised 16 December 2009
Accepted 17 December 2009
Available online 23 December 2009
Keywords:
NSAIDs
Phenylacetic acids
N-Difluoromethyl-1,2-dihydropyrid-2-one
pharmacophore
Cyclooxygenase isozyme and 5-
lipoxygenase inhibition
Molecular modeling
Anti-inflammatory activity
The design of agents to relieve pain and inflammation associ-
ated with inflammatory diseases has undergone continual evolu-
tion targeted toward the development of more efficacious classes
of drugs that exhibit fewer adverse side effects. In this regard, non-
steroidal anti-inflammatory drugs (NSAIDs) belonging to the
aryl(heteroaryl)acetic acid class became invaluable agents for the
treatment of arthritis and osteoarthritis. Inhibition of the cycloox-
ygenase (COX) enzyme, that catalyzes the biotransformation of
arachidonic acid (AA) to inflammatory prostaglandins and throm-
boxanes in the COX pathway, became a hallmark feature of virtu-
ally all marketed NSAIDs. However, NSAIDs such as indomethacin
(1), and many arylacetic acids, frequently cause mechanism based
side effects that include gastrointestinal bleeding and/or ulceration
(see structure in Fig. 1).1 It was subsequently discovered that there
are two isoforms of COX, each with a distinct physiological role.2,3
The COX-1 isozyme is constitutively produced in a variety of tis-
sues, and appears to be important to the maintenance of normal
physiological functions including gastric cytoprotection. A second
inducible isozyme, COX-2 is largely responsible for the production
of prostaglandins that cause inflammation.4,5 Accordingly, it was
proposed that a selective COX-2 inhibitor would have useful
anti-inflammatory (AI) activity without the ulcerogenic side effects
associated with the use of NSAIDs that inhibit both COX-1 and
COX-2.6 This concept was validated with the clinical introduction
of the selective COX-2 inhibitor celecoxib (2, CelebrexÒ) that is de-
void of adverse ulcerogenic effects.7 Alternatively, AA is also
metabolized by the lipoxygenase (5-LOX, 8-, 12-, 15-) enzyme fam-
ily to produce proinflammatory leukotrienes (LTs) that are associ-
ated with the production of inflammatory, bronchoconstrictor,
hypersensitivity, anaphylactic, and asthmatic actions.8
There is credence for the concept that a dual inhibitor of the
LOX/COX enzymatic pathways9 constitutes a logical approach for
the design of more efficacious AI agents with a superior safety pro-
file relative to non-selective COX inhibitory NSAIDs, and selective
COX-2 inhibitors that increase the incidence of adverse thrombotic
events.10,11 This supposition is based on the premise that inhibiting
only one of the COX/LOX pathways may shift the metabolism of AA
to the other uninhibited pathway that may culminate in undesir-
able side effects.12 In a recent study, the tolyl ring in celecoxib
(2) was replaced by a N-difluoromethyl-1,2-dihydropyrid-2-one
5-LOX pharmacophore13 to provide a novel class of dual COX/5-
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.073

G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 896–902
897
MeO
CH2CO2H
CF3
CF3
N
N
N
C
Me
N
N
O
N
F2CH
Me
O
SO2R1
3, R1 = NH2, Me
SO2NH2
Celecoxib (2)
Cl
Indomethacin (1)
Figure 1. Some representative examples of a highly ulcerogenic non-selective COX-1/COX-2 inhibitor indomethacin (1), the selective COX-2 inhibitor celecoxib (2), and dual
COX/5-LOX inhibitors having a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore (3).
LOX inhibitors (3) that exhibited effective AI activity.14 It was
anticipated that attachment of this N-difluoromethyl-1,2-dihydro-
pyrid-2-one 5-LOX pharmacophore to a classical arylacetic acid
NSAID template would furnish a hitherto unknown group of dual
5-LOX/COX inhibitory AI agents. Accordingly, we now describe
the synthesis of a novel class of arylacetic acid regioisomers having
a N-difluoromethyl-1,2-dihydropyrid-2-one moiety (9a–f), their
in vitro evaluation as COX-1/COX-2, 5-LOX inhibitors, some molec-
ular modeling studies, and their in vivo assessment as AI agents.
The methyl 2-, 3- and 4-iodophenylacetates (5a–c) were pre-
pared from the corresponding acid (4a–c) using a previously re-
ported procedure.15 Subsequent treatment of the acetates (5a–c)
with lithium diisopropylamide (LDA), followed by alkylation using
methyl iodide at À78 °C,16 afforded the respective methyl 2-(2-, 3-,
CONCHF2 fragment of the N-difluoromethyl-1,2-dihydropyrid-2-
one ring present in 9a–f can be viewed as a cyclic hydroxamic acid
mimetic. These N-difluoromethyl-1,2-dihydropyrid-2-ones 9a–f
could inhibit the 5-LOX enzyme by two possible mechanisms. In
this regard 9a–f, like acyclic hydroxamic acids, may act as effective
iron chelators to exhibit 5-LOX inhibitory activity. There is a sub-
stantial build-up of negative potential around the two fluorine
atoms of a CHF2 group.23 Even with this high electron-density, an
aliphatic fluorine rarely acts as a hydrogen-bond acceptor, most
likely due to its high electronegativity and low polarizability.24,25
Consequently, it is also possible 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.26
and 4-iodophenyl)propanoates (5d–f).
A
Suzuki–Miyaura17,18
In vitro COX-1 and COX-2 enzyme inhibition studies (see data in
Table 1) showed that the N-difluoromethyl-1,2-dihydropyrid-2-
one regioisomers 9a–f exhibited a more potent inhibition, and
hence selectively, for the COX-2 isozyme (COX-1 IC50 = 5.5 to
cross-coupling reaction was used to synthesize the biaryl
compounds 7a–f. Thus, reaction of an aryl iodide 5a–f with 2-chlo-
ropyridine-4-boronic acid (6) in the presence of tetrakis(triphenyl-
phosphine)palladium (0) catalyst and 2 M aqueous Na2CO3 in THF
afforded the respective diaryl product 7a–f in moderate yield
(45–55%).19 Transformation of the 2-chloropyridyl compounds
(7a–c or 7d–f) to the target methyl 2-, 3-, or 4-(1-difluoro-
methyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetates (8a–c) and
methyl 2-[2-, 3-, and 4-(1-difluoromethyl-2-oxo-1,2-dihydropyri-
din-4-yl)phenyl]propanoates (8d–f) were carried out using the
synthetic strategies shown in Scheme 1. In this regard, reaction
of a 2-chloropyridyl compound (7a–f) with 2,2-difluoro-2-(fluor-
osulfonyl)acetic acid (FSO2CF2COOH)20 in the presence of NaHCO3
in CH3CN gave the respective 1-difluoromethyl-2-oxo-1,2-dihydro-
pyridine product 8a–f in moderate to good yields (40–83%).
Hydrolysis of the 2-, 3-, and 4-(N-difluoromethyl-2-oxo-1,2-dihy-
dropyridin-4-yl) esters 8a–f using aqueous 2 N NaOH at
80–85 °C21 afforded the target 2-, 3-, and 4-(1-difluoromethyl-2-
oxo-1,2-dihydropyridin-4-yl)phenylacetic acids (9a–c) and 2-[2-,
3-, and 4-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phe-
nyl]propanoic acids (9d–f) in moderate to excellent yield
(51–90%).
Non-steroidal AI arylacetic acid derivatives possess several
common structural features that include a carboxyl group sepa-
rated by one-carbon atom from a flat aromatic nucleus, and one
or more large lipophilic groups attached to the aromatic nucleus
that is two, three or four carbon atoms removed from the point
of attachment of the acetic acid side chain.22 The rational for the
design of the 2-, 3-, and 4-(1-difluoromethyl-2-oxo-1,2-dihydro-
pyridin-4-yl)phenylacetic acids (9a–c) and 2-[2-, 3-, and 4-(1-
difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoic
acids (9d–f) was based on the expectation that attachment of a
lipophilic N-difluoromethyl-2-oxo-1,2-dihydropyridyl moiety to a
phenylacetic acid template may provide a hitherto unknown class
of compounds with dual COX-2/5-LOX inhibitory activities. The
>100 lM range; COX-2 IC50 = 0.86–11.1 lM range). The point of
attachment of the N-difluoromethyl-1,2-dihydropyrid-2-one ring
system to the phenylacetic acid moiety was not, with the exception
of 9b, a determinant of COX-2 inhibitory activity since the poten-
cies of 9a, 9c and 9d–f were similar to each other and to that of
the reference drug ibuprofen (IC50 = 1.1 lM). The COX-2 inhibitory
activity of the phenylacetic acids 9a and 9c (R1 = H) was marginally
more potent than that of the phenylpropionic acids 9d–f (R1 = Me).
In vitro 5-LOX inhibition studies showed that attachment of the
N-difluoromethyl-1,2-dhydropyrid-2-one ring system to a phenyl-
acetic acid moiety confers 5-LOX inhibitory activity that is absent
in traditional NSAIDs. This group of compounds 9a–f exhibited
more potent 5-LOX inhibitory activity (IC50 = 0.20–3.47
lM range)
than the reference drug caffeic acid (IC50 = 4.0 M). The point of
l
attachment of the N-difluoromethyl-1,2-dhydropyrid-2-one
moiety was a determinant of activity where the C-2 and C-3 regioi-
somers were more potent than the corresponding C-4 regioisomer
(9a–b > 9c; 9d–e > 9f). The R1 substituent was a determinant of
5-LOX inhibitory potency for the C-2 regioisomers (9a, R1 = H,
IC50 = 0.20 l lM), but not the C-3 and
M; 9d, R1 = Me, IC50 = 2.0
C-4 regioisomers (9b–c, R1 = H equipotent to 9e–f, R1 = Me).
Some physicochemical comparisons indicate that the phenyla-
cetic acids 9a–c (R1 = H) are less lipophilic (calculated
Log P = 1.07–1.37 range) and smaller in size (calculated volume
of 228 Å3) than the corresponding phenylpropionic acids 9d–f
(R1 = Me; calculated Log P = 1.38–1.68 range; calculated volume
of 244 Å3; see data in Table 1).
Molecular modeling (docking) studies were carried out to inves-
tigate binding interactions between a phenylacetic acid derivative
possessing the novel N-difluoromethyl-1,2-dihydropyrid-2-one
pharmacophore and the mammalian COX-2 and 15-LOX enzymes.
In this regard, the binding interaction of the most potent 2-(1-

898
G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 896–902
R1
2
R1
CO2Me
+
CO2H
CO2Me
B(OH)2
Cl
a
c
N
I
I
3
N
6
Cl
4
4a-c
7
5
7a: 2-(2-chloropyridin-4-yl), R1 = H
7b: 3-(2-chloropyridin-4-yl), R1 = H
7c: 4-(2-chloropyridin-4-yl), R1 = H
7d: 2-(2-chloropyridin-4-yl), R1 = Me
7e: 3-(2-chloropyridin-4-yl), R1 = Me
7f: 4-(2-chloropyridin-4-yl), R1 = Me
4a: 2-I
4b: 3-I
4c: 4-I
5a: 2-I, R1 = H
5d: 2-I, R1 = Me
5b: 3-I, R1 = H
5e: 3-I, R1 = Me
5c: 4-I, R1 = H
5f: 4-I, R1 = Me
b
b
b
R1
2
CO2H
R1
CO2Me
O
N
O
N
d
e
2
3
F2HC
F2HC
3
4
4
8
9
8a: 2-isomer, R1 = H
8b: 3-isomer, R1 = H
8c: 4-isomer, R1 = H
8d: 2-isomer, R1 = Me
8e: 3-isomer, R1 = Me
8f: 4-isomer, R1 = Me
9a: 2-isomer, R1 = H
9b: 3-isomer, R1 = H
9c: 4-isomer, R1 = H
9d: 2-isomer, R1 = Me
9e: 3-isomer, R1 = Me
9f: 4-isomer, R1 = Me
Scheme 1. Reagents and conditions: (a) MeOH, concd H2SO4, reflux, 3 h; (b) LDA, MeI, THF, À78 °C for 30 min, and then 0 °C for 1 h; (c) Pd(PPh3)4, 2 M Na2CO3, THF, reflux,
16 h; (d) FSO2CF2COOH, NaHCO3, reflux, 12 h; (e) aqueous 2 N NaOH, 80–85 °C, 4 h.
Table 1
In vitro COX-1, COX-2 and 5-LOX enzyme inhibition data for 2-, 3-, or 4-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetic acids (9a–c) and 2-[2-, 3-, or
4-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoic acids (9d–f)
R1
2
CO2H
O
N
F2HC
3
4
9: a,d = 2-isomer; b,e = 3-isomer; c,f = 4-isomer
R1
COX-1 IC50
(
lM)
COX-2 IC50
(l
M)
5-LOX IC50 (lM)
Log Pc
Volumed (Å3)
a
a
b
Compound
9a
9b
9c
9d
9e
9f
H
H
H
Me
Me
Me
5.5
>100
9.6
>100
13.9
15.3
2.9
0.86
11.1
0.89
2.6
0.20
0.37
3.47
2.0
0.37
3.0
1.07
1.37
1.37
1.38
1.68
1.68
3.68
228.2
228.9
228.9
244.9
244.8
244.7
211.8
1.2
2.4
Ibuprofen
1.1e
—
—
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
,
lM) is the mean of two
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.
The volume of the molecule, after minimization using the MM3 force field, was calculated using the Alchemy 2000 program, Tripos Inc.
Data acquired using ovine COX-2 (Catalog No. 560101, Cayman Chemicals Inc.).
Caffeic acid: 3,4-dihydroxycinnamic acid.
d
e
f
difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetic acid
(9a, COX-2 IC50 = 0.86 M) within the COX-2 binding site is illus-
trated in Figure 2. The most stable enzymeÀligand complex shows
that the N-difluoromethyl-1,2-dihydropyrid-2-one group is buried
deep inside the secondary pocket of COX-2 suggesting that the
N-difluoromethyl-1,2-dihydropyrid-2-one moiety is a potential
l

G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 896–902
899
Figure 2. Docking 2-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetic acid (9a) (ball-and-stick) in the active site of mammalian COX-2
(Eintermolecular = À22.46 kcal/mol). Red dotted line represents hydrogen bonding. Hydrogen atoms are not shown for clarity.
COX-2 pharmacophore. The 1,2-dihydropyrid-2-one ring is sur-
rounded by His90, Gln192, Arg513, Phe518 and Val523 (distance
<5 Å). The C@O (dihydropyrid-2-one) undergoes a favorable hydro-
gen bonding interaction with the NH of His90 (distance = 2.17 Å).
The NCHF2 (part of 1,2-dihydropyrid-2-one ring) is positioned in
the vicinity of polar amino acids such as His90, Gln192 and
Arg513 (distance <4.50 Å). The fluorine atoms (CHF2) are located
about 1.99 Å and 3.83 Å from the NH of His90, and the guanidino
NH2 of Arg513, respectively. This data confirms the preferential
interaction of the highly electronegative CHF2 substituent with a
polar electropositive environment. The phenylacetic acid
(PhCH2COOH) ring is oriented in the vicinity of Trp387, Tyr385,
Ser530, Leu359, Leu352, Ile345 and Val344 (distance <5 Å). Fur-
thermore, the distance between C@O of CH2COOH and OH of
Tyr385 at the apex of the COX-2 binding site is about 4.59 Å. This
observation is consistent with crystal structure data for the NSAID
diclofenac [ortho-(2,6-dichloroanilino)phenylacetic acid] bound to
the COX-2 enzyme where the carboxylate is oriented in the region
of Tyr385 and Ser530.27
The mammalian lipoxygenase isozymes (5, 12 and 15-LOX) are
closely related since they share 35–80% sequence identity. X-ray
data for mammalian 15-LOX has been reported,28 but is not avail-
able for the 5-LOX isozyme. Therefore, 15-LOX x-ray data was used
as a model to investigate the binding interactions of 2-(1-difluoro-
methyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetic acid (9a,
5-LOX IC50 = 0.20 lM) within a LOX binding site (see Fig. 3). This
docking study indicated that 9a is oriented such that the
phenylacetic acid (PhCH2COOH) moiety is oriented toward the
hydrophobic base of the 15-LOX active site in the vicinity of
Ile414, Phe415, Met419 and Ile593 (distance <5 Å). The CH2COOH
substituent appears in a region comprised of Gln548, Val594 and
Leu597 (distance <5 Å). A favorable hydrogen bonding interaction
was observed between the C@O (CH2COOH) and NH2 of Gln548
(distance = 1.98 Å). The N-difluoromethyl-1,2-dihydropyrid-2-one
LOX pharmacophore is oriented close to the region containing
the catalytic iron (His361 and His366). Furthermore, the distance
between the fluorine atoms (CHF2) and NHs of His361 and
His366 is about 4.61 Å and 4.67 Å, respectively. In addition, the
C@O (dihydropyrid-2-one) is located about 3.85 Å from the C@O
of Glu357. The distance between the CHF2 and NH2 (guanidine)
group of Arg403 that is closer to the mouth of the 15-LOX binding
site is about 10.78 Å. This molecular modeling data provides cre-
dence for our hypothesis that the CONCHF2 fragment present in
the N-difluoromethyl-1,2-dihydropyrid-2-one ring can be viewed
as a cyclic hydroxamic acid mimetic.
The acetic acid (9a, 9b), and propionic acid (9e) compounds,
based on in vitro enzyme inhibition data, were selected for
in vivo pharmacological evaluation to determine their anti-inflam-
matory (AI) activities. In a carrageenan-induced rat paw edema as-
say model, the acetic acid 2-regioisomer 9a exhibited weak AI
activity (19.9 0.9% inhibition) for a 150 mg/kg oral (po) dose at
3 h post-drug administration relative to the non-selective COX-1/
COX-2 inhibitor (ibuprofen, ED50 = 67.4 mg/kg po dose), 5-LOX
inhibitor (caffeic acid, 8.2% inhibition for a 30 mg/kg po dose),
and the 15-LOX inhibitor (nordihydroguaiaretic acid, NDGA,
15.1% inhibition for a 30 mg/kg po dose, ED50 = 205 mg/kg) refer-

900
G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 896–902
Figure 3. Docking 2-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetic acid (9a) (ball-and-stick) in the active site of mammalian 15-LOX
(Eintermolecular = À46.26 kcal/mol). Red dotted line represents hydrogen bonding. Hydrogen atoms are not shown for clarity.
ence drugs. The related acetic acid 3-regioisomer 9b was also a
weak AI agent (11.2 4.2% inhibition for a 100 mg/kg po dose). In
contrast, the propionic acid 3-regioisomer 9e was an inactive AI
agent at a 100 mg/kg po dose. The route of administration was
not a determinant of AI activity for 9a since a 150 mg/kg intraperi-
toneal (ip) dose provided similar AI activity (18.5 3.9% inhibition)
to that determined for the same oral dose. The in vivo AI potency
exhibited by compounds 9a, 9b and 9e was weaker than expected
based on their excellent in vitro inhibition of the COX-2 and/or 5-
LOX enzymes. One possible explanation is that the acetic acid moi-
ety in 9a is oriented toward the apex of the COX binding site as
indicated in the molecular modeling study (see Fig. 2) in compar-
ison to traditional arylacetic NSAIDs where the carboxyl group
generally interacts with Arg120 at the mouth of the COX binding
site.
coxib (2) to confer selectivity for the COX-2 isozyme, and that
the N-difluoromethyl-1,2-dihydropyrid-2-one group (9a) is ori-
ented close to the region of the 15-LOX enzyme containing the
catalytic iron (His361, His366). Accordingly, the N-difluoro-
methyl-1,2-dihyrdopyrid-2-one moiety possesses properties suit-
able for the design of dual COX-2/5-LOX inhibitory AI drugs.
Acknowledgments
We are grateful to the Canadian Institutes of Health Research
(CIHR) (MOP-14712) for financial support of this research. G.Y.
was a recipient of a Jiangsu Government Abroad Study Scholarship,
China.
References and notes
In conclusion, a new class of 2-, 3-, and 4-(1-difluoromethyl-2-
oxo-1,2-dihydropyridin-4-yl)phenylacetic acids (9a–c) and 2-[2-,
3-, and 4-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phe-
nyl]propanoic acids (9d–f) was synthesized29 for evaluation as
dual 5-LOX30 and COX-1/COX-231 isozyme inhibitors of inflamma-
tion. Biological data acquired indicate that compounds 9a–b and
9e exhibit the best combination of in vitro COX-2 and 5-LOX inhib-
itory potency. Among this group of compounds 9a–f, the C-2 phen-
ylacetic acid regioisomer (9a) exhibited the most potent AI
activity.32 Molecular modeling (docking) studies33 showed that
the N-difluoromethyl-1,2-dihydropyrid-2-one moiety present in
9a inserts into the secondary pocket present in COX-2 similar to
the sulfonamide (SO2NH2) COX-2 pharmacophore present in cele-
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7. Penning, T.; Talley, J.; Bertenshaw, S.; Carter, J.; Collins, P.; Docter, S.; Graneto,
M.; Lee, L.; Malecha, J.; Miyashiro, J.; Rogers, R.; Rogier, D.; Yu, S.; Anderson, G.;
Burton, E.; Cogburn, J.; Gregory, S.; Koboldt, C.; Perkins, W.; Seibert, K.;
Veenhuizen, A.; Zhang, Y.; Isakson, P. A. J. Med. Chem. 1997, 40, 1347.
8. Moreau, A.; Praveen Rao, P. N.; Knaus, E. E. Bioorg. Med. Chem. 2006, 14, 5340.
and references cited therein.
9. Praveen Rao, P. N.; Chen, Q.-H.; Knaus, E. E. J. Med. Chem. 2006, 49, 1668. and
references cited therein.

G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 896–902
901
10. Scheen, A. J. Rev. Med. Liege 2004, 59, 565–569.
Methyl 2-(2-iodophenyl)propanoate (5d): Yield, 72%; 1H NMR (CDCl3) d 1.49 (d,
J = 6.7 Hz, 3H, CHMe), 3.69 (s, 3H, OMe), 4.12 (q, J = 6.7 Hz, 1H, CHMe), 6.92–
6.98 (m, 1H, H-4), 7.26–7.35 (m, 2H, H-5, H-6), 7.85 (d, J = 7.9 Hz, H-3).
Methyl 2-(3-iodophenyl)propanoate (5e): Yield, 49%; 1H NMR (CDCl3) d 1.49 (d,
J = 6.7 Hz, 3H, CHMe), 3.68 (s, 3H, OMe), 4.12 (q, J = 6.7 Hz, 1H, CHMe), 7.07 (dd,
J = 7.9, 7.9 Hz, 1H, H-5), 7.29 (d, J = 7.9, Hz, 1H, H-6), 7.61 (d, J = 7.9 Hz, 1H, H-
4), 7.66 (d, J = 1.8 Hz, 1H, H-2).
11. Dogné, J.-M.; Supuran, C. T.; Pratico, D. J. Med. Chem. 2005, 48, 2251–2257.
12. Asako, H.; Kubes, P.; Wallace, J.; Gaginella, T.; Wolf, R. E.; Granger, N. Am. J.
Physiol. Gastrointest. Liver Physiol. 1992, 262, G903.
13. Chowdhury, M. A.; Abdellatif, K. R.; Dong, Y.; Rahman, M.; Das, D.; Suresh, M.
R.; Knaus, E. E. Bioorg. Med. Chem. Lett. 2009, 19, 584.
14. Chowdhury, M. A.; Abdellatif, K. R.; Dong, Y.; Das, D.; Suresh, M. R.; Knaus, E. E.
J. Med. Chem. 2009, 52, 1525.
15. Marchal, E.; Uriac, P.; Legouin, B.; Toupet, L.; Weghe, P. Tetrahedron 2007, 63,
9979.
16. Vasudevan, J.; Wang L. M.; Liu, X. X.; Tsang, K. Y.; Li, L.; Takeuchi, J. A.; Vu, T.;
Beard, R. L.; Bhat, S.; Vuligonda, V.; Chandraratna, R. A. PCT Int. Appl.
20050187298, issued June 30, 2005.
17. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
18. Stanforth, S. P. Tetrahedron 1998, 54, 263–303.
19. Chen, Q. H.; Rao, P. N. P.; Knaus, E. E. Bioorg. Med. Chem. 2005, 13, 2459.
20. Ando, M.; Wada, T.; Sato, N. Org. Lett. 2006, 8, 3805.
21. Sonawance, H. R.; Nanjundiah, B. S.; Kulkarni, D. G.; Ahuja, J. R. Tetrahedron
1988, 23, 7319.
22. Hamor, G. H. In Principles of Medicinal Chemistry; Foye, W. O., Ed., 2nd ed.; Lea &
Febiger Publishing C.: Philadelphia, 1981. Chapter 22.
23. Narjes, F.; Koehler, K. F.; Koch, U.; Gerlach, B.; Colarusso, S.; Steinkühler, C.;
Brunetti, M.; Altamura, S.; De Francesco, R.; Matassa, V. G. Bioorg. Med. Chem.
Lett. 2002, 12, 701. and references cited therein.
Methyl 2-(4-iodophenyl)propanoate (5f): Yield, 98%; 1H NMR (CDCl3) d 1.48 (d,
J = 6.7 Hz, 3H, CHMe), 3.66 (s, 3H, OMe), 4.06 (q, J = 6.7 Hz, 1H, CHMe), 7.03 (dd,
J = 7.9, 1.8 Hz, 2H, H-2, H-6), 7.65 (dd, J = 7.9, 1.8 Hz, 2H, H-3, H-5).
General procedure for the synthesis of methyl 2-, 3-, or 4-(2-chloropyridin-4-
yl)phenylacetates (7a–c) and methyl 2-[2-, 3-, or 4-(2-chloropyridin-4-
yl)phenyl]propanoates (7d–f): A methyl 2-, 3-, or 4-iodophenylacetate (5a–c)
(828 mg, 3.0 mmol), or a methyl 2-(2-, 3-, or 4-iodophenyl)propanoate (5d–f)
(870 mg, 3.0 mmol), and 2-chloropyridine-4-boronic acid (306 mg, 3.9 mmol)
were dissolved in THF (40 mL). Aqueous 2 M Na2CO3 (4.5 mL) and then
Pd(PPh3)4 (70 mg, 0.06 mmol) were added. The reaction was allowed to
proceed at reflux under argon for 16 h, cooled to 25 °C, water (150 mL) was
added, the mixture was acidified to pH 3 using 5% w/v HCl, and the mixture
was extracted with EtOAc (3 Â 100 mL). The combined EtOAc extracts were
washed with water (3 Â 50 mL), the organic fraction was dried (Na2SO4), and
the solvent was removed in vacuo. The dark brown residue obtained was
purified by silica gel column chromatography using hexanes/EtOAc (3:1, v/v)
as eluent to afford the respective product 7a–f. Spectroscopic data for 7a–f are
listed below.
24. Dunitz, J. D.; Taylor, R. Chem. A Eur. J. 1997, 3, 89.
25. Howard, J. A. K.; Hoy, V. J.; O’Hagan, D.; Smith, G. T. Tetrahedron 1996, 52,
12613.
Methyl 2-(2-chloropyridin-4-yl)phenylacetate (7a): Yield, 50%; pale yellow solid,
mp 63–64 °C; IR (film): 2919, 1735, 1589 cmÀ1 1H NMR (CDCl3) d 3.58 (s, 3H,
;
26. Chavatte, P.; Yous, S.; Marot, C.; Baurin, N.; Lesieur, D. J. Med. Chem. 2001, 44,
OMe), 3.67 (s, 2H, CH2), 7.22–7.25 (m, 2H, phenyl H-5, H-6), 7.33 (s, 1H, pyridyl
H-3), 7.35–7.40 (m, 3H, phenyl H-3, H-4 and pyridyl H-5), 8.44 (d, J = 4.7 Hz,
pyridyl H-6); 13C NMR (CDCl3) d 38.5, 52.2, 123.0, 124.7, 127.6, 129.1, 129.5,
130.8, 131.3, 138.4, 149.4, 151.6, 152.1, 171.6.
3223.
27. 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.
28. Gillmor, S. A.; Villasenor, A.; Fletterick, R.; Sigal, E.; Browner, M. F. Nat. Struct.
Biol. 1997, 4, 1003.
29. 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
Methyl 3-(2-chloropyridin-4-yl)phenylacetate (7b): Yield, 52%; yellow oil; IR
(film): 2920, 1736, 1589 cmÀ1 1H NMR (CDCl3) d 3.72 (s, 3H, OMe), 3.73 (s, 2H,
;
CH2), 7.39–7.55 (overlapping multiplets, 6H total, pyridyl H-3, H-5, phenyl H-2,
H-4, H-5, H-6), 8.44 (d, J = 5.5 Hz, pyridyl H-6); 13C NMR (CDCl3) d 41.0, 52.2,
120.5, 122.1, 125.8, 128.0 129.5, 130.6, 135.1, 137.2, 150.0, 151.2, 152.2, 171.5.
Methyl 4-(2-chloropyridin-4-yl)phenylacetate (7c): Yield, 54%; pale yellow solid,
mp 92–94 °C; IR (film): 2918, 1734, 1590 cmÀ1 1H NMR (CDCl3) d 3.71 (s, 3H,
;
OMe), 3.72 (s, 2H, CH2), 7.41 (dd, J = 4.9, 1.8 Hz, 1H, pyridyl H-5), 7.43 (dd,
J = 6.7, 1.8 Hz, 2H, phenyl H-2, H-6), 7.54 (s, 1H, pyridyl H-3), 7.59 (dd,
J = 6.7 Hz, 1.8 Hz, 2H, phenyl H-3, H-5), 8.43 (d, J = 4.9 Hz, pyridyl H-6); 13C
NMR (CDCl3) d 40.8, 52.2, 120.2, 121.9, 127.2, 130.2, 135.6, 135.7, 149.9, 151.1,
152.2, 171.5.
the internal standard. Microanalyses were performed for C, H,
N (Micro
Analytical Service Laboratory, Department of Chemistry, University of Alberta)
and were within 0.4% of theoretical values. Compounds 5a–f, 7a–f, and 8a–f
showed a single spot on Macherey-Nagel Polygram Sil G/UV254 silica gel
plates (0.2 mm) using a low, medium, and highly polar solvent system, and no
residue remained after combustion, indicative of high purity. Silica gel column
chromatography was performed using Merck Silica Gel 60 ASTM (70–
230 mesh). All reagents, purchased from the Aldrich Chemical Company
(Milwaukee, WI), with the exception of 2-chloropyridine-4-boronic acid
which was purchased from Combi-Blocks Inc., were used without further
purification. The in vivo anti-inflammatory assay was carried out using a
protocol approved by the Health Sciences Animal Welfare Committee at the
University of Alberta.
Methyl 2-[2-(2-chloropyridin-4-yl)phenyl]propanoate (7d): Yield, 50%; pale
yellow solid, mp 63–64 °C; IR (film): 2919, 1736, 1086 cmÀ1
;
1H NMR
(CDCl3) d 1.43 (d, J = 6.7 Hz, 3H, CHMe), 3.68 (s, 3H, OMe), 3.78 (q, J = 6.7 Hz,
1H, CHMe), 7.20–7.51 (overlapping multiplets, 6H total, pyridyl H-3, H-5,
phenyl H-3, H-4, H-5, H-6), 8.48 (d, J = 4.9 Hz, pyridyl H-6); 13C NMR (CDCl3) d
19.1, 41.0, 52.2, 123.3, 124.9, 127.2, 127.3, 129.3, 129.4, 137.5, 138.1, 149.4,
151.6, 152.2, 174.5.
Methyl 2-[3-(2-chloropyridin-4-yl)phenyl]propanoate (7e): Yield, 45%; yellow
oil; IR (film): 2945, 1736, 1088 cmÀ1 1H NMR (CDCl3) d 1.56 (d, J = 6.7 Hz, 3H,
;
General procedure for the synthesis of methyl 2-, 3-, and 4-iodophenylacetates
(5a–c): Concentrated sulfuric acid (97–98%, 1.10 mL) was added to a solution
of an iodophenylacetic acid (4a–c, 2.0 g, 7.6 mmol) in MeOH (5 mL), and the
reaction mixture was heated at reflux for 3 h. After cooling to 25 °C, the MeOH
was removed in vacuo, H2O (80 mL) was added to the residue, and this mixture
was extracted with CH2Cl2 (2 Â 50 mL). The combined organic extracts were
washed with saturated aqueous NaHCO3, water and then brine. The organic
fraction was dried (Na2SO4) and the solvent was removed in vacuo to furnish
the respective methyl ester product (5a–c) as a pale yellow oil that was
sufficiently pure for use in subsequent reactions. 1H NMR spectral data for 5a–c
are listed below.
CHMe), 3.69 (s, 3H, OMe), 3.81 (q, J = 6.7 Hz, 1H, CHMe), 7.38–7.56 (overlapping
multiplets, 6H total, pyridyl H-3, H-5, phenyl H-2, H-4, H-5, H-6), 8.44 (d,
J = 4.9 Hz, pyridyl H-6); 13C NMR (CDCl3) d 18.7, 45.4, 52.2, 120.5, 122.1, 125.9,
126.2 128.8, 129.5, 137.3, 141.7, 150.0, 151.3, 152.2, 174.5.
Methyl 2-[4-(2-chloropyridin-4-yl)phenyl]propanoate (7f): Yield, 55%; yellow oil;
IR (film): 2918, 1734, 1170 cmÀ1 1H NMR (CDCl3) d 1.55 (d, J = 6.7 Hz, CHMe),
;
3.70 (s, 3H, OMe), 3.80 (q, J = 6.7 Hz, 1H, CHMe), 7.41 (dd, J = 4.9, 1.2 Hz, 1H,
pyridyl H-5), 7.43 (dd, J = 8.5, 1.8 Hz, 2H, phenyl H-2, H-6), 7.54 (d, J = 1.2 Hz,
1H, pyridyl H-3), 7.59 (dd, J = 8.5, 1.8 Hz, 2H, phenyl H-3, H-5), 8.43 (d,
J = 4.9 Hz, pyridyl H-6); 13C NMR (CDCl3) d 18.5, 45.1, 52.2, 120.3, 121.9, 127.3,
128.4, 135.7, 142.2, 150.0, 151.1, 152.2, 174.5.
Methyl 2-iodophenylacetate (5a): Yield, 95%; 1H NMR (CDCl3) d 3.76 (s, 3H,
OMe), 3.84 (s, 2H, CH2), 6.97–7.03 (m, 1H, H-4), 7.29–7.36 (m, 2H, H-5, H-6),
7.88 (d, J = 7.9 Hz, H-3).
General procedure for the synthesis of methyl 2-, 3-, or 4-(1-difluoromethyl-2-oxo-
1,2-dihydropyridin-4-yl)phenylacetates (8a–c) and methyl 2-[2-, 3-, or 4-(1-
difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoates (8d–f): To
a
Methyl 3-iodophenylacetate (5b): Yield, 99%; 1H NMR (CDCl3) d 3.60 (s, 3H,
OMe), 3.73 (s, 2H, CH2), 7.09 (dd, J = 7.9, 7.9 Hz, 1H, H-5), 7.28 (d, J = 7.9 Hz, 1H,
H-6), 7.64 (d, J = 7.9 Hz, 1H, H-4), 7.68 (s, 1H, H-2).
solution of a methyl 2-, 3-, or 4-(2-chloropyridin-4-yl)phenylacetate (7a–c)
(261 mg, 1.0 mmol), or methyl 2-[2-, 3-, or 4-(2-chloropyridin-4-
yl)phenyl]propanoate (7d–f) (275 mg, 1.0 mmol), in acetonitrile (15 mL) was
added FSO2CF2CO2H (310 mL, 3.0 mmol) followed by NaHCO3 (93 mg,
1.1 mmol). This mixture was then heated at reflux under argon for 12 h,
cooled to 25 °C, a saturated solution of aqueous NaHCO3 (25 mL) was added,
and the mixture was extracted with CH2Cl2 (3 Â 30 mL). The combined organic
extracts were successively washed with 10 N HCl (2 Â 20 mL) to remove
unreacted starting material, the organic fraction was washed with water
(50 mL) and brine, and the organic fraction was dried (Na2SO4). Removal of the
solvent from the organic fraction in vacuo afforded the respective title product
8a–f. The physical and spectral data for 8a–f are listed below.
Methyl 4-iodophenylacetate (5c): Yield, 98%; 1H NMR (CDCl3) d 3.57 (s, 3H,
OMe), 3.69 (s, 2H, CH2), 7.03 (d, J = 8.5 Hz, 2H, H-2, H-6), 7.65 (d, J = 8.5 Hz, 2H,
H-3, H-5).
General procedure for the synthesis of methyl 2-(2-, 3-, and 4-
iodophenyl)propanoates (5d–f): n-Butyllithium (1.52 M in hexane, 2.4 mL)
was added to a solution of diisopropylamine (0.83 mL, 6.0 mmol) in THF
(6 mL) at À78 °C under argon, and the mixture was stirred for 30 min. A
solution of a methyl iodophenylacetate (5a–c, 1.38 g, 5.0 mmol) in THF (10 mL)
was added dropwise, the mixture was stirred for 30 min, iodomethane
(0.60 mL, 9.6 mmol) was added dropwise, the reaction was allowed to
proceed with stirring at À78 °C for 30 min prior to raising the temperature
to 0 °C and quenching the reaction with saturated aqueous NH4Cl (30 mL). The
aqueous layer was washed with EtOAc (2 Â 50 mL), the combined organic
phases were washed with brine, and the organic fraction was dried (Na2SO4).
Removal of the solvent in vacuo afforded a residue that was purified by silica
gel column chromatography (EtOAc/hexanes, 1:16, v/v) to give the respective
product (5d–f) as a yellow oil. 1H NMR spectral data for 5d–f are listed below.
Methyl 2-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetate (8a):
Yield, 40%; pale yellow solid, mp 63–64 °C; IR (film): 1734, 1676, 1064 cmÀ1
;
1H NMR (CDCl3) d 3.70 (s, 3H, OMe), 3.72 (s, 2H, CH2), 6.58 (dd, J = 7.9, 1.8 Hz,
1H, pyridone H-5), 6.76 (s, 1H, pyridone H-3), 7.38–7.50 (m, 4H, phenyl H-3, H-
4, H-5, H-6), 7.49 (d, J = 7.9 Hz, 1H, pyridone H-6), 7.73 (t, J = 60.4 Hz, 1H,
CHF2); 13C NMR (CDCl3) d 41.0, 52.2, 107.1, 107.5 (t, J = 255 Hz), 117.6, 125.6,
127.7, 129.3 (t, J = 4 Hz), 129.4, 131.1, 135.0, 136.9, 153.0, 161.3, 171.5.
Methyl 3-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetate (8b):

902
G. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 896–902
Yield, 73%; pale yellow oil; IR (film): 1735, 1681, 1058 cmÀ1
;
1H NMR (CDCl3) d
(t, J = 60.4 Hz, 1H, CHF2), 10.63 (br s, 1H, COOH); 13C NMR (CDCl3) d 40.9, 107.0,
107.4 (t, J = 255 Hz), 117.1, 126.8, 129.1, 130.2, 135.0, 136.8, 152.9, 161.3,
173.3. Anal. Calcd for C14H11F2NO3: C, 60.22; H, 3.97; N, 5.02. Found: C, 60.20;
H, 4.01; N, 5.03.
3.65 (s, 3H, OMe), 3.67 (s, 2H, CH2), 6.40 (dd, J = 7.3, 1.8 Hz, 1H, pyridone H-5),
6.53 (s, 1H, pyridone H-3), 7.23 (d, J = 7.3 Hz, phenyl H-6), 7.33–7.43 (m, 1H,
phenyl H-5), 7.36 (s, 1H, phenyl H-2), 7.39 (d, J = 7.3 Hz, 1H, phenyl H-4), 7.50
(d, J = 7.3 Hz, 1H, pyridone H-6), 7.74 (t, J = 60.4 Hz, 1H, CHF2); 13C NMR (CDCl3)
d 38.3, 52.1, 107.4 (t, J = 255 Hz), 109.5, 120.6, 127.6, 128.5, 128.9 (t, J = 4 Hz),
129.3, 131.0, 138.2, 154.4, 160.7, 171.5, 173.5.
2-[2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoic
(9d): Yield, 51%; pale yellow solid, mp 147–149 °C; IR (film): 2981, 1735,
1662, 1067 cmÀ1 1H NMR (CDCl3) d 1.42 (d, J = 6.7 Hz, 3H, CHMe), 3.88 (q,
acid
;
Methyl 4-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetate (8c):
J = 7.3 Hz, 1H, CHMe), 6.46 (d, J = 6.7 Hz, pyridone H-5), 6.63 (s, 1H, pyridone H-
3), 7.20 (d, J = 7.3 Hz, 1H, phenyl H-6), 7.33 (ddd, J = 7.3, 7.3, 1.2 Hz, 1H, phenyl
H-4), 7.43 (ddd, J = 7.3, 7.3, 1.2 Hz, 1H, phenyl H-5), 7.48 (dd, J = 7.3, 1.2 Hz, 1H,
phenyl H-3), 7.51 (d, J = 7.3 Hz, 1H, pyridone H-6), 7.76 (t, J = 60.4 Hz, 1H,
CHF2), 11.2 (br s, 1H, COOH); 13C NMR (CDCl3) d 18.9, 41.1, 107.4 (t, J = 255 Hz),
110.1, 120.9, 127.4 (t, J = 4 Hz), 127.5, 128.5, 128.7, 129.6, 137.5, 137.6, 154.8,
161.1, 178.9. Anal. Calcd for C15H13F2NO3: C, 61.43; H, 4.47; N, 4.78. Found: C,
61.47; H, 4.55; N, 4.58.
Yield, 80%; pale yellow solid, mp 65–67 °C; IR (film): 1734, 1675, 1059 cmÀ1
;
1H NMR (CDCl3) d 3.70 (s, 3H, OMe), 3.73 (s, 2H, CH2), 6.58 (dd, J = 7.3, 1.8 Hz,
1H, pyridone H-5), 6.76 (d, J = 1.8 Hz, 1H, pyridone H-3), 7.40 (d, J = 7.9 Hz, 2H,
phenyl H-2, H-6), 7.52 (d, J = 7.9 Hz, 1H, pyridone H-6), 7.56 (d, J = 7.9 Hz, 2H,
phenyl H-3, H-5), 7.73 (t, J = 60.4 Hz, 1H, CHF2); 13C NMR (CDCl3) d 40.8, 52.2,
107.0, 107.5 (t, J = 255 Hz), 117.3, 127.0, 129.2, 130.3, 135.4, 136.3, 152.7,
161.2, 171.4.
Methyl 2-[2-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoate
2-[3-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoic
(9e): Yield, 63%; pale yellow solid, mp 133–134 °C; IR (film): 2929, 1724,
1672, 1067 cmÀ1 1H NMR (CDCl3) d 1.54 (d, J = 6.7 Hz, 3H, CHMe), 3.80 (q,
J = 6.7 Hz, 1H, CHMe), 6.58 (dd, J = 7.3, 1.2 Hz, pyridone H-5), 6.77 (s, 1H,
pyridone H-3), 7.43–7.53 (m, 4H, phenyl H-3, H-4, H-5, H-6), 7.51 (d, J = 7.3 Hz,
1H, pyridone H-6), 7.72 (t, J = 60.4 Hz, 1H, CHF2), 8.96 (br s, 1H, COOH); 13C
NMR (CDCl3) d 18.2, 45.3, 107.3, 107.4 (t, J = 255 Hz), 125.8, 126.1, 127.5, 129.2,
129.4 (t, J = 4 Hz), 129.6, 136.9, 141.0, 153.1, 161.4, 179.0. Anal. Calcd for
C15H13F2NO3: C, 61.43; H, 4.47; N, 4.78. Found: C, 61.59; H, 4.40; N, 4.67.
acid
(8d): Yield, 48%; pale yellow oil; IR (film): 1735, 1683, 1057 cmÀ1 1H NMR
;
(CDCl3) d 1.43 (d, J = 6.7 Hz, 3H, CHMe), 3.67 (s, 3H, OMe), 3.87 (q, J = 6.7 Hz, 1H,
CHMe), 6.38 (dd, J = 7.3, 1.8 Hz, 1H, pyridone H-5), 6.55 (s, 1H, pyridone H-3),
7.20 (d, J = 7.9 Hz, 1H, phenyl H-6), 7.31–7.45 (m, 3H, phenyl H-3, H-4, H-5),
7.51 (d, J = 7.3 Hz, 1H, pyridone H-6), 7.76 (t, J = 60.4 Hz, 1H, CHF2); 13C NMR
(CDCl3) d 19.3, 41.2, 52.2, 107.5 (t, J = 255 Hz), 109.7, 121.0, 127.3, 127.4, 128.6
(t, J = 4 Hz), 128.7, 129.6, 137.5, 137.9, 154.5, 160.8, 174.5.
;
Methyl 2-[3-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoate
(8e): Yield, 83%; pale yellow solid, mp 94–96 °C; IR (film): 1735, 1678,
2-[4-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoic
(9f): Yield, 70%; pale yellow solid, mp 106–108 °C; IR (film): 2909, 1734,
1675, 1064 cmÀ1 1H NMR (CDCl3) d 1.59 (d, J = 7.3 Hz, 3H, CHMe), 3.85 (q,
acid
1072 cmÀ1 1H NMR (CDCl3) d 1.55 (d, J = 6.7 Hz, 3H, CHMe), 3.69 (s, 3H, OMe),
;
3.80 (q, J = 6.7 Hz, 1H, CHMe), 6.58 (dd, J = 7.3 Hz, 1.8 Hz, 1H, pyridone H-5),
6.76 (s, 1H, pyridone H-3), 7.43 (dd, J = 7.9, 2.4 Hz, 1H, phenyl H-6), 7.45 (d,
J = 2.4 Hz, 1H, phenyl H-2), 7.47 (t, J = 2.4 Hz, 1H, phenyl H-5), 7.50 (dd, J = 7.9,
2.4 Hz, 1H, phenyl H-4), 7.52 (d, J = 7.3 Hz, 1H, pyridone H-6), 7.73 (t,
J = 60.4 Hz, 1H, CHF2); 13C NMR (CDCl3) d 18.6, 45.3, 52.2, 107.1, 107.5 (t,
J = 255 Hz), 117.6, 125.6, 126.0, 129.2, 129.3 (t, J = 4 Hz), 129.6, 137.0, 141.6,
153.0, 161.2, 174.5.
;
J = 7.3 Hz, 1H, CHMe), 6.61 (dd, J = 7.3, 1.8 Hz, pyridone H-5), 6.79 (s, 1H,
pyridone H-3), 7.48 (dd, J = 7.9 Hz, 1.8 Hz, 2H, phenyl H-2, H-6), 7.55 (d,
J = 7.3 Hz, 1H, pyridone H-6), 7.58 (dd, J = 7.9, 1.8 Hz, 2H, phenyl H-3, H-5), 7.76
(t, J = 60.4 Hz, 1H, CHF2), 10.10 (br s, 1H, COOH); 13C NMR (CDCl3) d 18.1, 45.1,
107.2, 107.5 (t, J = 255 Hz), 117.2, 127.1, 128.5, 129.3, 135.4, 142.4, 152.9,
161.5, 178.9. Anal. Calcd for C15H13F2NO3: C, 61.43; H, 4.47; N, 4.78. Found: C,
61.33; H, 4.60; N, 4.72.
Methyl 2-[4-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoate
(8f): Yield, 70%; pale yellow oil; IR (film): 1735, 1677, 1064 cmÀ1
;
1H NMR
30. 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
(CDCl3) d 1.54 (d, J = 6.7 Hz, 3H, CHMe), 3.69 (s, 3H, OMe), 3.79 (q, J = 6.7 Hz, 1H,
CHMe), 6.57 (dd, J = 7.3, 1.8 Hz, 1H, pyridone H-5), 6.74 (s, 1H, pyridone H-3),
7.42 (dd, J = 7.9, 1.8 Hz, 2H, phenyl H-2, H-6), 7.55 (dd, J = 7.9, 1.8 Hz, 2H,
phenyl H-3, H-5), 7.56 (d, J = 7.3 Hz, 1H, pyridone H-6), 7.76 (t, J = 60.4 Hz, 1H,
CHF2); 13C NMR (CDCl3) d 18.5, 45.2, 52.2, 106.9, 107.5 (t, J = 255 Hz), 117.3,
127.0, 128.3, 129.2 (t, J = 4 Hz), 135.4, 142.8, 152.7, 161.2, 174.4.
Arbor, MI, USA) (IC50 values, lM) were determined using an enzyme immuno
assay (EIA) kit (Catalog No. 760700, Cayman Chemical, Ann Arbor, MI, USA)
according to our previously reported method.34
31. 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)
General procedure for the synthesis of 2-, 3-, or 4-(1-difluoromethyl-2-oxo-1,2-
dihydropyridin-4-yl)phenylacetic acids (9a–c) and 2-[2-, 3-, or 4-(1-
were determined using an enzyme immuno assay (EIA) kit (Catalog No.
560131, Cayman Chemical, Ann Arbor, MI, USA) according to our previously
reported method.35
difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoic acids (9d–f):
A
mixture of a methyl 2-, 3-, or 4-(1-difluoromethyl-2-oxo-1,2-dihydropyridin-
4-yl)phenylacetate (8a–c) (221 mg, 0.75 mmol), or a methyl 2-[2-, 3-, or 4-(1-
32. Anti-inflammatory assay: The test compounds 9a–b, 9e, and the reference drugs
ibuprofen, caffeic acid and nordihydroguaiaretic acid (NDGA) were evaluated
using the in vivo carrageenan-induced rat foot paw edema model reported
previously.36
difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl]propanoate
(8d–f)
(230 mg, 0.75 mmol), and aqueous 2 N sodium hydroxide (5 mL) was stirred
at 80–85 °C for 4 h. The reaction mixture was cooled to 25 °C and acidified to
pH 3 by addition of 3 N hydrochloric acid prior to extraction with EtOAc
(3 Â 50 mL). The combined EtOAc extracts were washed with water, brine, the
organic fractions was dried (Na2SO4) and the solvent was removed in vacuo.
The impure product was purified by silica gel column chromatography using
hexanes/EtOAc (2:1, v/v) as eluent to give the title product 9a–c. The physical
and spectral data for 9a–c are listed below.
33. 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
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 file1lox 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 ligand–enzyme assembly was then subjected to a molecular
2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetic acid (9a): Yield,
63%; pale yellow solid, mp 136–138 °C; IR (film): 3033, 1724, 1682, 1071 cmÀ1
;
1H NMR (CDCl3) d 3.66 (s, 2H, CH2), 6.37 (dd, J = 7.3, 1.8 Hz, 1H, pyridone H-5),
6.61 (s, 1H, pyridone H-3), 7.23 (dd, J = 7.3, 1.2 Hz, 1H, phenyl H-6), 7.34–7.40
(m, 3H, phenyl H-3, H-4, H-5), 7.51 (d, J = 7.3 Hz, 1H, pyridone H-6), 7.74 (t,
J = 59.8 Hz, 1H, CHF2), 10.22 (br s, 1H, COOH); 13C NMR (CDCl3) d 38.3, 107.4 (t,
J = 255 Hz), 109.9, 120.7, 127.8, 128.6, 128.9, 129.2, 129.5 (t, J = 4 Hz), 131.1,
138.3, 154.6, 161.1, 175.8. Anal. Calcd for C14H11F2NO3: C, 60.22; H, 3.97; N,
5.02. Found: C, 60.13; H, 4.30; N, 4.91.
3-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetic acid (9b): Yield,
70%; pale yellow solid, mp 160–161 °C; IR (film): 3453, 1733, 1675, 1027 cmÀ1
;
1H NMR (CDCl3 + DMSO) d 3.61 (s, 2H, CH2), 6.54 (dd, J = 7.9, 1.8 Hz, 1H,
pyridone H-5), 6.68 (s, 1H, pyridone H-3), 7.35–7.46 (m, 4H, phenyl H2, H-4, H-
5, H-6), 7.46 (d, J = 7.9 Hz, 1H, pyridone H-6), 7.66 (t, J = 60.4 Hz, 1H, CHF2),
10.40 (br s, 1H, COOH); 13C NMR (CDCl3 + DMSO) d 40.1, 107.1, 107.4 (t,
J = 255 Hz), 117.3, 125.2, 127.7, 129.0 (t, J = 4 Hz), 129.3, 131.0, 135.5, 136.5,
153.0, 161.1, 173.1. Anal. Calcd for C14H11F2NO3: C, 60.22; H, 3.97; N, 5.02.
Found: C, 59.94; H, 4.01; N, 4.99.
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 ligand–enzyme 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 ligand–enzyme assembly was evaluated.
34. Chowdhury, M. A.; Abdellatif, K. R. A.; Dong, Y.; Das, D.; Suresh, M. R.; Knaus, E.
E. Bioorg. Med. Chem. Lett. 2008, 18, 6138.
4-(1-Difluoromethyl- 2-oxo-1,2-dihydropyridin-4-yl)phenylacetic acid (9c): Yield,
90%; pale yellow solid, mp 133–134 °C; IR (film): 3080, 1734, 1653, 1053 cmÀ1
;
1H NMR (CDCl3) d 3.73 (s, 2H, CH2), 6.59 (dd, J = 7.3, 1.8 Hz, pyridone H-5), 6.77
(d, J = 1.2 Hz, 1H, pyridone H-3), 7.42 (d, J = 8.5 Hz, 2H, phenyl H-2, H-6), 7.55
(d, J = 8.5 Hz, 2H, phenyl H-3, H-5), 7.56 (d, J = 7.3 Hz, 1H, pyridone H-6), 7.73
35. Rao, P. N. P.; Amini, M.; Li, H.; Habeeb, A.; Knaus, E. E. J. Med. Chem. 2003, 46,
4872.
36. Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544.