Synthesis and structure-activity relationship studies of urea-containing pyrazoles as dual inhibitors of cyclooxygenase-2 and soluble epoxide hydrolase

Article abstract of DOI:10.1021/jm2001376

A series of dual inhibitors containing a 1,5-diarylpyrazole and a urea were designed, synthesized, and evaluated as novel COX-2/sEH dual inhibitors in vitro using recombinant enzyme assays and in vivo using a lipopolysaccharide (LPS) induced model of pain in rats. The best inhibition potencies and selectivity for sEH and COX-2 over COX-1 were obtained with compounds (21b, 21i, and 21j) in which both the 1,5-diaryl-pyrazole group and the urea group are linked with a three-methylene group. Compound 21i showed the best pharmacokinetic profiles in both mice and rats (higher AUC and longer half-life). Following subcutaneous administration at 10 mg/kg, compound 21i exhibited antiallodynic activity that is more effective than the same dose of either a COX-2 inhibitor (celecoxib) or a sEH inhibitor (t-AUCB) alone, as well as coadministration of both inhibitors. Thus, these novel dual inhibitors exhibited enhanced in vivo antiallodynic activity in a nociceptive behavioral assay.

Full text of DOI:10.1021/jm2001376

ARTICLE
pubs.acs.org/jmc
Synthesis and StructureꢀActivity Relationship Studies of
Urea-Containing Pyrazoles as Dual Inhibitors of Cyclooxygenase-2
and Soluble Epoxide Hydrolase
Sung Hee Hwang, Karen M. Wagner, Christophe Morisseau, Jun-Yan Liu, Hua Dong, Aaron T. Wecksler, and
Bruce D. Hammock*
Department of Entomology and Cancer Center, University of California, One Shields Avenue, Davis, California 95616-8584,
United States
S Supporting Information
b
ABSTRACT:
A series of dual inhibitors containing a 1,5-diarylpyrazole and a urea were designed, synthesized, and evaluated as novel COX-2/sEH
dual inhibitors in vitro using recombinant enzyme assays and in vivo using a lipopolysaccharide (LPS) induced model of pain in rats.
The best inhibition potencies and selectivity for sEH and COX-2 over COX-1 were obtained with compounds (21b, 21i, and 21j) in
which both the 1,5-diaryl-pyrazole group and the urea group are linked with a three-methylene group. Compound 21i showed the
best pharmacokinetic profiles in both mice and rats (higher AUC and longer half-life). Following subcutaneous administration at 10
mg/kg, compound 21i exhibited antiallodynic activity that is more effective than the same dose of either a COX-2 inhibitor
(celecoxib) or a sEH inhibitor (t-AUCB) alone, as well as coadministration of both inhibitors. Thus, these novel dual inhibitors
exhibited enhanced in vivo antiallodynic activity in a nociceptive behavioral assay.
’ INTRODUCTION
acid monoepoxides known as epoxy-eicosatrienoic acids
(EETs).10 The soluble epoxide hydrolase (sEH) enzyme cata-
lyzes the conversion of these EETs into the corresponding diols,
or dihydroxyeicosatrienoic acids (DHETs). EETs are known to
exhibit vasodilatory,11 cardioprotective,12 anti-inflammatory,13
and antihyperalgesic14 properties, while the DHETs have greatly
reduced activity in most assays.15
NSAIDs target cyclooxygenases which are key enzymes
involved in prostaglandin (PG) biosynthesis from AA.16 How-
ever, morbidity and mortality due to NSAID-induced gastro-
intestinal (GI) toxicity are so significant and frequent worldwide
to limit the therapeutic use of this drug class.17 To mitigate this
side effect caused primarily by COX-1 inhibition, COX-2 selec-
tive inhibitors, or coxibs such as celecoxib and rofecoxib, were
designed and developed. These coxibs were specialized to retain
the beneficial anti-inflammatory and antihyperalgesic properties
of NSAIDs but enhance GI tolerance.18 In spite of this design,
COX-2 selective inhibitors retain some GI toxicity at higher
The arachidonic acid (AA) cascade is the target of many
pharmaceutical therapies for various conditions such as cardio-
vascular, asthmatic, and inflammatory diseases. For example, non-
steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygen-
ase-2 (COX-2) selective inhibitors (coxibs) block the conversion
of AA to prostaglandins (PGs) to treat pain and inflammation.1
Lipoxygenase (LOX) inhibitors, in particular 5-LOX inhibitors,
block the conversion of AA to leukotrienes (LTs) to reduce
allergy.2 The concomitant inhibition of COX and LOX enzymes
seems advantageous in various cardiovascular diseases and cancer
therapy.3 Several dual inhibitors4 that inhibit cyclooxygenases
(either COX-2 or both COX-1 and COX-2) and 5-LOX have
been reported as potential agents for the treatment of arthritis.
Licofelone (ML-3000) is an example of such an arthritis drug.5
And such dual inhibitors also have been prepared to treat
inflammation,6 pain,7 and cancers.8
In addition to the COX and LOX pathways, there is a third
major metabolic pathway in the AA cascade involving cyto-
chrome P450 metabolism. This pathway leads to the formation
of 20-hydroxyeicosatetranoic acid (20-HETE)9 and arachidonic
Received: February 8, 2011
Published: March 24, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
polypharmacological action is derived from a single agent rather
than from combination therapies (coadministration). Therefore,
there has recently been a growing interest in designed multiple
ligands (DMLs).24 The aim of DMLs is to enhance drug efficacy
and improve drug safety by acting specifically on multiple targets
(“targeted polypharmacology”), as opposed to drugs that address
only a single target. DMLs have advantages over combination
drugs or combination therapies because they circumvent the
inherent problems associated with formulation of two or more
drugs used for coadministration. In addition, the distinct differ-
ences in the pharmacodynamic and pharmacokinetic properties
of individual drugs, which may raise safety concerns, do not apply
to DMLs.25 DMLs may also offer some advantage due to
regulation of intellectual property. For all of these reasons, dual
inhibition of COX-2 and sEH through a single molecule is likely
to be more advantageous than coadministration of the drugs
using combination therapy.
The therapeutic targeting of the P450 branch of the AA
cascade remains to be thoroughly explored and even less so
using dual inhibitors. To date, there is only one current example
of a dual inhibitor related to sEH in the literature, a sEH/
11β-HSD1 dual inhibitor designed by GlaxoSmithKline.26 Herein,
we report COX-2/sEH dual inhibitors as a new class of DMLs
active in the AA cascade.
Chemistry. Diarylheterocycles have been extensively studied
as COX-2 selective inhibitors (Figure 1). Among them, celecox-
ib, which has a 1,5-diarylpyrazole skeleton, is the most successful
compound. It is a potent COX-2 selective inhibitor with a good
pharmacokinetic profile. A methyl sulfone group as in rofecoxib
or a sulfonamide group as in celecoxib has been proven to be
essential for the selective inhibition of COX-2 over COX-1,27
while N,N0-disubstituted ureas as well as their corresponding
amides and carbamates have been thoroughly studied as sEH
inhibitors.28 From this knowledge base, we sought to develop
conjugates of pyrazole and urea having both COX-2 selective and
sEH inhibitory activities as a new class of DML (Figure 1).
Therefore we prepared the compounds 7, 11aꢀg, 15, and 21a
that are linked with noncleavable methylene chains between a
pyrazole ring as a COX-2 fragment and a urea as a sEH fragment
(Scheme 1) starting from compound 3a and 3b. These common
intermediates were prepared by regioselective cyclization of
either 4-methylsulfonylphenylhydrazine 1a or 4-amidosulfonyl-
phenylhydrazine 1b with β-diketone 2.29 Compounds 21b,dꢀj
were also prepared from the β-diketone 22 (Scheme 2). Com-
pounds 11h and 21c were also prepared using similar methods
(see Supporting Information). Compound 7 was prepared by the
reaction with 3-(trifluoromethyl)phenyl isocyanate and an ami-
nopyrazole 6, which was obtained by hydrolysis from correspond-
ing ethyl carbamate 5. The Curtius reaction of carboxylic acid 4 in
thepresenceofEtOHgave5. Compounds 11aꢀgwere prepared by
the reaction of various isocyanates with amine 10a or 10b, which
was obtained by LAH reduction, mesylation, and azide formation
followed by Pd/C catalyzed hydrogenation starting from the
common intermediate 3a or 3b. Compound 15 was prepared by
the reaction of 3-(trifluoromethyl)phenyl isocyanates with amine
14, which was obtained via Henry reaction, followed by LAH
reduction from aldehyde 12b. Compound 21a was obtained by the
reaction of 3-(trifluoromethyl)phenyl isocyanates with amine 20a,
which was obtained via sequential HornerꢀWadsworthꢀEmmons
reaction, Pd/C hydrogenation, LAH reduction, Mitsunobu reaction,
and hydrazine hydrolysis from aldehyde 12a, while compounds 21b,
dꢀj were prepared using the reactions shown in Scheme 2. The
Figure 1. Structures of reference COXs and sEH inhibitors and a
general structure for COX-2/sEH dual inhibitors.
doses and/or with long-term use. Moreover, COX-2 selective
inhibitors may lose selectivity and inhibit COX-1 in vivo at higher
doses, resulting in the undesirable side effects.19 High doses of
COX-2 selective inhibitors also shift plasma thromboxane/
prostacyclin ratio20,22 and increase the eicosanoid 20-HETE,
which could potentially lead to thrombic events and
hypertension.21 We have previously demonstrated that drug
combinations with low doses of NSAIDs and soluble epoxide
hydrolase inhibitors (sEHIs) produce synergistic effects when
measuring antihyperalgesia and anti-inflammation outcomes.
This observed sEHI synergy with NSAIDS reduces pain and
inflammation while prospectively decreasing the side effects of
coxibs such as cardiovascular toxicity.22
In general, there are safety concerns when administering
combination therapy. Two drugs which are safe when used
independently of each other cannot be assumed to be safe in
combination, as drugꢀdrug interaction warnings indicate. There
are several tests that are necessary to find the optimal dose
regiments including safety studies, a complex dosage ranging
investigation, and drugꢀdrug interaction analysis, all of which
may significantly raise the practical cost and complexity of
developing combination therapies.23 It is clear that this issue is
also not exclusively due to metabolic shunting effects. For
drug development, the prediction of pharmacodynamic and
pharmacokinetic relationships is substantially less complex if
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Scheme 1. Synthesis of sEH/COX-2 Dual Inhibitors 7, 11aꢀh, 15, 21aa
a Reagents and conditions: (a) EtOH, AcOH, reflux, 2 h; (b) LiOH, THF, water, rt, 12 h; (c) DPPA, Et3N, EtOH, 1,4-dioxane, reflux, 12 h; (d) 10%
NaOH, EtOH, reflux, 5 h; (e) R3-NCO, Et3N, DMF, 0 °C to rt, 12 h; (f) LAH, THF, rt, 6 h; (g) (i) MsCl, Et3N, O °C, 2 h, (ii) NaN3, 1,4-dioxane, water,
80 °C, 2 h; (h) Pd/C, H2, EtOAc, 2 h; (i) R3-NCO, DMF, rt, 12 h; (j) PCC, DCM, rt, 6 h; (k) (i) MeNO2, AcONH4, reflux, 1 h; (l) LAH, THF, rt, 6 h;
(m) R3-NCO, DMF, rt, 12 h; (n) triethyl phosphonoacetate, NaH, THF, 0 °C, 1 h; (o) Pd/C, H2, EtOAc, 2 h; (p) LAH, THF, 6 h; (q) PPh3,
phthalimide, DIAD, THF, rt, 12 h; (r) 35% hydrazine, CH2Cl2, MeOH, rt, 1 d; (s) R3-NCO, DMF, rt, 12 h. * R2 = ph (see Supporting Information for the
synthetic scheme of compounds having R2 = t-butyl or p-tolyl).
desired 3C-linker in compound 18b was introduced by the reaction
of p-amidosulfonylphenylhydrazine hydrochloride with 1-phenyl-1,
3-dione-3-propanol 22, which was obtained by the reaction of
acetophenone with γ-butyrolactone.30,41 The amine 20b was pre-
pared by hydrogenation on Pd/C from an azide 23. A series of dual
inhibitors 21b,dꢀj possessing a three-methylene group as a linker
were then obtained by reacting amine 20b with various isocyanates.
’ RESULTS AND DISCUSSION
Inhibition of COXs and sEH Activities. Known COX-2
selective inhibitors celecoxib and rofecoxib, the COX-1 preferred
inhibitor indomethacin, and the sEH inhibitor t-AUCB31
(Figure 1) were evaluated for their cross activities against
COX-2, COX-1, and sEH enzymes. Selected reference inhibitors
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ARTICLE
Scheme 2. Synthesis of sEH/COX-2 Dual Inhibitors 21b,dꢀja
a Reagents and conditions: (a) p-sulfoamidophenylhydrazine hydrochloride, EtOH, AcOH, reflux, 2 h; (b) (i) MsCl, Et3N, DCM, 0 °C, 2 h, (ii) NaN3,
1,4-dioxane, water, reflux, 6 h; (c) Pd/C, H2, EtOAc, rt, 2 h; (d) R3-NCO, DMF, rt, 12 h.
showed expected selective inhibitory activity against each targeted
enzyme (Table 1). To obtain dual COX-2/sEH inhibitors, we
linked the 1,5-diarylpyrazole moiety of celecoxib to the urea
pharmacophore of the sEH inhibitor. It has previously been shown
that the 3-position of the pyrazolyl ring in celecoxib has very few
steric restrictions in COX-2.32 Therefore, a series of compounds
11aꢀh were first synthesized by introducing urea groups at this
position. Compound 11a was synthesized and tested because the
methylsulfone group has been proven to give an excellent COX-2
selectivity over COX-1 as in rofecoxib. Furthermore, the adaman-
tyl urea group has shown excellent inhibitory activity against
recombinant human sEH and is detected at very low levels by
LC/MS analysis.33 However, such a simple connection between
two known pharmacophores (Figure 1) was not effective due to
the lack of COX-2 inhibition and only weak sEH inhibition by
compound 11a. Replacement of the adamantyl group of com-
pound 11a by a cylcloheptyl group, which has been proven to
exhibit excellent sEH inhibitory activity (11b), improved sEH
inhibition by 10-fold,but still lacked COX-2 inhibition. On the
basis of our previous SAR study for sEH inhibitors,34 replacement
by an aryl group, especially when there is a substituent at para- or
meta-position, showed excellent sEH inhibition and good phar-
macokinetics. Therefore aryl groups were introduced as R3 groups
such as compounds 11cꢀf. Surprisingly, introduction of a tri-
fluoromethoxy group at the para-position (11d) gave slight COX-
2 inhibition with moderate sEH inhibition. However, adding a
trifluoromethyl group at the para-position not only decreased
COX-2 inhibition but also unexpectedly decreased sEH inhibition
approximately 20-fold. When the trifluoromethyl group was
introduced on the meta-position on the phenyl ring, it regained
COX-2 inhibition but poorly inhibited sEH. In general, sulfona-
mides have slightly better affinity than sulfones for the COX-2
active site,35 hence the methylsulfone group of compound 11f was
replaced by an aminosulfone group 11g, which became 2-fold
more potent than the reference drug rofecoxib (COX-2 IC50 = 2
μM), but thepotencyforsEH inhibitiondidnot improve from11f.
Replacement of a rigid phenyl group by a tert-butyl group in 11h
increased sEH inhibition (sEH IC50 = 32 ( 3 nM) but lost its
COX-2 inhibitory activity.
Comparison of Dual Inhibitors Having Variable-Length
Spacer. These results suggested that 1,5-diarylpyrazole group is
sterically hindered, which has been proven to decrease sEH
inhibition.34 Subsequently, attempts to improve sEH inhibitory
activity were initiated by increasing the length of the linker between
the 1,5-diarylpyrazole group and the urea group to avoid steric
repulsion. Compound 7, in which a biarylpyrazole ring and a urea
group are directly connected, showed poor sEH inhibitory activity as
with compound 11g. A two-methylene spacer between the two
pharmacophores as in compound 15 improved the sEH inhibition
only 3-fold compared to that of compound 11g. A further 6-fold
improvement was obtained in compound 21b by the addition of
another methylene group (total of three methylene groups) between
a COX-2 pharmacophore and a sEH pharmacophore, yielding an
overall 20-fold improvement in potency against sEH compared to
compound 11g, without changing the efficacy against COX-2.
SAR for the Dual Inhibitors Having a Three-Methylene
Linker between Two Pharmacophores. The previous data
suggested that a three-methylene spacer between two pharma-
cophores is critical for sEH inhibition in this series of com-
pounds. A compound having an aminosulfonyl group as R1 in
21b was more potent as a COX-2 inhibitor than a methylsulfone
group as R1 in 21a while sEH inhibition remained the same.
Interestingly, compound 21c having a methyl group at para-
position on a phenyl group at C-5 (R2) on the pyrazole ring
similar to celecoxib diminished activities 3-fold for both COX-2
and sEH compared to compound 21b. Compound 21d that has
two sterically hindered ortho-isopropyl groups next to a urea
group (R3) was a poor sEH inhibitor in accordance of our
previous SAR study for sEH inhibitors.34 Having developed
compounds with reasonable COX-2 inhibitory, we began an
effort to improve sEH inhibitory activity of the compounds.
Again, we started introducing various groups at R3 based on our
previous SAR study.34 This study showed that, generally, a
relatively bulky carbocyclic group and a meta- or para-substituted
phenyl group increased the inhibitory activity of sEH inhibitors.
In this case, compounds followed our previous SAR, unlike
compounds 11aꢀg. Compounds 21f became as potent as t-
AUCB. On the basis of these results, we next further optimized
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Table 1. Inhibitory Activities of Compounds 7, 11aꢀh, 15, and 21aꢀj
entry
R1
R2
n
R3
COX-1a inhibition (%)c
COX-2a IC50 (μM)d
sEHb IC50 (nM)
celecoxib
rofecoxib
Indomethacin
t-AUCB
11a
41
17
84
25
13
3
0.01
2
>10000
>10000
17
>10000
>100
>10
>10
>10
7
0.5 ( 0.1
25 ( 1
Me
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
t-butyl
phenyl
phenyl
phenyl
phenyl
p-tolyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
1
1
1
1
1
1
1
1
0
2
3
3
3
3
3
3
3
3
3
3
1-adamantyl
c-heptyl
11b
Me
2.6 ( 0.3
47 ( 4
11c
Me
phenyl
10
10
11
8
11d
Me
p-CF3O-phenyl
p-CF3-phenyl
m-CF3-phenyl
m-CF3-phenyl
m-CF3-phenyl
m-CF3-phenyl
m-CF3-phenyl
m-CF3-phenyl
m-CF3-phenyl
m-CF3-phenyl
2,6-diiPr-phenyl
phenyl
6.0 ( 0.5
110 ( 5
72 ( 8
11e
Me
>10
2.5
1
11f
Me
11g
NH2
Me
17
2
84 ( 6
11h
>10
2
32 ( 3
7
NH2
NH2
NH2
Me
34
22
27
6
88 ( 5
15
1
26 ( 3
21b
0.7
3
4.1 ( 0.4
3.4 ( 0.2
10 ( 1
21a
21c
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
12
6
2.8
>10
>10
7
21d
1550 ( 70
0.8 ( 0.1
0.5 ( 0.1
0.5 ( 0.1
0.8 ( 0.1
0.9 ( 0.1
0.5 ( 0.1
21e
16
7
21f
1-adamantyl
c-heptyl
21g
10
15
23
14
2
21h
p-Cl-phenyl
p-CF3-phenyl
p-CF3O-phenyl
6
21i
1.3
0.9
21j
a Values are the means ( SD of three independent experiments with COX Fluorescent Inhibitor Screening Assay Kit (catalogue no. 700100, Cayman
Chemicals Inc., Ann Arbor, MI). b Determined via a kinetic fluorescent assay, results are means ( SD of three separate experiments. c Percent inhibition
at 100 μM concentration. d Data points are triplicate average. We observed coefficient variation between 5 and 10%.
their sEH inhibition activity by changing R3 group while fixing a
group for COX-2 inhibition (Scheme 2). On the basis of our
previous SAR study for sEH inhibitors, several groups that
increased sEH inhibition were introduced, e.g., a bulky carbo-
cyclic group as in 21f and 21g and a para-substituted phenyl
group as in compounds 21j, 21i, and 21h. Not surprisingly,
compounds 21fꢀj in which R3 groups are carbocyclic or para-
substituted phenyl groups inhibited sEH at picomolar concen-
trations. In addition, those compounds also showed good
potency against COX-2 except compounds having a rigid ring
as in 21f or a simple phenyl group 21e (or p-Cl phenyl group
21h). However, compounds having a relatively flexible cyclo-
heptyl group 21g and phenyl group with a substitution at the
para-position such as a trifluoromethyl group in 21i or a
trifluoromethoxy group in 21j kept their COX-2 inhibitory
activity while retaining their sEH inhibitory activity. Among
them, compounds having a CF3 group as in 21b, 21i, and 21j
gave better COX-2 inhibition. They showed IC50 values better
than rofecoxib for COX-2, good to excellent sEH inhibition, and
showed reasonable COX-1/COX-2 selectivity. In addition, it has
been reported that celecoxib possesses inhibitory activity against
5-LOX, an enzyme that produces proinflammatory leukotrienes
(LTs).36 Our compounds showed similar potency with celecoxib
against 5-LOX (i.e., % inhibition of at 10 μM: celecoxib inhibits
80% of human 5-LOX while 21i yields 83% inhibition; Table S1
of the Supporting Information).
Docking Compound 21i with COX-2 and sEH Enzymes. To
understand the observation that the dual inhibitors inhibit both
sEH and COX-2, we manually docked inhibitor 21i into the
active site of sEH and COX-2 (Figure 2). For this, we used the
published X-ray crystal structure of human sEH complexed with
a urea-based ligand N-cyclohexyl-N0-(4-iodophenyl)urea, CIU
(PDB accession number 1VJ5).37 Between two plausible binding
modes for 21i, the orientation given in Figure 2A was favorable.
The other binding mode resulted in steric clashes between the
two aryl groups of the inhibitor and the residues of the binding
site, such as Leu395, Leu406, Leu427, Pro266, and Phe265. These
results might explain why we obtained unexpectedly poor sEH
inhibition with compounds having a single methylene linker such
as 11a vs 21f, 11e vs 21i, and 11g vs 21b, suggesting that these
compounds, and, presumably, other compounds in the same
series (Table 1), could not orient themselves to avoid an
unfavorable interactions with the residues at the active site. As
seen in Figure 2A, compound 21i was bound primarily through
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ARTICLE
Figure 2. Compound 21i docked into (A) the cocrystal structure of human soluble epoxide hydrolase and (B) the cocrystal structure of murine COX-2.
Black lines indicate possible hydrogen bonds.
interactions with Tyr381, Tyr465, and Asp333 with the urea
pharmacophore. We further manually docked inhibitor 21i into
the active site of COX-2 (Figure 2B) by using the published X-ray
crystal structure (PDB accession number 1CX2) of murine
COX-2 complexed with a biarylpyrazole-based ligand 1-phenyl-
sulfonamide-3-trifluoromethyl-5-parabromophenylpyrazole 24
(SC-558).38 One of the oxygen atoms in the sulfonamide group
of compound 21i forms a hydrogen bond to His90 in a similar
fashion to celecoxib. In addition, we found that one of the NH
groups in the urea of compound 21i could form the other
H-bonding with Tyr365. Presumably, the presence of extra
H-bonding in 21i might explain the slightly increased activity
of compounds having a three-methylene linker compared to
others in that the linker could provide enough space the urea
group to interact with this residue.
Pharmacokinetic Screening in Mice and Rats. Having
developed compounds with the desired inhibitor effects, we then
investigated in vivo properties of selected inhibitors 21b,eꢀj.
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Table 2. PK Parameters of the Compounds 21b,eꢀj in Mice
by Oral Cassette Dosing at 5 mg/kg (n = 1) and in Rats by Oral
Administration at 1 mg/kg (n = 4)
species inhibitors Tmax (h) Cmax (nM) AUC (nM h) t1/2 (h)
3
mouse
21h
21i
21j
21e
21b
21g
21f
21i
21j
0.8
0.8
0.8
0.5
0.5
0.5
0.5
190
260
290
100
19
375
2.4
980
2.6
1335
2.4
125
3.1
35
1.0
42
100
3.5
20
22
1.7
rat
1.5 ( 0.6 415 ( 139
1.0 ( 0.7 180 ( 71
2764 ( 804
1230 ( 521
13 ( 4
12 ( 6
Pharmacokinetic screening was performed by oral cassette dos-
ing in mice. As can be seen in Table 2, compounds 21i and 21j
showed good PK parameters by demonstrating higher Cmax and
AUC among selected inhibitors 21b,eꢀj. Even though the
compound 21b exhibited similar inhibitory activities compared
to 21i and 21j, it showed 15-fold lower Cmax and 8-fold decrease
in AUC, respectively (Table 2). On the basis of these murine PK
data, we further investigated two compounds, 21i and 21j, in rats
(Table 2). Comparing these compounds, 21i displayed better PK
parameters in rats with a 2-fold higher Cmax and greater AUC.
Therefore, compound 21i was selected for in vivo study to
determine its antinociceptive activity in a model of pain in rats.
In Vivo Antiallodynic Effects of COX-2/sEH Dual Inhibitors.
A LPS induced model of inflammatory pain39 was used to
compare the pain reducing activity of the COX-2/sEH dual
inhibitors to individual and combination therapies. Specifically, a
von Frey mechanical nociceptive assay was used to measure
allodynia induced by LPS and the attenuation of this pain
(Figure 3). Here we compared the effects of the dual inhibitor
21i to the individual and coadministration of celecoxib and the t-
AUCB from different rat groups. Male SpragueꢀDawley rats
(n = 6) injected with LPS (10 μg per rat; intraplantar) developed
allodynia, indicated by a decrease in paw withdrawal threshold to
a non-noxious mechanical stimulus. Post LPS challenge, the
average threshold for hindpaw withdrawal dropped to 54 ( 4%
(SEM) within 60 min and remained at or below this score
compared to vehicle controls, which did not vary significantly
from 100% for a 6 h time-course. Prophylactic subcutaneous
administration of the dual inhibitor 21i (10 mg/kg), a combina-
tion of celecoxib and t-AUCB (10 mg/kg, respectively), celecox-
ib alone (10 mg/kg), and t-AUCB alone (10 mg/kg) all showed
antiallodynic effects (Figure 3). While t-AUCB or celecoxib
exerted a slight antiallodynic effect, both the dual inhibitor 21i
and coadministration of t-AUCB with celecoxib synergized
effects in reducing allodynia. Remarkably, the compound 21i at
10 mg/kg exerted a stronger effect than the combination
treatment at 10 mg/kg of each drug (celecoxib þ t-AUCB),
suggesting that the dual inhibitor could compensate for different
pharmacokinetic properties of the coadministered individual
compound. This high efficacy also is interesting because 21i is
a stronger COX-2 inhibitor than rofecoxib but a far weaker
inhibitor than celecoxib (Table 1.). Additional quantitative
measurements of changes in animal behavior were not made
beyond the von Frey mechanical assay. However, the animals
were observed and handled over nine hours on the test day and
there were no overt signs of any behavioral changes or acute
Figure 3. Effect of sEH, COX-2 and dual inhibitor (21i) in a model of
inflammatory pain. Male SpragueꢀDawley rats (n = 6 per group) were
pretreated with inhibitors (Inh) 60 min before LPS injection (10 μg/rat
hindpaw intraplantar injection). (A) Time course of allodynia using Von
Frey mechanical assay. Results are expressed as percentage of baseline
(Bl) that was measured prior to inhibitor treatment. LPS (open circle);
t-AUCB (10 mg/kg sc) (gray upward-pointing triangle); celecoxib (10
mg/kg sc) (black solid square); celecoxib þ t-AUCB (10 mg/kg each
injected separately sc) (gray tilted square); 21i (10 mg/kg sc) (black
downard-pointing triangle). (B) Overall response estimated by calculat-
ing area under the curve (AUC) from (A).
observable symptoms of toxicity such as lacrimation, exuding
porphyrin, frozen or hunched postures, gasping, or chattering
from the rats treated by the compound 21i. In addition, we did
not observe any acute toxicity on U937 cell line.
’ CONCLUSION
A series of novel COX-2/sEH dual inhibitors were rationally
designed and synthesized to provide the basis for a structureꢀ
activity relationship (SAR) study. The compounds synthesized
as DMLs demonstrate significant inhibition to both COX-2
selectively and sEH. This study has demonstrated that a grouping
of diarylpyrazole (COX-2 pharmacophore) and urea (sEH
pharmacophore) can improve the inhibitory activity of the
resulting molecule on both COX-2 and sEH in vitro and
in vivo. This study has demonstrated that the COX-2/sEH dual
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Journal of Medicinal Chemistry
ARTICLE
inhibitor 21i displays improved in vivo efficacy as compared to
both the individual and combination therapies of celecoxib and t-
AUCB, as observed using a pain model in rats. Furthermore, the
dual inhibitor is also expected to show a better safety profile than
that of the combination therapy.
chromatography to give the titled compound (0.48 g, 62%) as a white
solid; mp 120.9ꢀ127.5 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.30 (s,
1H), 7.79 (d, J = 8 Hz, 2H), 7.50ꢀ7.34 (m, 7H), 7.33ꢀ7.25 (m, 2H),
6.74 (s, 1H), 4.14 (q, J = 7 Hz, 2H), 1.24 (t, J = 7 Hz, 3H). MS (ESI) m/
z: 387.11 (M þ Hþ).
4-(3-Amino-5-phenyl-pyrazol-1-yl)-benzenesulfonamide (6). To a
solution of an ethyl carbamate 5 (0.39 g, 1 mmol) in EtOH (10 mL) was
added 10% NaOH solution (3.2 mL) at room temperature. The reaction
mixture was refluxed for 5 h. The reaction mixture was cooled to room
temperature and water was added. The resulting white precipitate was
filtered and washed with water thoroughly to give the titled compound
(0.22 g, 70%) as a white solid. mp 286.3ꢀ290.2 °C. 1H NMR (DMSO-
d6): δ 7.70 (d, J = 9 Hz, 2H), 7.43ꢀ7.20 (m, 9H), 5.89 (s, 1H), 5.17 (br
s, 2H). MS (ESI) m/z: 315.09 (M þ Hþ).
’ EXPERIMENTAL SECTION
General. Celecoxib was a gift from Pfizer Corporation, Indometha-
cin was purchased from TCI America Fine Chemicals (Portland, USA),
rofecoxib was purchased from Fisher Scientific (Chicago, USA). All
other reagents and solvents were obtained from commercial suppliers
and were used without further purification. All reactions, unless other-
wise described, were performed under an inert atmosphere of dry
nitrogen. Melting points were determined on an OptiMelt melting
4-{5-Phenyl-3-[3-(3-trifluoromethyl-phenyl)-ureido]-pyrazol-1-yl}-
benzenesulfonamide (7). To a solution of amine 6 (0.1 g, 0.32 mmol) in
5 mL of DMF was added 3-(trifluoromethyl)phenyl isocyanate (0.12 g,
0.62 mmol) followed by triethylamine (0.05 mL, 0.32 mmol) at 0 °C.
The reaction mixture was warmed up to room temperature and stirred
overnight. The reaction mixture was poured into water, and the resulting
precipitates were collected and washed with water. The crude product
was purified by column chromatography by dry loading method to give
the titled compound (0.13 g, 81% yield); mp 195.1ꢀ197.0 °C. 1H NMR
(300 MHz, DMSO-d6): δ 9.48 (s, 1H), 9.21 (s, 1H), 8.07 (s, 1H), 7.80
(d, J = 9 Hz, 2H), 7.61ꢀ7.26 (m, 12H), 6.81 (s, 1H). MS (ESI) m/z:
1
point apparatus and are uncorrected. H NMR and 13C NMR spectra
were recorded at 300 and 75 MHz, respectively. Elemental analyses were
determined at Midwest Microlab, Indianapolis, IN. Mass spectra were
measured by LC-MS equipped with a Waters 2790 and a Waters PDA
996 using electrospray (þ) ionization. Flash chromatography was
performed on silica gel. All compounds were characterized by NMR
and LC/MS. All final compounds, 7, 15, 11aꢀh, and 21aꢀj, had a purity
of g95%. The purity of final compounds was determined by elemental
analysis (within (0.4% of the calculated value).
5-Phenyl-1-(4-sulfamoyl-phenyl)-1H-pyrazole-3-carboxylic Acid
Ethyl Ester (3b). To a solution of potassium bis(trimethylsilyl)amide
(8.88 g, 44.5 mmol) in 400 mL of THF was added dropwise acetophe-
none (4.85 mL, 41.6 mmol) at ꢀ78 °C. After 1 h, diethyl oxalate
(6.2 mL, 45.8 mmol) was added dropwise at the same temperature. The
reaction mixture was allowed to warm up to room temperature and
stirred overnight. The solvent was removed in vacuo. The resulting
yellowish solid was filtered and washed with Et2O to give the title
compound 2 (10.5 g, 98%) as a potassium salt of ethyl 2-hydroxy-4-oxo-
4-phenyl-2-butenoate (2).40 To a solution of potassium salt of 2 in
EtOH (300 mL) was added 4-amidosulfonylphenylhydrazine 1b (10 g,
44.7 mmol) at room temperature. The reaction mixture was stirred for 1
h, and then 2.4 mL of AcOH was added. The reaction mixture was
refluxed for 2 h. After cooling, the solvent was removed in vacuo. The
resulting solid was purified by recrystallization with MeOH to afford the
titled compound (13.5 g, 89% yield) as a white solid; mp
502.12 (M þ Hþ). Anal. (C23H18F3N5O3S 0.36CH4O) C, H, N.
3
The Preparation of the Compounds 11aꢀg. 1-(4-Methane-
sulfonyl-phenyl)-5-phenyl-1H-pyrazole-3-carboxylic Acid Ethyl Ester
(3a). Compound 3a was synthesized in a manner similar to the synthesis
of 3b using 4-(methylsulfonyl)phenylhydrazine hydrochloride (10 g,
44.7 mmol) to give 3a (13.6 g, 82% yield) as a white solid; mp
1
199.4ꢀ202.9 °C. H NMR (300 MHz, DMSO-d6): δ 7.99 (d, J = 9
Hz, 2H), 7.58 (d, J = 9 Hz, 2H), 7.43ꢀ7.38 (m, 3H), 7.34ꢀ7.27 (m,
2H), 7.17 (s, 1H), 4.34 (q, J = 7, 2H), 3.27 (s, 3H), 1.31 (t, J = 7, 3H). MS
(ESI) m/z: 371.11 (M þ Hþ).
General Procedure for the Synthesis of Hydroxymethylpyrazoles 8a
and 8b. To a solution of compound 3a or 3b in THF was added LiAlH4
(3.2 equiv) at room temperature. The reaction mixture was stirred for 6
h. EtOAC was added, and excess LiAlH4 was quenched by adding
minimum amounts of water. The solution was dried with MgSO4, and
white precipitates were filtered off. The filtrate was concentrated in
vacuo. The crude product was purified by column chromatography
(EtOAc/hexanes in aequate proportions) to give the compound 8a
or 8b.
1
191.5ꢀ194.6 °C. H NMR (300 MHz, DMSO-d6): δ 7.87 (d, J = 8
Hz, 2H), 7.58ꢀ7.48 (m, 4H), 7.44ꢀ7.38 (m, 3H), 7.34ꢀ7.27 (m, 2H),
7.16 (s, 1H), 4.35 (q, J = 7 Hz, 2H), 1.32 (t, J = 7 Hz, 3H). MS (ESI) m/
z: 372.10 (M þ Hþ).
[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-yl]-meth-
anol (8a). Synthesized as a white solid (3.25 g, 76% yield) from the ester
3a (5 g, 17.1 mmol) by the General Procedure for the Synthesis of
Hydroxymethylpyrazoles 8a and 8b and purified by column chroma-
The Preparation of the Compound 7. 5-Phenyl-1-(4-sulfa-
moyl-phenyl)-1H-pyrazole-3-carboxylic Acid (4). To a solution of
compound 3b (0.37 g, 1 mmol) in THF (10 mL) was added LiOH
(35 mg, 1.5 mmol) followed by 2 mL of water at room temperature. The
reaction mixture was stirred overnight. The reaction was quenched by
adding 1N HCl. The solvent was evaporated in vacuo. The resulting
white solids were collected by suction filtration and washed with water.
The crude product was recrystallized from MeOH to give the titled
compound (0.33 g, 96% yield) as a white solid; mp 188.5ꢀ190.9 °C. 1H
NMR (300 MHz, DMSO-d6): δ 13.10 (s, 1H), 7.87 (d, J = 8 Hz, 2H),
7.55ꢀ7.48 (m, 4H), 7.44ꢀ7.37 (m, 3H), 7.34ꢀ7.26 (m, 2H), 7.10 (s,
1H). MS (ESI) m/z: 344.07 (M þ Hþ).
1
tography with hexanes/EtOAc (1:1); mp 126.5ꢀ128.5 °C. H NMR
(300 MHz, DMSO-d6): δ 7.88 (d, J = 9 Hz, 2H), 7.49 (d, J = 9 Hz, 2H),
7.41ꢀ7.34 (m, 3H), 7.26ꢀ7.21 (m, 2H), 6.56 (s, 1H), 4.81 (d, J = 6 Hz,
2H), 3.05 (s, 3H), 2.13 (t, J = 6 Hz, 1H). MS (ESI) m/z: 329.10
(M þ Hþ).
4-(3-Hydroxymethyl-5-phenyl-pyrazol-1-yl)-benzenesulfona-
mide (8b). Synthesized as a white solid (2.8 g, 75% yield) from ester 3b
(4.18 g, 11.3 mmol) by the General Procedure for the Synthesis of
Hydroxymethylpyrazoles 8a and 8b and purified by column chroma-
[5-Phenyl-1-(4-sulfamoyl-phenyl)-1H-pyrazol-3-yl]-carbamic Acid
Ethyl Ester (5). To a solution of acid 4 (0.74 g, 2 mmol) in 10 mL of
EtOH and 10 mL of 1,4-dioxane was added diphenylphosphonylazide
(0.52 mL, 2.4 mmol) followed by Et3N (0.34 mL, 2.4 mmol). The
reaction mixture was refluxed overnight. After cooling the reaction
mixture to room temperature, EtOAc and water were added. The
organic layer was separated, washed with brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by column
1
tography with hexanes/EtOAc (4:6);. mp 173.8ꢀ180.5 °C. H NMR
(300 MHz, DMSO-d6): δ 7.81 (d, J = 8 Hz, 2H), 7.50ꢀ7.35 (m, 7H),
7.30ꢀ7.23 (m, 2H), 6.64 (s, 1H), 5.28 (t, J = 6 Hz, 1H), 4.53 (d, J = 6 Hz,
2H). MS (ESI) m/z: 330.09 (M þ Hþ).
General Procedure for the Synthesis of Azidomethylpyrazoles 9a
and 9b. To a solution of alcohol 8a or 8b in THF was added
methanesulfonyl chloride (1.3 equiv) followed by Et3N (1.6 equiv) at
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ARTICLE
0 °C. The reaction mixture was stirred for 2 h. After adding water, the
product was extracted with EtOAc. The organic layer was dried with
MgSO4, and the solvent was removed in vacuo. To a solution of a
mesylate obtained, in 1,4-dioxane/water (1:1) was added NaN3 (2.5
equiv). The reaction mixture was heated at 80 °C for 3 h. After cooling,
the solvent were removed in vacuo. The residue was purified by column
chromatography (EtOAc/hexanes in adequate proportions) to give the
compound 9a or 9b as a viscous liquid.
was prepared in 68% yield from 4-(trifluoromethoxy)phenyl isocyanate
using the procedure detailed for compound 7; mp 152.9ꢀ156.3 °C. 1H
NMR (300 MHz, DMSO-d6): δ 8.87 (s, 1H), 7.94 (d, J = 9 Hz, 2H),
7.55ꢀ7.46 (m, 4H), 7.43ꢀ7.37 (m, 3H), 7.31ꢀ7.19 (m, 4H), 6.73 (t, J =
6 Hz, 1H), 6.62 (s, 1H), 4.39 (d, J = 6 Hz, 2H), 3.25 (s, 3H). MS (ESI)
m/z: 531.13 (M þ Hþ). Anal. (C25H21F3N4O4S) C, H, N.
1-[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-ylmet-
hyl]-3-(4-trifluoromethyl-phenyl)-urea (11e). The titled compound
was prepared in 68% yield from 4-(trifluoromethyl)phenyl isocyanate
using the procedure detailed for compound 7; mp 129.3ꢀ131.7 °C. 1H
NMR (300 MHz, DMSO-d6): δ 9.09 (s, 1H), 7.94 (d, J = 9 Hz, 2H),
7.65ꢀ7.54 (m, 4H), 7.50 (d, J = 9 Hz, 2H), 7.43ꢀ7.38 (m, 3H),
7.31ꢀ7.25 (m, 2H), 6.83 (t, J = 6 Hz, 1H), 6.64 (s, 1H), 4.41 (d, J = 6 Hz,
2H), 3.25 (s, 3H). MS (ESI) m/z: 515.14 (M þ Hþ). Anal.
3-Azidomethyl-1-(4-methanesulfonyl-phenyl)-5-phenyl-1H-pyrazole
(9a). Synthesized as a viscous liquid (0.7 g, 99% yield) from alcohol 8a (0.5
g, 2 mmol) by the General Procedure for the Synthesis of Azidomethylpyr-
azoles 9a and 9b and purified by column chromatography with hexanes/
EtOAc (6:4). 1H NMR (300 MHz, DMSO-d6): δ 7.95 (d, J = 9 Hz, 2H),
7.51 (d, J = 9 Hz, 2H), 7.45ꢀ7.39 (m, 3H), 7.33ꢀ7.26 (m, 2H), 6.76 (s,
1H), 4.53 (s, 2H), 3.26 (s, 3H). MS (ESI) m/z: 354.10 (M þ Hþ).
4-(3-Azidomethyl-5-phenyl-pyrazol-1-yl)-benzenesulfonamide (9b).
Synthesized as a viscous liquid (0.48 g, 90% yield) from ester 8b (0.5 g,
1.52 mmol) by the General Procedure for the Synthesis of Azidomethyl-
pyrazoles 9a and 9b and purified by column chromatography with
hexanes/EtOAc (1:1). 1H NMR (300 MHz, DMSO-d6): δ 7.83 (d, J = 9
Hz, 2H), 7.51ꢀ7.37 (m, 7H), 7.31ꢀ7.24 (m, 2H), 6.75 (s, 1H), 4.53 (s,
2H). MS (ESI) m/z: 355.10 (M þ Hþ).
(C25H21F3N4O3S 0.375CH4O) C, H, N.
3
1-[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-ylmet-
hyl]-3-(3-trifluoromethyl-phenyl)-urea (11f). The titled compound
was prepared in 68% yield from 3-(trifluoromethyl)phenyl isocyanate
using the procedure detailed for compound 7; mp 107.5ꢀ113.0 °C. 1H
NMR (300 MHz, DMSO-d6): δ 9.05 (s, 1H), 8.00 (s, 1H), 7.94 (d, J = 9
Hz, 2H), 7.56ꢀ7.36 (m, 7H), 7.32ꢀ7.21 (m, 3H), 6.82 (t, J = 6 Hz, 1H),
6.63 (s, 1H), 4.40 (d, J = 6 Hz, 2H), 3.25 (s, 3H). MS (ESI) m/z: 515.13
(M þ Hþ).
General Procedure for the Synthesis of Aminomethylpyrazoles 10a
and 10b. To a solution of 9a or 9b in EtOAc was added 10% palladium
on carbon. The solution was filled with H2, and the reaction mixture was
stirred for 2 h. After the solution was filtered through Celite, the filtrate
was concentrated in vacuo. The crude product was used for the next step
without further purification.
4-{5-Phenyl-3-[3-(3-trifluoromethyl-phenyl)-ureidomethyl]-pyr-
azol-1-yl}-benzenesulfonamide (11g). The titled compound was
prepared in 68% yield from 3-(trifluoromethyl)phenyl isocyanate using
the procedure detailed for compound 7; mp 184.6ꢀ189.6 °C. 1H NMR
(300 MHz, DMSO-d6): δ 9.04 (s, 1H), 8.00 (s, 1H), 7.81 (d, J = 9 Hz,
2H), 7.56ꢀ7.35 (m, 9H), 7.30ꢀ7.21 (m, 3H), 6.81 (t, J = 6 Hz, 1H),
6.61 (s, 1H), 4.39 (d, J = 6 Hz, 2H). MS (ESI) m/z: 516.13 (M þ Hþ).
For the preparation of compound 11h see Supporting Information.
The Preparation of the Compound 15. 4-(3-Formyl-5-phen-
yl-pyrazol-1-yl)-benzenesulfonamide (12b). To a solution of alcohol
8b (3 g, 9.1 mmol) and 3 g of powdered 4 Å molecular sieves in 150 mL
of DCM was added PCC (2.94 g, 13.7 mmol) at room temperature.
After 6 h, the reaction mixture was filtered through a 0.75 in. pad of
Celite. The filtrate was concentrated in vacuo, and then the residue was
purified by column chromatography to afford the titled compound (2.1
C-[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-yl]-
methylamine (10a). Yield 91%. 1H NMR (300 MHz, CDCl3): δ 7.87
(d, J = 9 Hz, 2H), 7.48 (d, J = 9 Hz, 2H), 7.40ꢀ7.32 (m, 3H), 7.26ꢀ7.19
(m, 2H), 6.48 (s, 1H), 3.97 (s, 2H), 3.04 (s, 3H), 1.65 (s, 2H).
4-(3-Aminomethyl-5-phenyl-pyrazol-1-yl)-benzenesulfonamide
1
(10b). Yield 90%. H NMR (300 MHz, CDCl3): δ 7.87 (d, J = 9 Hz,
2H), 7.48 (d, J = 9 Hz, 2H), 7.39ꢀ7.31 (m, 3H), 7.27ꢀ7.19 (m, 2H),
6.48 (s, 1H), 3.98 (s, 2H), 3.05ꢀ3.02 (m, 4H).
1-Adamantan-1-yl-3-[1-(4-methanesulfonyl-phenyl)-5-phenyl-
1H-pyrazol-3-ylmethyl]-urea (11a). The title compound was prepared
in 68% yield from 1-adamantyl isocyanate using the procedure detailed
1
g, 70%) as a white solid; mp 177.3ꢀ179.8 °C. H NMR (300 MHz,
1
for compound 7; mp 150.0ꢀ155.3 °C. H NMR (300 MHz, DMSO-
DMSO-d6): δ 10.03 (s, 1H), 7.88 (d, J = 8 Hz, 2H), 7.61ꢀ7.49 (m, 4H),
7.48ꢀ7.37 (m, 3H), 7.35ꢀ7.26 (m, 2H), 7.20 (s, 1H). MS (ESI) m/z:
328.08 (M þ Hþ).
d6): δ 7.93 (d, J = 9 Hz, 2H), 7.48 (d, J = 9 Hz, 2H), 7.44ꢀ7.38 (m, 3H),
7.30ꢀ7.23 (m, 2H), 6.54 (s, 1H), 6.13 (t, J = 6 Hz, 1H), 5.71 (s, 1H),
4.22 (d, J = 6 Hz, 2H), 3.25 (s, 3H), 2.04ꢀ1.96 (m, 3H), 1.90ꢀ1.85 (m,
6H), 1.63ꢀ1.57 (m, 6H). MS (ESI) m/z: 505.22 (M þ Hþ). Anal.
(C28H32N4O3S) C, H,N.
4-[3-((E)-2-Nitro-vinyl)-5-phenyl-pyrazol-1-yl]-benzenesulfona-
mide (13). To a solution of aldehyde 10 (0.96 g, 2.9 mmol) in MeNO2
(5.9 mL, 109 mmol) was added AcONH4 (0.34 g, 4.4 mmol). The
reaction mixture was refluxed for 1 h. After cooling, MeNO2 was
evaporated in vacuo. The residue was purified by column chromatog-
raphy with hexanes/EtOAc (7: 3) to give the corresponding nitroethy-
lene compound (0.5 g, 46%) as a yellow solid; mp 189.2ꢀ191.8 °C. 1H
NMR (300 MHz, DMSO-d6): δ 8.24 (d, J = 14 Hz, 1H), 8.01 (d, J = 14
Hz, 1H), 7.85 (d, J = 9 Hz, 2H), 7.54ꢀ7.47 (m, 4H), 7.46ꢀ7.40 (m,
3H), 7.37 (s, 1H), 7.32ꢀ7.26 (m, 2H). MS (ESI) m/z: 371.08 (M þ
Hþ).
1-Cycloheptyl-3-[1-(4-methanesulfonyl-phenyl)-5-phenyl-1H-
pyrazol-3-ylmethyl]-urea (11b). The title compound was prepared in
68% yield from cycloheptyl isocyanate using the procedure detailed for
compound 7; mp 103.9ꢀ109.9 °C. 1H NMR (300 MHz, DMSO-d6): δ
7.93 (d, J = 9 Hz, 2H), 7.48 (d, J = 9 Hz, 2H), 7.44ꢀ7.38 (m, 3H),
7.29ꢀ7.24 (m, 2H), 6.54 (s, 1H), 6.18 (t, J = 6 Hz, 1H), 5.94 (d, J = 8 Hz,
1H), 4.26 (d, J = 6 Hz, 2H), 3.66ꢀ3.54 (m, 1H), 3.25 (s, 3H), 1.82ꢀ1.70
(m, 2H), 1.61ꢀ1.29 (m, 10H). MS (ESI) m/z: 467.21 (M þ Hþ). Anal.
(C25H30N4O3S 0.25CH4O) C, H, N.
4-[3-(2-Amino-ethyl)-5-phenyl-pyrazol-1-yl]-benzenesulfonamide
(14). To a solution of the compound above (0.42 g, 1.1 mmol) in 10 mL
of THF was added LiAlH4 (0.21 g, 5.7 mmol) at room temperature. The
reaction mixture was stirred for 6 h and quenched by adding water
(10 mL). The mixture was extracted with EtOAc, and the combined
organic layers were dried with MgSO4. The solution was evaporated in
vacuo. The crude product was used next step without further purification
(0.3 g, 77% yield); mp 189.2ꢀ191.8 °C. 1H NMR (300 MHz, DMSO-
d6): δ 7.79 (d, J = 9 Hz, 2H), 7.43ꢀ7.36 (m, 5H), 7.28ꢀ7.23 (m, 2H),
6.55 (s, 1H), 5.23ꢀ3.88 (m, 4H), 2.88 (t, J = 7 Hz, 2H), 2.71 (t, J = 7 Hz,
2H).
3
1-[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-ylmet-
hyl]-3-phenyl-urea (11c). The title compound was prepared in 68%
yield from phenyl isocyanate using the procedure detailed for compound
7; mp 95.7ꢀ104.2 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.62 (s, 1H),
7.94 (d, J = 9 Hz, 2H), 7.50 (d, J = 9 Hz, 2H), 7.43ꢀ7.37 (m, 5H),
7.31ꢀ7.18 (m, 4H), 6.89 (t, J = 7 Hz, 1H), 6.67ꢀ6.61 (m, 1H), 6.62 (s,
1H), 4.38 (d, J = 6 Hz, 2H), 3.25 (s, 3H). MS (ESI) m/z: 447.15 (M þ
Hþ). Anal. (C24H22N4O3S) C, H, N.
1-[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-ylmet-
hyl]-3-(4-trifluoromethoxy-phenyl)-urea (11d). The title compound
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4-(5-Phenyl-3-{2-[3-(3-trifluoromethyl-phenyl)-ureido]-ethyl}-pyr-
azol-1-yl)-benzenesulfonamide (15). The title compound was pre-
pared in 84% yield from 3-trifluoromethylphenyl isocyanate using the
1-{3-[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-yl]-
propyl}-3-(3-trifluoromethyl-phenyl)-urea (21a). Hydrazine hydrate
(35 wt %, 0.16 g, 1.73 mmol) was added to a solution of compound 19
(0.42 g, 0.86 mmol) in DCM (15 mL) followed by MeOH (15 mL) at
room temperature. The reaction mixture was stirred for 1 day. The white
precipitates were filtered off, and the solvent was removed in vacuo. The
residue was dissolved in aqueous 1N HCl solution and washed with
DCM. Aqueous layer was basified with excess 1N NaOH solution and
then extracted with DCM. After drying with MgSO4, the solvent was
evaporated affording the amine 20a. To a solution of amine obtained was
an added 3-trifluoromethylphenyl isocyanate (0.16 g, 0.86 mmol). The
reaction mixture was stirred overnight. After adding water, precipitates
were collected by suction filter. The white solid was purified by column
chromatography with hexanes/EtOAc (1:1) to give the title compound
(0.37 g, 80 mmol) as a white solid; mp 150.1ꢀ151.8 °C. 1H NMR (300
MHz, DMSO-d6): δ 8.86 (s, 1H), 7.99 (s, 1H), 7.91 (d, J = 9 Hz, 2H),
7.53ꢀ7.36 (m, 7H), 7.31ꢀ7.25 (m, 2H), 7.24ꢀ7.19 (m, 1H), 6.59 (s,
1H), 6.39 (t, J = 6 Hz, 1H), 3.26ꢀ3.17 (m, 2H), 3.24 (s, 3H), 2.70 (t, J =
8 Hz, 2H), 1.94ꢀ1.80 (m, 2H). MS (ESI) m/z: 543.17 (M þ Hþ). Anal.
(C27H25F3N4O3S) C, H, N.
1
procedure detailed for compound 7; mp 121.9ꢀ124.7 °C. H NMR
(300 MHz, DMSO-d6): δ 8.97 (s, 1H), 8.00 (s, 1H), 7.80 (d, J = 8 Hz,
2H), 7.52ꢀ7.36 (m, 8H), 7.29ꢀ7.20 (m, 4 H), 6.61 (s, 1H), 6.41 (t, J = 6
Hz, 1H), 3.49 (dd, J = 13 and 7 Hz, 2H), 2.85 (t, J = 7 Hz, 2H). MS (ESI)
m/z: 530.15 (M þ Hþ).
The Preparation of the Compound 21a. 1-(4-Methanesulfo-
nyl-phenyl)-5-phenyl-1H-pyrazole-3-carbaldehyde (12a). The title
compound was prepared in 65% yield from the compound 3a using
1
the procedure detailed for compound 12b; mp 133.0ꢀ135.5 °C. H
NMR (300 MHz, DMSO-d6): δ 10.03 (s, 1H), 8.02 (d, J = 9 Hz, 2H),
7.63 (d, J = 9 Hz, 2H), 7.47ꢀ7.39 (m, 3H), 7.37ꢀ7.29 (m, 2H), 7.22 (s,
1H), 3.29 (s, 3H). MS (ESI) m/z: 327.08 (M þ Hþ).
(E)-3-[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-yl]-
acrylic Acid Ethyl Ester (16). To a solution of aldehyde 12a (1.4 g, 4.3
mmol) and triethyl phosphonoacetate (0.94 mL, 4.73 mmol) in 50 mL of
THF was added 60% sodium hydride in oil (0.19 g, 4.73 mmol) at 0 °C.
The reaction mixture was stirred for 1 h. The reaction was quenched by
adding 5 mL of water, and then the mixture was extracted with EtOAc. After
drying with MgSO4, the solvent was removed in vacuo. The residue was
purified by column chromatography with 7:3 (hexanes/EtOAc) to give the
titled compound (1.32 g, 77% yield) as a white solid; mp 163.5ꢀ168.4 °C.
1H NMR (300 MHz, DMSO-d6): δ 7.96 (d, J = 9 Hz, 2H), 7.58 (d, J = 16
Hz, 1H), 7.55 (d, J = 9 Hz, 2H), 7.47ꢀ7.40 (m, 3H), 7.34ꢀ7.27 (m, 2H),
7.28 (s, 1H), 6.72 (d, J = 16 Hz, 1H), 4.21 (q, J = 7 Hz, 2H), 3.27 (s, 3H),
1.27 (t, J = 7 Hz, 3H). MS (ESI) m/z: 397.12 (M þ Hþ).
The Preparation of the Compound 21b and 21dꢀj.
4-[3-(3-Hydroxy-propyl)-5-phenyl-pyrazol-1-yl]-benzenesulfonamide
(18b).40. Dispersion of sodium hydride (60%) in mineral oil (4.0 g, 100
mmol) was washed twice with hexane (100 mL each) and dried under a
stream of nitrogen. Ether (300 mL) was added followed by dropwise
addition of ethanol (0.25 mL) and γ-butyrolactone (4.0 mL, 52 mmol).
The mixture was cooled to 10 °C and acetophenone (5.8 mL, 50 mmol)
in ether (40 mL) was added dropwise over 1 h. The mixture was warmed
to 25 °C and stirred overnight. The mixture was cooled to 0 °C and
quenched with ethanol (5 mL) followed by 10% aqueous ammonium
sulfate (100 mL). The organic solution was separated, dried over
Mg2SO4, and concentrated. The residue was chromatographed on silica
gel with 1:1 hexane/ethyl acetate to give the desired diketone 22 (3.4 g,
33%) as oil. Pyridine (0.34 mL, 4.2 mmol) and the diketone 22 (700 mg,
3.4 mmol) in methanol (3 mL) were added to slurry of 4-sulfonamido-
phenylhydrazine-HCl (750 mg, 3.4 mmol) in methanol (8 mL). The
mixture was stirred at 25 °C overnight and concentrated in vacuo. The
residue was dissolved in DCM and the solution washed with 1N HCl.
The organic solution was separated, dried, and concentrated. The
residue was chromatographed on silica gel using EtOAc to give the
desired alcohol 18b (1 g, 90%) as a white solid; mp 203.5ꢀ205.4 °C. 1H
NMR (300 MHz, DMSO-d6): δ 7.79 (d, J = 9 Hz, 2H), 7.45ꢀ7.35 (m,
7H), 7.28ꢀ7.23 (m, 2H), 6.53 (s, 1H), 4.51 (t, J = 5 Hz, 1H), 3.50 (dd, J
= 12 and 6 Hz, 2H), 2.84ꢀ2.56 (m, 2H), 1.88ꢀ1.77 (m, 2H). MS (ESI)
m/z: 358.12 (M þ Hþ).
3-[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-yl]-pro-
pionic Acid Ethyl Ester (17). To a solution of 16 (0.17 g, 0.42 mmol) in
5 mL of EtOAc was added 10% palladium on carbon. The solution was
filled with H2, and the reaction mixture was stirred for 2 h. After the
solution was filtered through Celite, the filtrate was concentrated in
vacuo. Purification by column chromatography (3:7 EtOAc/hexanes)
gave the title compound, 0.15 g (90%) as a white solid. mp
96.5ꢀ99.1 °C. 1H NMR (300 MHz, DMSO-d6): δ 7.91 (d, J = 9 Hz,
2H), 7.45 (d, J = 9 Hz, 2H), 7.44ꢀ7.37 (m, 3H), 7.29ꢀ7.23 (m, 2H),
6.57 (s, 1H), 4.09 (q, J = 7 Hz, 2H), 3.24 (s, 3H), 2.93 (t, J = 7 Hz, 2H),
2.74 (t, J = 7 Hz, 2H), 1.18 (t, J = 7 Hz, 3H). MS (ESI) m/z: 399.14
(M þ Hþ).
3-[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-yl]-pro-
pan-1-ol (18a). To a solution of ester (1.27 g, 4 mmol) in 40 mL of THF
was added LiAlH4 (0.24 g, 6.4 mmol). The reaction mixture was stirred
for 6 h at room temperature. EtOAC (5 mL) and water (10 mL) were
added successively. The solvent was removed in vacuo, and the resulting
solid was filtered. The crude product was purified by column chroma-
tography (EtOAc/hexanes = 7:3) to give the titled compound (0.97 g,
4-[3-(3-Azido-propyl)-5-phenyl-pyrazol-1-yl]-benzenesulfonamide
(23). The titled compound was prepared in 88% yield using the
compound above using the procedure detailed for compound 9b; mp
114.0ꢀ115.6 °C. 1H NMR (300 MHz, DMSO-d6): δ 7.80 (d, J = 9 Hz,
2H), 7.48ꢀ7.36 (m, 7H), 7.28ꢀ7.23 (m, 2H), 6.58 (s, 1H), 3.46 (t, J = 7
Hz, 2H), 2.72 (t, J = 8 Hz, 2H), 2.00ꢀ1.89 (m, 2H). MS (ESI) m/z:
383.13 (M þ Hþ).
1
85%) as a white solid; mp 119.5ꢀ123.4 °C. H NMR (300 MHz,
DMSO-d6): δ 7.91 (d, J = 9 Hz, 2H), 7.46 (d, J = 9 Hz, 2H), 7.43ꢀ7.37
(m, 3H), 7.31ꢀ7.25 (m, 2H), 6.56 (s, 1H), 4.52 (t, J = 5 Hz, 1H),
3.54ꢀ3.46 (m, 2H), 3.24 (s, 3H), 2.79ꢀ2.60 (m, 2H), 1.88ꢀ1.77
(m, 2H). MS (ESI) m/z: 357.12 (M þ Hþ).
2-{3-[1-(4-Methanesulfonyl-phenyl)-5-phenyl-1H-pyrazol-3-yl]-
propyl}-isoindole-1,3-dione (19). To a solution of alcohol 18 (0.3 g,
0.84 mmol), PPh3 (0.22 g, 0.84 mmol), and phthalimide (0.12 g, 0.84
mmol) in 10 mL of THF was added dropwise DIAD (0.17 g, 0.84
mmol), at room temperature. The reaction mixture was stirred over-
night. The solvent was evaporated, and the residue was purified by
column chromatography with hexanes/EtOAc (1:1) to afford 0.37 g
3-[5-Phenyl-1-(4-sulfamoyl-phenyl)-1H-pyrazol-3-yl]-propyl-am-
monium Chloride (20b). Synthesized as a white solid (68% yield) from
compound 23 using the General Procedure for the Synthesis of
Aminomethylpyrazoles 10a and 10b, except the titled compound was
prepared as an HCl salt with anhydrous HCl (g); mp 152.3ꢀ270.0 °C
dec 1H NMR (300 MHz, TFA-d): δ 11.81ꢀ11.52 (m, 3H), 8.07 (d, J = 8
Hz, 2H), 7.57 (d, J = 8.18 Hz, 2H), 7.50ꢀ7.43 (m, 1H), 7.40ꢀ7.32 (m,
2H), 7.26ꢀ7.21 (m, 2H), 7.05 (br s, 2H), 6.88 (s, 1H), 3.48ꢀ3.36 (m,
2H), 3.26ꢀ3.06 (m, 2H), 2.51ꢀ2.35 (m, 2H). MS (ESI) m/z: 357.14
(M þ Hþ).
1
(90%) of the title compound as a white solid; mp 62.4ꢀ72.2 °C. H
NMR (300 MHz, DMSO-d6): δ 7.88 (d, J = 8 Hz, 2H), 7.83ꢀ7.74 (m,
5H), 7.43ꢀ7.29 (m, 4H), 7.17ꢀ7.09 (m, 2H), 6.51 (s, 1H), 3.72 (t, J = 7
Hz, 2H), 3.24 (s, 3H), 2.71 (t, J = 7 Hz, 2H), 2.15ꢀ2.02 (m, 2H). MS
(ESI) m/z: 486.15 (M þ Hþ).
4-(5-Phenyl-3-{3-[3-(3-trifluoromethyl-phenyl)-ureido]-propyl}-
pyrazol-1-yl)-benzenesulfonamide (21b). The title compound was
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Journal of Medicinal Chemistry
ARTICLE
prepared in 84% yield from 3-(trifluoromethyl)phenyl isocyanate using
the procedure detailed for compound 7; mp 175.5ꢀ176.4 °C. 1H NMR
(300 MHz, DMSO-d6): δ 8.85 (s, 1H), 7.98 (s, 1H), 7.79 (d, J = 9 Hz,
2H), 7.53ꢀ7.35 (m, 9H), 7.29ꢀ7.18 (m, 3H), 6.57 (s, 1H), 6.38 (t, J = 6
Hz, 1H), 3.25ꢀ3.17 (m, 2H), 2.69 (t, J = 8 Hz, 2H), 1.94ꢀ1.80 (m, 2H).
MS (ESI) m/z: 544.16 (M þ Hþ). Anal. (C26H24F3N5O3S) C, H, N.
4-(3-{3-[3-(2,6-Diisopropyl-phenyl)-ureido]-propyl}-5-phenyl-pyr-
azol-1-yl)-benzenesulfonamide (21d). The title compound was pre-
pared in 86% yield from 2,6-diisopropylphenyl isocyanate using the
Molecular Modeling. Molecular modeling was performed
using Scigress Explorer standard version 7.7.0.49 software (Fujitsu
Computer Systems Corporation). The atomic coordinates of the crystal
structures of human sEH complex with CIU (N-cyclohexyl-N0-(4-
iodophenyl)urea) and of murine COX-2 complexed with 2438 were
retrieved from Protein Data Bank (entry 1VJ5 and 1CX2, respectively).
21i was docked into the ligand-binding pocket manually by super-
position with the parent molecule (CIU or 2438) and minimized on MM
geometry (MM3). The image was produced using freewares VMD 1.8.6
(www.ks.uiuc.edu/Research/vmd) and POV-Ray 3.6 (www.povray.
org).
sEH IC50 Assay. For the recombinant affinity purified sEHs
(human, mouse, and rat), we used a fluorescent-based assay to deter-
mine IC50s.5 Enzymes (∼1 nM human sEH) were incubated with
inhibitors for 5 min in 25 mM Bis-Tris/HCl buffer (200 μL; pH 7.0) at
30 °C before substrate (cyano(2-methoxynaphthalen-6-yl)methyl
trans-(3-phenyl-oxyran-2-yl)methyl carbonate (CMNPC) was added
([S]final = 5 μM). Activity was assessed by measuring the appearance of
the fluorescent 6-methoxynaphthaldehyde product (λem . = 330 nm,
λex = 465 nm) at 30 °C during a 10 min incubation (Spectramax M2;
Molecular Device, Inc., Sunnyvale, CA). The IC50s were calculated from
at least three separate runs, each in triplicate, to obtain the standard
deviation given in the Results section. The IC50 was determined from at
least four points in the linear region of the inhibition curve with at least
one point above and one below the IC50.
Cyclooxygenase Inhibition Assay. The ability of the test
compounds 7, 11aꢀh, 15, and 21aꢀj to inhibit ovine COX-1 and
human recombinant COX-2 (% inhibition at 100 μM and IC50 values
(μM), respectively) was determined using an COX Fluorescent Inhi-
bitor Screening Assay Kit (catalogue no. 700100, Cayman Chemical,
Ann Arbor, MI) according to the manufacturer’s instructions. Stock
solutions of test compounds were dissolved in a minimum volume of
DMSO. Briefly, to a series of supplied reaction buffer solutions (150 μL,
100 mM Tris-HCl, pH 8.0) with either COX-1 or COX-2 (10 μL)
enzyme in the presence of Heme (10 μL) and fluorometric substrate (10
μL) were added 10 μL of various concentrations of the test compound
solutions ([I]final between 0.01 and 100 μM). The reactions were
initiated by quickly adding 10 μL of arachidonic acid solution and then
incubated for 2 min at room temperature. Fluorescence of resorufin that
is produced by the reaction between PGG2 and the fluorometric
substrate, 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), were analyzed
with an excitation wavelength of 535 nm and an emission wavelength of
590 nm. The intensity of this fluorescence is proportional to the amount
of resorufin, which is proportional to the amount of PGG2 present in the
well during the incubation. Percent inhibition was calculated by com-
parison from the 100% initial activity sample value (no inhibitor). The
concentration of the test compound causing 50% inhibition of COX-2
(IC50, μM) was calculated from the concentrationꢀinhibition response
curve (triplicate determinations).
Pharmacokinetic Protocol in Mice. All the animal experiments
were performed according to protocols approved by the Animal Use and
Care Committee of University of California, Davis. The PK study of
compounds 21b,eꢀj in a murine model was conducted according to the
protocol previously reported by Liu et al.42 Male CFW mice (8 week old,
24ꢀ30 g) were purchased from Charles River Laboratories. Compounds
21b,eꢀj (1 mg each) were dissolved in 1 mL of oleic ester-rich
triglyceride containing 20% polyethylene glycol (average molecular
weight: 400) to give a clear solution for oral administration. Cassette
1 contained compounds 21e, 21b, 21g, and rofecoxib, cassette 2,
compounds 21i, 21j, 21f, and celecoxib, cassette 3 compounds 21h,
and t-AUCB. Each cassette was orally administered to four different mice
at a dose of 5 mg/kg in 120ꢀ150 μL of vehicle depending on animal
weight. Blood (10 μL) was collected from the tail vein using a pipet tip
rinsed with 7.5% EDTA(K3) at 0, 0.5, 1, 1.5, 2, 4, 6, 8, and 24 h after oral
1
procedure detailed for compound 7; mp 137.9ꢀ140.7 °C. H NMR
(300 MHz, DMSO-d6): δ 7.79 (d, J = 9 Hz, 2H), 7.46ꢀ7.34 (m, 9H),
7.29ꢀ7.16 (m, 3H), 7.14ꢀ7.07 (m, 2H), 6.55 (s, 1H), 3.22ꢀ3.10 (m,
4H), 2.75ꢀ2.61 (m, 2H), 1.90ꢀ1.73 (m, 2H), 1.12 (d, J = 7 Hz, 12H).
MS (ESI) m/z: 560.27 (M þ Hþ). Anal. (C31H37N5O3S) C, H, N.
4-{5-Phenyl-3-[3-(3-phenyl-ureido)-propyl]-pyrazol-1-yl}-benze-
nesulfonamide (21e). The title compound was prepared in 90% yield
from phenyl isocyanate using the procedure detailed for compound 7;
mp 171.0ꢀ172.1 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.43 (s, 1H),
7.79 (d, J = 9 Hz, 2H), 7.47ꢀ7.36 (m, 9H), 7.29ꢀ7.16 (m, 4H), 6.87 (t, J =
7 Hz, 1H), 6.57 (s, 1H), 6.23 (t, J = 6 Hz, 1H), 3.25ꢀ3.14 (m, 2H), 2.69
(t, J = 8 Hz, 2H), 1.92ꢀ1.78 (m, 2H). MS (ESI) m/z: 476.18 (M þ Hþ).
Anal. (C25H25N5O3S 0.3CH4O) C, H, N.
3
4-{3-[3-(3-Adamantan-1-yl-ureido)-propyl]-5-phenyl-pyrazol-1-
yl}-benzenesulfonamide (21f). The title compound was prepared in
84% yield from 1-adamantyl isocyanate using the procedure detailed for
1
compound 7; mp 139.4ꢀ143.5 °C. H NMR (300 MHz, CDCl3): δ
7.75 (d, J = 9 Hz, 2H), 7.37ꢀ7.28 (m, 5H), 7.21ꢀ7.15 (m, 2H), 6.36 (s,
1H), 5.70ꢀ5.64 (m, 2H), 4.68ꢀ4.60 (m, 1H), 4.29 (s, 1H), 3.23 (q, J = 7
Hz, 2H), 2.78 (t, J = 7, 2H), 2.08ꢀ1.87 (m, 9H), 1.67ꢀ1.59 (m, 6H),
1.29ꢀ1.22 (m, 2H). MS (ESI) m/z: 534.25 (M þ Hþ).
4-{3-[3-(3-Cycloheptyl-ureido)-propyl]-5-phenyl-pyrazol-1-yl}-
benzenesulfonamide (21g). The title compound was prepared in 91%
yield from cycloheptyl isocyanate using the procedure detailed for
compound 7; mp 164.2ꢀ169.8 °C. 1H NMR (300 MHz, DMSO-d6):
δ 7.79 (d, J = 9 Hz, 2H), 7.47ꢀ7.36 (m, 7H), 7.29ꢀ7.22 (m, 2H), 6.55
(s, 1H), 5.82ꢀ5.70 (m, 2H), 3.63ꢀ3.49 (m, 1H), 3.13ꢀ3.02 (m, 2H),
2.63 (t, J = 8 Hz, 2H), 1.84ꢀ1.66 (m, 4H), 1.60ꢀ1.27 (m, 10H). MS
(ESI) m/z: 496.24 (M þ Hþ).
4-(3-{3-[3-(4-Chloro-phenyl)-ureido]-propyl}-5-phenyl-pyrazol-1-
yl)-benzenesulfonamide (21h). The title compound was prepared in
92% yield from 4-chlorophenyl isocyanate using the procedure detailed
1
for compound 7; mp 103.2ꢀ110.4 °C. H NMR (300 MHz, DMSO-
d6): δ 8.60 (s, 1H), 7.79 (d, J = 9 Hz, 2H), 7.47ꢀ7.35 (m, 9H),
7.29ꢀ7.21 (m, 4H), 6.57 (s, 1H), 6.29 (t, J = 6 Hz, 1H), 3.26ꢀ3.13 (m,
2H), 2.68 (t, J = 8 Hz, 2H), 1.92ꢀ1.77 (m, 2H). MS (ESI) m/z: 510.14
(M þ Hþ). Anal. (C25H24ClN5O3S CH4O) C, H, N.
3
4-(5-Phenyl-3-{3-[3-(4-trifluoromethyl-phenyl)-ureido]-propyl}-
pyrazol-1-yl)-benzenesulfonamide (21i). The title compound was
prepared in 85% yield from 3-trifluoromethylphenyl isocyanate using
the procedure detailed for compound 7; mp 184.8ꢀ185.6 °C. 1H NMR
(300 MHz, DMSO-d6): δ 8.90 (s, 1H), 7.79 (d, J = 9 Hz, 2H),
7.63ꢀ7.53 (m, 4H), 7.46ꢀ7.36 (m, 7H), 7.28ꢀ7.22 (m, 2H), 6.57 (s,
1H), 6.41 (t, J = 6 Hz, 1H), 3.27ꢀ3.15 (m, 2H), 2.69 (t, J = 8 Hz, 2H),
1.93ꢀ1.78 (m, 2H). MS (ESI) m/z: 544.16 (M þ Hþ). Anal.
(C26H24F3N5O3S) C, H, N.
4-(5-Phenyl-3-{3-[3-(4-trifluoromethoxy-phenyl)-ureido]-propyl}-
pyrazol-1-yl)-benzenesulfonamide (21j). The title compound was
prepared in 84% yield from 3-trifluoromethylphenyl isocyanate using
the procedure detailed for compound 7; mp 205.7ꢀ206.6 °C. 1H NMR
(300 MHz, DMSO-d6): δ 8.67 (s, 1H), 7.79 (d, J = 9 Hz, 2H), 7.49 (d, J
= 9 Hz, 2H), 7.45ꢀ7.36 (m, 7H), 7.29ꢀ7.18 (m, 4H), 6.57 (s, 1H), 6.30
(t, J = 5 Hz, 1H), 3.27ꢀ3.15 (m, 2H), 2.68 (t, J = 8 Hz, 2H), 1.92ꢀ1.79
(m, 2H). MS (ESI) m/z: 560.16 (M þ Hþ). Anal. (C26H24F3N5O4S)
C, H, N.
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Journal of Medicinal Chemistry
ARTICLE
administration. The extraction of compounds from blood was per-
formed by following our previous method except that ethyl acetate
(200 μL) was added after the addition of internal solution I (10 μL, 500
nM solution of 1-(4-chloro-3-trifluoromethanylphenyl-3-(1-cyclopro-
panecarboxylpiperidin-4-yl)urea in methanol) to each thawed blood
sample instead of prior to the addition of internal solution I. Blood
samples were analyzed using an Agilent 1200 Series HPLC equipped
with a 4.6 mm ꢁ 150 mm Inertsil ODS-4 3 μm column (GL Science
Inc., Japan) held at 40 °C and coupled with an Applied Biosystems
4000 QTRAP hybrid, triple-quadrupole mass spectrometer. The
instrument was equipped with a linear ion trap and a Turbo V ion
source and was operated in negative ion MRM mode. The solvent
system consisted of water/acetic acid (999/1 v/v, solvent A) and
acetonitrile/acetic acid (999/1 v/v; solvent B). The gradient was
begun at 30% solvent B and was linearly increased to 100% solvent
B in 5 min. This was maintained for 3 min and then returned to 30%
solvent B in 2 min. The flow rate was 0.4 mL/min. The injection
volume was 10 μL, and the samples were kept at 4 °C in the auto
sampler. The PK parameters of individual mice were calculated by
fitting the time dependent curve of blood inhibitor concentration to a
noncompartmental analysis with the WinNonlin software (Pharsight,
Mountain View, CA). Parameters determined include the time of
maximum concentration (Tmax), maximum concentration (Cmax), half-
life (t1/2), and area under the concentrationꢀtime curve to terminal
time (AUCt).
’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analysis data and
b
HRMS data, Synthesis of compounds 11h and 21c, human
5-LOX activity data, PK analysis in mice and rats, and data for
LPS pain model. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 530-752-7519 (office). Fax: 530-752-1537. E-mail:
bdhammock@ucdavis.edu.
’ ACKNOWLEDGMENT
This work was supported in part by NIEHS grant ES02710,
NIEHS Superfund grant P42 ES04699, and NIHLB grant
HL059699. Karen M. Wagner was supported by award no.
T32ES007059 from the National Institute of Environmental
Health Sciences. Aaron T. Wecksler was support by award no.
T32CA108459 from the National Institutes of Health. Bruce D.
Hammock is a George and Judy Senior Fellow of the American
Asthma Society. We gratefully acknowledge T. R. Holman for
generously providing the recombinant human 5-LOX enzyme.
Pharmacokinetic Protocol in Rats. Four male SpragueꢀDaw-
ley rats (8 week old, 250ꢀ300 g) were used for pharmacokinetic study
for dual inhibitors. Compounds 21i and 21j were given by oral
administration at the dose of 1 mg/kg. Inhibitors were dissolved in
oleic ester rich triglyceride containing 10% PEG 400 to form a clear
solution. Blood (10 μL) was collected from the tail vein by using a pipet
tip rinsed with 7.5% EDTA(K3) at 0, 0.5, 1, 1.5, 2, 4, 6, 8, and 24 h after
oral dosing with the inhibitor. Each blood sample was immediately
transferred to a tube containing 50 μL of water and mixed by Vortex for 1
min, all samples were stored at ꢀ80 °C until analysis. Blood sample
preparation and drug level quantification by LC/MS/MS were the same
as previous study.42 The pharmacokinetic parameters of individual rat
were calculated by fitting the data from blood concentrationꢀtime
dependent curve to a noncompartmental analysis with the WinNonlin
software (Pharsight, Mountain View, CA). Cmax, Tmax, t1/2, and AUC
were analyzed to characterize the pharmacokinetic profile of compounds
21i and 21j.
Von Frey Mechanical Nociceptive Assay. Male Spra-
gueꢀDawley rats weighing approximately 250ꢀ300 g were obtained
from Charles River Laboratories. On the day of the test, rats were
brought to the testing apparatus and allowed to acclimate. After 30
min, the rats were tested with a von Frey aesthesiometer for their
nontreated baseline withdrawal threshold to mechanical stimulation.
The von Frey apparatus is a raised metal mesh platform that has clear
acrylic chambers to enclose rats but allow them to move freely. A rigid
tip von Frey probe was used to probe the plantar surface of the rat
hind paw though the mesh floor to elicit a withdrawal response. The
measurements recorded are grams of force applied to the hind paw
required to elicit a withdrawal. The pretreatment baseline score was
considered the pain-free baseline (BL) and was assigned 100% for
further response calculations. For the pain assay, one of three
compounds sEHI, celecoxib, compound 21i, or a combination of
sEHI þ celecoxib formulated in 100% PEG400 was injected sub-
cutaneously 60 min prior to the intraplantar LPS injection. After the
10 μg LPS injection, rats were tested at 15, 30, 60, 120, 180, 240, 300,
and 360 min with the von Frey aesthesiometer for their withdrawal
threshold. Reported scores are an average of six rats (three trials per
rat) with SEM for the group. The measures were then calculated as a
percent of the pretreatment baseline score.
’ ABBREVIATIONS USED
t-AUCB, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-
benzoic acid; sEH, soluble epoxide hydrolase; COX-1, cycloox-
ygenase-1; COX-2, cyclooxygenase-2; 5-LOX, 5-lipoxygenase;
LPS, lipopolysaccharide; AUC, area under the concentrationꢀ
time curve; AA, arachidonic acid; PGs, prostaglandins; EETs,
epoxyeicosatrienoic acids; DHETs, dihydroxyeicosatrienoic acids;
20-HETE, 20-hydroxyeicosatetranoic acid; DML, designed
multiple ligand
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