Synthesis and biological evaluation of acyclic triaryl (Z)-olefins possessing a 3,5-di-tert-butyl-4-hydroxyphenyl pharmacophore: Dual inhibitors of cyclooxygenases and lipoxygenases

Article abstract of DOI:10.1016/j.bmc.2006.03.054

A new class of regioisomeric acyclic triaryl (Z)-olefins possessing a 3,5-di-tert-butyl-4-hydroxyphenyl (DTBHP) 5-lipoxygenase (5-LOX) pharmacophore that is vicinal to a para-methanesulfonylphenyl cyclooxygenase-2 (COX-2) pharmacophore were designed for evaluation as selective COX-2 and/or 5-LOX inhibitors. The target compounds were synthesized via a highly stereoselective McMurry olefination cross-coupling reaction. This key synthetic step afforded a (Z):(E) olefinic mixture with a predominance for the (Z)-olefin stereoisomer. Structure-activity studies for the (Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4-methanesulfonylphenyl)-1-phenylalk-1-ene regioisomers showed that COX-1 inhibition decreased, COX-2 inhibition increased, and the COX-2 selectivity index (SI) increased as the chain length of the alkyl substituent attached to the olefinic double bond was increased (Et → n-butyl → n-heptyl). In this group of compounds, inhibition of both 5-LOX and 15-LOX was dependent upon the length of the alkyl substituent with the hex-1-ene compound 9c having a n-butyl substituent exhibiting potent inhibition of both 5-LOX (IC₅₀ = 0.3 μM) and 15-LOX (IC₅₀ = 0.8 μM) relative to the inactive (IC₅₀ > 10 μM) Et and n-heptyl analogs. Compound 9c is of particular interest since it also exhibits a dual inhibitory activity against the COX (COX-1 IC₅₀ = 3.0 μM, and COX-2 IC₅₀ = 0.36 μM, COX-2 SI = 8.3) isozymes. A comparison of the relative inhibitory activities of the two groups of regioisomers investigated shows that the regioisomers in which the alkyl substituent is attached to the same olefinic carbon atom (C-2) as the para-methanesulfonylphenyl moiety generally exhibit a greater potency with respect to COX-2 inhibition. The 4-hydroxy substituent in the 3,5-di-tert-butyl-4-hydroxyphenyl moiety is essential for COX and LOX inhibition since 3,5-di-tert-butyl-4-acetoxyphenyl derivatives were inactive inhibitors. These structure-activity data indicate acyclic triaryl (Z)-olefins constitute a suitable template for the design of dual COX-2/LOX inhibitors.

Full text of DOI:10.1016/j.bmc.2006.03.054

Bioorganic & Medicinal Chemistry 14 (2006) 5340–5350
Synthesis and biological evaluation of acyclic triaryl (Z)-olefins
possessing a 3,5-di-tert-butyl-4-hydroxyphenyl pharmacophore:
Dual inhibitors of cyclooxygenases and lipoxygenases
Anne Moreau, P. N. Praveen Rao and Edward E. Knaus*
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alta., Canada T6G 2N8
Received 30 January 2006; revised 20 March 2006; accepted 22 March 2006
Available online 3 May 2006
Abstract—A new class of regioisomeric acyclic triaryl (Z)-olefins possessing a 3,5-di-tert-butyl-4-hydroxyphenyl (DTBHP) 5-lipoxy-
genase (5-LOX) pharmacophore that is vicinal to a para-methanesulfonylphenyl cyclooxygenase-2 (COX-2) pharmacophore were
designed for evaluation as selective COX-2 and/or 5-LOX inhibitors. The target compounds were synthesized via a highly stereo-
selective McMurry olefination cross-coupling reaction. This key synthetic step afforded a (Z):(E) olefinic mixture with a predomi-
nance for the (Z)-olefin stereoisomer. Structure–activity studies for the (Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4-
methanesulfonylphenyl)-1-phenylalk-1-ene regioisomers showed that COX-1 inhibition decreased, COX-2 inhibition increased,
and the COX-2 selectivity index (SI) increased as the chain length of the alkyl substituent attached to the olefinic double bond
was increased (Et ! n-butyl ! n-heptyl). In this group of compounds, inhibition of both 5-LOX and 15-LOX was dependent upon
the length of the alkyl substituent with the hex-1-ene compound 9c having a n-butyl substituent exhibiting potent inhibition of both
5-LOX (IC50 = 0.3 lM) and 15-LOX (IC50 = 0.8 lM) relative to the inactive (IC50 > 10 lM) Et and n-heptyl analogs. Compound 9c
is of particular interest since it also exhibits a dual inhibitory activity against the COX (COX-1 IC50 = 3.0 lM, and COX-2
IC50 = 0.36 lM, COX-2 SI = 8.3) isozymes. A comparison of the relative inhibitory activities of the two groups of regioisomers
investigated shows that the regioisomers in which the alkyl substituent is attached to the same olefinic carbon atom (C-2) as the
para-methanesulfonylphenyl moiety generally exhibit a greater potency with respect to COX-2 inhibition. The 4-hydroxy substituent
in the 3,5-di-tert-butyl-4-hydroxyphenyl moiety is essential for COX and LOX inhibition since 3,5-di-tert-butyl-4-acetoxyphenyl
derivatives were inactive inhibitors. These structure–activity data indicate acyclic triaryl (Z)-olefins constitute a suitable template
for the design of dual COX-2/LOX inhibitors.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
inflammation, fever, and pain are produced via the
inducible COX-2 isozyme whereas, PGs that regulate
desirable gastrointestinal cytoprotective and renal func-
tions are produced via the constitutive COX-1 isozy-
me.1,3 The initial postulate that a selective COX-2
inhibitor would reduce inflammation without causing
gastric irritation was validated following the introduc-
tion of selective COX-2 inhibitors such as celecoxib
and rofecoxib. However, it was subsequently observed
that selective COX-2 inhibitors may alter the balance
in the cyclooxygenase pathway resulting in a decrease
in the level of the vasodilatory and anti-aggregatory
prostacyclin (PGI2), relative to an increase in the level
of the prothrombotic tromboxane A2 (TxA2), leading
to increased incidences of an adverse cardiovascular
thrombotic event.1
Arachidonic acid (AA) is the most abundant polyunsat-
urated fatty acid present in cell membranes. Following
the phospholipase A2 (PLA2) catalyzed release of AA
from membrane-bound phospholipids, AA is metabo-
lized by two major enzyme families, the cyclooxygenases
(COX-1, -2, and -3) and the lipoxygenases (5-, 8-, 12-,
and 15-LOX). In this regard, proinflammatory prosta-
glandins (PGs), produced via the COX pathway, and
leukotrienes (LTs), produced via the LOX pathway,
are implicated in physiologic processes such as inflam-
mation, fever, arthritis, bronchospasm,1 and the etiolo-
gy of cancer.2 PGs that induce undesirable
Keywords: Di-tert-butylphenol; Cyclooxygenase/lipoxygenase inhibi-
tion; Anti-inflammatory and analgesic activity.
Two of the three major isoforms (5-, 12-, and 15-LOX)
observed in humans induce undesirable physiological
effects. For example, 5-LOX is implicated in the
*
Corresponding author. Tel.: +1 780 492 5993; fax: +1 780 492
1217; e-mail: eknaus@pharmacy.ualberta.ca
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.054

A. Moreau et al. / Bioorg. Med. Chem. 14 (2006) 5340–5350
5341
production of LTs that are associated with inflammatory,
bronchoconstrictor, hypersensitivity, anaphylactic, and
asthmatic actions.1,4 15-LOX is involved in atherosclero-
sis since it catalyzes the oxidation of lipoproteins (LDL,
HDL) to atherogenic forms.5 It is generally agreed that
a dual inhibitor of the COX/LOX enzymatic pathways
offers an alternative approach for the design of more effi-
cacious anti-inflammatory agents with an improved safe-
ty profile relative to ulcerogenic nonsteroidal anti-
inflammatory drugs (NSAIDs) and selective COX-2
inhibitors. Accordingly, compounds possessing a 3,5-di-
tert-butyl-4-hydroxyphenyl (DTBHP) moiety that can
act as a redox inhibitor, or antioxidant, to interfere with
the redox cycle of the 5-LOX isozyme have been investi-
gated extensively in the search for new dual COX/LOX
inhibitors.6–15 DTBHP derivatives have been identified
that exhibit oral anti-inflammatory activity and are de-
void of ulcerogenic potential. Some representative com-
pounds (1a–d), in which the 3,5-di-tert-butyl-4-
hydroxyphenyl moiety is linked through a vinyl bridge
to a heterocyclic ring, that exhibit a dual activity against
COX and 5-LOX8,13–15 are illustrated in Figure 1. S-2474
(1c) was selected as an antiarthritic drug candidate that is
now undergoing clinical trials.13
the unsubstituted-phenyl ring present in the acyclic tria-
ryl (Z)-ethene regioisomers 2 and 3, that is cis to the
para-methanesulfonylphenyl substituent (COX-2 phar-
macophore), is replaced by a DTBHP moiety (5-LOX
pharmacophore). Accordingly, we now describe the
synthesis and biological evaluation of a group of
(Z)-2-(3,5-di-tert-butyl-4-hydroxyphenyl)-1-(4-metha-
nesulfonylphenyl)-1-phenylalk-1-enes (6a–e), (Z)-1-(3,5-
di-tert-butyl-4-hydroxyphenyl)-2-(4-methanesulfonyl-
phenyl)-1-phenylalk-1-enes (9a–d), and some 4-acetoxy-
3,5-di-tert-butylphenyl derivatives (11a–b and 12a–b).
2. Chemistry
The (Z)-2-(4-hydroxy-3,5-di-tert-butylphenyl)-1-(4-metha-
nesulfonylphenyl)-1-phenylalk-1-enes (6a–e) (R1 = Me,
Et, n-butyl, n-heptyl, n-pentadecyl) were synthesized
using a McMurry olefination reaction by Zn–TiCl4
catalyzed reductive cross-coupling of 4-methanesulfo-
nylbenzophenone (4) and
a
1-(3,5-di-tert-butyl-4-
hydroxyphenyl)alkan-1-one (5a–e) as illustrated in
Scheme 1. This key synthetic step afforded a (Z):(E) ole-
finic mixture with a predominant selectivity for the (Z)-
olefin stereoisomer. Subsequent consecutive recrystalli-
zations provided the respective target (Z)-olefins 6a–e
in 30–37% isolated yield. The (Z)-1-(4-hydroxy-3,5-di-
tert-butylphenyl)-2-(4-methanesulfonylphenyl)-1-phen-
ylalk-1-ene regioisomers (9a–d) (R1 = Me, Et, n-butyl,
n-heptyl) were prepared in a similar way by reaction of
a 4-(methanesulfonyl)alkanophenone (7a–d) with 3,5-
di-tert-butyl-4-hydroxybenzophenone (8) in 22–41%
yield. Although the product 9a (R1 = Me) was obtained
as an inseparable (Z):(E) 73:27 olefinic mixture, the
related compounds 9b–d (R1 = Et, n-Bu, n-heptyl) were
obtained as a single (Z)-olefinic stereoisomer after sever-
al fractional recrystallizations. In contrast, the cross-
coupling reaction between the ketone 8 and the 4-(meth-
anesulfonyl)hexadecanophenone (7e) did not proceed
and the two starting materials were recovered. The
explanation for the (Z)-stereoselectivity observed in this
McMurry olefination reaction by coupling two aryl ke-
tones having respective methanesulfonyl and hydroxyl
substituents was described in a previous study where it
was shown that a sulfonyl moiety in one carbonyl com-
pound and a hydroxyl moiety in the other carbonyl
Recently, we reported that the triaryl (Z)-ethene regioi-
somers (216a and 316b, Fig. 1) having a COX-2 metha-
nesulfonyl (MeSO2) pharmacophore at the para
position of a phenyl ring in conjunction with a R1-alkyl
substituent of appropriate chain length exhibited
selective COX-2 inhibition. It was therefore of interest
to design a new class of hybrid compounds in which
O
O
N
t-Bu
t-Bu
N
S
S
HO
HO
NH2
t-Bu
Darbufelone (1a)
OH
O
t-Bu
CI-987 (1b)
O
O
N
t-Bu
HO
t-Bu
HO
S
O
t-Bu
t-Bu
compound are
stereoselectivity.17
a
requirement for high (Z)-
KME-4 (1d)
S-2474 (1c)
4-Methanesulfonylbenzophenone (4) and the 4-(metha-
nesulfonyl)alkanophenones (7a–e) were prepared by Fri-
edel–Crafts acylation of thioanisole followed by a
subsequent oxidation of the respective intermediate
methylthio compounds using Oxoneꢁ (potassium
peroxymonosulfate) in 50–60% overall yield. 3,5-Di-
tert-butyl-4-hydroxybenzophenone (8) and the 1-(3,5-
di-tert-butyl-4-hydroxyphenyl)alkan-1-ones (5a–e) were
also synthesized by Friedel–Crafts acylation of 2,6-di-
tert-butylphenol using benzoyl chloride, or an appropri-
ate alkanoyl chloride, in 45–64% yield.
MeO2S
MeO2S
R1
R1
3, R1 = n-alkyl
2, R1 = n-alkyl
Figure 1. Some representative examples of the 3,5-di-tert-butyl-4-
hydroxyphenyl (DTBHP) class that are dual inhibitors of cyclooxy-
genase and 5-lipoxygenase (1a–d), and acyclic triaryl (Z)-olefins that
exhibit selective cyclooxygenase-2 inhibitory activity (2–3).
The acetates 11a–b and 12a–b were synthesized in
45–57% yield via
a McMurry coupling reaction

5342
A. Moreau et al. / Bioorg. Med. Chem. 14 (2006) 5340–5350
t-Bu OH
MeO2S
t-Bu
OH
MeO2S
MeO2S
t-Bu
OAc
t-Bu
t-Bu
t-Bu
+
+
+
O
O
O
O
O
O
R1
R1
R2
R1
7a, R1 = Me
7b, R1 = Et
5a, R1 = Me
5b, R1 = Et
4, R2 = Ph
7c, R2 = n-Butyl
7d, R2 = n-Heptyl
10a, R1 = n-Butyl
10b, R1 = n-Heptyl
10c, R1 = Ph
8
4
7c, R1 = n-Butyl
7d, R1 = n-Heptyl
5c, R1 = n-Butyl
5d, R1 = n-Heptyl
7e, R1 = n-Pentadecyl
5e, R1 = n-Pentadecyl
MeO2S
t-Bu
OH
MeO2S
t-Bu
OH
MeO2S
t-Bu
OAc
t-Bu
t-Bu
t-Bu
R1
R1
R2
R1
6a, R1 = Me
6b, R1 = Et
11a, R1 = n-Butyl and R2 = Ph (Z/E: 94/6)
11b, R1 = n-Heptyl and R2 = Ph (Z/E; 83/17)
12a, R1 = Ph and R2 = n-Butyl (Z pure)
12b, R1 = Ph and R2 = n-Heptyl (Z/E: 78/22)
9a, R1 = Me (Z/E: 73/27)
9b, R1 = Et
9c, R1 = n-Butyl
9d, R1 = n-Heptyl
6c, R1 = n-Butyl
6d, R1 = n-Heptyl
6e, R1 = n-Pentadecyl
Scheme 1. Reagents and conditions: Zn, TiCl4, THF, reflux, 4.5 h.
(Scheme 1) between 4-methanesulfonylbenzophenone
(4) or a 4-(methanesulfonyl)alkanophenone (7c, d) and
the respective acetoxy compounds (10a, 10b or 10c) that
were previously obtained by acetylation of the respective
parent-phenol compounds 5c, 5d or 8. Initial attempts to
synthesize the acetates 11a–b and 12a–b by acetylation
of the respective (Z)-olefinic compounds 6c, 6d, 9c or
9d using a variety of acylation conditions such as (i)
TEA (1.5 equiv), AcCl (1.5 equiv) in diethyl ether, (ii)
TEA (3 equiv), AcCl (3 equiv) in freshly distillated
THF, or (iii) n-butyllithium (1.2 equiv), AcCl (1.5 equiv)
in freshly distillated THF, as well as varying the reaction
temperature and time, afforded only traces of the target
acetylated products. To circumvent this low reactivity, it
was decided to acetylate the phenolic substituent prior
to performing the McMurry olefination reaction.
Accordingly, the acetoxy compounds 10a–c were
prepared by reaction of the respective 1-(3,5-di-tert-bu-
tyl-4-hydroxyphenyl)alkan-1-one (5c or 5d), or 3,5-di-
tert-butyl-4-hydroxybenzophenone (8), with acetyl
chloride (3 equiv) in the presence of TEA (3 equiv) in
66–78% yield. However, the subsequent McMurry
olefination reaction using the acetoxy compounds
(10a–c) and a methanesulfonyl ketone (4, 7c or 7d)
was less (Z)-stereoselective. In this regard, the product
was isolated as a mixture of (Z)- and (E)-olefins for
11a (Z:E ratio = 94:6), 11b (Z:E ratio = 83:17), and
12b (Z:E ratio = 78:22; oil). On the other hand, 12a
was obtained as a single (Z)-stereoisomer after several
recrystallizations.
The structure of the methanesulfonyl compounds 7a–e,
the acetates 10a–c, and the target olefins 6a–e, 9a–d,
11a–b, and 12a–b was consistent with their spectral
and/or microanalytical data. The stereochemistry of
the target olefins possessing a phenolic moiety (6a–e,
9a–d) was not determined by 1H NMR difference nucle-
ar Overhauser effect (NOE) studies since these com-
pounds undergo slow isomerization in solution (CDCl3
or DMSO-d6) where new resonances appear that are
attributed to formation of the more stable (E)-stereoiso-
mer. This observation in conjunction with X-ray crystal
structure data previously used to establish the high
(Z)-stereoselectivity observed in the McMurry olefin-
ation reaction17 provides credence for the (Z)-stereo-
selectivity observed in this study.
3. Results and discussion
In vitro structure–activity relationships acquired for
these (Z)-2-(3,5-di-tert-butyl-4-hydroxyphenyl)-1-4-meth-
anesulfonylphenyl)-1-phenylalk-1-ene
(6a–e)
and
(Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4-metha-
nesulfonylphenyl)-1-phenylalk-1-ene (9b–d) regioisom-
ers showed that they exhibit a broad range (potent-
to-inactive) of COX/LOX inhibitory activities (COX-1
IC50 = 1 to >100 lM range; COX-2 IC50 = 0.1 to
>100 lM range; 5-LOX IC50 = 0.3 to >10 lM range;
15-LOX IC50 = 0.6 to >10 lM range; see data in
Table 1). Alk-1-enes 6a–d having a shorter R1 alkyl

A. Moreau et al. / Bioorg. Med. Chem. 14 (2006) 5340–5350
5343
Table 1. In vitro COX-1/COX-2 and 5-LOX/15-LOX enzyme inhibition assay data for (Z)-2-(3,5-di-tert-butyl-4-hydroxyphenyl)-1-(4-metha-
nesulfonylphenyl)-1-phenylalk-1-enes (6a–e), (Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4-methanesulfonylphenyl)-1-phenylalk-1-enes (9b–d), and
some 4-acetoxy-3,5-di-tert-butylphenyl derivatives (11a and 12a)
R1
R2
IC50 (lM)
Selectivity index (SI)b
IC50 (lM)
a
a
Compound
COX-1
COX-2
15-LOX
5-LOX
6a
6b
Me
Et
—
—
>100
1.0
3.6
32.0
3.3
>27.7
—
>10
0.6
0.5
>10
>10
>10
>10
>10
0.3
6c
6d
n-Butyl
n-Heptyl
n-Pentadecyl
Et
—
—
>100
4.1
>30.3
4.1
—
3.8
1.0
>10
>10
>10
0.8
6e
9b
—
—
>100
1.0
3.0
>100
1.77
0.36
0.1
—
9c
9d
n-Butyl
n-Heptyl
n-Butyl
—
—
—
8.3
310
—
31.0
>100
>100
>10
>10
>10
3.2
>10
>10
>10
—
11ac
—
n-Butyl
>100
>100
12ad
—
Luteolin
Caffeic acid
NDGA
Celecoxib
Rofecoxib
—
3.5
3.0
>10
—
33.1
>100
0.07
0.5
472
>200
—
—
—
a Values are means of two determinations acquired using an ovine COX-1/COX-2 and potato 5-LOX/soyabean 15-LOX assay kits (Catalog Nos.
560101, 60401, and 760700, Cayman Chemicals Inc., Ann Arbor, MI, USA) and the deviation from the mean is <10% of the mean value.
b In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
c Mixture of isomers, (Z):(E) ratio = 94:6.
d Pure (Z)-isomer.
substituent (Me, Et, n-butyl, n-heptyl) exhibited the
highest COX-2 inhibitory potency (IC50 = 1.0–32 lM
range) relative to the inactive R1 pentadecyl
[–(CH2)14CH3] analog 6e (IC50 > 100 lM). The require-
ment for a shorter R1 alkyl chain was more stringent for
inhibition of 5-LOX and 15-LOX since active inhibitors
(6a–c) possess a R1-alkyl chain length of 1–4 carbon
atoms. Among the group of compounds 6a–e, the
prop-1-ene (6a) having a R1 Me substituent exhibited
modest COX-2 inhibition (IC50 = 3.6 lM) and selectivi-
ty [COX-2 selectivity index (SI) > 27] in conjunction
with a potent inhibition of 5-LOX (IC50 = 0.5 lM).
By comparison, the related hex-1-ene (6c) having a R1
n-butyl substituent showed a similar COX-2 potency
(IC50 = 3.3 lM) and selectivity (SI > 30), but with a
IC50 = 0.36 lM, COX-2 SI = 8.3), in conjunction with
well-balanced inhibition of the 15-LOX
a
(IC50 = 0.8 lM) and 5-LOX (IC50 = 0.3 lM), isozymes.
A comparison of the relative inhibitory activities of
the two groups of regioisomers 6 and 9 shows that
regioisomers 9 in which the R1 alkyl substituent is at-
tached to the same olefinic carbon atom (C-2) as the
para-methanesulfonylphenyl moiety generally exhibit a
greater potency with respect to COX-2 inhibition
(R1 = Et, 9b > 6b; R1 = n-Bu, 9c > 6c; R1 = n-heptyl,
9d > 6d), 15-LOX inhibition (R1 = n-butyl, 9c > 6c but
R1 = Et is an exception where 6b ꢀ 9b), and 5-LOX
inhibition (R1 = n-butyl, 9c ꢀ 6c). The 4-hydroxy sub-
stituent in the 3,5-di-tert-butyl-4-hydroxyphenyl moiety
(such as in 6c and 9c) is essential for COX and LOX
inhibition since the 3,5-di-tert-butyl-4-acetoxyphenyl
derivatives 11a and 12a were inactive inhibitors.
simultaneous potent inhibition of 15-LOX (IC50
=
3.8 lM). These LOX inhibitory data for 6a–e show that
the small R1 Me substituent provides 5-LOX selectivity
whereas, compounds having a slightly longer R1 Et or n-
Bu substituent show 15-LOX selectivity. In contrast,
compound 6e having a long R1 pentadecyl chain is an
inactive inhibitor of both COX and LOX isozymes.
A molecular modeling study of the most potent and
selective COX-2 inhibitor, (Z)-1-(3,5-di-tert-butyl-4-
hydroxyphenyl)-2-(4-methanesulfonylphenyl)-1-phenyl-
non-1-ene (9d) (COX-2 IC50 = 0.10 lM; SI = 310)
docked in the COX-2 active site (Fig. 2), shows that it
binds in the primary binding-site such that the p-SO2Me
COX-2 pharmacophore on the C-2 phenyl ring inserts
Similar structure–activity in vitro enzyme inhibition
data for the alk-1-ene regioisomers (9b–d) showed that
COX-1 inhibition decreased, COX-2 inhibition
increased, and the COX-2 SI increased as the chain
length of the R1 alkyl substituent was increased
(Et ! n-butyl ! n-heptyl). In the group of compounds
9b–d, inhibition of both 5-LOX and 15-LOX was highly
dependent upon the length of the R1 substituent since 9c
(R1 = n-Bu) was a potent inhibitor of both 5-LOX
(IC50 = 0.3 lM) and 15-LOX (IC50 = 0.8 lM) relative
to the inactive (IC50 > 10 lM) R1 Et (9b) and n-heptyl
(9d) analogs. The hex-1-ene (9c, R1 = n-butyl) is of par-
ticular interest since it exhibits a dual inhibitory activity
against the COX (COX-1 IC50 = 3.0 lM, and COX-2
˚
into the 2ꢂ-pocket present in COX-2 (distance = 1.50 A)
where it undergoes interactions with Val523, Phe518,
Ile517, Thr94, and His90. One of the O-atoms of SO2Me
moiety forms a H-bond with the OH of Thr94 (dis-
˚
tance = 1.69 A), whereas the distance between O-atom
˚
and the NH2 (guanidine) of Arg513 was about 7.59 A.
The other O-atom (of SO2Me) forms a weak hydrogen
bond with the amide bond (NH) of Ile517 (dis-
˚
tance = 3.62 A). The Me of the SO2Me group is within
˚
van der Waals contact range of Phe518 (distance < 5 A),
and the C-2 phenyl ring (of the p-MeSO2-phenyl) is in-
volved in a p–p stacking interaction with the phenyl ring

5344
A. Moreau et al. / Bioorg. Med. Chem. 14 (2006) 5340–5350
Figure 2. Docking (Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4-methanesulfonylphenyl)-1-phenylnon-1-ene (9d) (ball and stick) in the active site of
murine COX-2. Hydrogen atoms of the amino acid residues have been removed to improve clarity.
of Phe518. The C-1 di-tert-butylphenol moiety is
oriented toward a hydrophobic pocket at the apex of
the COX-2 binding site where it is surrounded by
Leu525, Met522, Trp387, Leu384, and Phe381. The
OH of the di-tert-butylphenol group is positioned in
the vicinity of the backbone C@O of Glu524‘ (dis-
A molecular modeling (docking) simulation was
performed to investigate the binding interaction of the
most potent 15-LOX inhibitor, (Z)-2-(3,5-di-tert-butyl-
4-hydroxyphenyl)-1-(4-methanesulfonylphenyl)-1-phe-
nylbut-1-ene (6b), (15-LOX IC50 = 0.6 lM) within the
15-LOX binding site (Fig. 3, some amino acid residues
such as His356, Gln548, and Phe415 listed in the text be-
low are not shown in Fig. 3 to improve clarity). The C-1
p-MeSO2-phenyl COX-2 pharmacophore is oriented to-
ward the catalytic center of 15-LOX where it is able to
interact with amino acid residues such as Phe552,
His545, Ile544, Leu362, His361, Glu357, and His356.
The Me of the SO2Me group is located within van der
Waals contact range of Phe552, Gln548, and Glu357
˚
tance = 5.20 A), whereas the C-1 unsubstituted phenyl
ring is oriented toward a region comprised of Leu534,
Leu531, Ser530, Tyr385, Tyr348, and Val344. The dis-
tance between the center of the C-1 phenyl ring and
˚
the OH of Ser530 is about 4.97 A. The C-2 n-heptyl
substituent is oriented closer to the mouth of the
COX-2 binding site which allows it to interact with
amino acid residues such as Leu359, Tyr355, Val349,
Ser121, and Val116. The terminal Me of the n-heptyl
substituent is within van der Waals contact range of
˚
(distance < 5 A). The phenyl ring (of p-MeSO2-phenyl)
is involved in a p–p stacking interaction with the imidaz-
ole ring of His361. It is interesting to note that the OH
of the C-2 di-tert-butylphenol moiety participates in a
hydrogen bonding interaction with the OH of Glu357
˚
the Me side chain of Leu359 (distance < 5 A). This
computational study shows that the stereochemical dis-
position of alkyl, p-MeSO2-phenyl, or di-tert-butylphe-
nol substituents about the C@C bond controls the
optimal protein–ligand binding interactions in the ac-
tive site of COX-2.
˚
(distance = 1.74 A). The di-tert-butylphenol moiety is
oriented toward the base of the 15-LOX binding site
where it is positioned in a hydrophobic pocket

A. Moreau et al. / Bioorg. Med. Chem. 14 (2006) 5340–5350
5345
Figure 3. Docking of (Z)-2-(3,5-di-tert-butyl-4-hydroxyphenyl)-1-(4-methanesulfonylphenyl)-1-phenylbut-1-ene (6b) (ball and stick) in the active site
of soyabean 15-LOX. Hydrogen atoms of the amino acid residues have been removed to improve clarity.
comprised of Leu597, Val594, Ile593, Phe552, Met419,
Ile418, Phe415, Leu358, and Phe353. The C-1 unsubsti-
tuted phenyl ring is oriented in the vicinity of amino acid
residues such as His545, Gly598, Ile400, His366, and
His361, and it undergoes a p–p stacking interaction with
the imidazole ring of His366. The C-2 ethyl substituent
that is cis to the unsubstituted phenyl ring is oriented to-
ward a hydrophobic pocket closer to the entrance of the
15-LOX binding site (Arg403) where it is within van der
Waals contact range of Leu597, Leu408, and Ala394.
compound (12a, 8.1% inhibition) with that of the parent
3,5-di-tert-butyl-4-hydroxyphenyl compound (9c, 17.9%
inhibition) indicates that in vivo cleavage of the acetoxy
substituent to a hydroxyl group following oral adminis-
tration must not be rapid or complete. Further studies
showed that the most active compounds 6b
(ED50 = 131.9 mg/kg) and 9d (ED50 = 70.2 mg/kg) were
more potent anti-inflammatory agents than the 15-
LOX inhibitor NDGA (ED50 = 205 mg/kg), but less po-
tent than the selective COX-2 inhibitor celecoxib
(ED50 = 10.8 mg/kg).
The (Z)-olefins 6a, 6b, 9c, 9d and the acetate 12a (acet-
oxy derivative of 9c), based on in vitro enzyme inhibi-
tion data, were selected for further in vivo
pharmacological evaluation to determine their anti-in-
flammatory (AI) and analgesic activities (see data in
Table 2). In a carrageenan-induced rat paw edema assay
model, qualitative assays using a 30 mg/kg oral dose
showed that compounds 6a, 6b, 9c, and 9d inhibited
inflammation at 3 h post-drug administration in the
13–33% range relative to the 5-LOX inhibitor caffeic
acid (8.2% inhibition) and the 15-LOX inhibitor
nordihydroguaiaretic acid (NDGA, 15.1% inhibition)
reference drugs. A comparison of the anti-inflammatory
In a rat 4% NaCl-induced abdominal constriction (anal-
gesic) assay, a 30 mg oral dose of the (Z)-olefins 6a, 6b,
9c, and 9d exhibited good analgesic activities (40–70%
range) compared to the reference drugs caffeic acid,
NDGA, and celecoxib at 30 and 60 min post-drug
administration (see data in Table 2).
4. Conclusions
A new class of acyclic triaryl (Z)-olefin regioisomers were
designed that possess para-methanesulfonylphenyl
(COX-2) and 3,5-di-tert-butyl-4-hydroxyphenyl (5-
activity
of
the
3,5-di-tert-butyl-4-acetoxyphenyl

5346
A. Moreau et al. / Bioorg. Med. Chem. 14 (2006) 5340–5350
Table 2. In vivo anti-inflammatory and analgesic activities for (Z)-2-(3,5-di-tert-butyl-4-hydroxyphenyl)-1-(4-methanesulfonylphenyl)-1-phenylalk-
1-enes (6a–b), (Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4-methanesulfonylphenyl)-1-phenylalk-1-enes (9c–d), and the 4-acetoxy-3,5-di-tert-
butylphenyl derivative (12a)
Compound
R1
R2
AI activitya
Analgesic activityb
% inhibition (30 mg/kg)
ED50 (mg/kg)
% inhibition (30 min)
% inhibition (60 min)
6a
6b
Me
Et
—
—
13.0 4.6
33.3 4.0
17.9 1.4
25.0 3.0
8.1 3.1
–
40.0 18.9
70.7 6.5
64.7 3.6
41.3 8.3
27.3 4.8
47.2 10.6
45.8 9.2
69.3 12.1c
43.3 14.6
68.3 12.2
61.9 6.8
63.3 1.6
39.4 13.8
58.3 12.8
62.5 4.8
79.5 2.0c
131.9
—
9c
9d
n-Butyl
n-Heptyl
—
—
—
70.2
—
12a
n-Butyl
Caffeic acid
NDGA
Celecoxib
8.2 2.5
15.1 1.7
79.9 1.9c
—
205.0
10.8
—
—
a Inhibitory activity in a carrageenan-induced rat paw edema assay. The results are expressed as means SEM (n = 4) at 3 h following a 30 mg/kg
oral dose of the test compound or as the ED50 value when it was determined.
b Inhibitory activity in the rat 4% NaCl-induced abdominal constriction assay. The results are expressed as means SEM (n = 3–4) following a
30 mg/kg oral dose of the test compound.
c 5 mg/kg oral dose.
LOX) pharmacophores for evaluation as dual acting
COX/LOX inhibitors. Structure–activity data acquired
identified (Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-
(4-methanesulfonylphenyl)-1-phenylnon-1-ene (9d) as a
highly potent (IC50 = 0.1 lM) and a particularly selective
(SI = 310) COX-2 inhibitor that showed good anti-in-
flammatory activity (ED50 = 70.2 mg/kg) even though it
did not inhibit either 5-LOX or 15-LOX. In contrast,
(Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4-metha-
nesulfonylphenyl)-1-phenylhex-1-ene (9c) was found to
be a potent COX-2 inhibitor (IC50 = 0.36 lM) with a
modest COX-2 selectivity (SI = 8.3) that also effectively
inhibited both 15-LOX (IC50 = 0.8 lM) and 5-LOX
(IC50 = 0.3 lM). The structure–activity data acquired in
this investigation show that optimum dual COX/LOX
inhibitory activity is observed when the acyclic triaryl
(Z)-olefin possesses a hex-1-ene (R1 n-butyl substituent)
moiety in combination with a para-methanesulfonylphe-
nyl COX-2 pharmacophore and a 3,5-di-tert-butyl-4-
hydroxyphenyl LOX pharmacophoric group.
purchased from Animal Health Services at the University
of Alberta, and experiments were carried out using proto-
cols approved by the Animal Welfare Committee, Univer-
sity of Alberta.
5.1. General procedure for the synthesis of 4-(metha-
nesulfonyl)benzophenone (4) and 4-(methanesulfo-
nyl)alkanophenones (7a–e)
A solution of Oxoneꢁ (potassium peroxymonosulfate)
(4.06 g, 6.6 mmol) in water (20 mL) was added dropwise
at 0 ꢂC to a solution of 4-(methylthio)benzophenone
or the corresponding 4-(methylthio)alkanophenone
(3.3 mmol) in THF/MeOH (1:1, v/v, 10 mL). The
reaction was allowed to proceed for 15 h at 25 ꢂC with
stirring and the solvent was removed in vacuo. Water
(20 mL) was added to the residue, this mixture was
extracted with EtOAc (3 · 30 mL), the combined organic
extracts were washed with water, and the organic layer
was dried (Na2SO4). Removal of the solvent in vacuo
afforded a solid residue which was recrystallized from
petroleum ether/dichloromethane. Some physical and
spectral data for compounds 4 and 7a–e are listed below.
5. Experimental section
Melting points were determined using a Thomas–
Hoover capillary apparatus and are uncorrected. Infrared
(IR) spectra were recorded as films on NaCl plates using a
5.2. 4-(Methanesulfonyl)benzophenone (4)
Yield, 82%; white crystals; mp 140–141 ꢂC (lit.18
141 ꢂC); IR (film) 1661 (C@O), 1599 (C@C), 1319
1
Nicolet 550 Series II Magna FT-IR spectrometer. H
NMR spectra were measured on a Bruker AM-300 spec-
trometer in CDCl3 with TMS as the internal standard,
where J (coupling constant) values are estimated in Hz.
Spin multiples are given as s (singlet), d (double), t (trip-
let), q (quartet), m (multiplet), and br (broad). Microanal-
yses were performed for C, H (Microanalytical Service
Laboratory, Department of Chemistry, University of
Alberta) and were within 0.4% of theoretical values. Sil-
ica gel column chromatography was performed using
Merck silica gel 60 ASTM (70–230 mesh). Luteolin, caf-
feic acid, and nordihydroguaiaretic acid (NDGA) were
purchased from Cayman Chemicals Inc., Ann Arbor,
MI, USA. All other reagents were purchased from the Al-
drich Chemical Company (Milwaukee, WI) and used
without further purification. Male Sprague–Dawley rats,
used in the anti-inflammatory–analgesic screens, were
(SO2), 1156 cmꢁ1 1H NMR (CDCl3) d 3.13 (s, 3H,
,
SO2CH3), 7.53 (t, J = 7.6 Hz, 1H, phenyl H-4), 7.66 (t,
J = 7.6 Hz, 2H, phenyl H-3, H-5), 7.81 (d, J = 7.6 Hz,
2H, phenyl H-2, H-6), 7.96 (d, J = 8.2 Hz, 2H, metha-
nesulfonylphenyl H-3, H-5), 8.09 (d, J = 8.2 Hz, 2H,
methanesulfonylphenyl H-2, H-6).
5.3. 1-(4-Methanesulfonylphenyl)ethan-1-one (7a)
Yield, 79%; white crystals; the spectral data were identi-
cal to those previously reported.19
5.4. 1-(4-Methanesulfonylphenyl)propan-1-one (7b)
Yield, 84%; white crystals; mp 109–110 ꢂC (lit.20
108 ꢂC); IR (film) 1686 (C@O), 1317 (SO2),

A. Moreau et al. / Bioorg. Med. Chem. 14 (2006) 5340–5350
5347
1151 cmꢁ1
;
1H NMR (CDCl3) d 1.25 (t, J = 7.3 Hz,
5.10. 1-(3,5-Di-tert-butyl-4-acetoxyphenyl)octan-1-one
(10b)
3H, CH2CH3), 3.05 (t, J = 7.3 Hz, 2H, COCH2),
3.08 (s, 3H, SO2CH3), 8.04 (d, J = 8.5 Hz, 2H, phen-
yl H-3, H-5), 8.13 (d, J = 8.5 Hz, 2H, phenyl H-2,
H-6).
Yield, 78%; viscous pale yellow oil (lit.21 bp 234–236 ꢂC/
10 Torr); IR (film) 1762 (C@O of OAc), 1686 (C@O),
1367, 1200, 1110 cmꢁ1 1H NMR (CDCl3) d 0.90 (t,
;
5.5. 1-(4-Methanesulfonylphenyl)pentan-1-one (7c)
J = 7.3 Hz, 3H, CH2CH3), 1.30–1.82 [m, 28H,
(CH2)5CH3 + two (CH3)3C], 2.38 (s, 3H, OCOCH3),
2.94 (t, J = 7.3 Hz, 2H, COCH2), 7.96 (s, 2H, phenyl
H-2, H-6).
Yield, 82%; white foam; mp 84–85 ꢂC; IR (film) 1686
(C@O), 1315 (SO2), 1156 cmꢁ1 1H NMR (CDCl3) d
;
0.97 (t, J = 7.3 Hz, 3H, CH2CH3), 1.43 (sextet,
J = 7.3 Hz, 2H, CH2CH2CH3), 1.74 (quintet,
J = 7.3 Hz, 2H, CH2CH2CH2), 3.02 (t, J = 7.3 Hz, 2H,
COCH2), 3.09 (s, 3H, SO2CH3), 8.05 (d, J = 8.2 Hz,
2H, phenyl H-3, H-5), 8.14 (d, J = 8.2 Hz, 2H, phenyl
H-2, H-6).
5.11. 3,5-Di-tert-butyl-4-acetoxybenzophenone (10c)
Yield, 78%; viscous pale yellow oil; IR (film) 1765 (C@O
of OAc), 1659 (C@O), 1367, 1186, 1110 cmꢁ1; 1H NMR
(CDCl3) d 1.37 [s, 18H, two (CH3)3C], 2.40 (s, 3H,
OCOCH3), 2.97 (s, 3H, OCOCH3), 7.47–7.52 (m, 2H,
phenyl H-3, H-5), 7.57–7.60 (m, 1H, phenyl H-4),
7.81–7.84 (m, 4H, acetoxyphenyl H-2, H-6, phenyl H-
2, H-6).
5.6. 1-(4-Methanesulfonylphenyl)octan-1-one (7d)
Yield, 81%; white crystals; mp 104–106 ꢂC; IR (film)
1689 (C@O), 1293 (SO2), 1149 cmꢁ1
;
1H NMR
(CDCl3) d 0.88 (t, J = 7.3 Hz, 3H, CH2CH3), 1.29–
1.38 [m, 8H, (CH2)4CH3], 3.00 (t, J = 7.3 Hz, 2H,
COCH2), 3.09 (s, 3H, SO2CH3), 8.05 (d, J = 8.5 Hz,
2H, phenyl H-3, H-5), 8.13 (d, J = 8.5 Hz, 2H, phenyl
H-2, H-6).
5.12. General procedure for the synthesis of (Z)-2-(3,5-di-
tert-butyl-4-hydroxyphenyl)-1-(4-methanesulfonylphe-
nyl)-1-phenylalk-1-enes (6a–e), (Z)-1-(3,5-di-tert-butyl-4-
hydroxyphenyl)-2-(4-methanesulfonylphenyl)-1-phen-
ylalk-1-enes (9a–d), and the 4-acetoxy-3,5-di-tert-butyl-
phenyl derivatives (11a–b, 12a–b)
5.7. 1-(4-Methanesulfonylphenyl)hexadecane-1-one (7e)
Titanium tetrachloride (1.30 mL, 11.7 mmol) was add-
ed dropwise to a stirred suspension of Zn powder
(1.57 g, 24.0 mmol) in dry THF (30 mL), under Ar,
at ꢁ10 ꢂC, and the reaction was allowed to proceed
at reflux for 2 h. A solution of the first ketone 4,
7a–d, 4 or 7c–d (3.0 mmol), respectively, and the sec-
ond ketone 5a–e, 8, 10a–b or 10c (3.0 mmol), respec-
tively, in THF (65 mL) was added dropwise to the
cooled suspension of the titanium reagent at 0 ꢂC,
and the reaction was allowed to proceed at reflux
for 2.5 h. The reaction mixture was cooled to 25 ꢂC,
poured into 10% aqueous K2CO3 solution (100 mL),
and after vigorous stirring for 5 min, the dispersed
insoluble material was removed by vacuum filtration
using a Celite 545ꢁ pad. The organic layer was sepa-
rated, the aqueous layer was extracted with EtOAc
(3 · 50 mL), the combined organic fractions were
washed with water (100 mL), and the organic fraction
was dried (Na2SO4). Evaporation of solvent in vacuo
left a residue which was purified by silica gel column
chromatography using petroleum ether/ethyl acetate
(3:1, v/v) as eluant to afford a white solid which was
recrystallized several times from 95% EtOH.
Yield, 76%; bright white crystals; mp 85–87 ꢂC; IR
(film) 1681 (C@O), 1473, 1397 cmꢁ1
;
1H NMR
(CDCl3) d 0.89 (t, J = 6.6 Hz, 3H, CH2CH3), 1.29-
1.38 [m, 26H, (CH2)13CH3], 2.53 (s, 3H, SO2CH3),
2.92 (t, J = 7.5 Hz, 2H, COCH2), 7.27 (d, J = 8.5 Hz,
2H, phenyl H-3, H-5), 7.88 (d, J = 8.5 Hz, 2H, phenyl
H-2, H-6).
5.8. General procedure for the synthesis of acetates (10a–
c)
Acetyl chloride (1.47 mL, 20.7 mmol) was added drop-
wise at 0 ꢂC with stirring to a solution of the appro-
priate phenol 5c, 5d or 8 (6.9 mmol) and TEA
(2.89 mL, 20.7 mmol) in freshly distilled THF
(40 mL). The reaction was allowed to proceed for
24 h at 25 ꢂC with stirring, the mixture was diluted
with water (30 mL), extracted with EtOAc (3 ·
30 mL), and the combined organic layers were dried
(Na2SO4). Removal of the solvent in vacuo furnished
a residue which was purified by silica gel column chro-
matography using petroleum ether/ethyl acetate (9:1,
v/v) as eluant.
5.13. (Z)-2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-1-(4-
methanesulfonylphenyl)-1-phenylprop-1-ene (6a)
5.9. 1-(3,5-Di-tert-butyl-4-acetoxyphenyl)pentan-1-one
(10a)
Yield, 30%; white solid; mp 185–186 ꢂC; IR (film) 3628
(OH), 1590 (C@C), 1436, 1315 (SO2), 1231,
Yield, 69%; viscous yellow oil; IR (film) 1766 (C@O of
1
OAc), 1684 (C@O), 1367, 1186, 1109 cmꢁ1; H NMR
1150 cmꢁ1 1H NMR (CDCl3) d 1.28 [s, 18H, two
;
(CDCl3) d 0.97 (t, J = 7.3 Hz, 3H, CH2CH3), 1.33–1.47
[m, 20H, CH2CH3 + two (CH3)3C], 1.73 (quintet,
J = 7.3 Hz, 2H, CH2CH2CH2), 2.38 (s, 3H, OCOCH3),
2.95 (t, J = 7.3 Hz, 2H, COCH2), 7.96 (s, 2H, phenyl
H-2, H-6).
(CH3)3C], 2.19 (s, 3H, CH3C@C), 2.97 (s, 3H, CH3SO2),
5.15 (s, 1H, OH), 6.90 (s, 2H, phenolic H-2, H-6), 7.05
(d, J = 8.5 Hz, 2H, phenyl H-2, H-6), 7.22–7.41 (m,
5H, phenyl H-3, H-4, H-5, 4-methanesulfonylphenyl
H-2, H-6), 7.61 (d, J = 8.5 Hz, 2H, 4-methanesulfonyl-

5348
A. Moreau et al. / Bioorg. Med. Chem. 14 (2006) 5340–5350
phenyl H-3, H-5). Anal. Calcd for C30H36O3S: C, 75.59;
H, 7.61. Found: C, 75.41; H, 8.00.
5.18. (Z, E)-1-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-(4-
methanesulfonylphenyl)-1-phenylprop-1-ene (9a)
5.14. (Z)-2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-1-(4-
methanesulfonylphenyl)-1-phenylbut-1-ene (6b)
Yield, 38%; (Z):(E) ratio = 73:27 (after several recrystal-
lizations); white solid; mp 113–115 ꢂC; IR (film) 3629
(OH), 1590 (C@C), 1436, 1314 (SO2), 1236,
1152 cmꢁ1; major (Z)-isomer; 1H NMR (CDCl3) d
1.17 [s, 18H, two (CH3)3C], 2.13 (s, 3H, CH3C@C),
3.01 (s, 3H, CH3SO2), 5.05 (s, 1H, OH), 6.60 (s, 2H, phe-
nolic H-2, H-6), 6.86-7.05 (m, 2H, phenyl H-2, H-6),
7.28–7.41 (m, 5H, phenyl H-3, H-4, H-5, 4-metha-
nesulfonylphenyl H-2, H-6), 7.67-7.76 (m, 2H, 4-metha-
nesulfonylphenyl H-3, H-5). Anal. Calcd for
C30H36O3S: C, 75.59; H, 7.61. Found: C, 75.24; H, 7.63.
Yield, 35%; white foam; mp 210–211 ꢂC; IR (film) 3629
(OH), 1590 (C@C), 1435, 1314 (SO2), 1228, 1150 cmꢁ1
;
1H NMR (CDCl3) d 1.02 (t, J = 7.3 Hz, 3H, CH3CH2),
1.28 [s, 18H, two (CH3)3C], 2.52 (q, J = 7.3 Hz, 2H,
CH3CH2), 2.95 (s, 3H, CH3SO2), 5.12 (s, 1H, OH),
6.84 (s, 2H, phenolic H-2, H-6), 6.95 (d, J = 8.5 Hz,
2H, phenyl H-2, H-6), 7.22–7.41 (m, 5H, phenyl H-3,
H-4, H-5, 4-methanesulfonylphenyl H-2, H-6), 7.58 (d,
J = 8.5 Hz, 2H, 4-methanesulfonylphenyl H-3, H-5).
Anal. Calcd for C31H38O3S: C, 75.88; H, 7.81. Found:
C, 75.49; H, 8.03.
5.19. (Z)-1-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-(4-
methanesulfonylphenyl)-1-phenylbut-1-ene (9b)
5.15. (Z)-2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-1-(4-
methanesulfonylphenyl)-1-phenylhex-1-ene (6c)
Yield, 22%; bright white crystals; mp 194–195 ꢂC; IR
(film) 3635 (OH), 1590 (C@C), 1436, 1315 (SO2), 1236,
1152 cmꢁ1 1H NMR (CDCl3) d 0.92 (t, J = 7.0 Hz,
;
Yield, 32%; white foam; mp 192–194 ꢂC; IR (film)
3629 (OH), 1591 (C@C), 1435, 1315 (SO2), 1234,
3H, CH3CH2), 1.17 [s, 18H, two (CH3)3C], 2.50 (q,
J = 7.0 Hz, 2H, CH3CH2), 3.03 (s, 3H, CH3SO2), 5.04
(s, 1H, OH), 6.59 (s, 2H, phenolic H-2, H-6), 7.26-7.41
(m, 7H, phenyl hydrogens, 4-methanesulfonylphenyl
H-2, H-6), 7.76 (d, J = 8.2 Hz, 2H, 4-methanesulfonyl-
phenyl H-3, H-5). Anal. Calcd for C31H38O3S: C,
75.88; H, 7.81. Found: C, 75.52; H, 8.11.
1151 cmꢁ1
3H,
;
1H NMR (CDCl3) d 0.83 (t, J = 7.0 Hz,
CH3CH2),
1.24–1.40
[m,
22H,
two
(CH3)3C + CH3(CH2)2], 2.47 (t, J = 7.0 Hz, 2H,
C@CCH2CH2), 2.95 (s, 3H, CH3SO2), 5.12 (s, 1H,
OH), 6.84 (s, 2H, phenolic H-2, H-6), 7.02 (d,
J = 8.2 Hz, 2H, phenyl H-2, H-6), 7.21–7.40 (m, 5H,
phenyl H-3, H-4, H-5, 4-methanesulfonylphenyl H-2,
H-6), 7.58 (d, J = 8.2 Hz, 2H, 4-methanesulfonylphe-
nyl H-3, H-5). Anal. Calcd for C33H42O3S: C, 76.41;
H, 8.16. Found: C, 76.26; H, 8.34.
5.20. (Z)-1-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-(4-
methanesulfonylphenyl)-1-phenylhex-1-ene (9c)
Yield, 25%; white foam; mp 195–196 ꢂC; IR (film) 3634
(OH), 1590 (C@C), 1436, 1315 (SO2), 1236, 1152 cmꢁ1
;
5.16. (Z)-2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-1-(4-
methanesulfonylphenyl)-1-phenylnon-1-ene (6d)
1H NMR (CDCl3) d 0.78 (t, J = 7.0 Hz, 3H, CH3CH2),
1.16–1.26 [m, 22H, two (CH3)3C + CH3(CH2)2], 2.45 (t,
J = 7.0 Hz, 2H, C@CCH2CH2), 3.03 (s, 3H, CH3SO2),
5.03 (s, 1H, OH), 6.55 (s, 2H, phenolic H-2, H-6),
7.26-7.41 (m, 7H, phenyl hydrogens, 4-methanesulfonyl-
phenyl H-2, H-6), 7.76 (d, J = 8.2 Hz, 2H, 4-metha-
nesulfonylphenyl H-3, H-5). Anal. Calcd for
C33H42O3S: C, 76.41; H, 8.16. Found: C, 76.09; H, 8.16.
Yield, 34%; white foam; mp 163–165 ꢂC; IR (film) 3635
(OH), 1590 (C@C), 1434, 1315 (SO2), 1231, 1150 cmꢁ1
;
1H NMR (CDCl3) d 0.85 (t, J = 7.3 Hz, 3H, CH3CH2),
1.19–1.39 [m, 28H, two (CH3)3C + CH3(CH2)5], 2.46 (t,
J = 7.3 Hz, 2H, C@CCH2CH2), 2.95 (s, 3H, CH3SO2),
5.11 (s, 1H, OH), 6.83 (s, 2H, phenolic H-2, H-6), 7.02
(d, J = 8.2 Hz, 2H, phenyl H-2, H-6), 7.21–7.40 (m,
5H, phenyl H-3, H-4, H-5, 4-methanesulfonylphenyl
H-2, H-6), 7.58 (d, J = 8.2 Hz, 2H, 4-methanesulfonyl-
phenyl H-3, H-5). Anal. Calcd for C36H48O3S: C,
77.10; H, 8.63. Found: C, 76.78; H, 9.01.
5.21. (Z)-1-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-(4-
methanesulfonylphenyl)-1-phenylnon-1-ene (9d)
Yield, 27%; white foam; mp 183–184 ꢂC; IR (film) 3635
(OH), 1590 (C@C), 1436, 1313 (SO2), 1236, 1152 cmꢁ1
;
1H NMR (CDCl3) d 0.85 (t, J = 7.0 Hz, 3H, CH3CH2),
1.16–1.34 [m, 28H, two (CH3)3C + CH3(CH2)5], 2.44 (t,
J = 7.0 Hz, 2H, C@CCH2CH2), 3.03 (s, 3H, CH3SO2),
5.03 (s, 1H, OH), 6.58 (s, 2H, phenolic H-2, H-6),
7.25–7.40 (m, 7H, phenyl hydrogens, 4-methanesulfo-
nylphenyl H-2, H-6), 7.75 (d, J = 8.2 Hz, 2H, 4-metha-
nesulfonylphenyl H-3, H-5). Anal. Calcd for
C36H48O3S: C, 77.10; H, 8.63. Found: C, 76.80; H, 8.61.
5.17. (Z)-2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-1-(4-
methanesulfonylphenyl)-1-phenylheptadec-1-ene (6e)
Yield, 37%; white foam; mp 149–150 ꢂC; IR (film) 3629
(OH), 1591 (C@C), 1434, 1312 (SO2), 1233, 1151 cmꢁ1
;
1H NMR (CDCl3) d 0.89 (t, J = 7.0 Hz, 3H, CH3CH2),
1.16–1.43 [m, 44H, two (CH3)3C + CH3(CH2)13], 2.46 (t,
J = 7.0 Hz, 2H, C@CCH2CH2), 2.95 (s, 3H, CH3SO2),
5.10 (s, 1H, OH), 6.83 (s, 2H, phenolic H-2, H-6), 7.02
(d, J = 8.2 Hz, 2H, phenyl H-2, H-6), 7.21–7.40 (m,
5H, phenyl H-3, H-4, H-5, 4-methanesulfonylphenyl
H-2, H-6), 7.57 (d, J = 8.2 Hz, 2H, 4-methanesulfonyl-
phenyl H-3, H-5). Anal. Calcd for C44H64O3S: C,
78.52; H, 9.58. Found: C, 78.31; H, 9.92.
5.22. (Z, E)-2-(3,5-Di-tert-butyl-4-acetoxyphenyl)-1-(4-
methanesulfonylphenyl)-1-phenylhex-1-ene (11a)
Yield, 57%; ratio (Z):(E) = 94:6 (after several recrystalli-
zations); bright white crystals; mp 157–158 ꢂC; IR (film)
1759 (C@O), 1591 (C@C), 1428, 1367, 1316 (SO2), 1152,

A. Moreau et al. / Bioorg. Med. Chem. 14 (2006) 5340–5350
5349
1
1109 cmꢁ1; major (Z)-isomer; H NMR (CDCl3) d 0.83
coordinates for the X-ray crystal structure of the en-
zymes COX-2 and 15-LOX were obtained from the
RCSB Protein Data Bank and hydrogens were added.
The ligand molecules were constructed using the Builder
module and energy minimized for 1000 iterations reach-
(t, J = 7.3 Hz, 3H, CH3CH2), 1.17–1.47 [m, 22H, two
(CH3)3C + CH3(CH2)2], 2.30 (s, 3H, CH3CO), 2.48 (t,
J = 7.3 Hz, 2H, C@CCH2CH2), 2.93 (s, 3H, CH3SO2),
6.97-7.02 (m, 4H, acetoxyphenyl H-2, H-6, phenyl H-
2, H-6), 7.22–7.41 (m, 5H, phenyl H-3, H-4, H-5, 4-
methanesulfonylphenyl H-2, H-6), 7.59 (d, J = 8.5 Hz,
2H, 4-methanesulfonylphenyl H-3, H-5). Anal. Calcd
for C35H44O4S: C, 74.96; H, 7.91. Found: C, 74.58; H,
8.24.
˚
ing a convergence of 0.01 kcal/mol A. The docking
experiment on COX-2 was carried out by superimposing
the energy minimized ligand on SC-558 in the PDB file
1cx2 after which SC-558 was deleted. The coordinates
for 15-LOX were obtained from PDB file1lox and the
energy minimized ligand was superimposed on the
inhibitor RS75091 after which RS75091 was deleted.
In all these experiments the resulting ligand–enzyme
complex was subjected to docking using the Affinity
command in the Docking module of Insight II after
defining subsets of the enzyme such that residues within
5.23. (Z, E)-2-(3,5-Di-tert-butyl-4-acetoxyphenyl)-1-(4-
methanesulfonylphenyl)-1-phenylnon-1-ene (11b)
Yield, 57%; ratio (Z):(E) = 83:17 (after several recrystal-
lizations); white solid; mp 114–115 ꢂC; IR (film) 1761
(C@O), 1592 (C@C), 1427, 1367, 1316 (SO2), 1151,
1109 cmꢁ1; major (Z)-isomer; 1H NMR (CDCl3) d
0.86 (t, J = 7.0 Hz, 3H, CH3CH2), 1.12–1.52 [m, 28H,
two (CH3)3C + CH3(CH2)5], 2.30 (s, 3H, CH3CO),
2.48 (t, J = 7.0 Hz, 2H, C@CCH2CH2), 2.93 (s, 3H,
CH3SO2), 6.97-7.02 (m, 4H, acetoxyphenyl H-2, H-6,
phenyl H-2, H-6), 7.22–7.41 (m, 5H, phenyl H-3, H-4,
H-5, 4-methanesulfonylphenyl H-2, H-6), 7.59 (d,
J = 8.5 Hz, 2H, 4-methanesulfonylphenyl H-3, H-5).
Anal. Calcd for C38H50O4S: C, 75.71; H, 8.36. Found:
C, 75.47; H, 8.61.
˚
10 A of the ligand were allowed to relax, while the
remainder of the enzyme residues were fixed. The consis-
tent valence force field (CVFF) was employed for all
docking purposes. The ligand–enzyme assembly was
then subjected to a molecular dynamics (MD) simula-
tion using the Discover module Version 2.98 at a con-
stant temperature of 300 K with
a
100-step
equilibration for over 1000 iterations and a time step
of 1 fs using a distance-dependent dielectric constant
4r. The optimal binding orientation of the ligand–en-
zyme assembly obtained after docking was further min-
imized for 1000 iterations using the conjugate gradient
˚
5.24. (Z)-1-(3,5-Di-tert-butyl-4-acetoxyphenyl)-2-(4-
methanesulfonylphenyl)-1-phenylhex-1-ene (12a)
method until a convergence of 0.001 kcal/mol A was
reached after which Eintermolecular (kcal/mol) of the li-
gand–enzyme assembly was evaluated.
Yield, 55%; white solid; mp 116–117 ꢂC; IR (film) 1760
(C@O), 1592 (C@C), 1427, 1367, 1315 (SO2), 1151,
5.27. In vitro cyclooxygenase (COX) inhibition assays
1109 cmꢁ1
3H,
;
1H NMR (CDCl3) d 0.78 (t, J = 7.0 Hz,
CH3CH2),
1.07–1.32
[m,
22H,
two
The ability of the test compounds listed in Table 1 to
inhibit ovine COX-1 and COX-2 (IC50 value, lM) was
determined using an enzyme immuno assay (EIA) kit
(catalog number 560101, Cayman Chemical, Ann Ar-
bor, MI, USA) according to our previously reported
method.16a The solution of the test compound was pre-
pared immediately before starting the assay to minimize
the potential isomerization reaction.
(CH3)3C + CH3(CH2)2], 2.25 (s, 3H, CH3CO), 2.45 (t,
J = 7.0 Hz, 2H, C@CCH2CH2), 3.00 (s, 3H, CH3SO2),
6.76 (s, 2H, acetoxyphenyl H-2, H-6), 7.28–7.41 (m,
7H, phenyl hydrogens, 4-methanesulfonylphenyl H-2,
H-6), 7.75 (d, J = 8.5 Hz, 2H, 4-methanesulfonylphenyl
H-3, H-5). Anal. Calcd for C35H44O4S: C, 74.96; H,
7.91. Found: C, 74.69; H, 8.10.
5.25. (Z, E)-1-(3,5-Di-tert-butyl-4-acetoxyphenyl)-2-(4-
methanesulfonylphenyl)-1-phenylnon-1-ene (12b)
5.28. In vitro lipoxygenase (LOX) inhibition assays
The ability of the test compounds to inhibit potato 5-
LOX (catalog number 60401, Cayman Chemical, Ann
Arbor, MI, USA) and soybean 15-LOX (catalog num-
ber 760700, Cayman Chemical, Ann Arbor, MI, USA)
(IC50 values, lM) was determined using an enzyme
immuno assay (EIA) kit according to the manufactur-
er’s instructions. The Cayman Chemical lipoxygenase
inhibitor screening assay detects and measures the
hydroperoxides produced in the lipoxygenation reaction
using a purified lipoxygenase. Stock solutions of test
compounds, prepared immediately before use, were dis-
solved in a minimum volume of DMSO and were diluted
using the supplied buffer solution (0.1 M, Tris–HCl, pH
7.4). To a 90 ll solution of 5- or 15-LOX enzyme in
0.1 M, Tris–HCl, pH 7.4, buffer, 10 ll of various con-
centrations of test drug solutions (0.001, 0.01, 0.1, 1,
and 10 lM in a final volume of 210 ll) was added and
the lipoxygenase reaction was initiated by the addition
Yield, 45%; ratio (Z):(E) = 78:22; viscous pale yellow
oil; IR (film) 1759 (C@O), 1592 (C@C), 1427, 1367,
1316 (SO2), 1152, 1110 cmꢁ1; major (Z)-isomer; 1H
NMR (CDCl3) d 0.83 (t, J = 7.3 Hz, 3H, CH3CH2),
1.08–1.33 [m, 28H, two (CH3)3C + CH3(CH2)5], 2.25
(s, 3H, CH3CO), 2.46 (t, J = 7.3 Hz, 2H, C@CCH2CH2),
2.99 (s, 3H, CH3SO2), 6.77 (s, 2H, acetoxyphenyl H-2,
H-6), 7.28–7.39 (m, 7H, phenyl hydrogens, 4-metha-
nesulfonylphenyl H-2, H-6), 7.76 (d, J = 8.2 Hz, 2H, 4-
methanesulfonylphenyl H-3, H-5). Anal. Calcd for
C38H50O4S: C, 75.71; H, 8.36. Found: C, 75.34; H, 8.42.
5.26. Molecular modeling (docking) studies
Docking experiments were performed using Insight II
software Version 2000.1 (Accelrys Inc.) running on a Sil-
icon Graphics Octane 2 R14000A workstation. The

5350
A. Moreau et al. / Bioorg. Med. Chem. 14 (2006) 5340–5350
R. B.; Chan, K.; Schrier, D. J.; Guglietta, A.; Bornemeier,
D. A.; Dyer, R. B. J. Med. Chem. 1999, 42, 1161; (g) Song,
Y.; Connor, D. T.; Doubleday, R.; Sorenson, R. J.; Sercel,
A. D.; Unangst, P. C.; Roth, B. D.; Gilbertsen, R. B.;
Chan, K.; Schrier, D. J.; Guglietta, A.; Bornemeier, D. A.;
Dyer, R. B. J. Med. Chem. 1999, 42, 1151.
of 10 ll (100 lM) of linoleic acid (LA). After maintain-
ing the 96-well plate on a shaker for 5 min, 100 ll of
chromogen was added and the plate was retained on a
shaker for 5 min. The lipoxygenase activity was deter-
mined after measuring absorbance at a wavelength of
490 nm. Percent inhibition was calculated by the com-
parison of compound-treated to various control incuba-
tions. The concentration of the test compound causing
50% inhibition (IC50, lM) was calculated from the con-
7. Cuadro, A. M.; Valenciano, J.; Vaquero, J. J.; Alvarez-
Builla, J.; Sunkel, C.; Fau de Casa-Juana, M.; Pilar
Ortega, M. Bioorg. Med. Chem. 1998, 6, 173.
8. Unangst, P. C.; Connor, D. T.; Cetenko, W. A.; Sorenson,
R. J.; Kostlan, C. R.; Sircar, J. C.; Wright, C. D.; Schrier,
D. J.; Dyer, R. D. J. Med. Chem. 1994, 37, 322.
centration–inhibition
determinations).
response
curve
(duplicate
9. Unangst, P. C.; Shrum, G. P.; Connor, D. T.; Dyer, R. D.;
Schrier, D. J. J. Med. Chem. 1992, 35, 3691.
5.29. Anti-inflammatory assay
10. Lazer, E. S.; Wong, H.-C.; Possanza, G. J.; Graham, A.
G.; Farina, P. R. J. Med. Chem. 1989, 32, 100.
11. Flynn, D. L.; Belliotti, T. R.; Boctor, A. M.; Connor, D.
T.; Kostlan, C. R.; Nies, D. E.; Ortwine, D. F.; Schrier, D.
J.; Sircar, J. C. J. Med. Chem. 1991, 34, 518.
Anti-inflammatory activity was performed using a meth-
od described by Winter et al.22
5.30. Analgesic assay
´
12. Ruiz, J.; Perez, C.; Pouplana, R. Bioorg. Med. Chem.
2003, 11, 4207.
Analgesic activity was determined using a 4% sodium
chloride-induced writhing (abdominal constriction) as-
say previously reported.23
13. (a) Inagaki, M.; Tsuri, T.; Jyoyama, H.; Ono, T.; Yamada,
K.; Kobayashi, M.; Hori, Y.; Arimura, A.; Yasui, K.;
Ohno, K.; Kakudo, S.; Koizumi, K.; Suzuki, R.; Kato,
M.; Kawai, S.; Matsumoto, S. J. Med. Chem. 2000, 43,
2040; (b) Yagami, T.; Ueda, K.; Asakura, K.; Sakaeda, T.;
Kuroda, T.; Hata, S.; Kambayashi, Y.; Fujimoto, M. Br.
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Acknowledgment
14. Hidaka, T.; Hosoe, K.; Ariki, Y.; Takeo, K.; Yamashita,
T.; Katsumi, I.; Kondo, H.; Yamashita, K.; Watanabe, K.
Jpn. J. Pharmacol. 1984, 36, 77.
We are grateful to the Canadian Institutes of Health Re-
search (CIHR) (MOP-14712) for financial support of
this research.
15. (a) Janusz, J. M.; Young, P. A.; Ridgeway, J. M.; Scherz,
M. W.; Enzweiler, K.; Wu, L. I.; Gan, L.; Darolia, R.;
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