Synthesis and biological evaluation of fluorinated 1,5-diarylpyrrole-3-alkoxyethyl ether derivatives as selective COX-2 inhibitors endowed with anti-inflammatory activity

Article abstract of DOI:10.1016/j.ejmech.2015.12.044

A series of substituted 1,5-diarylpyrrole-3-alkoxyethyl ethers were previously synthesized and the potential anti-inflammatory and antinociceptive activities of these compounds were evaluated in vivo. The compounds displayed a very good activity against b

Full text of DOI:10.1016/j.ejmech.2015.12.044

Accepted Manuscript
Synthesis and Biological Evaluation of Fluorinated 1,5-Diarylpyrrole-3-Alkoxyethyl
Ether Derivatives as Selective COX-2 Inhibitors Endowed with Anti-inflammatory
Activity
Angela Di Capua, Claudia Sticozzi, Simone Brogi, Margherita Brindisi, Andrea
Cappelli, Lidia Sautebin, Antonietta Rossi, Simona Pace, Carla Ghelardini, Lorenzo
Di Cesare Mannelli, Giuseppe Valacchi, Gianluca Giorgi, Antonio Giordani, Giovanna
Poce, Mariangela Biava, Maurizio Anzini
PII:
S0223-5234(15)30430-X
10.1016/j.ejmech.2015.12.044
EJMECH 8284
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 October 2015
Revised Date: 22 December 2015
Accepted Date: 23 December 2015
Please cite this article as: A. Di Capua, C. Sticozzi, S. Brogi, M. Brindisi, A. Cappelli, L. Sautebin, A.
Rossi, S. Pace, C. Ghelardini, L. Di Cesare Mannelli, G. Valacchi, G. Giorgi, A. Giordani, G. Poce, M.
Biava, M. Anzini, Synthesis and Biological Evaluation of Fluorinated 1,5-Diarylpyrrole-3-Alkoxyethyl
Ether Derivatives as Selective COX-2 Inhibitors Endowed with Anti-inflammatory Activity, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2015.12.044.
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ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of Fluorinated 1,5-Diarylpyrrole-3-Alkoxyethyl Ether
Derivatives as Selective COX-2 Inhibitors Endowed with Anti-inflammatory Activity
Angela Di Capua, Claudia Sticozzi, Simone Brogi, Margherita Brindisi, Andrea Cappelli, Lidia Sautebin,
Antonietta Rossi, Simona Pace, Carla Ghelardini, Lorenzo Di Cesare Mannelli, Giuseppe Valacchi, Gianluca
Giorgi, Antonio Giordani, Giovanna Poce, Mariangela Biava, and Maurizio Anzini

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Synthesis and Biological Evaluation of Fluorinated 1,5-
Diarylpyrrole-3-Alkoxyethyl Ether Derivatives as
Selective COX-2 Inhibitors Endowed with Anti-
inflammatory Activity
Angela Di Capua,a,# Claudia Sticozzi,b Simone Brogi,a Margherita Brindisi,a Andrea Cappelli,a Lidia
Sautebin,c Antonietta Rossi,c Simona Pace,c Carla Ghelardini,d Lorenzo Di Cesare Mannelli,d Giuseppe
Valacchi,b,e Gianluca Giorgi,a Antonio Giordani,f Giovanna Poce,g Mariangela Biava,g and Maurizio
Anzinia,*
aDepartment of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy.
bDepartment of Life Sciences and Biotechnology, University of Ferrara, Via Luigi Borsari 46, 44121 Ferrara, Italy.
cDepartment of Pharmacy, University of Naples “Federico II”, Via D. Montesano 49, I-80131 Napoli, Italy.
dDepartment of Pharmacology Neuroscience, Psychology, Drug Research and Child Health – Neurofarba – Pharmacology
and Toxicology Section, University of Florence, Viale G. Pieraccini 6, I-50139 Firenze, Italy.
eDepartment of Food and Nutrition, Kyung Hee University, Seoul, South Korea.
fRottapharm Biotech, Via Valosa di Sopra 7, 20052 Monza, Italy.
gDepartment of Chemical Studies and Technologies of Drugs, University of Rome "La Sapienza”, Piazzale Aldo Moro 5,
00185 Roma, Italy
.
*Corresponding author. Tel. +390577 234173; fax +39 0577 234254; e-mail: maurizio.anzini@unisi.it
#Present address: Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland,
Australia.

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Abstract
A series of substituted 1,5-diarylpyrrole-3-alkoxyethyl ethers were previously synthesized and the
potential anti-inflammatory and antinociceptive activities of these compounds were evaluated in vivo.
The compounds displayed a very good activity against both carrageenan-induced hyperalgesia and
oedema in the rat paw test. Therefore, in a very preliminary test, compounds (8a,b) showed
antiproliferative activity in the HaCaT (aneuploid immortal keratinocyte from adult human skin) cell
models. On these basis, our research continued with the synthesis of fluorinated derivatives (8c,d, 9b-
d, and 10b-d) with the aim of improving the pharmacokinetic profile of the previous active
compounds. Substitution of a hydrogen atom by a fluorine atom may change the conformational
preferences of the molecules due to stereoelectronic effects and also fluorine atom may be able to exert
the metabolic obstruction reducing the “first-pass effect”. Compound 10b exhibited a prominent in vivo
anti-inflammatory and antinociceptive activities, in addition its antiproliferative power in an in vitro
model of human skin cancer is herein described.
Keywords: COX-2 inhibitors, 1,5-diarylpyrrole derivatives, anti-inflammatory agents, antinociceptive
agents, antiproliferative activity, molecular modelling
1. Introduction
Prostaglandin G/H synthase enzymes (PGHS), commonly known as cyclooxygenases (COX), are
responsible for the formation of important biological mediators called prostanoids, which play a critical
role in various biological processes [1]. Arachidonic acid (AA) is a 20 carbon unsaturated fatty acid
distributed throughout the lipid bilayer of the cell membranes. Phospholipase A2 (PLA2) enzymes
cleave membrane bound arachidonate for the conversion into bioactive precursors. AA can be

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metabolized through COX pathway via a two-step process: the first step involves conversion of AA to
prostaglandin G2 (PGG2), a 9,11-endoperoxide-15-hydroperoxide, and in the second step peroxidase
reduces PGG2 to prostaglandin H2 (PGH2). Specific PG synthases further metabolize PGH2 to give
structurally related bioactive lipids, including PGs PGE2, PGD2, PGF2α, prostacycline PGI2 and
thromboxane A2 (TxA2) [2].
Non-Steroidal Anti-Inflammatory Drugs (NSAID), which include both traditional NSAIDs (tNSAIDs)
and selective inhibitors of COX-2 (COXib) (Figure 1), relieve pain and inflammation suppressing the
COX function of PGHS and the consequent formation of PGE2 and prostacyclin (PGI2), along with
other prostanoids [1].
Long-term therapy with tNSAIDs generates adverse gastrointestinal complications ranging from
stomach erosions and silent intestinal ulcerations to life-threatening complications [3]. Selective
inhibitors of COX-2 depress prostacyclin (PGI2), an atheroprotective agent, but not COX-1-derived
thromboxane A2 (TXA2), a pro-aggregatory and vasoconstrictor mediator, which might predispose
patients to heart attack and stroke [4]. In 2004, Rofecoxib, (4-[4-(methylsulfonyl)phenyl]-3-phenyl-
2(5H)-furanone), marketed as Vioxx was withdrawn from the market due to increased risk for
cardiovascular thrombotic events in a long term-therapy [5].
Moreover, the involvement of COX-2 in cancer development was previously evidenced by
pharmacological analysis of PGs in different human cancers. PGE2 specifically exerts carcinogenic
effects in the human body. It was found that premalignant lesions and established cancers produce
excessive quantities of PGE2 and this enhances tumor cell growth and increases tumor invasiveness [1].
COX-2 is up-regulated in a number of epithelial cancers, including those originating in the colon and
rectum, stomach, breast, prostate, and lung [6]. In the context of cutaneous tissues, increased
expression of COX-2 and production of its primary product PGE2 have been reported to increase cell

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growth and decrease apoptosis. PGE2 is also thought to be responsible for different skin cancers, such
as squamous cell and basal cell carcinoma [7].
During the last decade a large number of COX inhibitors have been described. In particular, very
recently novel chemical entities have been identified by different approaches such as: i) rational design
providing novel scaffolds based on 4-phenylpyrimidine-2(1H)-thiones [8] and, indole based
peptidomimetics [9], or ii) computational procedures providing novel and structurally unrelated COX-2
inhibitors [10].
Within a large programme devoted to the design and synthesis of new anti-inflammatory agents based
on a diaryl heterocyclic scaffold, we previously synthesized different series of 1,5-diarylpyrrole-3-
acetic esters 7 [11] and 3-alkoxyethyl ethers 8a,b [12] as new selective COX-2 inhibitors, in which the
pyrroloacetic subunit, reminiscent of indomethacine and clopirac was conjugated with vicinal diaryl
moiety mimicking the main feature of COXib (Figure 1).

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Figure 1. Structures of reference compounds.
These compounds were shown to be potent and highly selective COX-2 inhibitors in in vitro cell
culture assays [12-14]. The potential anti-inflammatory and antinociceptive activities of these
compounds were also evaluated in vivo, demonstrating a very good activity against both carrageenan
induced hyperalgesia and oedema in rat paw test. Furthermore, compounds 8a,b showed, in a very
preliminary screening, a clear inhibition of cell proliferation on HaCaT cells with non-toxic effects in
the range between 10 and 100 ꢀM [7].
Encouraged by these data, we focused our attention on the preparation of two homologous series of
differently fluorinated 3-substituted-1,5-diarylpyrrole alkoxyethyl ethers (8c,d; 9b-d and 10b-d)
(Figure 2) with the aim of investigating the biological effects that can be elicited by the insertion of a

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fluorine atom on different portion of the previously synthesized COX-2 inhibitors. Compound 10b,
which displays the best biological profile in terms of affinity and selectivity toward COX-2, as well as
percent inhibition of the enzyme (cell culture assay), was also investigated for its in vivo anti-
inflammatory, analgesic and antiproliferative activity, displaying interesting results.
Figure 2. Structure of fluorinated compounds 8c,d, 9b-d and 10b-d.
2. Results and Discussion
2.1. Chemistry
The synthetic procedure used to obtain final compounds 8c,d, 9b-d and 10b-d is depicted in Scheme1.
Briefly, the suitable alcohols 11c-d were treated with propyl iodide in NaOH 50% (w/w) solution,
following the previously reported procedure [12] to obtain final compounds 8c,d. Using the same
procedure, derivatives 11b-d were treated with the proper hydroxyalkyl bromide in its
tetrahydropyranyl protected form [15] (see Supplementary Material) to yield compounds 12b-d and
13b-d that were successively deprotected to give hydroxyalkyl ethers 14b-d and 15b-d. These
derivatives were then activated by tosylation and treated with Bu4N+F- to afford fluorinated compounds
9b-d and 10b-d in satisfactory yield. Purity of compounds 8c,d, 9b-d, 10b-d was assessed by RP-
HPLC and was found to be higher than 95% (see Supplementary Material for further details).

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Scheme 1. Synthesis of compounds 8c,d, 9b-d, and 10b-d. Reagents and conditions:(i) Propyl iodide,
(Bu4N+)Br-, Sodium hydroxide, 50% (w/w) aqueous solution, 60 °C, 72 h; (ii) Br(CH2)nOTHP,
(Bu4N+)Br-, Sodium hydroxide, 50% (w/w) aqueous solution , 60 °C, 72 h (iii) PPTS, MeOH, 55 °C,
20 h; (iv) DMAP, DIPEA, TsCl, DCM, r.t., 4 h; (v) (Bu4N+)F-in THF, 1 M; dry THF, 80 °C, 3 h.
2.2. Structure-activity relationship, molecular modeling studies and biological evaluation
In this limited series of compounds the presence of an additional fluorine atom in the 3-alkoxyalkyl
side-chain with respect to compounds 8a,b would allow us to assess if the contemporary presence of

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fluorine atoms in two different portions of the molecule could influence the interaction of these new
inhibitors with the COX-2 active site. In particular, compounds 8c,d were synthesized in order to
evaluate if the number and the different position of fluoro substituent in the pendant N1 aromatic ring
could lead to biological results comparable with those previously reported for compounds 8a and 8b
[12].
The effects of a fluorine atom as a substituent on physical and chemical properties, as lipophilicity,
electronic properties, hydrogen bonding, and metabolic stability are well known [16, 17]. The
difference in electronegativity between carbon and fluorine generates a large dipole moment in this
bond that, when combined with the electrostatic distribution of a specific molecule, may contribute to
the molecule’s ability to establish intermolecular interactions.
By means of in vitro cell culture (J774 murine macrophage) assay, the title compounds were evaluated
for their inhibitory potency and selectivity on both COX-isoforms and the results are resumed in Table
1.
Table 1. In vitro inhibition (J774 murine macrophage essay) of COX-1 and COX-2 by compounds 8a-
d, 9b-d, and 10b-d.
Cpd
8a[c]
8b[c]
8c
X
H
F
Y
F
F
F
F
n
-
COX-1 IC50 (µM)a
COX-2 IC50 (µM)a COX-1/COX-2 (SI)b
>100
>100
>100
>100
0.018
0.030
0.013
0.018
>5555
>3333
>7692
>5555
-
H
F
-
-
8d

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F
H
F
H
F
F
H
F
F
2
2
2
3
3
3
>100
>100
>100
>100
>100
>100
5.1
0.013
>7692
>714
9b
9c
0.140
0.029
0.007
0.135
0.019
0.079
>3448
>14285
>2857
>5263
65
9d
F
10b
H
F
10c
10d
Celecoxib[d]
aResults are expressed as the mean (n = 3) of the % inhibition of PGE2 production by the test compounds
with respect to control sample and IC50 values were calculated by GraphPad Instat program.
bIC50 (COX-1)/IC50 (COX-2). cSee ref. [12]. dSee ref.[13].
Results showed that in compounds 8c,d, the presence of a fluoro substituent in meta and in meta/para
position of the pendant N1 ring did not alter the inhibitory potency and selectivity with respect to
previously reported analogues 8a,b. In compounds sharing general structures 9 and 10, the replacement
of a hydrogen atom with fluorine in the side chain coupled with the presence of mono- or difluoro-
substitution on the N1 pendant ring led to contrasting effects on the inhibitory activity of the resulting
compounds. Accordingly, for meta-substituted compounds 9c and 10c, a considerable decrease of
COX-2 inhibition was observed whereas the para-fluoro derivative 10b, showed a significantly
improved inhibitory activity (IC50 = 0.007 µM) when compared with that of 1,5-diarylpyrrole-3-
propoxyethyl-, 3-fluoroethoxyethyl-, and 3-fluoropropoxyethyl ethers 8a-d, 9c,d, and 10c,d,
respectively, the IC50 of which ranges from 0.018 to 0.030 µM. The in vitro potency and selectivity
showed by compound 10b appears more striking when compared to that of celecoxib (5) the IC50 of
which being 0.079 µM (11-fold less potent and much more less selective than 10b).

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Next, the new fluorinated derivatives were analysed by means of molecular docking calculations using
Glide software (Glide version 5.7, Schrödinger, LLC, New York, NY, 2011), with the aim of
investigating the potential binding modes of such inhibitors within the COX-2 binding site. The
selected compounds 9b and 10b differing for one carbon atom in the fluorinated tail, showed a similar
binding mode to those found for a previously reported COX-2 inhibitors [18]. In particular, compound
9b strongly interacts with the COX-2 active site by a series of polar contacts with the backbone of
Phe518 and with the side-chain of Arg120. Moreover, one π-π stacking was established between
Tyr355 and 9b (Figure 3). The fluorophenyl moiety interacts with a series of hydrophobic residues as
depicted in Figure 3B. These interactions of 9b into COX-2 binding site result in docking XPscore of -
8.47 kcal/mol and in estimated free binding energy (ꢁGbind) of -97.74 kcal/mol.
Figure 3. (A) Best docked pose for compound 9b (green sticks) into COX-2 (orange cartoon PDB ID:
6COX) binding site. The key residues in the active site were represented by lines. H-bonds were
represented by black dotted lines. The hydrogens were omitted for the sake of clarity. The picture was
generated by means of PyMOL (The PyMOL Molecular Graphics System, v1.6-alpha; Schrodinger,
LLC, New York, 2013). (B) Ligand interaction diagram (Maestro version 9.3, Schrödinger, LLC, New
York, NY, 2012

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As depicted in Figure 4, compound 10b showed a similar binding mode with respect to that found for
9b. In fact, 10b established the same hydrophobic/polar contacts previously described. Also, the
docking score and the estimated free binding energy resulted comparable to those calculated for 9b
(XPscore -9.27 kcal/mol; ꢁGbind -100.97 kcal/mol). Remarkably, all the newly synthesized compounds
reported in Table 1 showed a comparable binding mode as well as the docking score and the estimated
ꢁGbind (data not shown), confirming the strong affinity of this kind of scaffold for COX-2 enzyme.
Figure 4. (A) Best docked pose for compound 10b (grey sticks) into COX-2 (orange cartoon PDB ID:
6COX) binding site. The key residues in the active site were represented by lines. H-bonds were
represented by black dotted lines. The hydrogens were omitted for the sake of clarity. (B) Ligand
interaction diagram.
On the basis of its very encouraging in vitro COX-2 inhibitory activity, compound 10b was evaluated
in a rat model of acute inflammatory pain. As shown in Table 2, inflammatory pain was induced by the
intraplantar administration of carrageenan.

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Table 2. Effect of 10b on carrageenan-induced acute inflammatory pain hypersensitivity (paw-pressure
test) and paw edema.
PAW-PRESSURE (g)
PAW VOLUME
(mL)
PRE-
TREATMENT
Intraplantar
TREATMENT DOSE
Before
per os
mg kg-1 Treatment
After treatment
30 min
60 min
90 min
120 min
60 min
57. 6 ± 2.2
33.9 ± 3.1
63.8 ± 2.9
59.4 ± 3.3
62.7 ± 2.8
64.6 ± 3.2
34.5 ± 4.4
36.2 ± 3.3
1.45 ± 0.08
VEHICLE
VEHICLE
34.1 ± 2.3
35.4 ± 3.6
37.2 ± 3.4
44.8 ± 3.0
2.73 ± 0.07
2.38 ± 0.06*
2.15 ± 0.08**
2.06 ± 0.05**
CARRAGEENAN
CARRAGEENAN
CARRAGEENAN
CARRAGEENAN
VEHICLE
10b
3
30.5 ± 2.6
31.8 ± 2.7
30.5 ± 2.0
45.2 ± 4.6** 49.2 ± 3.7**
51.7 ± 4.1** 53.5 ± 3.9**
53.9 ± 3.7** 56.3 ± 4.6**
10b
10
20
47.2 ± 4.4** 38.1 ± 2.9
44.9 ± 3.7** 35.5 ± 2.6
10b
Carrageenan (Car, 1%, 100 ꢀL) was injected in the left posterior sole 2 h before pain evaluation by the paw
pressure test. Molecule or vehicle (1% carboxymethylcellulose) was administered intraplantarly at time 0 and
measures were performed at time 30, 60, 90 and 120 min. The volume of the paw edema was measured at time
60 min. Values are the mean ± s.e.m. of 6 animals. *P < 0.05 and **P < 0.01 vs the carrageenan + vehicle group.
The anti-inflammatory effect of the new compounds emerged also by the measurement of the paw
edema volume, it was significantly decreased by 10b (3-20 mg kg-1) (Table 2). To evaluate the pain
reliever profile of the active compound 10b, its antinociceptive efficacy and potency was assessed in
the model of visceral pain induced by the intraperitoneal (i.p.) injection of a 0.6% acetic acid solution
(Writhing test). The abdominal contractions induced by the acid solution (about 30 writhing in vehicle-
treated animals, Table 3), were dose-dependently prevented by the administration p.o. of 10b. The
measurement performed 30 min after 20 mg kg-1 10b treatment showed a number of writhing reduced
to 10.7 ± 2.5 (Table 3).

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Table 3. Effect of 10b in the mouse abdominal contraction test (acetic acid 0.6%).
TREATMENT per os
DOSE mg kg-1 Number of writhes 30 min Number of writhes 60 min
VEHICLE
30.5 ± 1.7
21 .8 ± 2.5**
11.4 ± 3.1**
10.7 ± 2.5**
32.5 ± 2.4
24.6 ± 2.7*
17.7 ± 3.1**
16.9 ± 2.7**
3
10
20
10b
10b was administered 30 or 60 min before the experiment. 0.6% acetic acid was administered intraperitoneally 5
min before the observation. Each value represents the mean of 12 mice per group. *P< 0.05 and **P <0.01
versus vehicle-treated mice.
The efficacy of compound 10b was also evaluated in the rat osteoarthritis model induced by the intra-
articular injection of monoiodoacetate (MIA) (Table 4). The injection of MIA in the knee joint induces
necrosis of chondrocytes with decrease of cartilage thickness and osteolysis [19]. Kobayashi et al.
showed that MIA is able to disorganize chondrocytes and to promote cartilage erosion [20]. These
alterations are comparable with joint damages typical of humans affected by osteoarthritis [21-23].
Briefly, on day 14 after MIA injection the pain threshold on the ipsilateral paw was decreased to 39.2 ±
3.2 g in comparison to 62.9 ± 2.3 g of control rats as evaluated by Paw pressure test. Compound
administered p.o. was able to reduce articular pain starting from the dosage of 3 mg kg-1. Twenty mg
kg-1 of 10b were effective up to 60 min after treatment reaching the threshold of 54.5 ± 3.5 g (60 min,
Table 4). The repeated treatment with 10b significantly prevented MIA dependent hyperalgesia (before
treatment). A further compound administration (after treatment, 30 min) did not induce an additive
acute antihyperalgesic effect, suggesting that a repeated treatment with the present anti-inflammatory
compounds could be able to prevent the joint damage and, consequently, pain [21-24].

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In addition to the canonical anti-inflammatory and anti-nociceptive activities, many studies highlighted
the potential of COX-2 inhibitors in the prevention of several cancer types such as colon, breast, lung
and prostate cancers [25-28] most likely by modulating the cell proliferation pathways. So, title
compounds were evaluated to assess their effects on cell toxicity by means of LDH release assay and
potential antiproliferative activity using HaCaT, aneuploid immortal keratinocyte cell line from adult
human skin [7].
Table 4. Effect of 10b on monoiodoacetate-induced osteoarthritis hypersensitivity in the rat paw-
pressure test.
PAW-PRESSURE (g)
PRE-TREATMENT TREATMENT DOSE
Before
After treatment
Intraplantar
per os
mg kg-1 treatment
30 min
60 min
90 min
120 min
VEHICLE
MIA
VEHICLE
VEHICLE
62. 9 ± 2.3 60.5 ± 2.4
58.3 ± 4.0 66.3 ± 4.1 61.5 ± 3.2
36.1 ± 3.5 37.8 ± 3.1 39.2 ± 4.0
39.2 ± 3.2
37.8 ± 3.3
3
36.5 ± 2.5 47.9 ± 4.2** 45.4 ± 3.8* 42.4 ± 3.5 39.0 ± 3.1
38.8 ± 2.8 50.3 ± 4.4** 51.8 ± 3.7** 44.3 ± 3.8 40.6 ± 3.5
37.8 ± 2.2 51.3 ± 3.3** 54.5 ± 3.5** 43.8 ± 3.1 37.1 ± 2.9
MIA
10
20
10b
Effect of 10b on monoiodoacetate (MIA)-induced unilateral osteoarthritis. MIA (2 mg/25 µL) was injected in
the left posterior tibiotarsal articulation 14 days before pain evaluation by the paw pressure test. Molecules or
vehicle were administered intraperitoneally at time 0 and measures were performed at time 30, 60, 90 and 120
min. Values are the mean ± s.e.m. of 6 rats. *P<0.05 and **P< 0.01 in comparison with MIA + vehicle treated
rats.

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2.3. Effect of compounds 8c,d, 9b-d, and 10b-d on cell toxicity
Cytotoxicity of compounds was evaluated in HaCaT cells after treatment with doses ranging from 1 to
50 ꢀM by means of LDH release. As shown in Figure 5, 24 h after treatment no cytotoxicity was
detected.
Figure 5. Cytotoxicity of compounds 8c,d, 9b-d, and 10b-d ranging from 1 to 50 ꢀM after 24 h was
evaluated in HaCaT cell line by LDH release assay. Data are expressed as percentage of Triton X-100
(100%) (averages of three different experiments, *p < 0.05).
As shown in Figure 6, the treatment with compounds 8c,d, 9b-d, and 10b-d for 24 h, had a clear dose-
dependent effect on cellular proliferation as measured by 5-bromo-2'-deoxyuridine (BrdU)
incorporation. Indeed, while 1 ꢀM did modify cellular proliferation, the treatment with higher albeit not
toxic doses (10 and 50 ꢀM) evidenced a drastic and significant cellular growth inhibition for 8d, 9b, 9c
9d, 10b and 10d compared to the control cells (DMSO 0.1%). Cells treated with 8c and 10c did not
show any significant changes in BrdU incorporation. However, further investigation is required to
confirm the antiproliferative activity showed by previously reported compounds 8a,b [7].

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Figure 6. The effect on cell proliferation of compounds 8c,d, 9b-d, and 10b-d on HaCaT cells was
evaluated by means of BrdU incorporation after 24 h of treatment. Data are expressed as percentage of
control (averages of three different experiments, *p < 0.05).
3. Conclusions
In this work, we have designed, synthesized and tested a new series of fluorinated 1,5-diarylpyrrole-3-
alkoxyethyl ether derivatives as COX-2 selective inhibitors. In vitro biological results are in full
agreement with all the previously published data, demonstrating the high affinity of the 1,5-
diarylpyrrole scaffold for COX-2 active site. The affinity was also confirmed by molecular docking
calculations, proving that replacement of a hydrogen atom by fluorine in the alkyl side chain didn’t
modify steric and electronic properties of the newly conceived chemical entities. Compound 10b
demonstrated efficacy in the model of rat acute inflammatory pain and in the rat osteoarthritis model;
thus, highlighting its favourable potential to turn into a novel anti-inflammatory and antinociceptive
agents. Furthermore, the anti-proliferative but not toxic effect of the most promising compounds in the
presented series on HaCaT cell suggests a possible application in the therapy of human skin disorders.

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4. Methods
4.1. Biological evaluation
4.1.1. In Vitro Inhibition of COX-1 and COX-2 enzymes
The in vitro COX-1 and COX-2 inhibition of compounds 8a-d, 9b-d, and 10b-d was evaluated through
cell-based assay employing murine monocyte/macrophage J774 cell lines. The 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 x 105 cells/mL and allowed to adhere at 37 °C in 5% CO2 for 2 h.
Immediately before the experiments, culture medium was replaced with fresh medium and cells were
stimulated as described previously [12]. The evaluation of COX-1 inhibitory activity was achieved pre-
treating cells with test compounds (100 µM) for 15 min and then incubating them 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 PGE2 levels by a radioimmunoassay (RIA). 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 (0.001–10 µM). The
supernatants were collected for the measurement of PGE2 by means of RIA. Celecoxib was utilized as
a reference compound for the selectivity index. 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 experiments, 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).

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4.1.2. Animals
For all the experiments described below, male CD1 albino mice (23-25 g) or male Sprague–Dawley
rats (Harlan, Varese, Italy) weighing approximately 200–250 g at the beginning of the experimental
procedure, were used. Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio,
University of Florence) and used at least one week after their arrival. Four rats were housed per cage
(size 26 × 41 cm); animals were fed a standard laboratory diet and tap water ad libitum, and kept at 23
± 1 °C with a 12 h light/dark cycle, light at 7 a.m. All animal manipulations were carried out according
to the European Community guidelines for animal care (DL 116/92, application of the European
Communities Council Directive of 24 November 1986 (86/609/EEC). The ethical policy of the
University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US
National Institutes of Health (NIH Publication No. 85-23, revised 1996; University of Florence
assurance number: A5278-01). Formal approval to conduct the experiments described was obtained
from the Italian Ministry of Health (N°54/2014-B) and from the Animal Subjects Review Board of the
University of Florence. Experiments involving animals have been reported according to ARRIVE
guideline. All efforts were made to minimize animal suffering and to reduce the number of animals
used.
4.1.3. Carrageenan-induced acute inflammatory pain
One hundred µL of carrageenan solution (Sigma-Aldrich; 1% in saline) was injected intraplantarly into
the rat left hind paw. Two h after carrageen injection, compounds were per os (p.o.) administered and
their anti-hypersensitive effect was measured over time by the paw pressure test. Control rats received
100 µL of vehicle (saline) solution intraplantarly and saline i.p..

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4.1.4. Abdominal contraction pain
At predetermined time intervals (30 and 60) after drug administration, mice were injected
intraperitoneally with a 0.6 % (w/w) solution of acetic acid (10 mL kg-1), to induce pain sensation
evident from typical abdominal contractions of animals named 'writhing'. The number of induced
writhings was counted for 10 min, starting 5 min after acetic acid injection [29].
4.1.5. Monoiodoacetate-induced osteoarthritis
Unilateral osteoarthritis was also induced by injection of monoiodoacetate (MIA, Sigma-Aldrich) into
the rat tibio-tarsal joint [30]. On day 0, rats were slightly anesthetized by 2% isoflurane, the left leg
skin was sterilized with 75% ethyl alcohol and the lateral malleolus located by palpation; then, a 28-
gauge needle was inserted vertically to penetrate the skin and turned distally for insertion into the
articular cavity at the gap between the tibiofibular and tarsal bone until a distinct loss of resistance was
felt. 2 mg MIA in 25 ꢀL saline were delivered into the left articular cavity. The paw pressure was
performed at day 14. The tested compounds were p.o. administered. Control rats received 25 µL of
saline solution (day 0) in the tibiotarsal joint and saline i.p. at day 14.
4.1.6. Paw pressure test
The pain threshold in the rat was determined with an analgesimeter (Ugo Basile, Varese, Italy) as
described.[31] Briefly, a constantly increasing pressure was applied to a small area of the dorsal surface
of the paw using a blunt conical probe. Pressure was increased until a vocalization or a withdrawal
reflex occurred. The withdrawal threshold was expressed in grams, the test was repeated twice and the
mean was considered as the value for each paw.

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4.1.7. Edema volume measurement
Rat paw volumes were measured using a plethysmometer. Sixty min after the injection of carrageenan,
the paw volume of the ipsilateral hind paw was measured and compared with carrageenan + vehicle-
treated controls. The results are reported as paw volume expressed in mL.
4.1.8. Statistical analysis of behavioral measurements
Results were expressed as means ± s.e.m. and the analysis of variance was performed by one way
ANOVA. A Bonferroni's significant difference procedure was used as post-hoc comparison. P values
of less than 0.05 or 0.01 were considered significant. Data were analyzed by means of the “Origin 9”
software (OriginLab, Northampton, USA).
4.1.9. Cell culture and treatments.
HaCaT cells, (a cell line gift from Dr. F. Virgili), were grown in Dulbecco’s modified Eagle’s medium
High Glucose (Lonza, Milan, Italy), supplemented with 10% FBS, 100 U/ml penicillin, 100 lg/ ml
streptomycin and 2 mM L-glutamine as previously described.[32] Cell suspension containing 10 or 1
105 viable cells/ml were used. Cells were incubated at 37 C for 24 h in 95% air/5% CO2 until 80%
confluency. HaCaT cells were treated with different doses of title compounds at different time points
(30 min, 1, 3 and 24 h). After treatment, cells or medium were collected for the several assays
described below. Compounds were dissolved in DMSO at a concentration of 10 mM as a stock
solution. The stock was diluted to the required concentrations directly in the medium. The final
concentration of DMSO in culture medium during compounds treatment did not exceed 0.1% (v/v).

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4.2. Computational details
All calculations performed in this work were carried out on Cooler Master Centurion 5 (Intel Core-i5
Quad CPU Q6600 @ 2.40 GHz) with Ubuntu 10.04 LTS (long-term support) operating system running
Maestro 9.2 and Maestro 9.3 (Schrödinger, LLC, New York, NY, 2011 and Maestro version 9.3,
Schrödinger, LLC, New York, NY, 2012, respectively).
4.2.1. Molecular Docking studies
4.2.1.1. Molecules preparation
Three-dimensional structures of compounds were built by means of Maestro (Maestro version 9.2,
Schrödinger, LLC, New York, NY, 2011). Molecular energy minimizations were performed by means
of MacroModel using the Optimized Potentials for Liquid Simulations-all atom (OPLS-AA) force field
2005 [33, 34]. The solvent effects are simulated using the analytical Generalized-Born/Surface-Area
(GB/SA) model [35], and no cutoff for nonbonded interactions was selected. Polak-Ribiere conjugate
gradient (PRCG) method with 1000 maximum iterations and 0.001 gradient convergence threshold was
employed. The compounds were treated by LigPrep application (LigPrep version 9.2, Schrödinger,
LLC, New York, NY, 2011), implemented in Maestro suite 2011, generating the most probable
ionization state of any possible enantiomers and tautomers at cellular pH value (7 ± 0.5).
4.2.1.2. Protein Preparation
The three-dimensional structures of the COX-2 were taken from PDB (PDB ID: 6COX) and imported
into Schrödinger Maestro molecular modeling environment. The structures were submitted to protein
preparation wizard implemented in Maestro suite 2011 (Protein Preparation Wizard workflow 2011) as
previously reported [36, 37]. This protocol through a series of computational steps, allowed us to obtain
a reasonable starting structure of the proteins for molecular docking calculations by a series of

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computational steps. In particular, we performed diverse steps to (1) add missing residues or side-
chains, (2) add hydrogens, (3) optimize the orientation of hydroxyl groups, Asn, and Gln, and the
protonation state of His, and (4) perform a constrained refinement with the impref utility, setting the
max RMSD of 0.30. The impref utility consists of a cycles of energy minimization based on the impact
molecular mechanics engine and on the OPLS_2005 force field [33, 34].
4.2.1.3. Molecular Docking
Docking studies were carried out by Glide (Grid-Based Ligand Docking with Energetics) using the
ligands and the protein prepared as above-mentioned, applying Glide extra precision (XP) method.[38,
39] Energy grid was prepared using default value of protein atom scaling factor (1.0 Å) within a cubic
box centered on the crystallized inhibitor. After grid generation, the ligands and the crystallized
inhibitor were docked into the enzymes with default parameters (no constraints were added). The
number of poses entered to post-docking minimization was set to 50. Glide XP score was evaluated.
The XP method correctly accommodated the crystallized inhibitors into the binding site (data not
shown).
4.2.1.4. Estimated free binding energy
The Prime/MM-GBSA method implemented in Prime software (Prime version 3.0, Schrödinger, LLC,
New York, NY, 2011) consists in computing the change between the free and the complex state of both
the ligand and the protein after energy minimization. The technique was used on the docking
complexes of the selected compounds presented in this study. The software was employed to calculate
the free-binding energy (ꢁGbind) as previously reported by some of us [40-42].

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Acknowledgements
The authors are grateful to Dr Francesco Berrettini (CIADS, Siena University) for the X-ray data
collection.
Supplementary Material
Supplementary data associated with this article (supplementary experimental procedures, X-Ray
1
13
crystallography data for compounds 9b,d, 10b,d, and H- and C-NMR spectra for compounds 8c,d,
9b-d, 10b-d) can be found, in the online version, at….
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