Improving the solubility of a new class of antiinflammatory pharmacodynamic hybrids, that release nitric oxide and inhibit cycloxygenase-2 isoenzyme

Article abstract of DOI:10.1016/j.ejmech.2012.10.014

The development of a novel class of pharmacodynamic hybrids that inhibits COX-2 isoform is reported. These molecules display enhanced nitric oxide releasing properties due to the presence of an ionisable moiety. The in vivo analgesic/anti-inflammatory activity was maintained in relation to the parent compounds.

Full text of DOI:10.1016/j.ejmech.2012.10.014

European Journal of Medicinal Chemistry 58 (2012) 287e298
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Improving the solubility of a new class of antiinflammatory pharmacodynamic
hybrids, that release nitric oxide and inhibit cycloxygenase-2 isoenzyme
,
a
a
a
a
Mariangela Biava a , Claudio Battilocchio , Giovanna Poce , Salvatore Alfonso , Sara Consalvi ,
Giulio Cesare Porretta a, Silvia Schenone b, Vincenzo Calderone c, Alma Martelli c, Lara Testai c,
Carla Ghelardini d, Lorenzo Di Cesare Mannelli d, Lidia Sautebin e, Antonietta Rossi e, Antonio Giordani f,
*
Paola Patrignani g, Maurizio Anzini h
,
i
a Dipartimento di Chimica e Tecnologie del Farmaco, Università “La Sapienza”, Piazzale Aldo Moro 5, I-00185 Roma, Italy
b Dipartimento di Scienze Farmaceutiche, University of Genoa, Viale Benedetto XV 3, I-16132 Genova, Italy
c Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, I-56126 Pisa, Italy
d Dipartimento di Farmacologia, Università di Firenze, viale G. Pieraccini 6, I-50139 Firenze, Italy
e Dipartimento di Farmacologia Sperimentale, Università di Napoli “Federico II”, via D. Montesano 49, I-80131 Napoli, Italy
f Rottapharm S.p.A., via Valosa di Sopra 7, I-20052 Monza, Italy
g Department of Medicine and Center of Excellence on Aging, “G. d’Annunzio” University and CeSI, Via dei Vestini 31, I-66013 Chieti, Italy
h Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy
i European Research Centre for Drug Discovery and Development, via Banchi di Sotto 55, I-53100 Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of a novel class of pharmacodynamic hybrids that inhibits COX-2 isoform is reported.
These molecules display enhanced nitric oxide releasing properties due to the presence of an ionisable
moiety. The in vivo analgesic/anti-inflammatory activity was maintained in relation to the parent
compounds.
Received 3 August 2012
Received in revised form
8 October 2012
Accepted 10 October 2012
Available online 18 October 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Cyclooxygenases
CINODS
Coxibs
1,5-Diarylpyrroles
Glycine derivatives
Nitric oxide
1. Introduction
Side effects related to Non-Steroidal Anti-inflammatory Drugs
(NSAIDs) (Fig. 1) have been extensively documented by numerous
clinical trials. Long-term therapy with these medicines is often
associated with gastrointestinal (GI), renal and haematological
effects which reduce patient compliance [1,2].
The development of COX-2 selective NSAIDs (coxibs) (Fig. 2) was
meant to circumvent these side effects, by selectively inhibiting the
isoenzyme involved in the production of pro-inflammatory media-
tors. Though clinical trials have shown a reduction of gastrointes-
tinal and renal side effects, an increase in cardiovascular (CV) events
was observed suggesting that an exclusive inhibition of COX-2
enzyme could be associated with heart failure and stroke [3,4].
In the last few years many research groups have focused their
efforts on the discovery and development of “molecular hybrids”
Abbreviations: NSAID, nonsteroidal anti-inflammatory drug; GI, gastrointes-
tinal; COXIB, cyclooxygenase-2 inhibitor; CV, cardiovascular; COX, cyclooxygenase;
NO, nitric oxide; NOS, nitric oxide synthase; CINOD, cyclooxygenase-inhibiting
nitric oxide donor; SAR, structure activity relationship; Boc, tert-butyl-oxy
carbonyl; Cbz, benzyl-oxy carbonyl; SGF, simulated gastric fluid; PBS, phosphate
buffered saline; c-GMP, cyclic guanosine monophosphate; ODQ, 1H-[1,2,4]oxadia-
zolo[4,3-a]quinoxalin-1-one; Emax, maximal vasorelaxing response; EDCI, 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide; DMAP, dimethylaminopyridine; DMEM,
Dulbecco’s modified Eagle’s medium; HEPES, 4-(2-hydroxyethyl)piperazine-1-
ethanesulfonic acid; FBS, fetal bovine serum; PGE2, prostaglandin E2; RIA, radio-
immunoassay; LPS, lipopolysaccharide; Ach, acetylcholine; ipl, intraplantar; ip,
intraperitoneal; po, per os.
* Corresponding author. Tel.: þ39 06 4991 3812; fax: þ39 06 4991 3133.
E-mail address: mariangela.biava@uniroma1.it (M. Biava).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.014

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M. Biava et al. / European Journal of Medicinal Chemistry 58 (2012) 287e298
(HTC-1026) were the first molecules developed as COX-Inhibiting
Nitric Oxide Donors namely CINODs (Fig. 3) [8e12].
As part of our ongoing development of analgesic/antiin-
flammatory candidates, we focused our attention on the design of
“Pharmacodynamic Hybrids” endowed with both NO-releasing and
COX-2 selective inhibiting properties, with the aim of reducing the
side-effects due to a selective COX-2 inhibition [13e21]. In this class
of drugs the NO-mediated effect is not directed to the reduction of
GI side effects because COX-2 selectivity is a factor of GI safety itself.
The NO-releasing properties are addressed to limit the CV toxicity
due to COX-2 selective inhibition, which leads to reduced levels of
prostacyclin, a vasorelaxing and antiplatelets agents [22]. Indeed
the strategy of introducing a NO-releasing moiety to obtain
a pharmacodynamics hybrid has been widely used to improve the
overall CV profile of these drugs [23]. Several examples involving
the “hybridisation” of NSAIDs have been reported; nevertheless
very few of these, concern the modification of a coxib-like structure
[24e26]. We identified a first class of molecules (Fig. 4) with
significant analgesic and antiinflammatory properties, charac-
terised by an ideal NO release.
Fig. 1. Chemical structures of Meloxicam, Nimesulide, Diclofenac and Indometacin.
This class of molecules is characterised by a central core
responsible for COX-2 inhibitory activity and a nitric oxide donating
moiety which should be able to suppress the side-effects. These
molecules showed selective inhibition towards COX-2 isoenzyme
with activities in the nanomolar range, lower than that shown by
already marketed coxibs (i.e. Celecoxib). Nonetheless their in vivo
profile, even though describing a very good efficacy, was somehow
reduced (in relation to the in vitro one), indicating that the phar-
macokinetic behaviour plays a crucial role in the fate of this class of
molecules [21]. We then moved forward to analyse the solubility of
those compounds and as predicted, these molecules were charac-
terised by low water solubility and this feature could heavily affect
their absorption [27]. There are two main factors responsible for
poor solubility: high crystallinity (zwitterion formation, insoluble
salts, strong H-bonding networks, etc) and hydrophobicity related
to high log P (absence of ionisable moiety, high molecular weight).
In our case the problem revealed to be the latter. Formulation
strategies represent a fashionable and successful way to get
improvement of solubility and bioavailability of active compounds;
nevertheless, the study of these systems adds further parameters
which results in complexity and expensiveness of the development
at early stages [28]. We decided therefore to proceed through
chemical modification of the previously identified core (Fig. 5).
An amino group (ionisable moiety) was introduced at the alpha
position of the acetic side chain which structure activity relation-
ship (SAR) studies has shown to be one of the few positions where
change could be tolerated (compounds 1aed). The meta-fluorine
and para-fluorine substituents at the N1 phenyl ring were chosen
for a higher degree of interaction with the target. Since we expected
that the NO release could give rise to the nitro-free intermediates as
metabolites, we decided to further synthesise (2aed) and analyse
the activity of these molecules to better understand the biological
profile of this class of derivatives.
characterised by a nitric oxide (NO) releasing moiety, in order to
enhance the pharmacological profile of the parent drug. NO is an
important endogenous mediator generated by Nitric Oxide Syn-
thase (NOS) enzymes. While at high concentrations NO is cytotoxic
and forms a part of the defence and immunological system, at low
concentrations it has important functions in the overall homeo-
stasis of the organism. NO, biosynthesised by endothelial cells, is
mainly considered as a fundamental modulator of the cardiovas-
cular function, where it acts as a powerful vasodilator, inhibits
platelets activation/aggregation and is involved in ischemic pre-
conditioning, thus ensuring important cardioprotective effects.
Increasing evidence suggests a considerable “modulating” effect of
nitric oxide as pleiotropic agent, acting in several districts of the
body [5e7]. In the GI tract, NO modulates the blood flow contrib-
uting to mucosal defence against luminal irritants and it also
modulates mucus and bicarbonate secretion. It has a striking role in
mucosal healing processes and modulates vascular permeability.
The prevention of GI toxicity, by means of nitric oxide, was firstly
explored by NicOx combining a nitric oxide releasing moiety with
existing NSAIDs. Naproxcinod (AZD3582) and NO-flurbiprofen
2. Chemistry
Compounds 1aed and 2aed were prepared, using a common
route to the synthesis of the main core, namely derivative 9
(Scheme 1) and then following two different strategies for the
generation of nitro-oxy and nitro-free compounds (Scheme 2). The
synthesis commenced with a Stetter reaction between the 4-
methythiobenzaldehyde 3 and methyl vinyl ketone in the pres-
ence of a thiazolium salt [13e21]; the obtained compound 4 was
oxidised by means of Oxone and subsequently a PaaleKnorr reac-
tion afforded the 1,5-diarylpyrroles 6aeb [13e21]. Acylation of
Fig. 2. Chemical structures of Celecoxib, Lumiracoxib, Etoricoxib and Parecoxib
sodium.

M. Biava et al. / European Journal of Medicinal Chemistry 58 (2012) 287e298
289
Fig. 3. Chemical structures of NO-flurbiprofen and Naproxcinod.
pyrroles was achieved by a titanium-catalysed regioselective
FriedeleCraft on the pyrrole core to obtain compounds 7aeb [13e
21]. The installation of the protected amino functionality was
achieved by the formation of the oxime, its subsequent reduction to
the amine and protection, giving 10aeb and 13aeb in very good
yields. Hydrolysis of the ethyl ester was achieved employing really
mild conditions and through a subsequent coupling we obtained
compounds 12aeb and 15aeb. A final deprotection step yielded
for 18 and 1.1 mM for 19). These data could therefore explain the
reduced activity found when compounds are tested in vivo since it
is well known that solubility is a crucial parameter in determining
the biological effect, due to the relation between dissolution rate
(hence solubility) and absorption. The introduction of an ionisable
moiety (amino group) within that scaffold determined a huge
increase of solubility as expected; however, we were aware that,
even though changes on that position were tolerated, alteration of
activity could occur. Analysis of the solubility profiles for 1a and 1c
underlined a 200-fold improvement of solubility in SGF and around
100-fold in PBS.
nitro-free and nitro-oxy compounds. The installation of the
a-
functionality was afforded using the same strategy (oxime forma-
tion, reduction and protection) but employing two different pro-
tecting strategies. Indeed, we envisaged these two different routes
could be easily converted to the final products, namely the nitro-
free and the nitro-oxy derivatives reducing the problem that
3.2. Cell-based cyclooxygenases inhibition assay
could have risen using
a common protection/deprotection
The cell-based assay showed a drop in activity for compounds
1aed and 2aed in relation to 18 and 19 (Table 2) [21].
We speculate this drop of activity to be related to the presence of
the amino group. However, an average micromolar activity asso-
ciated to all compounds is still present and selectivity towards COX-
2 isoenzyme is maintained. Amongst the nitro-oxy derivatives, 1a
approach. We chose the tert-butyl-oxy carbonyl (Boc) protecting
group for the synthesis of the nitro-oxy compounds 1aed, whose
deprotection in mild acid conditions did not affect the stability of
the nitro-oxy side chain. The benzyl-oxy carbonyl (Cbz) protecting
group was selected for the route to the nitro-free derivatives since
a late stage double deprotection was envisaged to yield compounds
2aed. Indeed compounds 15aed represent a double protected
scaffold where catalytic hydrogenation can get rid of both the nitro
and Cbz functionalities.
proved to exert the best inhibition towards COX-2 (IC50 0.82
while the nitro-free compound 2c inhibited COX-2 isoform at the
lowest IC50 value (0.22 M).
mM)
m
3.3. Reduction of acetic acid-induced abdominal constrictions in mice
3. Results and discussion
The issues related to the acetic acid-induced inflammation
indicate that compounds 1a and 1c are endowed with activities
comparable to compounds 18 and 19 (Table 3). In particular, the
presence of an ionisable moiety enhancing solubility seems to
overcome the relatively lower activity underlined in the cell-based
assay shown by compounds 1a and 1c. Compound 1a bears a three-
atom side chain and its activity is higher than the parent compound
19 both at 20 and 40 mg kgꢀ1 and comparable to 18 (which has
a smaller side chain of two atoms). Compound 1c shows an activity
which is higher than 19 even though the latter presents a smaller
side chain. We can assess that though the molecular interaction
characterising the amino derivatives are less favourable (cell-based
assay) the solubility enhancement gained with the introduction of
an ionisable group may be responsible for the slight increase of
activity when comparing the side chain length.
3.1. Solubility assessment in SGF and PBS solutions
Simulated gastric fluid (SGF-without pepsin) and phosphate
buffered saline (PBS) were chosen as ideal media to evaluate the
solubility of compounds in acid (pH 1.5 for SGF) and neutral
conditions (7.4 pH for PBS) to simulate the gastric and the body
fluid environment respectively (Table 1) [29].
Solubility of compounds was assessed using a multi-pulse vor-
texer (Glass-Col), essential for shaking the samples prepared using
stock solutions of compounds in DMSO (20 mM), and then ana-
lysing the samples obtained through HPLC. Previously, we deter-
mined the solubilities of compounds 18 and 19 (Fig. 6); we found
that in SGF these molecules were scantly soluble with concentra-
tion ranging below the micromolar values. Similarly, solubility of
compounds in PBS ranged the same values of concentration (1.6 mM
Fig. 4. General structure for selective COX-2 inhibiting nitric oxide donors, previously
Fig. 5. Novel selective COX-2 inhibiting nitric oxide donors bearing an amino group on
developed in our research group.
the side chain.

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M. Biava et al. / European Journal of Medicinal Chemistry 58 (2012) 287e298
O
O
O
O
O
R
O
O
O
H
N
i
ii
iv
iii
N
O
S
O
O
R
O
S
S
S
O
S
O
6a-b
3
4
5
7a-b
v
OEt
OEt
N
H2N
HO
O
O
vi
N
N
O
S
O
S
R
O
R
9a-b
O
8a-b
Compounds. 6a: R = 4-F; 6b: R = 3-F; 7a: R = 4-F; 7b: R = 3-F; 8a: R = 4-F; 8b: R= 3-F; 9a: R = 4-F; 9b: R = 3-F;
Reagents and conditions: (i) CH2=CHCOMe, TEA, 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide, MW; (ii)
Oxone, MeOH/H2O; (iii) RPhNH2, p-toluensulfonic acid, EtOH, MW; (iv) ClCOCOOEt, TiCl4, DCM; (v) NH2OH HCl,
NaAcO, dioxane; vi) Zn dust, EtOH, formic acid.
Scheme 1. Synthesis of compounds 9aeb.
If we compare the activities of compounds 1a and 1c we can
induced activation of guanylate cyclase and the consequent
increase of cytosolic cyclic guanosine monophosphate (cGMP),
1 mM ODQ (inhibitor of guanylate cyclase) was used to identify the
speculate that side chain dimension inversely relates to the
analgesic/anti-inflammatory potency of these molecules when
performing the writhing test as observed in the previous series of
compounds.
mechanism of action. ODQ significantly antagonized the vaso-
relaxing activity of these derivatives, indicating that these effects
can be attributed to the release of NO. In particular, the different
levels of potency exhibited by the four derivatives
(18 > 1c > 1a > 19) are likely to reflect different rates of the NO-
releasing process. In order to have a further confirmation of the
NO-releasing properties, 18 (the compound showing the highest
level of potency) was incubated in rat liver homogenate in the
presence of opportune cofactors such as glutathione, NADH, and
NADPH (all at the concentration 1 mM), i.e. in a biological sample
able to metabolize organic nitrates, leading to the production of NO
and of the corresponding alcohol derivative. In turn, NO is quickly
oxidized to inorganic nitrites and nitrates, the latter being the
principal species. As shown in Fig. 7, the incubation of 1 mM 18 was
followed by a time-dependent slow formation of the inorganic
stable metabolites of NO. The concentrations of nitrite and nitrate
reached after 2 h after the incubation of the above derivative were
6 ꢁ 1 mM and 281 ꢁ 80, respectively. This experimental result
further demonstrates that these derivatives are endowed with the
pharmacological feature of slow NO-releasing agents.
3.4. Reduction of carrageenan-induced inflammatory condition in rats
Compound 1a was selected amongst all derivatives for the
carrageenan test. The results were collected every 15 min until
activity of compounds could be still detected. The results display an
activity which is present already after 15 min (Table 4). The data
show the max activity being at 30 min (70% pressure reduction at
40 mg kgꢀ1) and then it decreases after 45 min (53% reduction). The
same trend is observed for 18 and 19, indicating that pharmaco-
kinetics plays a similar role whether or not we introduce an amino
group at the alpha position of the acetic side chain. As observed in
the acetic acid induced contractions test, the solubility enhance-
ment for compound 1a seems to be favourable being this
compound more active than the corresponding parent compound
19. Moreover, 1a is endowed with an activity which is very close to
the one displayed by 18.
3.5. Nitric oxide releasing properties
4. Conclusions
In order to evaluate the NO-releasing properties, compounds 1a,
1c, 18 and 19 were submitted to a functional assay on isolated
preparations of vascular smooth muscle; in particular, their vaso-
relaxing effects were tested on endothelium-denuded rat aortic
rings, pre-contracted with 30 mM KCl. As shown in Table 5, all the
tested compounds exhibited vasodilator effects. Since the effects of
NO on the vascular smooth muscle are mediated by the NO-
In this paper, we report a further structural development of
a novel class of pharmacodynamic hybrids, able to produce signif-
icant analgesic/anti-inflammatory effects, due to the preferential
inhibition of COX-2, and to act as moderate donor of NO. The
presence of the latter ancillary mechanism is likely to be a useful
pharmacological strategy for attenuating the cardiovascular risk

M. Biava et al. / European Journal of Medicinal Chemistry 58 (2012) 287e298
291
i
ii
9a-b
10a-b
11a-b
iii
v
16a-b
n= 1, 2
17a-b
13a-b
iv
vi
viii
vii
14a-b
2a-d
15a-d
1a-d
Scheme 2. Synthesis of compounds 1aed and 2aed.
potentially associated with the selective inhibition of COX-2, and
mainly due to the reduced biosynthesis of prostacyclin. The
experimental results demonstrated that the synthetised
compounds were endowed with significant inhibiting activity on
the COX-2 isoform, and lower activity on COX-1. Moreover, their
nitrooxy function is effectively converted to NO, leading to addi-
tional cardiovascular effects, such as the vasorelaxing one, which
has been clearly observed in this study. Noteworthy, the related
alcoholic derivatives deriving from the biotransformation of the
nitrooxy function still behave as COX-2 inhibitors. Moreover, in the
new series of derivatives developed in this study, a further struc-
tural change has been introduced. Indeed, the analogous
compounds previously reported [21] showed some limiting phys-
icochemical aspects, able to negatively influence their pharmaco-
kinetics, and in particular, a very poor levels of water solubility. In
order to improve such a potentially negative feature, in these
compounds an amino group was introduced. In line with the aims
of the work, this structural modification actually led to a clear
improvement of the water solubility of the target compounds,
without affecting significantly the two cardinal pharmacodynamic
properties (i.e., the COX-2 inhibition and the NO-release). There-
fore, these novel compounds seem to exhibit promising feature as
COX-2-inhibiting antiinflammatory/analgesic agents, endowed
with an improved cardiovascular safety.
5. Experimental protocols
5.1. Chemistry
Discovery Microwave System apparatus (CEM Srl, Cologno al
Serio, Italy) was used to perform the Stetter and PaaleKnorr reac-
tions. 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 a Perkine
Elmer 240C or a PerkineElmer Series II CHNS/O Analyzer 2400.
Fluka silica gel 60 (230e400 mesh) was used for column chroma-
tography. Fluka TLC plates were used for TLC. Fluka aluminium
oxide (activity IIeIII, according to Brockmann) was used for chro-
matographic purifications. Fluka Stratocrom aluminium oxide
Table 1
Solubility assessment in SGF and PBS for compounds 1a, 1c, 18 and 19.
Compound
SGF (uM)
PBS (uM)
1a
1c
18
19
>200
>200
<1
80.0
70.8
1.6
<1
1.1
Fig. 6. Chemical structures of compounds 18 and 19.

292
M. Biava et al. / European Journal of Medicinal Chemistry 58 (2012) 287e298
Table 2
5 min. After 2 h the reaction mixture was diluted with water
(400 mL) and extracted with dichloromethane (3 ꢃ 100 mL). The
organic layer was washed with brine (200 mL) and dried over
sodium sulphate. After filtration and concentration, the crude
material was chromatographed (silica gel, 3:1 hexane/ethyl
acetate) to give 5 (90% yield) as a white solid. Analytical data,
melting point, and the 1H NMR spectrum were consistent with
those reported in the literature [13e21].
In vitro assessment of COX-1 and -2 activity for compounds 1aed, 2aed.
Compound
% Inhib
COX-1
(10 uM)
% Inhib
COX-2
(10 uM)
IC50
IC50
COX-1/COX-2
(S.I.)b
(COX-1)
(COX-2)
(uM)a
(uM)a
1a
1b
1c
1d
2a
2b
2c
2d
0
5
1
15
23
30
10
23
95
100
82
59
100
79
>10
>10
>10
>10
>10
>10
>10
>10
0.82
1.51
1.00
1.41
0.76
1.19
0.22
1.50
>12.20
>6.62
>10.00
>1.35
>13.16
>8.40
>45.45
>6.67
5.1.3. General procedure for the preparation of 1,5-diarylpyrroles
(6aeb)
100
100
A solution of 5 (0.58 g, 2.28 mmol) in ethanol (2 mL), the suitable
amine (2.28 mmol) and p-toluenesulfonic acid (30 mg, 0.17 mmol)
was microwave irradiated at 160 ꢂC for 45 min (power 150 W,
pressure 150 psi). The crude reaction mixture was concentrated
and purified by chromatography on aluminium oxide with a 3:1
cyclohexane/ethyl acetate mixture to give the expected 1,5-
diarylpyrroles 6aed as solids in satisfactory yield. Analytical data,
melting points, and the 1H NMR spectra were consistent with those
reported in the literature [13e21].
a
Results are expressed as the mean (n ¼ 3 experiments) of the % inhibition of
PGE2 production by test compounds with respect to control samples and the IC50
values were calculated by GraphPad Instat program; data fit was obtained using the
sigmoidal doseeresponse equation (variable slope) (GraphPad software).
b
In vitro COX-2 Selectivity Index [IC50 (COX-1)/IC50(COX-2)].
plates with fluorescent indicator were used for TLC to check the
purity of the compounds. 1H NMR and 13C spectra were recorded
with a Bruker AC400 spectrometer in the indicated solvent (TMS as
internal standard): the values of the chemical shifts are expressed
in ppm. Infrared spectra were recorded neat on a PerkinElmer
Spectrum One FT-IR spectrometer using Universal ATR sampling
accessories. Purity of compounds was assessed with elemental
analysis; analyses which are indicated by the symbols of the
elements or functions were within ꢁ0.4% of the theoretical values.
5.1.4. Generalprocedurefor the preparationofethyl (1,5diaryl-pyrrol)
glyoxylate (7aeb)
To a solution of pyrrole 6aeb (4.82 mmol) in anhydrous
dichloromethane, under nitrogen flow, TiCl4 (0.53 mL, 4.82 mmol)
and ethyl oxalyl chloride (0.54 mL, 4.82 mmol) were added at 0 ꢂC.
The solution was stirred for 4 h at rt. The mixture was then poured
into cold water and extracted with CHCl3. The organic solution was
washed with a saturated aqueous NaCl solution, dried and evapo-
rated in vacuo. Purification of the residue by flash chromatography
with CHCl3 as the eluent gave a solid which, after re-crystallization
from hexane, afforded the expected products 7aeb. Analytical data,
melting points, and the 1H NMR spectra were consistent with those
reported in the literature [21].
5.1.1. Procedure for the synthesis of 1-[4-(methylthio)phenyl]
pentane-1,4-dione (4)
A solution of 4-methylthiobenzaldehyde 3 (11.97 mL, 0.09 mol),
triethylamine (19.5 mL, 0.14 mol), methyl vinyl ketone (5.8 mL,
0.09 mol), and 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium
bromide (3.53 g, 0.014 mol) was microwave irradiated for 15 min
at 70 ꢂC (power 150 W, pressure 60 psi) [13e21]. The reaction
mixture was treated with 2 M HCl (10 mL). After extraction with
ethyl acetate, the organic layer was washed with aqueous sodium
bicarbonate and brine. The organic fractions were dried over
sodium sulphate, filtered, and concentrated to give a crude orange
liquid. After crystallization from cyclohexane, intermediate 4 was
isolated as white needles (78% yield). Analytical data, the melting
point, and the 1H NMR spectrum were consistent with those re-
ported in the literature [13e21].
5.1.5. General procedureforthepreparation ofethyl-2-(hydroxyimino)-
(1,5-diarylpyrrol) acetate (8aeb)
To a suspension of glyoxylate derivative 7aeb (0.0582 mol) in
a mixture of ethanol and 1,4-dioxan (100 mL), hydroxylamine
hydrochloride (6.07 g, 0.0874 mol) and sodium acetate (11.9 g,
0.1456 mol) were added. The resulting solution was heated at 90 ꢂC
under stirring for 40 h. The solvents were removed in vacuo and the
mixture was extracted with ethyl acetate (500 mL) and washed with
water (100 mL). The organic phase was dried over sodium sulphate,
filteredandconcentratedtoaffordtheoximes8aebasyellowishsolid.
5.1.2. Procedure for the synthesis of 1-[4-(methylsulfonyl)phenyl]
pentane-1,4-dione (5)
To a solution of 4 (7.8 g, 35 mmol) in methanol (150 mL) Oxone
was added (37.7 g, 61.4 mmol dissolved in 150 mL of water) over
5.1.5.1. Ethyl
phenyl]-1-(4-fluorophenyl)-1H-pyrrol-3-yl]acetate
solid, m.p.103.ꢂC (80% yield); 1H NMR (400 MHz, DMSO-d6):
2-(hydroxyimino)-2-[2-methyl-5-[4-(methylsulfonyl)
(8a). Yellowish
(ppm)
d
Table 3
7.73 (d, 2H, J ¼ 8.5 Hz), 7.33 (m, 4H), 7.18 (d, 2H, J ¼ 8.5 Hz), 6.55 (s,
Activity of compounds 1a, 1c, 18 and 19 assessed with the acetic acid-induced
writhing test on the mice.
1H), 5.67 (s, 1H), 4.18 (q, 2H, J ¼ 7.4 Hz), 3.14 (s, 3H), 2.05 (s, 3H), 1.18
(t, 3H, J ¼ 7.4 Hz); 13C NMR (100 MHz, DMSO-d6):
d (ppm) 170.02,
Compound
Dose per p.o.
N. writhes
% Of writhes reduction
159.70, 149.49, 144.50, 141.33, 135.20, 130.25, 129.20, 128.22, 126.11,
123.45, 122.50, 118.03, 115.34, 65.84, 43.15, 27.60, 10.96; ESI-Mass: m/
z 467.105 [M þ Na]þ; Anal. C22H21FN2O5S (C, H, N).
mg kgꢀ1
nmol kgꢀ1
CMC
1a
33.4 ꢁ 2.5
26.9 ꢁ 2.6
21.3 ꢁ 2.5*
18.1 ꢁ 3.0*
13.5 ꢁ 2.8*
24.5 ꢁ 3.2
19.8 ꢁ 2.4*
23.1 ꢁ 2.6
17.1 ꢁ 2.3*
9.4 ꢁ 2.5*
25.7 ꢁ 2.8
24.9 ꢁ 2.2
e
3
10
20
40
20
40
10
20
40
20
40
5.5
18.4
36.8
73.6
35.9
71.8
21.0
42.0
84.0
40.8
81.6
19.0
36.2
46.1
59.6
26.9
40.7
30.8
48.8
71.2
23.3
26.4
5.1.5.2. Ethyl
phenyl]-1-(3-fluorophenyl)-1H-pyrrol-3-yl]acetate
solid, m.p.104 ꢂC (79% yield); 1H NMR (400 MHz, DMSO-d6):
7.73 (d, 2H, J ¼ 8.5 Hz), 7.33 (m, 4H), 7.18 (d, 2H, J ¼ 8.5 Hz), 6.55 (s,
1H), 5.67 (s, 1H), 4.18 (q, 2H), 3.14 (s, 3H), 2.05 (s, 3H), 1.18 (t, 3H); 13
NMR (100 MHz, DMSO-d6): (ppm) 170.10, 159.66, 149.51, 144.67,
2-(hydroxyimino)-2-[2-methyl-5-[4-(methylsulfonyl)
(8b). Yellowish
(ppm)
1c
18
19
d
C
d
141.33, 138.78, 135.00, 130.75, 129.20, 128.22, 126.06, 123.40, 122.50,
120.66, 118.34, 115.37, 65.99, 43.10, 27.62, 11.06; ESI-Mass: m/z
467.105 [M þ Na]þ; Anal. C22H21FN2O5S (C, H, N).
*P < 0.01 in comparison with CMC-treated group.

M. Biava et al. / European Journal of Medicinal Chemistry 58 (2012) 287e298
293
Table 4
Activity of compounds 1a, 18, 19 and Celecoxib in the carrageenan-induced inflammation.
Pre-treatment i.pl. Treatment p.o. Dose Before treatment Paw-pressure
Oedema volume
(mL)
After treatment
15 min
30 min
45 min
60 min
Saline
CMC
CMC
1a
1a
18
18
19
Celecoxib
e
61.2 ꢁ 2.5
36.1 ꢁ 3.3
33.8 ꢁ 2.8
32.9 ꢁ 3.0
35.7 ꢁ 3.1
29.8 ꢁ 3.1
33.5 ꢁ 2.6
31.7 ꢁ 2.7
65.6 ꢁ 3.4
40.2 ꢁ 3.6 ˇ (25%)
58.9 ꢁ 3.8
43.7 ꢁ 3.7 ˇ (44%)
40.1 ꢁ 3.4 ˇ (35%)
61.4 ꢁ 4.1
63.6 ꢁ 4.0
1.44 ꢁ 0.05
2.71 ꢁ 0.08
Carrageenan
Carrageenan
Carrageenan
Carrageenan
Carrageenan
Carrageenan
Carrageenan
e
32.8 ꢁ 2.9
30.6 ꢁ 3.2
31.8 ꢁ 2.2
34.8 ꢁ 3.1
20
40
20
40
40
10
38.3 ꢁ 3.1 (27%)
38.7 ꢁ 3.4 ˇ (29%)
40.7 ꢁ 2.6 ˇ (40%)
37.1 ꢁ 3.0 (15%)
2.58 ꢁ 0.05 (15%)
2.11 ꢁ 0.09* (45%)
2.52 ꢁ 0.06 (17%)
2.17 ꢁ 0.08* (40%)
2.35 ꢁ 0.07* (35%)
45.4 ꢁ 3.2* (40%) 50.2 ꢁ 4.4* (70%) 45.6 ꢁ 4.3* (53%) 37.5 ꢁ 2.8 (16%)
38.7 ꢁ 3.3 (18%)
35.2 ꢁ 2.8 (10%)
48.3 ꢁ 3.3* (50%) 54.1 ꢁ 3.7* (80%) 49.7 ꢁ 3.0* (60%) 38.7 ꢁ 3.1 (23%)
41.2 ꢁ 2.2 ˇ (40%)
42.3 ꢁ 3.8 ˇ (41%)
37.9 ꢁ 3.0 (17%)
45.7 ꢁ 4.2 ˇ (45%)
52.9 ꢁ 3.1* (75%) 48.3 ꢁ 3.4* (56%) 42.5 ꢁ 2.9 ˇ (39%) 2.53 ꢁ 0.05* (17%)
ˇ
P < 0.05; *P < 0.01 in comparison with carrageenan-treated group.
5.1.6. General procedure for the preparation of ethyl 2-amino-(1,5-
diarylpyrrol)acetate (9aeb)
EtOAc. The combined organic fractions were washed with water,
dried over sodium sulphate and evaporated to give the crude
product. The residue was purified by column chromatography
(silica gel) eluting with EtOAc/hexane (1:20) (v/v) to afford the
desired product as white solid.
To a solution of the crude oxime 8aeb in ethanol (100 mL)
formic acid (100 mL) was added. The solution was then cooled to
0 ꢂC and zinc dust (16.4 g, 0.2522 mol) was added in portions over
a period of 1 h. The mixture was allowed to warm up to rt very
slowly and then stirred for 19 h. Afterwards, zinc salts were filtered
off and the solvents evaporated under reduced pressure. The
residue obtained was dissolved in water and the pH adjusted to 10
with 10% NaOH solution and extracted with EtOAc. The combined
organic fractions were dried over sodium sulphate, filtered and
concentrated in vacuo to afford the crude product. The material was
then purified by column chromatography (silica gel) eluting with
MeOH/CHCl3 (1:20) (v/v) to afford compounds 9aeb.
5.1.7.1. tert-Butyl (ethoxycarbonyl)-[1-(4-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]methyl carbamate (10a).
White solid, m.p. 105 ꢂC (63% yield); 1H NMR (400 MHz, CDCl3):
d
(ppm) 7.70 (d, 2H, J ¼ 8.5 Hz), 7.20 (m, 4H), 7.05 (d, 2H, J ¼ 8.5 Hz),
6.51 (s, 1H), 5.67 (d, 1H, J ¼ 7.8 Hz), 5.40 (d, 1H, J ¼ 7.8 Hz), 4.15 (q,
2H, J ¼ 6.8 Hz), 3.15 (s, 3H), 2.05 (s, 3H), 1.45 (s, 9H), 1.13 (t, 3H,
J ¼ 6.8 Hz). 13C NMR (100 MHz, CDCl3):
d (ppm) 171.21, 163.43,
158.20, 140.28, 139.37, 135.81, 132.96, 128.87, 127.93, 126.75, 122.47,
121.34, 118.92, 117.55, 81.68, 65.79, 54.20, 44.25, 28.61, 27.22, 11.20.
ESI-Mass: m/z 553.597 [M þ Na]þ. Anal. C27H31FN2O6S (C, H, N).
5.1.6.1. Ethyl 2-amino-2-[1-(4-fluorophenyl)-2-methyl-5-[4-(meth-
ylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate (9a). White powder, m.p.
95 ꢂC (50% yield); 1H NMR (400 MHz, DMSO-d6):
d
(ppm) 7.73 (d,
5.1.7.2. 1H-Pyrrol-3-yl]methyl carbamate (10b). White solid, m.p.
2H, J ¼ 8.5 Hz), 7.30 (m, 4H), 7.18 (d, 2H, J ¼ 8.5 Hz), 6.55 (s, 1H), 4.60
(s, 1 H), 4.04 (q, 2H, J ¼ 7.3 Hz), 3.14 (s, 3H), 2.05 (s, 3H), 1.74 (s
broad, 2H), 1.18 (t, 3H, J ¼ 7.3 Hz); 13C NMR (100 MHz, DMSO-d6):
109 ꢂC (67% yield); 1H NMR (400 MHz, CDCl3):
d (ppm) 7.69 (d, 2H,
J ¼ 8.4 Hz), 7.35 (m, 1H), 7.13 (d, 2H, J ¼ 8.4 Hz), 7.09 (m, 1H), 6.90
(m, 2H), 6.59 (s, 1H), 5.65 (d, 1H, J ¼ 7.8 Hz), 5.39 (d, 1H, J ¼ 7.8 Hz),
4.13 (d, 2H, J ¼ 6.8 Hz), 3.17 (s, 3H), 2.06 (s, 3H), 1.44 (s, 9H), 1.14 (t,
d
(ppm) 169.77, 159.42, 144.50, 141.33, 135.20, 130.25, 129.20,
128.22, 126.11, 123.45, 122.50, 115.34, 108.59, 65.84, 58.23, 43.33,
27.60, 10.96; ESI-Mass: m/z 453.126 [M þ Na]þ; Anal. C22H23FN2O4S
(C, H, N).
3H, J ¼ 6.8 Hz). 13C NMR (100 MHz, CDCl3):
d (ppm) 171.32, 168.35,
154.93, 139.72, 137.21, 135.81, 132.86, 128.98, 127.84, 126.75, 122.47,
121.44, 118.92, 117.76, 117.58, 108.43, 81.46, 63.87, 53.96, 43.82,
29.04, 27.27, 11.10. ESI-Mass: m/z 553.597 [M þ Na]þ. Anal.
5.1.6.2. Ethyl 2-amino-2-[1-(3-fluorophenyl)-2-methyl-5-[4-(meth-
ylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate (9b). White powder, m.p.
C27H31FN2O6S (C, H, N).
97 ꢂC (47% yield); 1H NMR (400 MHz, DMSO-d6):
d
(ppm) 7.73 (d,
5.1.8. Procedure for the preparation of benzyl (ethoxycarbonyl)(1,5-
diarylpyrrol)carbamate (13aeb)
To a biphasic solution of amino ester 9aeb (2.326 mmol) in DCM
(10 mL), sodium carbonate (0.481 g, 4.6 mmol) and benzyl
2H), 7.32 (m, 4H), 7.19 (d, 2H), 6.59 (s, 1H), 4.76 (t, 1H), 4.09 (q, 2H),
3.16 (s, 3H), 2.05 (s, 3H), 1.74 (s, broad 2H), 1.16 (t, 3H); 13C NMR
(100 MHz, DMSO-d6):
d (ppm) 169.89, 159.27, 143.85, 140.59,
135.72, 130.93, 128.96, 128.13, 125.89, 124.91, 123.92, 122.48, 117.02,
114.84, 108.04, 65.95, 57.99, 42.97, 27.31, 11.02; ESI-Mass: m/z
453.126 [M þ Na]þ; Anal. C22H23FN2O4S (C, H, N).
5.1.7. General procedure for the preparation of tert-butyl
(ethoxycarbonyl)(1,5-diarylpyrrol)carbamate (10aeb)
To a solution of the amino ester 9aeb (2.326 mmol) in MeOH
(15 mL), Boc anhydride (1.014 g, 4.651 mmol) was added and the
reaction mixture stirred for 18 h. The mixture was then diluted with
water, adjusted the pH to 2e3 with 1 M HCl and extracted with
Table 5
Nitric oxide releasing efficacy and potency for compounds 1a, 1c, 18 and 19.
Compound
Emax
pIC50
þODQ 1
mM
1a
1c
18
19
84.0 ꢁ 2.0
93.0 ꢁ 1.0
69.0 ꢁ 4.0
39.0 ꢁ 1.0
5.66 ꢁ 0.03
5.81 ꢁ 0.03
6.48 ꢁ 0.06
n.c.
þ
þ
þ
þ
Fig. 7. Time-dependent increase of the concentrations of NOx (nitrites þ nitrates)
following the incubation of 1 mM 18 in rat hepatic homogenate, containing the
opportune cofactors.

294
M. Biava et al. / European Journal of Medicinal Chemistry 58 (2012) 287e298
chloroformate (0.401 g, 2.352 mmol) were added at 0 ꢂC, the
reaction mixture was allowed to warm up to rt and stirred for
further 4 h. The mixture was then diluted with water and the two
phases separated. The organic fraction was washed with water
(50 mL ꢃ 2), dried over sodium sulphate and evaporated to give the
crude product. The residue was purified by column chromatog-
raphy (silica gel) eluting with EtOAc/hexane (1:20) (v/v) to afford
the desired product as white solid.
White solid, m.p. 130 ꢂC (>95% yield); 1H NMR (400 MHz, DMSO-
d6):
d
(ppm) 12.81 (s, 1H), 7.81 (d, 1H, J ¼ 7.8 Hz), 7.79 (d, 2 H,
J ¼ 8.5 Hz), 7.68e7.37 (m, 9H), 7.16 (d, 2H, J ¼ 8.5 Hz), 6.63 (s, 1H),
5.09 (d,1H, J ¼ 7.8 Hz), 5.06 (s, 2H), 3.21 (s, 3H), 2.03 (s, 3H). 13C NMR
(100 MHz, DMSO-d6): d (ppm) 171.03, 169.27, 156.98, 143.08, 140.23,
136.91, 134.21, 133.09, 130.88, 127.97, 127.34, 126.32, 124.55, 122.91,
119.00,118.23,117.43,106.99, 63.21, 53.92, 43.71,11.18. ESI-Mass: m/z
559.132 [M þ Na]þ. Anal. C28H25FN2O6S (C, H, N).
5.1.8.1. N-Benzyl-(ethoxycarbonyl)-[1-(4-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]methylcarbamate (13a).
White solid, m.p. 95 ꢂC (65% yield); 1H NMR (400 MHz, DMSO-d6):
5.1.9.4. N-(Benzyloxy)carbonyl-2-[1-(3-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]-2-amino-acetic
(14b). White solid, m.p. 127 ꢂC (>95% yield); 1H NMR (400 MHz,
DMSO-d6):
acid
d
(ppm)7.89(d,1H, J¼ 7.8Hz), 7.74 (d, 2H,J¼ 8.5Hz), 7.51 (m, 5H), 7.19
d
(ppm) 12.81 (s, 1H), 7.89 (d, 1H, J ¼ 7.8 Hz), 7.70 (d, 2H,
(m, 6H), 6.50 (s, 1H), 5.16 (d, 1H, J ¼ 7.8 Hz), 5.08 (s, 2H), 4.11 (q, 2H,
J ¼ 8.6 Hz), 7.52 (m, 1H), 7.47 (m, 7H), 7.32 (d, 2H, J ¼ 8.6 Hz), 7.21
J ¼ 6.9 Hz), 3.10 (s, 3H), 2.00 (s, 3H), 1.15 (t, 3H, J ¼ 6.9 Hz). 13C NMR
(m,1H), 6.64 (m,1H), 5.11 (d,1H, J ¼ 7.8 Hz), 5.09 (s, 2H), 3.15 (s, 3H),
(100 MHz, DMSO-d6):
d
(ppm) 168.45,159.71,156.04,142.96,132.28,
2.09 (s, 3H). 13C NMR (100 MHz, DMSO-d6):
d (ppm) 171.22, 168.97,
138.86, 137.12, 135.98, 133.27, 130.34, 127.96, 126.51, 126.23, 125.38,
123.91, 122.54, 118.07, 117.44, 67.45, 64.31, 54.11, 43.89, 15.46, 12.01.
ESI-Mass: m/z 587.163 [M þ Na]þ. Anal. C30H29FN2O6S (C, H, N).
155.85, 141.02, 139.18, 138.12, 136.58, 132.93, 128.56, 128.02, 127.90,
126.76, 126.54, 126.00, 124.21, 124.00, 117.11, 116.50, 115.99, 106.97,
63.74, 55.05, 42.46, 11.02. ESI-Mass: m/z 559.132 [M þ Na]þ. Anal.
C28H25FN2O6S (C, H, N).
5.1.8.2. tert-Butyl (ethoxycarbonyl)-[1-(3-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-N-benzyl-(ethoxycarbonyl)-[1-(3-
fluorophenyl)-2-methyl-5-[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-
yl]methylcarbamate (13b). White solid, m.p. 97 ꢂC (61% yield); 1H
5.1.10. General procedure for the preparation of protected (Cbz and
Boc) -(1,5-diarylpyrrole)-glycine nitroxyalkyl esters (12aed,15aed)
To a solution of the suitable protected -1,5-diarylpyrrole-
a
a
NMR (400 MHz, DMSO-d6):
d
(ppm) 8.01 (d, 1H, J ¼ 7.8 Hz), 7.89 (d,
glycine (11aeb and 14aeb) (0.1 mmol) in dichloromethane (5 mL),
under nitrogen atmosphere, an excess of hydroxyalkyl nitrate
(17aeb) (0.3 mmol), DMAP (0.1 mmol), and EDCI (0.2 mmol) were
added in sequence. The reaction was quenched with water after 3 h
and extracted with chloroform. The organic layer was washed with
1 M HCl, NaHCO3 saturated solution, brine and dried over sodium
sulphate. The filtration and concentration of the organic phase gave
a crude material, which was purified by chromatography on silica
gel using petroleum ether/chloroform/ethyl acetate, 4:4:1 (v/v/v),
as the eluent to give the desired product in good yield.
2H, J ¼ 8.5 Hz), 7.59 (m, 6H), 7.46 (m, 3H), 7.06 (m, 2H), 6.48 (s, 1H),
5.18 (d, 1H, J ¼ 7.8 Hz), 5.15 (s, 2H), 4.16 (q, 2H, J ¼ 6.8 Hz), 3.15 (s,
3H), 2.04 (s, 3H), 1.17 (t, 3H, J ¼ 6.8 Hz). 13C NMR (100 MHz, DMSO-
d6):
d (ppm) 170.23, 169.27, 155.08, 140.24, 139.23, 137.21, 136.03,
132.78, 130.58, 129.05, 128.04, 127.70, 127.12, 126.65, 124.55, 123.09,
119.25, 118.33, 117.67, 107.28, 67.98, 64.77, 54.05, 44.66, 15.62, 12.03.
ESI-Mass: m/z 587.163 [M þ Na]þ. Anal. C30H29FN2O6S (C, H, N).
5.1.9. Procedure for the preparation of protected (Boc and Cbz) (1,5-
diarylpyrrol)glycine (11aeb, 14aeb)
To a solution of the amino ester 9aeb (2.326 mmol) in MeOH
(15 mL), a solution of NaOH (0.2367 g, 5.917 mmol) in water (3 mL)
was added and the reaction mixture stirred for 17 h. The mixture
was then concentrated in vacuo, diluted with water (2 mL) and the
acid precipitated through the addition of HCl. The solid was
collected to give the product as off white solid.
5.1.10.1. 3-(Nitroxy)propyl N-tert-butyl-(ethoxycarbonyl)-2-(1-(4-
fluorophenyl)-2-methyl-5-(4-(methylsulfonyl)phenyl)-1H-pyrrol-3-
yl)-2-amino-acetate (12a). White solid, m.p. 112 ꢂC (73% yield) 1H
NMR (400 MHz, CDCl3):
d
(ppm) 7.75 (d, 2H, J ¼ 8.4 Hz), 7.35 (m,
4H), 7.15 (d, 2H, J ¼ 8.4 Hz), 6.63 (s, 1H), 5.59 (d, 1H, J ¼ 6.8 Hz), 5.33
(d,1H, J ¼ 6.8 Hz), 4.64 (t, 2H, J ¼ 7.0 Hz), 4.28 (t, 2H, J ¼ 7.0 Hz), 3.05
(s, 3H), 2.15 (s, 3H), 1.98 (m, 2H), 1.46 (s, 9H). 13C NMR (100 MHz,
5.1.9.1. N-tert-Butyl-2-[1-(4-fluorophenyl)-2-methyl-5-[4-(methyl-
sulfonyl)phenyl]-1H-pyrrol-3-yl]-2-amino-acetic acid (11a). White
CDCl3): d (ppm) 171.24, 168.72, 155.24, 138.72, 135.83, 134.88,
132.50, 129.21, 127.17, 126.74, 123.02, 123.33, 118.86, 117.32, 81.97,
64.05, 61.34, 52.94, 44.03, 29.50, 27.82, 11.24. ESI-Mass: m/z 628.174
[M þ Na]þ. Anal. C27H32FN3O9S (C, H, N).
solid, m.p. 120 ꢂC (60% yield); 1H NMR (400 MHz, DMSO-d6):
d (ppm)
12.70 (s, 1H), 7.81 (d, 1H, J ¼ 7.8 Hz), 7.75 (d, 2H, J ¼ 8.5 Hz), 7.35 (m,
4H), 7.15 (d, 2H, J ¼ 8.5 Hz), 6.63 (s, 1H), 5.09 (d, 1H, J ¼ 7.8 Hz), 3.17 (s,
3H), 2.02 (s, 3H), 1.43 (s, 9H). 13C NMR (100 MHz, DMSO-d6):
d
(ppm)
5.1.10.2. 3-(Nitroxy)propyl] N-tert-butyl-(ethoxycarbonyl)-2-(1-(3-
fluorophenyl)-2-methyl-5-(4-(methylsulfonyl)phenyl)-1H-pyrrol-3-
yl)-2-amino-acetate (12b). White solid, m.p. 114 ꢂC (71% yield) 1H
170.23, 162.38, 157.75, 140.28, 139.77, 136.23, 133.06, 128.71, 127.86,
126.88, 121.98, 122.01, 119.32, 117.33, 82.01, 53.87, 44.15, 28.53, 11.11.
ESI-Mass: m/z 525.147 [M þ Na]þ. Anal. C25H27FN2O6S (C, H, N).
NMR (400 MHz, CDCl3):
d
(ppm) 7.83 (d, 2H, J ¼ 8.4 Hz), 7.40 (m,
1H), 7.12 (m, 3H), 6.90 (m, 2H), 6.65 (s, 1H), 5.55 (d, 1H, J ¼ 7.8 Hz),
5.30 (d, 1H, J ¼ 7.8 Hz), 4.71 (t, 2H, J ¼ 7.0 Hz), 4.30 (t, 2H, J ¼ 7.0 Hz),
3.05 (s, 3H), 2.17 (s, 3H), 1.99 (m, 2H), 1.40 (s, 9H). 13C NMR
5.1.9.2. N-tert-Butyl-2-[1-(3-fluorophenyl)-2-methyl-5-[4-(methyl-
sulfonyl)phenyl]-1H-pyrrol-3-yl]-2-amino-acetic
White solid, m.p. 128 ꢂC (64% yield); 1H NMR (400 MHz, DMSO-d6):
(ppm) 12.75 (s, 1H), 7.85 (d, 1H, J ¼ 7.8 Hz), 7.72 (d, 2H, J ¼ 8.4 Hz),
acid
(11b).
(100 MHz, CDCl3):
d (ppm) 170.27, 168.88, 154.23, 141.40, 138.07,
d
136.29, 134.52, 130.01, 128.13, 126.68, 123.12, 121.43, 117.32, 117.06,
118.11, 108.31, 81.93, 62.85, 60.56, 54.82, 43.83, 28.51, 27.31, 10.90.
ESI-Mass: m/z 628.174 [M þ Na]þ. Anal. C27H32FN3O9S (C, H, N).
7.37 (m, 1H), 7.16 (d, 2H, J ¼ 8.4 Hz), 7.12 (m, 1H), 6.90 (m, 1H), 6.64 (s,
1H), 5.10 (d, 2H, J ¼ 7.8 Hz), 3.15 (s, 3H), 2.10 (s, 3H), 1.45 (s, 9H). 13
C
NMR (100 MHz, DMSO-d6):
d (ppm) 170.84, 169.17, 155.62, 140.23,
138.12, 136.04, 133.11, 129.01, 128.13, 126.88, 123.98, 122.21, 117.45,
117.32, 116.82, 108.99, 82.31, 63.74, 55.05, 42.46, 29.00, 18.26, 10.90.
ESI-Mass: m/z 525.147 [M þ Na]þ. Anal. C25H27FN2O6S (C, H, N).
5.1.10.3. 4-(Nitroxy)butyl N-tert-butyl-(ethoxycarbonyl)-2-(1-(4-
fluorophenyl)-2-methyl-5-(4-(methylsulfonyl)phenyl)-1H-pyrrol-3-
yl)-2-amino-acetate (12c). White solid, m.p. 114 ꢂC (75% yield) 1H
NMR (400 MHz, CDCl3):
4H), 7.15 (d, 2H, J ¼ 8.5 Hz), 6.65 (s, 1H), 5.59 (d, 1H, J ¼ 6.8 Hz), 5.33
d
(ppm) 7.75 (d, 2H, J ¼ 8.5 Hz), 7.36 (m,
5.1.9.3. N-(Benzyloxy)carbonyl-2-[1-(4-fluorophenyl)-2-methyl-5-[4-
(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]-2-amino-acetic acid (14a).
(d, 1H, J ¼ 6.8 Hz), 4.65 (t, 2H, J ¼ 7.6 Hz), 4.10 (t, 2H, J ¼ 7.6 Hz), 3.05

M. Biava et al. / European Journal of Medicinal Chemistry 58 (2012) 287e298
295
(s, 3H), 2.15 (s, 3H), 1.73 (m, 2H), 1.58 (m, 2H), 1.46 (s, 9H). 13C NMR
(100 MHz, CDCl3): (ppm) 170.82, 169.04, 156.23, 139.01, 135.82,
134.65, 132.46, 129.02, 126.89, 125.38, 124.21, 123.13, 118.15, 116.85,
109.95, 81.88, 65.82, 63.00, 53.28, 44.03, 25.24, 23.82, 10.98. ESI-
Mass: m/z 642.190 [M þ Na]þ. Anal. C28H34FN3O9S (C, H, N).
2H); 13C NMR (100 MHz, CDCl3):
d
(ppm) 170.92, 169.00, 155.20,
d
142.45, 142.21, 138.07, 136.29, 134.31, 130.83, 128.79, 127.64, 127.32,
127.03, 126.41, 124.08, 121.89, 117.51, 117.08, 116.97, 108.09, 67.20,
63.44, 61.45, 54.82, 43.83, 27.33, 28.51, 10.91. ESI-Mass: m/z 676.184
[M þ Na]þ; Anal. C32H32FN3O9S (C, H, N).
5.1.10.4. 4-(Nitroxy)butyl N-tert-butyl-(ethoxycarbonyl)-2-[1-(3-
fluorophenyl)-2-methyl-5-(4-(methylsulfonyl)phenyl)]-1H-pyrrol-3-
yl]-2-amino-acetate (12d). White solid, m.p. 118 ꢂC (68% yield) 1H
5.1.11. General procedure for the preparation of
glycine hydroxyalkyl esters (2aed)
a
-(1,5-diarylpyrrole)-
To
a
solution of the opportune Cbz-protected
a
-1,5-
NMR (400 MHz, CDCl3):
d
(ppm) 7.83 (d, 2H, J ¼ 8.5 Hz), 7.40 (m,
diarylpyrrole-glycine (15aed) (0.32 mmol) in isopropanol (2 mL),
ammonium formate (0.081 g, 1.3 mmol) and Pd/C (0.07 g) were
added. The solution was microwave irradiated at 80 ꢂC for 5 min
(power 150 W, pressure 170 psi). The reaction mixture was passed
through CeliteÒ and then poured into water (20 mL). The pH was
adjusted to 10e12 (using NaOH 10% solution) and the mixture
extracted with chloroform. The organic layer was then dried over
sodium sulphate. The filtration and concentration of the organic
phase gave a material, which was identified to be the product
without the need of any further purification.
1H), 7.12 (m, 3H), 6.90 (m, 2H), 6.65 (s, 1H), 5.55 (d, 1H, J ¼ 7.8 Hz),
5.30 (d, 1H, J ¼ 7.8 Hz), 4.70 (t, 2H, J ¼ 7.6 Hz), 4.11 (t, 2H, J ¼ 7.6 Hz),
3.05 (s, 3H), 2.17 (s, 3H), 1.71 (m, 2H), 1.63 (m, 2H), 1.40 (s, 9H). 13
C
NMR (100 MHz, CDCl3):
d (ppm) 169.82, 167.54, 153.90, 142.04,
138.75, 135.81, 133.81, 129.85, 128.00, 127.41, 122.31, 123.56, 117.20,
117.06, 117.33, 107.02, 79.68, 65.79, 62.09, 54.82, 44.28, 27.94, 26.82,
22.54, 12.03. ESI-Mass: m/z 642.190 [M þ Na]þ. Anal. C28H34FN3O9S
(C, H, N).
5.1.10.5. 3-(Nitroxy)propyl N-benzyl-(ethoxycarbonyl)-2-(1-(4-
fluorophenyl)-2-methyl-5-(4-(methylsulfonyl)phenyl)-1H-pyrrol-3-
yl)-2-amino-acetate (15a). White solid, 118 ꢂC (62% yield); 1H NMR
5.1.11.1. 3-Hydroxypropyl 2-amino-2-[1-(4-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate
(2a). Yellowish
(400 MHz, CDCl3):
d
(ppm) 7.69 (d, 2 H, J ¼ 8.4 Hz), 7.55 (m, 9H), 7.16
powder, m.p.107 ꢂC (>95% yield); FT-IR (neat, cmꢀ1) v: 3340 (m), 3320
(d, 2 H, J ¼ 8.4 Hz), 6.64 (s, 1H), 5.54 (d, 1H, J ¼ 7.7 Hz), 5.32 (d, 1H,
J ¼ 7.7 Hz), 5.10 (s, 2H), 4.66 (t, 2H, J ¼ 6.8 Hz), 4.39 (t, 2H,
J ¼ 6.8 Hz), 3.00 (s, 3H), 2.20 (s, 3H), 1.95 (m, 2H). 13C NMR
(m), 2852 (w), 1736 (s), 1593 (s), 1302 (s), 1140 (s), 1098 (s); 1H NMR
(400 MHz, DMSO-d6):
d
(ppm) 7.90 (d, 2H, J ¼ 8.4 Hz), 7.70 (d, 2H,
J ¼ 8.4 Hz), 7.29 (d,2H, J ¼ 8.4 Hz), 7.08 (m, 2H), 6.56(s,1H), 4.78 (tapp.,
(100 MHz, CDCl3):
d
(ppm) 170.83, 168.72, 154.98, 143.23, 139.01,
1H), 4.45 (s,1H), 4.15 (m, 2H), 3.57 (m, 2H), 3.06 (s, 3H), 2.24 (s br., 2H),
135.82, 134.65, 132.43, 131.80, 127.98, 127.49, 127.29, 127.17, 126.89,
123.45, 123.22, 119.05, 116.92, 115.88, 66.82, 66.24, 60.45, 44.03,
27.8, 11.24. ESI-Mass: m/z 662.158 [M þ Na]þ; Anal. C31H30FN3O9S
(C, H, N).
2.18 (s, 3H), 1.90 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
d (ppm)
171.30, 159.87, 147.30, 146.78, 141.51, 135.00, 130.32, 129.25, 128.78,
126.20, 123.48, 122.53, 117.12, 64.10, 61.00, 58.88, 43.07, 27.62, 11.06;
ESI-Mass: m/z 483.137 [M þ Na]þ; Anal. C23H25FN2O5S (C, H, N).
5.1.10.6. 3-(Nitroxy)propyl N-benzyl-(ethoxycarbonyl)-2-(1-(3-
fluorophenyl)-2-methyl-5-(4-(methylsulfonyl)phenyl)-1H-pyrrol-3-
yl)-2-amino acetate (15b). White solid, 115 ꢂC (66% yield); 1H NMR
5.1.11.2. 3-Hydroxypropyl 2-amino-2-[1-(3-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate
(2b). Yellowish
powder, m.p. 110 ꢂC (>95% yield); FT-IR (neat, cmꢀ1) v: 3345 (m),
(400 MHz, CDCl3):
d
(ppm) 7.70 (d, 2H, J ¼ 8.5 Hz), 7.52 (m, 1H), 7.47
3322 (m), 2858 (w), 1741 (s), 1590 (s), 1300 (s), 1145 (s), 1090 (s); 1H
(m, 7H), 7.32 (d, 2H, J ¼ 8.5 Hz), 7.21 (m, 1H), 6.64 (m, 1H), 5.50 (d,
1H, J ¼ 7.8 Hz), 5.29 (d, 1H, J ¼ 7.8 Hz), 5.10 (s, 2H), 4.67 (t, 2H,
J ¼ 6.9 Hz), 4.29 (t, 2H, J ¼ 6.9 Hz), 3.00 (s, 3H), 2.20 (s, 3H), 1.95 (m,
NMR (400 MHz, DMSO-d6):
d
(ppm) 7.71 (d, 2H, J ¼ 8.4 Hz), 7.41 (m,
1H), 7.15 (d, 2H, J ¼ 8.4 Hz), 7.05 (m, 1H), 6.97 (m, 2H), 6.59 (s, 1H),
4.79 (t app., 1H), 4.40 (s, 1H), 4.13 (m, 2H), 3.56 (m, 2H), 3.11 (s, 3H),
2.20 (s br., 2H), 2.19 (s, 3H), 1.92 (m, 2H); 13C NMR (100 MHz, DMSO-
2H). 13C NMR (100 MHz, CDCl3):
d (ppm) 170.83, 169.04, 155.28,
142.47, 142.06, 138.07, 136.29, 134.31, 130.83, 128.79, 127.64, 127.32,
127.13, 126.40, 124.08, 121.31, 117.51, 117.08, 116.92, 108.09, 67.20,
63.04, 60.93, 54.82, 43.83, 28.51, 10.90. ESI-Mass: m/z 662.158
[M þ Na]þ; Anal. C31H30FN3O9S (C, H, N).
d6): d (ppm) 171.44, 158.57, 148.78, 144.00, 139.50, 138.56, 137.66,
135.54, 131.92, 128.90, 128.66, 126.20, 123.44, 122.59, 117.11, 64.09,
60.88, 58.80, 43.10, 27.60, 11.01; ESI-Mass: m/z 483.137 [M þ Na]þ;
Anal. C23H25FN2O5S (C, H, N).
5.1.10.7. 4-(Nitroxy)butyl N-benzyl-(ethoxycarbonyl)-2-(1-(4-
fluorophenyl)-2-methyl-5-(4-(methylsulfonyl)phenyl)-1H-pyrrol-3-
yl)-2-amino acetate (15c). White solid, 121 ꢂC (61% yield); 1H NMR
5.1.11.3. 4-Hydroxybutyl 2-amino-2-[1-(4-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate
(2c). Yellowish
powder, m.p.101 ꢂC (>95% yield); FT-IR (neat, cmꢀ1) v: 3349 (m), 3324
(400 MHz, CDCl3):
d
(ppm) 7.65 (d, 2 H, J ¼ 8.5 Hz), 7.53 (m, 9H), 7.17
(m), 2861 (w), 1731 (s), 1588 (s), 1298 (s), 1132 (s), 1079 (s); 1H NMR
(d, 2 H, J ¼ 8.5 Hz), 6.64 (s, 1H), 5.55 (d, 1H, J ¼ 7.6 Hz), 5.34 (d, 1H,
J ¼ 7.6 Hz), 5.12 (s, 2H), 4.65 (t, 2H, J ¼ 7.0 Hz), 4.11 (t, 2H, J ¼ 7.0 Hz),
3.05 (s, 3H), 2.15 (s, 3H), 1.73 (m, 2H), 1.58 (m, 2H). 13C NMR
(400 MHz, DMSO-d6):
d
(ppm) 7.72 (d, 2H, J ¼ 8.5 Hz), 7.37 (d, 2H,
J ¼ 8.5 Hz), 7.15 (d, 2H, J ¼ 8.5 Hz), 7.09 (m, 2H), 6.60 (s,1H), 4.78 (t app.,
1H), 4.41 (s,1H), 4.17 (m, 2H), 3.55 (m, 2H), 3.10 (s, 3H), 2.20 (s br., 2H),
(100 MHz, CDCl3):
d
(ppm) 171.00, 168.66, 154.99, 143.21, 139.34,
2.19 (s, 3H), 1.89 (m, 4H); 13C NMR (100 MHz, DMSO-d6):
d (ppm)
135.04, 134.09, 132.40, 131.81, 128.22, 127.39, 127.21, 127.07, 126.09,
123.31, 123.20, 118.95, 117.55, 116.09, 66.67, 66.04, 60.05, 44.03,
27.81, 27.55, 11.21. ESI-Mass: m/z 676.184 [M þ Na]þ; Anal.
171.31, 159.90, 147.40, 146.71, 141.50, 135.02, 130.32, 129.67, 128.80,
126.21, 123.43, 122.51, 117.03, 74.34, 64.10, 60.90, 43.11, 24.62, 22.88,
11.16; ESI-Mass: m/z 497.152 [M þ Na]þ; Anal. C24H27FN2O5S (C, H, N).
C
32H32FN3O9S (C, H, N).
5.1.11.4. 3-Hydroxybutyl 2-amino-2-[1-(3-fluorophenyl)-2-methyl-5-
5.1.10.8. 4-(Nitroxy)butyl N-benzyl-(ethoxycarbonyl)-2-(1-(3-
fluorophenyl)-2-methyl-5-(4-(methylsulfonyl)phenyl)-1H-pyrrol-3-
yl)-2-amino acetate (15d). White solid, 119 ꢂC (64% yield); 1H NMR
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate
(2d). Yellowish
powder, m.p. 98 ꢂC (>95% yield); FT-IR (neat, cmꢀ1) v: 3353 (m), 3311
(m), 2861 (w), 1744 (s), 1590 (s), 1300 (s), 1147 (s), 1092 (s); 1H NMR
(400 MHz, CDCl3):
d
(ppm) 7.70 (d, 2H, J ¼ 8.4 Hz), 7.55 (m, 1H), 7.47
(400 MHz, DMSO-d6):
d
(ppm) 7.74 (d, 2H, J ¼ 8.5 Hz), 7.43 (m, 1H),
(m, 7H), 7.29 (d, 2H, J ¼ 8.4 Hz), 7.21 (m,1H), 6.64 (s,1H), 5.48 (d,1H,
J ¼ 7.7 Hz), 5.31 (d,1H, J ¼ 7.7 Hz), 5.12 (s, 2H), 4.60 (t, 2H, J ¼ 7.0 Hz),
4.07 (t, 2H, J ¼ 7.0 Hz), 3.05 (s, 3H), 2.10 (s, 3H), 1.75 (m, 2H),1.60 (m,
7.15 (d, 2H, J ¼ 8.5 Hz), 7.11 (m, 1H), 6.97 (m, 2H), 6.52 (s, 1H), 4.80 (t
app., 1H), 4.39 (s, 1H), 4.18 (m, 2H), 3.53 (m, 2H), 3.13 (s, 3H), 2.21 (s
br., 2H), 2.18 (s, 3H), 1.86 (m, 4H); 13C NMR (100 MHz, DMSO-d6):

296
M. Biava et al. / European Journal of Medicinal Chemistry 58 (2012) 287e298
d
(ppm) 171.39, 158.60, 148.90, 144.05, 139.54, 138.56, 137.66, 135.50,
128.88, 128.60, 126.20, 123.44, 122.59, 117.18, 74.70, 64.10, 61.20,
131.91, 128.88, 128.60, 126.20, 123.44, 122.59, 117.18, 74.70, 64.10,
61.20, 43.34, 25.69, 23.44, 11.09; ESI-Mass: m/z 497.152 [M þ Na]þ;
Anal. C24H27FN2O5S (C, H, N).
43.88, 25.69, 23.44, 11.09; ESI-Mass: m/z 542.137 [M þ Na]þ; Anal.
C24H26FN3O7S (C, H, N).
5.1.13. General procedure for the preparation of hydroxylalkyl
nitrates (17aeb)
5.1.12. Generalprocedureforthepreparationof
a-(1,5-diarylpyrrole)-
glycine nitroxyalkyl esters (1aed)
To a solution of diol (20 mmol) in ethyl acetate (100 mL) nitric
acid (65% w/v, 16 mmol), glacial acetic acid (97 mmol), and acetic
anhydride (97% w/v, 59 mmol) were added. The mixture was
allowed to react overnight. The reaction was quenched with 20%
KOH solution. The pH was adjusted to 12e13, and the aqueous
phase was extracted with ethyl acetate (3 ꢃ 100 mL), washed with
NaCl saturated solution, and dried over Na2SO4. After filtration, the
organic layers were concentrated in vacuo, and the crude product
was purified by chromatography on silica gel, using petroleum
ether/chloroform/ethyl acetate, 3:1:1 (v/v/v), as eluent, to obtain
the expected nitroester in good yield. Analytical data, melting
points, and the 1H NMR spectra were consistent with those re-
ported in the literature [21].
To a solution of the opportune Boc-protected
a-1,5-diarylpyrrole-
glycine (12aed) (0.40 mmol) in dioxane (2 mL), an excess of tri-
fluoroacetic acid (4 mL) was added dropwise at 0 ꢂC. The solutionwas
microwave irradiated at 60 ꢂC for 40 min (power 150 W, pressure
170 psi). The reaction mixture was poured into ice/water (20 mL) and
the pH adjusted to 12e13 (using ammonia solution) and the mixture
extracted with chloroform. The organic layer was then dried over
sodium sulphate. The filtration and concentration of the organic
phase gave a crude material, which was purified through column
chromatography(alumina)usingMeOH/DCM1:20(v/v)toobtainthe
product as a yellowish powder.
5.1.12.1. 3-(Nitroxy)propyl 2-amino-2-[1-(4-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate
(1a). Yellowish
5.2. Bio-pharmacological testing
powder, m.p. 121 ꢂC (75% yield); FT-IR (neat, cmꢀ1) v: 3344 (m), 2890
(w), 1736 (s), 1685 (s), 1590 (s), 1302 (s), 1147 (s), 1090 (s), 880 (m); 1H
5.2.1. Pharmacology
NMR (400 MHz, CDCl3):
d
(ppm) 7.70 (d, 2H, J ¼ 8.4 Hz), 7.29 (d, 2H,
In Vitro Anti-Inflammatory Study. The in vitro profiles of
compounds 1aed and 2aed, related to their inhibitory activity
towards both COX-1 and COX-2 isoenzymes, were evaluated
through cell-based assay employing murine monocyte/macro-
phage J774 cell lines. The cell line was grown in DMEM supple-
mented with 2 mM glutamine, 25 mM HEPES, 100 units/mL
J ¼ 8.4 Hz), 7.11 (d, 2H, J ¼ 8.4 Hz), 7.08 (m, 2H), 6.61 (s, 1H), 4.65 (t,
2H, J ¼ 7.2 Hz), 4.50 (s,1H), 4.39 (t, 2H, J ¼ 7.2 Hz), 3.01 (s, 3H), 2.20 (s,
3H), 1.95 (m, 2H), 1.80 (s br., 2H); 13C NMR (100 MHz, CDCl3):
d (ppm)
171.33, 159.91, 147.33, 146.80, 141.53, 135.00, 130.32, 129.22, 128.76,
126.19, 123.45, 122.50, 117.09, 74.34, 64.08, 60.98, 53.07, 27.62, 11.06;
ESI-Mass: m/z 528.122 [M þ Na]þ; Anal. C23H24FN3O7S (C, H, N).
penicillin, 100 mg/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 ꢃ 105 cells/mL or in 60 mm diameter
culture dishes (3 ꢃ 106 cells per 3 mL per dish) and allowed to
adhere at 37 ꢂC in 5% CO2 for 2 h. Immediately before the experi-
ments, the culture medium was replaced with fresh medium and
cells were stimulated as described previously [15].
5.1.12.2. 3-(Nitroxy)propyl 2-amino-2-[1-(3-fluorophenyl)-2-
methyl-5-[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate
(1b).
Yellowish powder, m.p. 117 ꢂC (73% yield); FT-IR (neat, cmꢀ1) v:
3340 (m), 2887 (w), 1738 (s), 1681 (s), 1592 (s), 1300 (s), 1149 (s),
1091 (s), 891 (m); 1H NMR (400 MHz, CDCl3):
d (ppm) 7.70 (d, 2H,
J ¼ 8.5 Hz), 7.39 (m,1H), 7.18 (d, 2H, J ¼ 8.5 Hz), 7.10 (m,1H), 7.03 (m,
The evaluation of COX-1 inhibitory activity was achieved by
2H), 6.60 (s, 1H), 4.67 (t, 2H, J ¼ 7.2 Hz), 4.48 (s, 1H), 4.38 (t, 2H,
pretreating cells with test compounds (10
incubating them at 37 ꢂC for 30 min with 15
activate the constitutive COX. For the compounds with COX-1
inhibition higher than 50% (at 10 M), the cells were also treated
with lower concentrations (0.01e1 M). At the end of the incuba-
mM) for 15 min and then
J ¼ 7.2 Hz), 3.07 (s, 3H), 2.23 (s, 3H), 1.98 (m, 2H), 1.90 (s br., 2H); 13
C
mM arachidonic acid to
NMR (100 MHz, CDCl3):
d (ppm) 171.44, 158.57, 148.78, 144.00,
139.50, 138.56, 137.66, 135.54, 131.92, 128.90, 128.66, 126.20, 123.44,
122.59, 117.11, 74.58, 64.11, 61.03, 43.00, 27.69, 11.11; ESI-Mass: m/z
528.122 [M þ Na]þ; Anal. C23H24FN3O7S (C, H, N).
m
m
tion, the supernatants were collected for the measurement of
prostaglandin E2 (PGE2) levels by a radioimmunoassay (RIA). To
evaluate COX-2 activity, cells were stimulated for 24 h with
5.1.12.3. 4-(Nitroxy)butyl 2-amino-2-[1-(4-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate
(1c). Yellowish
Escherichia coli lipopolysaccharide (LPS, 10
in the absence or presence of test compounds (0.01e10
m
g/mL) to induce COX-2,
powder, m.p. 102 ꢂC (58% yield); FT-IR (neat, cmꢀ1) v: 3347 (m), 2894
mM). The
(w), 1742 (s), 1689 (s), 1587 (s),1298 (s), 1157 (s), 1099 (s), 877 (m); 1H
supernatants were collected for the measurement of PGE2 by
means of RIA. Throughout the time the experiments lasted, tripli-
cate wells were used for the various conditions of treatment.
Results are expressed as the mean, for three experiments, of the
percent inhibition of PGE2 production by test compounds with
respect to control samples. The IC50 values were calculated with
GraphPad Instat, and the data fit was performed using the
sigmoidal doseeresponse equation (variable slope) (GraphPad).
NMR (400 MHz, CDCl3):
d
(ppm) 7.69 (d, 2H, J ¼ 8.5 Hz), 7.25 (d, 2H,
J ¼ 8.5 Hz), 7.15 (d, 2H, J ¼ 8.5 Hz), 7.07 (m, 2H), 6.60 (s, 1H), 4.68 (t,
2H, J ¼ 7.2 Hz), 4.48 (s,1H), 4.39 (t, 2H, J ¼ 7.2 Hz), 3.07 (s, 3H), 2.23 (s,
3H), 2.01 (m, 4H), 1.90 (s br., 2H); 13C NMR (100 MHz, CDCl3):
d (ppm)
171.31, 159.90, 147.40, 146.71, 141.50, 135.02, 130.32, 129.67, 128.80,
126.21, 123.43, 122.51, 117.03, 74.34, 64.10, 60.90, 43.11, 24.62, 22.88,
11.16; ESI-Mass: m/z 542.137 [M þ Na]þ; Anal. C24H26FN3O7S (C, H, N).
5.1.12.4. 4-(Nitroxy)butyl 2-amino-2-[1-(3-fluorophenyl)-2-methyl-5-
5.2.2. Ex vivo vasorelaxing activity
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate
(1d). Yellowish
All the experimental procedures were carried out following the
guidelines of the European Community Council Directive 86e609.
The effects of nitro-oxy compounds were tested on isolated
thoracic aortic rings of male normotensive Wistar rats (250e350 g).
After a light ether anaesthesia, rats were sacrificed by cervical
dislocation and bleeding. The aortae were immediately excised,
freed of extraneous tissues, and the endothelial layer was removed
by gently rubbing the intimal surface of the vessels with
powder, m.p. 113 ꢂC (57% yield); FT-IR (neat, cmꢀ1) v: 3350 (m), 2892
(w), 1740 (s), 1693 (s), 1580 (s), 1292 (s), 1150 (s), 1088 (s), 870 (m); 1H
NMR (400 MHz, CDCl3):
d
(ppm) 7.68 (d, 2H, J ¼ 8.4 Hz), 7.42 (m, 1H),
7.18 (d, 2H, J ¼ 8.4 Hz), 7.10 (m, 1H), 7.05 (m, 2H), 6.61 (s, 1H), 4.70 (t,
2H, J ¼ 7.2 Hz), 4.50 (s,1H), 4.37 (t, 2H, J ¼ 7.2 Hz), 3.12 (s, 3H), 2.20 (s,
3H), 2.03 (m, 4H),1.90 (s br., 2H); 13C NMR (100 MHz, CDCl3):
d (ppm)
171.39, 158.60, 148.90, 144.05, 139.54, 138.56, 137.66, 135.50, 131.91,

M. Biava et al. / European Journal of Medicinal Chemistry 58 (2012) 287e298
297
a hypodermic needle. The 5 mm wide aortic rings were suspended,
under a preload of 2 g, in 20 mL organ baths containing Tyrode
solution (composition of saline in mM: NaCl 136.8; KCl 2.95; CaCl2
1.80; MgSO4 1.05; NaH2PO4 0.41; NaHCO3 11.9; glucose 5.5) ther-
mostated at 37 ꢂC and continuously gassed with a mixture of O2
(95%) and CO2 (5%). Changes in tension were recorded by means of
an isometric transducer (Grass FTO3) connected to a computerized
system (Biopac). After an equilibration period of 60 min, the
endothelium removal was confirmed by the administration of
and 120 min) and added into a beaker containing 1.9 mL of an
aqueous solution of H2SO4 (0.1 M) and KI (0.1 M). This solution
allow nitrite ions to be exhaustively and instantaneously reduced to
NO, which is amperometrically titrated by a NO-selective electrode
connected to an Apollo 4000 free radical analyser (World Precision
Instrument), allowing us to determine the concentration of the
nitrites previously formed in the biological sample. In parallel
experiments, a Nitrate Reductor (World Precision Instrument) was
constantly kept immerged into the vial. This tool allowed nitrates to
be constantly converted to nitrites. The concentration of nitrites in
these samples is defined as NOx, which reflects the sum of all the
nitrites and nitrates previously derived from NO. Opportune cali-
bration curves were previously obtained with standard solutions of
sodium nitrite.
acetylcholine (ACh) (10 mM) to KCl (30 mM) precontracted vascular
rings. A relaxation of <10% of the KCl-induced contraction was
considered representative of an acceptable lack of the endothelial
layer, while the organs, showing a relaxation of ꢄ10% (i.e., signifi-
cant presence of the endothelium), were discarded. From 30 to
40 min after the confirmation of the endothelium removal, the
aortic preparations were contracted by a single concentration of KCl
(30 mM). When the contraction reached a stable plateau, 3-fold
5.2.4. In vivo analgesic and anti-inflammatory study
In vivo anti-inflammatory activity of the new compounds was
also assessed. Male Swiss albino mice (23e25 g) and Spraguee
Dawley or Wistar rats (150e200 g) were used. The animals were
fed with a standard laboratory diet and tap water ad libitum and
kept at 23 ꢁ 1 ꢂC with a 12 h light/dark cycle, light on at 7 a.m. The
paw pressure test was performed by inducing an inflammatory
process by the intraplantar (ipl) carrageenan administration 4 h
before the test. The carrageenan induced paw oedema test was also
performed, evaluating the paw volume of the right hind paw 4 h
after the injection of carrageenan and comparing it with saline/
carrageenan-treated controls. The analgesic activity of compounds
was also assessed by performing the abdominal constriction test,
using mice into which a 0.6% solution of acetic acid (10 mL/kg) had
been injected intraperitoneal (ip). The number of stretching move-
ments was counted for 10 min, starting 5 min after administration.
Statistical analysis. Results were expressed as the means ꢁ S.E.M.
and an analysis of variance was performed by ANOVA. A Fisher’s
protected least significant difference procedure was used as post-
hoc comparison. P values of less than 0.05 or 0.01 were consid-
ered significant. Data were analysed using the “Origin 7.5” software.
increasing concentrations of compounds (1 nMe10
mM) were
added. Preliminary experiments showed that the KCl (30 mM)
induced contractions remained in a stable tonic state for at least
40 min. The same experiments were carried out also in the
presence of a well-known GC inhibitor: 1
mM ODQ which was
incubated in aortic preparations after the endothelium removal
confirmation. The vasorelaxing efficacy was evaluated as maximal
vasorelaxing response (Emax), expressed as a percentage of the
contractile tone induced by 30 mM KCl. When the limit concen-
tration of 10 mM (the highest concentration that could be admin-
istered) of the tested compounds did not produce the maximal
effect, the parameter of efficacy represented the vasorelaxing
response, expressed as a percentage of the contractile tone induced
by 30 mM KCl, evoked by this limit concentration. The parameter of
potency was expressed as pIC50, calculated as the negative loga-
rithm of the molar concentration of the tested compounds evoking
a half reduction of the contractile tone induced by 30 mM KCl. The
pIC50 could not be calculated for those compounds showing an
efficacy parameter lower than 50%. The parameters of efficacy and
potency were expressed as the mean ꢁ standard error for 6e10
experiments. Two-way ANOVA was used for statistical analysis,
and P < 0.05 was considered representative of significant statistical
differences. Experimental data were analysed by a computer fitting
procedure (software: GraphPad Prism, version 4.0).
5.3. Solubility assessment in phosphate buffered saline (PBS) and
simulated gastric fluid (SGF)
Stock solutions of compounds 1a, 1c, 18 and 19 of concentration
20 mM were prepared in DMSO. A standard phosphate buffered
saline (PBS) 0.01 M (pH 7.4) was used to determine the solubility in
neutral pH conditions; SGF 0.05 M solution without pepsin (pH was
adjusted to 1.5 using concentrated HCl) was instead used to check
the solubility of compounds in acid medium. Seven point calibra-
5.2.3. Determination of the formation of nitrites and nitrates, in rat
liver homogenate
The experimental procedures were carried out following the
guidelines of the European Community Council Directive 86e609.
The liver homogenates were obtained from male normotensive
Wistar rats (250e350 g). After a light ether anaesthesia, rats were
sacrificed by cervical dislocation and bleeding. The portal vein was
immediately cannulated and the liver was perfused with 4 ꢂC-cold
homogenization buffer (composition: K2HPO4 100 mM; EDTA
1 mM; KCl 15 mM; sucrose 0.25 M and Ethanol 0.1%, pH 7.4). After
about 5 min of slow perfusion, the liver was minced with scissors
and washed with cold homogenation buffer. Then, it was dried with
filter paper, weighted and resuspended (1:5 w/v) in cold homo-
genation buffer. The sample was finally homogenated in Turrax. In
tion standards (1, 5,10, 25, 50,100 and 200
each 20 mM solution stock by serial dilution. 2
compound (stock solution 20 mM in DMSO) were added to 198
m
M) were prepared from
m
L of each test
mL
of PBS and SGF in two duplicate wells in multiscreen solubility filter
plate, covered and shaken for 90 min at 300 rpm using a Multi-
pulse Vortexer (Glas-Col). The sample was filtered using a Milli-
pore manifold filter assembly and collected in an acceptor plate and
then analysed on HPLC together with the standards solutions
(HPLC-Waters Separations Module 2695 with photodiode array
detector).
a vial, 400 mL of the above homogenate were mixed with 500 mL of
assay buffer (composition: K2HPO4 100 mM; EDTA 1 mM; pH 7.4),
containing glutathione, NADH and NADPH (all at the final
concentration of 1 mM). The vials were then thermostated at 37 ꢂC,
Acknowledgements
We are thankful to Rottapharm S.p.A. for financial support.
and 100 mL of a DMSO solution of the tested NO-donor were added
(final concentration of the tested compound ¼ 1 mM), allowing the
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