Syntheses and characterization of nimesulide derivatives for dual enzyme inhibitors of both cyclooxygenase-1/2 and 5-lipoxygenase

Article abstract of DOI:10.1016/j.bmc.2011.01.043

Cyclooxygenase-1/2 (COX-1/2) and 5-lipoxygenase (5-LOX) are enzymes in two different pathways in the inflammatory process. In the present study, a variety of new nimesulide derivatives were synthesized through incorporation of a 5-LOX pharmacophore into nimesulide followed with some structural modifications, which were then characterized for dual enzyme inhibitors for these two types of enzymes. Their structure-activity relationships (SARs) were studied, and compound 20f was found to be an excellent dual enzyme inhibitor. Its binding conformation and interaction mode were studied with molecular docking experiments. Compound 20f could become a lead compound for further development for potential anti-inflammatory drugs.

Full text of DOI:10.1016/j.bmc.2011.01.043

Bioorganic & Medicinal Chemistry 19 (2011) 2074–2083
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Syntheses and characterization of nimesulide derivatives for dual enzyme
inhibitors of both cyclooxygenase-1/2 and 5-lipoxygenase
⇑
Yue Li, Shu-Han Chen, Tian-Miao Ou, Jia-Heng Tan, Ding Li, Lian-Quan Gu, Zhi-Shu Huang
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclooxygenase-1/2 (COX-1/2) and 5-lipoxygenase (5-LOX) are enzymes in two different pathways in the
inflammatory process. In the present study, a variety of new nimesulide derivatives were synthesized
through incorporation of a 5-LOX pharmacophore into nimesulide followed with some structural modi-
fications, which were then characterized for dual enzyme inhibitors for these two types of enzymes. Their
structure–activity relationships (SARs) were studied, and compound 20f was found to be an excellent
dual enzyme inhibitor. Its binding conformation and interaction mode were studied with molecular
docking experiments. Compound 20f could become a lead compound for further development for poten-
tial anti-inflammatory drugs.
Received 24 November 2010
Revised 20 January 2011
Accepted 21 January 2011
Available online 1 February 2011
Keywords:
Nimesulide derivatives
Cyclooxygenase
Lipoxygenase
Ó 2011 Elsevier Ltd. All rights reserved.
Dual enzyme inhibitor
Inflammatory
1. Introduction
In addition to the COX pathway, AA is also metabolized via the
lipoxygenase (LOX) pathway. Lipoxygenases (LOXs) belong to a
Cyclooxygenases (COXs) or prostaglandin endoperoxide syn-
thases (PGHSs) are key enzymes in the first step for the biosynthe-
sis of the prostaglandins (PGs) from the substrate arachidonic acid
(AA) during the inflammatory process.1,2 Two isoforms of COX en-
zymes, COX-1 and COX-23 are known to catalyze the rate-limiting
step of prostaglandins synthesis, which are the targets of nonste-
roidal anti-inflammatory drugs (NSAIDs).4–6 However, NSAIDs
have been reported to be associated with gastrointestinal adverse
effects, and many users of traditional NSAIDs have been suffering
from gastrointestinal ulcers and bleeding.7–9 The COX-2 hypothesis
has suggested that selective COX-2 inhibitors share the anti-
inflammatory properties of traditional NSAIDs without the gastro-
intestinal toxicity,10 which has been validated by the successful
human clinical trials of celecoxib (1) and other selective COX-2
inhibitors.11 However, selective COX-2 inhibitors have also been
failed to fully satisfy the criteria of new safer anti-inflammatory
agents for the increased risk of cardiovascular complications,12–14
for example, rofecoxib (2) has been withdrawn from the global
market. Meanwhile, the post-marketing surveillances have re-
vealed that nimesulide (3) may cause more severe adverse effect
of hepatic damage.15,16 Therefore, recent studies are mainly focus-
ing on strategies towards reducing the adverse effects of anti-
inflammatory drugs and searching for new safer drugs.
class of non-heme iron-containing enzymes, which catalyze the
hydroperoxidation reaction of fatty acids to peroxides.17 5-LOX is
associated with the production of leukotrienes (LTs) from AA,
which causes inflammatory, bronchoconstrictor, hypersensitivity,
anaphylactic, and asthmatic actions.18,19 Thus, 5-LOX inhibitors
have also been used as potential therapeutic agents for the treat-
ment of inflammatory and allergic diseases.20 Furthermore, among
three types of known 5-LOX inhibitors (redox, iron chelators, and
non redox), non-redox compounds such as licofelone (4) and ZD-
2138 (5), which are the active-site directed inhibitors, have shown
fewer side effects than redox and iron chelating agents.21
It has been believed that the design and discovery of a dual
inhibitor for both COX and LOX enzymatic pathways should pro-
vide a new rational approach for effective anti-inflammatory
agents with better safety profiles than NSAIDs and selective COX-
2 inhibitors.22 The inhibition of only one of the enzymatic path-
ways can possibly shift the metabolism of AA toward the other
pathway, which causes side effects. Therefore, searching for a bal-
ance in the inhibition of two enzymatic pathways is essential in the
development of superior and safer anti-inflammatory agents.23
One of the successful strategies to develop COX/5-LOX inhibitors
is to modify NSAIDs and selective COX-2 inhibitors by introducing
a 5-LOX pharmacophore, and several successful examples are
shown in Figure 1. A potent hybrid COX/5-LOX inhibitor (6) has
been reported, in which the C-3 trifluoromethyl substituent pres-
ent in the COX-2 inhibitor celecoxib (1) has been replaced by a
non-redox competitive 5-LOX pharmacophore, 4-(3-fluoro-
⇑
Corresponding author. Tel./fax: +86 20 39943056.
E-mail address: ceshzs@mail.sysu.edu.cn (Z.-S. Huang).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.043

Y. Li et al. / Bioorg. Med. Chem. 19 (2011) 2074–2083
2075
SO2Me
SO2NH2
O2N
O
N
N
N
Me
NH
SO2Me
O
COOH
F3C
Cl
O
Rofecoxib (2)
Licofelone (4)
Nimesulide (3)
Celecoxib (1)
SO2Me
SO2Me
F
OMe
O
O
O
O
N
N
N
OMe
N
N
CHF2
N
Me
O
O
ZD-2138 (5)
6
7
Figure 1. Chemical structures of the selective COX-2 inhibitors celecoxib (1), rofecoxib (2), nimesulide (3), non-redox 5-LOX inhibitors licofelone (4) and ZD-2138 (5), and
representative COX/LOX inhibitors (6 and 7).
5-oxymethyl)phenyl-4-methoxytetrahydropyran.24,25 Another type
of novel dual COX/5-LOX inhibitor celecoxib analogues (7)26
that possess N-difluoromethyl-1,2-dihydropyrid-2-one 5-lipoxyge-
nase pharmacophore, has also shown acceptable in vitro COX-2
inhibitory potency and selectivity, and potent inhibition of the
enzyme 5-LOX. These dual COX/5-LOX inhibitors have key pharma-
cophoric elements in their structures for optimal interactions with
binding sites of both enzyme systems, and have shown certain
antitumor activity. Accordingly, we anticipated that introduction
of a 5-LOX pharmacophore to the selective COX-2 inhibitor
nimesulide (3) may provide a type of effective dual COX/5-LOX
inhibitory anti-inflammatory agents. In this paper, we report
our syntheses of nimesulide derivatives with incorporation of a
5-LOX pharmacophore, 4-methoxytetrahydropyran, and their
characterization through dual enzyme inhibition and molecular
docking experiments.
functional groups at different positions might give good dual COX/
5-LOX inhibitors. As shown in Figure 2, nimesulide derivative was
systematically modified in both position A and position B, which
include the replacement of a nitro group in position A with various
other substitutive groups, and the change of the linkage between
5-LOX pharmacophore and nimesulide analogue in position B.
The preparation of synthetic intermediate compounds 11 and
12 was shown in Scheme 1. The Grignard reagent was prepared
from 1-bromo-3-methylbenzene, which was then reacted with tet-
rahydro-4H-pyran-4-one to give a tertiary alcohol compound 9. Its
methylation followed with bromination afforded intermediate
compound 11. Finally, another intermediate compound 12 with a
phenylmethanesulfonyl chloride functional group was prepared
following a general method reported previously.28
The synthetic pathway for compounds 16a–f and 17a–e is
shown in Scheme 2. Various substituted o-aminophenols were re-
acted with phthalic acid anhydride to afford compounds 14a–f,
which could protect their amino group from reacting with benzyl
bromide in the next step. After the reactions of compounds
14a–f with benzyl bromide and subsequent deprotection reactions
with hydrazine, compounds 16a–f were obtained in good yield.
Then, the reactions of compounds 16a–f with methanesulfonyl
chloride gave products 17a–e as methane sulfonamides.
2. Results and discussion
2.1. Syntheses of nimesulide derivatives
A new type of COX/5-LOX dual enzyme inhibitors was synthe-
sized through incorporation of a 5-LOX pharmacophore to a selec-
tive COX-2 inhibitor, nimesulide. According to the structural
requirement for selective COX-2 inhibition by nimesulide reported
previously, the nitro group and methane sulfonamide group are
crucial to its selectivity and inhibition activity.27 Therefore, modi-
fications of these two groups may be helpful to make a balance be-
tween COX and 5-LOX inhibition. On the other hand, our binding
models showed that the replacement of the nitro group with other
The preparations of compounds 19a–d are shown in Scheme 3.
The reactions of pyrocatechols with benzyl bromide gave com-
pounds 19a and 19d. The following chlorination and bromination
of 2-(benzyloxy) phenol (19a) gave compounds 19b and 19c,
respectively.
The synthetic scheme for the preparation of final products
20a–f, 21a–e, 22a–e, and 23a–d with different linkages are shown
O2N
O
O2N
O
O
O
OMe
NH
SO2CH3
S
A
N
O
H
B
Nimesulide (3)
Figure 2. Design of dual COX/5-LOX enzyme inhibitors through incorporation of a 5-LOX pharmacophore to nimesulide followed with some structural modifications in
position A and B.

2076
Y. Li et al. / Bioorg. Med. Chem. 19 (2011) 2074–2083
a
b
OH
Me
Me
Br
Me
O
8
9
d
c
OMe
OMe
OMe
ClO2S
Br
O
O
O
12
10
11
Scheme 1. Organic syntheses of compounds 11 and 12. Reagents and conditions: (a) (i) Mg, THF, 65 °C; (ii) tetrahydro-4H-pyran-4-one, THF, reflux, 2 h yield 71%; (b) NaH,
CH3I, THF, 50 °C, 2 h, yield 89%; (c) NBS, CCl4, reflux, 2 h, yield 85%; (d) (i) Na2SO3, H2O, C2H5OH, reflux, overnight; (ii) SOCl2, DMF, CH2Cl2, rt, 3 h yield 69%.
R1
R2
O
N
R1
R2
OH
N
O
O
a
R1
R2
OH
b
NH2
O
O
13a-f
15a f
-
14a f
-
R1
R2
a
b
c
d
e
f
H
H
Cl
H
H
NO2
H
d
R1
R2
O
c
R1
R2
O
H
Cl
SO2Me
Me
N
H
NH2
H
COOEt
16a-f
17a-e
Scheme 2. Synthetic pathway for the preparations of compounds 16a–f and 17a–e. Reagents and conditions: (a) phthalic acid anhydride, HOAc, reflux, 3 h, yield 91–98%; (b)
benzyl bromide, K2CO3, acetone, reflux, 3 h, yield 79–89%; (c) NH2NH2ÁH2O, EtOH, reflux, 2 h, yield 57–86%; (d) MeSO2Cl, pyridine, CH2Cl2, rt, 1 h, yield 46–88%.
Cl
O
b
OH
OH
OH
O
a
19b
OH
c
19a
18
Br
O
OH
19c
19d
EtOOC
OH
OH
EtOOC
O
HOOC
OH
OH
e
d
OH
Scheme 3. Synthetic pathways for the preparation of compounds 19a–d. Reagents and conditions: (a) benzyl bromide, K2CO3, acetone, reflux, 3 h, yield 90%; (b) SO2Cl2,
toluene, rt, 1 h, yield 76%; (c) Br2, HOAc, 0 °C, 1 h, yield 55%; (d) H2SO4, EtOH, 50 °C, 3 h, yield 98%; (e) benzyl bromide, K2CO3, acetone, reflux, 3 h, yield 89%.

Y. Li et al. / Bioorg. Med. Chem. 19 (2011) 2074–2083
2077
a
R1
R2
O
O
O
16a f
OMe
-
S
N
H
O
20a f
-
R1
R2
O
R1
R2
O
N
b
a
b
c
d
e
f
H
H
MeO
17a e
-
Cl
H
H
Cl
H
Me
NO2
H
MeO2S
21a e
H
-
COOEt
O
c
R1
R2
O
MeO
16a-e
19a-d
N
H
22a-e
R1
O
23a
, R1 = H
O
d
MeO
23b, R1 = Cl
23c, R1 = Br
O
23d
, R1 = COOEt
23a-d
Scheme 4. Syntheses of compounds 20a–f, 21a–e, 22a–e, and 23a–d. Reagents and conditions: (a) compound 12, pyridine, CH2Cl2, rt, overnight, yield 71–75%; (b) compound
11, K2CO3, MeCN, reflux, 3 h, yield 43–66%; (c) compound 11, K2CO3, acetone, reflux, 3 h, yield 33–77%; (d) compound 11, K2CO3, acetone, reflux, 3 h, yield 43–88%.
in Scheme 4. Compounds 20a–f were obtained through the reac-
tions of compounds 16a–f with compound 12, while compounds
21a–e were synthesized through the reactions of compounds
17a–e with compound 11. The reactions of compounds 16a–e with
compound 11 gave products 22a–e, while the reactions of com-
pounds 19a–d with compound 11 afforded compounds 23a–d.
of their COX inhibition activity. The primary structure–activity
relationship study suggested that the combination of a sulfon-
amide linkage and a polar substitutive group in position A gave
compounds 20e and 20f with strong inhibition activity for COX.
However, the selectivity of compound 20f between COX-1 and
COX-2 was slightly lower than that of nimesulide, and most of
above compounds did not show significant selectivity between
COX-1 and COX-2.
2.2. Enzyme inhibition studies of nimesulide derivatives
For 5-LOX inhibition activity, the linkage of 5-LOX pharmaco-
phore was found to be less important, and most above compounds
showed certain inhibition activity towards 5-LOX, except the com-
pounds with secondary amine linkage (22a, 22c, 22d and 22e).
However, the compounds with sulfonamide and ether linkage
had better 5-LOX inhibition activity. Interestingly, like the COX
inhibition, the combination of a sulfonamide or an ether linkage
with a polar substitutive group in position A showed good 5-LOX
inhibition. Compounds 23d and 20f were found to be the best 5-
LOX inhibitors, which indicated that a proper polar group such as
ester group in the position A could effectively improve 5-LOX inhi-
bition activity.
Compound 20a–f with a sulfonamide linkage showed dual inhi-
bition activity against COX and 5-LOX, and the inhibition activity
varied among different substituted groups in position A. It was
obviously that polar group like nitro (20e) and benzoate (20f) were
better than other groups (methyl or halogen) in both COX and
5-LOX inhibition activity. Meanwhile, benzoate substituted in
position A led to certain selectivity between COX-1 and COX-2
inhibition activity, though the selective index was still lower than
nimesulide. It suggested that further modifications in position A
may result in a good selectivity between COX-1 and COX-2 inhibi-
tion activity.
Compounds 20a–f, 21a–e, 22a–e, and 23a–d were character-
ized for their COX-1/2 and 5-LOX inhibitory activity using an ovine
COX-1/COX-2 assay kit (Catalog No. 560101, Cayman Chemicals
Inc., Ann Arbor, MI, USA) and a LOX assay kit (Catalog No.
760700, Cayman Chemicals Inc., Ann Arbor, MI, USA), and the re-
sults are shown in Table 1. The IC50 values were measured, which
indicated the required concentration of tested compound for 50%
inhibition of the enzymatic activity. The average of two experi-
mental data was used, and the deviation from the mean was about
10%.
In general, sulfonamide group was found to be essential for
their COX inhibition activity. The substitution of methane sulfon-
amide with other linkages caused almost complete loss of their
COX inhibition activity, except the compounds with polar substitu-
tion in position A, which showed weak COX inhibition activity.
Compounds 21a–e modified in position B lost their COX inhibition
activity, which was consistent with previous reports that the ioni-
zation process of N–H in position B is important for their COX-2
inhibitory activity.29,30 Compounds 23a–d with an ether linkage
also had much weak inhibition for COX-1 and COX-2. Polar substi-
tutions in position A gave good inhibition activity for compounds
20e and 20f, while other substitutions generally caused certain loss

2078
Y. Li et al. / Bioorg. Med. Chem. 19 (2011) 2074–2083
Table 1
six good COX inhibitors (20a–f). Two hydrogen bonds were crucial
for selective COX-2 inhibition, including one hydrogen bond
formed between methanesulfonyl and Arg120, and another one be-
tween ethyl benzoate and Ser530. The interaction between the
benzyloxy group and the selective COX-2 pocket formed by
Arg513, His90, and Val523, was also important. Therefore, it was
reasonable that compounds 20e and 20f with polar substituent
group on their aryl ring were the best COX-2 inhibitors. Further
modifications on the benzyloxy group, which interacted with the
selective COX-2 pocket, might improve the selectivity of these
compounds.
Meanwhile, another possible binding mode was also provided
by AUTODOCK VINA. The additional 5-LOX pharmacophoric group got
into the upper part of the channel, and the substituted aryl ring
was close to Ser530. The amide linkage of the compounds 20e
and 20f formed hydrogen bonds with residues Arg120 and
Tyr355. These key interactions might provide an explanation
why introduction of any other linkage in the position B caused al-
most complete loss of their COX inhibition activity. The difference
between these two binding modes was the location of the benzyl-
oxy group and 5-LOX pharmacophoric group. The two key interac-
tions were similar, including the interaction of methanesulfonyl
with Arg120 and the interaction of ethyl benzoate with Ser530.
There was no key interaction observed in the COX-2 pocket for
selective binding in both binding modes, which may explain why
these compounds had relatively low selectivity between COX-1
and COX-2.
Inhibition study results for compounds 20a–f, 21a–e, 22a–e, and 23a–d
Compound
R1
R2
IC50
(
l
M)
Selectivity COX-1/
COX-2
COX-
1a
COX-
2a
5-
LOXb
20a
20b
20c
20d
20e
20f
21a
21b
21c
21d
21e
22a
22b
22c
22d
22e
23a
23b
23c
23d
Celecoxib
Nimesulide
Caffeic acid
H
Cl
H
H
NO2
H
H
H
Cl
H
NO2
H
Cl
H
H
NO2
H
Cl
Br
COOEt
H
H
Cl
Me
H
14.8
24.4
15.6
16.5
7.9
2.3
3.7
2.9
4.9
3.4
2.4
3.3
1.9
1.1
0.87
7.5
6.9
8.5
4.6
3.2
>10
7.8
>10
>10
>10
3.6
1.6
1.9
0.98
>10
>10
4.5
6.4
6.6
5.4
3.4
5.3
12
—
—
—
—
—
—
—
—
—
—
—
—
—
1.5
COOEt 11.6
H
Cl
H
Me
H
H
H
Cl
Me
H
0.97
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
38.8
0.05
0.4
>100
>100
>100
>100
13.5
>100
34.6
>100
>100
88.8
>100
>100
>100
44.2
8.1
1.1
160
15
—
6.2
—
—
a
The concentration of in vitro tested compound required to produce 50% inhi-
bition of ovine COX-1 or COX-2. The result (IC50 M) is the mean of two deter-
,
l
minations using an ovine COX-1/COX-2 assay kit (Catalog No. 560101, Cayman
Chemicals Inc., Ann Arbor, MI), and the deviation from the mean is 10% of the mean
value.
The three dimensional structure of the enzyme 5-LOX has been
well studied. The 5-LOX active site is consisted of a deep bent-
shaped cleft containing the non-heme iron cofactor. Two hydrogen
bond acceptors exist in the entrance and exit of cleft. It’s interest-
ing that a small side pocket also exists, which could accommodate
lipophilic groups.34 In our study, two binding modes for the excel-
b
The concentration of in vitro tested compound required to produce 50% inhi-
bition of potato 5-LOX (Cayman Chemicals Inc. Catalog No. 60401). The result (IC50
,
lM) is the mean of two determinations using a LOX assay kit (Catalog No. 760700,
Cayman Chemicals Inc., Ann Arbor, MI), and the deviation from the mean is 10% of
the mean value.
lent 5-LOX inhibitors (20f and 23d) were provided by AUTODOCK
,
which were slightly different according to the site of substitutions
in aryl ring (Fig. 4), while their general binding modes were very
similar. The 5-LOX pharmacophore of the inhibitor filled the en-
trance of the active site, while the polar substituent formed hydro-
gen bonds with hydrogen bond acceptors. The difference between
compounds 20f and 23d was the orientation of their benzoxy
group, which was deepen into the non-heme iron cofactor site
for compound 20f and a small side pocket for compound 23d,
respectively. They both had the hydrogen bonds formed between
ethyl benzoate and hydrogen bond acceptors at the bottom of
the active site, leading to their stronger 5-LOX inhibition activity
than other compounds. It should be noted that an ester substitu-
tive group in position A might form hydrogen bond with Ser530
in the COX active site, which could also form hydrogen bonds with
the hydrogen bond acceptors at the bottom of the 5-LOX active site
in our docking studies. This could be a key substitutive group for
further modifications for dual enzyme inhibitors. On the other
hand, the benzoxy group, which got deep into the COX-2 active site
in our docking studies, might be further modified to improve the
selectivity of the inhibitors.
Among these synthetic compounds, compound 20f had the best
overall inhibition activity and selectivity. The modifications of po-
sition A and B for nimesulide derivatives successfully made some
dual COX/5-LOX inhibitors. Based on the result of our structure–
activity relationship study, the sulfonamide part should be kept
due to its key contribution to COX inhibition, and further modifica-
tions at position A with addition of more polar groups might give
better inhibitors.
2.3. Docking studies of nimesulide derivatives
High-resolution structure of COX–inhibitor complex has been
solved, which provides detailed information about the COX active
site.31 The COX channel narrows at the interface between the
membrane binding domain and the catalytic domain, to form a
constriction composed of three residues (Arg-120, Tyr-355, and
Glu-524) that separate the ‘lobby’ from the active site. The solvent
accessible surface in the COX-2 active site is larger than that of
COX-1, because of the Val-523 to Ile substitution in the active site
and several key substitutions in the secondary shell (Arg-513 to
His and Val-434 to Ile). This difference in the side pocket is very
essential for the selective COX-2 inhibition.31
3. Conclusions
In conclusion, a novel class of dual COX/5-LOX inhibitors de-
rived from selective COX-2 inhibitor nimesulide was synthesized,
and their structure–activity relationship was studied. Compounds
20e and 20f were found to be overall the best dual enzyme inhib-
itors. Further computational docking studies provided rational
explanation for their binding affinity with above two enzymes.
These studies shed light on their further modifications for better
dual COX/5-LOX inhibitors as anti-inflammatory drugs.
One main binding mode was considered for the inhibitors
20a–f, which was very similar to that of nimesulide, as shown in
Figure 3.27 The benzyloxy group was inserted into the selective
side pocket of COX-2, and the aryl ring substituent was close to
Ser530 that was the key amino acid for the interaction between
NSAIDs and COXs.31 The additional 5-LOX pharmacophoric group
filled a hydrophobic cavity at the entrance of the active site. This
32
was the most favorable binding mode given by AUTODOCK VINA for

Y. Li et al. / Bioorg. Med. Chem. 19 (2011) 2074–2083
2079
Figure 3. Two different favorable docking modes of compound 20f in the COX-2 active site were provided by AUTODOCK VINA. Hydrogen bonds were marked with dashed green
lines. Pictures were generated with PyMOL.33
Figure 4. Docking of compounds 20f (left) and 23d (right) inside the 5-LOX active site, given by AUTODOCK VINA. Hydrogen bonds were marked with dashed green lines. Pictures
were generated with PyMOL.33
4.1.1. 4-m-Tolyltetrahydro-2H-pyran-4-ol (9)
4. Experimental
4.1. Chemistry
1-Bromo-3-methylbenzene (1.7 g, 10 mmol) was added drop-
wise to Mg (0.36 g, 15 mmol) in dry THF under N2 at such a rate
that the solution was kept at gentle reflux. After the addition
was completed, the mixture was heated under reflux for an addi-
Commercially available chemicals and solvents were used with-
out further purification, unless otherwise stated. NMR was re-
corded on a Bruker AvanceIII 400 spectrometer, and TMS was
used as an internal standard for recorded spectra. HRMS was re-
corded on an Agilent Iron-TOF-LC/MS spectrometer. Melting points
(mp) were determined using a SRS-OptiMelt automated melting
point instrument without correction. ESI-MS spectra were ob-
tained using a Thermo LCQ DECA XP LC–MS spectrometry. HPLC
analysis was performed on a HP1100 system (Hewlett–Packard,
Palo Alto, CA), and the purity of all target compounds used in the
biophysical and biological studies was more than 95%. All reactions
were routinely monitored using TLC with Merck Silica Gel 60F-254
glass plates.
tional 1–3 h. Then
a solution of tetrahydro-4H-pyran-4-one
(1.0 g, 10 mmol) in THF was added slowly to keep the reaction
solution at a gentle reflux. The mixture was heated under reflux
for 1 h after the addition was finished. The reaction was then
stopped with the addition of satd NH4Cl followed with extraction
using EtOAc (3 Â 50 mL). The organic layers were combined,
washed with brine (3 Â 50 mL), dried over MgSO4, filtered, and
concentrated under reduced pressure to afford a crude product
as oil. The product was purified using flash column chromatogra-
phy (EtOAc/hexane, 10:90) to give a white solid (1.4 g, 71%). 1H
NMR (400 MHz, CDCl3) d 7.28–7.16 (m, 3H), 7.11 (m, 1H), 3.94–
3.78 (m, 4H), 2.96 (s, 3H), 2.08–1.94 (m, 4H), 1.60 (s, 1H).

2080
Y. Li et al. / Bioorg. Med. Chem. 19 (2011) 2074–2083
4.1.2. 4-Methoxy-4-m-tolyltetrahydro-2H-pyran (10)
4.1.5.4. 2-(Benzyloxy)-4-chloroaniline (16b). Following the
above procedure, the desired product was obtained as a red oil
with a yield of 79%. 1H NMR (400 MHz, CDCl3) d 7.48–7.30 (m,
5H), 6.74 (s, 1H), 6.57 (d, J = 1.6 Hz, 1H), 6.50 (d, J = 1.6 Hz, 1H),
5.05 (s, 2H), 3.60 (s, 2H).
A solution of alcohol 9 (1.9 g, 10 mmol) in THF (100 mL) were
added sodium hydride (60% dispersion in mineral oil, 0.44 g,
11 mmol) and methyl iodide (1.0 mL, 11 mmol). The mixture was
stirred for 15 h at room temperature before water (100 mL) was
added. The aqueous phase was extracted with EtOAc (3 Â 50 mL),
and the combined organic phases were washed with brine, dried
with MgSO4, and evaporated. The crude product was purified using
flash chromatography with hexane–EtOAc (80:20) elution to give a
colorless liquid (1.9 g, 89%). 1H NMR (400 MHz, CDCl3) d 7.29–7.23
(m, 1H), 7.22–7.16 (m, 2H), 7.10 (d, J = 7.4 Hz, 1H), 3.92–3.77 (m,
4H), 2.97 (s, 3H), 2.37 (s, 3H), 2.05–1.92 (m, 4H).
4.1.5.5. 2-(Benzyloxy)-5-chloroaniline (16c). Following the
above procedure, the desired product was obtained as a red oil
with a yield of 68%. 1H NMR (400 MHz, CDCl3) d 7.50–7.30 (m,
5H), 6.76 (s, 1H), 6.57 (d, J = 1.8 Hz, 1H), 6.51 (d, J = 1.8 Hz, 1H),
5.07 (s, 2H), 3.65 (s, 2H).
4.1.5.6. 2-(Benzyloxy)-5-methylaniline (16d). Following the
above procedure, the desired product was obtained as a brown
oil with a yield of 73%. 1H NMR (400 MHz, CDCl3) d 7.38–7.25
(m, 5H), 6.68 (s, 1H), 6.54 (d, J = 2.0 Hz, 1H), 6.46 (d, J = 2.0 Hz,
1H), 5.08 (s, 2H), 3.65 (s, 2H), 2.17 (s, 3H).
4.1.3. 4-(3-(Bromomethyl)phenyl)-4-methoxytetrahydro-2H-
pyran (11)
A mixture of compound 10 (2.1 g, 10 mmol), N-bromosuccini-
mide (2.0 g, 11 mmol), and azoisobutyronitrile (100 mg, 0.6 mmol)
in carbon tetrachloride (80 mL) was heated under reflux for 1.5 h.
Then, the reaction mixture was cooled to room temperature
and filtered. The filtrate was evaporated to give a crude product,
which was purified using flash chromatography with hexane–
EtOAc (80:20) elution to give a colorless liquid (2.4 g, 85%). 1H
NMR (400 MHz, CDCl3) d 7.29–7.22 (m, 3H), 7.10 (m, 1H), 4.51
(s, 2H), 3.94–3.77 (m, 4H), 2.98 (s, 3H), 2.36 (s, 3H), 2.06–1.94
(m, 4H).
4.1.5.7. Ethyl 3-amino-4-(benzyloxy) benzoate (16e). Following
the above procedure, the desired product was obtained as a red oil
with a yield of 57%. 1H NMR (400 MHz, CDCl3) d 7.48–7.30 (m, 5H),
6.70 (d, J = 8.7 Hz, 1H), 6.66 (d, J = 2.5 Hz, 1H), 6.63 (dd, J = 8.7,
2.5 Hz, 1H), 5.06 (s, 2H), 4.39–4.27 (q, J = 2.3 Hz, 2H), 3.72 (s, 2H)
,1.42 (t, J = 2.3 Hz, 3H).
4.1.5.8. 2-(Benzyloxy)-5-nitroaniline (16f). Following the above
procedure, the desired product was obtained as a yellow solid with
a yield of 86%. 1H NMR (400 MHz, CDCl3) d 7.45–7.30 (m, 5H), 6.74
(d, J = 8.5 Hz, 1H), 6.70 (d, J = 2.4 Hz, 1H), 6.63 (dd, J = 8.5, 2.5 Hz,
1H), 5.05 (s, 2H), 3.84 (s, 2H).
4.1.4. (3-(4-Methoxytetrahydro-2H-pyran-4-yl)phenyl)meth-
anesulfonyl chloride (12)
A mixture of compound 11 (1.0 equiv) and sodium sulfite
(1.2 equiv) in H2O (0.1 M) and EtOH (0.2 M) was heated under re-
flux overnight. The mixture was cooled to room temperature, and
concentrated until a precipitate began to form. The product was
collected through filtration to give a white solid. To a suspension
of this white solid (1.0 equiv) in CH2Cl2 (0.1 M) were added DMF
(0.1 equiv) and SOCl2 (5 equiv). After 1.5 h, the white suspension
was concentrated. The sulfonyl chloride compound 12 thus formed
was used for the next step without further purification (yield 69%).
4.1.6. General procedure for the preparation of methane
sulfonamides 17a–e
4.1.6.1. N-(2-(Benzyloxy)phenyl)methanesulfonamide (17a).
A
solution of pyridine (1 mL) and compound 16a (1.0 g, 5 mmol)
was stirred at room temperature. The solution of MeSO2Cl
(1.6 mL, 20 mmol) in CH2Cl2 was added slowly to keep the reaction
solution at a gentle reflux. After the addition was finished, the reac-
tion mixture was stirred at room temperature for 1 h. Then the
mixture was washed with saturated NaHCO3, 1 M HCl, and brine,
which was then dried with MgSO4 and evaporated to give a crude
product. This product was purified through recrystallization in eth-
anol to give a white crystal with a yield of 88%. Mp: 78–82 °C; 1H
NMR (400 MHz, CDCl3) d 7.45–7.31 (m, 7H), 6.95–6.85 (m, 2H),
6.70 (s, 1H), 5.08 (s, 2H), 2.89 (s, 3H).
4.1.5. General procedure for the preparation of anilines 16a–f
4.1.5.1. 2-(2-Hydroxyphenyl) isoindoline-1,3-dione (14a).
A
mixture of 2-aminophenol 13a (2.2 g, 20 mmol) and phthalic anhy-
dride (3.0 g, 20 mmol) in acetic acid (100 mL) was heated under re-
flux for two hours, and put in cooled water. The suspension was
filtered, and the aqueous filtrate was extracted with chloroform.
The organic phase was dried with MgSO4, and the solvent was re-
moved. The residue and the solid, obtained through filtration, were
used in the next step without further purification (yield 93%).
4.1.6.2. N-(2-(Benzyloxy)-4-chlorophenyl)methanesulfonamide
(17b). Following the above procedure, the desired product was
obtained as a white solid with a yield of 86%. Mp: 81–83 °C; 1H
NMR (400 MHz, CDCl3) d 7.54–7.39 (m, 6H), 6.95 (s, 1H), 6.92 (d,
J = 2.4 Hz, 1H), 6.75 (d, J = 2.4 Hz, 1H), 5.15 (s, 2H), 2.83 (s, 3H).
4.1.5.2. 2-(2-(Benzyloxy)phenyl) isoindoline-1,3-dione (15a).
K2CO3 (1.4 g, 10 mmol) and benzyl bromide (2.1 g, 12 mmol) were
sequentially added to a solution of compound 14a (2.4 g, 10 mmol)
in acetone, and the mixture was stirred under reflux for 3 h. The
reaction mixture was cooled to room temperature and filtered.
The filtrate was evaporated to give the desired product, which
was used for the next step without further purification (yield 86%).
4.1.6.3. N-(2-(Benzyloxy)-5-chlorophenyl)methanesulfonamide
(17c). Following the above procedure, the desired product was
obtained as a white solid with a yield of 76%. Mp: 80–83 °C; 1H
NMR (400 MHz, CDCl3) d 7.50–7.29 (m, 6H), 6.97 (s, 1H), 6.96 (d,
J = 2.4 Hz, 1H), 6.70 (d, J = 2.4 Hz, 1H), 5.17 (s, 2H), 2.89 (s, 3H).
4.1.5.3. 2-(Benzyloxy) aniline (16a). A solution of dioxolan 15a
(1.7 g, 5 mmol) and NH2NH2ÁH2O (2 mL) in ethanol (30 mL) was
heated under reflux for 2 h. The reaction mixture was cooled to
room temperature, and the mixture was filtered through Celite.
The solvent was removed, and the crude product was purified
using column chromatography with hexane–EtOAc (90:10) elution
to give a red oil (0.83 g, 83%). 1H NMR (400 MHz, CDCl3) d 7.52–
7.27 (m, 5H), 6.91–6.77 (m, 2H), 6.76–6.64 (m, 2H), 5.08 (s, 2H),
3.70 (s, 2H).
4.1.6.4. N-(2-(Benzyloxy)-5-methylphenyl)methanesulfonamide
(17d). Following the above procedure, the desired product was
obtained as a white solid with a yield of 77%. Mp: 78–81 °C; 1H
NMR (400 MHz, CDCl3) d 7.43–7.33 (m, 6H), 6.93–6.87 (m, 2H),
6.77 (s, 1H), 5.10 (s, 2H), 2.88 (s, 3H), 2.29 (s, 3H).
4.1.6.5. N-(2-(Benzyloxy)-5-nitrophenyl)methanesulfonamide
(17e). Following the above procedure, the desired product was

Y. Li et al. / Bioorg. Med. Chem. 19 (2011) 2074–2083
2081
obtained as a pale yellow solid with a yield of 46%. Mp 89–93 °C;
1H NMR (400 MHz, CDCl3) d 7.56 (d, J = 2.5 Hz, 1H), 7.55–7.28 (m,
5H), 7.06 (dd, J = 8.8, 2.5 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 6.86 (s,
1H), 5.09 (s, 2H), 2.95 (s, 3H).
2.01–1.95 (m, 4H); 13C NMR (101 MHz, CDCl3) d 150.81, 141.37,
140.74, 137.09, 136.10, 130.07, 129.62, 129.36, 128.12, 128.19,
128.17, 125.82, 124.47, 122.45, 119.82, 109.27, 75.64, 71.33,
67.32, 61.00, 52.22, 34.78; ESI-MS: m/z 527 [M+H]+.
4.1.7. General procedure for the preparation of sulfonamides
20a–f
4.1.7.6. Ethyl 4-(benzyloxy)-3-((3-(4-methoxytetrahydro-2H-
pyran-4-yl)phenyl)methylsulfonamido) benzoate (20f). Fol-
lowing the above procedure, the desired product was obtained as
a white solid with a yield of 73%; mp: 120–123 °C; 1H NMR
(400 MHz, CDCl3) d 7.52–7.35 (m, 5H), 7.28–7.20 (m, 3H), 7.19 (s,
1H), 7.11 (d, J = 4.8 Hz, 1H), 6.98 (d, J = 4.8 Hz, 2H), 5.10 (s, 2H),
4.97 (s, 1H), 4.51 (s, 2H), 4.37–4.21 (q, J = 2.7 Hz, 2H), 3.90–3.69
(m, 4H), 2.87 (s, 3H), 1.96–1.84 (m, 4H) ,1.35 (t, J = 2.7 Hz, 3H);
13C NMR (101 MHz, CDCl3) d 167.21, 150.43, 148.25, 143.73,
136.56, 134.54, 128.46, 127.97, 127.30, 125.88, 124.66, 124.12,
123.45, 122.59, 114.77, 112.79, 73.91, 70.38, 69.89, 62.70, 59.77,
48.80, 34.27, 13.33; HRMS (ESI): [M+Na]+ (C29H33NNaO7S) calcd
562.1875, found 562.1861.
To a solution of the amine (1.0 mmol) in CH2Cl2 (10 mL) were
added sulfonyl chloride (1.2 mmol) and saturated NaHCO3
(10 mL). The resulting suspension was stirred until the amine
was consumed through TLC analysis. The mixture was diluted with
CH2Cl2 (20 mL), washed with H2O (20 mL) and brine, dried, and
concentrated. Purification of the crude product using flash chroma-
tography (EtOAc–hexanes) afforded the sulfonamide.
4.1.7.1. N-(2-(Benzyloxy)phenyl)-1-(3-(4-methoxytetrahydro-
2H-pyran-4-yl)phenyl)methanesulfonamide (20a). Following
the above procedure, the desired product was obtained as a white
solid with a yield of 73%; mp: 130–133 °C; 1H NMR (400 MHz,
CDCl3) d 7.45–7.26 (m, 5H), 7.25–7.11 (m, 5H), 7.05–6.86 (m,
3H), 5.11 (s, 2H), 4.92 (s, 1H), 4.57 (s, 2H), 3.85–3.74 (m, 4H),
2.81 (s, 3H), 1.94–1.83 (m, 4H); 13C NMR (101 MHz, CDCl3) d
151.71, 140.77, 137.19, 136.10, 134.69, 131.62, 129.36, 128.32,
128.23, 128.17, 128.16, 125.72, 125.32, 123.02, 122.45, 114.21,
75.14, 71.13, 67.62, 61.10, 50.19, 34.95; ESI-MS: m/z 480 [MÀH]À.
4.1.8. General procedure for the preparation of compounds
21a–e, 22a–e, and 23a–d
K2CO3 (1 equiv) and substituted benzyl bromine (1.1 equiv)
were sequentially added to a solution of phenols 19a–d, anilines
16a–e, or methanesulfonamides 17a–e (1.0 equiv) in acetone/ace-
tonitrile, and the mixture was stirred under reflux from 3 h to over-
night. Then, the reaction mixture was cooled to room temperature
and filtered. The filtrate was evaporated to give a crude product,
which was purified using flash chromatography with hexane–
EtOAc to give the desired final product.
4.1.7.2. N-(2-(Benzyloxy)-4-chlorophenyl)-1-(3-(4-methoxytet-
rahydro-2H-pyran-4-yl)phenyl)methane-sulfonamide (20b).
Following the above procedure, the desired product was obtained
as a white solid with a yield of 71%; mp: 132–135 °C; 1H NMR
(400 MHz, CDCl3) d 7.44–7.12 (m, 9H), 6.78–6.53 (m, 3H), 5.12 (s,
2H), 4.94 (s, 1H), 4.59 (s, 2H), 3.89–3.78 (m, 4H), 2.92 (s, 3H),
2.03–1.94 (m, 4H); 13C NMR (101 MHz, CDCl3) d 154.18, 144.57,
136.19, 135.49, 134.08, 129.58, 128.95, 128.90, 128.87, 128.08,
127.98, 127.78, 126.67, 125.45, 125.78, 113.55, 74.70, 70.91,
63.56, 55.44, 49.83, 35.24; ESI-MS: m/z 514 [MÀH]À.
4.1.8.1. 2-(Benzyloxy)-N-(3-(4-methoxytetrahydro-2H-pyran-4-
yl) benzyl) aniline (22a). Following the above procedure, the de-
sired product was obtained as a brown oil with a yield of 66%. 1H
NMR (400 MHz, CDCl3) d 7.46–7.26 (m, 9H), 6.88–6.81 (m, 2H),
6.70–6.56 (m, 2H), 5.10 (s, 2H), 4.73 (s, 1H), 4.38 (s, 2H), 3.89–
3.76 (m, 4H), 2.92 (s, 3H), 2.05–1.91 (m, 4H); 13C NMR (101 MHz,
CDCl3) d 144.83, 144.56, 139.52, 136.72, 128.81, 128.62, 128.16,
127.59, 127.54, 126.85, 126.31, 124.91, 124.88, 115.72, 111.92,
110.36, 74.95, 70.87, 63.65, 49.77, 47.86, 35.31; ESI-MS: m/z 402
[MÀH]À.
4.1.7.3. N-(2-(Benzyloxy)-5-chlorophenyl)-1-(3-(4-methoxytet-
rahydro-2H-pyran-4-yl)phenyl)methane-sulfonamide (20c).
Following the above procedure, the desired product was obtained
as a white solid with a yield of 75%; mp: 134–136 °C; 1H NMR
(400 MHz, CDCl3) d 7.54–7.15 (m, 9H), 6.88–6.55 (m, 3H), 5.11 (s,
2H), 4.98 (s, 1H), 4.63 (s, 2H), 3.82–3.75 (m, 4H), 2.94 (s, 3H),
1.99–1.90 (m, 4H); 13C NMR (101 MHz, CDCl3) d 149.10, 140.54,
136.09, 135.10, 134.48, 130.62, 129.56, 128.72, 128.67, 128.16,
127.90, 127.66, 126.72, 125.75, 125.45, 113.40, 74.54, 70.43,
63.32, 55.00, 50.19, 34.98; ESI-MS: m/z 514 [MÀH]À.
4.1.8.2. 2-(Benzyloxy)-4-chloro-N-(3-(4-methoxytetrahydro-2H-
pyran-4-yl) benzyl) aniline (22b). Following the above proce-
dure, the desired product was obtained as red oil with a yield of
63%. 1H NMR (400 MHz, CDCl3) d 7.45–7.24 (m, 9H), 6.78 (s, 1H),
6.68 (d, J = 4.9 Hz, 1H), 6.63 (d, J = 4.9 Hz, 1H), 5.08 (s, 2H), 4.68
(s, 1H), 4.43 (s, 2H), 3.85–3.76 (m, 4H), 2.93 (s, 3H), 2.05–1.95
(m, 4H); 13C NMR (101 MHz, DMSO) d 154.21, 144.78, 144.23,
136.16, 135.57, 129.16, 129.34, 128.32, 127.97, 127.47, 127.61,
126.56, 125.87, 119.09, 117.23, 116.67, 74.35, 73.79, 70.56, 63.04,
48.97, 34.63; HRMS (ESI): [M+Na]+ (C26H28ClNNaO3) calcd
460.1655, found 460.1641.
4.1.7.4. N-(2-(Benzyloxy)-5-methylphenyl)-1-(3-(4-methoxytet-
rahydro-2H-pyran-4-yl)phenyl)methane-sulfonamide (20d).
Following the above procedure, the desired product was obtained
as a white solid with a yield of 74%; mp 123–125 °C; 1H NMR
(400 MHz, CDCl3) d 7.46–7.27 (m, 8H), 6.85–6.77 (m, 2H), 6.70–
6.63 (m, 2H), 5.11 (s, 2H), 4.84 (s, 1H), 4.51 (s, 2H), 3.84–3.76 (m,
4H), 2.95 (s, 3H), 2.21 (s, 3H), 2.06–1.95 (m, 4H); 13C NMR
4.1.8.3. 2-(Benzyloxy)-5-chloro-N-(3-(4-methoxytetrahydro-2H-
pyran-4-yl)benzyl)aniline (22c). Following the above proce-
dure, the desired product was obtained as red oil with a yield of
43%. 1H NMR (400 MHz, CDCl3) d 7.44–7.22 (m, 9H), 6.71 (s, 1H),
6.58–6.53 (m, 2H), 5.10 (s, 2H), 4.74 (s, 1H), 4.39 (s, 2H), 3.88–
3.78 (m, 4H), 2.91 (s, 3H), 2.07–1.92 (m, 4H); 13C NMR (101 MHz,
DMSO) d 154.22, 144.71, 144.25, 136.12, 135.47, 129.15, 129.34,
128.31, 127.95, 127.44, 127.60, 126.57, 125.88, 119.11, 117.22,
116.61, 74.38, 73.78, 70.55, 63.04, 48.98, 34.62; ESI-MS: m/z 460
[M+Na]+.
(101 MHz, CDCl3)
d 149.91, 140.74, 136.89, 135.10, 134.62,
129.76, 129.36, 128.32, 128.17, 128.16, 128.15, 127.72, 126.63,
125.45, 125.14, 112.61, 75.54, 71.43, 63.42, 55.19, 52.19, 34.88,
21.21; ESI-MS: m/z 518 [M+Na]+.
4.1.7.5. N-(2-(Benzyloxy)-5-nitrophenyl)-1-(3-(4-methoxytetra-
hydro-2H-pyran-4-yl)phenyl)methanesulfonamide
(20e).
Following the above procedure, the desired product was obtained
as a white solid with a yield of 72%; mp 135–137 °C; 1H NMR
(400 MHz, CDCl3) d 7.64–7.43 (m, 9H), 7.08–6.83 (m, 3H), 5.12 (s,
2H), 4.99 (s, 1H), 4.64 (s, 2H), 3.90–3.88 (m, 4H), 2.98 (s, 3H),
4.1.8.4. 2-(Benzyloxy)-N-(3-(4-methoxytetrahydro-2H-pyran-4-
yl)benzyl)-5-methylaniline (22d). Following the above proce-

2082
Y. Li et al. / Bioorg. Med. Chem. 19 (2011) 2074–2083
dure, the desired product was obtained as brown oil with a yield of
55%. 1H NMR (400 MHz, CDCl3) d 7.46–7.26 (m, 8H), 6.84–6.80 (m,
2H), 6.75–6.66 (m, 2H), 5.10 (s, 2H), 4.75 (s, 1H), 4.41 (s, 2H), 3.87–
3.75 (m, 4H), 2.92 (s, 3H), 2.17 (s, 3H), 2.06–1.91 (m, 4H); 13C NMR
(101 MHz, CDCl3) d 144.84, 144.55, 139.42, 136.72, 128.81, 128.62,
128.16, 127.59, 127.54, 126.85, 126.31, 124.91, 124.88, 115.72,
111.92, 110.36, 74.95, 70.87, 63.65, 49.77, 47.86, 35.31, 20.15;
ESI-MS: m/z 418 [M+H]+.
128.46, 127.95, 127.68, 126.61, 126.18, 125.20, 112.40, 74.73,
70.52, 63.60, 53.62, 49.59, 39.69, 35.20, 20.11; ESI-MS: m/z 496
[M+H]+.
4.1.8.10. N-(2-(Benzyloxy)-5-nitrophenyl)-N-(3-(4-methoxytet-
rahydro-2H-pyran-4-yl)benzyl)methanesulfonamide (21e).
Following the above procedure, the desired product was obtained
as a pale yellow solid with a yield of 33%. Mp: 102–105 °C; 1H
NMR (400 MHz, CDCl3) d 7.45–7.36 (m, 5H), 7.29–7.21 (m, 5H),
7.15 (s, 1H), 7.02 (d, J = 5.1 Hz, 1H), 6.86 (d, J = 5.1 Hz, 1H), 5.08
(s, 2H), 4.98 (s,2H), 3.98–3.72 (m, 4H), 2.86 (s, 3H), 2.81 (s, 3H),
1.98–1.85 (m, 4H); 13C NMR (101 MHz, CDCl3) d 154.13, 144.54,
136.23, 135.50, 134.08, 129.61, 128.97, 128.77, 128.73, 127.98,
127.80, 127.53, 126.61, 125.62, 125.53, 113.52, 74.73, 70.96,
63.59, 53.54, 49.73, 39.95, 35.18; ESI-MS: m/z 527 [M+H]+.
4.1.8.5. 2-(Benzyloxy)-N-(3-(4-methoxytetrahydro-2H-pyran-4-
yl)benzyl)-5-nitroaniline (22e). Following the above procedure,
the desired product was obtained as a yellow solid with a yield of
56%. 1H NMR (400 MHz, CDCl3) d 7.44–7.22 (m, 9H), 6.71 (s, 1H),
6.58 (d, J = 5.2 Hz, 1H), 6.53 (d, J = 5.2 Hz, 1H), 5.08 (s, 2H), 4.78
(s, 1H), 4.40 (s, 2H), 3.89–3.78 (m, 4H), 2.91 (s, 3H), 2.06–1.92
(m, 4H); 13C NMR (101 MHz, CDCl3) d 150.11, 141.77, 140.71,
137.19, 136.11, 130.11, 129.67, 129.99, 128.12, 128.19, 128.24,
125.67, 124.57, 122.46, 119.88, 109.29, 75.64, 71.33, 67.52, 61.09,
52.48, 34.77; ESI-MS: m/z 461 [M+Na]+.
4.1.8.11. 4-(3-((2-(Benzyloxy)phenoxy)methyl)phenyl)-4-meth-
oxytetrahydro-2H-pyran (23a). Following the above procedure,
the desired product was obtained as a white oil with a yield of 78%.
1H NMR (400 MHz, CDCl3) d 7.53–7.22 (m, 10H), 7.05–6.79 (m, 3H),
5.16 (s, 2H), 5.11 (s, 2H), 3.94–3.75 (m, 4H), 2.97 (s, 3H), 2.04–1.92
(m, 4H); 13C NMR (101 MHz, CDCl3) d 148.23, 147.98, 143.64,
136.59, 127.58, 127.44, 126.75, 126.28, 125.48, 124.41, 124.25,
124.01, 120.82, 120.61, 120.03, 114.54, 73.91, 70.49, 70.25, 62.64,
48.75, 34.28; ESI-MS: m/z 427 [M+Na]+.
4.1.8.6. N-(2-(Benzyloxy)phenyl)-N-(3-(4-methoxytetrahydro-
2H-pyran-4-yl)benzyl)methanesulfonamide (21a). Following
the above procedure, the desired product was obtained as a white
solid with a yield of 77%. Mp: 98–103 °C; 1H NMR (400 MHz,
CDCl3) d 7.45–7.36 (m, 5H), 7.29–7.21 (m, 5H), 7.05–6.86 (m,
3H), 5.10 (s, 2H), 4.77 (s, 2H), 3.88–3.74 (m, 4H), 2.87 (s, 3H),
2.82 (s, 3H), 1.96–1.84 (m, 4H); 13C NMR (101 MHz, CDCl3) d
154.16, 144.56, 136.24, 135.52, 134.05, 129.57, 128.94, 128.74,
128.70, 127.96, 127.77, 127.63, 126.61, 125.64, 125.52, 113.54,
74.73, 70.98, 63.59, 53.57, 49.69, 39.96, 35.20; ESI-MS: m/z 482
[M+H]+.
4.1.8.12. 4-(3-((2-(Benzyloxy)-4-chlorophenoxy)methyl)phenyl)-
4-methoxytetrahydro-2H-pyran (23b). Following the above
procedure, the desired product was obtained as a colorless solid
with a yield of 81%. Mp: 55–57 °C; 1H NMR (400 MHz, CDCl3) d
7.55–7.33 (m, 12H), 5.22 (s, 2H), 4.95 (s, 2H), 3.70–3.66 (m, 4H),
3.36 (s, 3H), 1.94–1.86 (m, 4H); 13C NMR (101 MHz, CDCl3) d
153.23, 144.39, 144.10, 136.08, 135.46, 128.46, 128.44, 128.14,
127.87, 127.55, 127.51, 126.29, 125.74, 118.08, 116.99, 116.49,
74.35, 73.83, 70.69, 62.79, 49.17, 34.73; HRMS (ESI): [M+Na]+
(C26H27ClNaO4) calcd 461.1496, found 461.1481.
4.1.8.7. N-(2-(Benzyloxy)-4-chlorophenyl)-N-(3-(4-methoxytet-
rahydro-2H-pyran-4-yl)benzyl)methanesulfonamide (21b).
Following the above procedure, the desired product was obtained
as a white solid with a yield of 64%. Mp 90–93 °C; 1H NMR
(400 MHz, CDCl3) d 7.50–7.36 (m, 5H), 7.36–7.09 (m, 5H), 6.98
(d, J = 5.8 Hz, 1H), 6.92 (d, J = 5.8 Hz, 1H), 5.10 (s, 2H), 4.75 (s,
2H), 3.99–3.71 (m, 4H), 2.88 (s, 3H), 2.83 (s, 3H), 1.96–1.85 (m,
4H); 13C NMR (101 MHz, CDCl3) d 153.13, 144.14, 136.88, 136.22,
134.73, 130.55, 130.12, 128.83, 128.48, 128.46, 127.98, 127.69,
126.61, 126.19, 125.23, 112.46, 74.75, 70.53, 63.61, 53.63, 49.61,
39.78, 35.44; HRMS (ESI): [M+H]+ (C27H31ClNO5S) calcd 516.1533,
found 516.1511.
4.1.8.13. 4-(3-((2-(Benzyloxy)-4-bromophenoxy)methyl)phenyl)-
4- methoxytetrahydro-2H-pyran (23c). Following the above
procedure, the desired product was obtained as a white solid with
a yield of 88%. Mp: 58–61 °C; 1H NMR (400 MHz, CDCl3) d 7.58–
7.30 (m, 12H), 5.22 (s, 2H), 4.94 (s, 2H), 3.72–3.68 (m, 4H), 3.33
(s, 3H), 1.97–1.88 (m, 4H); 13C NMR (101 MHz, CDCl3) d 153.35,
144.29, 144.14, 136.09, 135.56, 128.56, 128.47, 128.11, 127.81,
127.56, 127.53, 126.49, 125.78, 118.11, 117.08, 116.47, 74.37,
73.79, 70.69, 62.78, 49.20, 34.75; ESI-MS: m/z 505 [M+Na]+.
4.1.8.8. N-(2-(Benzyloxy)-5-chlorophenyl)-N-(3-(4-methoxytet-
rahydro-2H-pyran-4-yl)benzyl)methanesulfonamide (21c).
Following the above procedure, the desired product was obtained
as a white solid with a yield of 62%. Mp: 94–97 °C; 1H NMR
(400 MHz, CDCl3) d 7.49–7.34 (m, 5H), 7.35–7.09 (m, 5H), 6.88
(d, J = 5.6 Hz, 1H), 6.72 (d, J = 5.6 Hz, 1H), 5.10 (s, 2H), 4.75 (s,
2H), 3.96–3.70 (m, 4H), 2.88 (s, 3H), 2.85 (s, 3H), 1.98–1.86 (m,
4H); 13C NMR (101 MHz, CDCl3) d 153.11, 144.15, 136.89, 136.22,
134.73, 130.54, 130.12, 128.81, 128.49, 128.46, 127.96, 127.69,
126.61, 126.19, 125.20, 112.45, 74.77, 70.53, 63.61, 53.63, 49.61,
39.77, 35.45; ESI-MS: m/z 516 [M+H]+.
4.1.8.14. Ethyl 3-(benzyloxy)-4-(3-(4-methoxytetrahydro-2H-
pyran-4-yl)benzyloxy)benzoate (23d).
Following the above
procedure, the desired product was obtained as a colorless oil with
a yield of 43%. 1H NMR (400 MHz, CDCl3) d 7.69–7.62 (m, 2H),
7.56–7.30 (m, 9H), 6.95 (d, J = 4.9 Hz,1H), 5.2 (s, 4H), 4.39–4.28
(q, J = 2.8 Hz ,2H), 3.94–3.73 (m, 4H), 2.92 (s, 3H), 2.11–1.83 (m,
4H), 1.37 (t, J = 2.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 165.20,
151.93, 147.23, 143.72, 136.10, 135.54, 127.64, 127.57, 127.00,
125.48, 124.56, 124.05, 123.04, 122.50, 114.72, 112.12, 73.90,
70.34, 69.84, 62.64, 59.75, 48.76, 34.28, 13.36; HRMS (ESI):
[M+Na]+ (C29H32NaO6) calcd 499.2097, found 499.2071.
4.1.8.9. N-(2-(Benzyloxy)-5-methylphenyl)-N-(3-(4-methoxytet-
rahydro-2H-pyran-4-yl)benzyl)methanesulfonamide (21d).
Following the above procedure, the desired product was obtained
as a white solid with a yield of 58%. Mp: 88–91 °C; 1H NMR
(400 MHz, CDCl3) d 7.49–7.35 (m, 5H), 7.29–7.20 (m, 3H), 7.15 (s,
1H), 7.01 (d, J = 4.8 Hz, 1H), 6.86 (d, J = 4.8 Hz, 2H), 5.10 (s, 2H),
4.78 (s, 2H), 3.90–3.69 (m, 4H), 2.87 (s, 3H), 2.82 (s, 3H), 2.12 (s,
3H), 1.96–1.84 (m, 4H); 13C NMR (101 MHz, CDCl3) d 153.08,
144.19,136.90, 136.22, 134.70, 130.54, 130.13, 128.81, 128.48,
4.2. Cyclooxygenase-1/2 inhibition assay
All compounds were tested for their activity to inhibit COX-1
and COX-2 using a COX (ovine) inhibitor screening kit (Catalog
No. 560101, Cayman Chemical, Ann Arbor, MI) according to
the manufacturer’s instructions. The COX (ovine) Inhibitor
Screening Assay directly measures PGF2 through SnCl2 reduction
a

Y. Li et al. / Bioorg. Med. Chem. 19 (2011) 2074–2083
2083
of COX-derived PGH2 produced in COX reaction. The prostanoid
product was measured with enzyme immunoassay (EIA) using a
broadly specific antiserum that binds to all major PG compounds.
COX reagent preparation was carefully performed according to
instructions before COX reaction. Reaction buffer solution
their ligands were used to check the AUTODOCK program for the bind-
ing modes of compounds in the binding sites of the enzymes.38 For
this purpose, the complexes were manipulated to extract the
inhibitors, which were in turn computationally re-docked by using
AUTODOCK within the binding sites. The grid was narrowed to an area
slightly larger than the enzyme binding sites (15 Â 18 Â 18 Å) and
was centered on the mass center of the crystallographic inhibitors
for both COX-2 and 15-LOX. Docking was carried out with a max-
imum output of 25 structures. By using these settings, the program
successfully reproduced the X-ray coordinates of the inhibitor’s
binding conformations. The same set of parameters was also used
to perform docking simulations of all the remaining inhibitors. The
most favorable conformations of inhibitors were chosen based on
the combination of affinity energy and structures given by the pro-
gram. The candidate models were energy-minimized with Discov-
ery Studio to get the final models.
(960
2 mM phenol) with either COX-1 or COX-2 (10
presence of heme (10 L) was added 10 L of various concentra-
tions of testing drug solutions (0.1, 1, 10, 50 M) in a final volume
of 1 mL. This solution was incubated for a period of 2 min at 37 °C,
and then 10 L of AA (100 M) was added. The COX reaction was
stopped through the addition of 50 L of 1 M HCl after 2 min.
lL, 0.1 M Tris–HCl, pH 8.0, containing 5 mM EDTA and
lL) enzyme in the
l
l
l
l
l
l
PGF2 , which was produced from PGH2 through reduction with
a
stannous chloride, was measured with enzyme immunoassay.
After the addition of EIA reagent, the plate was covered with plastic
film and incubated for 18 h at room temperature on an orbital sha-
ker. The plate was then washed to remove any unbound reagents,
and Ellman’s Reagent was added to the well. The product of this
enzymatic reaction had a yellow color. The COX activity was deter-
mined by measuring the absorbance at a wavelength of 410 nm.
The percentage inhibition was calculated through comparison of
enzymatic activity with and without inhibitor. The IC50 value was
determined as the concentration of the testing compound causing
50% inhibition of enzymatic activity, and the average of two mea-
sured data was reported.
Acknowledgements
We thank the Natural Science Foundation of China (Grants
U0832005, 90813011) and the Specialized Research Fund for the
Doctoral Program of Higher Education (20090171110050) for
financial support of this study.
References and notes
1. Hamberg, M.; Samuelsson, B. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 899.
2. Rouzer, C. A.; Marnett, L. Chem. Rev. 2003, 103, 2239.
4.3. 5-Lipoxygenase inhibition assay
3. Vane, J. R.; Bakhle, Y. S.; Botting, R. M. Annu. Rev. Pharmacol. Toxicol. 1998, 38,
97.
4. Vane, J. R. Nature (London), New Biol. 1971, 231, 232.
5. Smith, J. B.; Willis, A. L. Nature (London), New Biol. 1971, 231, 235.
6. Ferreira, S. H.; Moncada, S.; Vane, J. R. Nature (London), New Biol. 1971, 231, 237.
7. Hawkey, C. J. Gastroenterology 2000, 119, 521.
8. Barrier, C. H.; Hirschowitz, B. I. Arthritis Rheum. 1989, 32, 926.
9. Fries, J. F.; Miller, S. R.; Spitz, P. W.; Williams, C. A.; Hubert, H. B.; Bloch, D. A.
Gastroenterology 1989, 96, 647.
10. Masferrer, J. L.; Zweifel, B. S.; Manning, P. T.; Hauser, S. D.; Leahy, K. M.; Smith,
W. G.; Isakson, P. C.; Seibert, K. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3228.
11. Flower, R. J. Nat. Rev. Drug Disc. 2003, 2, 179.
12. Dogne, J. M.; Supuran, C. T.; Pratico, D. J. Med. Chem. 2005, 48, 2251.
13. Solomon, D. H. Arthritis Rheum. 2005, 52, 1968.
14. McAdam, R. F.; Catella-Lawson, F.; Nardini, I. A.; Fitzgerald, G. A. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 272.
15. Van Steenbergen, W.; Rosksams, T.; Desmet, V. J. Hepatol. 1998, 29, 135.
16. Rainsford, K. D. Rheumatology 1999, 38, 4.
The IC50 values of all compounds was determined using potato
5-LOX (Catalog No. 60401, Cayman Chemical, Ann Arbor, MI, USA)
and an enzyme immuno assay (EIA) kit (Catalog No. 760700, Cay-
man Chemical, Ann Arbor, MI, USA) according to manufacturer’s
instructions. The lipoxygenase inhibitor screening assay detects
and measures the hydroperoxides produced in the lipoxygenation
reaction. Pre-assay preparation was carefully performed according
to the instructions. To a 90
l
L solution of 5-LOX enzyme in 0.1 M
L of various concentrations of testing
M in a final volume of 210 L)
were added, and the lipoxygenase reaction was initiated by the
addition of 10 L (100 M) of linoleic acid (LA). After putting the
96-well plate on a shaker for 5 min, 100 L of chromogen was
Tris–HCl buffer, pH 7.4, 10
l
drug solutions (0.1, 1, 10 and 50
l
l
l
l
l
17. Lewis, R. A.; Austen, K. F.; Soberman, R. N. Eng. J. Med. 1990, 323, 645.
18. Vila, L. Med. Res. Rev. 2004, 24, 399.
added, and the plate was on the shaker for another 5 min. The
lipoxygenase activity was determined by measuring absorbance
at a wavelength of 490 nm. The percentage inhibition was calcu-
lated through the comparison of enzymatic activity with and with-
out inhibitor. The IC50 value was the concentration of the testing
compound causing 50% inhibition of enzymatic activity, and the
average of two measured data was reported.
19. Zhao, L.; Funk, C. D. Trends Cardiovasc. Med. 2004, 14, 191.
20. Jampilek, J.; Dolezal, M.; Opletalova, V.; Hartl, J. Curr. Med. Chem. 2006, 13, 117.
21. McMillan, R. M.; Walker, E. R. Trends Pharmacol. Sci. 1992, 13, 323.
22. Charlier, C.; Michaux, C. Eur. J. Med. Chem. 2003, 38, 645.
23. Rao, P. N. P.; Chen, Q.-H.; Knaus, E. E. J. Med. Chem. 2006, 49, 1668.
24. Barbey, S.; Goossens, L.; Taverne, T.; Cornet, J.; Choesmel, V.; Rouaud, C.;
Gimeno, G.; Yannic-Arnoult, S.; Michaux, C.; Charlier, C.; Houssin, R.;
Henichart, J.-P. Bioorg. Med. Chem. Lett. 2002, 12, 779.
25. Pommery, N.; Taverne, T.; Telliez, A.; Goossens, L.; Charlier, C.; Pommery, J.;
Goossens, J.; Houssin, R.; Durant, F.; Hnichart, J. J. Med. Chem. 2004, 47,
6195.
4.4. Molecular modeling
26. Chowdhury, M. A.; Abdellatif, K. R. A.; Dong, Y.; Das, D.; Suresh, M. R.; Knaus, E.
E. J. Med. Chem. 2009, 52, 1525.
Since the 3D structures of human COX-2 and 5-LOX were not
available in the Protein Data Bank, the model structure of human
5-LOX was obtained through homology modeling using rabbit
15-LOX (PDB entry LOX1)35 and the docking study of COX-2 used
murine COX-2 (PDB entry 6COX)36 instead. We repeated the
homology modeling with Discovery Studio (DS 2.1 Accelrys, San
Diego, CA)37 to get a human 5-LOX model. With the highest se-
quence similarity, rabbit 15-LOX was used as template for the
homology model of human 5-LOX. Five models were done with
slightly difference and model with the best verify score was chosen
as the candidate. Then the candidate model was energy-minimized
using the CVFF force field to make the final model.
27. Fabiola, G. F.; Pattabhi, V.; Nagarajan, K. Bioorg. Med. Chem. 1998, 13, 6810.
28. Procopiou, P. A.; Barrett, V. J. J. Med. Chem. 2009, 52, 5280.
29. Su, B.; Tian, R.; Darby, M. V.; Brueggemeier, R. W. J. Med. Chem. 2008, 51, 1126.
30. Julemont, F.; de Leval, X. J. Med. Chem. 2004, 47, 6749.
31. Blobaum, A. L.; Marnett, L. J. J. Med. Chem. 2007, 50, 1425.
32. Trott, O.; Olson, A. J. J. Comput. Chem. 2009, 31, 455.
33. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San
Carlos, CA; 2003.
34. Charlier, C.; Hnichart, J.-P.; Durant, F.; Wouters, J. J. Med. Chem. 2006, 49, 186.
35. Gillmor, S. A.; Villasenor, A.; Fletterick, R.; Sigal, E.; Browner, M. F. Nat. Struct.
Biol. 1997, 4, 1003.
36. Kurumbail, R. G.; Stevens, A. M.; Gierse, J. K.; McDonald, J. J.; Stegeman, R. A.;
Pak, J. Y.; Gildehaus, D.; Miyashiro, J. M.; Penning, T. D.; Seibert, K.; Isakson, P.
C.; Stallings, W. C. Nature 1997, 385, 555.
Docking studies were performed using the program AUTODOCK
VINA 1.0.3. The crystallographic structures of murine COX-2 (PDB
entry 6COX) and rabbit 15-LOX (PDB entry LOX1) complexed with
37. Discovery Studio, version 2.1; Accelrys: San Diego, CA, 2008.
38. Harrak, Y.; Casula, G.; Basset, J.; Rosell, G.; Plescia, G.; Raffa, D.; Cusimano, M.
G.; Pouplana, R.; Pujol, M. D. J. Med. Chem. 2010, 53, 6560.