Synthesis, biological evaluation, and enzyme docking simulations of 1,5-diarylpyrrole-3-alkoxyethyl ethers as selective cyclooxygenase-2 inhibitors endowed with anti-inflammatory and antinociceptive activity

Article abstract of DOI:10.1021/jm800084s

A series of substituted 1,5-diarylpyrrole-3-alkoxyethyl ethers (6, 7, and 8) has been synthesized with the aim to assess if in the previously reported 1,5-diarylpyrrole derivatives (5) the replacement of the acetic ester moiety with an alkoxyethyl group s

Full text of DOI:10.1021/jm800084s

4476
J. Med. Chem. 2008, 51, 4476–4481
Synthesis, Biological Evaluation, and Enzyme Docking Simulations of
1,5-Diarylpyrrole-3-Alkoxyethyl Ethers as Selective Cyclooxygenase-2 Inhibitors Endowed with
Anti-inflammatory and Antinociceptive Activity
Maurizio Anzini,*,† Michele Rovini,† Andrea Cappelli,† Salvatore Vomero,† Fabrizio Manetti,† Maurizio Botta,† Lidia Sautebin,‡
Antonietta Rossi,‡,§ Carlo Pergola,‡ Carla Ghelardini,| Monica Norcini,| Antonio Giordani, Francesco Makovec,
Paola Anzellotti,# Paola Patrignani,# and Mariangela Biava
Dipartimento Farmaco Chimico Tecnologico, UniVersita` di Siena, Via A. Moro, 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 Preclinica e Clinica “M. Aiazzi Mancini”,
UniVersita` degli Studi di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy, Rottapharm S.p.A., Via Valosa di Sopra 7, 20052 Monza, Italy,
Dipartimento di Medicina e Scienze dell’InVecchiamento, Sezione di Farmacologia, UniVersita` di Chieti “G. D’Annunzio” e CeSI,
Via dei Vestini 31, 66013 Chieti, Italy, Dipartimento di Chimica e Tecnologie del Farmaco, UniVersita` “La Sapienza”, Ple A. Moro 5,
I-00185 Roma, Italy
ReceiVed January 28, 2008
A series of substituted 1,5-diarylpyrrole-3-alkoxyethyl ethers (6, 7, and 8) has been synthesized with the
aim to assess if in the previously reported 1,5-diarylpyrrole derivatives (5) the replacement of the acetic
ester moiety with an alkoxyethyl group still led to new, highly selective and potent COX-2 inhibitors. In the
in vitro cell culture assay, all the compounds proved to be potent and selective COX-2 inhibitors. In the
human whole blood (HWB) assay, compound 8a had a comparable COX-2 selectivity to valdecoxib, while
it was more selective than celecoxib but less selective than rofecoxib. The potential anti-inflammatory and
antinociceptive activities of compounds 7a, 8a, and 8d were evaluated in vivo, where they showed a very
good activity against both carrageenan-induced hyperalgesia and edema in the rat paw test. In the abdominal
constriction test compound 7a, 8a, and 8d were able to reduce the number of writhes in a statistically
significant manner. Furthermore, the affinity data of these compounds have been rationalized through enzyme
docking simulations in terms of interactions with a crystallographic model of the COX-2 binding site by
means of the software package Autodock 3.0.5, GRID 21, and MacroModel 8.5 using the complex between
COX-2 and SC-558 (1b), refined at a 3 Å resolution (Brookhaven Protein Data Bank entry: 6cox)
Introduction
COX-2 oxygenates neutral fatty acid derivatives such as
endocannabinoids, which are poor substrates for COX-1.4
COX-1 is the major form expressed in healthy tissues and
plays a role in thrombogenesis and in the homeostasis of the
gastrointestinal tract.5 COX-2 is induced in many cell types in
response to bacterial endotoxin, cytokines, including IL-1,
TNFR, and growth factors, and is down-regulated by dexam-
ethasone.5 Both enzymes are inhibited by traditional (t)nonste-
roidal anti-inflammatory drugs (NSAIDs).5–7 Inhibition of
COX-2 is involved in the therapeutic effects of (t)NSAIDs, while
the inhibition of COX-1 is associated with gastrointestinal
toxicity.7,8
The identification of the selective COX-2 inhibitory prototype
“sulide” compound NS-398, and the “coxib” compound DuP 697,
the first diarylheterocycle, served as the rationale for the original
concept that a drug having a greater selectivity for the pro-
inflammatory inducible COX-2 isozyme, relative to the cytopro-
tective constitutive COX-1 isozyme, would reduce the incidence
of gastrointestinal bleeding. Subsequent drug discovery programs
led to the clinical development of diarylheterocycles as the major
class of selective COX-2 inhibitors such as celecoxib (1a),
rofecoxib (2), and valdecoxib (3) (Chart 1).9
Cyclooxygenase [(prostaglandin (PGa) H synthase] catalyzes
the oxygenation of arachidonic acid (AA) to prostaglandin H2
as a first step in the synthesis of PGs, prostacyclin and
thromboxane (TX)A2.1,2 The enzyme is expressed in mammalian
cells as two distinct isozymes (named COX-1 and COX-2) that
show about 60% amino acid sequence. COX-1 and COX-2 share
the same catalytic activities, i.e., cyclooxygenase and peroxi-
dase,1 but they are differently regulated. In fact, it has been
shown that COX-2 requires considerably lower levels of
hydroperoxides to initiate cyclooxygenase catalysis than those
required by COX-1.2 Moreover, COX-1 seems to prefer
exogenous AA, while COX-2 prefers endogenous AA.3 Finally,
* To whom correspondence should be addressed. Phone: +39-0577
234173. Fax: +39-0577 234333. E-mail: anzini@unisi.it.
†
Dipartimento Farmaco Chimico Tecnologico, Universita` di Siena.
Dipartimento di Farmacologia Sperimentale, Universita` di Napoli
‡
“Federico II”.
§
IRCCS Centro Neurolesi ”Bonino-Pulejo”.
Dipartimento di Farmacologia Preclinica e Clinica “M. Aiazzi Mancini”,
|
Universita` degli Studi di Firenze.
Rottapharm S.p.A.
The gastrointestinal benefit of coxibs is obliterated by the
finding of increased incidence of vascular events.10 However,
the results of observational studies and randomized clinical trials
have shown that the cardiovascular hazard is not restricted to
selective COX-2 inhibitors but also attached to some (t)NSAIDs,
such as diclofenac.11,12 The most plausible mechanism is the
suppression of COX-2-dependent prostacyclin, leaving uncon-
#
Dipartimento di Medicina e Scienze dell’Invecchiamento, Sezione di
Farmacologia, Universita` di Chieti ”G. D’Annunzio” e CeSI.
Dipartimento di Chimica e Tecnologie del Farmaco, Universita` “La
Sapienza”.
a Abbreviations: PG, prostaglandin; AA, arachidonic acid, TX, trom-
boxane; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; IL-1, in-
terleukin-1, TNFR, tumor necrosis factor-R; (t)NSAIDs, traditional non-
steroidal anti-inflammatory drugs; HWB, human whole blood.
10.1021/jm800084s CCC: $40.75
2008 American Chemical Society
Published on Web 07/04/2008

1,5-Diarylpyrrole-3-Alkoxyethyl Ethers as COX-2 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4477
Chart 1. Structures of Compounds 1-8
Scheme 1a
a Compounds: 6a, X ) H, R ) Me; 7a, X ) H, R ) Et; 8a, X ) H, R ) n-Pr; 6c, X ) CF3, R ) Me; 7c, X ) CF3, R ) Et; 8c, X ) CF3, R ) n-Pr;6d,
X ) F, R ) Me; 7d, X ) F, R ) Et; 8d, X ) F, R ) n-Pr. Reagents and conditions: (i) LiAlH4, THF, room temp, 20 min; (ii) KOH, RI, DMSO, room
temp, 30 min.
strained the intricate network of stimuli predisposing to throm-
bosis, atherogenesis, and hypertension, such as TXA2.10,12 The
finding of a marked variability in how each person reacts to
these drugs, mainly based on their genetic background,13
encourages the development of novel compounds to increase
the spectrum of therapeutic opportunities for each individual
patient.14
ate ethyl acetate ester 5 prepared as previously reported.15 By
treatment with lithium aluminum hydride in dry THF at room
temperature, compounds 5 were reduced to the respective
alcohol derivatives 9. These compounds were alkylated by the
suitable alkyl iodide in the presence of powdered KOH in
dimethylsulfoxide (DMSO), to afford the expected 1,5-diaryl-
3-(2-alkoxyethyl)pyrroles 6, 7, and 8 in satisfactory yield.
Within a wide research program focused on the synthesis of
new 3-substituted-1,5-diarylpyrrole derivatives 515–17 as selective
COX-2 inhibitors in which the pyrroleacetic and vicinal diaryl
heterocyclic moieties were reminiscent of indomethacin (4) and
of the above “coxib” family, respectively, the synthesis, the
biological evaluation, and enzyme docking simulations of the
hitherto unknown 1,5-diarylpyrrole-3-alkoxyethyl ethers 6, 7,
and 8 are here reported.
Compounds 7a, 8a, and 8d, with the best biological profile
in terms of affinity and selectivity toward COX-2, as well as
percent inhibition of the enzyme (cell culture assay), were also
investigated for their in vivo anti-inflammatory and analgesic
activity. Moreover, the selectivity of compound 8a was also
determined by the human whole blood assay (HWB) in vitro,
which assesses the pattern of relative inhibition for human
platelet COX-1 and monocyte COX-2.17
In the in vitro cell culture assay, biological evaluation of the
title compounds showed that, in 1,5-diarylpyrrole derivatives
5, the replacement of the acetic ester moiety with an alkoxyethyl
group still led to new, highly selective, and potent COX-2
inhibitors, the activities of which are presented in Table 1.
The transformation of the ester moiety at position 3 of the
pyrrole scaffold into an ether substituent produces a significant
difference in terms of inhibitory activity. Actually, compounds
6, 7, and 8 are in general more potent than the corresponding
previously reported esters 5.15
At a first glance, these data seem to be surprising if we take
into consideration the different stereoelectronic properties of
the acetic ester chain with respect to the alkoxyethyl moiety
and in view of their mode of interaction with one of the pockets
of the COX-2 binding site.
In fact, as it has previously been reported,15 the ester chain
of compounds 5a-c is clearly involved with its oxygen atoms
in a network of three hydrogen bonds with the guanidino group
of Arg120, in a portion of the COX-2 binding site referred to
as the ”classical NSAIDs carboxylate binding site” by Kurum-
bail and co-workers.18 On the other hand, it is less clear how
Results and Discussion
Scheme 1 summarizes the synthetic procedures used to obtain
the 1,5-diaryl-3-(2-alkoxyethyl)pyrroles considered in this study.
The target compounds were synthesized starting from appropri-

4478 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Anzini et al.
Table 1. In Vitro COX-1 and COX-2 Inhibitory Activity of Compounds
5, 6, 7, and 8
COX-1 IC50 COX-2 IC50 COX-1/COX-2
compd
X
R
(µM)a
(µM)a
(SI)b
5ac
5bc
5cc
6a
6c
6d
7a
7c
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.040
0.480
0.060
0.048
0.049
0.018
0.015
0.085
0.047
0.018
0.110
0.030
>2500
>208
CH3
CF3
H
CF3
F
H
CF3
F
>1600
>2083
>2040
>5555
>6666
>1176
>2127
>5555
>909
Me
Me
Me
Et
Et
Et
n-Pr
n-Pr
n-Pr
7d
8a
8c
H
CF3
F
Figure 1. Graphical representation of the best pose found for
compound 7a (ball and stick notation) within the binding site of COX-
2. Hydrogen bond interactions (involving the ether oxygen atom and
one of the sulfone oxygens) are coded by black dashed lines. For the
sake of clarity, only a few amino acids of the binding site are displayed.
8d
>3333
a Results are expressed as the mean of three experiments of the %
inhibition of PGE2 production by the test compounds with respect to control
samples. b In vitro COX-2 selectivity index [IC50(COX-1)/IC50(COX-2)].
c See ref 15.
which shows an activity (0.030 µM) higher than that of the
corresponding ethyl ether analogue 7d (0.047 µM). Interestingly,
the less bulky ether in the subseries of the 4′-fluoro derivatives
(compound 6d) shows an IC50 of 0.018 µM. Moreover, activity
of fluoro derivatives 6d, 7d, and 8d was better than that of the
corresponding CF3 analogues 6a, 7a, and 8a, respectively. This
result appears to be in full agreement with the analysis of the
Grid map generated for the fluoride probe that suggested for
the 4′-fluorine substituent (as it occurs in the case of the bromine
atom of SC-558, 1b) an improving effect on the interaction with
the COX-2 binding site, involving Trp387, Tyr348, and Tyr385
of the hydrophobic cavity15 with respect to the CF3 substituent.
Finally, the 1-aryl substituent showed the usual interaction
pathway with the hydrophobic pocket of protein, as well as the
aryl moiety at C5 that is in contact with the selectivity site with
both hydrophobic interactions and hydrogen bonds.
On the basis of their very encouraging COX-2 inhibitory
activity evidenced in the in vitro tests, compounds 7a, 8a, and
8d were selected and submitted to further pharmacological tests
to assess their in vivo anti-inflammatory and antinociceptive
activities by carrying out the carrageenan-induced rat paw test,
the paw volume test and abdominal constriction test (Tables 2
and 3).
These compounds, administered at a dose of 20 mg/kg po,
showed a very good activity against carrageenan-induced
hyperalgesia 30 and 60 min after administration, disappearing
almost completely 2 h after treatment (Table 2). At the same
time, a very good activity was demonstrated against carrageenan-
induced edema in the rat paw (Table 2), with a complete
remission 1 h after the administration of all compounds at the
same dose of 20 mg/kg po. In the abdominal constriction test
compounds 7a, 8a, and 8d were able to reduce the number of
writhes in a statistically significant manner at the dose of 20
mg/kg po, while at the dose of 10 mg/kg po, they were devoid
of any antinociceptive efficacy (Table 3).
the alkoxyethyl derivatives 6, 7, and 8, even in the absence of
the carbonyl group, are able to exploit profitable interactions,
eliciting, in the in vitro using the cell culture test, a highly
selective and potent COX-2 inhibitory activity. In particular,
compounds 7a and 8a showed IC50 values of 0.015 and 0.018
µM, respectively, (Table 1) which are comparable to the IC50
of rofecoxib (0.012 µM, ref 19). This behavior may be probably
due to the presence of an oxygen atom in the side chain along
with the more lipophilic character of the alkoxyethyl moiety
capable of tighter interactions with the hydrophobic environment
of the COX-2 “carboxylate binding site”. In fact, the binding
mode of such compounds was characterized by the ether group
located within the carboxylate site with its oxygen atom involved
in hydrogen bonds with the guanidino moiety of Arg120 (Figure
1). Moreover, the alkyl portion of the ether side chain was
embedded into a lipophilic region defined by Leu93, Val116,
and Leu359, where the best interaction point with a C3 probe
was found by means of GRID software. In fact, GRID
calculations using a C3 probe, intended to analyze hydrophobic
interactions between a methyl group (the probe) and the surface
of COX-2,15 found a region of very profitable interactions
between the probe and the protein close to the location of the
terminal methyl group of the ethyl ether chain.
Significant differences in inhibitory activity are observed
when the substitution pattern is considered (e.g., the presence
of alkoxy or the 4′-phenyl substituent at N1 position of the
pyrrole ring or the contemporary presence of both moieties).
In fact, compounds 7a and 8a appear to be the most active
compounds in the in vitro and in vivo tests. This means that
the contemporary presence of an ethoxy or a propoxy group
and that of the unsubstituted 1-phenyl ring represents the best
requisite in order to fit both the “carboxylate binding site” and
the hydrophobic binding site.
On the contrary, in the presence of a 4′-trifluoromethyl or a
4′-fluoro substituent at 1-phenyl ring as in compounds 6c,d, 7c,d,
and 8c,d, the COX-2 inhibitory activity slightly decreases with
the increase in the side chain length, with the exception of 8d,
Among the title compounds, 8a was selected and submitted
to a further biochemical selectivity test. In particular, the HWB
assay was performed to predict the actual extent of isozyme

1,5-Diarylpyrrole-3-Alkoxyethyl Ethers as COX-2 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4479
Table 2. Effect of Compounds 7a, 8a, and 8d on Hyperalgesia and Edema Induced by Carrageenan in the Rat Paw-Pressure Testa
paw pressure in rats (g)
after treatment
60 min
paw volume (mL)
pre-treatment
saline
before treatment
30 min
120 min
before treatment
60 min after treatment
saline
saline
7a
63.5 ( 4.7
62.6 ( 4.3
60.5 ( 4.3
59.2 ( 5.2
63.5 ( 4.3
60.2 ( 5.0
36.4 ( 5.2
59.6 ( 4.7b
56.3 ( 5.6b
52.4 ( 5.2b
58.9 ( 6.3
38.4 ( 5.2
53.8 ( 4.2b
59.4 ( 5.1b
56.8 ( 4.9b
61.4 ( 5.1
41.2 ( 4.8
49.1 ( 5.4
50.6 ( 5.3
52.7 ( 4.0
1.29 ( 0.08
1.25 ( 0.09
1.29 ( 0.10
1.32 ( 0.06
1.25 ( 0.09
1.34 ( 0.10
2.48 ( 0.07
1.37 ( 0.11b
1.41 ( 0.14b
1.45 ( 0.12b
carrageenan
carrageenan
carrageenan
carrageenan
8a
8d
a All compounds were administered at the dose of 20 mg kg-1 po. Carrageenan (100 µL, 1%) was administered ipl. 4 h before test. Test was performed
30 min after the compound injection. There were 5-6 rats per group. b P < 0.01 versus the corresponding carrageenan-treated rat.
Table 3. Effect of Compounds 7a, 8a, and 8d in the Mouse Abdominal
Constriction Test (Acetic Acid 0.6%)
treatmenta
no. of mice
dose per os mg kg-1
no. of writhes
CMC
7a
7a
8a
8a
8d
8d
20
10
8
9
9
34.7 ( 3.9
26.5 ( 3.7
11.4 ( 3.6b
27.43 ( 4.1
13.6 ( 4.2b
38.1 ( 3.4
16.7 ( 3.5b
10
20
10
20
10
20
10
10
a All drugs were administered per os 30 min before test. b P < 0.01
versus vehicle-treated mice.
inhibition achievable in vivo by circulating drug levels because
of a number of variables are potentially able to affect
drug-enzyme interaction. In particular, the use of exogenous
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 conformation change in the enzyme
binding site.19 The results showed that, in HWB assay,
compound 8a was 66-fold more potent toward COX-2 than
COX-1 (Figure 2B). Interestingly, it had a comparable COX-2
selectivity to valdecoxib (Figure 2A, COX-1/COX-2 IC50 ratio
of 52), while it was more selective than celecoxib15 but less
selective than rofecoxib (which was previously shown to be
>200-fold more potent toward COX-2).20 In fact, we have
previously found that celecoxib showed a COX-1/COX-2 IC50
ratio of 23-29.6 in the HWB assay.17,20 The lower selectivity
showed by compound 8a in the HWB assay versus the cell
culture assay was plausible due to the use of exogenous AA
and/or murine COX-isozymes.
Figure 2. In vitro inhibition (whole blood assay) of COX-1 and COX-2
by valdecoxib (A) and 8a (B). Results are expressed as average percent
inhibition of three donors of prostanoid production assessed in the
absence of the test compounds (control). COX-2 selectivity index
[IC50(COX-1)/IC50(COX-2)].
Conclusion
the dose of 20 mg/kg po. In conclusion, compounds 6-8 can
be considered interesting candidates for further preclinical
studies.
A small set of novel 1,5-diarylpyrrole-3-alkoxyethyl ethers
has been designed, synthesized, and tested. All of them have
proved to be potent and highly selective COX-2 inhibitors in
in vitro tests. In particular, compounds 7a and 8a appear to be
equipotent with rofecoxib. In the HWB, compound 8a was
demonstrated to be as selective as valdecoxib. Enzyme docking
simulations demonstrate that the binding mode of such com-
pounds is characterized by the ether group located into the
“carboxylate site” with its oxygen atom involved in a hydrogen
bond with the guanidino moiety of Arg120. Moreover, the alkyl
portion of the ether side chain was embedded into a lipophilic
region defined by Leu93, Val116, and Leu359. The potential
anti-inflammatory and antinociceptive activities of compounds
7a, 8a, and 8d were evaluated in vivo, where they showed a
very good activity against both carrageenan-induced hyperal-
gesia and carrageenan-induced edema in the rat paw, with a
complete remission 1 h after the administration. In the abdominal
constriction test compound 7a, 8a, and 8d were able to reduce
the number of writhes in a statistically significant manner at
Experimental Section
Chemistry. 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 uncorrected. Microanalyses were carried out by means of
Perkin-Elmer 240C or a Perkin-Elmer series II CHNS/O analyzer,
model 2400. Merck silica gel 60 (230-400 mesh) was used for
column chromatography. Merck TLC plates and 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). The values of the chemical shifts are expressed in ppm,
and the coupling constants (J) are expressed in Hz. Mass spectra
were recorded on a Varian Saturn 3 or a ThermoFinningan LCQ-
deca spectrometer.
General Procedure for the Preparation of 1,5-Diaryl-
3-(2-hydroxyethyl)pyrroles (9a,c,d). A solution of the suitable ethyl
acetate ester 5a,c,d (1.3 mmol) in dry THF (5 mL) was added

4480 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Anzini et al.
dropwise to a stirred suspension of lithium aluminum hydride (2.8
mmol) in dry THF (20 mL). After stirring under nitrogen
atmosphere for 20 min, the excess of reducing agent was decom-
posed by careful addition of H2O (2 mL). The inorganic material
was filtered off and washed with THF. The filtrate was dried
(Na2SO4) and evaporated under reduced pressure. The residue,
purified by flash-chromatography (EtOAc/hexane 6:4 v/v), gave the
expected compound.
General Procedure for the Preparation of 1,5-Diaryl-
3-(2-alkoxyethyl)pyrroles (6a,c,d, 7a,c,d, 8a,c,d). A suspension of
powdered KOH (0.7 mmol) in DMSO (2 mL) was stirred for 5
min. The suitable alcohol 9a,c,d (0.2 mmol) and alkyl iodide (0.3
mmol) were added in sequence, and the mixture, after being stirred
for an additional 30 min, was poured into water and extracted with
dichloromethane. The organic extracts were washed with water,
dried over Na2SO4, and evaporated to dryness in vacuo. The residue,
purified by flash chromatography (EtOAc/hexane 4:6 v/v), gave
the expected compound as an oil.
Biology. Compounds 6a-d, 7a-d, and 8a-d were all evaluated
for their inhibitory activity toward both COX-2 and COX-1
enzymes. Arachidonic acid was obtained from SPIBIO, Paris,
France. [3H-PGE2] was from Perkin-Elmer Life Sciences (Milan,
Italy). All other reagents and compounds used were obtained from
Sigma-Aldrich, Milan, Italy.
Cellular Assay: Cell Culture. The murine monocyte/macroph-
age J774 cell line was grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 2 mM glutamine, 25 mM
Hepes, penicillin (100 u/mL), streptomycin (100 µg/mL), 10% fetal
bovine serum (FBS), and 1.2% Na pyruvate (Bio Whittaker,
Europe). Cells were plated in 24-well culture plates at a density of
2.5-105 cells/mL or in 10 cm diameter culture dishes (1-107 cells/
10 mL per dish) and allowed to adhere at 37 °C in 5% CO2/95%
O2 for 2 h. Immediately before the experiments, the culture medium
was replaced by a fresh medium without FBS in order to avoid
interference with radioimmunoassay,21 and cells were stimulated
as described.
Assessment of COX-1 Activity. The cells were pretreated with
the reference standard or the test compounds (0.01-10 µM) for
15 min and incubated at 37 °C for 30 min with 15 µM arachidonic
acid in order to activate the constitutive COX.21 Stock solutions
of the reference standard or test compounds 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 by
radioimmunoassay.
Assessment of COX-2 Activity. The cells were stimulated for
24 h with E. coli lipopolysaccharide (LPS, 10 µg/mL) to induce
COX-2 in the absence or as well as in the presence of the test
compounds at the concentrations previously reported. The super-
natants were collected for the measurement of PGE2 by radioim-
munoassay.
samples were drawn from the same donors when they had not taken
any NSAID during the 2 weeks preceding the study. Aliquots (1
mL) of whole blood were immediately transferred into glass tubes
and allowed to clot at 37 °C for 1 h. Serum was separated by
centrifugation (10 min at 3000 rpm) and kept at -80 °C until
assayed for TXB2. Whole blood TXB2 production was measured
as a reflection of maximally stimulated platelet COX-1 activity in
response to endogenously formed thrombin.23
Analysis of PGE2 and TXB2. PGE2 and TXB2 concentrations
weremeasuredbypreviouslydescribedandvalidatedradioimmunoassays.22,23
Unextracted plasma and serum samples were diluted in the standard
diluent of the assay (0.02 M phosphate buffer, pH 7.4) and assayed
in a volume of 1.5 mL at a final dilution of 1:50-1:30000.
[3H]PGE2 or [3H]TXB2 (4000 dpm, specific activity >100 Ci/mmol,
1:100000 dilution) and anti-TXB2 (1:120000 dilution) sera were
used. The least detectable concentration was 1-2 pg/mL for both
prostanoids22,23
Animals.MaleSwissalbinomice(23-25g)andSprague-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 kept
at 23 ( 1 °C with a 12 h light/dark cycle, light on at 7 a.m. 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.
Paw-Pressure Test. The nociceptive threshold in the rat was
determined with an analgesimeter, according to the method
described by Leighton et al.24 Threshold pressure was measured
before and 30, 60, and 120 min after treatment. An arbitrary cutoff
value of 250 g was adopted. To induce an inflammatory process in
the rat, paw carrageenan (0.1 mL, 1%) was administered ipl 4 h
before test.
Carrageenan-Induced Edema. Rat paw volumes were measured
using a plethysmometer. Four hours after the injection of carrag-
eenan (0.1 mL injection of 1.0%), the paw volume of the right
hind paw was measured and compared with saline/carrageenan-
treated controls. Rats received test compounds 3 h 30 min after
carrageenan. The results are reported as paw volume expressed in
mL.
Antinociceptive Assay. Antinociceptive activity was determined
by means of a 0.6% acetic acid-induced writhing in the mouse-
abdominal constriction test according to Koster.25 The number of
stretching movements was counted for 10 min, starting 5 min after
acetic acid injection.
Statistical Analysis. Triplicate wells were used for the various
conditions of the treatment in the cell culture assay throughout the
experiments. Results are expressed as the mean of three experi-
ments, of the % 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). The IC50 values were calculated by the GraphPad Instat
program (GraphPad software).
Human Whole Blood (HWB) Assay. Compound 8a was also
evaluated for COX-1 versus COX-2 selectivity by means of the
HWB assay.
Subjects. Three healthy volunteers (2 females 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.
Results from paw-pressure and writhing tests are given as the
mean ( SEM; analysis of variance (ANOVA), followed by Fisher’s
PLSD procedure for post hoc comparison, was used to verify the
significance between two means. P values of less than 0.05 were
considered significant. The data were analyzed by the StatView
for the Macintosh computer program.
Computational Details. All calculations and graphical manipu-
lations were performed on Silicon Graphics computers (Origin 300
server and Octane workstations) using the software package
Autodock 3.0.5,26 GRID 21,27 and MacroModel 8.528 (equipped
with an implemented version of the AMBER94 force field kindly
provided by Soliva R. and co-workers29). The program Autodock
was used to evaluate the binding mode of the new inhibitors and
to explore their binding conformations within the COX-2 structure.
Because Autodock does not perform any structural optimization
and energy minimization of the complexes found, a molecular
mechanics/energy minimization (MM/EM) approach was applied
Effects of COX-2 Inhibitors on Whole Blood COX-2 and
COX-1 Activities. Valdecoxib (0.005-50 mM) and 8a (0.5-500
mM) were dissolved in DMSO. Aliquots of the solutions (2 µL) or
vehicle were pipetted directly into test tubes to give final concentra-
tions of 0.01-1000 µM in whole blood samples. To evaluate
COX-2 activity, 1 mL aliquots of peripheral venous blood samples
containing 10 IU of sodium heparin were incubated in the presence
of LPS (10 µg/mL) or saline for 24 h at 37 °C, as previously
described.22 The contribution of platelet COX-1 was suppressed
by pretreating the subjects with aspirin (300 mg, 48 h) before
sampling. Plasma was separated by centrifugation (10 min at 2000
rpm) and kept at -80 °C until assayed for PGE2 as an index of
LPS-induced monocyte COX-2 activity. Peripheral venous blood

1,5-Diarylpyrrole-3-Alkoxyethyl Ethers as COX-2 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4481
Cyclooxygenase-2. Gastroenterology 2006, 130, 55–64.
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the COX-2 binding site onto a three-dimensional grid and to
calculate for each grid point the interaction energy (including
contributions from the van der Waals, electrostatic, and hydrogen
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Further details on computational protocols are in Supporting
Information.
Acknowledgment. We thank Rottapharm SpA (Monza,
Italy) for financial support in this research. Prof. Gabriele
Cruciani (University of Perugia, Italy) is also acknowledged
for kindly providing us with the program GRID. We are also
grateful to Dr. Laura Salvini (C.I.A.D.S., University of Siena,
Italy) for the recording of the mass spectra and to Prof. Stefania
D’Agata D’Ottavi for the careful reading of the manuscript. The
in vitro experiments using the HWB assay were supported by
a grant from MIUR (Fondi Ateneo) to Prof. Paola Patrignani
(16) Di Bari, L.; Pescitelli, G.; Salvadori, P.; Rovini, M.; Anzini, M.;
Cappelli, A.; Vomero, S. Synthesis, Resolution, and Absolute Con-
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(17) Biava, M.; Porretta, G. C.; Poce, G.; Supino, S.; Forli, S.; Rovini,
M.; Cappelli, A.; Manetti, F.; Botta, M.; Sautebin, L.; Rossi, A.;
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Supporting Information Available: Physicochemical, spectro-
scopic, and analytical data of compounds 6-8 along with compu-
tational details (Autodock calculations and optimization of the COX-
2-inhibitor complexes). This material is available free of charge
via the Internet at http://pubs.acs.org.
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