Synthesis, in vitro, and in vivo biological evaluation and molecular docking simulations of chiral alcohol and ether derivatives of the 1,5-diarylpyrrole scaffold as novel anti-inflammatory and analgesic agents

Article abstract of DOI:10.1016/j.bmc.2008.07.058

Following our previous research on anti-inflammatory drugs (NSAIDs), we report here the synthesis of chiral 1,5-diarylpyrroles derivatives that were characterized for their in vitro inhibitory effects toward cyclooxygenase (COX) isozymes. Analysis of enzy

Full text of DOI:10.1016/j.bmc.2008.07.058

Bioorganic & Medicinal Chemistry 16 (2008) 8072–8081
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, in vitro, and in vivo biological evaluation and molecular
docking simulations of chiral alcohol and ether derivatives of the
1,5-diarylpyrrole scaffold as novel anti-inflammatory and analgesic agents
a
a
a
b,
b
Mariangela Biava a, , Giulio C. Porretta , Giovanna Poce , Sibilla Supino , Fabrizio Manetti , Stefano Forli ,
Maurizio Botta b, Lidia Sautebin d, Antonietta Rossi d,e, Carlo Pergola d, Carla Ghelardini f, Monica Norcini f,
Francesco Makovec g, Antonio Giordani g, Paola Anzellotti h, Roberto Cirilli i, Rosella Ferretti i,
Bruno Gallinella i, Francesco La Torre i, Maurizio Anzini b,c, Paola Patrignani h
*
*
a Dipartimento di Studi di Chimica e Tecnologie del Farmaco, Università ‘La Sapienza’, Piazzale Aldo Moro, 5, I-00185 Roma, Italy
b Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy
c European Research Centre for Drug Discovery and Development, via Banchi di Sotto 55, I-53100 Siena, Italy
d Dipartimento di Farmacologia Sperimentale, Università di Napoli ‘Federico II’, via D. Montesano 49, I-80131 Napoli, Italy
e IRCCS Centro Neurolesi ‘Bonino-Pulejo’, Via Provinciale Palermo—C.da Casazza, I-98124 Messina, Italy
f Dipartimento di Farmacologia, Università di Firenze, viale G. Pieraccini 6, I-50139 Firenze, Italy
g Rottapharm S.p.A., via Valosa di Sopra 7, I-20052 Monza, Italy
h Department of Medicine and Center of Excellence on Aging, ‘G. d’Annunzio’ University and CeSI, Via dei Vestini 31, I-66013 Chieti, Italy
i Istituto Superiore di Sanità, Dipartimento del Farmaco, Viale Regina Elena 299, I-00161 Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Following our previous research on anti-inflammatory drugs (NSAIDs), we report here the synthesis of
chiral 1,5-diarylpyrroles derivatives that were characterized for their in vitro inhibitory effects toward
cyclooxygenase (COX) isozymes. Analysis of enzymatic affinity and COX-2 selectivity led us to the selec-
tion of one compound (+/À)-10b that was further tested in vitro in the human whole blood (HWB) and
in vivo for its anti-inflammatory activity in mice. The affinity data have been rationalized through dock-
ing simulations.
Received 17 March 2008
Revised 16 July 2008
Accepted 22 July 2008
Available online 24 July 2008
Keywords:
Anti-inflammatory
NSAIDs
Ó 2008 Elsevier Ltd. All rights reserved.
Analgesic agents
COX-1 inhibitors
COX-2 inhibitors
Coxibs
Celecoxib
1. Introduction
COX-2-dependent prostanoids.1 However, they are associated with
side-effects for the gastrointestinal (GI) (i.e., dyspepsia, ulcer, per-
Nonsteroidal anti-inflammatory drugs (NSAIDs) [traditional
tNSAIDs and NSAIDs selective for cyclooxygenase (COX)-2, named
coxibs] are a chemically heterogeneous group of agents to treat
symptoms of acute pain and chronic inflammatory and degenera-
tive joint diseases, which act mainly through the inhibition of
foration, occlusion, and bleeding) and cardiovascular (CV) system
(myocardial infarction, stroke, hypertension, sodium retention
with edema and heart failure), which plausibly involve the inhibi-
tion of COX-1 and COX-2, respectively.2–4
Selectivity for COX-2 in vitro (a chemical property of a COX
inhibitor) is described as the ratio of the concentrations required
to inhibit the activity of the isozymes by 50% (IC50 for COX-1/IC50
for COX-2).4 Using the whole blood assays in vitro, Patrignani P.
et al. evidenced that the biochemical selectivity of COX inhibitors
is a continuous variable.5,6 It was shown that, among traditional
tNSAIDs, there is a cluster of compounds, such as etodolac7 (2),
meloxicam8 (3), nimesulide9 (4), and diclofenac10 (5, Chart 1),
which are from 5- to 29-fold more potent toward COX-2 in vitro.
Abbreviations: NSAIDs, nonsteroidal anti-inflammatory drugs; COX, cyclooxy-
genase; HWB, human whole blood; tNSAIDs, traditional nonsteroidal anti-inflam-
matory drugs; GI, gastrointestinal; CV, cardiovascular; DMEM, Dulbecco’s modified
Eagle’s medium; FBS, fetal bovine serum; LPS, lipopolysaccharide.
* Corresponding authors. Tel.: +39 06 49913812; fax: +39 06 49913133 (M.B),
tel.: +39 0577 234330; fax: +39 0577 234333 (F.M).
E-mail addresses: mariangela.biava@uniroma1.it (M. Biava), manettif@unisi.it
(F. Manetti).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.058

M. Biava et al. / Bioorg. Med. Chem. 16 (2008) 8072–8081
8073
CF3
N
N
O
Me
O
O
N
H2NO2S
H2NO2S
MeO2S
rofecoxib, 1a
celecoxib, 1b
COOH
valdecoxib, 1c
CH3
Me
N
CH3
Et
N
H
N
COOH
Et
N
Cl
Cl
Cl
O
SO2Me
etoricoxib, 1d
lumiracoxib, 1e
etodolac, 2
O
Cl
S
OH
O
S
HN
O
O
NH
COONa
COOH
N
N
H
Cl
N
MeO
S
O
O
NO2
nimesulide, 4
naproxen, 6
meloxicam, 3
diclofenac, 5
EtOOC
MeO
X
COOH
Me
7: X = CO; CH2; R = H; F; 3,4-diF; Me;
OMe; CF3; R1 = SO2CH3.
8: X = CO; CH2; R = SO2CH3; R1 = H; F;
3,4-diF; Me; OMe; CF3
Me
N
R
N
R1
O
Cl
indometacin, 9
Chart 1. Chemical structure of NSAIDs: coxib derivatives, traditional NSAIDs and previous pyrrole compounds.
Among NSAIDs selective for COX-2 (commonly named coxibs:
rofecoxib,11 1a; celecoxib,12 1b; valdecoxib,13 1c; etoricoxib,14
1d; and lumiracoxib,15 1e, Chart 1), developed to reduce the inci-
dence of serious GI effects, a wide spectrum of COX-1/COX-2 IC50
ratio was reported, which ranges from 30 for 1b to 433 for 1e.
However, efficacy and side-effects in vivo are dependent on the
achieved selectivity (i.e., the ratio of degree of suppression of
COX-1 and COX-2 at circulating drug concentrations), which is dri-
ven by pharmacokinetic features and dose-potency, but also influ-
enced by marked variability in how each person reacts to these
drugs based on its genetic background.4,16
It has been shown that 80% inhibition of COX-2 ex vivo (in
whole blood) by circulating drug concentrations seems sufficient
to translate into efficacy (analgesia).17 High degrees of inhibition
of COX-2 seem to be associated with increased incidence of vascu-
lar events (with myocardial infarction that exceeds over stroke), an
effect that can be mitigated, but not obliterated, by complete and
persistent (throughout dosing interval) inhibition of platelet
COX-1 activity (i.e., >95%, necessary to inhibit platelet function).3,4
Among tNSAIDs, only naproxen18 (6, Chart 1), a balanced inhibitor
of COX-1 and COX-2 with long half-life (>12 h), seems to have
these pharmacokinetic and pharmacodynamic features.19,20 Thus,
most of tNSAIDs and coxibs are functionally selective for COX-2
in vivo in respect of the platelet COX-1, at therapeutic doses. This
explains why coxibs but also some tNSAIDs are thrombogenic.21,22
Differently from platelets, in the kidney, an incomplete inhibition
of COX-1 might attenuate the deleterious functional effect derived
from inhibition of COX-2-dependent prostacyclin.3,22 Thus, selec-
tive and profound inhibition of COX-2 by coxibs may have a greater
propensity to increase blood pressure than concomitant inhibition
of both COX-1 and COX-2 by tNSAIDs.
The results of human pharmacology and clinical trials have
enlightened that a NSAID with 5- to 10-fold higher selectivity for
COX-2 versus COX-1 in vitro might translate into acceptable GI
safety profile when administered to a dose causing 70–80% inhibi-
tion of COX-2 ex vivo appropriate for efficacy. Presumably, this will
leave sufficient COX-2-dependent generation of prostacyclin to sus-
tain vascular function. Of course, this strategy will not obliterate the
incidence of CV and GI toxicity in susceptible patients. The only way
to reduce the incidence of these effects is by the development of ge-
netic or biochemical biomarkers predictive of toxicity to select the
individuals that should avoid the use of these pain killers.
Despite several NSAIDs with these pharmacodynamic features
available (e.g., 3–5), most of them have short half-life, which leads
to their administration at high doses (often overshooting) to ex-
tend the pharmacodynamic effects, and importantly some are
associated with hepatic toxicity (i.e., 5). It should be pointed out
that the Food and Drug Administration has recently rejected 1e
(a 5-derived coxib) as a treatment for patients with osteoarthrosis
pain. The coxib was recently withdrawn in Australia, placed under
restrictions in the EU and undergoing review by Health Canada,
following reports of serious liver damage. Thus, there is the need

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M. Biava et al. / Bioorg. Med. Chem. 16 (2008) 8072–8081
to have novel NSAIDs with improved pharmacokinetic features and
metabolism.
parable to that of the reference compound 1b. Moreover, one of the
most active compounds, (+/À)-10b, was also tested for its in vivo
anti-inflammatory activity in the carrageenan-induced paw edema
test. Finally, the human whole blood (HWB) in vitro assay was uti-
lized to assess the pattern of the relative inhibition for platelet
COX-1 and monocyte COX-2 by compound (+/À)-10b in compari-
son with the coxib 1b and the tNSAID 2.
In the context of COX inhibitors, we recently reported several
investigations describing the design, synthesis, and anti-inflamma-
tory properties for a class of novel pyrrole-containing anti-inflam-
matory agents.23,24 In particular, we focused our attention on the
synthesis of 1,5-diarylpyrrol-3-acetic and -glyoxylic acid and es-
ters 7 and 8 (Chart 1) as new COX-2 selective inhibitors in which
the pyrroleacetic and vicinal diaryl heterocyclic moieties were
reminiscent of both indomethacin25 (9) and the above-cited ‘coxib’
family, respectively. A structure–activity relationship (SAR) analy-
sis of such compounds, supported by molecular docking simula-
tions of inhibitors into the COX-2 binding site, allowed us to rule
out several considerations: (i) the position of the p-methylsulfonyl
substituent was very important for activity, compounds 7 being
more active than 8, (ii) the acetic group at C3 of the pyrrole ring
led to compounds more active than the corresponding glyoxylic
analogues, (iii) substituents and substitution pattern on the phenyl
ring at N1 influenced activity in the following order: 3-F > 4-
F > 3,4-diF > 4-OMe > H > 4-CF3 > 4-CH3.24 On this basis, we
planned the synthesis of new derivatives, keeping the most conve-
nient fluorine substitution (namely the 3-F substituent at the N1
phenyl ring, previously suggested by docking calculations), as well
as the 4-methoxyphenyl group and an unsubstituted phenyl ring at
position 1 of the pyrrole. On the other hand, a hydroxy or an ethoxy
group was introduced into the methylene moiety of the C3 alkyl
chain (leading to the corresponding alcohol and ether chiral deriv-
atives 10a–c and 11a–c, respectively, Table 1) to investigate on the
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds is described in Scheme 1.
Briefly, compounds 12a–c, obtained by a procedure previously de-
scribed,23,24 were reduced with sodium cyanoborohydride in the
presence of tert-butyl alcohol to give
esters 10a–c as mixture of enantiomers, or with sodium cyano-
borohydride in the presence of ethanol to give -ethoxy pyrrole-
a-hydroxy pyrroleacetic ethyl
a
acetic ethyl esters 11a–c, as mixture of enantiomers.
Enantiomerically pure samples were obtained, at mg-scale, by
HPLC on the amylose-based Chiralpak IA chiral stationary phase.
In particular, the absolute configuration of (À)-10b was deter-
mined by single-crystal X-ray diffraction analysis. The molecular
structure and the crystallographic data are reported elsewhere.26
The findings of X-ray structure analysis allowed to assign unam-
biguously the (R)-configuration to the (À)-10b enantiomer. Then,
the stereochemistry of remaining pyrrole derivatives was empiri-
cally assigned by circular dichroism spectroscopy.26
influence that different substituents at the
a-position of this side
chain could exert on the activity, and to check the possible varia-
2.2. Biology
tion of activity induced by different enantiomers.
We performed on all the new compounds a preliminary screen-
ing for COX-isozyme inhibition using a cell-based assay to identify
the compounds with IC50 values in the submicromolar range, com-
Compounds 10a–c and 11a–c were all evaluated for their anti-
inflammatory activity toward both COX-2 and COX-1 enzymes fol-
lowing protocols previously described.24
Table 1
Structural properties and in vitro inhibition of COX-1 and COX-2 by compounds 10a–c, 11a–c, and 1b
RO
COOEt
N
H3CO2S
R1
R1
IC50
(l
M)
% Inhibition
M)
COX-1/COX-2b (SI)
a
Compound
R
COX-1
COX-2
COX-2 (10
l
COX-2 (1 lM)
(+/À)-10a
H
H
H
H
H
H
H
H
H
H
H
3-F
3-F
3-F
4-OMe
4-OMe
4-OMe
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.7
0.30
0.18
0.15
0.12
1.10
0.075
0.12
0.079
0.15
0.18
0.10
0.14
0.10
0.25
0.81
0.13
0.14
0.09
0.06
100
92
92
100
84
92
100
100
93
100
100
86
100
100
100
—
100
100
100
78
79
86
70
56
86
82
88
86
90
81
77
74
86
73
—
>600
>550
>650
>800
>90
(+)-(S)-10a
(À)-(R)-10a
(+/À)-10b
(+)-(S)-10b
(À)-(R)-10b
(+/À)-10c
>1300
>800
>1200
>650
>800
>1000
>700
>1000
>400
>100
>800
>700
>1100
61.7
(+)-(S)-10c
(À)-(R)-10c
(+/À)-11a
H
Et
Et
Et
Et
Et
Et
Et
Et
Et
(+)-(S)-11a
(À)-(R)-11a
(+/À)-11b
(+)-(S)-11b
(À)-(R)-11b
(+/À)-11c
H
H
3-F
3-F
3-F
4-OMe
4-OMe
4-OMe
(+)-(S)-11c
(À)-(R)-11c
Celecoxib, 1b
100
87
80
a
Results are expressed as the mean, for three experiments, of the % inhibition of PGE2 production by test compounds with respect to control samples.
In vitro COX-2 Selectivity Index (IC50(COX-1)/IC50(COX-2)).
b

M. Biava et al. / Bioorg. Med. Chem. 16 (2008) 8072–8081
8075
compound (+/À)-10b had a comparable COX-2 selectivity to 2
while it was about 5-fold less selective than 1b (the selectivity in-
dex, expressed as COX-1/COX-2 IC50 ratio, was 5.6, 4.9, and 29.6,
respectively, Fig. 1A–C). Differently, in the in vitro cell-based assay,
1b showed a selectivity index of about 62, lower than that found
for (+/À)-10b (>300). The discrepancy of these results could be
due to several matters. In particular, the use of exogenous arachi-
donic acid (AA) in the assay for COX-1 activity in vitro, in murine
monocyte/macrophage J774 cell line, might have caused a loss in
COX-1 affinity for an AA-dependent allosteric activation of COX-
1, which induces a conformational change in the enzyme binding
site.27
The same compound was also checked for its in vivo anti-
inflammatory (carrageenan-induced paw edema test) and analge-
sic (paw-pressure test and abdominal writhing test) activity. Thirty
minutes after a 20 mg/kg po administration, it showed a good
activity against carrageenan-induced hyperalgesia (paw-pressure
test, Table 2), very similar to that obtained with 1b (10 mg/kg
po). However, different from what was observed with 1b, the activ-
ity tended to fade at longer time-points (60 min), disappearing al-
HO
N
COCOOEt
(i)
COOEt
N
H3CO2S
MeO2S
R
R
12a-c
10a-c
(ii)
EtO
N
COOEt
MeO2S
R
11a-c
Scheme 1. Compounds: 10a, R = H; 10b, R = 3-F; 10c, R = OMe; 11a, R = H; 11b,
R = 3-F; 11c, R = OMe; 12a, R = H; 12b, R = 3-F; 12c, R = OMe. Reagents and
conditions: (i) ZnCl2, sodium cyanoborohydride, CH2Cl2, tert-butyl alcohol, rt, 2 h;
(ii) ZnCl2, CH2Cl2, ethyl alcohol, rt, 2 h.
Moreover, compound (+/À)-10b was also evaluated for COX-1
versus COX-2 selectivity by the use of the HWB assay, as reported
elsewhere.24 Its in vivo anti-inflammatory and analgesic activity
was also checked on Male Swiss albino mice (23–25 g) and Spra-
gue–Dawley or Wistar rats (150–200 g). In detail, the paw-pres-
sure test, the carrageenan-induced paw edema test, and the
abdominal writhing test were performed as previously reported.24
2.3. Results and discussion
Analysis of biological data from the cell-based assay showed
IC50 values toward COX-2 in the submicromolar concentration
(with the only exception of (+)-10b, Table 1), while compounds
were all inactive toward COX-1 at the maximum tested dose
(100 lM).
All the new compounds showed a very good activity, even
though, in general they are less active than corresponding previ-
ously synthesized derivatives lacking a stereogenic center at the
a
-position of the side chain.23,24
Regarding the influence of the alcohol and ether groups on
activity, a comparison of racemates or corresponding enantiomers
of compounds 10a and 11a revealed comparable IC50 values, while
less than a 2-fold difference was the maximum value found be-
tween compounds 10c and 11c. The major variation in affinity
was found comparing (+)-10b and (+)-11b (1.10 vs 0.25
lM), and
(À)-10b and (À)-11b (0.075 vs 0.81
l
M), while their racemates
10b and 11b showed very similar activity (0.12 vs 0.10 lM,
respectively).
Analysis of the influence of the substituent on the N1 phenyl
ring showed that ether derivatives 11a and 11c (both racemates
and enantiomers) were characterized by comparable IC50 values,
while the 3-F derivatives (+)- and (À)-11b were slightly less ac-
tive. All of the alcohol derivatives had a good biological profile
in terms of both IC50 values and percent inhibition values. In fact,
COX-2 affinity was ranging from 0.075 through 0.30
l
M, and
M dose),
lM, 84% enzyme
COX-2 percent inhibition was higher than 92% (at a 10
l
with the exception of (+)-10b (IC50 1.10
inhibition).
Compound (+/À)-10b was submitted to further pharmacologi-
cal tests. In particular, the HWB assay was performed to evaluate
the actual extent of isozyme inhibition achievable in vivo by circu-
lating drug levels, because of a number of variables potentially
affecting inhibitor–enzyme interaction. Results indicated that
Figure 1. In vitro inhibition (human whole blood assay) of COX-1 and COX-2 by 1b
(A), 2 (B), and (+/À)-10b (C).

8076
M. Biava et al. / Bioorg. Med. Chem. 16 (2008) 8072–8081
Table 2
tion points for the methyl probe, while the carbonyl oxygen of
the ester moiety occupied a pocket (close to the terminal portion
of the Arg120 side chain) identified by the carbonyl probe of
Grid. As a consequence, also in terms of interactions with the
COX-2 binding site, (À)-(R)-10b made expected contacts (Fig.
2A). In fact, one of the sulfone oxygens interacted by a hydrogen
bond with the NH group of Tyr518, while the carbonyl oxygen
contacted the basic portion of Arg120. A very similar orientation
and interactions were also found for the corresponding S-enan-
tiomer of 10b, with only a major difference involving the alcohol
group (Fig. 2B). In particular, in the case of the S-enantiomer,
such a residue was directed toward a large hydrophobic region
delimited by Val349, Leu359, and Leu531, without any interac-
tion with the protein. Differently, the same group of the R-enan-
tiomer pointed toward Ser353 and made an additional hydrogen
bond contact with its hydroxy group, probably accounting for
the higher affinity of the R-enantiomer in comparison to the S-
enantiomer. Moreover, the N1 phenyl ring of the S-enantiomer
underwent a conformational rearrangement (Fig. 2B), in compar-
ison with the same moiety of the R-enantiomer, leading the fluo-
rine substituent in a region of space not perfectly corresponding
to the profitable interaction point found by Grid for the fluorine
probe.
Effect of (+/À)-10b in the rat paw-pressure test, in comparison to 1ba
Pre-treatment Treatment
Paw pressure (g)
After treatment (min)
Before
pre-treatment
30
60
120
Saline
Saline
Saline
63.4 4.6
61.9 5.1
60.2 5.1
38.7 5.3
62.9 4.9
35.8 4.5
60.7 5.4
40.1 5.0
47.5 4.6
Carrageenan
Carrageenan
Carrageenan
(+/À)-10b 59.6 2.8
54.8 3.8* 45.2 4.6
1b 62.7 3.9
56.5 3.8* 58.2 4.4* 55.2 5.1*
a
(+/À)-10b and 1b were administered at the dose of 20 and 10 mg/kg po,
respectively.
*
P < 0.01 versus carrageenan/saline-treated group.
Table 3
Effect of compounds (+/À)-10b and 1b in the edema induced by carrageenana
Pre-treatment
Treatment
Paw volume (mL)
Before pre-treatment
60 min after treatment
Saline
Saline
Saline
(+/À)-10b
1b
1.25 0.09
1.22 0.08
1.37 0.12
1.28 0.14
1.29 0.10
2.33 0.08
1.40 0.13*
1.33 0.10*
Carrageenan
Carrageenan
Carrageenan
Transforming 10b into the corresponding ethyl ether deriva-
tive 11b, the overall orientation within the binding site remained
quite unaltered (Fig. 3). Moreover, enantiomers of 11b showed a
very similar pose (in both cases, we chose the first conformation
of the first ranked cluster, corresponding to the most populated
docking cluster) also in terms of orientation of the side chain
at position 3 of the pyrrole. In fact, the ether side chain of both
enantiomers was located within the hydrophobic region defined
by Val349, Leu359, and Leu531 and pointed toward Ile345, while
the ester portions were accommodated within the carboxylate
site. The only difference was that the S-enantiomer contacted
with its carbonyl oxygen the terminal part of Arg120 side chain,
accordingly to Grid simulations for the oxygen probe. Differently,
the ether oxygen of the ester moiety of the R-enantiomer was
able to make a hydrogen bond with the same residue. However,
such an oxygen atom was located in a region quite far from the
pocket identified by Grid for profitable interactions with Arg120,
accounting for the decreased affinity (about 3-fold) with respect
to S-enantiomer.
Regarding the p-methoxy derivative 10c, its R-enantiomer
showed a binding mode and interactions with the protein very
similar to (À)-(R)-10b. As a consequence, although additional
hydrophobic contacts involving the methoxy group and the
hydrophobic region of the protein were found, affinity of (À)-
(R)-10c was comparable to that of (À)-(R)-10b. On the other
hand, (+)-(S)-10c, whose hydroxy group lacked the contact with
Ser353, showed an unexpected high affinity for the enzyme.
However, it gained van der Waals interactions by means of its
terminal methyl group (part of the p-methoxy substituent) that
a
(+/À)-10b and 1b were administered at the dose of 20 and 10 mg/kg po,
respectively.
*
P < 0.01 versus carrageenan/saline-treated group.
most completely 120 min after administration. In parallel, a good
activity was demonstrated against carrageenan-induced edema in
the rat paw (Table 3), with a complete remission 60 min after the
administration (20 mg/kg po). Moreover, a dose-dependent antin-
ociceptive activity was observed in the abdominal writhing test
(Table 4).
2.4. Computational studies on the binding mode
Following a computational protocol previously described,23
molecular docking simulations (software Autodock 3.0)28 were
performed on the enantiomeric pairs of alcohol derivatives 10b
and 10c, as well as on the enantiomers of the ether derivative
11b. Results of calculations showed that all the compounds as-
sumed the same orientation within the COX-2 binding site, with
the methylsulfonyl group located into the selectivity site
(according to the notation used by Kurumbail et al.),29 the 5-
phenyl ring accommodated into the hydrophobic pocket, and
the side chain at position 3 of the pyrrole ring matching the car-
boxylate site (Fig. 2A). In further detail, the best docked confor-
mation of (À)-(R)-10b, belonging to the first ranked and most
populated docking cluster, was also characterized by expected
features. In fact, its fluorine substituent was located at close con-
tact with the portion of the hydrophobic pocket previously iden-
tified by Grid30 as the best interaction region for the fluoride
probe.23,24 Moreover, the methyl group at position 2 of the pyr-
role approximately corresponded to one of the profitable interac-
was able to fill
a hydrophobic cavity defined by Phe381,
Leu384, and Tyr385 (Fig. 4).
In summary, docking simulations showed that the new chiral
derivatives adopted a binding mode very similar to that previ-
ously reported for the corresponding achiral parent compounds,
in terms of both orientation within the binding site and interac-
tions with the protein. Hydrogen bonds involving the sulfonyl
oxygens and the carbonyl group of the ligands, which interacted
with Tyr518 and Arg120, respectively, were confirmed as impor-
tant anchor points for the binding of COX-2 inhibitors. More-
over, additional interactions, such as a hydrogen bond with
Ser353 and van der Waals contacts with the hydrophobic region,
could serve to modulate affinity of compounds toward the
enzyme.
Table 4
Effect of compound (+/À)-10b in the mouse abdominal writhing test (acetic acid 0.6%)
Treatmenta
Number of mice
Dose (po mg kgÀ1
)
Number or writhes
CMC
25
8
8
38.1 2.6
26.3 3.6
16.1 2.7*
(+/À)-10b
10
20
(+/À)-10b
a
All compounds were administered per os 30 min before test.
P < 0.01 versus vehicle-treated mice.
*

M. Biava et al. / Bioorg. Med. Chem. 16 (2008) 8072–8081
8077
Figure 2. (A) Stereographical representation of the binding mode of (À)-R-10b, taken as the representative compound of the new pyrrole derivatives, into the binding pocket
of COX-2. Hydrogen bonds involving the inhibitor and Arg120, Ser353, and Phe518, are shown as black dashed lines. (B) Stereographical representation of the binding mode of
(À)-R-10b (green, thick lines) and (+)-S-10b (magenta, thick lines). The major difference involves the orientation of the hydroxy group on the stereogenic center. A
conformational rearrangement also occurs in the N1 phenyl ring of the S-enantiomer with respect to the corresponding R-enantiomer.
The values of the chemical shifts are expressed in ppm and the
coupling constants (J) in Hertz. Mass spectra were recorded on
3. Experimental
3.1. Chemistry
either
a Varian Saturn 3 or a ThermoFinnigan LCQ-deca
spectrometer.
All chemicals used were of reagent grade. Yields refer to puri-
fied products, and are not optimized. Melting points were deter-
mined in open capillaries on a Gallenkamp apparatus, and are
uncorrected. Microanalyses were carried out by means of a Per-
kin-Elmer 240 C or a Perkin-Elmer Series II CHNS/O Analyzer
2400. Merck silica gel 60 (230–400 mesh) was used for column
chromatography. Merck TLC plates, silica gel 60 F254, were used
for TLC. 1H NMR spectra were recorded with a Bruker AC 200
spectrometer in the indicated solvent (TMS as internal standard).
3.2. Preparation of ethyl 1,5-diarylpyrrol-3-ylglyoxylic esters
(12a–c)
These compounds were prepared starting from the correspond-
ing 1,5-diaryl-2-methylpyrroles, by regioselective acylation (at po-
sition 3) with ethoxalyl chloride in the presence of pyridine.
Analytical data, mp, and 1H NMR spectra were consistent with
those reported in the literature.23,24

8078
M. Biava et al. / Bioorg. Med. Chem. 16 (2008) 8072–8081
Figure 3. Stereographical representation of the binding mode of (À)-R-11b (green, thick lines) and (+)-S-11b (magenta, thick lines) showing the different hydrogen bonding
patterns: the terminal portion of Arg120 side chain interacts with the carbonyl oxygen of the S-enantiomer, as well as with the ether oxygen of the ester chain of the R-
enantiomer.
3.3.1. Ethyl-[2-hydroxy-2-[2-methyl-5-(4-methylsulfonyl)-
Analytical data
Compound
phenyl-1-phenyl-1H-pyrrol-3-yl]]acetate (10a)
Mp 145 °C (0.30 g, yield 50%); 1H NMR (CDCl3) 7.62–7.67 (m,
2H), 7.24–7.27 (m, 3H), 7.12–7.17 (m, 4H), 6.47–6.50 (m, 1H),
5.21 (s, 1H), 4.24–4.37 (m, 2H), 3.20–3.22 (br, 1H), 2.96–3.01 (s,
3H), 2.13–2.18 (s, 3H), 1.28–1.35 (t, 3H). Anal. (C22H23NO5S) C, H,
N.
Formula
C
H
N
Calcd %
Found%
Calcd%
Found%
Calcd%
Found%
10a
10b
10c
11a
11b
11c
C22H23NO5S
63.90
63.95
5.61
4.57
3.39
3.40
(+)-(S)-Ethyl-[2-hydroxy-2-[2-methyl-5-(4-methylsulfonyl)phenyl-
1-phenyl-1H-pyrrol-3-yl]]acetate ((+)-(S)-10a). [
a
]
D
+70° (c 0.049,
C22H22FNO5S
C23H25NO6S
C23H25NO5S
C23H24FNO5S
C24H27NO6S
61.24
61.22
5.14
5.10
4.40
4.45
EtOH).
(À)-(R)-Ethyl-[2-hydroxy-2-[2-methyl-5-(4-methylsulfonyl)phenyl-
1-phenyl-1H-pyrrol-3-yl]]acetate. (À)-(R)-10a. [a D À60° (c 0.049,
EtOH).
62.29
62.30
5.68
5.65
3.16
3.18
]
64.62
64.58
5.89
5.90
3.28
3.22
3.3.2. Ethyl-[2-hydroxy-2-[1-(3-fluoro)phenyl-2-methyl-5-(4-
methylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate (10b)
Mp 173 °C (0.315 g, yield 50%); 1H NMR (CDCl3): 7.66–7.72 (d,
2H), 7.37–7.38 (m, 1H), 7.24–7.25 (d, 2H), 6.90–6.93 (m, 3H),
6.46–6.47 (s, 1H), 5.18 (s, 1H), 4.22–4.35 (m, 2H), 2.99–3.20 (br,
1H) 2.98 (s, 3H), 2.15 (s, 3H), 1.24–1.32 (m, 3H). Anal.
(C22H22FNO5S) C, H, N.
62.01
62.00
5.43
5.40
4.26
4.20
63.00
63.02
5.65
5.63
3.06
3.00
(+)-(S)-Ethyl-[2-hydroxy-2-[1-(3-fluoro)phenyl-2-methyl-5-(4-
methylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate ((+)-(S)-10b).
+88° (c 0.019, EtOH).
[a]
D
3.3. General procedure for the preparation of 1,5-diarylpyrrole-
3-( -hydroxy) acetic esters (10a–c)
(À)-(R)-Ethyl-[2-hydroxy-2-[1-(3-fluoro)phenyl-2-methyl-5-(4-
a
methylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate ((À)-(R)-10b). [
a] =
D
À81° (c 0.046, EtOH).
To a solution of the appropriate ethyl 1,5-diarylpyrrol-3-ylgly-
oxylic ester 12a–c (1.46 mmol) in CH2Cl2 (9 mL) stirred at rt, ZnI2
(0.67 g, 2.17 mmol) was added. Sodium cyanoborohydride
(0.07 g, 1.08 mmol) was added after 5 min, the mixture was left
to react for 2 h, under nitrogen atmosphere, stirring at rt. At the
end, tert-butyl alcohol (100 mL) was added. The mixture was fil-
tered on Celite and the solution obtained was acidified with a solu-
tion of ammonium chloride (10%) in HCl 6 N and extracted with
CHCl3. The organic solution was washed, dried, and evaporated in
vacuo. The resulting residue was chromatographed on silica gel
eluting with ether/ethyl acetate (1:1) to give a solid, which, after
re-crystallization from ethyl acetate, afforded the required prod-
uct. Enantiomers of each compound described have been separated
by chromatography, using a Chiral Pack IA, chiral stationary phase,
with n-hexane/CH2Cl2/ethanol (50:50:1) as the eluant.
3.3.3. Ethyl-[2-hydroxy-2-[1-(4-methoxy)phenyl-2-methyl-5-
(4-methylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate (10c)
Mp 130 °C (0.324 g, yield 50%); 1H NMR (CDCl3): 7.65–7.67 (d,
2H), 7.16–7.18 (d, 2H), 7.12–7.15 (m, 2H), 7.06–7.11 (m, 2H),
6.46 (s, 1H), 5.02 (s, 1H), 4.19 (m, 2H), 3.85 (s, 3H), 3.20–3.25 (br,
1H), 3.00 (s, 3H), 2.14 (s, 3H), 1.29–133 (t, 3H). Anal. (C23H25NO6S)
C, H, N.
(+)-(S)-Ethyl-[2-hydroxy-2-[1-(4-methoxy)phenyl-2-methyl-5-(4-
methylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate ((+)-(S)-10c).
+57° (c 0.086, EtOH).
[a
]
D
(À)-(R)-Ethyl-[2-hydroxy-2-[1-(4-methoxy)phenyl-2-methyl-5-
(4-methylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate ((À)-(R)-10c). [
a
]
D
À58° (c 0.091, EtOH).

M. Biava et al. / Bioorg. Med. Chem. 16 (2008) 8072–8081
8079
Figure 4. Graphical representation of two different views of the complex between COX-2 and (À)-R-10c. Analysis of the molecular surface of inhibitor and portion of the
hydrophobic pocket of the binding site shows a perfect complementarity between hydrophobic amino acid side chains (Phe381, Tyr385, Leu384, and Trp387) and the 4-
methoxy group of the inhibitor (labeled as Me for the methyl portion and as O for the oxygen atom). The picture of the left panel shows the inhibitor in an orientation
comparable to that of Figure 2A, while in the right panel the complex was rotated 180° on the Y-axis.
3.4. General procedure for the preparation of 1,5-diarylpyrrole-
3-( -ethoxy) acetic esters (11a–c)
6.55–6.57 (s, 1H), 4.79–4.82 (s, 1H), 4.23–4.27 (m, 2H), 3.43–3.45
(m, 2H) 2.96–3.00 (s, 3H), 2.16 (s, 3H), 1.28–1.32 (t, 6H). Anal.
(C24H26FNO5S) C, H, N.
a
To a solution of the appropriate ethyl 1,5-diarylpyrrol-3-ylgly-
(+)-(S)-Ethyl-[2-ethoxy-2-[1-(3-fluoro)phenyl-2-methyl-5-(4-meth-
oxylic esters 12a–c (1.46 mmol) in CH2Cl2 (9 mL) stirred at rt, ZnCl2
(0.67 g, 2.17 mmol) was added. Sodium cyanoborohydride (0.07 g,
1.08 mmol) was added after 5 min, the mixture was left to react for
2 h, under nitrogen atmosphere, stirring at rt. At the end, ethyl
alcohol (100 mL) was added. The mixture was filtered on Celite,
and the solution obtained was acidified with a solution of ammo-
nium chloride (10%) in HCl 6 N and extracted with CHCl3. The or-
ganic solution was washed, dried, and evaporated in vacuo. The
resulting residue was chromatographed on silica gel eluting with
ether/ethyl acetate (1:1) to give a solid, which, after re-crystalliza-
tion from ethyl acetate, afforded the required product. Enantio-
mers of the compound described were separated by
chromatography, using a Chiral Pack IA, chiral stationary phase,
with n-hexane/CH2Cl2/ethanol (70:25:0.2) as the eluant.
ylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate ((+)-(S)-11b). [a D
0.041, EtOH).
]
+45° (c
(À)-(R)-Ethyl-[2-ethoxy-2-[1-(3-fluoro)phenyl-2-methyl-5-(4-methyl
sulfonyl)phenyl-1H-pyrrol-3-yl]]acetate ((À)-(R)-11b).
[
a D
]
À34° (c
0.020, EtOH).
3.4.3. Ethyl-[2-ethoxy-2-[1-(4-methoxy)phenyl-2-methyl-5-(4-
methylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate (11c)
Mp 159 °C (0.344 g, yield 50%); 1H NMR (CDCl3): 7.65–7.67 (d,
2H), 7.14–7.16 (d, 2H), 7.04–7.08 (m, 2H), 6.88–6.90 (m, 2H),
6.56 (s, 1H), 4.89–490 (s, 1H), 4.18–4.28 (m, 2H), 3.77–3.83 (s,
3H), 3.56–3.65 (m, 2H), 2.92–2.98 (s, 3H), 2.14 (s, 3H), 1.23–131
(t, 6H). Anal. (C25H29NO6S) C, H, N.
(+)-(S)-Ethyl-[2-ethoxy-2-[1-(4-methoxy)phenyl-2-methyl-5-(4-
methylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate ((+)-(S)-11c).
+32° (c 0.040, EtOH).
[a]
D
3.4.1. Ethyl-[2-ethoxy-2-[-2-methyl-5-(4-methylsulfonyl)-
phenyl-1-phenyl-1H-pyrrol-3-yl]]acetate (11a)
(À)-(R)-Ethyl-[2-ethoxy-2-[1-(4-methoxy)phenyl-2-methyl-5-(4-
Mp 165 °C (0.322 g, yield 50%); 1H NMR (CDCl3) 7.62–7.64 (m,
2H), 7.24–7.27 (m, 3H), 7.12–7.15 (m, 4H), 6.47–6.60 (m, 1H),
4.92 (s, 1H), 4.15–4.40 (m, 2H), 3.50–3.70 (m, 2H), 2.97–3.00 (s,
3H), 2.13–2.18 (s, 3H), 1.28–1.32 (t, 6H). Anal. (C24H27NO5S) C, H,
N.
methylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate ((À)-(R)-11c).
À43° (c 0.043, EtOH).
[a]
D
3.5. Biology
(+)-(S)-Ethyl-[2-ethoxy-2-[-2-methyl-5-(4-methylsulfonyl)phenyl-
Arachidonic acid was obtained from SPIBIO (Paris, France).
[3H]PGE2 and [3H]TXB2 were from Perkin-Elmer Life Sciences (Mi-
lan, Italy). Compound 1b was kindly provided by Merck (Darms-
tadt, Germany), 2 and all other reagents and compounds used
were obtained from Sigma–Aldrich (Milan, Italy).
1-phenyl-1H-pyrrol-3-yl]]acetate ((+)-(S)-11a). [
a]
D
+41° (c 0.027,
EtOH).
(À)-(R)-Ethyl-[2-ethoxy-2-[-2-methyl-5-(4-methylsulfonyl)phenyl-
1-phenyl-1H-pyrrol-3-yl]]acetate ((À)-(R)1-1a). [a D À52° (c 0.022,
EtOH).
]
3.5.1. Cellular assay
3.4.2. Ethyl-[2-ethoxy-2-[1-(3-fluoro)phenyl-2-methyl-5-(4-
methylsulfonyl)phenyl-1H-pyrrol-3-yl]]acetate (11b)
3.5.1.1. Cell culture. The murine monocyte/macrophage J774 cell
line was grown in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 2 mM glutamine, 25 mM Hepes, penicillin
Mp 165 °C (0.369 g, yield 55%); 1H NMR (CDCl3): 7.66–7.72 (d,
2H), 7.37–7.38 (m, 1H), 7.23–7.25 (d, 2H), 6.90–6.93 (m, 3H),
(100 U/mL), streptomycin (100 lg/mL), 10% fetal bovine serum

8080
M. Biava et al. / Bioorg. Med. Chem. 16 (2008) 8072–8081
(FBS), and 1.2% sodium pyruvate (Bio Whittaker, Europe). Cells were
plated in 24-well culture plates at a density of 2.5 Â 105 cells/mL or
in 10 cm-diameter culture dishes (1 Â 107 cells/10 mL/dish) and
were allowed to adhere at 37 °C in 5% CO2/95% O2 for 2 h. Immedi-
ately before the experiments, the culture medium was replaced by
a fresh medium without FBS in order to avoid interference with
radioimmunoassay,31 and cells were stimulated as described.
3.5.2.5. Analysis of PGE2 and TXB2. PGE2 and TXB2 concentrations
were measured by previously described and validated radioimmu-
noassays.5,32 Unextracted plasma and serum samples were diluted
in the standard diluent of the assay (0.02 M phosphate buffer, pH
7.4), and were assayed in a volume of 1.5 mL at a final dilution of
1:50–1:30,000. [3H]PGE2 or [3H]TXB2 (4000 dpm, specific activity
>100 Ci/mmol, Perkin-Elmer Life Science Products, Brussels, Bel-
gium) and specific anti-PGE2 (1:100,000 dilution) and anti-TXB2
(1:120,000 dilution) sera were used. The least detectable concen-
tration was 1–2 pg/mL for both prostanoids.
3.5.1.2. Assessment of COX-1 activity. Cells were pre-treated with
the reference standard (1b) or the test compounds (0.01–100
lM)
for 15 min, and were further incubated at 37 °C for 30 min with
15
l
M arachidonic acid in order to activate the constitutive
3.5.2.6. Statistical analysis. Results are expressed as the mean,
for three experiments (triplicate wells were used for the various
conditions of treatment, in the cell culture assay), of the percent
inhibition of prostanoid production assessed in the absence of
the test compounds (control). Concentration–response curves
were fitted, and IC50 values were analyzed with PRISM (Graph-
Pad, San Diego, CA, USA) and ALLFIT, a basic computer program
for simultaneous curve-fitting based on a four-parameter logistic
equation.
COX.31 Stock solutions of the reference standard or of the test com-
pounds were prepared in dimethyl sulfoxide, and an equivalent
amount of dimethyl sulfoxide was included in control samples.
At the end of the incubation, the supernatants were collected for
the measurement of PGE2 levels by radioimmunoassay.
3.5.1.3. Assessment of COX-2 activity. Cells were stimulated for
24 h with Escherichia coli lipopolysaccharide (LPS, 10 lg/mL) to in-
duce COX-2, in the absence or in the presence of test compounds,
at the concentrations previously reported. The supernatants were
collected for the measurement of PGE2 levels by
radioimmunoassay.
3.5.3. In vivo anti-inflammatory activity
3.5.3.1. Animals. Male Swiss albino mice (23–25 g) and Sprague–
Dawley or Wistar rats (150–200 g) were used. Fifteen mice and
four rats were housed per cage. The cages were placed in the
experimental room 24 h before the test for acclimatization. The
animals were fed in a standard laboratory diet and tap water ad
libitum, and were kept at 23 1 °C with a 12 h light/dark cycle,
light on at 7 am. All experiments were carried out in accordance
with the NIH Guide for the Care and Use of Laboratory animals.
All efforts were made to minimize animal suffering, and to reduce
the number of animals used.
3.5.1.4. Statistical analysis. 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. Data fit was
obtained using the sigmoidal dose–response equation (variable
slope) (GraphPad software).
3.5.2. Human whole blood (HWB) assay
3.5.2.1. Subjects. Three healthy volunteers (2 female and 1 male,
aged 29 3 years) were enrolled to participate in the study after
its approval by the Ethical Committee of the University of Chieti.
Informed consent was obtained from each subject.
3.5.3.2. Paw-pressure test. The nociceptive threshold in the rat
was determined with an analgesimeter, according to the method
described by Leighton.33 Threshold pressure was measured before
and 30, 60, and 120 min after treatment. An arbitrary cut-off value
of 250 g was adopted. In order to induce an inflammatory process
in the rat, paw carrageenan (0.1 mL, 1%) was administered ip 4 h
before test.
3.5.2.2. COX-2 assay. To evaluate COX-2 activity, 1 mL aliquots of
peripheral venous blood samples containing10 iu of sodium heparin
were incubated in thepresence ofLPS (10 lg/mL)or saline for 24 h at
37 °C as previously described.5 The contribution of platelet COX-1
was suppressed by pre-treating the subjects with aspirin (300 mg,
48 h) before sampling. Plasma was separated by centrifugation
(10 min at 2000 rpm), and was kept at À80 °C until assayed by RIA
for PGE2, as an index of LPS-induced monocyte COX-2 activity.
3.5.3.3. Carrageenan-induced paw edema. Rat paw volumes
were measured using a plethysmometer. Five hours after the injec-
tion of carrageenan (0.1 mL injection of 1.0%) the paw volume of
the right hind paw was measured and compared with saline/carra-
geenan-treated controls.34 Rats received test compounds 4 h after
carrageenan. Results are reported as paw volume expressed in
milliliter.
3.5.2.3. COX-1 assay. Peripheral venous blood samples were
drawn from the same donors when they had not taken any NSAID
during the 2 weeks preceding the study. One-milliliter aliquots of
whole blood were immediately transferred into glass tubes, and
were allowed to clot at 37 °C for 1 h. Serum was separated by cen-
trifugation (10 min at 3000 rpm), and was kept at À80 °C until as-
sayed for TXB2. Whole blood TXB2 production was measured by
RIA as a reflection of maximally stimulated platelet COX-1 activity
in response to endogenously formed thrombin.32
3.5.3.4. Abdominal writhing test. Mice were injected ip with a
0.6% solution of acetic acid (10 mL/kg), according to Koster.35 The
number of stretching movements was counted for 10 min, starting
5 min after acetic acid injection.
Acknowledgment
Rottapharm S.p.A., Monza, Italy, is acknowledged for partial
financial support.
3.5.2.4. Effects of COX-2 Inhibitors on whole blood COX-2 and
COX-1 activities. Compounds 1b (0.005–50 mM),
200 mM), and 10b (0.5–50 mM) were dissolved in DMSO. Aliquots
of the solutions (2 L) were pipetted directly into test tubes to give
final concentrations of 0.01–100 M in heparinized whole blood
samples in the presence of LPS (10 g/mL) for 24 h or with whole
2 (0.005–
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