Properties and synthesis of 2-{2-fluoro (or bromo)-4-[(2-oxocyclopentyl) methyl]phenyl}propanoic acid: Nonsteroidal anti-inflammatory drugs with low membrane permeabilizing and gastric lesion-producing activities

Article abstract of DOI:10.1021/jm101116s

We previously proposed that membrane permeabilization activity of NSAIDs is involved in NSAID-induced gastric lesions. We here synthesized derivatives of loxoprofen that have lower membrane permeabilization activity than other NSAIDs. Compared to loxoprofen, the derivatives 10a and 10b have lower membrane permeabilization activity and their oral administration produced fewer gastric lesions but showed an equivalent anti-inflammatory effect. These results suggest that 10a and 10b are likely to be therapeutically beneficial as safer NSAIDs.

Full text of DOI:10.1021/jm101116s

J. Med. Chem. 2010, 53, 7879–7882 7879
DOI: 10.1021/jm101116s
Properties and Synthesis of 2-{2-Fluoro (or Bromo)-4-[(2-oxocyclopentyl)methyl]phenyl}propanoic
Acid: Nonsteroidal Anti-inflammatory Drugs with Low Membrane Permeabilizing and
Gastric Lesion-Producing Activities
Naoki Yamakawa,† Shintaro Suemasu,† Masaaki Matoyama,† Ayumi Kimoto,† Miho Takeda,† Ken-ichiro Tanaka,†
Tomoaki Ishihara,† Takashi Katsu,‡ Yoshinari Okamoto,† Masami Otsuka,† and Tohru Mizushima*,†
†Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan, and
‡Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan
Received August 27, 2010
We previously proposed that membrane permeabilization activity of NSAIDs is involved in NSAID-
induced gastric lesions. We here synthesized derivatives of loxoprofen that have lower membrane
permeabilization activity than other NSAIDs. Compared to loxoprofen, the derivatives 10a and 10b
have lower membrane permeabilization activity and their oral administration produced fewer gastric
lesions but showed an equivalent anti-inflammatory effect. These results suggest that 10a and 10b are
likely to be therapeutically beneficial as safer NSAIDs.
Introduction
recently suggested that COX-independent NSAID-induced
cell death is also involved in NSAID-induced gastric lesions
and that this direct cytotoxicity of NSAIDs is due to their
membrane permeabilization activity.16,17 Thus, we proposed
that NSAIDs with lower membrane permeabilization activity
would be safer on stomach tissue even without the selectivity
for COX-2.17
Loxoprofen has been used clinically for a long time as a
standard NSAID in Japan, and clinical studies have suggested
that it is safer than other NSAIDs, such as indomethacin.18,19
Loxoprofen is a pro-drug, which is converted (by reduction of
the cyclopentanone moiety) to its active metabolite (the trans-
alcohol metabolite of loxoprofen) by aromatic aldehyde-
ketone reductase only after absorption by the gastrointestinal
tract.20 We recently reported that loxoprofen has relatively lower
membrane permeabilization activity than other NSAIDs.21
In this study, we synthesized a series of its derivatives and
examined their membrane permeabilization, ulcerogenic
and anti-inflammatory activities.
Nonsteroidal anti-inflammatory drugs (NSAIDsa) are a
useful family of therapeutics, accounting for nearly 5% of all
prescribed medications.1 Their inhibitory effect on cyclooxy-
genase (COX) activity and resulting decrease in prostaglan-
dins (PGs) such as PGE2 has been shown to be responsible for
their anti-inflammatory actions. On the other hand, NSAID
use is associated with gastrointestinal complications, with
about 15-30% of chronic users of NSAIDs suffering from
gastrointestinal ulcers and bleeding.2-4
In 1991, two subtypes of COX, COX-1, and COX-2, which
are responsible for the majority of COX activity at the gastro-
intestinal mucosa and in tissues with inflammation, respec-
tively, were identified,5,6 and a greatly reduced incidence of
gastroduodenal lesions has been reported for selective COX-2
inhibitors (such as celecoxib and rofecoxib).7-9 However, a
recently raised issue concerning the use of selective COX-2
inhibitors is their potential risk for cardiovascular thrombotic
events.10,11 This may be due to the fact that prostacyclin, a
potent antiaggregator of platelets and a vasodilator, is mainly
produced by COX-2 in vascular endothelial cells, while throm-
boxane A2, a potent aggregator of platelets and a vasocon-
strictor, is mainly produced by COX-1 in platelets.12-14
Because of this concern, rofecoxib was withdrawn from the
worldwide market. Therefore, NSAIDs exhibiting gastroin-
testinal safety, other than selective COX-2 inhibitors, need to
be developed.
Results and Discussion
We considered that loxoprofen sodium (1) (Scheme 1)
could be a lead compound to obtain NSAIDs with even lower
membrane permeabilization activity, and on this basis, we
synthesized a series of its derivatives, including the position
isomer which is chemically modified at an aromatic ring of
lead structure by halogens or modified aryl groups (Chart 1).
We then examined their membrane permeabilization activities
and selected two compounds, fluoro loxoprofen (10a) and
bromo loxoprofen (10b). The synthesis of these compounds is
outlined in Scheme 1 (experimental details are available in
Supporting Information).
Although PGE2 has a strong protective effect on the
gastrointestinal mucosa, it is now believed that the inhibition
of COX by NSAIDs is not thought to be the sole explanation
for the gastrointestinal side effects of NSAIDs.15 We have
We previously established an assay system for assessing the
membrane permeabilization activity of NSAIDs using cal-
cein-loaded liposomes. Calcein fluorescence is very weak at
high concentrations due to self-quenching, so the addition of
*To whom correspondence should be addressed. Phone and fax: 81-
96-371-4323. E-mail: mizu@gpo.kumamoto-u.ac.jp.
aAbbreviations: COX, cyclooxygenase; EIA, enzyme immunoassay;
NSAID, nonsteroidal anti-inflammatory drug; PG, prostaglandin.
r
2010 American Chemical Society
Published on Web 10/18/2010
pubs.acs.org/jmc

7880 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Yamakawa et al.
Scheme 1. Synthesis of Halogeno-Loxoprofen Derivatives (10a,b)a
a Reagents and conditions: (a) 3 m HCl aq, NaNO2, CuSO4, Na2SO3, AcONa, H2O, 0 °C; (b) NH2OH HCl, (HCHO)n, AcONa, H2O; (c) conc HCl,
3
reflux; (d) MeOCH2P(Ph3)Cl, C6H18KNSi2, toluene; (e) 3 M HCl aq, acetone, reflux; (f ) PFC (2 mol %), H5IO6, acetonitrile; (g) conc HCl, CH3OH,
reflux; (h) 2.0 M LDA, CH3I, dry THF, -78 °C to -40 °C; (i) NBS, AIBN, CCl4, reflux; ( j) dry Na2CO3, methyl 2-oxocyclopentanecaroxylate, dry
acetone, reflux; (k) conc HCl, AcOH, reflux; (l) 1 M NaOH aq, C2H5OH, reflux.
Chart 1. Synthesized Loxoprofen Derivatives
Figure 1. Membrane permeabilization by loxoprofen sodium and
its derivatives. Calcein-loaded liposomes were incubated in the
presence of indicated concentrations of 1, 10a and 10b. The release
of calcein from the liposomes was determined by measuring fluor-
escence intensity. Triton X-100 (10 μM) was used to establish the
100% level of membrane permeabilization. Values are mean ( SEM
(n=3).
Table 1. In Vitro Human Whole Blood Assay for Inhibition of COX-1-
and COX-2-Derived PG Biosynthesisa
IC50 (μM)
compd
COX-1
COX-2
COX-1/COX-2
1
23.5 ( 4.8
24.2 ( 8.6
30.3 ( 4.1
6.3 ( 1.2
1.3 ( 0.3
7.7 ( 0.3
7.2 ( 0.9
0.6 ( 0.1
2.2 ( 1.6
10.1 ( 1.3
14.3 ( 6.8
65.3 ( 11.4
12.2 ( 3.9
2.4 ( 0.4
48.0 ( 3.2
34.0 ( 4.9
3.1 ( 1.5
8.5 ( 0.4
2.5 ( 0.5
2.5 ( 1.5
0.5 ( 0.1
0.6 ( 0.1
0.6 ( 0.1
0.2 ( 0.02
0.2 ( 0.1
0.4 ( 0.2
0.3 ( 0.2
10a
10b
17
18
membrane-permeabilizing drugs to a medium containing
calcein-loaded liposomes causes an increase in fluorescence
by diluting the calcein.17 Using this assay system, we com-
pared the membrane permeabilization activity of 10a and 10b
to 1. Neither 10a nor 10b apparently released calcein into the
medium at concentrations less than 1 M (Figure 1), showing
that these compounds have much lower membrane perme-
abilization activity than 1.
We then examined their inhibitory effects on COX-1 (or
COX-2), using a human whole blood COX assay. As shown in
Table 1, both 10a and 10b showed IC50 values for COX-1-
derived PG biosynthesis that are similar to 1. For COX-2-
derived PG biosynthesis, 10a showed a similar result, but 10b
showed a little weaker inhibitory effect compared to that of 1.
These results suggest that, as for 1, 10a and 10b did not exhibit
apparent selectivity for COX-2 (The COX-1/COX-2 value of
19a
19b
20a
20b
a Inhibitory effect of each compound on COX-1- and COX-2-derived
PG biosynthesis was measured and the IC50 value (concentration of each
compound required for 50% inhibition), and the COX-1/COX-2 ratio of
that for COX-1 and COX-2 are shown. We estimated the values of IC50
from the sigmoid-like dose-response curve (four-parameter logistic
curve model) drawn by the logistic-curve fitting software (ImageJ
1.43u; National Institutes of Health, USA). Values are mean ( SEM
(n=3).
celecoxib was reportedtobe 22.7withthe same methodas that
used in Table 122).
As described above, 1 is a pro-drug and the trans-alcohol
form is the active metabolite. To test whether or not 10a and

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7881
10b maintain this characteristic, we examined the COX-
inhibitory activities of the trans-alcohol forms of 10a and
10b (20a and 20b, respectively). The synthesis of these com-
pounds is outlined in Scheme 2 (experimental details are
available in Supporting Information). The trans-alcohol form
of1 (18) showed a more potentinhibitoryeffect onbothCOX-
1- and COX-2-derived PG biosynthesis than 1 or its cis-
alcohol form (17) (Table 1), as described previously.20 Both
20a and 20b also showed more potent inhibitory effects on
both COX-1- and COX-2-derived PG biosynthesis than 10a
and 10b or their cis-alcohol forms (19a and 19b, respectively)
(Table 1). These results suggest that 10a and 10b may also act
as pro-drugs and that their trans-alcohol forms are the active
for inhibition of COX.
We then evaluated the activities of 10a and 10b in vivo.
Compound 1 (40 or 50 mg/kg) and equivalent molar amounts
of 10a and 10b were orally administered to rats and the lesion
index was calculated. Administration of 1 produced gastric
lesions in a dose-dependent manner (Figure 2A), as described
previously.18,19 Both 10a and 10b produced fewer gastric
lesions than 1 (Figure 2A). We also measured the gastric level
of PGE2 by enzyme immunoassay (EIA) after oral admin-
istration of these compounds. As shown in Figure 2B,
administration of 10a decreased the level to an extent
similar to that of 1. On the other hand, administration of
10b decreased the gastric level of PGE2, although not to
the extent of that seen with 1 (Figure 2B). Considering our
hypothesis that both a decrease in the gastric level of PGE2
and gastric mucosal damage due to membrane permeabiliza-
tion activity of NSAIDs are involved in the production of
NSAID-induced gastric lesions, the lower lesion-producing
activity of 10a seems to be due to its lower membrane perme-
abilizing activity. On the other hand, that of 10b may involve
both its lower membrane permeabilization activity and
decreased inhibitory effect on the gastric production of
PGE2.
Figure 2. (A) Production of gastric lesions or (B) gastric PGE2 level
in the presence of loxoprofen sodium and its derivatives. (A,B) Rats
were orally administered a low (40, 43, or 52 mg/kg) or high (50, 53,
or 65 mg/kg) dose of 1, 10a, or 10b, respectively, and their stomachs
were removed after 8 h. (A) Stomachs were scored for hemorrhagic
damage. (B) Gastric PGE2 level was determined by EIA. Values are
mean ( SEM (n = 3). **P < 0.01; *P < 0.05; n.s., not significant
(vs, 1 (A); vs, vehicle (B)).
Figure 3. Anti-inflammatory activities of loxoprofen and its deri-
vatives. Rats were orally administered 10, 11, or 13 mg/kg of 1, 10a,
or 10b, respectively, and 1 h later they received an intradermal
injection of carrageenan (1%) into the left hindpaw. Paw edema
were measured 3 h and 6 h after the administration of carrageenan.
Values are mean ( SEM (n = 3-4). **P < 0.01; n.s., not significant
(vs, vehicle).
Scheme 2. Synthesis of Loxoprofen Alcohol (17, 18) and its Halogeno-Derivatives (19a,b, 20a,b)a
a Reagents and conditions: (a) 6 M HCl aq, CH2Cl2 extract; (b) 4-DMAP, EDC, CH3OH; (c) NaBH4, C2H5OH; (d) KOH, C2H5OH, H2O, reflux.

7882 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Yamakawa et al.
Finally, we compared the anti-inflammatory effects of 10a
and 10b to 1 by employing a rat carrageenan-induced footpad
edema assay. As shown in Figure 3, the volume of carragee-
nan-induced footpad edema was significantly decreased after
oral administration of 1, confirming its previously described
anti-inflammatory activity.20,23 The effects of 10a and 10b
were much the same as that of 1, showing that 10a and 10b
have anti-inflammatory activity equivalent to 1.
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Conclusions
In this study, we showed that two derivatives of 1, 10a and
10b, have even lower membrane permeabilization activity and
produced fewer gastric lesions after oral administration to
rats, compared to 1. This is chemical evidence in support of
our proposal that the membrane permeabilization activity of
NSAIDs is involved in their induction of gastric lesions.
We also found that 10a and 10b showed anti-inflammatory
effects equivalent to that of 1, suggesting that the membrane
permeabilization activity of NSAIDs is not involved in their
anti-inflammatory effect. Compounds 10a and 10b showed very
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Acknowledgment. This work was supported by Grants-in-
Aid of Scientific Research from the Ministry of Health,
Labour, and Welfare of Japan, Grants-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, and Grants-in-Aid of the
Japan Science and Technology Agency.
Supporting Information Available: Experimental details and
elemental analysis results. This material is available free of
charge via the Internet at http://pubs.acs.org.
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