Novel Ester and acid derivatives of the 1,5-diarylpyrrole Scaffold as anti-inflammatory and analgesic agents. Synthesis and in vitro and in vivo biological evaluation

Article abstract of DOI:10.1021/jm901269y

A new generation of selective cyclooxygenase-2 (COX-2) inhibitors (coxibs) was developed to circumvent the major side effects of cyclooxygenase-1 (COX-1) and COX-2 inhibitors (stomach ulceration and nephrotoxicity). As a consequence, coxibs are extremely valuable in treating acute and chronic inflammatory conditions. However, the use of coxibs, such as rofecoxib (Vioxx), was discontinued because of the high risk of cardiovascular adverse events. More recent clinical findings highlighted how the cardiovascular toxicity of coxibs could be mitigated by an appropriate COX-1 versus COX-2 selectivity. We previously reported a set of substituted 1,5-diarylpyrrole derivatives, selective for COX-2. Here, we describe the synthesis of new1,5-diarylpyrroles along with their inhibitory effects in vitro, ex vivo, and in vivo toward COX isoenzymes and their analgesic activity. Isopropyl-2-methyl-5-[4- (methylsulfonyl)phenyl]-1-phenyl-1H-pyrrole-3-acetate (10a), a representative member of the series, was selected for pharmacokinetic and metabolic studies. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901269y

J. Med. Chem. 2010, 53, 723–733 723
DOI: 10.1021/jm901269y
Novel Ester and Acid Derivatives of the 1,5-Diarylpyrrole Scaffold as Anti-Inflammatory and
Analgesic Agents. Synthesis and in Vitro and in Vivo Biological Evaluation
Mariangela Biava,*,† Giulio C. Porretta,† Giovanna Poce,† Claudio Battilocchio,† Fabrizio Manetti,*,‡ Maurizio Botta,‡
Stefano Forli,‡,O Lidia Sautebin, Antonietta Rossi,^ Carlo Pergola, Carla Ghelardini,@ Nicoletta Galeotti,@
Francesco Makovec,# Antonio Giordani,# Paola Anzellotti,r Paola Patrignani,r and Maurizio Anzini‡,§
†
ꢀ
Dipartimento di Studi di Chimica e Tecnologie del Farmaco, Universita “La Sapienza”, piazzale Aldo Moro 5, I-00185 Roma, Italy,
‡
§
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Siena, via Alcide de Gasperi 2, I-53100 Siena, Italy, European Research
ꢀ
Centre for Drug Discovery and Development, via Banchi di Sotto 55, I-53100 Siena, Italy, Dipartimento di Farmacologia Sperimentale,
Universita di Napoli “Federico II”, via D. Montesano 49, I-80131 Napoli, Italy, ^IRCCS Centro Neurolesi “Bonino-Pulejo”, via Provinciale
ꢀ
Palermo, C. da Casazza, I-98124 Messina, Italy, @Dipartimento di Farmacologia, Universita di Firenze, viale G. Pieraccini 6, I-50139 Firenze,
ꢀ
Italy, #Rottapharm SpA, via Valosa di Sopra 7, I-20052 Monza, Italy, and rDepartment of Medicine and Center of Excellence on Aging,
“G. d’Annunzio” University and CeSI, Via dei Vestini 31, I-66013 Chieti, Italy. OPresent address: Department of Molecular Biology, MB-5,
The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037-1000.
Received August 25, 2009
A new generation of selective cyclooxygenase-2 (COX-2) inhibitors (coxibs) was developed to
circumvent the major side effects of cyclooxygenase-1 (COX-1) and COX-2 inhibitors (stomach
ulceration and nephrotoxicity). As a consequence, coxibs are extremely valuable in treating acute and
chronic inflammatory conditions. However, the use of coxibs, such as rofecoxib (Vioxx), was
discontinued because of the high risk of cardiovascular adverse events. More recent clinical findings
highlighted how the cardiovascular toxicity of coxibs could be mitigated by an appropriate COX-1
versus COX-2 selectivity. We previously reported a set of substituted 1,5-diarylpyrrole derivatives,
selective for COX-2. Here, we describe the synthesis of new 1,5-diarylpyrroles along with their inhibitory
effects in vitro, ex vivo, and in vivo toward COX isoenzymes and their analgesic activity. Isopropyl-
2-methyl-5-[4-(methylsulfonyl)phenyl]-1-phenyl-1H-pyrrole-3-acetate (10a), a representative member
of the series, was selected for pharmacokinetic and metabolic studies.
Introduction
European Medicines Agency (EMEA) has recommended
the withdrawal of the marketing authorizations for all lumira-
coxib-containing medicines, because of the risk of serious side
effects affecting the liver.7
Selective COX-2 inhibitors were developed to reduce gas-
trointestinal (GI)a side effects associated with the use of tradi-
tional nonsteroidal anti-inflammatory drugs (tNSAIDs).1,2
A reduced incidence of serious GI adverse effects compared
to tNSAIDs has been demonstrated for two highly selective
COX-2 inhibitors (namely, rofecoxib and lumiracoxib) in large
randomized clinical trials.3,4 This was a proof of concept that
sparing COX-1 in the GI tract and possibly in platelets
translates into a safer GI profile.5 Some coxibs, such as
rofecoxib and valdecoxib, were withdrawn from the market
because of an increased incidence of thrombotic events
presumably associated with selective inhibition of COX-2-
dependent generation of prostacyclin in endothelial cells, with-
out the concomitant complete suppression of COX-1-dependent
platelet thromboxane (TX) A2 generation.6 Furthermore, the
In vitro selectivity toward COX-2 is described as the ratio
of the concentration required to inhibit the activity of
the isoenzymes by 50% (IC50 toward COX-1/IC50 toward
COX-2).8 Selectivity at the circulating drug concentration
impacts both efficacy and side effects. It was shown that,
among tNSAIDs, there is a cluster of compounds, such as
etodolac9 (2a), meloxicam10 (3), nimesulide11 (4), and diclo-
fenac12 (5) (Chart 1), that are 5-29-fold more potent in vitro
toward COX-2, in comparison to COX-1. Among coxibs,
rofecoxib13 (1a), celecoxib14 (1b), valdecoxib15 (1c), etoricoxib16
(1d), and lumiracoxib17 (1e) (Chart 1) showed a wide range
of COX-1/COX-2 IC50 ratio values (from 30 for 1b to 433
for 1e).
In the in vitro human whole blood (HWB) assay, the
biochemical selectivity of COX inhibitors is a continuous
variable.18,19 It is worth mentioning that 80% ex vivo inhibi-
tion of COX-2 (in whole blood) by circulating drug concen-
trations seems to be sufficient to translate into efficacy
(analgesia).20
Moreover, a high degree of COX-2 inhibition seems to be
associated with an increased incidence of cardiovascular
events (with myocardial infarction that exceeds over stroke),
and such an effect could be mitigated by an appropriate
*To whom correspondence should be addressed. M. Biava: phone,
þ39 06 49913812; fax, þ39 06 49913133; e-mail, mariangela.biava@
uniroma1.it. F. Manetti: phone, þ39 0577 234330; fax, þ39 0577
234333; e-mail, manettif@unisi.it.
a Abbreviations: NSAIDs, nonsteroidal anti-inflammatory drugs;
COX, cyclooxygenase; AA, arachidonic acid; PGH2, prostaglandin
H2; PGE2, prostaglandin E2; HWB, human whole blood; tNSAIDs,
traditional nonsteroidal anti-inflammatory drugs; GI, gastrointestinal;
CV, cardiovascular; DMEM, Dulbecco’s modified Eagle’s medium;
FBS, fetal bovine serum; LPS, lipopolysaccharide; RIA, radioimmu-
noassay; TX, thromboxane; MPE, maximum percent effect; CMC,
carboxymethylcellulose; MSMS, maximal speed molecular surface.
r
2009 American Chemical Society
Published on Web 12/03/2009
pubs.acs.org/jmc

724 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Biava et al.
Chart 1. Structure of NSAIDs, Including Coxib Derivatives, Traditional NSAIDs, and Previously Described Pyrrole Compounds
inhibition of plateletCOX-1 activity(i.e., >95%, necessary to
inhibit platelet function).6,18 Accordingly, the fact that most
tNSAIDs and coxibs are selective in vivo for COX-2 at
therapeutic doses could account for their thrombogenic
effects.21,22 Conversely, an incomplete inhibition of COX-2
in the kidney might attenuate the deleterious functional effect
derived from the lack of COX-2-dependent prostacyclin.6,22
Thus, selective and profound inhibition of COX-2 by coxibs
may have a greater propensity to increase side effects than
balanced inhibition of both COX-1 and COX-2 by tNSAIDs.
In addition, the approach to identifying biochemical and/or
genetic biomarkers to select the patients who are most sus-
ceptibletoa hazardorbenefit by selectiveinhibition of COX-2
should be taken into account as well.5,9 In fact, a marked
intersubject variability in COX-2 inhibition by NSAIDs, in
part due to a genetic background, has been shown.10,11
Despite the fact that several NSAIDs with suitable phar-
macodynamic features are available (e.g., 3-5), most of them
have short half-life, which leads to their administration
at high doses (often overshooting) to extend the pharmaco-
dynamic effects, and in addition, some of them displayed
hepatic toxicity (i.e., 5).
the “coxib” family 1a-e cited above and of indomethacin
(2c), respectively. A structure-activity relationship (SAR)
analysis of such compounds, supported by molecular docking
simulations of these inhibitors within the COX-2 binding
site,23-26 allowed us to rule out several considerations.
(i) The position of the p-methylsulfonyl substituent was very
important for activity (compounds 6 being more active than
7). (ii) Surprisingly, the acetic ester chain at C3 of the pyrrole
ring led to compounds more active than the corresponding
acids.23,24,26 (iii) Substituents and the substitution pattern
on the phenyl ring at N1 influenced activity in the follow-
ing order: 3-F = 4-F > 3,4-diF > 4-OMe > H > 4-CF3 >
4-CH3.23-27
In this paper, we report the synthesis and biological activity
of new derivatives 10a-h (Scheme 1 and Table 1), in which
longer side chains were introduced at C3 on the basis of the
hypothesis that such chains could better interact with hydro-
phobicresidues of the carboxylate pocket of the receptor.23-26
In particular, compounds 10a-h, bearing an isopropyl and an
n-butyl ester function, a N1 phenyl ring with various substi-
tuents previously found to be important for activity (namely,
3-F, 3,4-diF, and 4-OCH3), and a C5 p-methylsulfonyl phenyl
moiety, showed outstanding activity. The new compounds
were evaluated in vitro for their ability to inhibit COX-1
and COX-2 in a J774 murine macrophage cell line, and bio-
chemical data were rationalized through docking simulations.
In addition, their inhibitory effects on platelet COX-1 and
monocyte COX-2 activity were compared in an in vitro HWB
assay.18,19 Their in vivo anti-inflammatory and analgesic
We have previously reported design, synthesis, and anti-
inflammatoryproperties of a class of novel pyrrole-containing
anti-inflammatory agents.23-27 In particular, we focused our
attention on the synthesis of 1,5-diarylpyrrole-3-acetic acids,
esters, R-hydroxy esters, and ethers 6-9 (Chart 1) as new
COX-2 selective inhibitors in which the pyrroleacetic and
vicinal diaryl heterocyclic moieties were reminiscent both of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 725
Scheme 1a
a Compounds: 10a, R = H, R1 = isopropyl, 55% yield; 10b, R = H, R1 = n-butyl, 50% yield; 10c, R = 3-F, R1 = isopropyl, 96% yield; 10d, R =
3-F, R1 = n-butyl, 40% yield; 10e, R = 3,4-diF, R1 = isopropyl, 45% yield; 10f, R = 3,4-diF, R1 = n-butyl, 40% yield; 10g, R = 4-OCH3, R1 =
isopropyl, 40% yield; 10h, R = 4-OCH3, R1 = n-butyl, 40% yield; 6a, R = H, 71% yield; 11c, R = 3-F, 60% yield; 11e, R = 3,4-diF, 75% yield; 11g,
R = 4-OCH3, 70% yield; 14a, R = H, 80% yield; 14c, R = 3-F, 80% yield; 14e, R = 3,4-diF, 74% yield; 14g, R = 4-OCH3, 70% yield; 15a, R = H,
R1 = isopropyl, 76% yield; 15b, R = H, R1 = n-butyl, 90% yield; 15c, R = 3-F, R1 = isopropyl, 40% yield; 15d, R = 3-F, R1 = n-butyl, 55% yield; 15e,
R = 3,4-diF, R1 = isopropyl, 77% yield; 15f, R = 3,4-diF, R1 = n-butyl, 65% yield; 15g, R = 4-OCH3, R1 = isopropyl, 40% yield; 15h, R = 4-OCH3,
R1 = n-butyl, 60% yield; 16a, R = H, 76% yield; 16c, R = 3-F, 57% yield; 16e, R = 3,4-diF, 77% yield; 16g, R = 4-OCH3, 75% yield; 17a, R = H, 71%
yield; 17c, R = 3-F, 45% yield; 17e, R = 3,4-diF, 60% yield; 17g, R = 4-OCH3, 30% yield. Reagents and conditions: (i) CH2dCHCOMe, TEA, 3-ethyl-
5-(2-hydroxyethyl)-4-methylthiazolium bromide, MW, 15 min; (ii) Oxone, MeOH/H2O, room temperature, 2 h; (iii) RPhNH2, p-toluenesulfonic acid,
EtOH, MW, 45 min; (iv) EtOCOCOCl, TiCl4, CH2Cl2, room temperature, 4 h; (v) Et3SiH, TFA, room temperature, 2 h; (vi) 1 N NaOH, MeOH, 1 h;
(vii) ClCOCOCl, 2,6-lutidine, CH2Cl2, R1OH; (viii) Et3SiH, TFA, room temperature, 2 h.
Table 1. In Vitro Inhibition (J774 murine macrophage assay) of COX-1
and COX-2 by Compounds 6a,d, 10a-h, and 11c,e,g, and Celecoxib
derivatives could give rise in vivo to the formation of corres-
ponding acids, the anti-inflammatory and analgesic activities of
COX-2
inhibition (%)
acid derivatives 6a and 11c,e,g were also evaluated, using the
same protocols applied for the corresponding esters.
IC50 (μM)a
selectivity
indexb
compd
10a
10b
COX-1
COX-2
10 μM
1 μM
Chemistry
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10
0.0073
0.014
0.043
0.024
0.021
0.030
0.022
0.038
1.0
100
88
100
85
85
87
85
86
88
89
43
89
70
62
79
80
>13600
>7100
>2300
>4100
>4700
>3300
>4500
>2600
>100
>3500
38
The synthesis of the target compounds is described in
Scheme 1. Briefly, the reaction of 4-(methylthio)benzaldehyde
with methyl vinyl ketone for 15 min using the micro-
wave apparatus gave the intermediate 12,28 which was trans-
formed into the corresponding 4-methylsulfonyl derivative
13 by means of Oxone oxidation.29 Next, following the
Paal-Knoor condensation in the presence of the appropriate
arylamine (45 min by using the microwave apparatus),28 13
cyclized to yield the expected 1,5-diarylpyrroles 14a,c,e,g. The
construction of the C3 side chain was achieved by regioselec-
tive acylation of 14a,c,e,g with oxalyl chloride in the presence
of 2,6-lutidine to give corresponding R-keto acid chlorides
that, by the successive reaction in the presence of the proper
alcohol, gave the corresponding R-keto ester derivatives
15a-h in a one-pot reaction. Compounds 15a-h were in turn
reduced by means of triethylsilane in trifluoroacetic acid to
give the pyrrole esters 10a-h.30 Construction of the C3 acetic
acid chain of 6a and 11c,e,g was achieved by regioselective
acylation of 14a,c,e,g with ethoxalyl chloride in the presence
of titanium tetrachloride to give corresponding R-keto esters
16a,c,e,g in turn reduced (triethylsilane in trifluoroacetic acid)
10c
10d
91
100
90
10e
10f
90
10g
10h
6ac
100
92
100
96
11c
11e
0.028
0.26
0.17
98
90
11g
6dc
celecoxib, 1b
>100
>100
>580
>2500
65
0.04
0.079
84
95
5.1
a Results are expressed as the mean (n = 3) of the % inhibition of
PGE2 production by test compounds with respect to control samples.
b In vitro COX-2 selectivity index [IC50(COX-1)/IC50(COX-2)]. c See ref 23.
activity was also evaluated in several animal models. A repre-
sentative member of the series (10a) was selected to perform
pharmacokinetic studies. After both oral and intravenous
administrations of 10a, the formation of the corresponding
acid 6a was observed. Accordingly, considering that the ester

726 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Biava et al.
Figure 1. Graphical representation of the binding mode of 10a within the COX-2 binding site (A), in comparison to the cocrystallized inhibitor
SC-558 (B) (SC-558, red; 10a, yellow). For the sake of clarity, only the most important residues flanking the binding site are shown as thin lines.
Images have been generated by means of ADT version 1.5.1.
to the pyrrole acetic esters 17a,c,e,g that led to the correspond-
ing acids 6a and 11a,c,e,g by treatment with 1 N NaOH in
MeOH for 1 h.
analogues (Figure 1B). Moreover, following a trendin activity
already found, ester derivatives 10a-h were more active than
the corresponding acid analogues (with the exception of 11c),
because of additional hydrophobic interactions between the
terminal alkyl chain and residues of the carboxylate site
(Leu93, Val116, Tyr355, and Leu359). GRID calculations
with the C3 probe seemed to suggest that a two-carbon atom
group was the optimal substituent to be inserted into the ester
chain, although additional space for longer alkyl groups was
available in the region where the ester alkyl chain is accom-
modated. Inspection of the binding mode of the new deriva-
tives showed that both the isopropyl and the butyl chains
are able to satisfy this geometrical constraint. In fact, the
isopropyl moiety, having the same length of an ethyl group
although larger in size, matched the GRID minimum point of
the C3 probe. On the other hand, the butyl group assumed a
folded conformation, leading its carbon atoms to be located
around the C3 best interaction point found by GRID, and
thus making similar hydrophobic contacts with enzyme
residues. Moreover, both ester and acid derivatives were also
able to interact by hydrogen bonds with the guanidino moiety
of Arg120.
In vitro results and a SAR analysis of acids 6a and 11c,e,
g, synthesized and tested on the basis of the hypothesis that
hydrolysis of the ester moiety can occur in vivo to at least
some extent, confirmed the dependence of the activity
from the N1 phenyl ring substitution pattern (Table 1).
Moreover, ester derivatives 10a-h proved to be more active
than the corresponding acids, exhibiting IC50 values from
4.5- to 143-fold better (compare 10a,b vs 6a, 10e,f vs 11e,
and 10g,h vs 11g). The sole exception to this trend was
represented by 3-F derivatives that exhibited comparable
activity (compare 10c,d vs 11c). Esters also had a higher
COX-1/COX-2 selectivity in comparison to the acids.
Finally, all the esters 10a-h were found to be more effective
in inhibiting COX-2 than their ethyl analogues previously
described (as an example, compare 10a and 10b with 6d)
(Chart 1)23,24 or celecoxib while maintaining or even
increasing COX-1/COX-2 in vitro selectivity.
Results and Discussion
Previous molecular docking simulations, in combination
with GRID calculations, led us to hypothesize the binding
mode of 1,5-diarylpyrrole derivatives within the COX-2 bind-
ing site and to identify the interactions between ligands and
the protein that allowed us to account for the major SARs.
In particular, the docked orientation of pyrroles showed
the N1 phenyl ring, the C3 side chain, and the C5 phenyl ring
embedded in the hydrophobic pocket, the carboxylate site,
and the selectivity site of the enzyme, respectively (Figure 1A),
in correspondence with the bromophenyl moiety, the triflu-
oromethyl group, and the N1 phenyl ring of the cocrystallized
inhibitor SC-558, respectively (Figure 1B). The structure
of SC-558 is depicted in Chart 1. Moreover, the identification
of points of best interaction between different GRID probes
and COX-2 residues provided suggestions for appropriately
decorating positions 1 and 3 of the pyrrole nucleus. Accord-
ingly, the insertion of various substituents at meta and
para positions of the N1 phenyl ring yielded derivatives with
high affinity for (in the range of two-digit nanomolar con-
centrations) and selective toward (up to 10000) COX-2.23,24,26
On the other hand, inspection of the binding mode of pyrrole
derivatives bearing an acetic acid ethyl ester moiety at C3 led
to the suggestion that its terminal alkyl moiety is very
important for hydrophobic interactions with Val116 and
Tyr355 and contributes to the strength of the complex with
COX-2.23,24,26 To further support this hypothesis, the new
pyrrole derivatives 10a-h were designed and synthesized,
keeping fixed the SO2Me group at the C5 phenyl ring and
several substituents at the N1 phenyl ring (previously found to
be key determinants of potency and COX-2 selectivity), while
the size and length of the ester chain at C3 were modified by
insertion of an isopropyl or butyl moiety. As expected,
resulting compounds showed very strong inhibition of
COX-2, with IC50 values ranging from 0.007 [10a (Table 1)]
to 0.043 μM (10c) and a selectivity index of >2300. Docking
calculations showed that the new compounds bind the COX-2
binding site with the same orientation of their previous
In summary, analysis of the influence of substituents and
substitution pattern on the activity and selectivity toward
COX-2 showed that current substituents at positions N1
and C3 are optimal cooperative determinants for potent and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 727
selective inhibitors and should be considered as structural
elements for fine-tuning of affinity toward the enzyme.
Pharmacological Evaluation. Among the most interesting
compounds found in the cell-based assay, 10a and 10c,
as well as the corresponding acid derivatives, 6a and 11c,
were selected for evaluation of the inhibitory effects toward
the HWB assay for COX-1 and COX-2. Results indicated
a dramatic change in terms of COX selectivity under
these experimental conditions (Table 2), since celecoxib
remained as selective as 6a (selectivity indexes of 23 and 19,
respectively), while the remaining compounds, 10a, 10c, and
11c, showed a selectivity index of <12. The discrepancy
between these results and those found in the cell-based
assay could be due to a different inhibitor sensitivity exhi-
bited by the mouse and human COX isozymes, as already
reported for other anti-inflammatory compounds.10 As a
matter of the fact, such a dramatic loss in COX inhibitory
potency and a drop in selectivity under the HWB assay
conditions are common to other selective COX-2 inhibitors,
such as valdecoxib,31,32 etoricoxib,31,32 lumiracoxib,33 and
rofecoxib,34,35 as well as tNSAIDs such as nimesulide16,31
and meloxicam.36,37
It should be pointed out that, according to the test, 10a,
10c, and 11c exhibited an affinity for COX-2 5-10-fold
higher than that for COX-1, which should translate clinically
into an acceptable GI safety, and allowing for a sufficient
prostacyclin generation that should mitigate the CV effects
showed by overly selective COX-2 inhibitors, as previously
discussed.
Table 2. Ex Vivo Inhibition (HWB) of COX-1 and COX-2 by Com-
pounds 10a,c, 6a, and 11c and Celecoxib
To assess in vivo anti-inflammatory and analgesic activity
of 10a-h, 6a, and 11c,e,g, the rat paw pressure test and the
paw volume and abdominal constriction tests were per-
formed (Tables 3-5, respectively). Compounds, adminis-
tered at different dosages (from 10 to 20 mg/kg po), showed a
very good activity against carrageenan-induced hyperalge-
sia. The activity of 10a-h was better than that of both the
corresponding acids and ethyl esters.23,24 Esters also proved
IC50 (μM)
compd
10a
10c
COX-1
COX-2
selectivity index
20
24
2.5
2
8
12
19
7
6a
11c
132
125
12.5
7
17
celecoxib, 1b
0.54
23
Table 3. Effect of Compounds 6a, 10a-h, and 11c,e,g and Celecoxib in the Rat Paw Pressure Test
paw pressure (g)
pretreatment
treatment
saline
before pretreatment
30 min after treatment
60 min after treatment
120 min after treatment
saline
63.4 ( 4.6
61.9 ( 5.1
60.8 ( 4.3
57.5 ( 3.7
63.2 ( 3.8
62.4 ( 3.2
59.5 ( 2.9
58.3 ( 3.6
58.6 ( 4.1
60.5 ( 3.0
63.1 ( 2.9
62.7 ( 4.2
59.2 ( 3.4
61.2 ( 3.6
62.7 ( 3.9
60.2 ( 5.1
38.7 ( 5.3
56.2 ( 4.8
52.5 ( 4.9
59.5 ( 5.4
49.7 ( 4.1
64.8 ( 3.8
52.7 ( 5.0
51.0 ( 5.5
53.4 ( 4.8
48.6 ( 4.3
54.8 ( 3.7
58.3 ( 3.4
52.3 ( 3.7
56.5 ( 3.8
62.9 ( 4.9
35.8 ( 4.5
51.8 ( 5.3
46.3 ( 5.3
57.9 ( 4.1
46.9 ( 5.5
52.3 ( 4.9
59.2 ( 6.3
56.7 ( 4.4
51.4 ( 5.0
51.8 ( 3.6
55.2 ( 3.8
54.1 ( 4.4
39.8 ( 4.1
58.2 ( 4.4
60.7 ( 5.4
40.1 ( 5.0
48.8 ( 3.7
42.6 ( 3.8
45.2 ( 4.3
45.2 ( 4.8
58.3 ( 3.4
56.6 ( 4.5
41.7 ( 4.1
50.8 ( 5.2
41.6 ( 3.7
43.1 ( 3.9
48.6 ( 3.4
36.2 ( 3.2
55.2 ( 5.1
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
saline
10aa
10bb
10cb
10db
10eb
10fb
10gb
10hb
6ab
11cb
11eb
11gb
celecoxib, 1ba
a Compounds were administered po at a dose of 10 mg/kg. b Compounds were administered po at a dose of 20 mg/kg.
Table 4. Effect of Compounds 6a, 10a-h, and 11c,e,g in Comparison to Celecoxib in the Edema Induced by Carrageenan
paw volume (mL)
pretreatment
treatment
saline
before pretreatment
60 min after treatment
% of paw edema suppression
saline
1.26 ( 0.09
1.22 ( 0.08
1.29 ( 0.09
1.31 ( 0.10
1.17 ( 0.11
1.35 ( 0.09
1.29 ( 0.10
1.23 ( 0.08
1.16 ( 0.07
1.33 ( 0.08
1.23 ( 0.10
1.28 ( 0.05
1.32 ( 0.10
1.27 ( 0.08
1.28 ( 0.14
1.31 ( 0.10
2.33 ( 0.08
1.26 ( 0.12
1.68 ( 0.15
1.31 ( 0.09
1.71 ( 0.11
1.39 ( 0.12
1.52 ( 0.09
1.41 ( 0.08
1.45 ( 0.14
1.86 ( 0.07
1.35 ( 0.09
1.42 ( 0.09
1.81 ( 0.12
1.33 ( 0.10
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
saline
10aa
100
10bb
66.3
87.2
67.2
89.0
73.6
77.2
89.1
42.7
93.6
90.9
50.9
95.5
10cb
10db
10eb
10fb
10gb
10hb
6ab
11cb
11eb
11gb
celecoxib, 1ba
a Compounds were administered po at a dose of 10 mg/kg. b Compounds were administered po at a dose of 20 mg/kg.

728 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Biava et al.
to be very effective and long-lasting, in this test a relevant
analgesia being still present 2 h after administration.
The most active compounds were 10e, 10f, and 10h
(Table 3). Conversely, the corresponding acids 6a and 11c,
e,g had a remarkably lower activity (especially after 2 h),
except for 11e whose activity was comparable to that of
10a. Moreover, a very good activity was found toward
carrageenan-induced edema in the rat paw (Table 4), with
a complete remission 1 h after administration (20 mg/kg po).
Among the test compounds, 10a, 10c, and 11c showed the best
activity. Finally, a dose-dependent antinociceptive activity was
also observed in the abdominal constriction test (Table 5), 10f,
10h, 11c, and 11e exhibiting remarkable activity.
The metabolic stability of the compounds was also evalu-
ated using human liver microsomes (Table 6). Acids 6a and
11c,e had high stability, thus indicating that the 1,5-diaryl-
pyrrole scaffold is not liable to metabolic attack, even with
an unsubstituted N1 phenyl ring as in 6a. The lower stability
of 11g suggests that the 4-methoxy group can give rise to
some metabolic liability. Conversely, esters 10a-h were
liable to hepatic metabolism at concentrations of both 10-5
and 10-7 M, thus suggesting hydrolysis at the ester moiety as
the major metabolic pathway for these compounds, even
when hindered esters (such as isopropyl) are taken into
account.
Compound 10a was then selected for further studies,
because of the low activity of its acid analogue 6a. This
choice was made to avoid issues caused by a possible in vivo
metabolism to an overly active metabolite, as in the case of
11c and 11e. Compound 10a was evaluated in comparison to
celecoxib in the Randall-Selitto model of zymosan-induced
hyperalgesia38 in rats. Compounds were administered orally
as a 0.5% suspension in methocel at doses of 1, 3, and
5 mg/kg. Table 7 reports results of a 3 mg/kg dose adminis-
tration, expressed as the maximum percentage effect (MPE).
In this model, ED50 values for 10a and celecoxib were 2.2
and 9.0 mg/kg, respectively. Accordingly, 10a was more
potent than celecoxib and was endowed with a longer dura-
tion of action. To exclude the possibility that these results
were due to any impairment effect, 10a was finally evaluated
in the rota-rod test. Mice treated with test compound at a
dose of 25 mg/kg po did not exhibit a larger number of falls
from the rotating rod in comparison with controls (Table 8).
Therefore, 10a did not cause any alteration in mouse motor
coordination.
Pharmacokinetic Study. The pharmacokinetics of 10a was
assessed in rats after po and iv administration of the com-
pound. Profiles of the plasma concentration versus time after
po and iv administration of 10a (10 mg/kg) are reported in
Figure 2. Compound 10a was sufficiently absorbed, its
absolute bioavailability being 42% (F%). The clearance
after iv treatment (∼2 L/h) was higher than the rate of blood
flow in the rat (0.8 L/h),32 thus indicating that 10a is a high-
clearance drug. The volume of distribution after iv treatment
(∼5.6 L) was higher than the total volume of body fluid in the
rat (0.17 L),32 indicating extensive distribution of 10a into
extravascular compartments. When compared to the clear-
ance data, this result could explain the steady state observed
in the plasma concentration for up to 5 h. The calculated
Table 5. Effect of Compounds 6a, 10a-h, and 11c,e,g in Comparison to
Celecoxib in the Mouse Abdominal Constriction Test (0.6% acetic acid)
treatmenta
no. of mice
dose (mg/kg po)
no. or writhes
CMCb
10a
10a
10a
10b
10c
20
8
31.4 ( 4.3
33.6 ( 3.0
28.5 ( 3.3c
25.5 ( 2.8c
23.9 ( 3.4c
39.5 ( 3.8
17.7 ( 2.9c
24.4 ( 4.1c
35.1 ( 4.6
21.5 ( 3.9c
19.8 ( 3.2c
34.4 ( 3.5
13.7 ( 4.0c
42.2 ( 4.0
18.0 ( 3.5c
21.6 ( 2.9c
15.9 ( 3.3c
27.3 ( 3.2
22.2 ( 2.9
31.6 ( 3.2
17.5 ( 3.1
29.3 ( 3.0
15.7 ( 2.6
30.9 ( 3.3
21.8 ( 2.1
14.2 ( 2.3
1
5
8
8
15
20
5
8
12
9
10c
20
20
5
10d
10e
8
10
12
10
12
30
11
12
8
10e
10e
20
40
5
10f
10f
20
5
10g
10g
10h
10h
6a
20
10
20
10
20
10
20
10
20
10
20
10
10
11
10
9
11a
11c
11c
11e
8
12
10
10
10
12
11e
11g
11g
celecoxib, 1b
a All the compounds were administered po 30 min before the test.
b CMC is carboxymethylcellulose. c p < 0.01 vs vehicle-treated mice.
Table 6. Metabolic Stability of Compounds 6a, 10a-h, and 11c,e,g
mean parent
remaining (%)
mean parent
remaining (%)
compd
(test concentration, 10 μM)
(test concentration, 0.1 μM)
6a
97
100
100
84
1
94
100
100
77
11
7
11c
11e
11g
10a
10b
10c
10d
10e
10f
10g
10h
3
1
0
2
0
13
15
1
0
2
7
2
5
Table 7. Analgesic Activity of 10a and Celecoxib in the Randall-Selitto
Model of Zymosan-Induced Hyperalgesia in Rats
analgesia for
0-2 h MPE (%)
analgesia for
0-6 h MPE (%)
compd
10a
celecoxib, 1b
75
30
70
35
Table 8. Lack of an Effect of 10a on Mice in the Rota-Rod Testa
treatment dose (mg/kg po) before treatment 15 min after treatment 30 min after treatment 45 min after treatment 60 min after treatment
CMC
10a
4.2 ( 0.4
4.2 ( 0.5
2.9 ( 0.3
2.8 ( 0.4
1.7 ( 0.4
2.7 ( 0.5
0.9 ( 0.2
1.1 ( 0.3
0.7 ( 0.2
0.8 ( 0.2
25
a Each value represents the mean of values from eight mice. The test was performed 30 min after treatment.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 729
Figure 2. Profile of plasma concentration vs time after iv and po
administration of 10a (10 mg/kg).
Figure 4. Plasmatic concentrations vs time of 10a compared to the
corresponding plasmatic concentrations of 6a after oral adminis-
tration of 10a (10 mg/kg).
on the presence of ester 10a, assuming a similar protein
binding for both 10a and 6a. Beginning 6 h after adminis-
tration, the contribution of acid 6a to the residual activity
could become relevant.
Conclusions
On the basis of previously published data and suggestions
derived from both SAR analysis and molecular modeling
simulations on 1,5-diarylpyrrole derivatives, a new series of
compounds has been designed and synthesized. Biological
data showed a significant enhancement of their biological
activity with respect to the parent compounds. One of the new
entries (namely, 10a) was selected for pharmacokinetic and
pharmacodynamic studies. It was approximately 8-fold more
potent toward COX-2 than COX-1 in the HWB assay, and it
was characterized by a high volume of distribution, suggesting
its accumulation in target sites which may explain its long-
lasting pharmacological activity. This pharmacokinetic fea-
ture (i.e., high volume of distribution) might translate into fast
clearance from side effect compartments (including vascular
wall, heart, and kidney).31 This possibility will be explored in
further studies.
Figure 3. Concentrations (nanograms per milliliter) of 6a vs time
after iv and po administration of 10a (10 mg/kg).
concentration of the drug within the 1-5 h time frame
indicates a concentration of at least 240 nM, significantly
higher than the COX-2 IC50 of 10a. These pharmacokinetic
data fit well with the observed long-lasting pharmacological
activity in the rat. In addition, the plasmatic levels of the
expected metabolite 6a were evaluated to gain additional
information about impacts exerted by this possible meta-
bolic pathway on the overall pharmacological profile. Pro-
files of the plasma concentration versus time for 6a after
iv and po administration of 10a (10 mg/kg) are reported in
Figure 3. After both oral and iv administration of 10a, the
formation of 6a was observed, thus confirming hydrolysis of
the ester to the corresponding acid. The fact that the con-
centration of 6a reached the same level in a manner inde-
pendent of the administration route of 10a suggested that
10a was not largely hydrolyzed in the GI tract during the
absorption but rather was formed by either plasmatic or
hepatic metabolism due to esterase activity. Plasmatic
concentrations versus time of 10a and 6a are compared in
Figure 4. The circulating concentration of acid 6a is 1 order
of magnitude higher than that of ester 10a, within the 2-5 h
time frame. However, since the difference of the in vitro
potency between 10a and 6a is 3 orders of magnitude, in vivo
activity observed during the first 5 h should mostly depend
The remarkable activity of these new compounds, as well as
their pharmacokinetic profile, is meaningful for the develop-
ment of new compounds that could be effective and safe
agents for the pharmacological management of inflammation
and pain.
Experimental Section
Chemistry. A Discovery Microwave System apparatus (from
CEM) was used for the Stetter and Paal-Knorr reactions. All
chemicals used were of reagent grade. Yields refer to purified
products and are not optimized. Melting points were determined
in open capillaries on a Gallenkamp apparatus and are uncor-
rected. Microanalyses were conducted on a Perkin-Elmer 240C
or a Perkin-Elmer Series II CHNS/O Analyzer 2400 instrument.
Fluka silica gel 60 (230-400 mesh) was used for column
chromatography. Fluka TLC plates (silica gel 60 F254) were
used for thin-layer chromatography (TLC). Fluka aluminum
oxide (activity II-III, according to Brockmann) was used for
chromatographic purifications. Fluka Stratocrom aluminum
oxide plates with a fluorescent indicator were used for TLC to
1
check the purity of the compounds. 13C NMR and H NMR
spectra were recorded with a Bruker AC 400 spectrometer in the

730 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Biava et al.
indicated solvent (TMS as the internal standard). The values of
the chemical shifts are expressed in parts per million and the
coupling constants (J) in hertz.
dried, and evaporated in vacuo. Purification of the residue by
flash chromatography with ethyl acetate as the eluant gave a solid
which, after recrystallization from hexane, afforded the expected
products 15a-h.
HPLC Analysis. Analyses were conducted using a Waters
Alliance 2695 instrument, using a UV-vis Waters PDA 996
detector and working at 333 nm. Millennium Empower with
Windows XP was used. A Phenomenex LUNA C8, 5 μm
(150 mm ꢀ 4.6 mm) column (code 00F-4249-E0), at 40 ꢀC,
was used as the chromatographic column at a flow rate of
1.0 mL/min. The eluant was (A) a 65/15/20 (v/v/v) 10 mM
(pH 2.5) KH2PO4/MeOH/acetonitrile mixture or (B) a 15/75/10
(v/v/v) 10 mM (pH 2.5) KH2PO4/MeOH/acetonitrile mixture. A
gradient from 100% A to 100% B was run in 25 min, and then
isocratic conditions at 100% B were maintained for an addi-
tional 10 min. Using the analytical conditions reported above,
the retention times of the compounds are 19.8 min. All the
synthesized compounds were g95% pure.
Isopropyl-2-methyl-5-[4-(methylsulfonyl)phenyl]-1-phenyl-
1H-pyrrole-3-glyoxylate (15a): yellowish needles (76% yield);
1
mp 60 ꢀC; H NMR (CDCl3) δ 7.74-7.78 (m, 2H), 7.23-7.26
(m, 3H), 7.04-7.05 (m, 3H), 5.26 (m, 1H), 2.94-3.00 (s, 3H),
2.42- 2.46 (s, 3H), 1.33-1.41 (d, 6H).
General Procedure for the Preparation of 1,5-Diarylpyrrole-
3-acetic Esters 10a-h. To a solution of the suitable R-keto ester
[15a-h (2.3 mmol)] in TFA (9 mL, 0.12 mol) stirred at 0 ꢀC
under a nitrogen flow was slowly added triethylsilane (0.75 mL,
4.7 mmol), and the mixture was stirred for 2 h at room
temperature. At the end of the reaction, the mixture was made
alkaline with 40% aqueous ammonia (10 mL) and extracted
with CHCl3. The organic solution was dried and evaporated in
vacuo. The resulting residue was chromatographed on silica gel
eluting with CHCl3 to give a solid which, after recrystallization
from hexane, afforded the desired products 10a-h.
Isopropyl-2-methyl-5-[4-(methylsulfonyl)phenyl]-1-phenyl-
1H-pyrrole-3-acetate (10a). Yellowish needles (yield 55%). Ana-
lytical data, the melting point, and the 1H NMR spectrum were
consistent with those reported in the literature:27 13C NMR
δ 11.00 (CH3 pyrrolic), 21.82 (CH3 isopropyl), 32.62 (CH2),
44.41 (CH3 sulfonyl), 68.03 (CH isopropyl), 112.58 (CH pyrrole),
114.04 (Cq pyrrole), 127.12, 127.30, 128.10, 128.40, 129.40 (CH
aromatic), 131.10, 131.60, 136.55, 138.50, 138.92 (Cq aromatic),
171.67 (COO).
General Procedure for the Preparation of Ethyl 1,5-Diarylpyrrol-
3-ylglyoxylic Esters 16a,c,e,g. To a solution of the appropriate
pyrrole [14a, 14c, 14e, or 14g (4.82 mmol)] in anhydrous dichloro-
methane, under a nitrogen flow, were added TiCl4 (0.53 mL,
4.82 mmol) and etoxalyl chloride (0.54 mL, 4.82 mmol) at 0 ꢀC.
The solution was stirred for 4 h at room temperature. The mixture
was then washed with water and extracted with CHCl3. The
organic solution was washed with a saturated aqueous NaCl
solution, dried, and evaporated in vacuo. Purification of the
residue by flash chromatography with CHCl3 as the eluant gave
a solid which, after recrystallization from hexane, afforded the
expected products 16a,c,e,g.
1-[4-(Methylthio)phenyl]pentane-1,4-dione (12). A solution of
4-methylthiobenzaldehyde (11.97 mL, 0.09 mol), triethylamine
(19.5 mL, 0.14 mol), methyl vinyl ketone (5.8 mL, 0.09 mol), and
3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide (3.53 g,
0.014 mol) was put into a round-bottom flask equipped with a stir
bar. The flask was inserted into the cavity of the microwave
system apparatus and heated (150 W for 15 min, internal
temperature of 70 ꢀC, internal pressure of 60 psi).28 The residue
was treated with 2 N HCl (10 mL). After extraction with ethyl
acetate, the organic layer was washed with aqueous sodium
bicarbonate and water. The organic fractions were dried over
Na2SO4, filtered, and concentrated to give a crude orange liquid.
After crystallization from cyclohexane, intermediate 12 was
isolated as white needles (78% yield). Analytical data, the melting
1
point, and the H NMR spectrum were consistent with those
reported in the literature.23-26
1-[4-(Methylsulfonyl)phenyl]pentane-1,4-dione (13). To a solu-
tion of 12 (7.8 g, 35 mmol) in methanol (150 mL) was added
Oxone (37.7 g, 61.4 mmol), dissolved in water (150 mL), over
5 min. After being stirred at 25 ꢀC for 2 h, the reaction mixture
was diluted with water (400 mL) and extracted with dichloro-
methane. The organic layer was washed with brine (200 mL) and
water (200 mL) and dried (Na2SO4). After filtration and con-
centration, the crude material was chromatographed (silica gel,
3:1 hexane/ethyl acetate) to give 13 (90% yield) as a white solid.
Ethyl-2-methyl-5-[4-(methylsulfonyl)phenyl]-1-phenyl-1H-
pyrrole-3-glyoxylate (16a). White needles (76% yield). Ana-
1
Analytical data, the melting point, and the H NMR spectrum
1
were consistent with those reported in the literature.23-26
General Procedure for the Preparation of 1,5-Diarylpyrroles
14a,c,e,g. Following the Paal-Knorr reaction, 13 (0.58 g,
2.28 mmol) was dissolved in ethanol (2 mL) in a round-bottom
flask equipped with a stir bar. The suitable amine (2.28 mmol)
and p-toluenesulfonic acid (30 mg, 0.17 mmol) were added.
The flask was inserted into the cavity of the microwave
system apparatus and heated (150 W for 45 min, internal
temperature of 160 ꢀC, internal pressure of 150 psi).28 The
reaction mixture was cooled and concentrated. The crude
material was purified by chromatography on aluminum oxide
with a 3:1 cyclohexane/ethyl acetate mixture, as the eluant,
to give the expected 1,5-diarylpyrroles 14a,c,e,g as solids in
satisfactory yield.
lytical data, the melting point, and the H NMR spectrum
were consistent with those reported in the literature.25
General Procedure for the Preparation of Ethyl 1,5-Diarylpyrrole-
3-acetic Esters 17a,c,e,g. Compounds 17a,c,e,g were obtained from
16a,c,e,g, respectively, according to the procedure described for
10a-h.
Ethyl-2-methyl-5-[4-(methylsulfonyl)phenyl]-1-phenyl-1H-
pyrrole-3-acetate (17a). White solid (68% yield). Analytical
data, the melting point, and the 1H NMR spectrum were
consistent with those reported in the literature.25
General Procedure for the Preparation of 1,5-Diarylpyrrole-
3-acetic Acids 6a and 11c,e,g. To a solution of the appropriate
ethyl 1,5-diarylpyrrole-3-acetic esters 17a,c,e,g (0.30 g,
0.67 mmol) in methanol (4.83 mL) was added 4.83 mL of 1 N
NaOH. The resulting mixture was refluxed for 1 h, cooled,
and concentrated in vacuo. The residue was solubilized in
water and then acidified with concentrated HCl. The precipitate
was filtered to give the desired acids 6a and 11c,e,g as white
solids.
2-Methyl-5-[4-(methylsulfonyl)phenyl]-1-phenyl-1H-pyrrole
(14a). White needles (80% yield). Analytical data, the melting
point, and the 1H NMR spectrum were consistent with those
reported in the literature.29
General Procedure for the Preparation of 1,5-Diarylpyrrol-
3-glyoxylic Esters 15a-h. To a solution of the appropriate
pyrrole [14a, 14c, 14e, or 14g (4.82 mmol)] in anhydrous
dichloromethane, under a nitrogen flow, were added 2,6-lutidine
(0.56 mL, 4.82 mmol) andoxalyl chloride(0.40 mL, 4.82 mmol) at
0 ꢀC. The solution was stirred for 4 h at room temperature, and
thenthe suitablealcohol(4.82mmol) was added. The mixturewas
then washed with water and extracted with CHCl3. The organic
solution was washed with a saturated aqueous NaCl solution,
2-Methyl-5-[4-(methylsulfonyl)phenyl]-1-phenyl-1H-pyrrol-3-
acetic Acid (6a). White solid (71% yield). Analytical data, the
1
melting point, and the HNMR spectrum were consistent with
those reported in the literature:23 13C NMR δ 11.10 (CH3
pyrrolic), 32.90 (CH2), 44.41 (CH3 sulfonyl), 112.60 (CH pyrrole),
114.05 (Cq pyrrole), 127.14, 127.32, 128.15, 128.42, 129.44 (CH
aromatic), 131.15, 131.66, 136.55, 138.50, 138.98 (Cq aromatic),
177.67 (COO).

Article
Biology. In Vitro Anti-Inflammatory Study. Compounds
10a-h, 6a, and 11c,e,g were all evaluated for their inhibitory
activity toward both COX-2 and COX-1 enzymes.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 731
mice were tested simultaneously on the apparatus, with a rod
rotation of 16 rpm.
Zymosan-Induced Hyperalgesia. The interplantar injection of
zymosan-induced mechanical hyperalgesia is a model of inflam-
matory pain.38 In this model, a male Sprague-Dawely or Wistar
rat (200-250 g) receives an interplantar injection of 4 mg of
zymosan/100 μL into one hind paw; a marked inflammation
occurs. Compounds were administered orally for evaluation of
efficacy, 30 min before the inflammatory insult. The hyperalge-
sia induced by zymosan administration was evaluated using the
Randal-Selitto method.41 The quantitation of the analgesic
effect was achieved by an analgesimeter, which consists of
application to the inflamed paw an increasing weight (from
130-140 to 500 g). The difference in the mechanical pain
threshold between the basal value (generally, 230-250 g) and
the one tolerated by the animals treated with the drug, deter-
mined 4 h after the inflammatory challenge, is defined as
mechanical hyperalgesia. Mechanical hyperalgesia is expressed
as the maximum percent effect (MPE) that represents the
difference (percent) in pain threshold between the animals
treated with the drug and the controls that received only the
vehicle. Results are reported as MPE (reduction of the nocicep-
tive effect, due to paw loading with increasing weight, in
comparison to controls); 100% MPE means that the animal
treated with the compound and zymosan can tolerate the same
stimulus (weight) as the control animals which have not received
zymosan treatment. An MPE higher than 100% means that the
animal treated with the compound and zymosan can tolerate
stimuli (weight) more than the control animals, which has not
received zymosan treatment (hypoalgesia).
In Vitro Metabolic Stability in Human Liver Microsomes.
Metabolic stability was evaluated on human liver microsomes
incubated for 60 min at 37 ꢀC with test compounds (0.1 and
10 μM), NADP (1 mM), D-glucose 6-phosphate (G6P) (6 mM),
glucose-6-phosphate dehydrogenase (G6PDHase) (1 unit/mL),
0.6% methanol, 0.6% acetonitrile (n = 2), and phosphate buffer
(pH 7.4). At the end of the incubation at each of the time points,
an equal volume of an organic mixture [50:50 (v/v) acetonitrile/
methanol] was added to the incubation mixture followed by
centrifugation. Peak areas corresponding to the analytes were
determined by HPLC-MS/MS. The ratio of the precursor
compound remaining at the end of the incubation to the amount
remaining at time zero, expressed as a percent value, is reported
as metabolic stability.
Pharmacokinetic Study. Male Sprague-Dawley rats, three
animals per time point, were treated with each compound
(10 mg/kg), dissolved in DMSO either po or iv. Approximately
0.3 mL of blood per time point was taken from the retro-orbital
sinus using a capillary tube. Blood samples were collected
into sodium heparin-labeled tubes and treated with NaF to
minimize ester hydrolysis. The samples where then centrifugated
at 2500 rpm for 5 min at 4 ꢀC. Plasma was separated, placed in
prelabeled Eppendorf tubes, and stored at -20 ꢀC pending the
assay. The samples were analyzed by HPLC with UV detection,
and the LOQ was 1.0 ng/mL. Pharmacokinetic calculations
were performed using the kinetic software by noncompart-
mental methods.
Computational Details. Calculations were performed accord-
ing to a computational protocol previously described.23,24
Figure 1 has been generated by means of AutoDockTools
version 1.5.1. The MSMS (maximal speed molecular surface)
module of AutoDock has been used to graphically represent a
section of the enzyme molecular surface. In particular, a clipping
plan parallel to the ligand pyrrole ring has been applied to the
protein molecular surface to show regions of the binding site
accessible to ligands.
The murine monocyte/macrophage J774 cell line was grown in
DMEM supplemented with 2 mM glutamine, 25 mM HEPES,
100 units/mL penicillin, 100 μg/mL streptomycin, 10% fetal
bovine serum (FBS), and 1.2% sodium pyruvate. Cells were
plated in 24-well culture plates at a density of 2.5 ꢀ 105 cells/
mLorin60 mm diameterculturedishes(3 ꢀ 106 cells per 3 mL per
dish) and allowed to adhere at 37 ꢀC in 5% CO2 for 2 h.
Immediately before the experiments, culture medium was
replaced with fresh medium without FBS and cells were stimu-
lated as described previously.39
To evaluate COX-1 activity, cells were pretreated with test
compounds (0.01-10 μM) for 15 min and further incubated at
37 ꢀC for 30 min with 15 μM arachidonic acid to activate the
constitutive COX. At the end of the incubation, the supernatants
were collected for the measurement of prostaglandin E2 (PGE2)
levels by a radioimmunoassay (RIA). On the other hand, to
evaluate COX-2 activity, cells were stimulated for 24 h with
Escherichia coli lipopolysaccharide (LPS, 10 μg/mL) to induce
COX-2, in the absence or presence of test compounds, at the
concentrations previously reported. Celecoxib was utilized as a
reference compound for the selectivity index. The supernatants
were collected for the measurement of PGE2 by RIA. Through-
out the time the experiments lasted, triplicate wells were used for
the various conditions of treatment. Results are expressed as the
mean, for three experiments, of the percent inhibition of PGE2
production by test compounds with respect to control samples.
The IC50 values were calculated with GraphPad Instat, and
the data fit was obtained using the sigmoidal dose-response
equation (variable slope) (GraphPad).
Ex Vivo Anti-Inflammatory Study. Compounds 10a-h, 6a,
and 11c,e,g were also evaluated for COX-1 versus COX-2
selectivity by means of the HWB assay. To evaluate COX-2
activity, 1 mL aliquots of peripheral venous blood samples were
incubated in the presence of LPS (10 μg/mL) or saline for 24 h at
37 ꢀC as previously described.18 As an index of LPS-induced
monocyte COX-2 activity, PGE2 plasma levels were measured
by RIA.18 A COX-1 assay was performed on peripheral venous
blood samples drawn from the same donors when they had not
taken any NSAID during the 2 weeks preceding the study.
Whole blood thromboxane B2 production was measured by
RIA, as a reflection of maximally stimulated platelet COX-1
activity in response to endogenously formed thrombin.31
Celecoxib was tested at final concentrations of 0.01-100 μM
in both COX-2 and COX-1 assay.
In Vivo Anti-Inflammatory Study. In vivo anti-inflammatory
activity of the new compounds was also assessed. Male Swiss
albino mice (23-25 g) and Sprague-Dawley or Wistar rats
(150-200 g) were used. The animals were fed with a standard
laboratory diet and tap water ad libitum and kept at 23 ( 1 ꢀC
with a 12 h light/dark cycle, light on at 7 a.m. The paw pres-
sure test was performed by inducing an inflammatory process
in the administered ip carrageenan 4 h before the test. The
carrageenan-induced paw edema test was also performed, eval-
uating the paw volume of the right hind paw 5 h after the
injection of carrageenan and comparing it with saline/carragee-
nan-treated controls. The analgesic activity of compounds was
also assessed by performing the abdominal constriction test,
using mice into which a 0.6% solution of acetic acid (10 mL/kg)
had been injected ip. The number of stretching movements was
counted for 10 min, starting 5 min after administration.
The rota-rod test was performed as previously described.40
Potential motor incoordination/ataxia caused by compound
injection was evaluated using a rota-rod apparatus in which
animals were required to walk against the motion of a rotating
drum for 30 s. The time taken to fall off the rota-rod was
recorded as the number of falls in 30 s. The rod (30 cm long)
was divided into five equal section by six disks. Thus, up to five
Acknowledgment. We thank Rottapharm SpA (Monza,
Italy) for financial support of this research. The HWB assay
was supported by a grant from MIUR to P.P.

732 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Biava et al.
Supporting Information Available: Physicochemical, spectro-
scopical, and analytical data of compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
Boyce, S.; Visco, D.; Girad, Y.; Prasit, P.; Zamboni, R.; Rodger,
J. W.; Gresser, M.; Ford-Hutchinson, A. W.; Young, R. N.; Chan,
C.-C. Etoricoxib (MK-0663): Preclinical profile and comparison
with other agents that selectively inhibit cyclooxygenase-2.
J. Pharmacol. Exp. Ther. 2001, 296, 558–566.
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