The influence of double bond geometry in the inhibition of cyclooxygenases by sulindac derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.04.078

Sulindac sulfide is a benzylidene-indene that is a potent, time-dependent inhibitor of cyclooxygenases-1 and -2. Removal of the 2′-methyl group from the indene ring dramatically reduces time-dependent inhibition of both enzymes but also changes the geomet

Full text of DOI:10.1016/j.bmcl.2009.04.078

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3271–3274
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The influence of double bond geometry in the inhibition of cyclooxygenases
by sulindac derivatives
Matthew J. Walters, Anna L. Blobaum, Philip J. Kingsley, Andrew S. Felts, Gary A. Sulikowski,
*
Lawrence J. Marnett
A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in
Molecular Toxicology and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Sulindac sulfide is a benzylidene–indene that is a potent, time-dependent inhibitor of cyclooxygenases-1
and -2. Removal of the 20-methyl group from the indene ring dramatically reduces time-dependent inhi-
bition of both enzymes but also changes the geometry of the benzylidene double bond from Z to E.
Herein, we explore the importance of double bond geometry on cyclooxygenase inhibition. The Z-isomer
of 20-des-methyl sulindac sulfide was synthesized by reduction of a bromoindene precursor or by photo-
isomerization of the E-isomer. The Z-isomer inhibited both cyclooxygenases, but with diminished
potency compared to sulindac sulfide. Thus, although the 20-methyl group is a major determinant of
time-dependent cyclooxygenase inhibition, the geometry of the benzylidene double bond plays a role
as well.
Received 5 March 2009
Revised 15 April 2009
Accepted 20 April 2009
Available online 23 April 2009
Keywords:
COX inhibition
Photoisomerization
NSAIDs
20-des-Methyl sulindac sulfide
Ó 2009 Elsevier Ltd. All rights reserved.
Sulindac sulfide and indomethacin are non-steroidal anti-
inflammatory drugs (NSAIDs) that exert anti-inflammatory, anal-
gesic, and anti-pyretic activities by inhibition of cyclooxygenase
(COX) enzymes1,2 (Fig. 1). Both compounds are slow, tight-binding
inhibitors of COX-1 and COX-2 and the kinetics of inhibition are
consistent with a rapid reversible interaction with the enzymes
followed by a slow transition to a much more tightly bound com-
plex3,4 (Eq. 1). We have shown that an important determinant of
the tight binding of sulindac sulfide and indomethacin is insertion
of the 20-methyl group of the indene or indole rings into a hydro-
phobic depression in the side of the COX active site.5,6 Removal
of the methyl group nearly completely eliminates the time-depen-
dent inhibition of COX enzymes and transforms these compounds
into weaker competitive inhibitors.5,6 In the case of sulindac sul-
fide, removal of the 20-methyl group also changes the geometry
of the benzylidene double bond, which is introduced in the last
step of synthesis, from Z to E.6
geometric isomerism may also contribute significantly to the
reduction of inhibition. To determine the importance of the geom-
etry of the benzylidene double bond in COX inhibition, we have
synthesized the Z-isomer of 20-des-methyl sulindac sulfide and
compared the kinetics of its inhibition of COX-1 and COX-2 with
those of (E)-20-des-methyl sulindac sulfide.
O
O
4'
7'
4'
7'
3'
OH
3'
OH
F
O
2'
1'
2'
5'
6'
5'
6'
N
1'
O
Z
S
Cl
sulindac sulfide
indomethacin
O
k1
k2
E þ I ꢀ E—I ꢀ Eꢁ—I; K1 ¼ kꢀ1=k1
ð1Þ
4'
3'
OH
O
k
k
ꢀ2
F
ꢀ1
5'
2'
4'
OH
3'
Equation 1. Multi-step rate equation in which an initial rapid,
reversible step is followed by a slow reversible step for COX.
F
6'
7'
1'
5'
6'
2'
1'
7'
Models of sulindac sulfide–COX-2 interactions based on the
crystal structure of an indomethacin–COX-2 complex7 suggest this
Z
S
E
S
(Z)-2'-des-methyl sulindac sulfide
(E)-2'-des-methyl sulindac sulfide
* Corresponding author. Tel.: +44 1 615 343 7329; fax: +44 1 615 343 7534.
E-mail address: larry.marnett@vanderbilt.edu (L.J. Marnett).
Figure 1. The structures of sulindac sulfide and indomethacin along with the two
isomers of 20-des-methyl sulindac sulfide.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.078

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M. J. Walters et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3271–3274
6-Fluoro-1-indanone was treated with zinc and ethyl bromo-
acetate in a Reformatsky reaction to give the desired alcohol prod-
uct.6 The alcohol was then dehydrated under acidic conditions6
and the indene was treated with bromine to introduce a bromide
in the 20-position to serve as a temporary steric unit. Under basic
conditions, the ethyl ester was cleaved to the acid along with the
condensation of the bromoindene with p-methylthiobenzaldehyde
to give a single product. In the final step, debromination was
accomplished using tributyltin hydride. The final product resulted
in an 80:20 mixture of E and Z isomers (Fig. 2).
The E- and Z-isomers of 20-des-methyl sulindac sulfide were
separated by reverse-phase HPLC using a Phenyl–Hexyl column
(Phenomenex, Inc. 25 ꢂ 4.6 cm) with gradient elution (Fig. 3).
The mobile phase consisted of water with 0.5% acetic acid (v/v)
(A) and 1:1 acetonitrile/methanol with 0.5% acetic acid (B). Isomer
separation was also achieved on an analytical C8 column, but sep-
aration on the Phenyl–Hexyl column was more robust. The NMR
spectra of the two compounds was helpful in their structural
assignment. The triplet–doublet at 7.04 ppm on the aromatic ring
of the E-isomer shifted upfield to 6.89 ppm in the Z-isomer
whereas the indene proton at 7.11 ppm of the E-isomer shifted up-
field to 6.66 ppm and the external double bond proton shifted from
7.57 ppm to 7.31 ppm (Fig. 4). The two isomers have slightly differ-
ent UV properties (Fig. 3, inset). The E-isomer has a kmax of 370 nm
whereas the Z-isomer has a kmax at a lower wavelength (350 nm).
During HPLC purification of both isomers, it was observed that if
the Z-isomer was exposed to natural light it photoisomerized to
the E-isomer (Fig. 4). Upon further study, an equilibrium was found
to exist between the two isomers if exposed to natural light for
10 h (approximately a 70:30 ratio of E/Z). If kept in the dark, each
isomer remained stable.
Figure 3. HPLC separation of the E- and Z-isomers of 20-des-methyl sulindac sulfide.
the conversion under natural light (10–15%) and indicated that
photoisomerization has a low quantum efficiency at 370 nm
(Fig. 4). We also tried to photoisomerize the sulfoxide and sulfone
derivatives of 20-des-methyl sulindac sulfide8 and sulindac sulfide
itself under natural light and ultraviolet conditions but no inter-
conversion was observed.
(E)- and (Z)-20-des-methyl sulindac sulfide were evaluated as
inhibitors of murine COX-2 or ovine COX-1 in assays performed
with a saturating concentration of arachidonic acid substrate
(50 lM). The presence of saturating substrate concentrations pre-
cludes competitive inhibition and biases the assay to detect
time-dependent inhibitors. The data summarized in Figure 5 con-
firms our previous report that (E)-20-des-methyl sulindac sulfide
exhibits no time-dependent inhibition of COX-1 or COX-2.5,6 Very
weak COX-1 and COX-2 inhibition was detected with (Z)-20-des-
methyl sulindac sulfide, but the maximal extent of inhibition was
only 20%. The inability to achieve complete inhibition suggests that
the binding of the (Z)-isomer is readily reversible and consistent
We attempted to photoisomerize the E-isomer of 20-des-methyl
sulindac sulfide to the Z-isomer at 370 nm for 24 h using a high-
pressure mercury lamp (1000 W, Hanovia 528B-1) and a Spectral
Energy GM 252 high intensity grating monochromator. The per-
centage conversion was only 1–2%, which compared poorly to
O
O
O
HO
O
Zinc metal
F
F
F
p-TSA, CaCl2
O
O
Br
O
Br2/AcOH
O
OH
O
O
F
1 M NaOH
F
Br
Br
O
H
S
S
AIBN,
Bu3SnH
O
OH
O
F
OH
F
Z
S
E
(E)-2'-des-methyl sulindac sulfide
Figure 2. Synthesis of (Z)- and (E)-20-des-methyl sulindac sulfide.
S
(Z)-2'-des-methyl sulindac sulfide

M. J. Walters et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3271–3274
3273
Figure 4. Photoisomerization of (E)-20-des-methyl sulindac sulfide to (Z)-20-des-methyl sulindac sulfide under natural light for 10 h. The arrows indicate the appearance of
new peaks for (Z)-20-des-methyl sulindac sulfide.
100
80
60
40
20
0
0
1000
2000
3000
4000
Inhibitor, nM
Figure 6. Competitive inhibition of murine COX-2 and ovine COX-1 by the E- and Z-
isomers of 20-des-methyl sulindac sulfide under low arachidonate conditions. (s)
COX-2 and (E)-20-des-methyl sulindac sulfide, (d) COX-1 and (E)-20-des-methyl
sulindac sulfide, (h) COX-2 and (Z)-20-des-methyl sulindac sulfide, (j), COX-1 and
(Z)-20-des-methyl sulindac sulfide.
Figure 5. Time-dependent inhibition of murine COX-2 and ovine COX-1 under
saturating substrate conditions by sulindac sulfide and its 20-des-methyl sulindac
sulfide E- and Z-isomers. (s) COX-2 and (E)-20-des-methyl sulindac sulfide, (d)
COX-1 and (E)-20-des-methyl sulindac sulfide, (h) COX-2 and (Z)-20-des-methyl
sulindac sulfide, (j), COX-1 and (Z)-20-des-methyl sulindac sulfide, (}) COX-2 and
sulindac sulfide, (Ç) COX-1 and sulindac sulfide.
by the reduced substrate concentrations (data not shown). Thus,
whereas the 20-methyl group is a critical determinant of time-
dependent inhibition of COX enzymes by both (E)- and (Z)-isomers,
it is less important for competitive inhibition. The geometry of the
with competitive inhibition. The parent molecule, sulindac sulfide,
inhibits both COX enzymes potently (115 nM and 140 nM, for COX-
1 and COX-2, respectively), although it reaches a plateau around
75% inhibition. These results indicate that the 20-methyl group on
the indene nucleus is a major determinant of time-dependent inhi-
bition of both COX-1 and COX-2. The effect of removing the 20-
methyl group is greater for sulindac sulfide than for indometha-
cin.5 Indomethacin potently inhibits wild-type COXs with IC50 val-
ues of 40 nM and 250 nM against COX-1 and COX-2, respectively.5
However, the 20-des-methyl indomethacin has no activity against
COX-1, but is still able to inhibit COX-2 with an IC50 of 4 l
M.5
To evaluate the ability of (E)- or (Z)-20-des-methyl sulindac sul-
fide to act as competitive inhibitors, each isomer was tested in an
assay with reduced substrate concentrations (5
COX-1 and COX-2 for arachidonate. Interestingly, (E)-20-des-methyl
sulindac sulfide showed weak inhibition of COX-1 (IC50 of 1.8 M),
lM) near the Km of
l
but no inhibition of COX-2 at the concentration ranges tested
(Fig. 6). In contrast, (Z)-20-des-methyl sulindac sulfide strongly
inhibited both COX-1 and COX-2 under these conditions (IC50s of
147 nM and 375 nM, respectively) (Fig. 6). The ability of sulindac
sulfide to inhibit COX-1 and COX-2 was not significantly affected
Figure 7. Indomethacin bound in the active site of murine COX-2 (model based on
Kurumbail et al.).7 The figure illustrates where the 20-methyl of indomethacin
inserts into a small, hydrophobic depression in the COX active site comprised of
Ala527, Ser530, Leu531, and Val349. (PDB ID: 4COX).

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M. J. Walters et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3271–3274
benzylidene double bond seems important for competitive inhibi-
tion as isomerization from (Z) to (E) significantly reduces the po-
tency against COX-1 and nearly abolishes inhibition of COX-2.
The 20-methyl of the indole ring of indomethacin inserts into a
hydrophobic pocket of COX-2 comprised of residues Val349,
Ala527, Ser530, and Leu5315 (Fig. 7). These residues are responsi-
ble for the tight-binding and time-dependence of COX inhibition
by indomethacin.5 Whereas indomethacin is released only very
slowly once it is tightly-bound to COX-2, 20-des-methyl indometh-
acin is readily competed off the enzyme (kꢀ2 = 0.006 sꢀ1).5 The
binding of indomethacin results in the 4-chlorobenzoyl chloride
being out of the plane compared to the indole ring where the car-
bonyl forms a hydrogen bond with Ser530.5 The carboxylate of
indomethacin interacts with Arg120 while the o-methoxy group
protrudes into a large cavity surrounded by residues Ser353,
Tyr355, and Val523.5 It seems possible that (Z)-20-des-methyl sul-
indac sulfide is oriented similarly to the parent compound, sulin-
dac sulfide, and to 20-des-methyl indomethacin in the COX active
site. The carboxylate group and fluorine are likely oriented simi-
larly to that of indomethacin. The key differences between the
binding of the sulindac sulfide derivatives is the absence of a
hydrogen bond between the substrate and Ser530 as well as the
planarity of the indene ring and the benzylidene double bond.
The absence of the 20-methyl group makes the des-methyl deriva-
tives very poor time-dependent COX inhibitors. The E-isomer of 20-
des-methyl sulindac sulfide must bind in a completely different
orientation in the COX active site because the orientation of the
thiomethoxyphenyl group should introduce numerous steric
clashes as it attempts to adopt a configuration similar to that of
sulindac sulfide. There has been a recent report suggesting a novel
binding mode of indomethacin derived COX-1 selective inhibitors.9
In conclusion, we have found that the E- and Z-isomers of 20-
des-methyl sulindac have unique inhibitory profiles when tested
against COX-1 and COX-2. Importantly, we have shown that the
20-methyl group of sulindac sulfide is a major determinant of
tight-binding as it is for indomethacin.5 In addition, we have estab-
lished the importance of the geometry of the benzylidene double
bond in competitive inhibition of COX-1 and COX-2. Our discovery
that (E)-20-des-methyl sulindac sulfide is a selective COX-1 inhibi-
tor represents the first report of selective COX-1 inhibition by a
member of the arylacetic acid class of inhibitors. This may not only
be useful for evaluating the importance of COX-1 inhibition in the
pharmacological action of sulindac sulfide, but it may represent an
opportunity to define a new binding mode for an arylacetic acid to
a COX enzyme.
Acknowledgements
This work was supported by the National Institutes of Health
(CA89450).
Supplementary data
Procedures for the synthesis of (Z)-20-des-methyl sulindac sul-
fide under non-photoisomerization and photoisomerization condi-
tions. Enzymatic assay conditions for both time-dependent and
competitive inhibition of COX are available. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.04.078.
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