Sulfonilamidothiopyrimidone and thiopyrimidone derivatives as selective COX-2 inhibitors: Synthesis, biological evaluation, and docking studies

Article abstract of DOI:10.1016/j.ejmech.2012.09.005

Newly synthesized sulfonilamidothiopyrimidone derivatives and a subset of 14 sulfonilamidothiopyrimidones and thiopyrimidones selected by an MTT assays cell viability guided selection from an in house collection were evaluated to determine the inhibitory effect on the PGE₂ formation in human peripheral blood lymphocytes (PBLs) using commercial ELISA. The newly synthesized sulfonilamidothiopyrimidone derivatives showed interesting pharmacological activities. Preliminary in vitro assays showed that compounds 2-5 are endowed with very high activity. Compound 2 was the most active as hCOX-2 inhibitor. The observed effects were not due to an inhibition of cell proliferation as proved by the BrdU assay. Western blot of COX-2 confirmed the inhibition on the PGE₂ secretion. Further evidence on the inhibitory potential and selectivity as COX-2 inhibitors of the selected compounds came from the in vitro screening. In order to better rationalize the action and the binding mode of these compounds, docking studies were carried out. These studies were in agreement with the biological data. Compounds 2-5 were able to fit into the active site of COX-2 with highest scores and interaction energies. Furthermore, compound 2, which showed an inhibition of around 50% on PGE ₂ production, was the best scored in all the docking calculations carried out.

Full text of DOI:10.1016/j.ejmech.2012.09.005

European Journal of Medicinal Chemistry 57 (2012) 149e161
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Sulfonilamidothiopyrimidone and thiopyrimidone derivatives as selective COX-2
inhibitors: Synthesis, biological evaluation, and docking studies
Livia Basile a, Susana Álvarez b, Almudena Blanco b, Andrea Santagati c, Giuseppe Granata c,
,
c
,
a
b,
**
Patrizia Di Pietro c, Salvatore Guccione a , M Ángeles Muñoz-Fernández
*
a EtnaLead s.r.l., c/o Etnabuilding, Scuola Superiore di Catania, via S. Nullo 5/i, I-95123 Catania, Italy
b Lab. Inmuno-Biología Molecular, Hospital General Universitario Gregorio Marañón, 28007 Madrid, Spain
c Department of Drug Sciences, University of Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2012
Received in revised form
29 August 2012
Accepted 3 September 2012
Available online 10 September 2012
Newly synthesized sulfonilamidothiopyrimidone derivatives and a subset of 14 sulfonilamidothiopyr-
imidones and thiopyrimidones selected by an MTT assays cell viability guided selection from an in house
collection were evaluated to determine the inhibitory effect on the PGE2 formation in human peripheral
blood lymphocytes (PBLs) using commercial ELISA. The newly synthesized sulfonilamidothiopyrimidone
derivatives showed interesting pharmacological activities. Preliminary in vitro assays showed that
compounds 2e5 are endowed with very high activity. Compound 2 was the most active as hCOX-2
inhibitor. The observed effects were not due to an inhibition of cell proliferation as proved by the
BrdU assay. Western blot of COX-2 confirmed the inhibition on the PGE2 secretion. Further evidence on
the inhibitory potential and selectivity as COX-2 inhibitors of the selected compounds came from the
in vitro screening. In order to better rationalize the action and the binding mode of these compounds,
docking studies were carried out. These studies were in agreement with the biological data. Compounds
2e5 were able to fit into the active site of COX-2 with highest scores and interaction energies.
Furthermore, compound 2, which showed an inhibition of around 50% on PGE2 production, was the best
scored in all the docking calculations carried out.
Keywords:
Thiopyrimidone derivatives
Human PBLs
COX-2
Anti-inflammatory
Anti-cancer
PGE2
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
and maintenance of an inflammatory response, causing a change
from the acute phase to the chronic phase of inflammation [3].
Inflammation is a process involving multiple factors acting in
a complex network. The ingress of leukocytes into the site of
inflammation is crucial for the pathogenesis of inflammatory
conditions [1]. Neutrophils and macrophages are known to recruit
and play pivotal roles in acute and chronic inflammation, respec-
tively [2]. At the inflamed site, the recruited cells are activated to
release many inflammatory mediators which elicit the initiation
Chronic inflammation induced by biological, chemical and physical
factors has been associated with increased risk of human cancer at
various sites. Inflammation facilitates the initiation of normal cells
and their growth and progression to malignancy through produc-
tion of inflammatory oxidants. Appropriate treatment of inflam-
mation with anti-inflammatory agents, inhibitors of oxidant-
generating enzymes, and scavengers of oxidants should be
explored further for chemoprevention of human cancers especially
those known to be associated with chronic inflammation [4].
Non steroidal anti-inflammatory drugs (NSAIDs) are the first
choice therapeutic agents for the treatment of inflammation,
pain, and fever. Prostaglandins (PGs) are major proinflammatory
agents and the suppression of their synthesis at sites of
inflammation has been regarded as primarily responsible for the
beneficial properties of NSAIDs, although several cyclooxygenase
(COX)-independent effects have been described in recent years.
Prostaglandin synthase is a bi-functional enzyme that first
catalyzes the addition of two molecules of oxygen to arachidonic
acid to form the hydroperoxide prostaglandin G2, then reduces
Abbreviations: COX-2, cyclooxygenase-2; PG, prostaglandin; PGG2, hydroperoxy
endoperoxide; PGE2, prostaglandin E2; PBLs, peripheral blood lymphocytes; BrdU,
bromodeoxyuridine; NSAIDs, non steroidal anti-inflammatory drugs; PHA, phyto-
hemagglutinin; PBS, phosphate-buffered saline; BCA, bicinchoninic acid method;
NPS, Naproxen; PGH2, prostaglandin H2; PGF2
acetylcholinesterase.
a
,
prostaglandin; AChE,
* Corresponding author. Dipartimento di Scienze del Farmaco, V.le a. Doria 6 Ed.
2, Città Universitaria, I-95125 Catania, Italy. Tel.: þ39 095 738 4020; fax: þ39 095
222239.
** Corresponding author. Tel.: þ34 91 5868565; fax: þ34 91 586 8018.
E-mail addresses: guccione@unict.it (S. Guccione), mmunoz.hgugm@
madrid.salud.org (MaÁ. Muñoz-Fernández).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.005

150
L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
the hydroperoxide to the alcohol, PGH2, by a peroxidase activity
[5]. The identification of two different isoforms of the COX
enzyme known as COX-1 and COX-2, which are key enzymes in
the arachidonic acid metabolism, heralded the start of a new era
for research in anti-inflammatory therapy. COX-1, is constitu-
tively expressed in most cells and is responsible for the
production of prostaglandins that maintain homeostasis; COX-2
is an inducible isoform that is constitutively present in some
organs, such as brain and kidney, but is significantly upregulated
in response to a broad range of stimuli in inflammatory condi-
tions [6]. Moreover, the existence of COX-3, a variant of COX-1,
has been proposed and is considered to be another target for
anti-inflammatory agents [7].
Since COX isozymes discovery, many papers and reviews have
been published to describe the structural bases of COX inhibition,
and to debate on the therapeutic and adverse effects of worldwide
clinically used NSAIDs, included COX-2 selective inhibitors. COX-2
inhibition has been widely investigated, whereas the role of COX-
1 in human pathophysiology is mostly not yet well ascertained.
As time goes on, the cliché that the constitutively expressed iso-
form COX-1 is only involved in normal physiological functions, such
as platelet aggregation, gastric mucosa protection and renal elec-
trolyte homeostasis is going to be shattered. Recent data show that
COX-1, classically viewed as the homeostatic isoform, is actively
involved in brain injury induced by pro-inflammatory stimuli
It was thought that while the antiinflammatory effect of NSAIDs
occurs as a result of COX-2 inhibition, many of the undesirable side
effects including gastric irritation might be due to the inhibition of
the COX-1 isoform [8]. Therefore, several new inhibitors directed
toward COX-2 without interfering with COX-1 enzymatic activity at
therapeutical doses were developed during the last two decades.
These molecules, termed coxibs display improved gastrointestinal
safety profile compared to the traditional NSAIDs [9]. However,
coxibs were recently withdrawn from the market because of an
increased risk of cardiovascular side effects. Selective COX-2
inhibitors so far can be divided into three classes, namely (i)
vicinal diaryl heterocycle inhibitors (coxibs), (ii) sulfonanilide
inhibitors, and (iii) modified classical nonselective NSAIDs.
Nevertheless, the clinical use of selective COX-2 inhibitors
depends on the clinical situation of patients [10]. Till now celecoxib
is still available on the market and current evidence suggests that it
is an important therapeutic option because of its lower cardiovas-
cular toxicity potential compared to NSAIDs and other coxibs [11].
Recent achievements have also highlighted potential applications
of selective COX-2 inhibitors in treatments of cancers [12], and
neurodegenerative disorders, such as Alzheimer’s disease and
Parkinson’s disease [13].
The aim of this study was to determine the inhibition of sulfo-
nilamidothiopyrimidones 2e5, 7, 11, 12, 31 and thiopyrimidones 19,
23, 27, B41, B43, B45 [14] on the generation of COX-2 product in
PMAeIon-stimulated human peripheral blood lymphocytes (PBLs).
including Ab, lipopolysaccharide, IL-1b, and TNF-a. However, it is
clear that in immune system the isoform induced in response to
The newly designed and synthesized compounds 2e5,
7
inflammatory stimuli is mainly COX-2.
(Scheme 1) and the MTT-based small focused library 11, 12, 31, 19,
Scheme 1. Synthesis of sulfonilamidothiopyrimidone derivatives 2e5, 7.

L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
151
23, 27, B41, B43, B45 (Fig. 2) derived from a part of our collection of
polycondensed heterocycles (Fig. 2) were conceived as molecular
hybrids between the stilbene scaffold in which the C double bond
was bioisosterically substituted by sulfur [15] and a condensed
4-quinazolinone or thienopyrimidin-4-one moiety. The former
moiety is a motif in the coxibs where these latter two moieties were
previously reported as parts of polycondensed anti-inflammatory-
analgesic compounds endowed with no or low ulcerogenic effects
[16e19] (Fig. 1).
temperature 230 ꢀC, capillary voltage 46 V, gas flow rate 25 a.u.)
scanning from m/z 300 up to 800. Samples were dissolved in
H2O/CH3OH 20:80 (v:v) containing 1% triethylamine and directly
infused in the ion source at 5 mM/ml. Microanalyses for C, H, N and S
were obtained from an EA 1108 elemental analyzer Fisons Carlo-
Erba instrument. Analyses indicated by the symbols of the
elements were within ꢁ0.4% of the theoretical values. Melting
points with decomposition (dec.) were determined in open capil-
lary tubes on a SMP1 apparatus (Stuart Scientific Staffordshire) and
are uncorrected. The purity of substances was checked by thin layer
chromatography on Merck silica gel 60 F-254 plates. All commercial
chemicals were purchased from Aldrich, Fluka, Merck, Lancaster
and Carlo Erba and were used without further purification.
Compounds 2e5 were synthesized with satisfactory yields
heating the sulfamido-thioxo 1 with an aryl iodide under basic
conditions in the presence of copper powder and iodide
(Scheme 1).
2. Material and methods
2.1. Chemistry. Experimental
1H and 13C NMR spectra were recorded at 200 MHz on a Varian
Gemini 200 spectrometer; chemical shifts (d) are reported in ppm
from tetramethylsilane as an internal standard; coupling constants
(J) are in Hertz (Hz). IR spectra were recorded on a Perkin Elmer
1600 Series FT-IR in potassium bromide disks. ESIeMass spectra
were acquired on a Thermo Finnigan LCQ-DECA ion trap electro-
spray mass spectrometer operating in negative ion mode (capillary
The thio-cyclohexyl derivative
7 was obtained in dime-
thylformide (DMF) at 80 ꢀC in the presence of potassium carbonate.
The proposed structures were confirmed by spectral data (IR, 1H
NMR, 13C NMR, ESI-Mass) and elemental analysis.
CF3
SO2CH3
HN
N
N
O
NS398
NO2
CH3
H2NO2S
celecoxib
O
S
SO2CH3
SO2CH3
O
HN
HN
S
F
F
SO2CH3
nimesulide
L-745,337
HN
NO2
O
O
S
N
molecular
hybridization
R
N
S
SO2CH3
O
R
H5C6
N
HN
N
O
S
N
F
F
flosulide
S
O
H5C6
Fig. 1. Rationale for the design.

152
L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
Fig. 2. Dataset of compounds. Selected compounds for COX-2 inhibition assays are in the box.

L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
153
Fig. 2. (continued).

154
L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
Specifically, the 1H NMR spectra showed a typical singlet in the
and crystallized from ethanol/water to give product 4 as a pure light
green powder; yield 60%; m.p. < 100 ꢀC dec.; I.R. [cmꢂ1]: 3230 br
(NH), 3100 (CeH benzene), 1700 (C]O), 1345 and 1160 (NeSO2)
cmꢂ1 1H NMR (dimethylsulfoxide-d6):
3.25 (s, 3H, CH3), 7.35e
8.45 (m, 10H, AreH and Hethiophene), 11.40 (s, 1H, NH). 13C NMR
(TMS): 39.53,116.89,122.86,125.45,125.97,128.23,129.36,129.96,
132.86, 133.67, 134.00, 139.02, 141.03, 141.60, 150.87, 160.23. ESIe
MS [M ꢂ H]ꢂ: calcd for C19H14N405S3: m/z 474; found: 473. Anal.
Cald. for C19H14N405S3 (C, H, N, S): C, 48.10; H, 2.95; N, 11.81; S,
20.28. Found: C, 48.00; H, 2.75; N, 11.60; S, 20.00.
region of 11.0e11.45 ppm attributable to the methanesulfonamide
amino group and the multiplet aromatic signals; while that one of
the cyclohexyl derivative 7 showed the multiplet of the ten meth-
ylenes at 1.20e1.99 ppm and the signal of the proton bonded to the
carbon adjacent to sulfur atom appeared at 3.55e3.8 ppm,
respectively.
n
;
d
d
2.1.1. N-[2-[(2,4-difluorophenyl)thio]-4-oxo-6-phenylthieno[2,3-d]
pyrimidin-3(4H)-yl]methanesulfonamide (2)
To a stirred solution of compound 1 (0.180 g, 0.51 mmol) and
sodium hydroxide (40 mg, 1.0 mmol) in ethanol/water 1:1 (40 ml)
2,4-difluoroiodobenzene (0.13 g, 0.51 mmol) and copper powder
(30 mg) (I) iodide (20 mg) were added; the mixture was heated at
reflux under stirring for 4 h; after cooling to room temperature the
mixture was filtered and the filtrate poured in water (150 ml); the
resulting solution was filtered and acidified with hydrochloric acid
to pH 3e4: the solid separated was collected, washed with water,
dried and crystallized from water/dioxane to give pure product 2 as
2.1.4. N-[2-(2-nitrophenyl)thio]-4-oxo-6-phenylthieno[2,3-d]
pyrimidin-3(4H)-yl]methanesulfonamide (5)
To
a solution of appropriate methanesulfonamide-thioxo
(compound 1) [17] (0.180 g, 0.51 mmol), and sodium hydroxide
(30 mg, 0.75 mmol) water (20 ml) 2-nitroiodobenzene (0.131 g,
97%, 0.51 mmol) in ethanol (20 ml) and powdery copper (30 mg)/
copper (I) iodide were added; the mixture was heated at reflux
under stirring; after 6 h the mixture was filtered while hot and the
filtrate cooled to room temperature; the resulting solution was
filtered and acidified with concentrated hydrochloride acid to pH
3e4: the resulting solid was collected, washed with water, dried
and crystallized from ethanol/water to give product 5 as a pure
gray powder; yield 35%; m.p. 264e266 ꢀC dec.; I.R.
br (NH), 3090 (CeH benzene), 1690 (C]O), 1345 and 1155 (NeSO2)
cmꢂ1 1H NMR (DMSO-d6):
3.39 (s, 3H, CH3), 7.26e7.80 (m, 9H,
AreH and Hethiophene), 11.43 (s, 1H, NH).
13C NMR (TMS):
n
[cmꢂ1]: 3182
;
d
d
43.94, 105.20 (t, J ¼ 25.32), 112.70 (d,
yellow powder; yield 60%; m.p. < 125 ꢀC dec.; I.R.
(NH), 3090 (CeH benzene), 1700 (C]O), 1350 and 1160 (NeSO2)
cmꢂ1 1H NMR (dimethylsulfoxide-d6):
3.25 (s, 3H, CH3), 7.25e
8.50 (m, 10H, AreH and Hethiophene), 11.37 (s, br, 1H, NH). 13C
NMR (TMS): 39.49, 117.52, 122.25, 124.84, 125.32, 125.67, 128.71,
n
[cmꢂ1]: 3220 br
J ¼ 18.30), 116.21, 122.37, 124.00, 126.42, 129.00, 135.78, 137.65 (d,
J ¼ 9.68), 139.45, 155.88, 160.00, 160.20, 161.00, 163.10, 164.45. ESIe
MS [M ꢂ H]ꢂ: calcd for C19H13F2N3O3S3: m/z 465; found: m/z 464.
Anal. Cald. for C19H13F2N3O3S3 (C, H, N, S): C, 49.02; H, 2.79; N, 9.03;
S, 20.66. Found: C, 48.80; H, 2.65; N, 8.85; S, 20.30.
;
d
d
129.19, 129.47, 131.79, 132.26, 133.52, 138.02, 140.03, 141.06, 151.88,
160.88. Anal. Cald. for C19H14N4O5S3 (C,H,N,S): C, 48.10; H, 2.95; N,
11.81; S, 20.28. Found: C, 48.20; H, 2.80; N, 11.75; S, 20.00.
2.1.2. 2-({3-[(Methylsulfonyl)amino]-4-oxo-6-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidin-yl}thio)benzoic acid (3)
To a stirred solution of the appropriate methanesulphonamide
thioxo (compound 1) [17] (0.180 g, 0.51 mmol) and sodium
hydroxide (40 mg, 1.0 mmol) in water (20 ml) 2-iodobenzoic acid
(0.130 g 98%, 0.51 mmol), dissolved in a small amount of 10%
sodium hydroxide and powder copper (30 mg)/copper (I) iodide
(20 mg) were added. The mixture was heated at reflux under stir-
ring for 4 h and then filtered. The resulting solution was cooled to
room temperature and acidified with concentrated hydrochloride
acid to pH 3e4. The white solid separated was collected, washed
with water, dried and purified by chromatography on silica gel
column (ethyl acetate/methanol 1:3) to give product 3 as a white
2.1.5. N-[2-(cyclohexylthio)-4-oxo-6-phenylthieno[2,3-d]
pyrimidin-3(4H)-yl]methanesulfonamide (7)
A mixture of methanesulphonamide-thioxo 1 [17] (0.180 g,
0.51 mmol), cyclohexyl iodide (0.11 g, 98%, 0.53 mmol, 0.070 ml,
d ¼ 1.625) and potassium carbonate (0.145 g) in dimethylforma-
mide (2 ml) was heated at 80 ꢀC under stirring for 4 h; after cooling
to room temperature the mixture was treated with hydrochloride
acid and poured in water (100 ml): the resulting solid was collected,
washed with water, dried and crystallized from petroleum ether to
give compound 7 as a pure yellow powder with a 45% yield;
m.p.>125 ꢀC dec.; I.R.
(C]O), 1350 and 1155 (NeSO22); 1H NMR (dimethylsulfoxide-d6):
1.25e2.00 (m, 10H, cyclohexyl), 3.55e3.8 (m, 1H, SeCH), 7.33e
n
[cmꢂ1]: 3200 (NH), 2925 (H cicloesyl), 1690
powder; yield 50% m.p. 239e241ꢀ dec.; I.R.
1700 (C]O), 1345 and 1155 (SO2eN) cmꢂ1
thylformamide-d6): 3.38 (s, 3H, CH3), 7.32e7.95, (m, 10H, AreH
and Hethiophene), 11.45 (br s, 1H, NH), 13.15 (br s, 1H, COOH). 13
NMR (TMS): 43.44, 118.22, 125.21, 125.34, 125.76, 126.43, 127.21,
n
[cmꢂ1]: 3300 (NH),
;
1H NMR (dime-
d
d
7.90 (m, 6H, AreH and thiopheneeH), 11.22 (s, 1H, NH), Anal.
Cald. for C19H21N3O3S3 (C, H, N, S): C, 52.40; H, 4.82; N, 9.65; S,
22.09. Found: C, 52.10; H, 4.80; N, 9.35; S, 21.85.
C
d
128.10, 128.31, 129.18, 129.79, 131.99, 132.26, 132.83, 134.45, 135.69,
168.41, 170.32. ESIeMS [M ꢂ H]ꢂ: calcd for C20H15N3O5S3: m/z 473;
found: m/z 472. ESIeMS [M ꢂ H]ꢂ: calcd for C20H14N3O5S3Na m/z
494. Anal. Cald. for C20H15N3O5S3 (C, H, N, S): C, 50.73; H, 3.17; N,
8.88; S, 20.30. Found: C, 50.40; H, 3.22; N, 8.70; S, 19.90.
2.2. Cell cultures
Human PBLs were isolated by Ficoll-Hypaque centrifugation.
The PBLs were routinely grown in RPMI 1640 (Biochrom KG
Seromed, Berlin, Germany) containing 10% heat-inactivated fetal
2.1.3. N-[2-(4-nitrophenyl)thio]-4-oxo-6-phenylthieno[2,3-d]
pyrimidin-3(4H)yl]methane sulfonamide (4)
calf serum, 1% penicillin/streptomycin, and 2 mM
L-glutamine at
37 ꢀC. Cells were cultured with 2
mg/ml phytohemagglutinin (PHA)
To
a
solution of appropriate methanesulfonamide-thioxo
and 60 U/ml IL-2 (Murex Diagnostics Corp. Norcross, GA).
(compound 1) [17] (0.180 g, 0.51 mmol), and sodium hydroxide
(30 mg, 0.75 mmol) water (20 ml) 4-nitroiodobenzene (0.130 g,
98%, 0.51 mmol) in ethanol (20 ml) and powdery copper (30 mg)/
copper (I) iodide were added; the mixture was heated at reflux
under stirring; after 6 h the mixture was filtered while hot and the
filtrate, cooled to room temperature; the resulting solution was
filtered and acidified with concentrated hydrochloride acid to pH
3e4: the resulting solid was collected, washed with water, dried
2.3. Western blot analyses
Human PBLs were exposed to PMAeIon in the presence or
absence of the inhibitors tested. Cells were washed with
phosphate-buffered saline (PBS) and lysed with buffer lysis. Protein
contents were measured using the bicinchoninic acid method (BCA
protein assay kit from Pierce, Rockford, IL, USA) according to the

L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
155
manufacturer’s instructions. Samples were separated into a 10%
SDS polyacrylamide gel and blotted onto a polyvinylidene fluoride
membrane (Millipore, Bedford, MA, U.S.A.) by semidry transference
blotting. Membranes were blocked overnight at 4 ꢀC using Roti-
block (Roth, Karlsruhe) and for another hour at room temperature.
Blocked membranes were incubated with primary antibody,
Ellman’s Reagent is added to the well, containing the substrate to
AChE. The product of this enzymatic reaction was determined
spectrophotometrically at an absorbance of 405 nm by a Synergy
ht, biotek spectromether. It is proportional to the amount of PG
tracer in the well. This latter is in turn inversely proportional to the
amount of PGs present in the well during the incubation.
a rabbit anti-human COX-2 (Cayman; 1:500), or mouse anti-a-
tubulin (Sigma, St. Louis, MO), used as a protein loading control.
Visualization of protein bands was performed using enhanced
chemiluminescence (ECL) reagents (Amersham Biosciences, UK).
Western blot experiments were carried out at least three times.
2.8. Molecular modeling
Docking studies were carried out on an Intel Core i7, ram
memory of 16 GB operating under Linux/Ubuntu 10.04. Side chains
flexible ligandeprotein docking was carried out by the molecular
docking algorithm MolDock Optimizer and the scoring function
MolDock [GRID] as in the Molegro Virtual Docker software, version
5 [20,21]. Only torsion angles in the side chains were modified
during the minimization; all other properties (including bond
lengths and backbone atom positions) were held fixed and a new
receptor conformation was generated for each pose after side
chains flexible docking calculation. The ligands were built as .pdb
files by means of the server PRODRG2 (http://davapc1.bioch.
dundee.ac.uk/prodrg/) [22]. Docking of NPS into the hCOX-2
active site as obtained by homology modeling was used to cali-
brate the system. High scoring poses (Table 1) with the pattern of
interactions comparable to the mCOX-2-Naproxen were obtained.
Particularly the docking of Naproxen gave a high score pose with
high similarity to the entry crystal structure 3NT1 in PDB (Fig. 3);
the rmsd between the docked complex and the crystal structure
2.4. Cell viability measurement
To determine the optimum concentration of these novel
compounds as well as to assess that their suppressive effect on
PGE2 production was not due to their cytotoxicity, the cell viability
was assessed by MTT assay. The method is based on the ability of
living cells to reduce MTT tetrazolium salt into MTT formazan by
the mitochondrial enzyme succinate dehydrogenase, as reported
previously. Briefly, the cells were incubated with MTT (0.15 mg/ml)
for 4 h at 37 ꢀC. The amount of MTT formazan product was deter-
mined by measuring absorbance with a microplate reader at a test
wavelength of 570 nm and reference wavelength of 655 nm.
2.5. Cell proliferation
Human PBLs were grown in the absence or presence of
a concentration of 10 mM of the indicated compounds for 24 h in
ꢀ
(PDB entry: 3NT1) was around 0.16 A as calculated by Sybyl-X 1.3
a 37 ꢀC and 5% CO2 atmosphere. Cell proliferation was measured by
quantitating bromodeoxyuridine (BrdU) incorporated into the
newly synthesized DNA of replicating cells. In this method BrdU,
a thymidine analog, is incorporated into newly synthesized DNA
strands of actively proliferating cells. Following partial denatur-
ation of double stranded DNA, BrdU is detected immunochemically
allowing the assessment of the population of cells which are
synthesizing DNA.
(http://www.tripos.com). Typically, an RMSD value below 2.0 Å
represents a good solution for a redocked ligand of course
depending on factors such as the ligand size and flexibility.
Protein residues Arg(R) 89, Leu(L) 321, Tyr(Y) 324, Tyr(Y) 354
and Ser(S) 499 in hCOX-2 (human COX-2) were set as flexible; these
residues correspond to Arg(R) 120, Leu(L) 352, Tyr(Y) 355, Tyr(Y)
385, Ser(S) 530 which are involved in the inhibitors binding of the
homologous mCOX-2 (murine COX-2), as reported by Duggan et al.
[23].
Potentials were softened to a value of 1.5 and 0.5 for tolerance
and strength, respectively. Preliminary calculations with a toler-
ance value of 0.9 led to poses with low MolDock scores and unfa-
vorable steric interactions. Increasing the value up to 1.5 led to
poses with high scores and no significant unfavorable steric inter-
actions. 70 runs for each molecule were carried out with a pop-
ulation size of 50, maximum iteration of 1500, scaling factor of 0.50,
crossover rate of 0.90 and a variation-based termination scheme.
The results were in agreement with the experimental data.
2.6. PGE2 determination
Supernatants of control or treated human PBLs were tested for
PGE2 production using the PGE2 EIA kit (Cayman Chemical
Company, MI, USA), a competitive enzyme immunoassay.
2.7. Cyclooxygenase-2 (COX-2) assay
Selective Prostaglandin G/H synthase activity was measured
in vitro using purified COX-1 or COX-2 protein (assay kit No. 560101,
Cayman Chemical Company). Compounds 2e5 were analyzed with
celecoxib as the positive control. The assay is based on the chemical
ꢀ
Poses within a root-mean square deviation of 1.5 A were clus-
tered together by means of the “Tabu pose” clustering algorithm.
In order to improve the docking calculations taking into account
the interactions with the cofactors, the anion Clꢂ and protopor-
phyrin IX containing Fe as in the mCOX-2 pdb entry (3NT1) were
conversion of PGH2 to PGF2
a by stannous chloride followed by
a PGF2 immunoassay. The assay was carried out in 0.1 M Trise
a
HCl at pH 8. Overall compound to test was dissolved in dimethyl
sulfoxide. Prostaglandin synthesis was initiated by the addition of
arachidonic acid to the mixture of reaction. Afterward, stannous
Table 1
Energy contributions for compounds assayed.
chloride was added in order to convert PGH2 to PGF2
a and the
Compound
Total interaction
energy (a.u)
Proteineligand
interaction energy (a.u)
Score (a.u)
PGF2 generated was quantified by enzyme immunoassay, EIA. This
a
assay is based on the competition between PGs and a PG-AChE
conjugate (PG tracer) for a limited amount of PG antiserum. The
amount of PG tracer able to bind to the PG antiserum is inversely
proportional to the concentration of PGs in the wells, since the
concentration of PG tracer is constant while that of PGs varies. The
complex between the antibody and PG binds to a mouse antirabbit
monoclonal antibody that has been previously attached to the well.
The plate is washed to remove any unbound reagents and then
NPS
2
3
5
4
31
11
23
ꢂ127.26
ꢂ126.32
ꢂ111.36
ꢂ104.87
ꢂ86.78
ꢂ80.04
ꢂ78.29
ꢂ33.28
ꢂ126.31
ꢂ125.03
ꢂ109.30
ꢂ103.11
ꢂ86.01
ꢂ79.95
ꢂ77.26
ꢂ33.28
ꢂ120.19
ꢂ132.07
ꢂ122.90
ꢂ111.35
ꢂ89.26
ꢂ91.89
ꢂ79.39
ꢂ41.74

156
L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
Fig. 3. Light blue: X-ray structure of the complex mCOX-2-Naproxen. Magenta: Highest score pose of Naproxene docked into hCOX-2. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Cell death of human PBLs. Human cells were treated with IL-2 and different concentrations of the compounds tested (1, 10, and 50 mM). Cell toxicity was measured by MTT
assay after 24 h of treatment. DMSO is used as positive control of cell death.

L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
157
Fig. 5. A. PGE2 formation as parameter of COX-2 activity. A) PGE2 production of human PBLs treated with PMA (15 ng/ml)eIon (10 mM), alone or in combination with different
compounds (10 M) or NS398 (5 M) measured by ELISA after 24 h of treatment. B. PBLs proliferation in response to sulfonilamidothiopyrimidone derivatives. Human PBLs were
M of compounds 2, 3, 4, and 5 and 24 h after, cell proliferation was measured by incorporation of BrdU.
m
m
exposed to 10
m
added to the model [23]. The cofactors in the hCOX-2 model had the
Table 2
same coordinates as in mCOX-2 (see below). The structure of the
hCOX-2 used for the docking calculations was obtained according
to Michaux et al. [24,25].
All the docking results were counterchecked by the software
FLO (unpublished data) (QXP_FLO program: Thistlesoft, PO Box 227,
Colebrook, CT 06021) [26].
Favorable and unfavorable interactions of docked compounds.
Compound Favorable Energy (a.u)b Unfavorable Energy Steric interaction
contactsa
Val 318
contacts
(a.u)
energy (a.u)
2
3
ꢂ11.11
Arg 89
24.9
ꢂ9.71
Ala 496
Val 85
Val 318
ꢂ11.21
ꢂ12.01
ꢂ13.39
Arg 89
His 58
Tyr 324
Tyr 354
63.73 ꢂ20.63
7.57
5.65
18.13
5
Ala 496
Val 318
ꢂ13.84
ꢂ10.64
Arg 89
His 58
Tyr 324
Tyr 354
90.68 ꢂ13.86
2.04
26.99
1.43
4
Glu 493
Val 85
ꢂ13.15
ꢂ15.09
Arg 89
Tyr 324
Val 318
108.85 44.37
43.22
7.59
31
Ala 496
ꢂ10.07
Arg 89
His 58
124.83 37.53
0.41
Tyr 324
Val 492
2.78
2.27
11
23
Ser 322
ꢂ10.58
Arg 89
Leu 328
Tyr 324
0.79
4.39
67.42
ꢂ20.52
Arg 482
Glu 493
His 58
Phe 487
Ser 322
ꢂ10.52
ꢂ14.93
ꢂ12.98
ꢂ15.74
ꢂ16.31
Ala 485
Arg 89
Val 492
27.19 106.66
128.29
71.75
Fig. 6. COX-2 expression in human PBLs. Cell extracts were analyzed by Western blot
after treatment with PMA (15 ng/ml)eIon (10 mM), alone or in combination with the
numbered compounds (2, 3, 4, 5) (10
positive control of COX-2 inhibition. Cells were harvested, lysed, and proteins were
m mM) was used as
M) for 20 h at 37 ꢀC. NS398 (5
a
Only favorable contacts with energies below ꢂ10 a.u are listed.
separated by SDSePAGE and analyzed for COX-2. The membrane was reprobed with
tubulin antibody to confirm equal protein loading.
a-
b
a.u stands for arbitrary units.

158
L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
3.2. Effects of sulfonilamidothiopyrimidone derivatives on PGE2
secretion by human PBLs
The COX-2 activity was evaluated by measuring the PGE2 levels
in PMAeIon-treated human PBLs. Only, compounds 2e5, 7, 11, 12,
19, 23, 27, 31, B41, B43, and B45 which did not induce cell death at
dose of 10 mM were tested (Fig. 5A). The PMAeIon stimulated cells
secreted to the supernatant 2287 pg/ml. We observed that the
compounds synthesized by us either inhibited or stimulated PGE2
formation. Compounds 2, 3, 4, and 5 inhibited PMAeIon-induced
PGE2 secretion by 20e50%. Compound 2 was the most potent
among these inhibitors showing potency higher than 50%,
Compounds 23 [28] and B43 [17] were relatively less potent. In this
assay, NS398 (5
(Fig. 5A).
mM) was used as positive control of PGE2 inhibition
Fig. 7. Effect of sulfanilamidothiopirimidone derivatives on COX-1 enzyme activity
in vitro. Celecoxib (control) and sulfanilamidothiopirimidone derivatives (final
concentration of 5 nM) were added to purified COX-1. Prostaglandin synthesis was
initiated by the addition arachidonic acid substrate. The results, expressed as pico-
Interestingly, compounds 7, 11 [29], 31 [14], and B45 [17]
increased the PMAeIon-induced PGE2 formation. Between them,
compound 7 was the most potent among these derivatives and
stimulated PGE2 secretion 2.9-fold. Compounds 7, 11, and 31 [14]
(Scheme 1, Fig. 2) increased the PGE2 levels, suggesting an alter-
nate target on respect to hCOX-2. The docking results (Tables 1 and
2) were in agreement with this hypothesis being the interactions
with the hCOX-2 weak.
grams of PGF2 formed are the average of duplicate experiments.
a
3. Results
As shown in Fig. 5B, the subject COX-2 inhibitors did not alter
PHA-induced T cell proliferation, indicating that the observed
effects on PGE2 secretion are not associated with a decrease of cell
proliferation.
3.1. Assessment of cell cytotoxicity and effects on T cell activation
It was previously shown that COX-2 inhibitors as celecoxib were
able to act as immunosuppressive drugs modulating T cell activa-
tion events such as cell surface expression of activation markers,
cytokine production and cell proliferation [27]. Moreover, bioactive
molecules by and large are known to exhibit cytotoxic effect to
varying levels depending upon their concentration and duration of
exposure. To explore the influence of the treatment with these
novel compounds on T cell viability, we first addressed whether
these compounds were toxic for human PBLs treated with
increasing doses of these drugs for 24 h. The cytotoxicity of
compounds was estimated by determining the amount of MTT in
the culture.
3.3. Change in COX-2 expression
It has been previously described that high levels of PGE2 were
not only produced in cell lines as a result of strong COX-2 expres-
sion, but also in cells in which COX-2 expression was weak (Effects
of COX-2 inhibition on expression of vascular endothelial growth
factor and interleukin-8 in lung cancer cells [30]). This could
result from reduced catabolism of PGE2 due to low levels of
15-hydroxyprostaglandin dehydrogenase (15-PGDH), a primary
enzyme responsible for PGE2 metabolism [31]. To check whether
low levels of PGE2 produced by PBLs treated with the novel
compounds was due or not to an inhibition of COX-2 expression, we
performed Western blot analysis on lysate protein extracted from
PMAeIon-treated cells in presence or not of the four best
As shown in Fig. 4 at 1
mM most of the tested compounds did not
show cell toxicity in PBLs cultures, and at dose of 50
mM, a consid-
erable inhibition in cell viability was observed with the majority of
compounds. Having in account these results, we decided to use the
dose of 10
production.
mM to study the effects of these compounds on PGE2
compounds. NS398 (5 mM) was used again as positive control of
COX-2 inhibition. COX-2 expression was strongly detected after
PMAeIon treatment. All the compounds tested, especially
compound 2, were able to diminish COX-2 protein levels, sug-
gesting that the reduced secretion of PGE2 is due, at least in part, to
a lower expression of COX-2 (Fig. 6).
3.4. In vitro inhibitors COX-2 screening
Cyclooxygenase is the rate-limiting enzyme in prostaglandin
synthesis. Prostaglandin levels, particularly PGE2, are often used as
a measurement of cyclooxygenase activity. To test whether the
inhibition of serum-induced PGE2 synthesis by sulfonilamidothio-
pyrimidone derivatives was due to selective inhibition of COX-2
enzyme activity, the effect of these compounds on the activity of
purified COX-1 and COX-2 proteins in vitro was tested.
COX-1 and COX-2 proteins catalyze the conversion of arach-
idonic acid into PGG2 (via cyclooxygenase activity) and the
subsequent conversion of PGG2 into PGH2 (via peroxidase activity).
Cyclooxygenase activity was finally measured by an enzyme
Fig. 8. Effect of sulfanilamidothiopirimidone derivatives on COX-2 enzyme activity
in vitro. Celecoxib (control) and sulfanilamidothiopirimidone derivatives (final
concentration of 5 nM) were added to purified COX-1. Prostaglandin synthesis was
initiated by the addition arachidonic acid substrate. The results, expressed as pico-
immunoassay for PGF2a (see experimental for details).
As shown in Figs. 7 and 8, under identical conditions, compounds
grams of PGF2 formed are the average of duplicate experiments.
2e5 behave as the celecoxib, that reduce the cyclooxygenase-2
a

L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
159
Fig. 9. Flexible docking of compounds 2e5 into hCOX-2. Best scoring poses relative to R89, L321, Y324, Y354, Y324. a) Compound 2. b) Compound 3. c) Compound 4. d) Compound 5.
activity of about 50%, proving to be COX-2 selective inhibitors. In the
COX-2 assay the co-presence of these compounds with the ovine
COX-2 causes a minor production of PGF2a.
compounds 2, 3 and 5 (Fig. 9), show the best interaction
energies and highest scores; lowest interaction energies and
docking score characterize the poses of compounds 11, 23 and
31 (Tables 1 and 2). Those three compounds have shown the
ability to increase PGE2 levels probably due to the interaction
with another cellular target. Compound 7 which needs further
mechanistic studies to finally clarify on its activity (vide infra)
which is seemingly out of the COX-2 as proved from the low
in vitro inhibition was not docked.
3.5. Docking on hCOX-2
Overall the docking results were essentially in agreement
with the biological data in terms of evaluation of interaction
energy and docking score (Tables 1 and 2). The most active
Fig. 10. Compounds 23 (a) and 31 (b) docked into hCOX-2.

160
L. Basile et al. / European Journal of Medicinal Chemistry 57 (2012) 149e161
Docking of compound 23 on the hCOX-2, was carried out in
Further evidences on a COX-2 selective inhibition came from
in vitro screening assay. The cyclooxygenase-2 activity was notably
reduced from some of the tested compounds 2e5 compared with the
standard COX-2 selective inhibitor, celecoxib. These biological results
suggest that these new compounds (2e5) with anti-inflammatory
activities are the good candidate as selective anti-COX-2.
Developing drugs that interfere with signaling routes and gene
expression, aiming at attenuating the pro-inflammatory response
could prove to be a promising avenue.
Overall all the docking results were in agreement with the
biological data. Preliminary semi-flexible docking calculations were
carried out with MVD 4.2 (data not shown) but no relationship was
found with experimental data. Setting flexible residues involved in
the docking of Naproxen in mCOX-2 and softening the interaction
potential has led to a strong correlation between biological and
computational data suggesting a marked ligand-receptor adapting
in the binding process. Compound 2 is the most effective inhibitor
(inhibition over the 50%), and its predicted affinity is the highest
within all the compounds.
Our model was able to predict the ranking of biological data in
terms of docking score and binding energies. We found out that
hydrophobic interactions with apolar residues of binding site of
hCOX-2 are determinant for to have the final inhibitoreenzyme
complex. Compounds 7, 11, 23 and 31 have bad contacts with
polar residues in the pocket due to steric hindrance.
order to further test our docking protocol; the docking score and
the interaction energy were lower than that of the active
compounds 2, 3, 4 and 5 in agreement with the in vitro assays.
Taken all together the data (Table 2) show that the compounds
interact by a favorable interaction with Val 318 and Ala 496, which
significatively contribute formation of the binding. Hydrophobic
interactions involving residues such as Leu, Val, Phe are also
important. Binding modes of compounds 23 and 31 are character-
ized by a significative number of unfavorable interactions due to
steric hindrance (Fig. 10). The anomalous PGE2 promoting action of
compounds 11 and 31 might be explained by an interaction with an
alternate target.
4. Discussion
Inflammation is in the first instance a self-defense reaction that
may, under specific circumstances, develop into a chronic state and
become a causative factor in the pathogenesis of a broad range of
pathologies. In many chronic and disabling diseases, regardless of
their primary etiology, inflammation is the main therapeutic target
and, often, the best choice to treat the disease and alleviate the
symptoms. Although NSAIDs, which are non-selective COX inhibi-
tors, have been very often used as painkillers [32,33], their side
effects such as gastrointestinal hemorrhage remain a serious
problem. Although the role of COX-1 seems to be important during
neuroinflammation, and its inhibition could be a viable therapeutic
approach to treat CNS diseases with a marked inflammatory
component, we wanted to determine the effect of new derivatives
only on COX-2 activity since the search for new COX-2 inhibitors
without the increased risk of gastrointestinal events is a very actual
and challenging issue. Newly synthesized sulfonilamidothiopyr-
imidone 2e5 inhibit the COX-2 activity. The antiinflammatory
properties were assayed by evaluating the suppression of molecule
proinflammatory as PGE2 in human lymphocytes. PGE2 synthesized
from arachidonic acid by COX and synthases (PGES), acts as both an
inflammatory mediator and fibroblast modulator [34]. PGE2 is the
lipid mediator of inflammation in diseases, such as rheumatoid
arthritis and osteoarthritis, and is also involved in skin
inflammation.
Acknowledgments
We thank Dr. Catherine Michaux (Department of Chemistry,
University of Namur, Belgium) for the hCOX-2 structure and Dr.
René Thomsen (Molegro ApS Denmark) for the technical support,
the helpful suggestions and critically reading and correcting the
manuscript. Prof. Giovanni Li Volti (Department of Drug Sciences of
the University of Catania) is gratefully acknowledged for the
instrumental support and the helpful discussion in the COX assay.
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