Evaluation of loxoprofen and its alcohol metabolites for potency and selectivity of inhibition of cyclooxygenase-2

Article abstract of DOI:10.1016/j.bmcl.2003.12.047

Loxoprofen, its trans-alcohol and cis-alcohol metabolites were evaluated for selectivity of inhibition of COX-2 over COX-1. The (2S,1′R,2′S)- trans-alcohol derivative was found to be the most active metabolite and to be a potent and nonselective inhibitor of COX-2 and COX-1 in both enzyme and human whole blood assays.

Full text of DOI:10.1016/j.bmcl.2003.12.047

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1201–1203
Evaluation of loxoprofen and its alcohol metabolites for potency
and selectivity of inhibition of cyclooxygenase-2
Denis Riendeau,* Myriam Salem, Angela Styhler, Marc Ouellet,
Joseph A. Mancini and Chun Sing Li
Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Hwy, Kirkland, Quebec, Canada H9H 3L1
Received 20 October 2003; revised 11 December 2003; accepted 12 December 2003
Abstract—Loxoprofen, its trans-alcohol and cis-alcohol metabolites were evaluated for selectivity of inhibition of COX-2 over
COX-1. The (2S,10R,20S)-trans-alcohol derivative was found to be the most active metabolite and to be a potent and nonselective
inhibitor of COX-2 and COX-1 in both enzyme and human whole blood assays.
# 2004 Elsevier Ltd. All rights reserved.
Loxoprofen is a 2-arylpropionic acid anti-inflammatory
agent with analgesic and anti-pyretic properties. It has
been shown to be an effective inhibitor of the produc-
tion of inflammatory prostaglandins in vivo but only a
weak inhibitor of cyclooxygenase (COX) activity when
tested using enzyme and cell assays in vitro.1ꢀ3 Its inhi-
bitory effects on cyclooxygenase activity have been
attributed to its (2S,10R,20S)-trans-alcohol reduction
product (5a, see Scheme 1), the major metabolite of
loxoprofen detected after administration.1ꢀ6
Previous studies have shown that the metabolite 5a is
more potent at inhibiting COX activity in ram seminal
vesicle microsomes (a source of COX-1) than loxo-
profen.1 Limited data are available on selectivity. Inhi-
bition of COX-2 mediated PGE2 production by IL-1
stimulated human synovial cells (IC50=0.12 mM)
showed a 3-fold selectivity vs a COX-1 platelet assay for
5a, about 10-fold higher than indomethacin and 10-fold
lower than diclofenac.9 In the present study we have
prepared the four trans-alcohols and a mixture of the
cis-alcohol metabolites of loxoprofen and have eval-
uated them for their potency and selectivity of inhibi-
tion of human COX-2 over COX-1 using both enzyme
and whole blood assays.
Assessing the selectivity of loxoprofen as a cyclooxy-
genase inhibitor is a difficult task since the compound is
available as a mixture of 4 isomers, acts as pro-drug and
has a rather complex profile of metabolites. Loxoprofen
can be metabolized by stereoselective reduction, hydro-
xylation, chiral inversion, and conjugation to glucur-
onide derivatives.4ꢀ8 As described for several other
profens, the 2R propionic acid enantiomer of loxo-
profen undergoes rapid chiral inversion to 2S after oral
administration.7,8 A major route of metabolism for lox-
oprofen involves the stereoselective reduction of its
cyclopentanone moiety to generate the (2S,10R,20S)-
trans-alcohol derivative (5a). In addition to the trans-
alcohol metabolite, reduction of the cyclopentanone can
also yield the corresponding cis-alcohol (6), which has
been detected mainly with the 2S configuration at the
2-aryl propionic chiral centre.7,8
The four trans-cyclopentanol metabolites 5a–d and the
cis-cyclopentanol metabolite 6 were synthesized accord-
ing to Scheme 1.10 Loxoprofen 1 was derivatized with
(R)-pantolactone to give the ester intermediate 2, which
was reduced with sodium cyanoborohydride and sepa-
rated by flash column chromatography to yield the
trans-cyclopentanol 3 and the cis-cyclopentanol 4 in
aꢁ5:1 ratio. The four diastereomers of 3 were then
separated by chiral HPLC and the corresponding dia-
stereomeric intermediates were subsequently hydrolyzed
to provide the four trans-cyclopentanol metabolites 5a–
d.11 The cis-cyclopentanol 4 was hydrolyzed in a similar
manner to give the cis-cyclopentanol metabolite 6 as a
mixture of four diastereomers.
The structures of the various metabolites are provided
in Scheme 1 and were identified using the conventional
* Corresponding author. Tel.: +1-51-4428-2673; fax: +1-51-4428-
4930; e-mail: denis_riendeau@merck.com
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.047

1202
D. Riendeau et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1201–1203
Scheme 1. (a) (R)-Pantolactone, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfate (CMC), DMAP; (b) NaBH3CN, MeOH;
(c) HPLC separation on Prep Chiral Pak AD column using 30% EtOH in hexane; (d) 0.4 M Na2CO3 in 50% aq MeOH, then 2 M HCl in HOAc; or
2 M HCl in 50% aq 1,4-dioxane.
names described by Takasaki and Tanaka.8 The IC50
values for the inhibition of recombinant COX-1 and
COX-2 by loxoprofen and its alcohol metabolites are
the enzyme data, 5a was a potent inhibitor of COX-2
(IC50=0.3 mM) and showed no selectivity for the inhi-
bition of COX-2 over COX-1 (IC50=0.28 mM) (Table
2). As a comparison, the values for rofecoxib in these
assay are IC50=0.53ꢂ0.02 mM for COX-2 and
IC50=18.8ꢂ0.9 mM for COX-1.15
12
summarized in the Table 1. Indomethacin and MF-tri-
cyclic (a close analogue of rofecoxib13) were used as
comparators for nonselective and selective COX-2 inhi-
bitors. The (2S,10R,20S)-trans-alcohol derivative 5a was
found to be the most potent inhibitor and was equipo-
tent at inhibiting the enzymatic activities of COX-2
(IC50=0.39 mM) and of COX-1 (IC50=0.5 mM) assayed
under identical conditions. In contrast, the other trans-
alcohols (5a–d), or loxoprofen itself, were much weaker
or inactive as inhibitors of COX-2 with IC50>100 mM.
Similar data were obtained for COX-1, with a very
weak inhibition detected for the (2S,10S,20R)-trans-
alcohol (5b) (IC50=51 mM). The cis-alcohol isomers (6)
were not potent but gave an incomplete inhibition
(ꢁ50%) of COX-1 and COX-2 at high doses (<25 mM).
Loxoprofen and the other metabolites were also char-
acterized for their effect on COX-1 or COX-2 in human
whole blood assays. Although it is expected that the
enantiomeric composition of the tested compounds
might change during incubations in vitro,4,7,8,16 these
assays were performed to detect potential inhibitory
effects of loxoprofen and its metabolites in a more
physiologically relevant milieu with cells using endo-
genously generated arachidonic acid substrate for the
COX reaction. This assay would also detect alternative
mechanisms of inhibition (such as inhibition of PLA2).
In the whole blood assay, loxoprofen was a very weak
inhibitor of COX-2 (IC50 of 13.5ꢂ4.6 mM) and about
2-fold more potent against COX-1. Inhibitory effects on
COX-2 and COX-1 were also detected with other
metabolites (Table 2) but with a much lower potency
than 5a, possibly due to some isomerization to the most
active 5a metabolite during incubations with blood. The
chirality of the 2-aryl propionic moiety is clearly
important for the inhibitory effects on COX-2 or COX-
1, (as reported for other 2-arylpropionyl acids 16), with a
large decrease in potency for the corresponding 2R
enantiomers (5c,d). Considering their lower potency and
the fact that they represent more minor metabolites
than 5a in vivo, the other metabolites would not be
expected to contribute significantly to COX inhibition
at normal effective doses.
The selectivity of the (2S,10R,20S)-trans-alcohol deriva-
tive 5a, the most potent metabolite, was further char-
acterized using whole blood assays.14 In agreement with
Table 1. Inhibitory effects of loxoprofen and its alcohol metabolites
on the activities of recombinant human COX-1 and COX-2 (meanꢂSE)
COX-2
IC50 (mM)
COX-1
IC50 (mM)
MF-tricyclic
Indomethacin
0.23ꢂ0.04
0.73ꢂ0.07
>100
0.39ꢂ0.05
>100
0.11ꢂ0.02
>100
Loxoprofen (1)
(2S,10R,20S)-trans-alcohol (5a)
0.5ꢂ0.08
(2S,10S,20R)-trans-alcohol (5b) >100 (30% Inh.)
51ꢂ23
(2R,10R,20S)-trans-alcohol (5c)
(2R,10S,20R)-trans-alcohol (5d)
cis-alcohols
>100
>100
>25 (50% Inh.)
>100
>100
>25 (44% Inh.)
In conclusion, the (2S,10R,20S)-trans-alcohol reduction
product (5a) of loxoprofen is the most active major
(mixture of 4 isomers) (6)

D. Riendeau et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1201–1203
1203
Table 2. Potency of loxoprofen and its alcohol metabolites on COX-
1 and COX-2 in human whole blood assays (meanꢂSE)
7. Nagashima, H.; Tanaka, Y.; Watanabe, H.; Hayashi, R.;
Kawada, K. Chem. Pharm. Bull. (Tokyo) 1984, 32, 251.
8. Takasaki, W.; Tanaka, Y. Chirality 1992, 4, 308.
9. Kawai, S.; Nishida, S.; Kato, M.; Furumaya, Y.; Okamoto,
R.; Koshino, T.; Mizushima, Y. Eur. J. Pharmacol. 1998,
347, 87.
10. Synthesis of the eight possible metabolites of loxoprofen
has been reported in: Naruto, S.; Terada, A. Chem.
Pharm. Bull. 1983, 31, 4319.
COX-2
IC50 (mM)
COX-1
IC50 (mM)
MF-tricyclic
Loxoprofen (1)
0.48ꢂ0.07 (n=16) 15.3ꢂ2.2 (n=22)
13.5ꢂ4.6 (n=8)
6.5ꢂ1.3 (n=9)
0.28ꢂ0.14 (n=5)
4.7ꢂ1.4 (n=9)
20.2ꢂ4.3 (n=5)
>25
(2S,10R,20S)-trans-alcohol (5a) 0.30ꢂ0.07 (n=7)
(2S,10S,20R)-trans-alcohol (5b)
(2R,10R,20S)-trans-alcohol (5c)
(2R,10S,20R)-trans-alcohol (5d)
cis-alcohols
(mixture of 4 isomers) (6)
Indomethacin15
Rofecoxib15
2.0ꢂ1.3 (n=5)
49% at 30 mM
>33
11. trans-Cyclopentanol 5c contaminated with ꢁ5% of 5b
and trans-cyclopentanol 5d contaminated with ꢁ10% of
5c.
22ꢂ10( n=6)
1.9ꢂ0.3 (n=5)
12. Enzyme assays were performed in 50mM KPi pH 8.0, 1
mM heme, 1 mM phenol, with 10 mg/mL of COX-1 or
COX-2 microsomal fractions. Test compounds were
added as a 100-fold concentrated stock solution in DMSO
to 100 mL buffer. After a 15 min preincubation, the reac-
tion was initiated by the addition of 10 mL of 10 0mM
arachidonic acid. The enzyme reaction was allowed to
proceed for 5 min at room temperature before being
stopped by the addition of 10 mL 1 N HCl. PGE2 levels
were then determined by EIA. For details see: Percival,
M. D.; Ouellet, M.; Vincent, C. J.; Yergey, J. A.; Ken-
nedy, B. P.; O’Neill, G. P. Arch. Biochem. Biophys. 1994,
315, 111.
0.44ꢂ0.07
0.53ꢂ0.02
0.19ꢂ0.02
18.8ꢂ0.9
metabolite of loxoprofen with a potency of inhibition of
COX-2 comparable to that of indomethacin or of
potent selective COX-2 inhibitors. This result is con-
sistent with the high efficacy of loxoprofen as an anti-
inflammatory agent.1ꢀ3 However, 5a showed no selec-
tivity for the inhibition of COX-2 as compared to COX-
1 as assessed in both enzyme and whole blood assays.
Although the gastrointestinal tolerability of loxoprofen
has been shown to be superior to indomethacin,3,17 the
data on the nonselective inhibition are in agreement
with the ability of loxoprofen to cause gastric lesions
in animals at lower doses than selective COX-2
inhibitors.18,19
13. Oshima, M.; Dinchuk, J. E.; Kargman, S. L.; Oshima, H.;
Hancock, B.; Kwong, E.; Trzaskos, J. M.; Evans, J. F.;
Taketo, M. M. Cell 1996, 87, 803.
14. To study the inhibitory activities of these compounds on
the two isoforms of cyclooxygenase (COX-1 and COX-2),
human blood is either stimulated with lipopolysaccharide
(LPS) for 24 h to induce COX-2 or the blood is allowed to
clot spontaneously to activate COX-1. The production of
prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) are
measured by immunoassay at the end of the incubation as
readouts of COX-2 and COX-1 activity, respectively. For
details see: Brideau, C.; Kargman, S.; Liu, S.; Dallob,
A. L.; Ehrich, E. W.; Rodger, I. W.; Chan, C. C. Inflamm.
Res. 1996, 45, 68.
15. Riendeau, D.; Percival, M. D.; Brideau, C.; Charleson, S.;
Dube, D.; Ethier, D.; Falgueyret, J. P.; Friesen, R. W.;
Gordon, R.; Greig, G.; Guay, J.; Mancini, J.; Ouellet, M.;
Wong, E.; Xu, L.; Boyce, S.; Visco, D.; Girard, Y.; Prasit,
P.; Zamboni, R.; Rodger, I. W.; Gresser, M.; Ford-
Hutchinson, A. W.; Young, R. N.; Chan, C. C. J. Phar-
macol. Exp. Ther. 2001, 296, 558.
Acknowledgements
We thank C. K. Lau (Merck Frosst) for the chiral
HPLC separation of compound 3.
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