Methyl-monofluorination of ibuprofen selectively increases its inhibitory activity toward cyclooxygenase-1 leading to enhanced analgesic activity and reduced gastric damage in vivo

Article abstract of DOI:10.1016/j.bmcl.2011.04.114

Newly developed monofluoromethylation reaction provided access to various bioactive molecules with an interesting monofluoromethyl unit. An iridium-catalyzed asymmetric version was employed for large-scale methyl-monofluorination of widely used nonsteroid

Full text of DOI:10.1016/j.bmcl.2011.04.114

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3578–3582
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Methyl-monofluorination of ibuprofen selectively increases its inhibitory
activity toward cyclooxygenase-1 leading to enhanced analgesic activity
and reduced gastric damage in vivo
Hong Su a, Yuli Xie a,c, , Wen-Bo Liu b, Shu-Li You b,
⇑
⇑
a Shanghai Haini Pharmaceutical Co. Ltd., 3999 Hunan Road, Pudong District, Shanghai 201318, PR China
b State Key Laboratory of Organometallic Chemistry, Shanghai Institute of Organic Chemistry, 345 Lingling Lu, Shanghai 200032, PR China
c Department of Medicine, Columbia University, 630 West 168th street, New York, NY 10032, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Newly developed monofluoromethylation reaction provided access to various bioactive molecules with
an interesting monofluoromethyl unit. An iridium-catalyzed asymmetric version was employed for
large-scale methyl-monofluorination of widely used nonsteroidal anti-inflammatory drug ibuprofen
(the active S isoform). The methyl-monofluorinated ibuprofen was found to selectively inhibit cyclooxy-
genase-1 over cyclooxygenase-2 and surprisingly, the compound, with almost equal pharmacokinetic
profile, was shown to increase analgesic activity and diminish gastric damage in animal models compar-
ing to the parent drug ibuprofen. Therefore, methyl-monofluorination could be a useful strategy for
improving efficacy and safety profile of drugs from the ‘profen’ family.
Received 20 March 2011
Revised 20 April 2011
Accepted 25 April 2011
Available online 3 May 2011
Keywords:
Monofluoromethylation
Ibuprofen
Cyclooxylgenase-1
Analgesic
Ó 2011 Elsevier Ltd. All rights reserved.
Ibuprofen is a well-known nonsteroidal anti-inflammatory drug
(NSAID) widely used for the treatment of pain, fever and inflamma-
tion in clinic. However, long term use of NSAIDs including ibupro-
fen has been associated with gastrointestinal (GI) toxicities such as
ulceration and bleeding. Therefore, new NSAIDs without these side
effects have long been pursued. The major mechanism by which
ibuprofen and other NSAIDs exert their anti-inflammatory activity
is through the suppression of prostaglandin biosynthesis by inhib-
iting cyclooxygenases (COXs). Prostaglandins are major mediators
of inflammation response, but also play a cytoprotective role in
maintaining GI health and homeostasis. COXs, which catalyze the
synthesis of prostaglantins from arachidonic acid, have two major
subtypes COX-1 and COX-2.1 It is generally thought that COX-1 is
constitutively expressed to provide cytoprotection in GI tract while
COX-2 is inducible and mediates inflammation response, and thus
COX-1 inhibition is considered to be the main reason for gastric
damage caused by nonselective NSAIDs like ibuprofen.2 Several
selective COX-2 inhibitors have been developed as the second gen-
eration of NSAIDs with reduced GI side effects. Unfortunately,
these selective COX-2 inhibitors were found to increase incidence
of cardiovascular events such as heart attack and stroke, leading
to the withdrawal of Merck’s blockbuster rofecoxib (VioxxTM) from
the market. Thus, there remains a compelling need to search for
new NSAIDs with better efficacy and safety profile.
Fluorine substitution is a frequently employed strategy in
medicinal chemistry to improve a drug candidate’s physicochemi-
cal property, metabolic stability and binding affinity.3 There are
now a lot of marketed drugs containing fluorine atoms. Accord-
ingly, many efficient methodologies have been developed for the
specific introduction of fluorine into organic molecules, and among
these, fluoroalkylation reactions are one of the most versatile tools
and have continuingly been a research focus of organic chemists.
Although monofluoromethyl unit is of great interest in isostere-
based drug design, successful monofluoromethylation reactions,
especially their asymmetric versions are very rare resulting
in a limited number of known bioactive monofluoromethylated
compounds. Fukuzumi et al. recently reported a novel palladium-
catalyzed enantioselective allylic monofluoromethylation reaction
using FBSM as a fluoromethide(CH2F) equivalent and demon-
strated its application in the synthesis of enantiopure monofluo-
romethylated ibuprofen for the first time (Fig. 1), however, the
authors did not report any biological study of these previously
inaccessible molecules.4
Very recently, we developed a more practical synthetic route for
the preparation of (S)-F-ibuprofen in large scales based on our
observation that iridium/phosphoramidite complexes could effi-
ciently catalyze regio-and enantioselective alkylation of FBSM with
1,3-unsymmetrical allylic substrates to afford enantiopure mono-
fluorinated precursors of various ‘profens’.5 As outlined in Scheme
⇑
Corresponding authors.
E-mail addresses: yz2104@columbia.edu (Y. Xie), slyou@mail.sioc.ac.cn (S.-L.
You).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.114

H. Su et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3578–3582
3579
CH3
CH2F
O O
O
O
S
S
HOOC
HOOC
Ph
Ph
CH2F
H
F
(S)-ibuprofen
Fig. 1. The structures of FBSM and Ibuprofens.
(S)-F-ibuprofen
FBSM
1, the easily accessible starting material 1 smoothly reacted with
FBSM under optimized iridium catalytic system to provide the
highly enantiopure precursor 2 (ee, 94%) in almost quantitative
yield. Removal of phenylsulfonyl groups with activated magne-
sium in methanol followed by oxidation with RuCl3 conveniently
converted the precursor 2 to the final product (S)-F-ibuprofen in
high yield without loss of the optical purity (ee, 95%). This short-
ened synthetic route (two vs four steps compared with Fukuzumi’s
method) proved to be facile and reproducible for tens of grams
scale production of (S)-F-ibuprofen in high quality (>95% ee,
>99% purity) required for pharmacological studies.
fen (IC50 = 98.65 nM) in the parallel assays, and the increased po-
tency on COX-1 in cells, although much more pronounced, was
consistent with our observation in enzymatic assays. Interestingly,
COX-2 was inhibited almost equally by both compounds in LPS-
stimulated U937 cells, with IC50 values of 0.53
lM for F-ibuprofen
and 0.73 M for ibuprofen. Therefore, a single fluorine substitution
l
in the methyl group selectively increased ibuprofen’s inhibitory
activity against COX-1. Both compounds showed greater selectivity
for COX-1 than for COX-2, but two assays were conducted in differ-
ent cell lines and thus cell-type specific effects could not be ruled
out.
Given the importance and drawbacks of ‘profen’ drugs on the
market, and also the ‘magic effect’ of fluorine, it would be interest-
ing to know how specifically introducing a single fluorine atom to
the methyl group in ‘profens’ affects their activity and safety pro-
file. With convenient access to the material, we conducted phar-
macological evaluations of the novel methyl-monofluorinated
ibuprofen (the active S isoform) both in vitro and in vivo. We first
examined its inhibitory activities on COX in vitro. IC50 values
against COX-1 (ovine) and COX-2 (human recombinant) were as-
sessed with a newly developed COX inhibitory screening kit (Cay-
man Chemical’s ACETM EIA Kits, catalog No. 560131) according to
the supplier’s protocol. Possibly due to nonspecific interference,
both ibuprofen and F-ibuprofen were insensitive to this new assay
and exhibited IC50 values in micromolar range. The inactive R iso-
form of F-ibuprofen had no COX inhibitory effect at the concentra-
tions up to 1 mM. Notably, (S)-F-ibuprofen was about three times
Fluorination is well known to impact a compound’s pharmaco-
kinetic properties such as metabolic stability, bioavailability and
tissue penetration. We, therefore, conducted PK study and BBB
penetration of ibuprofen & F-ibuprofen in ICR mice following intra-
venous and oral administration (Supplementary data: Materials
and methods). Their selected pharmacokinetic parameters are pre-
sented in Table 1. Interestingly, F-ibuprofen and ibuprofen gave
very similar PK profiles following either intravenous administra-
tion at a dose of 5 mg/kg or oral administration at a dose of
10 mg/kg. The half-life (t1/2) of F-ibuprofen was less than one hour
(iv = 0.88 h and po = 0.75 h), which was slightly shorter than that
of ibuprofen (iv = 1.14 h and po = 1.12 h). Both compounds were
completely orally bio-available and reached maximum blood con-
centration in the range of 4000–5000 lg/L at given doses rapidly
(Tmax <0.25 h). F-ibuprofen was found to be able to cross blood
brain barrier to some extend with brain-plasma ratios greater than
0.025 mL/g, however, there was no significant improvement com-
paring to nonfluorinated ibuprofen. These results show that
methyl-monofluorination has no significant impact in ibuprofen’s
pharmacokinetic properties, suggesting that the methyl group is
not a metabolism site in vivo. Nevertheless, F-ibuprofen is orally
available and presents an excellent PK profile comparable to that
of ibuprofen.
We next investigated F-ibuprofen’s biological activities in vivo.
Its anti-inflammatory activity was evaluated by the classic carra-
geenan-induced rat paw edema method described by Winter
et al.8 F-ibuprofen (33.6 mg/kg) and ibuprofen (30 mg/kg) were
administered (i.g.) to rats as suspensions in 0.5% carboxyl methyl
more potent than (S)-ibuprofen against COX-1(IC50
, 0.5 vs
1.48 M), and more sensitive measurements may be needed to ob-
l
tain the accurate comparison. To more closely resemble the in vivo
situation, intact cell assays were employed to determine the effects
of F-ibuprofen on both COX isoenzymes. According to the protocols
described in the literature6, COX-1 and COX-2 inhibition were as-
sessed by measuring eicosanoid thromboxane B2 (TBX2) formation
in HEL cells and prostaglandin E2 (PEG2) production in LPS-stimu-
lated U937 cells, respectively.7 As shown in Figure 2, F-ibuprofen
dose-dependently and potently inhibited COX-1-mediated TBX2
formation in HEL cells with a striking IC50 value of 1.23 nM. It
was about 80 folds more potent than the parent compound ibupro-
2 mol% [Ir(COD)Cl]2
PhO2S
O O
O
O
MeO2CO
4 mol% (R,R,Ra)-1a
S
S
PhO2S
F
Ph
Ph
+
Cs2CO3,DCM
rt,12h, 97%
H
F
2
1
FBSM
94% ee
CH2F
CH2F
O
P
O
Ph
Ph
N
HOOC
RuCl3/NaIO4
Mg, MeOH
CCl4/CH3CN/H2O
rt, 24h, 92%
rt, 24h, 87%
(S)-F-ibuprofen
3
(R,R,Ra)-1a
95% ee
95% ee
Scheme 1. A practical synthesis of enantiopure (S)-F-ibuprofen.

3580
H. Su et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3578–3582
Ibuprofen--HEL92.1.7
F-ibuprofen--HEL92.1.7
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.4
0.3
0.2
0.1
-7
-6
-5
-4
-3
-2
-1
0
-7 -6 -5 -4 -3 -2 -1
0
1
2
Log10 concentration (µM)
Log10 concentration (µM)
IC50 / 0.001232
IC50 / 0.09865
Ibuprofen--U937
F-ibuprofen--U937
0.425
0.400
0.375
0.350
0.325
0.300
0.275
0.250
0.225
0.200
0.475
0.450
0.425
0.400
0.375
0.350
0.325
0.300
0.275
-2
-1
0
1
2
3
4
-2
-1
0
1
2
3
4
Log10 concentration (µM)
Log10 concentration (
µ
M)
IC50 / 0.7336
IC50 / 0.5285
Fig. 2. Inhibition of eicosanoid formation in HEL cells (COX-1) and LPS-stimulated U937 cells (COX-2). Drugs (10ꢀ6–10
l
M) were incubated with either cell types. After the
addition of arachidonic acid, COX-1-derived TXB2 or COX-2-derived PGE2 were assessed in cell-free supernatants. Values are presented as mean SD of the ratio of
eicosanoid production of cultures treated with vehicle only.
cellulose (CMC) 1 h before initiation of acute inflammation with
carrageenan.9 The volume of the edema was measured by the ple-
thismometric method in different time intervals and compared to
the control group (vehicle only). The results are shown in Figure
3. At tested doses, both compounds inhibited carrageenan induced
rat paw edema at all time points with the most pronounced effect
occurred at 4 h after carrageenan injection. F-ibuprofen was
slightly more potent than ibuprofen, but did not reach a significant
level as demonstrated by their inhibition of edema at the 4 h time
point (41.3% vs 29.5%). The compounds were further tested for
their analgesic activity at the same doses by using acetic acid in-
duced writhing method in mice.10The 1% (v/v) solution of acetic
acid was used to induce writhing and test compounds were admin-
istered (i.g.) to the mice 1 h before acetic acid injection.11Analgesic
activity was measured as percent inhibition of writhing in compar-
ison to control. As shown in Figure 4, acetic acid induced writhing
was effectively blocked by both ibuprofen and F-ibuprofen, and the
latter exhibited significantly higher analgesic potency (inhibition,
68.7% vs 50%). Among COX isotypes, COX-1 has been shown to play
an important role in pain processing and sensitize the spinal cord
and gracile nucleus.12 COX-1 inhibitors, thus, in many cases, tend
to show an analgesic effect rather than anti-inflammatory activity.
In fact, Mofezolac, a selective COX-1 inhibitor, has been used as
pain killer in clinic. Thus, the enhancement of F-ibuprofen’s pain-
relieving effect is likely resulted from its elevated inhibition of
COX-1 as seen in our cell-based assays.
The acute ulcerogenic effects of F-ibuprofen and ibuprofen were
studied at relatively high doses on Wistar rats.13 F-ibuprofen
(108.8 mg/kg) and ibuprofen (100 mg/kg) were orally adminis-
trated to the rats as 0.5% CMC suspensions and the gastric damage
at 17 h after drug or vehicle treatments was scored. Figure 5(a)–(c)
shows photographs of excised stomachs, showing that F-ibuprofen
had a tendency to induce less severe ulcer than ibuprofen. The gas-
tric damage score of ibuprofen, F-ibuprofen and control group was
3.00 0.25, 2.12 0.37 and 0.50 0.41, respectively (Fig. 5d). The
lower ulcerogenic activity of F-ibuprofen is somehow surprising
given its strong COX-1 inhibitory activity. COX-1 inhibition has
conventionally been considered the main reason for NSAID-
induced ulcer formation. However, emerging evidence suggests
that inhibition of COX-1 alone is not sufficient to induce gastric
damage. For example, Langenabach et al. reported that COX-1
knock out mice do not spontaneously develop gastric ulceration14
Table 1
Selected pharmacokinetics parameters of ibuprofen and F-ibuprofen in ICR mice following intravenous and oral administration
Drugs
AUC(0–t)
g/Lꢁh)
AUC(0–1)
g/Lꢁh)
MRT(0–1)
(h)
t1/2z
(h)
Tmax
(h)
Vz
(L/kg)
CLz
(L/h/kg)
Cmax
g/L)
Bio
(%)
B/P
(mL/g)
(
l
(
l
(
l
Ibuprofen
iv-5 mg/kg
po-10 mg/kg
iv-5 mg/kg
po-10 mg/kg
33947.26
81106.68
23287.97
50159.54
34528.94
81648.09
23345.61
50205.11
1.28
1.68
0.83
0.95
1.14
1.12
0.88
0.75
0.08
0.08
0.08
0.25
0.24
NA
0.27
NA
0.14
NA
0.21
NA
34594.65
52144.84
31627.39
42109.24
0.026–0.059
0.022–0.036
0.031–0.042
0.025–0.035
118.23
107.53
F-ibuprofen
NA: not applicable; Bio: bioavailability; B/P: brain-plasma ration

H. Su et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3578–3582
3581
CMC
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
50
Ibuprofen
Ibuprofen
F-ibuprofen
F-ibuprofen
40
**
**
**
**
**
**
30
20
10
0
**
**
24
24
1
2
3
4
5
6
0
1
2
3
4
5
6
-10
Time (h)
Time (h)
Fig. 3. Anti-inflammatory activity of ibuprofen and F-ibuprofen. Male Wistar rats weighing 140–160 g (eight for every compound and every measurement) was received
drugs or control 1 h before carrageenan injection. Paw volumes were measured every 1 h in 6 h and 24 h after Carrageenan treatment and compared to those of a control
group. Inhibition(%) = (Vc ꢀ Vt/Vc) ꢂ 100, where Vt represents the mean increase in paw volume in rats treated with test compounds, and Vc represents the mean increase in
paw volume in control group of rats. Data shown are means (n = 8) SEM (⁄p <0.05, ⁄⁄p <0.01).
##
70
60
50
40
30
20
10
0
30
25
20
15
10
5
**
##
**
0
Ibuprofen
F-ibuprofen
CMC
Ibuprofen F-ibuprofen
Fig. 4. Acetic acid-induced writhing tests. Ibuprofen and F-ibuprofen were administered (i.g.) at doses of 30 mg/kg and 33.6 mg/kg 1 h before injection of acetic acid,
respectively. The number of muscular contraction was counted starting at 10 min after injection of acetic acid for a period of 10 min. Data shown are the average of the total
writhes observed (n = 8) SEM. Significantly different from the vehicle ⁄⁄p <0.01 and significantly different from ibuprofen group, ##p <0.01.
a
b
F-ibuprofen
Ibuprofen
c
d
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
**
CMC
#
**
CMC
Ibuprofen F-ibuprofen
Fig. 5. Photographs and gastric damage scores of ibuprofen and F-ibuprofen; (a) treated with ibuprofen; (b) treated with F-ibuprofen; (c) vehicle; (d) gastric damage score
was calculated by measuring the lengths, in millimeters, and summing the values for each rat. F-ibuprofen and ibuprofen were orally administrated at the doses of 108.8 mg/
kg and 100 mg/kg, respectively. Data shown are the mean SEM (n = 8/group). The following indicates significantly different from the vehicle ⁄⁄p <0.01 and significantly
different from ibuprofen group #p <0.05.
and consistently, Wallace et al.15 reported that gastric ulcer forma-
tion required inhibition of both COX-1 and COX-2 in rats. These
findings were further supported by the observation that COX-1
inhibition could up-regulate COX-2 expression resulting in

3582
H. Su et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3578–3582
production of prostaglandins to protect mucosal integrity.16 It
seems that both COX-1 and COX-2 selective inhibitors provide
the advantage of reduced GI toxicity. Therefore, the high selectivity
toward COX-1 resulted from fluorine substitution may, on the con-
trary, contribute to F-ibuprofen’s reduced ulcerogenic effect. Of
course, other factors such as local stomach irritation by the carbox-
ylic acid moiety are also involved in GI side effects associated with
NSAIDs17, and should also be considered for F-ibuprofen’s im-
proved safety profile.
In summary, ‘profens’ are the most important drugs in the fam-
ily of NSAIDs, but associated with serious GI toxicity. New ‘profen’
drugs with reduced side effects are of great interest. Novel asym-
metrical monofluoromethylation reactions led to previously inac-
cessible enantiopure monofluoromethylated ‘profen’ molecules. A
practical synthetic route was developed by us to produce
methyl-monofluorinated (S)-ibuprofen in large scales for pharma-
cological studies. It was found that specifically adding a single fluo-
rine atom to the methyl group in (S)-ibuprofen did not affect its
pharmacokinetic property, but dramatically increased its inhibi-
tory activity toward COX-1. The fluorinated ibuprofen showed en-
hanced analgesic activity and reduced acute ulcerogenic effect in
animal studies, which was possibly attributed to its increased
selectivity against COX-1. Development of COX-1-selective inhibi-
tors has been hindered by the conventional view that COX-1 inhi-
bition causes gastric damage. However, our study supports the
notion that selective inhibition of COX-1 effectively elicits potent
analgesic effect with less severe gastric ulceration. Considering
the cardiovascular toxicity of selective COX-2 inhibitors, COX-1-
selective inhibitors should be paid more attention to, particularly
in the development of safe and effective analgesics. The monofluo-
rination of ‘profen’ molecules we described here could be a useful
tool to achieve this goal.
7. The inhibition of COX-1 and COX-2 activity in intact cells was performed as
described in literature with minor modifications. Briefly, cells of the human
erythroleukemic cell line HEL92.1.7 were harvested, suspended in fresh
medium (108/ml) and incubated with drug for 30 min at 37 °C. Arachidonic
acid (Sigma) was then added to a final concentration of 30 lM for a further
15 min at 37 °C. Thereafter, thromboxane B2 was measured in cell free culture
supernatants by ELISA (Cayman) according to the manufacturer’s instructions.
For COX-2 assays, cells of U937 (human leukemic monocyte lymphoma cell
line) were harvested and suspended in fresh medium (106/ml), and then
treated with PMA for 3d at 37 °C. The cells were then washed by PBS and
stimulated with LPS (100ng/ml) for 6 h. Then, arachidonic acid (30 lM; Sigma)
was added for 15 min at 37 °C. Thereafter, prostaglandin E2 was assessed in cell
free culture supernatants by ELISA (Cayman) according to the manufacturer’s
instructions.
8. Winter, C. A.; Risley, E. A.; Nus, G. N. Proc. Soc. Exp. Biol. 1962, 111, 544.
9. The experiment was performed on male Albino rats of Wistar strain, weighing
140–160 g. The animals were randomly divided into three groups (eight in
each group). Group I was kept as control, and received only 0.5% carboxy
methyl cellulose (CMC) solution. Groups II and III were kept as standard, and
received ibuprofen (30 mg/kg i.g.) and F-ibuprofen (33.6 mg/kg I.g.),
respectively. Carrageenan solution (Sigma, 0.1% in sterile 0.9% NaCl solution)
in a volume of 0.1 ml was injected subcutaneously into the sub-plantar region
of the right hind paw of each rat, 1 h after the administration of the test
compounds. The right hind paw volume was measured every 1 h in 6 h and
24 h after carrageenan treatment by means of a plethysmometer. The percent
anti-inflmmatory activity was calculated according to the following formula:
Percent anti-inflammatory activity = (Vc ꢀ Vt/Vc) ꢂ 100, where Vt represents
the mean increase in paw volume in rats treated with test compounds, and Vc
represents the mean increase in paw volume in control group of rats.
10. Seigmund, E.; Cadmus, R.; Lu, G. Proc. Soc. Exp. Biol. Med. 1957, 95, 729.
11. Three groups of male Swiss albino mice (22–24 g) were treated (i.g.) with the
CMC suspensions of ibuprofen (30 mg/kg) and F-ibuprofen (33.6 mg/kg), and
0.5% CMC (0.3 ml/10 g) 1 h prior to acetic acid injection. The muscular
contraction was induced by injection of 1% (v/v) acetic acid at a dose of
0.1 ml/10 g for 30 min after the treatment. Number of writhings for 10 min in
control and test compounds were counted and compared. Analgesic activity
was measured as percent decrease in writhings in comparison to control. All
the results are expressed as mean SEM. Statistical evaluation was performed
using analysis of variance followed by t-test for sub group comparison.
12. Patrignani, P.; Filabozzi, P.; Patrono, C. J. Clin. Invest. 1982, 69, 1266.
13. Wistar rats (150–200 g) were randomly divided into three groups. Each group
contained eight animals. They were deprived from food at least 8 h before drug
administration. Water was accessed ad libitum. CMC was dissolved in distilled
water to 0.5% solution (w/v). Ibuprofen and F-ibuprofen were suspended in
0.5% CMC, their concentration was 10 mg/ml and 10.88 mg/ml, respectively.
Rats were administrated orally with test compounds at the volume of 10 ml/kg.
Control rats received 10 ml/kg 0.5% CMC. Foods were provided after 30 min.
17 h after drug administration, rats were euthiuthetized with CO2 and
stomachs were taken. The stomachs were scissored along the greater
curvature and rinsed in PBS. They were fixed in 4% paraformaldehyde (in
0.1 M phosphate buffter, pH 7.4) for 30–60 min, and the severity of the ulcer
was then scored by an experimenter who did not know the treatment of the
animal.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.114.
References and notes
1. Perrone, M. G.; Scilimati, A.; Simone, L.; Vitale, P. Curr. Med. Chem. 2010, 17,
3769.
2. Allison, M. C.; Howatson, A. G.; Torrance, C. J.; Lee, F. D.; Russell, R. I. N. Engl. J.
Med. 1992, 327, 749.
3. Bohm, H. J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Muller, K.; Obst-
Sander, U.; Stahl, M. Chem. Biol. Chem. 2004, 5, 637.
4. Fukuzumi, T.; Shibata, N.; Sugiura, M.; Yaui, H.; Nakamura, S.; Toru, T. Angew.
Chem., Int. Ed. 2006, 45, 4973.
5. Liu, W.; Zheng, S.; He, H.; Zhao, X.; Dai, L.; You, S. Chem. Commun. 2009, 6604.
6. Berg, J.; Hristoph, T.; Widerna, M.; Bodenteich, A. J. Pharmacol. Toxicol. Methods
1997, 37, 179.
14. Langenbach, R.; Morhan, S. G.; Tiano, H. F.; Loftin, C. D.; Ghanayem, B. I.;
Chulada, P. C.; Mahler, J. F.; Lee, C. A.; Goulding, E. H.; Kluckman, K. D.; Kim, H.
S.; Smithies, O. Cell 1995, 83, 483.
15. Wallace, J. L.; McKnight, W.; Reuter, B. K.; Vergnolle, N. Gastroenterology 2000,
119, 706.
16. Tanaka, A.; Hase, S.; Miyazawa, T.; Takeuchi, K. J. Pharmacol. Exp. Ther. 2002,
300, 754.
17. Hawkey, C.; Laine, L.; Simon, T.; Beaulieu, A.; Maldonado-Cocco, J.; Acevedo, E.;
Shahane, A.; Quan, H.; Bolognese, J.; Mortensen, E. Arthritis Rheum. 2000, 43,
370.