Discovery of dual target inhibitors against cyclooxygenases and leukotriene A4 hydrolyase

Article abstract of DOI:10.1021/jm200063s

Dual target inhibitors against COX-2 and LTA₄H were designed by adding functional groups from a marketed COX-2 inhibitor, Nimesulide, to an existing LTA₄H inhibitor 1-(2-(4-phenoxyphenoxy) ethyl) pyrrolidine. A series of phenoxyphenyl pyrrolidine compounds were synthesized and tested for their inhibition activities using enzyme assays and human whole blood assay. Introduction of small electron withdrawing groups like NO₂ and CF₃ in the ortho-position of the terminal phenyl ring was found to change the original single target LTA₄H inhibitor to dual target LTA₄H and COX-2 inhibitors. Compound 5a and 5m showed dual LTA ₄H and COX-2 inhibition activities in the enzyme assays and the HWB assay with IC₅₀ values in the micromolar to submicromolar range. As their activities in HWB assay were comparable to the two starting single target inhibitors, the two compounds are promising for further studies. The strategy used in the current study may be generally applicable to other dual target drug designs.

Full text of DOI:10.1021/jm200063s

ARTICLE
pubs.acs.org/jmc
Discovery of Dual Target Inhibitors against Cyclooxygenases and
Leukotriene A4 Hydrolyase
Zheng Chen,§ Yiran Wu,§ Ying Liu,* Suijia Yang, Yunjie Chen, and Luhua Lai*
BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular
Engineering, Peking University, Beijing 100871, China
S Supporting Information
b
ABSTRACT: Dual target inhibitors against COX-2 and LTA4H
were designed by adding functional groups from a marketed
COX-2 inhibitor, Nimesulide, to an existing LTA4H inhibitor
1-(2-(4-phenoxyphenoxy) ethyl) pyrrolidine. A series of phe-
noxyphenyl pyrrolidine compounds were synthesized and
tested for their inhibition activities using enzyme assays and
human whole blood assay. Introduction of small electron with-
drawing groups like NO2 and CF3 in the ortho-position of the
terminal phenyl ring was found to change the original single
target LTA4H inhibitor to dual target LTA4H and COX-2 inhibitors. Compound 5a and 5m showed dual LTA4H and COX-2
inhibition activities in the enzyme assays and the HWB assay with IC50 values in the micromolar to submicromolar range. As their
activities in HWB assay were comparable to the two starting single target inhibitors, the two compounds are promising for further
studies. The strategy used in the current study may be generally applicable to other dual target drug designs.
’ INTRODUCTION
Nonsteriodal anti-inflammatory drugs (NSAIDs) have been
used for many years in treating acute and chronic inflammation.
It is believed that the main mechanism of action of NSAIDs is
blocking the formation of prostaglandins by inhibiting the
cyclooxygenase (COX) enzymes. As traditional NSAIDs cause
side effects such as gastrointestinal toxicity and mild bleeding by
inhibition of COX-1,1 selective inhibition of COX-2 was found to
be a safer way to gastrointestinal toxicity and mild bleeding.2 A
large number of selective COX-2 inhibitors have been developed
and approved for clinical use, such as Nimesulide (Figure 1),3
5-(4-fluorophenyl)-1-[4-(methylsulfonyl)phenyl]-3-(trifluoro-
methyl)-1H-pyrazole (SC-58125),4 and Rofecoxib (Figure 1),5
which represent significant advances in treating inflammatory
diseases. However, selective COX-2 inhibitors have been subse-
Figure 1. Chemical structures of COX-2 inhibitors Nimesulide, Rofe-
quently reported to have cardiovascular side effects in clinical
trials.6 Rofecoxib, Merck’s selective COX-2 inhibitor, was volun-
coxib, Celecoxib, and LTA4H inhibitor RB3041, 1-(2-(4-phenoxyphe-
noxy)ethyl)pyrrolidine (5z).
tarily withdrawn from the market in 2004,7 and other marketed
COX-2 inhibitors, like Celecoxib (Figure 1),8 were reappraised.9
in using single-target-based drugs in treating a complex disease.
The reasons for the toxicity of these drugs have not been fully
One possibility to overcome the limitations of single-target-
revealed. One hypothesis suggests that these inhibitors induce an
based drugs is to design and/or find multitarget drugs. Complex
imbalance between prostacyclin (PGI2), an antiaggregant and
vasodilator mainly synthesized via the action of COX-2, and
thromboxane A2 (TXA2), a molecule with strong pro-aggregant
diseases like cancer, AIDs, and atherosclerosis can benefit from
complex treatment that modulates multiple targets.11 In order to
understand the possible side effects of NSAIDs, we have built
and vasoconstrictor activity mainly produced through the COX-1
mathematical models for the inflammation-related arachidonic
pathway.10
acid metabolic network in human polymorphous neutrophil,12
The activity of drug molecules in the disease network can be
complex, and problems occur when a single-target-based mole-
cule is involved in multiple targets. The failure of unselective and
selective COX inhibitors provides an example of the limitations
Received: January 21, 2011
Published: May 04, 2011
r
2011 American Chemical Society
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Figure 3. Design strategy for COX-2/LTA4H dual inhibitors.
Among the multitarget control solutions, inhibitors that can
act on both COX-2 and 5-LOX have been explored for some time
and were shown to possess a wide range of anti-inflammatory
activity.14 Recent research has focused on the development of
COX-2/5-LOX dual inhibitors with a superior safety profile. One
of the most successful strategies is to design a hybrid molecule, a
COX-2 inhibitor analogue possessing a 5-LOX pharmacophore,
or a compound containing a COX-2 fragment and a 5-LOX
fragment, such as some celecoxib analogues15 and Tepoxalin.16
In the current study, we tried to design inhibitors that can
inhibit both COX-2/COX-1 and LTA4H at the same time. As
COX-1 and COX-2 have quite similar structures, most COX
inhibitors act on both of them. From our previous simulations
and literature, around 10ꢀ selectivity of COX-2 to COX-1 is
good for maintaining the balance between PGI2 and TXA2.13
To our knowledge, no COX-2/LTA4H dual target inhibitors
have been reported. A possible difficulty in designing dual target
inhibitors for these enzymes is the large difference between their
substrate binding pockets. A cavity search showed that the
LTA4H enzyme contains a long narrow substrate binding
channel (Figure 2, A), so most inhibitors have a long chain
without ramification; while the pocket of COX-2 enzyme is much
wider (Figure 2, B), and typical COX-2 inhibitors are in a
V-shape. However, some compounds with a branched structure
can also bind to LTA4H at the entrance of the substrate binding
pocket, such as RB3041 complexed to LTA4H in the crystal
structure (PDB code 2R59, Figure 1).17 This implies that it is
possible to design compounds that bind to both COX-2 and
LTA4H.
Figure 2. Substrate binding pockets of LTA4H (A) and COX-2 (B).
Multitarget inhibitor design has attracted much attention
recently.18ꢁ21 Both target structure-based and ligand-based de-
sign strategies have been tested. For target structure-based
design, sequential docking and multiple cross docking are the
straightforward strategies, which are all computationally de-
manding. We have developed a docking combined with a
common pharmacophore matching strategy for multitarget in-
hibitor design and successfully used it to discover dual functional
inhibitors for sPLA2 and LTA4H.22 Compared to target struc-
ture-based discovery, ligand-based multitarget inhibitor design
learns from the chemical structures of known inhibitors and tries
to combine them.23,24 The chances of deriving compounds that
are active for one of the targets are high, while it is difficult to
retain all the desirable activities at the same time.
and in a mixture of three cell types simulating a stretch of human
blood vessel.13 The two major inflammatory mediators, prosta-
glandins and leukotrienes, are produced through the COX
pathway and the 5-lipoxygenase (5-LOX) pathway, respectively.
Our simulation verified that there are five key enzymes for
inhibitor design to reduce the production of inflammatory
mediators, which include COX (COX-2/COX-1), 5-LOX, pros-
taglandin E synthase (PGES), LTA4H, and phospholipase A2
(PLA2). We also searched for multiple target optimum interven-
tion solutions that can simultaneously reduce the production of
both inflammatory mediators and keep a good balance of PGI2
and TXA2 (minimizing possible side effects). All of the solutions
include at least inhibition of two targets at the same time, for
example, the inhibition of COX-2/COX-1 and 5-LOX, the
inhibition of COX-2/COX-1 and LTA4H, and the inhibition of
PLA2, LTA4H, and COX-2/COX-1.
Our rational design of COX-2/LTA4H dual inhibitors
was mainly ligand-based, while molecular docking of the
designed compounds to both enzymes was also performed
to compare their interaction affinities. We started from the fusion
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Scheme 1a
a Reagents: (a) RX, KOH, 170 °C; (b) RX, K2CO3 or KOH, TDA, CuI or CuBr, Anisole or NMP, N2, 100ꢁ170 °C; (c) BBr3, CH2Cl2, 0 °C ∼rt,H2O;
(d) 1-(2-chloroethyl)pyrrolidine, KOH, DMF, 100 °C.
Scheme 2a
a Reagents: (a) 85% NH2NH2, IPA, Pd/C, reflux; (b) MeSO2Cl, TEA, CH2Cl2, rt; (c) MeSO2Cl, H2O, rt.
of 1-(2-(4-phenoxyphenoxy)ethyl) pyrrolidine (Figure 1, 5z), a
LTA4H inhibitor reported by Pennings,25 and Nimesulide
(Figure 1),3 a marketed NSAID targeting COX-2. Though these
two compounds are different in shape, they share the same
scaffold phenoxyphenyl. We used the slim-shaped LTA4H
inhibitor as the parent compound and added functional groups
in Nimesulide to it (Figure 3). Other substituted groups in o-, m-,
and p-position of the first phenyl were also considered.
A series of 1-(2-(4-phenoxyphenoxy)ethyl) pyrrolidine deri-
vatives were synthesized, and their inhibition activities to purified
enzymes and in human whole blood assay were tested. Molecular
docking of these compounds to COX-2, COX-1, and LTA4H was
done to analyze their structure and activity relationship.
Nitro groups in 5aꢁc could be further reduced to amine
groups to form (4-(2-(pyrrolidin-1-yl) ethoxy)phenoxy) aniline
(6aꢁc). N-(Methylsulfonyl)-N-(2-(4-(2-(pyrrolidin-1-yl) ethoxy)-
phenoxy)phenyl) methanesulfonamide (7a) and N-(methyl-
sulfonyl)-N-(3-(4-(2-(pyrrolidin-1-yl)ethoxy)phenoxy)phenyl)
methanesulfonamide (7b) were obtained by dimethylsulfonyla-
tion of 6a or 6b, and N-(4-(4-(2-(pyrrolidin-1-yl)ethoxy)phenoxy)-
phenyl) methanesulfonamide (8c) was provided by mono-
methylsulfonylation of 6c (Scheme 2).
N-Methyl-N-(2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenoxy)phenyl)
methanesulfonamide (13a) was achieved starting from 3a in five
steps (Scheme 3): 3a returned its 2-nitro group to 2-amine group
to form 2-(4-methoxyphenoxy) aniline (9a), then methylsulfo-
nylation of 2-amine group 9a produced N-(2-(4-meth-
oxyphenoxy)phenyl) methanesulfonamide (10a), methylation
of 2-methanesulfonamide group 10a produced N-(2-(4-meth-
oxyphenoxy)phenyl)-N-methylmethanesulfonamide (11a), de-
methylation of 11a produced N-(2-(4-hydroxyphenoxy)-
phenyl)-N-methylmethanesulfonamide (12a), and alkylation of
12a reached 13a.
In Vitro Enzyme Inhibition Study. The 25 synthesized target
compounds were tested for their inhibition activity against
purified COX-1, COX-2, and LTA4H (shown in Table 1).
The LTA4H hydrolase activity was measured using an ELISA
assay to quantify the amount of LTB4 generated. The known
LTA4H inhibitor, 1-(2-(4-phenoxyphenoxy)ethyl) pyrrolidine
(5z) was used as a positive control.
COX-2 is one of the two distinct isoforms of COX, which can
convert arachidonic acid to a hydroperoxy endoperoxide, PGG2,
and subsequently reduce it to corresponding alcohol, PGH2. The
enzyme activity of the purified COX-2 was measured by a
chromogenic assay based on monitoring the absorption of oxi-
dized N,N,N0,N0-tetramethyl-p-phenylenediamine (TMPD) dur-
ing the reduction of PGG2 to PGH2 at 610 nm. A known COX-2
inhibitor, Nimesulide, was used as a positive control. Suitable
’ RESULTS
Chemistry. The 1-(2-(4-phenoxyphenoxy)ethyl) pyrroli-
dine derivatives were synthesized by the routes shown in
Schemes 1,ꢁ3.
Substituted 1-phenoxyethylpyrrolidine compounds (5aꢁr)
were prepared according to Scheme 1 starting from commercially
available aryl halides or substituted phenoxyphenol. 1-(4-Meth-
oxyphenoxy)-2-nitrobenzene (3a) and 1-(4-methoxyphenoxy)-
4-nitrobenzene (3c) were obtained through direct heating to
couple the chloronitrobenzene with 4-methoxyphenol, and other
compounds need to be coupled through Goldberg reaction using
CuBr/TDA. Demethylation of 3 yielded a series of substituted
phenoxyphenol key intermediates, including 4-(2-nitrophenoxy)
phenol (4a) and similar compounds (4bꢁj) by boron tribro-
mide, and 4-(pyrazin-2-yloxy) phenol (4k) and similar com-
pounds 4lꢁn by Goldberg reaction through monocoupling
commercial aryl halide with hydroquinone. Then, these substi-
tuted phenoxyphenols were alkylated to produce 5aꢁn. 5z and
other 5 compounds were prepared directly from corresponding 4
compounds in one step.
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Scheme 3a
a Reagents: (a) Sn, HCl, EtOH, reflux; (b) MeSO2Cl, Pyridine, 90 °C; (c) (MeO)2SO2, K2CO3, acetone, reflux; (d) BBr3, CH2Cl2, 0 °C; (e) 1-(2-
chloroethyl)pyrrolidine, KOH, K2CO3, DMF, 100 °C.
absorption could be observed by adding surfactant Genapol
X-100.26 The IC50 value of Nimesulide derived under this condi-
tion was 23 nM, which was close to the reported value of 30 nM.27
Human Whole Blood Assay Study. We tested the inhibition
activity of the title compounds using human whole blood
(HWB) assay (shown in Table 1).
In order to monitor the production of both PGE2 and LTB4 at
the same time, we developed an assay that can simultaneously
stimulate both the COX and 5-LOX pathways.
E. coli lipopolysaccharide (LPS) was used to induce the COX-
2/PGES pathway in human whole blood, and the production of
PGE2 was monitored, while the 5-LOX/LTA4H pathway was
stimulated using calcium ionophore A23187 and the production
of LTB4 was measured. Heparinized HWB was first treated with
LPS for 23.5 h, then ionophore A23187 was added to the sample
for 0.5 h stimulation. As a control, we also tested whether LTB4
can be produced when stimulating with LPS and whether PGE2
can be produced with A23187, which turned out to be negligible
compared to their respective main products.
(5a), CF3 (5m), NMe (SO2Me) as the o-substituent, and NO2
(5c), NH2 (6c) as the p-substituent, pyridin-2-yl (5e), or
pyrazin-2-yl (5k) in the first phenyl ring, exhibited moderate
COX-2 inhibitory activities. Compound 5a having an o-NO2
showed selective COX-2 inhibitory activity (COX-2, IC50
=
7.7 μM; COX-1, IC50 = 61.0 μM). In contrast, compound 6c
having p-NH2 tends to be a selective COX-1 inhibitor (COX-2,
IC50 = 36.3 μM; COX-1, IC50 = 1.9 μM).
As the purpose of the current study was to design multitarget
inhibitors of COX-1, COX-2, and LTA4H, we compared the
inhibition activities of the title compounds against COX-1, COX-
2, and LTA4H. Three compounds (5a, 5c, and 6c) showed
significant inhibition activity toward the three enzymes. In
particular, 5a inhibits LTA4H with an IC50 of 0.7 μM and inhibits
COX-1 and COX-2 with IC50 of 61 μM and 7.7 μM, respectively.
We estimated possible side effects of 5a using the mathema-
tical model for the arachidonic acid metabolic network we
previously developed for the human blood vessel.13 Compound
5a was introduced into the network as inhibitor for LTA4H,
COX-1, and COX-2 with its IC50 values measured in the enzyme
assays. Concentration of 5a used could reduce the PGE2 and
LTB4 formation to less than 10% compared to no inhibition. The
ratio of PGI2/TXA2 was used as an index of side effects, which
was calculated to be 0.7820 here, quite close to the normal value
of 0.6817. Thus, 5a has good target selectivity and might
maintain the balance between PGI2 and TXA2 in vivo.
Human Whole Blood Assay Study. From the HWB assay,
compound 5a showed moderate inhibition to both PGE2 and
LTB4 with IC50 values of 8.4 μM and 6.9 μM, respectively,
indicating that the introduction of o-NO2 can successfully make
the compound inhibit the formation of PGE2, though its inhibi-
tion activity of LTB4 production was also slightly reduced. The
introduction of an o-CF3 (5m) led to a notable PGE2 forma-
tion inhibition, while keeping the inhibition activity to the
biosynthesis of LTB4. In fact, IC50 values of 5m, 5.0 μM for PGE2
and 0.73 μM for LTB4 formation, were comparable to those of
the two reference molecules. Though the introduction of a p-Br
moiety (5r) also led to a notable PGE2 formation inhibition, its
functions might be more complex, as we did not detect any
notable COX-1 and COX-2 inhibition in our enzyme assay; it is
possible that inhibition of upstream enzymes like PLA2, or
downstream enzyme PGES, may be involved.
’ DISCUSSION
In Vitro Enzyme Inhibition Study. For the first step, we tested
whether the title compounds could retain the LTA4H inhibition
activity. Inhibition testing against LTA4H hydrolase activity
indicated that most compounds maintained the LTA4H inhibi-
tion activity. IC50 values of 14 compounds were less than 1 μM
and that of 3 compounds were in the nanomolar range. This
encouraged us to do further study. From Table 1, we can see that
the three most potent inhibitors were 4-halogenphenoxy-sub-
stituted compounds (5p, 5q, and 5r), with IC50 values between
0.04 and 0.11 μM. In two series of compounds 5aꢁc (nitro-
substituted) and 6aꢁc (amino-substituted), which only differed
in the position of substituted group in the terminal aryl ring;
inhibition activities of p-substituted compounds (5c and 6c)
were stronger than the others; while for the trifluoromethyl-
substituted series 5mꢁo, o-substituted compound 5m had the
strongest inhibition effect. When the aryl group was pyridine
(5e and 5f), the compounds also had strong inhibition activity.
However, compounds with large substituted groups in the
terminal aryl ring had weak or no inhibition activity, except the
p-methanesulfonamide substituted compound 8c with moderate
inhibition activity.
This human whole blood assay can provide inhibition activity data
under more realistic conditions. However, the inhibition activities to
COX-1 cannot be assayed at the same time, since the measurement
We then tested the COX-2 and COX-1 inhibition activity of
the title compounds (Table 1). Seven compounds, with a NO2
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Table 1. COX-1, COX-2, and LTA4H Inhibition Activities of the Title Compounds
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Table 1. Continued
a The structures of these known inhibitors as positive control are provided in Figure 1.
Figure 4. Inhibitors in the LTA4H substrate binding pocket (predicted by molecular docking). A. 6c (magenta). B. 5a (green). C. 8c (cyan).
of COX-1 in HWB is typically based on TXB2 production through
the COX-1/TXAS pathway, either in spontaneously clotting, un-
heparinized blood28 or in only A23187 stimulated, heparinized
blood; however, the addition of LPS could also induce the produc-
tion of TXB2 through the COX-2/TXAS pathway.29
Combining the enzyme assay and HWB assay data, two
compounds, 5a and 5m, were found to meet the requirements
for dual COX-2 and LTA4H inhibition and a good selectivity for
COX-2 over COX-1.
alkyl group to His295, and the oxygen atom connected to
bibenzyl to Gly268 main-chain nitrogen atom (Figure 4B). Some
compounds may form extra hydrogen bonds to the enzyme using
their substituted groups. For example, NO2 of 5a forms a
hydrogen bond to Arg563. This binding mode corresponded
with other peoples’ docking results31 and can be seen in crystal
structures (PDB code 2R59 and 3B7R).17 (3) Compounds
possessing both o- and m-substituted groups on the terminal
aryl ring (5d, 5j) cannot enter the long and narrow substrate
channel of LTA4H, so they did not show inhibition activities to
LTA4H. (4) When the compounds have large substituted group
such as methylsulfonyl or aminomethanesulfonyl (5g, 5h, 5i, 7a,
7b, 8c, 13a), inhibition activities to LTA4H of the compounds
were lower except 8c gave an IC50 value 3.4 μM. Molecular
docking result showed that 5g, 5h, 5i, 7a, 7b, and 13a would
collide with the pocket, and only compound 8c entered into the
narrow cleft of the LTA4H pocket and stayed in the same
conformation as 6c (Figure 4C). The large methylsulfonyl group
of 8c was a little deeper than the position of Phe314. In
comparison, the methylsulfonyl group of compound 5i was
directly connected to the phenyl ring and would collide with
the side chain of Phe314. Compounds in case (1) and (2) all
maintained the inhibition activity of the parent compound 5z,
while those in cases (3) and (4) lost inhibition activity.
Molecular Docking. Molecular docking was performed to
study the structureꢁfunction relationship of the title compounds
toward the three enzymes.
For LTA4H, docking results showed that position and size of
substituted groups played a key role in the binding conformation
of the compounds. There are four cases: (1) the parent com-
pound 5z, compounds without branches (5e, 5f, 5k), and p-
substituted compounds (5c, 5o, 5p, 5q, 5r, 6c) tended to bind
LTA4H in the channel, forming a hydrogen bond between the
pyrrolidine nitrogen atom and Gly269 main-chain oxygen atom
(Figure 4A). Compounds may also interact with the residues at
the end of the channel, such as hydrogen bond between 6c amino
group and Trp311 main-chain oxygen atom. This binding mode
of Figure 4A and C is a typical conformation in LTA4H and can
be found in many crystal structures including the first reported
structure (PDB code 1HS6).30 (2) o- or m-Substituted com-
pounds (5a, 5b, 5l, 5m, 5n, 6a, 6b) can stay at the entrance part
of the pocket in a reversed orientation with three hydrogen
bonds (nitrogen atom to Gln136, the oxygen atom connected to
For COX enzymes, 7 compounds (5a, 5c, 5e, 5k, 5m, 6c, 13a)
showed inhibition activity. 5a, 5c, and 6c inhibit both COX-1
and COX-2, but with different selectivity. 5a was 8 times
more selective toward COX-2, with IC50 of 7.7 ( 0.3 μM and
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Figure 5. Inhibitors in the substrate binding pocket of COX enzymes (predicted by molecular docking). A. 5a (green) in COX-2. B. 6c (magenta) in
COX-1.
61.0 ( 0.4 for COX-2 and COX-1, respectively. 6c displayed
more inhibition activity toward COX-1, with IC50 of 1.9 ( 0.1
and 36.3 ( 1.1 μM for COX-1 and COX-2, respectively.
Molecular docking can well explain the activity difference due
to different interactions of 5a and 6c to COX-1 and COX-2. The
pyrrolidine group of 5a extended to a hydrophobic pocket
around Phe205 in COX-2 (Figure 5A) in a similar binding mode
with other reported COX inhibitors.32 Three hydrogen bonds
were formed between the inhibitor and the enzyme. The terminal
phenyl ring of 5a was close to Val523 and might collide with
Ile523 in COX-1, explaining COX-2 selectivity of 5a. Val523 also
prevents the binding of p-substituted compounds COX-2 in this
conformation. Compound 6c bound to COX-1 in a different
conformation with two hydrogen bonds (Figure 5B). The amino
group of 6c was close to Leu357, while in COX-2, the larger side
chain of Phe357 might collide with 6c and reduce its binding
affinity. There was no space for o- or m-substituted group on the
terminal ring so these compounds cannot bind to COX-1 in this
conformation. Thus, compounds with an electron-drawing sub-
stituted group in the p-position have the potential for COX-2
selectivity, while p-substituted compounds may bind to COX
enzyme in a different conformation that fit the pocket of COX-2
better and have COX-1 selectivity. In either situation, the
substituted group cannot be too large, for no compounds with
substituted groups larger than methylsulfonyl had high inhibition
activity to COX enzymes.
’ EXPERIMENTAL SECTION
Chemistry. The reagents and solvents were commercially available
and purified according to conventional methods. All reactions were
monitored by thin layer chromatography, carried out on silica gel 60
F-254 aluminum sheets using UV light (254 and 366 nm). All new
1
compounds gave satisfactory H NMR, elemental analyses, and mass
spectrometry analyses. 1H NMR spectra and 13C NMR were measured
on a Bruker-400 M spectrometer using TMS as internal standard.
Elemental analyses were preformed on an Elementar Vario EL instru-
ment. Mass spectra were recorded on a VG-ZAB-HS spectrometer. All
target compounds possessed a purity of g95% as verified by elemental
analyses by comparison with the theoretical values.
General Procedure for the Preparation of 1-(4-Methoxyphenoxy)-2-
nitrobenzene (3a). A mixture of 4-methoxyphenol (2.0 g, 16 mmol) and
anhydrous potassium hydroxide (1.0 g, 18 mmol) were heated at 150 °C
and stirred for 10 min. After the mixture was melted, 1-chloro-2-
nitrobenzene (2.0 g, 13 mmol) was added and the mixture was stirred
at 170 °C for 2 h (TLC monitor). The reaction mixture was poured into
50 mL of 3% potassium hydroxide aqueous solution and stirred at room
temperature for about 2 h and then cooled to 4 °C. The solid was filtered
with suction and washed with water. The crude product was recrystal-
1
lized by ethanol as a yellow solid and weighed 2.8 g (yield 90%). H
NMR (400 MHz, DMSO-d6) δ 7.93 (1H, d), 7.44 (1H, m), 7.26ꢁ6.90
(6H, m), 3.82 (3H, s).
3c was synthesized by a similar procedure.
1-(4-Methoxyphenoxy)-3-nitrobenzene (3b). A solution of 1-bro-
mo-2-nitrobenzene (2.0 g, 9.9 mmol), 4-methoxyphenol (3.7 g, 30
mmol), anhydrous potassium carbonate (7.4 g, 54 mmol), cuprous
bromide (0.15 g, 1 mmol), and TDA (0.34 g, 1 mmol) in 15 mL of
anisole was stirred at 170 °C under a nitrogen atmosphere for 48 h. The
solution was cooled and diluted with ethyl ether, washed with 1 M KOH
twice and water. The organic layer was evaporated on a rotary evaporator
and then vacuum distilled. The residue was purified by flash chroma-
tography (silica gel, the eluting solvent ranged from hexane to EtOAc/
hexane is 1:15) as a pale-yellow solid and weighed 1.3 g (yield 54%). 1H
NMR (400 MHz, DMSO-d6) δ 7.94 (1H, d), 7.64 (1H, t), 7.59 (1H, t),
7.42 (1H, d), 7.14 (2H, m), 7.04 (2H, m), 3.79 (3H, s).
General Procedure for the Preparation of 1-(4-Methoxyphenoxy)-
2,4-dinitrobenzene (3d). Under the protection of nitrogen, a solution of
1-chloro-2,4-dinitrobenzene (1 g, 4.9 mmol), 4-methoxyphenol (0.75 g,
6 mmol), anhydrous potassium carbonate (3.4 g, 24 mmol), cuprous
bromide (0.15 g, 1 mmol), and TDA (0.34 g, 1 mmol) in 10 mL NMP
was heated to 110 °C and stirred for 1 h. The reaction mixture was
cooled and diluted with ethyl ether, washed with 1 M KOH twice and
’ CONCLUSIONS
We have successfully designed and synthesized a series of
COX-2/LTA4H dual inhibitors by adding groups from known
COX-2 inhibitors to an existing LTA4H inhibitor 1-(2-(4-
phenoxyphenoxy)ethyl) pyrrolidine backbone. The inhibition
activities of the compounds indicate that introducing small
electron-withdrawing groups like NO2 and CF3 in the ortho-
position of the phenyl ring can make the original LTA4H
inhibitor turn into a dual functional COX-2/LTA4H inhibitor.
Compounds 5a and 5m showed dual COX-2/LTA4H inhibition
activities in the enzyme assay, as well as in the HWB assay with
good COX-2/COX-1 selectivity, which are promising for further
studies. The strategy used in the current study for designing dual
functional inhibitors may be generally applicable in dual func-
tional drug design against other targets.
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Journal of Medicinal Chemistry
ARTICLE
water, dried over Na2SO4, concentrated at reduced pressure, and the
residue was separated by flash chromatography (silica gel, the eluting
solvent ranged from hexane to EtOAc/hexane = 1:5) as a yellow solid
and weighed 1.0 g (yield 70%). 1H NMR (400 MHz, DMSO-d6) δ 8.88
(1H, d), 8.43 (1H, d), 7.24 (2H, m), 7.08 (3H, m), 3.77 (3H, s).
3eꢁj were synthesized using a similar procedure.
4.08 (2H, t), 2.74 (2H, t), 2.66 (4H, m), 1.74 (4H, s). 13C NMR (400
MHz, DMSO-d6) δ 163.75, 156.04, 147.25, 141.84, 126.11, 121.88,
116.55, 115.96, 67.12, 54.28, 53.98, 23.14. Anal. (C18H20N2O4).
1-(2-(4-(2,4-Dinitrophenoxy)phenoxy)ethyl) pyrrolidine (5d). The
title compound was prepared from 4d as a yellow oil (yield 36%). H
NMR (400 MHz, DMSO-d6) δ 8.88 (1H, d), 8.45 (1H, m), 7.24 (2H,
1
General Procedure for the Preparation of 4-(2-Nitrophenox-
y)phenol (4a). 3a (1.0 g, 4 mmol) was stirred in CH2Cl2 (20 mL) at
0 °C, and 0.57 mL (6 mmol) BBr3 in 20 mL CH2Cl2 was slowly dropped
into the solution above. After being stirred at room temperature for
about 3 h, the mixture was poured into H2O. The organic layer was
washed with brine, dried over Na2SO4, and concentrated at reduced
pressure and the residue was separated by flash chromatography (silica
gel, the eluting solvent ranged from hexane to EtOAc/hexane = 1:2) as a
m), 7.11 (3H, m), 4.22 (2H, t), 3.24 (2H, t), 2.99 (4H, m), 1.84 (4H, m).
Anal. (C18H19N3O6 0.4H2O).
3
2-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenoxy)pyridine (5e). The title
compound was prepared from 4e as a yellow oil (yield 48%). 1H
NMR (400 MHz, DMSO-d6) δ 8.12 (1H, d), 7.82 (1H, t), 7.07
(3H, m), 6.97 (3H, m), 4.08 (2H, t), 2.85 (2H, t), 2.59 (4H, s), 1.71
(4H, s). Anal. (C17H20N2O2 0.5H2O).
3
4-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenoxy)pyridine (5f). The title com-
pound was prepared from 4f as a yellow oil (yield 22%). 1H NMR (400
MHz, DMSO-d6) δ 8.42 (2H, d), 7.02 (4H, m), 6.82 (2H, d), 4.08 (2H, t),
1
yellow solid and weighed 0.87 g (yield 92%). H NMR (400 MHz,
DMSO-d6) δ 9.50 (1H, s), 8.00 (1H, d), 7.60 (1H, m), 7.24 (1H, m),
6.97ꢁ6.80 (5H, m).
2.76 (2H, t), 2.65 (4H, m), 1.73 (4H, s). Anal. (C17H20N2O2 0.3H2O).
1-(2-(4-(2-(Methylsulfonyl)phenoxy)phenoxy)ethyl) pyrrolidine
3
4bꢁj were synthesized by a similar procedure.
General Procedure for the Preparation of 4-(pyrazin-2-yloxy)phenol
(4k). Under the protection of nitrogen gas, a solution of 2-chloropyr-
azine (0.4 g, 3.5 mmol), hydroquinone (1.92 g, 17.5 mmol), anhydrous
potassium carbonate (3.0 g, 23.6 mmol), cuprous bromide (0.25 g, 1.8
mmol), and TDA (0.6 g, 1.8 mmol) in 25 mL of NMP was stirred at
120 °C for about 1 h (TLC monitor). The solution was diluted with
ethyl ether, washed with saturated KHCO3 aqueous solution and water,
dried over Na2SO4, and concentrated at reduced pressure, and the
residue was separated by flash chromatography (silica gel, the eluting
solvent ranged from hexane to EtOAc/hexane = 1:1) as a pale-yellow
solid and weighed 0.59 g (yield 90%). 1H NMR (400 MHz, DMSO-d6)
δ 9.34 (1H, s), 8.11 (1H, d), 7.79 (1H, t), 7.01 (2H, t), 6.91 (2H, m),
6.78 (2H, m).
(5g). The title compound was prepared from 4g as a white solid
1
(yield 80%). H NMR (400 MHz, DMSO-d6) δ 7.90 (1H, d), 7.64
(1H, t), 7.29 (1H, t), 7.11 (2H, d), 7.11 (2H, d), 7.03 (2H, d), 6.88 (1H, d),
4.07 (2H, t), 3.36 (3H, s), 2.79 (2H, t), 2.51 (4H, m), 1.69 (4H, m).
Anal. (C19H23NO4S).
1-(2-(4-(3-(Methylsulfonyl)phenoxy)phenoxy)ethyl) pyrrolidine
(5h). The title compound was prepared from 4h as a white solid
1
(yield 74%). H NMR (400 MHz, DMSO-d6) δ 7.96 (1H, s), 7.63
(2H, d), 7.34 (1H, m), 7.11ꢁ7.02 (4H, m), 4.08 (2H, t), 3.22 (3H, s),
2.81 (2H, t), 2.54 (4H, m), 1.69 (4H, m). ESI-HRMS m/z 362.1415
[M þ H]. Anal. (C19H23NO4S).
1-(2-(4-(4-(Methylsulfonyl)phenoxy)phenoxy)ethyl) pyrrolidine
(5i). The title compound was prepared from 4i as a white solid (yield
86%). 1H NMR (400 MHz, DMSO-d6) δ 7.88 (2H, d), 7.11ꢁ7.02 (6H,
m), 4.08 (2H, t), 3.36 (3H, s), 2.80 (2H, t), 1.69 (4H, s). ESI-HRMS m/
z 362.1418 [M þ H]. Anal. (C19H23NO4S).
4lꢁn were synthesized by a similar procedure.
General Procedure for the Preparation of 1-(2-(4-(2-Nitrophenoxy)-
phenoxy)ethyl) pyrrolidine (5a). A solution of 4a (0.2 g, 0.87 mmol)
and anhydrous potassium hydroxide (0.32 g, 5.7 mmol) in DMF was
1-(2-(4-(4-(Methylsulfonyl)-2-nitrophenoxy)phenoxy)ethyl) pyrroli-
dine (5j). The title compound was prepared from 4j as a yellow solid
(yield 86%). 1H NMR (400 MHz, DMSO-d6) δ 8.55 (1H, d), 8.11 (1H,
d), 7.20 (2H, m), 7.12ꢁ7.06 (3H, m), 4.10 (2H, t), 3.30 (3H, s), 2.85
(2H, t), 2.58 (4H, m), 1.71 (4H, m). Anal. (C19H22N2O6S).
2-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenoxy) pyrazine (5k). The title
stirred at 80ꢁ90 °C for 10 min. Then, 1-(2-chloroethyl) pyrrolidine
3
HCl (0.36 g, 2.1 mmol) was added and the solution was stirred at
100 °C for about 0.5 h (TLC monitor). The solution was cooled, poured
into water, and extracted with EtOAc twice. The combined organic
extracts were washed with 1 M KOH and water, dried over Na2SO4,
and concentrated in vacuo. The crude product was purified by flash
chromatography (silica gel, the eluting solvent ranged from CH2Cl2 to
CH2Cl2/MeOH = 9:1) as a yellow oil and weighed 0.19 g (yield 68%).
1H NMR (400 MHz, DMSO-d6) δ 8.02 (1H, d), 7.64 (1H, t), 7.29
(1H, t), 7.07ꢁ6.98 (5H, m), 4.06 (2H, t), 2.80 (2H, t), 2.53 (4H, m),
1.70 (4H, m). 13C NMR (400 MHz, DMSO-d6) δ 154.60, 150.31,
149.07, 140.61, 134.85, 125.50, 123.36, 120.64, 119.42, 116.25, 63.74,
53.52, 52.49, 22.62. ESI-HRMS m/z 329.1492 [M þ H]. Anal.
1
compound was prepared from 4k as a light red solid (yield 83%). H
NMR (400 MHz, DMSO-d6) δ 8.56 (1H, d), 8.40 (1H, d), 7.35
(1H, m), 7.13 (2H, d), 7.00 (2H, d), 4.08 (2H, t), 2.82 (2H, t), 2.53
(4H, m), 1.70 (4H, s). Anal. (C16H19N3O2 0.2H2O).
3
3-Nitro-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenoxy)pyridine (5l). The
title compound was prepared from 4l as a yellow solid (yield 73%).1H
NMR (400 MHz, DMSO-d6) δ 8.55 (1H, d), 8.39 (1H, t), 7.35 (1H, m),
7.13 (2H, d), 6.99 (2H, d), 4.08 (2H, t), 2.82 (2H, t), 1.70 (4H, s). Anal.
(C17H19N3O4).
(C18H20N2O4 0.3H2O).
3
1-(2-(4-(2-(Trifluoromethyl)phenoxy)phenoxy)ethyl) pyrrolidine
(5m). The title compound was prepared from 4m as a colorless oil (yield
75%).1H NMR (400 MHz, DMSO-d6) δ 7.58 (1H, t), 7.42 (1H, d),
7.22ꢁ7.18 (2H, m), 7.08ꢁ7.00 (4H, m), 4.07 (2H, t), 2.79 (2H, t), 2.53
(4H, m), 1.69 (4H, m). Anal. (C19H20F3NO2).
1-(2-(4-(3-(Trifluoromethyl)phenoxy)phenoxy)ethyl) pyrrolidine
(5n). The title compound was prepared from 4n as a colorless oil (yield
73%).1H NMR (400 MHz, DMSO-d6) δ 7.58 (1H, t), 7.42 (1H, d),
7.22ꢁ7.18 (2H, m), 7.08ꢁ7.00 (4H, m), 4.07 (2H, t), 2.80 (2H, t), 2.53
(4H, m), 1.69 (4H, m). Anal. (C19H20F3NO2).
1-(2-(4-(4-(Trifluoromethyl)phenoxy)phenoxy)ethyl) pyrrolidine
(5o). The title compound was prepared from 4-(4-(trifluoromethyl)
phenoxy) phenol as a white solid (yield 76%).1H NMR (400 MHz,
DMSO-d6) δ 7.69 (2H, d), 7.11ꢁ7.00 (6H, m), 4.08 (2H, t), 2.85
(2H, t), 2.58 (4H, m), 1.71 (4H, m). Anal. (C19H20F3NO2).
5z and 5bꢁr were synthesized by a similar procedure.
1-(2-(4-Phenoxyphenoxy)ethyl) pyrrolidine (5z). The title com-
pound was prepared from 4-phenoxyphenol as a light yellow oil (yield
82%). 1H NMR (400 MHz, DMSO-d6) δ 7.26 (2H, d), 7.08 (2H, d),
7.17ꢁ6.95 (5H, m), 4.06 (2H, t), 2.82 (2H, t), 2.57 (4H, m), 1.70 (4H,
m). Anal. (C18H21NO2).
1-(2-(4-(3-Nitrophenoxy)phenoxy)ethyl) pyrrolidine (5b). The title
compound was prepared from 4b as a yellow solid (yield 72%). 1H NMR
(400 MHz, DMSO-d6) δ 7.93 (1H, m), 7.64 (1H, m), 7.60 (1H, m),
7.43 (1H, m), 7.11 (2H, m), 7.05 (2H, m), 4.09 (2H, t), 2.81 (2H, t),
2.54 (4H, m), 1.69 (4H, m). ESI-HRMS m/z 329.1491 [M þ H]. Anal.
(C18H20N2O4 0.4H2O).
3
1-(2-(4-(4-Nitrophenoxy)phenoxy)ethyl) pyrrolidine (5c). The title
compound was prepared from 4c as a yellow solid (yield 72%). 1H NMR
(400 MHz, DMSO-d6) δ 8.20 (2H, m), 7.00 (4H, m), 6.82 (2H, m),
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ARTICLE
1-(2-(4-(4-Fluorophenoxy)phenoxy)ethyl) pyrrolidine (5p). The ti-
tle compound was prepared from 4-(4-fluorophenoxy) phenol as a white
solid (yield 89%). 1H NMR (400 MHz, DMSO-d6) δ 7.18 (2H, t), 6.96
(6H, m), 4.04 (2H, t), 2.77 (2H, t), 2.51 (4H, m), 1.68 (4H, m). Anal.
(1H, m), 7.03 (1H, m), 6.97 (2H, m), 6.85 (2H, m), 4.11 (2H, t), 3.52
(6H, s), 2.86 (2H, t), 2.59 (4H, s), 1.72 (4H, m). Anal.
(C20H26N2O6S2 0.3H2O).
3
General Procedure for the Preparation of N-(4-(4-(2-(Pyrrolidin-1-
yl)ethoxy)phenoxy)phenyl) methanesulfonamide (8c). A solution of
6c (0.23 g, 0.78 mmol) in 4 mL H2O was slowly added in methane-
sulfonyl chloride (0.12 g, 1.0 mmol) and stirred at room temperature for
2 h. The mixture was extracted with EtOAc twice. The combined organic
extracts were washed with brine and water, dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by flash
chromatography (silica gel, the eluting solvent ranged from CH2Cl2 to
CH2Cl2/MeOH = 9:1) as a yellow solid and weighed 0.20 g (yield 70%).
1H NMR (400 MHz, DMSO-d6) δ 7.18 (2H, d), 6.96ꢁ6.91 (6H, m),
4.04 (3H, t), 2.93 (3H, s), 2.79 (2H, t), 2.53 (4H, m), 1.69 (4H, m). ESI-
(C18H20FNO2 0.1H2O).
3
1-(2-(4-(4-Chlorophenoxy)phenoxy)ethyl) pyrrolidine (5q). The
title compound was prepared from 4-(4-chlorophenoxy) phenol as a
white solid (yield 60%). 1H NMR (400 MHz, DMSO-d6) δ 7.28 (2H, t),
7.06 (6H, m), 4.03 (2H, t), 2.78 (2H, t), 2.56 (4H, m), 1.68 (4H, m).
Anal. (C18H20ClNO2).
1-(2-(4-(4-Bromophenoxy)phenoxy)ethyl) pyrrolidine (5r). The title
compound was prepared from 4-(4-bromophenoxy) phenol as a light
brown solid (yield 90%). 1H NMR (400 MHz, DMSO-d6) δ 7.36
(2H, d), 7.26 (2H, d), 7.12 (2H, d), 7.04 (2H, d), 4.03 (2H, t), 2.78 (2H,
t), 2.56 (4H, m), 1.71 (4H, m). 13C NMR (400 MHz, DMSO-d6) δ
157.49, 155.17, 148.80, 132.54, 120.89, 119.22, 115.72, 114.00, 67.03,
54.27, 53.96, 23.12. ESI-HRMS m/z 362.0746 [M þ H]. Anal.
(C18H20BrNO2).
HRMS m/z 377.1528 [M þ H]. Anal. (C19H24N2O4S 0.1H2O).
3
General Procedure for the Preparation of N-Methyl-N-(2-(4-(2-
(pyrrolidin-1-yl)ethoxy)phenoxy)phenyl) methanesulfonamide (13a).
Step 1: Preparation of 2-(4-methoxyphenoxy) aniline (9a). Tin
granular (11 g) was added in the solution of 3a (1 g, 4 mmol) in ethanol
(18 mL). A mixture of concentrated HCl (9 mL) and ethanol (9 mL)
was then added into the solution above and refluxed for 16 h. The
solution was evaporated on a rotary evaporator to remove the ethanol,
alkalized with NaOH aqueous solution, and extracted with EtOAc. The
combined organic extracts were washed with brine and water, dried over
Na2SO4, and concentrated in vacuo to provide 0.74 g (86%) of a yellow
General Procedure for the Preparation of 2-(4-(2-(Pyrrolidin-1-
yl)ethoxy)phenoxy) aniline (6a). A solution of 5a (0.17 g, 0.52 mol)
and 10% Pd/C (0.008 g) in propan-2-ol was heated to 70 °C. 85%
Hydrazine hydrate solution (0.1 mol, 1 mol) was added and the solution
was further stirred for 0.5ꢁ1 h (TLC monitor). The solution was cooled,
filtered by 0.2 μm filter, and evaporated in vacuo. The product was a
white solid weighed 0.15 g (yield 97%) and was used without further
1
1
purification. H NMR (400 MHz, DMSO-d6) δ 6.92ꢁ6.84 (5H, m),
oil, which was used without further purification. H NMR (400 MHz,
6.79 (1H, d), 6.67 (1H, d), 6.50 (1H, t), 4.90 (2H, s), 4.00 (2H, t), 2.75
(2H, t), 2.49 (4H, m), 1.66 (4H, m). 13C NMR (400 MHz, DMSO-d6) δ
153.98, 150.78, 143.00, 140.04, 124.22, 118.95, 118.44, 116.24, 115.56,
115.36, 67.12, 54.39, 54.01, 23.14. Anal. (C18H22N2O2).
DMSO-d6) δ 6.94ꢁ6.88 (5H, m), 6.72 (1H, d), 6.57 (1H, d), 6.52 (1H,
t), 4.95 (2H, s).
Step 2: Preparation of N-(2-(4-Methoxyphenoxy)phenyl) metha-
nesulfonamide (10a). A solution of 9a (1 g, 4.7 mmol) in 20 mL
pyridine was heated to 70 °C. Methanesulfonyl chloride (0.69 g, 6.0
mmol) was slowly dropped into the solution above. After being stirred at
90 °C for 1 h, the mixture was poured into18% HCl aqueous solution,
filtered, and extracted with EtOAc twice. The combined organic extracts
were washed with brine and water, dried over Na2SO4, and concentrated
in vacuo. The crude product was purified by flash chromatography (silica
gel, the eluting solvent ranged from hexane to EtOAc/hexane = 1:5) as a
6bꢁc were synthesized by a similar procedure.
3-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenoxy) aniline (6b). The title
1
compound was prepared from 5b as a colorless oil (yield 96%). H
NMR (400 MHz, DMSO-d6) δ 6.94 (5H, m), 6.24 (1H, d), 6.09ꢁ6.03
(2H, m), 5.16 (2H, s), 4.04 (3H, t), 2.80 (2H, t), 2.54 (4H, m), 1.69
(4H, m). Anal. (C18H22N2O2).
4-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenoxy) aniline (6c). The title com-
1
1
pound was prepared from 5c as a colorless oil (yield 95%). H NMR
white solid and weighed 1.08 g (yield 73%). H NMR (400 MHz,
(400 MHz, DMSO-d6) δ 6.85 (2H, m), 6.34 (4H, d), 6.09 (2H, m), 5.26
(2H, s), 4.07 (3H, t), 2.85 (2H, t), 2.58 (4H, m), 1.70 (4H, m). 13C
NMR (400 MHz, DMSO-d6) δ 157.23, 154.20, 149.69, 144.89, 126.46,
124.80, 120.85, 116.17, 63.76, 53.57, 52.57, 22.60. ESI-HRMS m/z
299.1747 [M þ H]. Anal. (C18H22N2O2).
DMSO-d6) δ 9.26 (1H, s), 7.37 (1H, d), 7.12 (1H, t), 7.02 (1H, t), 6.90
(2H, d), 6.78 (2H, d), 6.70 (1H, d), 3.76 (3H, s), 3.03 (3H, s).
Step 3: Preparation of N-(2-(4-Methoxyphenoxy)phenyl)-N-
methylmethanesulfonamide (11a). A solution of 10a (1 g, 3.4 mmol),
dimethyl sulfate (0.86 g, 6.8 mmol) and anhydrous potassium carbonate
(2.6 g, 20 mmol) in 20 mL acetone was reflux for 3 h. 1 M KOH aqueous
solution was added and stirred at 60 °C for 1 h. The mixture was cooled
to room temperature and extracted with EtOAc twice. The combined
organic extracts were washed with brine and water, dried over Na2SO4,
and concentrated in vacuo. The crude product was purified by flash
chromatography (silica gel, the eluting solvent ranged from hexane to
EtOAc/hexane = 1:3) as a white solid and weighed 1.08 g (yield 72%).
1H NMR (400 MHz, DMSO-d6) δ 7.42 (1H, d), 7.28 (1H, t),
7.12ꢁ6.98 (5H, m), 6.74 (1H, s), 3.76 (3H, s), 3.22 (3H, s), 3.05
(3H, s).
General Procedure for the Preparation of N-(Methylsulfonyl)-
N-(2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenoxy)phenyl) methanesulfona-
mide (7a). A solution of 6a (0.15 g, 0.50 mol) and triethylamine
(1.5 mL) in 2.5 mL CH2Cl2 was cooled to 0 °C. Methanesulfonyl
chloride (0.14 g, 1.2 mmol) diluted with 1.5 mL CH2Cl2 in 0 °C was
slowly dropped into the solution above. After being stirred at 0 °C for 10
min and room temperature for 0.5 h, the mixture was poured into H2O,
and extracted with EtOAc twice. The combined organic extracts were
washed with brine and water, dried over Na2SO4, and concentrated in
vacuo. The crude product was purified by flash chromatography (silica
gel, the eluting solvent ranged from CH2Cl2 to CH2Cl2/MeOH = 9:1)
as a yellow solid and weighed 0.12 g (yield 53%). 1H NMR (400 MHz,
DMSO-d6) δ 7.59 (1H, d), 7.41 (1H, t), 7.13 (1H, t), 7.10ꢁ7.01 (4H,
m), 6.76 (1H, d), 4.10 (2H, t), 3.54 (6H, s), 2.93 (2H, t), 2.64 (4H, m),
Step 4: Preparation of N-(2-(4-Hydroxyphenoxy)phenyl)-N-
methylmethanesulfonamide (12a). The title compound was prepared
from 11a, using the general procedure for the preparation of 4a, to give
12a as a white solid (yield 81%). 1H NMR (400 MHz, DMSO-d6) δ 9.52
(1H, s), 7.32 (1H, d), 7.18 (1H, t), 7.11ꢁ6.94 (5H, m), 6.72 (1H, s),
3.22 (3H, s), 3.05 (3H, s).
1.73 (4H, m). ESI-HRMS m/z 455.1304 [M
þ
H]. Anal.
(C20H26N2O6S2 0.1H2O).
3
Step 5: Preparation of N-Methyl-N-(2-(4-(2-(pyrrolidin-1-
yl)ethoxy)phenoxy) phenyl) methanesulfonamide (13a). The title
compound was prepared from 12a, using the general procedure for the
7b was synthesized by a similar procedure.
N-(Methylsulfonyl)-N-(3-(4-(2-(pyrrolidin-1-yl)ethoxy)phenoxy)-
phenyl) methanesulfonamide (7b). The title compound was prepared
from 6b according to the procedure for 7a as a yellow solid (yield 36%).
1H NMR (400 MHz, DMSO-d6) δ 7.43 (1H, m), 7.23 (1H, m), 7.11
1
preparation of 5a, to give 13a as a light yellow solid (yield 86%). H
NMR (400 MHz, DMSO-d6) δ 7.43 (1H, d), 7.27 (1H, m), 7.11ꢁ6.98
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Journal of Medicinal Chemistry
ARTICLE
(5H, m), 6.74 (1H, d), 4.05 (2H, t), 3.20 (3H, s), 3.05 (3H, s), 2.79
(2H, t), 2.52 (4H, m), 1.69 (4H, m). ESI-HRMS m/z 391.1685
[M þ H]. Anal. (C20H26N2O4S).
codes: COX-1, 1Q4G;33 COX-2, 1PXX;34 LTA4H, 3CHR35). The
structures of ovine COX-1 and murine COX-2 were used, which have
conserved substrate binding sites compared to those in human enzymes.
3D models of the small molecules were built in Schro€dinger software
using the LigPrep command. Molecular docking with flexible ligands
and rigid receptors was performed using software AutoDock 4.00.
Conformational searching used the Lamarckian genetic algorithm
(LGA). Number of GA runs was set to 256, population size to 300,
maximum number of energy evaluations to 25 000 000, and maximum
number of generations to 27 000. Docked states of each small molecule
were clustered with rms of 2.0 Å. The lowest energy conformation in the
largest cluster was taken as the binding state.
’ BIOLOGICAL METHODS
Inhibition Assay of Human LTA4H Hydrolase. The LTA4H
hydrolase activity was measured using an ELISA assay to quantify the
amount of LTB4 produced. LTA4 substrate was prepared from hydro-
lyzation of LTA4 methyl ester (Cayman Chemical) in cold acetone with
50 mM NaOH (20%, v/v) under an inert atmosphere of nitrogen at
room temperature for 40 min, and the substrate solution was stored at
ꢁ80 °C until used. 300 ng of enzyme was diluted into 180 μL reaction
buffer (10 mM sodium phosphate, pH 7.4, 4 mg/mL BSA) and
incubated with test compounds (5% v/v DMSO, final) for 15 min at
37 °C. 20 μL LTA4 substrate (150 nM final concentrations) was added
to initiate the reaction and it was incubated for 10 min at 37 °C. A 25 μL
sample was diluted 20ꢁ100 times in EIA buffer to stop the reaction. The
concentration of LTB4 was quantified in the diluted sample by a
commercially available LTB4 ELISA kit (Cayman Chemical).
’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analyses data of all
b
the compounds; scanned spectrum of the H NMR and 13C
1
NMR data, and high-resolution mass spectra of representative
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Inhibition Assay of COX-2 and COX-1. The enzyme activity of
the purified COX was measured using a chromogenic assay based on the
oxidation of TMPD during the reduction of PGG2 to PGH2 in 96-well
plates (Costar, Corning Incorporated). The assay mixture (180 μL)
contained 100 mM Tris, pH 8, 2 mM genapol X-100,26 1 μM hematin,
and approx 20 units purified COX-1 (Cayman Chemical, Cat. No.
60100) or COX-2 (Cayman Chemical, Cat. No. 60120). One unit of
enzyme activity is defined as the amount of enzyme required to cause a
change in TMPD absorbance at 610 nm of 0.001 OD per min. The
TMPD solution was prepared in 0.1 M HCl with the concentration of
1 mM and the arachidonic acid was prepared in ethanol with the
concentration of 1.1 mM. Inhibitors were prepared in DMSO and
added for the final DMSO concentration was always 5% of the final
sample volume. The assay mixture was preincubated at 25 °C with
inhibitors for 15 min and 20 μL of the TMPD solution was added before
initiation of the enzymatic reaction by the addition of 20 μL of a solution
of arachidonic acid in assay buffer. The enzyme activity was measured by
estimation of the initial velocity of TMPD oxidation over the first 36 s
of the reaction as followed by the increase in absorbency at 610 nm
(Vmax; Molecular Devices). A low rate of nonenzymatic oxidation in the
absence of enzyme was subtracted before the calculation of the
percentage of inhibition. IC50 values were obtained from fitting the
data to a four-parameter logistical model of the graph of log dose against
percentage inhibition. All reported IC50 values are the results of averages
of independent experiments conducted at least twice.
’ AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed. Fax: (þ86)10-
62751725 E-mail: liuying@pku.edu.cn or lhlai@pku.edu.cn.
Author Contributions
§These authors contributed equally to this work.
’ ACKNOWLEDGMENT
This research was supported by the Ministry of Science
and Technology of China (grant numbers: 2009CB918503,
2009ZX09501) and the National Natural Science Foundation of
China (grant numbers: 20873003, 10721403)
’ ABBREVIATIONS
NSAIDs, nonsteriodal anti-inflammatory drugs; COX, cyclooxy-
genase; 5-LOX, 5-lipoxygenase; LTA4H, leukotriene A4 hydro-
lase; LTB4, leukotriene B4; PGES, prostaglandin E synthase;
PGE2, prostaglandin E2; PGG2, prostaglandin G2; PGH2, pros-
taglandin H2; TXB2,Thromboxane B2TXAS, Thromboxane
synthetase; PLA2, phospholipase A2
’ REFERENCES
Human Whole Blood Assay. Fresh blood was collected into
tubes containing heparin (10 IU/mL) and used within 1 h after
collection. 0.5 mL of blood were aliquoted into PP tubes preloaded
with either 2 μL of vehicle (DMSO) or 2 μL of test compounds DMSO
solutions at final concentrations ranging from 0.01 μM to 100 μM. The
tubes were vortexed gently and incubated for 15 min. 10 μL LPS (PBS
solution as the final concentration of 100 μg/mL) was added to the
blood and incubated for 23.5 h at 37 °C to induce COX-2 expression.
Calcium ionophore A23187 (final concentration of 20 μg/mL) was
added and the incubation was continued under the same conditions for
an additional 30 min. The incubation was terminated by centrifugation
(3200 g 15 min, 4 °C) to obtain plasma. Plasma was kept at ꢁ80 °C until
it was assayed for PGE2 and LTB4 production using an ELISA kit
(Cayman Chemical) according to the manufacturer’s instructions.
Molecular Docking. All the synthesized 1-(2-(4-phenoxy-
phenoxy)ethyl) pyrrolidine derivatives were docked into the cycloox-
ygenase sites of COX-1 and COX-2, and substrate binding site of
LTA4H. The crystal structures of the three enzymes were retrieved from
Protein Data Bank (RCSB PDB, http://www.rcsb.org/pdb/, entry
(1) Copeland, R. A.; Williams, J. M.; Giannaras, J.; Nurnberg, S.;
Covington, M. Mechanism of selective inhibition of the inducible
isoform of prostaglandin G/H synthase. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 11202–11206.
(2) Hunt, R. H.; Harper, S.; Watson, D. J.; Yu, C.; Quan, H.; Lee, M.;
Evans, J. K.; Oxenius, B. The gastrointestinal safety of the COX-2
selective inhibitor etoricoxib assessed by both endoscopy and analysis of
upper gastrointestinal events. Am. J. Gastroenterol. 2003, 98 (8),
1725–1733.
(3) Swingle, K. F.; Moore, G. G. I.; Grant, T. J. 4-Nitro-2-Phenox-
ymethanesulfonanilide (R-805) - chemically novel anti-inflammatory
agent. Arch. Int. Pharmacodyn. Ther. 1976, 221 (1), 132–139.
(4) Seibert, K.; Zhang, Y.; Leahy, K.; Hauser, S.; Masferrer, J.;
Perkins, W.; Lee, L.; Isakson, P. Pharmacological and biochemical
demonstration of the role of cyclooxygenase-2 in inflammation and
pain. Proc. Natl. Acad. Sci. U.S.A. 1994, 91 (25), 12013–12017.
(5) Prasit, P.; Wang, Z.; Brideau, C.; Chan, C. C.; Charleson, S.;
Cromlish, W.; Ethier, D.; Evans, J. F.; Ford-Hutchinson, A. W.; Gauthier,
J. Y.; Gordon, R.; Guay, J.; Gresser, M.; Kargman, S.; Kennedy, B.;
3659
dx.doi.org/10.1021/jm200063s |J. Med. Chem. 2011, 54, 3650–3660

Journal of Medicinal Chemistry
ARTICLE
Leblanc, Y.; Leger, S.; Mancini, J.; O’Neill, G. P.; Ouellet, M.; Percival,
M. D.; Perrier, H.; Riendeau, D.; Rodger, I.; Tagari, P.; Therien, M.;
Vickers, P.; Wong, E.; Xu, L. J.; Young, R. N.; Zamboni, R.; Boyce, S.;
Rupniak, N.; Forrest, N.; Visco, D.; Patrick, D. The discovery of
rofecoxib, [MK 966, Vioxx (R), 4-(4 -methylsulfonylphenyl)-3-phen-
yl-2(5H)-furanone], an orally active cyclooxygenase-2 inhibitor. Bioorg.
Med. Chem. Lett. 1999, 9 (13), 1773–1778.
(6) Psaty, B. M.; Furberg, C. D. COX-2 inhibitors - Lessons in drug
safety. N. Engl. J. Med. 2005, 352 (11), 1133–1135.
(7) Singh, D. Merck withdraws, arthritis drug worldwide. Br. Med. J.
2004, 329 (7470), 816–816.
(8) Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J. S.;
Collins, P. W.; Docter, S.; Graneto, M. J.; Lee, L. F.; Malecha, J. W.;
Miyashiro, J. M.; Rogers, R. S.; Rogier, D. J.; Yu, S. S.; Anderson, G. D.;
Burton, E. G.; Cogburn, J. N.; Gregory, S. A.; Koboldt, C. M.; Perkins,
W. E.; Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.; Isakson, P. C.
Synthesis and biological evaluation of the 1,5-diarylpyrazole class of
cyclooxygenase-2 inhibitors: Identification of 4-[5-(4-methylphenyl)-
3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-58635,
Celecoxib). J. Med. Chem. 1997, 40 (9), 1347–1365.
(23) Morphy, R.; Rankovic, Z. Designed multiple ligands. An
emerging drug discovery paradigm. J. Med. Chem. 2005, 48 (21), 6523–
6543.
(24) Morphy, R.; Rankovic, Z. Designing multiple ligands - medic-
inal chemistry strategies and challenges. Curr. Pharm. Des. 2009, 15 (6),
587–600.
(25) Penning, T. D.; Chandrakumar, N. S.; Chen, B. B.; Chen, H. Y.;
Desai, B. N.; Djuric, S. W.; Docter, S. H.; Gasiecki, A. F.; Haack, R. A.;
Miyashiro, J. M.; Russell, M. A.; Yu, S. S.; Corley, D. G.; Durley, R. C.;
Kilpatrick, B. F.; Parnas, B. L.; Askonas, L. J.; Gierse, J. K.; Harding, E. I.;
Highkin, M. K.; Kachur, J. F.; Kim, S. H.; Krivi, G. G.; Villani-Price, D.;
Pyla, E. Y.; Smith, W. G.; Ghoreishi-Haack, N. S. Structure-activity
relationship studies on 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine
(SC-22716), a potent inhibitor of leukotriene A(4) (LTA(4)) hydrolase.
J. Med. Chem. 2000, 43 (4), 721–735.
(26) Ouellet, M.; Falgueyret, J. P.; Percival, M. D. Detergents
profoundly affect inhibitor potencies against both cyclo-oxygenase iso-
forms. Biochem. J. 2004, 377, 675–684.
(27) Johnson, J. L.; Wimsatt, J.; Buckel, S. D.; Dyer, R. D.; Maddipati,
K. R. Purification and characterization of prostaglandin H synthase-2
from sheep placental cotyledons. Arch. Biochem. Biophys. 1995, 324
(1), 26–34.
0
(9) Brophy, J. M. Cardiovascular risk associated with celecoxib. N.
Engl. J. Med. 2005, 352 (25), 2648–2649.
(10) Wang, D. R.; Wang, M. A.; Cheng, Y.; FitzGerald, G. A.
Cardiovascular hazard and non-steroidal anti-inflammatory drugs. Curr.
Opin. Pharmacol. 2005, 5 (2), 204–210.
(11) Korcsmꢀaros, T.; Szalay, M.; B€ode, C.; Kovꢀacs, I. How to design
multi-target drugs:Target search options in cellular networks. Invit. Rev.
Expert Opin. Drug Discovery 2007, No. 2, 1–10.
(12) Yang, K.; Ma, W. Z.; Liang, H. H.; Qi, O. Y.; Tang, C.; Lai, L. H.
Dynamic simulations on the arachidonic acid metabolic network. PLoS
Comput. Biol. 2007, 3 (3), 523–530.
(13) Yang, K.; Bai, H. J.; Ouyang, Q.; Lai, L. H.; Tang, C. Finding
multiple target optimal intervention in disease-related molecular net-
work. Mol. Syst. Biol. 2008, 4, 228–228.
(14) Martel-Pelletier, J.; Lajeunesse, D.; Reboul, P.; Pelletier, J. P.
Therapeutic role of dual inhibitors of 5-LOX and COX, selective and
non-selective non-steroidal anti-inflammatory drugs. Ann. Rheum. Dis.
2003, 62 (6), 501–509.
(15) Chowdhury, M. A.; Abdellatif, K. R. A.; Dong, Y.; Das, D.;
Suresh, M. R.; Knaus, E. E. Synthesis of celecoxib analogues possessing a
N-difluoromethyl-1,2-dihydropyrid-2-one 5-lipoxygenase pharmaco-
phore: biological evaluation as dual inhibitors of cyclooxygenases and
5-lipoxygenase with anti-inflammatory activity. J. Med. Chem. 2009
52 (6), 1525–1529.
(28) Patrignani, P.; Panara, M. R.; Greco, A.; Fusco, O.; Natoli, C.;
Iacobelli, S.; Cipollone, F.; Ganci, A.; Creminon, C.; Maclouf, J.;
Patrono, C. Biochemical and pharmacological characterization of the
cyclooxygenase activity of human blood prostaglandin endoperoxide
synthases. J. Pharmacol. Exp. Ther. 1994, 271 (3), 1705–1712.
(29) Young, J. M.; Panah, S.; Satchawatcharaphong, C.; Cheung,
P. S. Human whole blood assays for inhibition of prostaglandin G/H
synthases-1 and ꢁ2 using A23187 and lipopolysaccharide stimulation of
thromboxane B2 production. Inflammation Res. 1996, 45 (5), 246–253.
(30) Thunnissen, M. M. G. M.; Nordlund, P.; Haeggstrom, J. Z.
Crystal structure of human leukotriene A(4) hydrolase, a bifunctional
enzyme in inflammation. Nat. Struct. Biol. 2001, 8 (2), 131–135.
(31) Ye, B.; Bauman, J.; Chen, M.; Davey, D.; Khim, S. K.; King, B.;
Kirkland, T.; Kochanny, M.; Liang, A.; Lentz, D.; Maya, K.; Mendoza, L.;
Phillips, G.; Selchau, V.; Schlyer, S.; Tseng, J. L.; Wei, R. G.; Ye, H.;
Parkinson, J.; Guilford, W. J. Synthesis of N-alkyl glycine amides as
potent inhibitors of leukotriene A(4) hydrolase. Bioorg. Med. Chem. Lett.
2008, 18 (14), 3891–3894.
(32) Rao, P. N. P.; Chen, Q. H.; Knaus, E. E. Synthesis and structure-
activity relationship studies of 1,3-diarylprop-2-yn-1-ones: Dual inhibi-
tors of cyclooxygenases and Lipoxygenases. J. Med. Chem. 2006, 49 (5),
1668–1683.
(16) Connolly, P. J.; Wetter, S. K.; Beers, K. N.; Hamel, S. C.; Chen,
R. H. K.; Wachter, M. P.; Ansell, J.; Singer, M. M.; Steber, M.; Ritchie,
D. M.; Argentieri, D. C. N-hydroxyurea and hydroxamic acid inhibitors
of cyclooxygenase and 5-lipoxygenase. Bioorg. Med. Chem. Lett. 1999, 9
(7), 979–984.
(17) Tholander, F.; Muroya, A.; Roques, B. P.; Fournie-Zaluski,
M. C.; Thunnissen, M. M. G. M.; Haeggstrom, J. Z. Structure-based
dissection of the active site chemistry of leukotriene A4 hydrolase:
implications for M1 aminopeptidases and inhibitor design. Chem. Biol.
2008, 15 (9), 920–929.
(18) Morphy, R.; Kay, C.; Rankovic, Z. From magic bullets to
designed multiple ligands. Drug Discovery Today 2004, 9 (15), 641–651.
(19) Csermely, P.; Agoston, V.; Pongor, S. The efficiency of multi-
target drugs: the network approach might help drug design. Trends
Pharmacol. Sci. 2005, 26 (4), 178–182.
(33) Gupta, K.; Selinsky, B. S.; Kaub, C. J.; Katz, A. K.; Loll, P. J. The
2.0 A resolution crystal structure of prostaglandin H2 synthase-1:
structural insights into an unusual peroxidase. J. Mol. Biol. 2004, 335
(2), 503–518.
(34) 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. A novel mechanism of cyclooxygenase-2 inhibition
involving interactions with Ser-530 and Tyr-385. J. Biol. Chem. 2003,
278 (46), 45763–45769.
(35) Kirkland, T. A.; Adler, M.; Bauman, J. G.; Chen, M.; Haegg-
strom, J. Z.; King, B.; Kochanny, M. J.; Liang, A. M.; Mendoza, L.;
Phillips, G. B.; Thunnissen, M.; Trinh, L.; Whitlow, M.; Ye, B.; Ye, H.;
Parkinson, J.; Guilford, W. J. Synthesis of glutamic acid analogs as potent
inhibitors of leukotriene A4 hydrolase. Bioorg. Med. Chem. 2008, 16 (9),
4963–4983.
(20) Morphy, R.; Rankovic, Z. Fragments, network biology and
designing multiple ligands. Drug Discovery Today 2007, 12 (3ꢁ4),
156–160.
(21) Hopkins, A. L. Network pharmacology: the next paradigm in
drug discovery. Nat. Chem. Biol. 2008, 4 (11), 682–690.
(22) Wei, D. G.; Jiang, X. L.; Zhou, L.; Chen, J.; Chen, Z.; He, C.;
Yang, K.; Liu, Y.; Pei, J. F.; Lai, L. H. Discovery of multitarget inhibitors
by combining molecular docking with common pharmacophore match-
ing. J. Med. Chem. 2008, 51 (24), 7882–7888.
3660
dx.doi.org/10.1021/jm200063s |J. Med. Chem. 2011, 54, 3650–3660