Enhancing the pharmacodynamic profile of a class of selective COX-2 inhibiting nitric oxide donors

Article abstract of DOI:10.1016/j.bmc.2013.12.008

We report herein the development, synthesis, physicochemical and pharmacological characterization of a novel class of pharmacodynamic hybrids that selectively inhibit cyclooxygenase-2 (COX-2) isoform and present suitable nitric oxide releasing properties.

Full text of DOI:10.1016/j.bmc.2013.12.008

Bioorganic & Medicinal Chemistry 22 (2014) 772–786
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enhancing the pharmacodynamic profile of a class of selective COX-2
inhibiting nitric oxide donors
a,
Mariangela Biava a, , Claudio Battilocchio , Giovanna Poce a, Salvatore Alfonso a, Sara Consalvi a,
⇑
Angela Di Capua b, Vincenzo Calderone c, Alma Martelli c, Lara Testai c, Lidia Sautebin d, Antonietta Rossi d,
Carla Ghelardini e, Lorenzo Di Cesare Mannelli e, Antonio Giordani f, Stefano Persiani f, Milena Colovic f,
Melania Dovizio g, Paola Patrignani g, Maurizio Anzini b
a Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma ‘‘La Sapienza’’, Piazzale Aldo Moro 5, 00185 Roma, Italy
b Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy
c Dipartimento di Farmacia, Università degli Studi di Pisa, Via Bonanno 6, 56126 Pisa, Italy
d Dipartimento di Farmacia, Università degli Studi di Napoli ‘‘Federico II’’, Via D. Montesano 49, 80131 Napoli, Italy
e Dipartimento di Neurologia, Psicologia, Area del Farmaco e Salute del Bambino, Università degli Studi di Firenze, Viale G. Pieraccini 6, I-50139 Firenze, Italy
f Rottapharm Madaus, Via Valosa di Sopra 7, 20052 Monza, Italy
g Neuroscienze e Imaging, Università ‘‘G. d’Annunzio’’ di Chieti and CeSI, Via dei Vestini 31, 66100 Chieti, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the development, synthesis, physicochemical and pharmacological characterization of a
novel class of pharmacodynamic hybrids that selectively inhibit cyclooxygenase-2 (COX-2) isoform and
present suitable nitric oxide releasing properties. The replacement of the ester moiety with the amide
group gave access to in vivo more stable and active derivatives that highlighted outstanding pharmaco-
logical properties. In particular, the glycine derivative proved to be extremely active in suppressing
hyperalgesia and edema.
Received 27 September 2013
Revised 29 November 2013
Accepted 5 December 2013
Available online 18 December 2013
Keywords:
CINODs
Ó 2013 Elsevier Ltd. All rights reserved.
Cyclooxygenases
Coxibs
1,5-Diarylpyrroles
Pharmacodynamic hybrids
Nitric oxide
1. Introduction
tologic disorders and other conditions. Long-term therapies with
traditional (t)NSAIDs are associated to mild-to-severe side effects,
Nonsteroidal anti-inflammatory drugs (NSAIDs) (Fig. 1) have
been used for decades to treat pain and inflammation in rheuma-
especially gastrointestinal (GI), renal and cardiovascular (CV) ones,
which can reduce the compliance of the patients.1–3 Albeit the
development of selective cyclooxygenases-2 (COX-2) inhibitors
(coxibs) has given access to more effective drugs for the treatment
of severe symptoms related to chronic diseases (rheumatoid arthri-
tis, RA, and osteoarthritis, OA) with reduced GI side effects, the
widely documented cardiovascular toxicity of these molecules
‘shrank’ their administration to a narrower population.4–7
‘Molecular hybrids’ characterized by a nitric oxide (NO) releas-
ing moiety have been explored by several research groups for
improving the pharmacological profile of the parent drugs.8,9 In-
deed, at physiological concentrations the ‘gasotransmitter’ NO is
a fundamental modulator of the homeostasis in many systems,
particularly active in the cardiovascular one, with its powerful
vasodilating and antiplatelet activities. Furthermore, NO is known
to be ‘cardioprotective’ against myocardial ischemia/reperfusion
injury.10–12 The pioneering ‘strategy’ adopted by NicOx led to the
development of the first candidate of the COX Inhibiting Nitric
Abbreviations: NSAIDs, nonsteroidal anti-inflammatory drug; (t)NSAIDs, tradi-
tional nonsteroidal anti-inflammatory drug; GI, gastrointestinal; CV, cardiovascu-
lar; COX-2, cyclooxygenase 2; RA, rheumatoid arthritis; OA, osteoarthritis; coxibs,
cyclooxygenase-2 inhibitors; NO, nitric oxide; CINODs, cyclooxygenase-inhibiting
nitric oxide donor; NOBA, NO releasing moiety (nitroxy)butanol; cNOS, constitutive
nitric oxide synthases; SAR, structure relationship studies; EDCI, 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide; DMAP, dimethylaminopyridine; HOBt, 1-
hydroxybenzotriazole; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate; CBz, carboxybenzyl; Boc, tert-butyloxycarbonyl; ODQ,
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; AA, arachidonic acid; HWB, human
whole blood; SGF, simulated gastric fluid; PBS, phosphate buffer solution; FBS, fetal
bovine serum; RIA, radioimmunoassay; PGE2, prostaglandin E2; LPS, lipopolysac-
charide; DMEM, Dulbecco’s modified eagle’s medium; TX, thromboxane; ipl,
intraplantar; ip, intraperitoneal; Emax, maximal vasorelaxing response.
⇑
Corresponding author. Tel.: +39 06 4991 3812; fax: +39 06 4991 3133.
E-mail address: mariangela.biava@uniroma1.it (M. Biava).
Present address: Innovative Technology Centre (for ACS), Department of Chem-
istry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, United Kingdom.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.008

M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
773
Me
O
CF3
N
N
N
N
OH
CH3
OH
NH
H2NO2S
Cl
Cl
Cl
O
H3CO
SO2Me
CH3
Etoricoxib
naproxen
Celecoxib
Diclofenac
Figure 1. Structure of some selective and non selective COX-2 inhibitors.
oxyalkyl ethers which were designed with the aim of preventing
the hydrolysis of the side chain (Fig. 3).28,29 However, despite their
enhanced stabilities, the nitro-oxyalkyl ethers were characterized
by low solubility.
O
O
ONO2
O
To complete the picture, the aim of this work is to generate
compounds endowed with greater stability and solubility as well
as improved pharmacokinetic properties. We report herein the
development of three different subclasses of compounds generated
through replacement of the ester moiety with the amide group. In
particular, we first explored a subclass of ‘linear’ acetic amides
(1a–d), through the replacement of the ester moiety of the first
generation of diarylpyrrolacetic esters with the amide group. Then,
with the aim of enhancing the solubility of the series, we have pre-
pared two subclasses of compounds decorated with either a free
carboxylic substituent (‘serine/homoserine’ acetic amides 2a–d)
or with a free amino group (glycine amides 3a–b) (Fig. 4).
Nowadays, evidences account for protective roles of NO (gener-
ated by the constitutive nitric oxide synthases (cNOS) isoform) in
the joint, thus suggesting that mild NO-donors could be an asset
in arthritis treatment. Accordingly, we speculated that an in-
creased penetration of our compounds in the cartilage could be
an additional target. Due to the presence of an amino group which
is protonated at physiological pH, the glycine derivatives (3) were
expected to better penetrate into the cartilage. Indeed, positively
charged compounds are reported to better diffuse into the cartilage
matrix due to electrostatic interaction with the negatively charged
GAG-sulphates.30–32
Naproxcinod
Figure 2. Chemical structure of naproxcinod.
Oxide Donors (CINODs) family (naproxcinod, AZD3582, Fig. 2),
which completed phase III trials (osteoarthritis of the knee and
hip).13–18 Interestingly, though the GI side-effects associated to
the administration of naproxcinod appeared slightly reduced when
compared to naproxen, a safer CV outcome was highlighted from
these studies.17,18 In addition to naproxcinod, many other exam-
ples have been reported regarding ‘NSAIDs hybridization’, even
though very few of them concern the hybridization of selective
COX-2 structures.19–24
In this context, our research was focused on the development of
a class of CINODs endowed with a diarylpyrrole scaffold25–28 and
our first report consisted of the synthesis of a first generation of
diarylpyrrolacetic esters showing very promising COX-2 inhibition
and nitric oxide donating properties (Fig. 3).26 The chemical
manipulation of this first generation gave access to a class of more
soluble derivatives (Fig. 3).27
However, the chemical and enzymatic liability of these esters
was speculated to be responsible of a potential gap between
in vitro and in vivo pharmacological profiles as discussed-above
for naproxcinod. Therefore, we focused our efforts on nitro-
The substitution pattern of compounds was based on the struc-
ture activity relationship (SAR) knowledge acquired during the
F
F
ONO2
O
ONO2
O
N
O
N
O
NH2
O
S
O
S
O
O
CH3
CH3
MAB124
MAB103
F
ONO2
N
O
O
S
O
CH3
VA694
Figure 3. Representative chemical structures of compounds previously generated.

774
M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
R
R
HOOC
O
O
(CH2)nONO2
(CH2)nONO2
N
N
H
N
NH
O
S
CH3
O
S
CH3
O
O
2a-d
1a-d
First generation of a mides
Second generation of a mides
R = 3-F, 4-F
R = 3-F, 4-F
n = 1, 2
n = 2, 3
F
O
(CH2)nONO2
N
N
H
NH2
O
O
S
3a,b
CH3
Third generation of a mides
n = 2, 3
Figure 4. Chemical structures of amides 1a–d, 2a–d and 3a,b.
studies of the previous series,25–28 thus fluorine substituted (meta or
para) compounds were initially explored. The phenyl ring in C5 of
the central core, is substituted with the methansulfonyl group
which was proven to contribute to selectivity towards COX-2.33–40
Since these nitro-esters can be metabolized to the corresponding
alcohols which in turn could be COX-2 inhibitors, these possible
metabolites (hydroxy-derivatives) (4a–d, 5a–d, and 6a–b) were
synthesized and tested for COX-2 inhibition (Table 1).
2. Results and discussion
2.1. Chemistry
Compounds 1a–d and 4a–d were obtained as shown in
Scheme 1. Access to the acetic acid derivatives 7a,b was achieved
as previously reported.25–27,33–39
Table 1
In vitro COXs inhibition and selectivity for compounds 1a–d, 2a–d, 3a,b, 4a–d, 5a–d and 6a,b
Compd
R
X
n
% Inhib COX-1 (10
lM)
% Inhib COX-2 (10
l
M)
IC50 (COX-1)a
(lM)
IC50 (COX-2)a
(lM)
COX-1/COX-2b (S.I.)
1a
1b
1c
1d
4a
4b
4c
4d
2a
2b
2c
2d
5a
5b
5c
5d
3a
3b
6a
6b
3-F
3-F
4-F
4-F
3-F
3-F
4-F
4-F
3-F
3-F
4-F
4-F
3-F
3-F
4-F
4-F
—
NO2
NO2
NO2
NO2
H
H
H
H
NO2
NO2
NO2
NO2
H
H
H
H
2
3
2
3
2
3
2
3
1
2
1
2
1
2
1
2
2
3
2
3
0
0
9
0
0
17
0
21
0
27
0
95
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.300
0.250
0.249
0.310
0.290
0.045
0.300
0.068
0.140
ND
0.310
1.600
0.068
ND
0.160
0.086
0.054
0.140
>10
>33
>40
>42
>32
>34
>222
>33
>147
>71
>6
>32
>6.2
>167
>10
>62.5
>16
>185
>71
ND
100
100
100
95
100
100
100
100
43
100
89
100
79
6
23
16
23
19
0
0
0
0
100
89
90
86
25
NO2
NO2
H
—
—
—
H
83
0.047
>213
a
Results are expressed as the mean (n = 3 experiments) of the percentage 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 dose–response equation (variable slope) (GraphPad software).
b
In vitro COX-2 selectivity index [IC50 (COX-1)/IC50(COX-2)].

M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
775
COOH
CH2CONHCH(CH2)nOH
CH2CONH(CH2)nOH
CH2COOH
(i)
(iv)
N
N
N
H3CO2S
H3CO2S
H3CO2S
R
R
R
5a-d
4a-d
7a-b
(v)
(ii)
HO(CH2)nNH2
8a-b
COOH
(iii)
n = 2-3
CH2CONHCH(CH2)nONO2
CH2CONH(CH2)nONO2
O2NO(CH2)nNH2
N
N
9a-b
H3CO2S
H3CO2S
R
R
COOH
HO(CH2)nCHNH2
2a-d
1a-d
10a-b
(vi)
n = 1, 2
COOH
O2NO(CH2)nCHNH2
11a-b
Scheme 1. Synthesis of acetic acid derivatives bearing a non-ionisable side chain. Reagents and conditions: (i) 8a–b, HOBt, EDCI, TEA, DCM, rt, 12 h, (ii) 9a–b, EDCl, DMAP,
TEA, DCM, rt, 12 h; (iii) HNO3 fuming, DCM, 1 h, then (CH3CO)2O 45 min; (iv) 10a–b, pyBOP, TEA, THF, rt, 4 h; (v) 11a–b, pyBOP, TEA, THF, rt, 4 h; (vi) HNO3 fuming, DCM, 1 h,
then (CH3CO)2O, 45 min.
The coupling stage which led to the nitro-oxy compounds 1a–d
was afforded by means of EDCI as activating agent and DMAP as
covalent nucleophilic catalyst, then the active species was reacted
with the nitro-oxyalkyl amines (9a,b). The nitro-oxyalkyl amine
9a,b were obtained via nitration of amino alcohols 8a,b by employ-
ing fuming nitric acid in dichloromethane and then precipitated as
nitrated salts after addition of acetic anhydride. The hydroxyl
derivatives 4a–d were obtained via EDCI/HOBt coupling, in order
to discriminate the nucleophilicity of the oxygen and the nitrogen.
Compounds 2a–d and 5a–d were obtained as depicted in
Scheme 1. Activation of the acid derivative with PyBOP and
subsequent reaction with the aminoacids (10a,b and 11a,b) gave
access to the final molecule (hydroxy or nitro-oxy derivative).
Compounds 11a,b were obtained following the same nitrating pro-
cedure adopted for compounds 9a,b. The used amino acids (10a,b)
were racemic serine and homoserine.
Glycine derivatives 3a,b and 6a,b were prepared using a com-
mon route to derivatives 1 and 2 and then following two different
strategies for the generation of nitro-oxy and hydroxy compounds
(Scheme 2). The installation of the amino functionality was
achieved via oxime formation and then its reduction gave the gly-
cine ethyl ester (14). A subsequent protection using two different
NOH
NHBoc
CHCOOEt
NH2
CHCOOEt
COCOOEt
CCOOEt
(i)
(ii)
(iii)
N
N
N
N
H3CO2S
H3CO2S
H3CO2S
H3CO2S
F
F
F
F
12
13
18
14
(iv)
(viii)
NHCbz
CHCONH(CH2)nONO2
NHCbz
CHCOOH
NH2
NHCbz
CHCOOEt
NHBoc
CHCOOH
CHCONH(CH2)nOH
(vi)
(v)
(vii)
N
N
N
N
N
H3CO2S
H3CO2S
H3CO2S
H3CO2S
H3CO2S
F
F
F
F
F
6a-b
17a-b
16
15
19
(ix)
NH2
NHBoc
CHCONH(CH2)nONO2
CHCONH(CH2)nONO2
(x)
N
N
H3CO2S
H3CO2S
F
F
20a-b
3a-b
Scheme 2. Synthesis of acetic acid derivatives bearing an amino acid side chain. Reagents and conditions: NH2OH, NaOAc, ethanol/1,4-dioxan (1:1 v/v), 90 °C, 40 h; (ii) Zn,
HCOOH, 0 °C, 1 h, then, rt, 19 h; (iii) (Boc)O2 rt, 18 h; (iv) CbzCl, Na2CO3 DCM, rt, 4 h; (v) NaOH, MeOH, rt, 17 h; (vi) 9a–b, EDCl, DMAP, TEA, DCM, rt, 12 h; (vii) Pd/c, NH4COO,
isopropanol, MW, 80 °C, 5 min, 150 W, 170 psi; (viii) NaOH, MeOH, rt, 17 h; (ix) 9a–b, EDCI, DMAP, TEA, DCM, rt, 12 h, (x) TFA, DCM, 0 °C then MW, 60 °C, 40 min, 150 W,
170 psi.

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M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
protecting groups (Cbz for 15, Boc for 18), hydrolysis of the ethyl
ester and coupling gave access to compounds 17a,b and 20a,b. A
final deprotection step yielded the expected nitro-oxy and hydroxy
compounds 3a,b and 6a,b, respectively.
2.2.4. Ex vivo nitric oxide releasing properties
The potential NO-mediated vasorelaxing effects of all the nitro-
oxy compounds were tested on rat aortic rings. Naproxcinod was
used as a reference NO-releasing agent. The compounds showed
variable NO-dependent vasorelaxing properties. In particular, nap-
roxcinod showed a high vasorelaxing efficacy (about 70%) and a
2.2. Pharmacological activity
potency value slightly lower than the
1a showed low vasorelaxing effect, compounds 1b and 1c proved
high vasorelaxing efficacy, with potency spanning in the M order
lM level. While compound
2.2.1. COX-2 inhibition in J774 cell line in vitro
Linear derivatives (1 and 4) displayed an average inhibition to-
wards COX-2 which ranges from 0.045 to 0.310 lM. Surprisingly
l
of magnitude. Derivatives 2a–d produced a modest vasorelaxing
effect. The introduction of the amide functionality within the gly-
cine skeleton, did not compromise the NO-mediated vasorelaxing
effects. Indeed, compounds 3a and 3b exhibited high levels of
hydroxyl-derivatives 4b and 4d showed significant COX-2 inhibi-
tion. This may be due to a non-specific cooperation between the
N1 substitution and the hydroxyl chain allowing very strong and
effective interactions with the enzyme. As far as the serine/homo-
serine derivatives are concerned, results show, in general, that the
homoserine derivatives are more active than the corresponding
serines. In particular, the best inhibition is obtained when a hydro-
xyl-derivative is allowed to interact with the enzyme (5a and 5c).
With respect to the glycine compounds 3a,b and 6a,b, nitro-oxyl-
derivative 3a and hydroxyl-derivative 6b showed the best
activities.
In general, outstanding COX-2 inhibitory potencies and high
selectivity values were obtained within the linear derivatives and
the glycine compounds (3a, 5a, 5c, and 6b), demonstrating that
the negative effect obtained in vivo with the previous class of gly-
cine esters was probably due to a mismatch-interaction rather
than to the presence of the amino group itself (Table 1).27
vasorelaxing efficacy as well as lM levels of potency. The vasore-
laxing activity of all the tested compounds were significantly
inhibited by ODQ, inhibitor of guanylate cyclase, indicating the
implication of the NO-cGMP pathway (Table 4).
2.2.5. In vitro nitric oxide releasing properties
The kinetics of NO-release for 1c, 3a and naproxcinod were also
evaluated. In particular, the time-dependent progressive increase
of the concentrations of inorganic nitrites and nitrates (i.e. the sta-
ble metabolites of NO), after incubation of the tested compounds
(1 mM) in rat liver homogenates (with appropriate co-factors),
was amperometrically detected. Two hours after incubation of 1c
the nitrite concentration was 27.0 12.1
l
M and the nitrate con-
centration was 38.3 4.3 lM
for 1c, while 3a showed
2.2.2. Writhes reduction in the acetic acid-induced abdominal
constrictions in mice
Table 2
All the nitro-oxyl-derivatives have been tested for evaluating
their analgesic effects in the writhes reduction test. All the tested
compounds highlighted a very good and dose-dependent activity
in reducing the abdominal writhes induced by acetic acid (Table 2).
In particular, nitro-oxyl-derivative 1c and its corresponding hydro-
xyl-derivative 4c, serine hydroxyl-derivative 5c and glycine nitro-
oxyl-derivative 3a are endowed with a higher extent of writhes
reduction compared to the other compounds. In particular, com-
pounds 1c and 3a were the best ones displaying a 77% and 79%
of reduction, respectively, when dosed at 20 mg/kg. Noteworthy
the introduction of the amino moiety (which is associated to high-
er solubility as discussed-below) gave rise to a compound (3a) with
outstanding properties. Derivative 3a was even active at 3 mg/kg
with a reduction of writhes around 23%; the reduction extent in-
creased with the dose and reached 56% and 79% when adminis-
tered at 10 mg/kg and 20 mg/kg, respectively.
Writhes reduction for compounds 1a–d, 4c, 2a–d, 5c, and 3a,b
Compound n°
Mice
Dose po mg/
kgÀ1
n° Writhes after
30 min
Writhes
reduction (%)
CMC
1a
43
—
32.6 2.1
26.8 2.1**
20.6 3.0*
14.7 2.1*
33.2 3.5
29.8 3.6
22.9 2.2*
12.2 2.3*
7.4 1.3*
—
10
10
10
9
9
10
9
9
10
8
10
20
40
10
20
40
10
20
40
3
18
37
55
0
1b
1c
1d
8.6
30
63
77
75
17
37
34
37
61
12
47
50
10
23
0
8.1 1.2*
27.2 2.7
20.4 2.2*
21.6 2.8*
20.7 3.5*
12.6 2.7*
28.3 2.5
17.1 3.1*
16.4 2.9*
29.2 2.4
25.2 3.3**
33.1 3.5
23.2 2.3*
16.3 3.2*
33.7 3.0
22.8 2.7*
14.9 2.8*
23.7 3.0
13.4 2.8*
12.9 2.1*
25.1 2.3**
14.2 2.9*
6.7 2.0*
8
8
10
20
10
20
10
20
40
20
40
10
20
40
3
10
20
10
20
40
3
4c
2a
10
10
10
10
10
8
10
8
9
10
10
10
11
14
15
15
13
10
13
9
2.2.3. Reduction of hyperalgesia and edema in the carrageenan
model
2b
2c
The most active nitro-oxy compounds 1c and 3a were selected
for a further assessment of their in vivo profile through the carra-
geenan induced edema and hyperalgesia. Surprisingly, the data on
the carrageenan induced inflammation showed that 1c is associ-
ated with a good but not outstanding activity. Indeed, dosing the
compound at 40 mg/kg the maximum activity was reached after
60 min with a reduction of hyperalgesia of 60%. A negligible activ-
ity (10%) was found when testing the compound for its anti-edemi-
genic properties. On the other hand, compound 3a proved to be
fast and highly active, with a reduction of hyperalgesia of 80% after
30 min. The activity was maintained for 60 min (70%) and after
90 min a satisfactory activity (50%) was still displayed. After 2 h,
some analgesic activity could be still detected (26%). An anti-
edemigenic effect was observed with the administration of this
compound at 20 mg/kg, with the edema volume reduced of about
76% (Table 3).
29
50
0
2d
5c
3a
3b
30
54
27
59
60
23
56
79
6
10
20
10
20
40
10
30.5 2.6
21.4 3.7*
18.8 3.1*
13.5 3.0*
10
10
13
34
42
59
Celecoxib
*
P < 0.01.
**
P < 0.05 in comparison with CMC treated animals.

M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
777
Table 4
42.9 6.6 lM and 56.1 16.0 lM for nitrites and nitrates, respec-
Vasorelaxing properties for compounds 1a–d, 2a–c and 3a,b in comparison with
MAB103 and MAB124. significant antagonism by ODQ, inhibitor of guanylate
cyclase, is also indicated (+)
tively. The corresponding concentration/time curves (Fig. 5)
indicate that both the compounds are endowed with slow
NO-releasing rate, which is thought to be a fundamental feature
for the development of well-balanced hybrids. Naproxcinod exhib-
ited a different behavior. Indeed, two hours after incubation of
naproxcinod, the highest concentration of NOx was detected
A
Compound
Emax
pIC50
+ODQ 1 lM
Naproxcinod
68.0 3.0
49.0 2.0
69.0 8.0
68.0 0.5
18.0 9.0
25.0 2.0
25.0 1.9
42.0 9.0
74.1 1.0
86.0 2.0
6.33 0.06
n.c.
5.78 0.07
5.31 0.05
n.c.
n.c.
n.c.
n.c.
5.38 0.20
5.38 0.03
+
+
+
+
+
+
+
+
+
+
1a
1b
1c
2a
2b
2c
2d
3a
3b
(367.7 11.4
gely due to the amount of nitrates released (340.1 5.0
a lower concentration of nitrites was detected (27.6 6.2
l
M); noteworthy, the concentration of NOx was lar-
M), while
M),
l
l
which was almost comparable with those detected for 1c and 3a.
A similar feature of naproxcinod was already observed in human
liver fractions.41 Such a lower ratio nitrites/NOx would suggest
that only a small part of the nitro-oxy group of naproxcinod is con-
verted to NO (and in turn, to nitrites and nitrates) and that a direct
hydrolysis to inorganic nitrate is prevalent.
P < 0.05) (Fig. 7A and B). Altogether these results show that com-
pound 3a is a more potent inhibitor of COX-2 than COX-1 up to
concentrations which affect COX-2 activity by approximately
80%, considered appropriate to translate into an analgesic/anti-
inflammatory effect.45 The hydroxy derivative 6a maintains a
similar inhibitory potency towards COX-2 but it is less potent to in-
hibit COX-1 thus resulting in improved COX-2 selectivity (Table 5)
versus the nitroxy-parent compound (3a). As shown in Table 5, in
HWB assays celecoxib showed a COX-2 selectivity comparable to
6a. The finding that 6a had a potency to affect COX-2 similar to that
of compound 3a is an advisable feature because it may lead to a
long-lasting therapeutic effect. The higher COX-2 selectivity of 6a
with respect to 3a might be useful to maintain a good GI safety also
when the nitrooxy compound loses its NO-releasing capacity.
2.2.6. Human whole blood (HWB) assays
We studied COX-2 selectivity of the most active nitro-oxy
amides 1c and 3a along with the respective hydroxy derivatives
4c and 6a, in the HWB assays. The use of these assays allows to
gain information of the potency and COX-isozyme selectivity of
COX inhibitors in the presence of plasma proteins and in clinically
relevant cells (i.e., monocytes, expressing COX-2 in response to
LPS,42 a cellular target for anti-inflammatory effects; platelets
expressing only COX-1,43 a cellular target for gastrointestinal tox-
icity44) in response to endogenously released AA. It has been sug-
gested that HWB COX-2 inhibition in vitro can be used as a
marker to predict drug efficacy in humans.45
Compound 1c caused a concentration-dependent inhibition of
COX-1 and COX-2 activities with IC50 values of 0.6 (95% CI:
0.38–0.97) and 1.7 (0.87–3.5) lM, respectively, which were not dif-
2.2.7. Solubility assessment
Solubility of compounds was assessed in simulated gastric fluid
(SGF-without pepsin) and phosphate buffered solution (PBS), using
a multi-pulse vortexer (Glass-Col) to shake the samples. Concen-
tration of the compounds was assessed by HPLC.
The replacement of the ester functionality with the amide moi-
ety gave rise to more soluble molecules. Surprisingly, the introduc-
tion of a carboxylate group (2c) led to compounds with a lower
solubility even at pH 7.4 (Table 6). This could be due to a participa-
tion of the carboxylic acid moiety to intramolecular H-bonding
networks that stabilize the folding of the molecule leading to re-
duced solubility. On the other hand ‘linear’ amides showed good
solubility in both SGF and PBS. The best solubility profile was dis-
played by glycine amide 3a the solubility of which was higher than
ferent, in a statistically significant fashion (Fig. 6A and Table 5). Its
hydroxy-derivative, 4c, inhibited HWB COX-2 and COX-1 activities
with similar IC50 values of 1.4 (1.1–1.8) and 1.9 (1.1–3.6)
lM,
respectively (Fig. 6B and Table 5). Thereby, 1c and its hydroxy
derivative 4c proved to be potent COX-2 inhibitors even if they
did not show COX-2 selectivity in the HWB assay.
As shown in Figure 7A, compound 3a inhibited COX-2 and
COX-1 activities in a concentration-dependent fashion with IC50
values of 21 (14.6–30) and 140 (117–168)
Compound 3a was significantly more potent towards COX-2 than
COX-1 (COX-1/COX-2 IC50 ratio was 6.7) (Table 5). At 100 M, 3a
lM, respectively.
l
inhibited COX-2 activity by 80.2 3.8% mean+SEM (n = 3) (consid-
ered appropriate to obtain a therapeutic effect45), while it inhibited
only marginally COX-1 activity (36.8 7.8%, n = 3) (Fig. 7A). The
corresponding hydroxyl-derivative 6a inhibited COX-2 activity
with an IC50 value that was comparable to that of 3a. In contrast,
6a was less potent to inhibit COX-1 activity than the nitro-oxy
200 lM in both conditions.
2.2.8. Stability insimulated gastric fluid (SGF-without pepsin),
phosphate buffered solution (PBS) and rat plasma
Stability studies on MAB103 and MAB124 revealed some liabil-
ity of these compounds to hydrolysis. It should be pointed out that
amide 3a. At 300 lM, COX-1 activity was reduced by 55.7 6.8%
and 84 1.8%, by 6a and 3a, respectively (mean SEM, n = 3,
Table 3
Hyperalgesia and oedema reduction in the carrageenan induced inflammation for compounds 1c and 3a in comparison with celecoxib
Pretreat. (i.pl.)
Compd
Dose
Before treatment
Paw-pressure
Edema volume (mL)
After treatment
30 min
60 min
90 min
120 min
Saline
CMC
CMC
1c
1c
3a
—
—
20
40
20
10
61.2 2.5
36.1 3.3
33.8 2.9
32.9 3.3
33.8 2.9
31.7 2.7
61.6 3.4
32.8 2.9
58.9 3.8
30.6 3.2
61.4 4.1
31.8 2.2
37.8 2.6 (20%)
41.5 3.7** (29%)
47.3 3.7* (50%)
48.3 3.4* (56%)
61.4 4.1
31.8 2.2
32.7 2.6 (0%)
31.5 2.6 (0%)
39.5 3.0 (26%)
42.5 2.9 (39%)
1.44 0.05
2.71 0.08
2.63 0.05 (4%)
2.50 0.09 (10%)
1.79 0.09* (76%)
2.53 0.05* (17%)
Carrageenan
Carrageenan
Carrageenan
Carrageenan
Carrageenan
40.7 3.5** (28%)
44.2 4.0* (32%)
55.2 3.3* (80%)
45.7 4.2* (45%)
42.8 3.4** (30%)
48.3 4.6* (60%)
52.1 4.2* (70%)
52.9 3.1* (75%)
Celecoxib
*
P < 0.01.
**
P < 0.05 in comparison with CMC treated animals.

778
M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
Figure 5. Time-dependent increase of the concentrations of nitrites (NO2) nitrates (NO3) and NOx (nitrites + nitrates) following the incubation of 1 mM 1c, 3a or naproxcinod
in rat hepatic homogenate, containing the opportune cofactors. The vertical bars indicate the standard error.
Table 5
IC50 values and COX-2 selectivity of compounds 1c, 4c, 3a and 6a, in HWB assays
Compound
COX-1 IC50
l
Ma (95% CI)
COX-2 IC50 a(95% CI)
S.I.*
1c
4c
3a
6a
0.6 (0.38–0.97)
1.9 (1.1–3.6)
140 (117–168)
ꢀ300
1.7 (0.87–3.5)
1.4 (1.1–1.8)
21 (15.6–30)
13.7 (7.9–24)
0.54 (0.29–0.52)
0.3
1.3
6.7
21.9
23
Celecoxib
12.47 (8.6–17.9)
*
Selectivity index (SI): selectivity of compounds towards COX-2 were determined
as IC50 ratios for HWB COX-1 and COX-2.
a
For IC50 values, 95% confidence intervals (CI) are shown in round brackets.
enzymatically. Thus the amide bond enhanced the overall stability
to hydrolysis especially for compounds belonging to the first and
third subclass of amides (1c and 3a) (Table 7). Compound 1c was
completely resistant to hydrolysis (in the tested conditions),
whereas compound 3a was relatively more liable in rat plasma
(even though after 120 min of incubation 77% of the compound
was still detected).
The serine derivative 2a displayed the lower stability in the
amide series. Again, this could be explained by means of intra-
molecular interactions related to the presence of the carboxylic
acid moiety; indeed, intra-molecularly, the acid group could ease
any structural transformation (enzyme-mediated or not).
2.2.9. Pharmacokinetic studies
The pharmacokinetics of the most active compounds 1c and 3a
were assessed in rats after po and iv administration of the com-
pounds. Profiles of the plasma concentration versus time after sin-
gle po and iv administration of 1c and 3a (10 mg/kg) are reported
in Figures 8 and 9. After iv administration, 1c was detected in plas-
ma up to 6 h. The average clearance value was about 0.6 L/h, rep-
resenting 72% of rat liver blood flow (0.83 L/h). This suggests
moderate to high liver extraction, while the average final volume
of distribution (Vss on AUCt) value was 367 mL which is twice the
rat total body water, suggesting moderate compound distribution
in tissues.
Figure 6. Effects of Compounds 1c and 4c on in vitro platelet COX-1 and monocyte
COX-2 activity in HWB assay. Results are expressed as average percent of inhibition
(n = 3–5, mean SEM).
the presence of the amino moiety, decreased this liability when
comparing the two esters,26 especially in SGF (100% of parent com-
pound detected at 120 min). Replacing the ester with the amide led
to a striking stabilization of the compounds both chemically and

M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
779
Figure 8. Profile of plasma concentration versus time after iv and po administration
of 1c (10 mg/kg).
Figure 7. Effects of Compounds 3a and 6a on in vitro platelet COX-1 and monocyte
COX-2 activity in HWB assay. Results are expressed as average percent of inhibition
(n = 3–5, mean SEM).
Table 6
Solubility assessment in SGF and PBS for compounds 1a, 1c, 2c, 3a, MAB103 and
MAB124
Figure 9. Profile of plasma concentration versus time after iv and po administration
of 3a (10 mg/kg).
Compound
SGF (pH 1.5) (
lM)
PBS (pH 7.4) (lM)
MAB103
<1
1.6
MAB124
>200
150.0
147.0
112.0
>200
80.0
>200
>200
90.5
>200
1a
1c
2c
3a
Table 7
Stability assessment in SGF and PBS for compounds MAB103, MAB124, 1c, 2a, and 3a
Compound
MAB103
MAB124
1c
Time (min)
Parent molecule remained (%)
PBS (pH 7.4)
SGF (pH 1.5)
Rat plasma
30
60
120
30
60
120
30
60
120
30
60
74.1
53.6
0.15
75.0
75.0
75.0
100
100
100
75
80.2
65.7
42.8
100
100
100
100
100
100
100
93.8
87.5
100
100
100
0.00
0.00
0.00
0.00
0.00
0.00
100
100
100
71.4
57.1
28.6
94
Figure 10. Mean plasma concentration versus time profiles of 4c after iv and po
administration of 1c.
2a
50
After iv administration, 3a was detected in plasma up to 6 h.
Average clearance value was about 1.12 L/h representing 136% of
rat liver blood flow, this suggests high liver extraction, while final
volume of distribution value (Vss on AUCt) was 1.85 L that is 11
120
30
60
37.5
100
100
100
3a
93
77
120

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M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
additional evidence about the effectiveness of the approach in
counteracting long-term CV toxicity triggered by COX-2 inhibitors,
will be addressed in further appropriate studies.
4. Experimental section
4.1. Chemistry
All chemicals used were of reagent grade. Yields refer to puri-
fied products and are not optimized. A CEM Discovery microwave
system apparatus was used for microwaved reactions. Melting
points were determined in open capillaries on a Gallenkamp appa-
ratus and are uncorrected. Sigma–Aldrich silica gel 60 (230À400
mesh) was used for column chromatography. Merck TLC plates (sil-
ica gel 60 F 254) were used for thin-layer chromatography (TLC).
Sigma–Aldrich aluminum oxide (activity IIÀIII, according to Brock-
mann) was used for chromatographic purifications. Sigma–Aldrich
Stratocrom aluminum oxide plates with a fluorescent indicator
were used for TLC to check the purity of the compounds. 13C
NMR and 1H NMR spectra were recorded with a Bruker AC 400
spectrometer in the indicated solvent (TMS as the internal stan-
dard). The values of the chemical shifts are expressed in ppm
and the coupling constants (J) in hertz. Mass spectra were recorded
on a API-TOF Mariner by Perspective Biosystem (Stratford, Texas,
USA).
Figure 11. Mean plasma concentration versus time profiles of 6a after iv and po
administration of 3a.
times rat total body water suggesting extensive compound distri-
bution in tissues.
In conclusion, both 1c and 3a appear to be characterized by
moderate to high rate of clearance and high volume of distribution.
In accordance with lower liver extraction ratio, 1c showed higher
absolute bioavailability (24%) than 3a (9%).
4.1.1. General procedure for the preparation of 1,5-diarylpyrrole-
3-acetic acids (7a,b)
Considering the possibility that nitro-esters can be metabolized
to the corresponding alcohols, the presence of the hydroxyl metab-
olites of 1c and 3a (4c and 6a, respectively) was also measured.
Profiles of the plasma concentration versus time for 4c and 6a after
iv and po administration of 1c and 3a (10 mg/kg) are reported in
Figures 10 and 11. Compound 4c was detected in rat plasma up
to 6 h after both iv and po administrations, while 6a was detected
in rat plasma at least up to 6 h after intravenous administrations
and only up to 2 h after oral administration. For 1c, after intrave-
nous administration at least 39% of the parent compound is con-
verted into metabolite 4c (calculated on a molar base) while
after oral administration 99% of the parent compound is converted
into the metabolite. This suggests that after oral administration 1c
is readily metabolized to 4c. For 3a, after intravenous administra-
tion only 10% of the parent compound was converted to metabolite
6a (calculated on a molar bases), while the conversion after oral
administration is 12.8%, indicating that the percentage of the first
pass metabolism is negligible.
1 N Sodium hydroxide solution (4.83 mL) was added to a solu-
tion of ethyl 1,5-diarylpyrrole-3-acetic ester (0.67 mmol) in meth-
anol (4.83 mL). The resulting mixture was refluxed for 1.5 h,
cooled, and concentrated in vacuo. The residue was solubilized in
water (5 mL) and then concentrated HCl was added dropwise until
a precipitate was formed. The precipitate was filtered off to give
the expected acid as a white solid. Physicochemical, spectroscopic,
and analytical data were consistent with those reported in the
literature.26
4.1.2. General procedure for the preparation of 1,5-diarylpyrrole-
3-acetic nitroxyalkyl amides (1a–d)
Nitroxyalkylamine (9a,b nitrate salt) (0.3 mmol), DMAP
(0.1 mmol), and EDCI (0.2 mmol) were added in sequence to a solu-
tion of the acid partner (7a,b) (0.1 mmol) in dichloromethane
(5 mL), under nitrogen atmosphere. An excess of triethylamine
(0.35 mmol) was added dropwise and the reaction was stirred at
rt for 12 h. Then the mixture was quenched with water (10 mL)
and extracted with chloroform (50 mL Â 3). The organic layer
was washed with 1 N HCl (50 mL), NaHCO3 saturated solution
(50 mL), brine (50 mL) and dried over Na2SO4. After filtration and
concentration of the organic phase a crude material was obtained.
The material was then purified by chromatography on silica gel/
alumina (1:1) using petroleum ether/chloroform/ethyl acetate,
4:4:1 (v/v/v), as the eluent to give the desired product in good
yield.
3. Conclusions
The replacement of the ester moiety with an amide group gave
access to a stable and soluble class of hybrid compounds. Nitro-
oxy derivatives highlighted a very good COX-2 inhibiting profile
both in vivo and in HWB. The NO-dependent vasorelaxing proper-
ties and the slow kinetic of NO release make these compounds a
very promising class of pharmacodynamic hybrids. Among all
derivatives, 3a is endowed with an outstanding in vivo profile
(comparable and in some extent greater than celecoxib). In HWB
assays, 3a was more potent to inhibit COX-2 than COX-1. The find-
ing that its hydroxyl-derivative 6a had higher COX-2 selectivity
than 3a might be a useful feature to maintain a good GI safety pro-
file even when the nitrooxy compound loses its NO-releasing
capacity.
4.1.3. N-[2-(Nitroxy)ethyl]-2-[1-(3-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3yl]acetamide (1a)
Yellow powder, mp 133 °C (yield 80%). FT-IR cmÀ1: 3300, 1640,
1595, 1520, 1278, 865. 1H NMR (400 MHz, CDCl3) d (ppm): 7.71 (d,
2H, J = 8.6 Hz), 7.44–7.40 (m, 1H), 7.17 (d, 2H, J = 8.6 Hz), 7.14–7.12
(m, 1H), 6.98–6.96 (m, 1H), 6.93–6.90 (m, 1H), 6.43 (s, 1H), 6.05 (s
br, 1H), 4.59 (t, 2H, J = 7.6 Hz), 3.63 (t, 2H, J = 7.6 Hz), 3.49 (s, 2H),
3.02 (s, 3H), 2.04 (s, 3H). 13C NMR (100 MHz, CDCl3) d (ppm):
169.45, 138.23, 136.12, 131.83, 131.30, 130.71, 130.66, 128.71,
In vivo assessment of the NO releasing properties and effects of
selected compounds in suitable animal models, aimed at providing

M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
781
127.10, 125.59, 120.71, 115.90, 113.44, 111.18, 110.42, 69.77,
44.61, 38.41, 32.21, 11.89. MS-ESI: m/z 476.12 [M+H+]. HPLC:
>97% pure (tR = 7.3 min).
heated at 90 °C under stirring for 40 h. The solvents were removed
in vacuum and the mixture was extracted with ethyl acetate
(500 mL) and washed with water (100 mL). The organic phase
was dried over sodium sulfate, filtered and concentrated to afford
the oxime 13 as a yellowish solid. Physicochemical, spectroscopic,
and analytical data were consistent with those reported in the
literature.27
4.1.4. General procedure for the preparation of 1,5-
diarylpyrrole-3-acetic hydroxylalkyl amides (4a–d)
Hydroxyalkylamine (8a–b) (1.0 mmol), HOBt (0.9 mmol), and
EDCI (1.2 mmol) were added in sequence to
a solution of
1,5-diarylpyrrole-3-acetic acid (7a,b) (0.7 mmol) in dichlorometh-
ane (20 mL), under nitrogen atmosphere. An excess of triethyl-
amine (2.0 mmol) was added dropwise and the reaction was
stirred at rt for 12 h. Then the mixture was quenched with water
(10 mL) and extracted with chloroform (50 mL Â 3). The organic
layer was washed with 1 N HCl (50 mL), NaHCO3 saturated solu-
tion (50 mL), brine (50 mL) and dried over Na2SO4. After filtration
and concentration of the organic phase a crude material was
obtained. The material was then purified by chromatography on
silica gel using petroleum ether/chloroform/ethyl acetate, 3:2:1
(v/v/v), as the eluent to give the desired product in good yield.
4.1.9. General procedure for the preparation of ethyl 2-amino-
2-[2-methyl-5-[4-(methylsulfonyl)phenyl]-1-(4-fluorophenyl)-
1H-pyrrol-3-yl]acetate (14)
Formic acid (100 mL) was added to a solution of the crude
oxime 13 in ethanol (100 mL) and the solution was cooled to
0 °C. Then zinc dust (252.2 mmol) 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 resi-
due obtained was dissolved in water (200 mL) and the pH adjusted
to 10 with 10% NaOH solution and extracted with EtOAc (500 mL).
The combined organic fractions were dried over sodium sulfate, fil-
tered 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 the product
14 (50% yield). Physicochemical, spectroscopic, and analytical data
were consistent with those reported in the literature.27
4.1.5. N-[2-(Hydroxy)ethyl]-2-[1-(3-fluorophenyl)-2-methyl-5-
[4-(methylsulfonyl)phenyl]-1H-pyrrol-3yl]acetamide (4a)
Yellowish powder, mp 97 °C (yield 77%). FT-IR cmÀ1: 3623,
3315, 1645, 1520, 1280, 1100. 1H NMR (400 MHz, CDCl3) d
(ppm): 7.69 (d, 2H, J = 8.6 Hz), 7.43–7.37 (m, 1H), 7.19 (d, 2H,
J = 8.6 Hz), 7.14–7.10 (m, 1H), 6.98-6.96 (m, 1H), 6.92–6.89 (m,
1H), 6.47 (s, 1H), 6.40 (s br, 1H), 3.70 (t, 2H, J = 7.4 Hz), 3.48 (s,
2H), 3.40 (m, 2H), 3.00 (s, 3H), 2.70 (s br, 1H), 2.04 (s, 3H). 13C
NMR (100 MHz, CDCl3) d (ppm): 169.47, 138.63, 136.32, 131.85,
131.37, 130.44, 130.56, 129.46, 127.12, 125.61, 120.31, 115.88,
113.54, 113.08, 111.29, 60.17, 44.61, 36.41, 32.21, 11.89. MS-ESI:
m/z 431.14 [M+H+]. HPLC: >98% pure (tR = 4.7 min).
4.1.10. General procedure for the synthesis of N-benzyl-
(ethoxycarbonyl)-[1-(4-fluorophenyl)-2-methyl-5-
[4(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]methylcarbamate
(15)
To a biphasic solution of amino ester 14 (2.326 mmol) in DCM
(10 mL), sodium carbonate (4.6 mmol) and benzyl chloroformate
(2.352 mmol) were added at 0 °C, the reaction mixture was al-
lowed to warm up to rt and stirred for further 4 h. The mixture
was then diluted with water (20 mL) and the two phases sepa-
rated. The organic fraction was washed with water (50 mL Â 2),
dried over sodium sulfate and evaporated to give the crude prod-
uct. 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 (65% yield). Physicochemical, spectroscopic,
and analytical data were consistent with those reported in the
literature.27
4.1.6. General procedure for the preparation of 1,5-
diarylpyrrole-3-acetic amides (2a–d, 5a–d)
PyBOP (0.93 mmol) and triethylamine (3 mmol) were added in
sequence to a solution of 7a,b (0.78 mmol) in THF (20 mL) at rt and
the mixture stirred for 1 h. Then the amino acid (10a,b, 11a–b)
(1.5 mmol) was added over 5 min. After 4 h the mixture was con-
centrated in vacuo and the oily residue was treated with 1 N
KOH (10 mL). The solution was filtered through a paper filter.
The liquid was collected, cooled to 0 °C and 5 N HCl was added
dropwise till a white precipitate was formed. The solid obtained
was filtered and dried. After re-crystallization from ethanol/petro-
leum ether the product was obtained in quite good yield.
4.1.11. General procedure for the synthesis of N-protected (CBz)
2-[1-(4-fluorophenyl)-2-methyl-5-[4-(methylsulfonyl)phenyl]-
1H-pyrrol-3-yl]-2-amino-acetic acid (16)
4.1.7. (R,S)-2-[2-[1-(3-Fluorophenyl)-2-methyl-5-[4-
(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetamide]-3-
nitroxypropanoic acid (2a)
A solution of NaOH (5.917 mmol) in water (3 mL) was added to
a solution of amino ester 15 (2.236 mmol) in MeOH (15 mL), and
the reaction mixture was stirred at rt for 17 h. The mixture was
then concentrated in vacuo, diluted with water (2 mL) and the acid
precipitated through the cautious addition of concentrated HCl.
The solid was collected to give the product as off white solid. Phys-
icochemical, spectroscopic, and analytical data were consistent
with those reported in the literature.27
Yellow powder, mp 110 °C (yield 80%). FT-IR cmÀ1: 3310, 1734,
1640, 1595, 1520, 1278, 865. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 11.13 (s br, 1H), 8.53 (s, br, 1H),7.70 (d, 2H, J = 8.5 Hz),
7.49–7.47 (m, 1H), 7.22–7.20 (m, 2H), 7.18–7.16 (m, 2H),
7.03–7.01 (m, 1H), 6.50 (s, 1H), 4.79–4.76 (m, 1H), 4.62–4.60 (m,
2H), 3.51 (s, 2H), 3.10 (s, 3H), 2.09 (s, 3H). 13C NMR (100 MHz,
DMSO-d6)
d
(ppm): 172.02, 169.67, 138.50, 136.13, 131.80,
4.1.12. General procedure for the Preparation of 1,5-
131.30, 130.71, 130.66, 129.48, 127.00, 125.59, 120.56, 115.98,
113.43, 113.22, 111.25, 70.18, 55.71, 38.41, 32.21, 11.80. MS-ESI:
m/z 520.11 [M+H+]. HPLC: >98% pure (tR = 4.3 min).
diarylpyrrole-3-acetic nitro-oxyalkyl amides (17a–b)
Nitroxyalkylamine (22a–b, nitrate salt) (0.3 mmol), DMAP
(0.1 mmol), and EDCI (0.2 mmol) were added in sequence to a solu-
tion of the acid partner (16) (0.1 mmol) in dichloromethane (5 mL),
under nitrogen atmosphere. An excess of triethylamine
(0.35 mmol) was added dropwise and the reaction was stirred at
rt for 12 h. Then the mixture was quenched with water (10 mL)
and extracted with chloroform (50 mL Â 3). The organic layer was
washed with 1 N HCl (50 mL), NaHCO3 saturated solution (50 mL),
brine (50 mL) and dried over Na2SO4. After filtration and concentra-
4.1.8. Preparation of ethyl 2-(hydroxyimino)-2-[2-methyl-5-[4-
(methylsulfonyl)phenyl]-1-(4-fluorophenyl)-1H-pyrrol-3-
yl]acetate (13)
Hydroxylamine hydrochloride (87.4 mmol) and sodium acetate
(145.6 mmol) were added to a suspension of 12 (58.2 mmol) in
ethanol/1,4-dioxan (100 mL, 1:1 v/v). The resulting solution was

782
M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
tion of the organic phase a crude material was obtained. The mate-
rial was then purified by chromatography on silica gel/alumina
(1:1) using petroleum ether/chloroform/ethyl acetate, 4:4:1
(v/v/v), as the eluent to give the desired product in good yield.
reaction mixture stirred at rt for 17 h. The mixture was then con-
centrated in vacuo, diluted with water (2 mL) and the acid precip-
itated through the caution addition of concentrated HCl. The solid
was collected to give the product as off white solid. Physicochem-
ical, spectroscopic, and analytical data were consistent with those
reported in the literature.27
4.1.13. N-[2-(Nitroxy)ethyl]-(R,S)-[(benzyloxy)carbonyl]amino-
2-(1-(3-fluorophenyl)-2-methyl-5-(4-(methylsulfonyl)phenyl)-
1H-pyrrol-3-yl)acetamide (17a)
4.1.18. General procedure for the preparation of 1,5-
diarylpyrrole-3-acetic nitro-oxyalkyl amides (20a,b)
White solid, 138 °C (75% yield); 1H NMR (400 MHz, CDCl3): d
(ppm) 7.71 (d, 2H, J = 8.4 Hz), 7.30–7.24 (m, 9H), 7.16 (d, 2H,
J = 8.4 Hz), 6.88 (s br, 1H) 6.60 (s, 1H), 5.54 (d, 1H, J = 7.7 Hz),
5.32 (d, 1H, J = 7.7 Hz), 5.10 (s, 2H), 4.70 (t, 2H, J = 6.8 Hz),
3.49–3.46 (m, 2H), 3.03 (s, 3H), 2.14 (s, 3H). 13C NMR (100 MHz,
CDCl3): d (ppm) 169.23, 168.66, 159.78, 143.23, 139.01, 135.82,
134.65, 132.43, 131.80, 128.92, 127.98, 127.49, 127.29, 127.17,
126.89, 123.45, 123.22, 119.05, 117.41, 116.92, 115.88, 70.82,
60.09, 58.32, 44.03, 32.80, 11.04. MS-ESI: m/z 625.17 [M+H+].
Nitroxyalkylamine (9a,b, nitrate salt) (0.3 mmol), DMAP
(0.1 mmol), and EDCI (0.2 mmol) were added in sequence to a solu-
tion of acid partner 19 (0.1 mmol) in dichloromethane (5 mL), un-
der nitrogen atmosphere. An excess of triethylamine (0.35 mmol)
was added dropwise and the reaction was stirred at rt for 12 h.
Then the mixture was quenched with water (10 mL) and extracted
with chloroform (50 mL Â 3). The organic layer was washed with
1 N HCl (50 mL), NaHCO3 saturated solution (50 mL), brine
(50 mL) and dried over Na2SO4. After filtration and concentration
of the organic phase a crude material was obtained. The material
was then purified by chromatography on silica gel/alumina (1:1)
using petroleum ether/chloroform/ethyl acetate, 4:4:1 (v/v/v), as
the eluent to give the desired product in good yield.
4.1.14. General procedure for the preparation of 1,5-
diarylpyrrol-glycine hydroxyalkyl amides (6a,b)
Ammonium formate (1.3 mmol) and Pd/C (0.07 g) were added
to a solution of 17a,b (0.32 mmol) in isopropanol (2 mL). The solu-
tion was microwave irradiated at 80 °C 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 10–12
(using NaOH 10% solution) and the mixture extracted with chloro-
form (100 mL Â 3). The organic layer was then dried over Na2SO4.
Filtration and concentration of the organic phase gave a material,
which was identified to be the product without the need of any fur-
ther purification.
4.1.19. N-[2-(Nitroxy)ethyl]-(R,S)-2-[(tert-butyl)oxycarbonyl]
amino-[2-(1-(3-fluorophenyl)-2-methyl-5-(4-(methylsulfonyl)
phenyl)-1H-pyrrol-3-yl)]acetamide (20a)
White solid, mp 102 °C (70% yield). FT-IR cmÀ1: 333 0, 2895,
1688, 1645, 1579, 1228, 1155, 1101, 867. 1H NMR (400 MHz,
CDCl3): d (ppm) 7.68 (d, 2H, J = 8.4 Hz), 7.43–7.40 (m, 1H),
7.30–7.28 (m, 1H,), 7.22–7.19 (m, 2H), 7.13–7.09 (m, 2H), 6.63 (s,
1H), 6.52 (s br, 1H), 5.59–5.56 (m, 1H), 5. 40 (s br, 1H), 4.69 (t,
2H, J = 7.3 Hz), 4.19 (t, 2H, J = 7.3 Hz), 3.05 (s, 3H), 2.15 (s, 3H),
1.46 (s, 9H). 13C NMR (100 MHz, CDCl3): d (ppm) 169.24, 168.70,
159.20, 138.70, 135.63, 134.18, 132.43, 129.18, 128.42, 127.11,
126.66, 123.00, 123.13, 120.18, 117.33, 81.91, 60.05, 58.34, 44.03,
32.21, 30.50, 27.82, 11.24. MS-ESI: m/z 591.18 [M+H+].
4.1.15. N-[3-(Hydroxy)propyl]-2-amino-2-[1-(3-fluorophenyl)-
2-methyl-5-[4-methylsulfonyl)phenyl]-1H-pyrrol-3-
yl]acetamide (6a)
Yellowish powder, mp 115 °C (>95% yield). FT-IR cmÀ1: 3400,
1750, 1620, 1518, 1350, 990. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 8.50 (s, br, 1H), 7.66 (d, 2H, J = 8.4 Hz), 7.26 (d, 2H,
J = 8.4 Hz), 7.12–7.08 (m, 3H, J = 8.4 Hz), 7.04–7.02 (m, 1H), 6.51 (s,
1H), 4.65 (s br, 1H), 4.26 (s, 1H), 4.15 (t, 2H, J = 7.4 Hz), 3.48–3.45
(m, 2H), 3.09 (s, 3H), 2.17 (s, 3H), 1.92–1.89 (m, 2H), 1.80 (s, br, 2
H). 13C NMR (100 MHz, DMSO-d6) d (ppm): 169.40, 159.12, 145.31,
144.71, 141.49, 138.06, 131.44, 129.23, 128.41, 127.00, 126.29,
122.42, 121.47, 116.10, 111.29, 62.10, 58.11, 44.07, 33.23, 30.12,
11.06. MS-ESI: m/z 460.16 [M+H+]. HPLC: >96% pure (tR = 4.31 min).
4.1.20. General procedure for the preparation of 1,5-
diarylpyrrol-glycine nitro-oxyalkylamides (3a,b)
An excess of trifluoroacetic acid (4 mL) was added dropwise at
0 °C to a solution of the Boc-protected 20a,b (0.40 mmol) in diox-
ane (2 mL). The solution was microwave irradiated at 60 °C for
40 min (power 150 W, pressure 170 psi). The reaction mixture
was poured into ice/water (20 mL) and the pH adjusted to 12–13
(using ammonia solution) and the mixture extracted with chloro-
form. The organic layer was then dried over sodium sulfate. The fil-
tration and concentration of the organic phase gave a crude
material, which was purified through column chromatography
(alumina) using MeOH/DCM 1:20 (v/v) to obtain the product as a
yellowish powder.
4.1.16. General procedure for the synthesis of tert-butyl
(ethoxycarbonyl)-[1-(4-fluorophenyl)-2-methyl-5-[4-
(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]methyl carbamate (18)
Boc anhydride (4.65 mmol) was added to a solution of amino
ester 14 (2.23 mmol) in MeOH (15 mL) and the reaction mixture
stirred at rt for18 h. The mixture was then diluted with water
(20 mL), adjusted the pH to 2–3 with 1 N HCl and extracted with
EtOAc (100 mL Â 3). The combined organic fractions were washed
with water (100 mL Â 2). Dried over sodium sulfate and evapo-
rated to give the crude product. The residue was purified by col-
umn chromatography (silica gel) eluting with EtOAc/hexane
(1:20) (v/v) to afford the desired product as white solid (63% yield).
Physicochemical, spectroscopic, and analytical data were consis-
tent with those reported in the literature.27
4.1.21. N-[2-(Nitroxy)ethyl]-2-amino-2-[1-(3-fluorophenyl)-2-
methyl-5-[4-(methylsulfonyl)phenyl]-1H-pyrrol-3-
yl]acetamide (3a)
Yellowish powder, mp 144 °C (81% yield). FT-IR (neat, cmÀ1
) m:
3330, 2890, 1654, 1590, 1300, 1147, 1102, 899. 1H NMR
(400 MHz, CDCl3) d (ppm): 7.67 (d, 2H, J = 8.6 Hz), 7.45–7.40 (m,
1H), 7.23 (d, 2H, J = 8.6 Hz), 7.09–7.07 (m, 1H), 6.96–6.94 (m,
1H), 6.84–6.81 (m, 1H), 6.40 (s br, 1H), 6.51 (s, 1H), 4.67 (t, 2H,
J = 7.4 Hz), 4.23 (s, 1H), 3.47–3.44 (m, 2H), 3.00 (s, 3H), 2.20 (s,
4.1.17. General procedure for the synthesis of n-tert-butyl-
(ethoxycarbonyl)-[2-[1-(4-fluorophenyl)-2-methyl-5-[4-
(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]-2-amino-acetic acid
(19)
A solution of NaOH (5.917 mmol) in water (3 mL) was added to
a solution of amino ester 18 (2.23 mmol) in MeOH (15 mL), and the
3H), 1.80 (s, br, 1H). 13C NMR (100 MHz, CDCl3)
d (ppm):
169.33, 159.91, 145.23, 144.51, 141.22, 138.00, 131.32, 129.55,
128.42, 127.91, 126.19, 122.45, 121.20, 117.26, 116.09, 68.34,
58.18, 43.07, 33.15, 11.06. MS-ESI: m/z 491.13 [M+H+]. HPLC:
>98% pure (tR = 4.89 min).

M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
783
4.1.22. General procedure for the preparation of nitro-oxyalkyl
amines (9a,b) and amino acids (11a,b)
cells were treated also with lower concentrations (0.01–1
l
M). At
the end of the incubation, the supernatants were collected for
the measurement of prostaglandin E2 (PGE2) levels by a radioim-
munoassay (RIA). To evaluate COX-2 activity, cells were stimulated
Fuming nitric acid (3 mL) was added to dichloromethane in a
100 mL round-bottomed flask. The solution was allowed to reach
0 °C and then hydroxyalkylamine (8a–b) (or amino acid 10a–b)
(16 mmol) was added dropwise. After 50 min of stirring, acetic
anhydride (2 mL) was added dropwise over 2 min. After 45 min a
precipitate was formed and then filtered. The solid was then crys-
tallized with hot chloroform/ethanol to give the product as nitrate
salt.
for 24 h with Escherichia coli lipopolysaccharide (LPS, 10
induce COX-2, in the absence or presence of test compounds
(0.01–10 M). Celecoxib was utilized as a reference compound
lg/mL) to
l
for the selectivity index. The supernatants were collected for the
measurement of PGE2 by means of RIA. Throughout the time the
experiments lasted, triplicate wells were used for the various con-
ditions 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 GraphPadInstat, and the data fit was obtained
using the sigmoidal dose-response equation (variable slope)
(GraphPad).
4.1.23. 2-(Nitro-oxy)ethyl amine nitrate salt (9a)
Off-white crystals, mp 89 °C (yield 90%). FT-IR cmÀ1: 1636, 889.
1H NMR (400 MHz, MeOH-d4) d (ppm): 4.80 (t, 2H), 3.40 (t, 2H). 13
C
NMR (100 MHz, CDCl3) d (ppm): 69.17, 38.49. MS-ESI: m/z 107.05
[M+H+].
4.1.24. HPLC methods
4.2.2. Ex vivo vasorelaxing activity
The purity of the title compounds was assessed by means of a
Waters Alliance 2695 instrument equipped with an UVÀvis Waters
PDA 996 as the detector. Millennium Empower with Windows XP
was used. For compounds 1a–d, 3a,b,4a–d, and 6a,b a Gemini-NX
All the experimental procedures were carried out following the
guidelines of the European Community Council Directive 86-609.
The effects of the compounds were tested on isolated thoracic
aortic rings of male normotensive Wistar rats (250–350 g). After
a light ether anaesthesia, rats were sacrificed by cervical disloca-
tion 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 a hypoder-
mic needle. Five 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), thermo-
stated at 37 °C 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 with a com-
puterised system (Biopac). After an equilibration period of
60 min, the endothelium removal was confirmed by the adminis-
C18 (100 Â 2 mm, 3
lm) column at 35 °C was used at a flow rate of
0.3 mL/min. The eluent was (A) H2O plus 0.1% formic acid or (B)
CH3CN plus 0.1% formic acid and a gradient from 90% A to 90% B
was run in 13 min for 1a–d and 4a–d. Using the analytical condi-
tions reported above, the retention times of the compounds ranged
between 4.3 and 8.6 min. The eluent was (A) ammonium bicarbon-
ate 10 mM (pH 9) or (B) CH3CN and a gradient from 50% A to 90% B
was run in 13 min for 3a,b and 6a,b. Using the analytical conditions
reported above, the retention times of the compounds ranged be-
tween 2.7 and 4.2 min.
For compounds 2a–d and 5a,b
a
Acquity BEH C18
(100 Â 2.1 mm, 1.7 m) column at 30 °C was used at a flow rate
l
of 0.3 mL/min. The eluent was (A) 5 mM ammonium acetate or
(B) CH3CN and a gradient from 90% A to 90% B was run in 9 min.
Using the analytical conditions reported above, the retention times
of the compounds ranged between 4.7 and 8.5 min. The eluent was
(A) ammonium bicarbonate 10 mM (pH 9) or (B) CH3CN and a gra-
dient from 50% A to 90% B was run in 13 min for 3a,b and 6a,b.
Using the analytical conditions reported-above, the retention times
of the compounds ranged between 4.23 and 8.57 min. All the syn-
thesized compounds were generally more than 95% pure.
tration of acetylcholine (ACh) (10 lM) to KCl (30 mM)-precon-
tracted vascular rings. A relaxation <10% of the KCl-induced
contraction was considered representative of an acceptable lack
of the endothelial layer, while the organs, showing a relaxation
P10% (i.e. significant presence of the endothelium), were dis-
carded. From 30 to 40 min after the confirmation of the endothe-
lium removal, the aortic preparations were contracted by a single
concentration of KCl (30 mM) and when the contraction reached a
stable plateau, 3-fold increasing concentrations of the tested com-
4.2. Biology and pharmacology
pounds (1 nM–10 lM) 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 inhibi-
4.2.1. In vitro anti-inflammatory studies
The in vitro profiles of compounds 1a–d, 2a–d, 3a,b, 4a–d, 5a–d,
and 6a,b related to their inhibitory activity towards both COX-1
and COX-2 isoenzymes, were evaluated through cell-based assay
employing murine monocyte/macrophage J774 cell lines. The cell
line was grown in DMEM supplemented with 2 mM glutamine,
tor: ODQ 1 lM which was incubated in aortic preparations after
the endothelium removal confirmation. The vasorelaxing efficacy
was evaluated as maximal vasorelaxing response (Emax), ex-
pressed as a percentage (%) of the contractile tone induced by
25 mM HEPES, 100 units/mL penicillin, 100
l
g/mL streptomycin,
KCl 30 mM. When the limit concentration 10 lM (the highest con-
10% fetal bovine serum (FBS), and 1.2% sodium pyruvate. Cells
were plated in 24-well culture plates at a density of 2.5 Â 105
cells/mL or in 60 mm diameter culture dishes (3 Â 106 cells per
3 mL per dish) and allowed to adhere at 37 °C in 5% CO2 for 2 h.
Immediately before the experiments, culture medium was re-
placed with fresh medium and cells were stimulated as described
previously.26–28,33–40 The evaluation of COX-1 inhibitory activity
centration, which could be administered) of the tested compounds
did not reach the maximal effect, the parameter of efficacy repre-
sented the vasorelaxing response, expressed as a percentage (%) of
the contractile tone induced by KCl 30 mM, evoked by this limit
concentration. The parameter of potency was expressed as pIC50
,
calculated as negative logarithm of the molar concentration of
the tested compounds evoking a half reduction of the contractile
tone induced by KCl 30 mM. 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
mean standard error, for 6-10 experiments. Two-way ANOVA
was achieved pre-treating cells with test compounds (10
15 min and then incubating them at 37 °C for 30 min with 15
arachidonic acid to activate the constitutive COX. For the com-
pounds with COX-1% inhibition higher than 50% (at 10 M), the
lM) for
lM
l

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M. Biava et al. / Bioorg. Med. Chem. 22 (2014) 772–786
was selected as statistical analysis, P < 0.05 was considered repre-
sentative of significant statistical differences. Experimental data
were analysed by a computer fitting procedure (software: Graph-
Pad Prism 4.0).
sayed for TXB2. Whole blood TXB2 production was measured as a
reflection of maximally platelet COX-1 activity in response to
endogenously formed thrombin.43
4.2.5. Analysis of PGE2 and TXB2
4.2.3. Determination of the formation of nitrites and nitrates, in
rat liver homogenate
PGE2 and TXB2 concentrations were measured by previously de-
scribed and validated radioimmunoassays.42,43
The experimental procedures were carried out following the
guidelines of the European Community Council Directive 86-609.
The liver homogenates were obtained from male normotensive Wis-
tar rats (250À350 g). After a light ether anaesthesia, rats were sacri-
ficed by cervical dislocation and bleeding. The portal vein was
immediately cannulised and the liver was perfused with 4 °C-cold
homogenation 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 homogenation
buffer. The sample was finally homogenated in Turrax. In a vial,
4.2.6. Statistical analysis
For the HWB assays, results are expressed as the mean SEM of
three to five experiments. The % inhibition of TXB2 (for COX-1 HWB
assay) and PGE2 (for COX-2 HWB assay) production by test com-
pounds was calculated respect to control samples. Data fit was ob-
tained using the sigmoidal dose–response equation (GraphPad
software). The IC50 values were calculated with GraphPad Instat,
and the data fit was obtained using the sigmoidal dose-response
equation (GraphPad). P values of less than 0.05 were considered
significant.
400
ll of the above homogenate were mixed with 500
ll of assay
4.2.7. In vivo analgesic and anti-inflammatory study
buffer (composition: K2HPO4 100 mM; EDTA 1 mM; pH 7.4), con-
taining glutathione, NADH and NADPH (all at the final concentration
of 1 mM). The vials were then thermostated at 37 °C, and 100 ll of a
DMSO solution of the tested NO-donor were added (final concentra-
tion of the tested compound = 1 mM), allowing the release of NO,
which in turn is rapidly converted to the stable inorganic metabo-
In vivo anti-inflammatory activity of the new compounds was
also assessed. Male Swiss albino mice (23–25 g) and Sprague-Daw-
ley or Wistar rats (150–200 g) were used. The animals were fed
with a standard laboratory diet and tap water ad libitum and kept
at 23 (1 °C with a 12 h light/dark cycle, light on at 7 a.m.) The paw
pressure test was performed by inducing an inflammatory process
by the intraplantar (ipl) carrageenan administration 4 h before the
test. In the administered intraplantar (ipl) carrageenan 4 h before
the test. The carrageenan-induced paw oedema test was also per-
formed, evaluating the paw volume of the right hind paw 4 h after
the injection of carrageenan and comparing it with saline/carra-
geenan-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 intra-peritoneal (ip). The number of stretching
movements was counted for 10 min, starting 5 min after
administration.
lites, i.e. nitrites and nitrates. Aliquots (100 ll) of the above medium
were collected at selected intervals (5, 15, 30, 60 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 ampero-
metricallytitrated by a NO-selective electrode connected to an Apol-
lo 4000 free radical analyzer (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 ni-
trites. The concentration of nitrites in these sample is defined as
NOx, which reflects the sum of all the nitrites and nitrates previously
derived from NO. Opportune calibration curves were previously ob-
tained with standard solutions of sodium nitrite.
4.2.8. Solubility assessment in phosphate buffered saline and
simulated gastric fluid
Stock solutions of test compounds (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;
a simulated gastric fluid (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
calibration standards (1, 5, 10, 25, 50, 100 and 200 lM) were pre-
pared from each 20 mM solution stock by serial dilution. Two
microlitres of each test compound (stock solution 20 mM in DMSO)
4.2.4. Ex vivo human whole blood (HWB) assay
Compounds 1c, 4c, 3a, and 6a were evaluated for COX-1 versus
COX-2 selectivity in HWB assay. Three to five healthy volunteers
(aged 29 3 years) were enrolled to participate in the study after
its approval by the Ethical Committee of the University of Chieti.
Informed consent was obtained from each subject. Compounds
(0.005–150 mM) were dissolved in DMSO. Aliquots of the solutions
(2
lL) or vehicle were pipetted directly into test tubes to give final
were added to 198 lL of PBS and SGF in two duplicate wells in
concentrations of 0.01–300
ate COX-2 activity, 1 mL aliquots of peripheral venous blood sam-
ples containing 10 IU of sodium heparin were incubated in the
presence of LPS (10 lg/mL) or saline for 24 h at 37 °C, as previously
l
M in whole blood samples. To evalu-
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 Millipore manifold filter assembly and col-
lected in an acceptor plate and then analysed on HPLC together
with the standards solutions (HPLC-Waters Separations Module
2695 with photodiode array detector).
described.42 The contribution of platelet COX-1 was suppressed by
pretreating the subjects with aspirin (300 mg, 48 h) before sam-
pling. Plasma was separated by centrifugation (10 min at
2000 rpm) and kept at À80 °C until assayed for PGE2 as an index
of monocyte COX-2 activity. Moreover, peripheral venous blood
samples were drawn from the same donors when they had not ta-
ken any NSAID during the 2 weeks preceding the study. Aliquots
(1 mL) of whole blood were immediately transferred into glass
tubes and allowed to clot at 37 °C for 1 h. Serum was separated
by centrifugation (10 min at 3000 rpm) and kept at À80 °C until as-
4.2.9. Stability assessment in phosphate buffered saline,
simulated gastric fluid and rat plasma
Stock solutions of test compounds (1 mM) were prepared in
DMSO. A standard phosphate buffered saline 0.01 M (pH 7.4) was
used to determine the stability in neutral pH conditions; a simu-
lated gastric fluid 0.05 M solution without pepsin (pH was adjusted
to 1.5 using concentrated HCl) was instead used to check the

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stability of compounds in acid medium. Rat plasma (Sprague-Daw-
ley rat) was used to underline the sensitivity of test compounds to
plasma enzymes; in this case, Na2EDTA dehydrate was used as
anticoagulant for the experiment. An aliquot of each medium of
588uL is taken and stored in a 2 mL Eppendorf tube. Afterwards,
12
medium. The samples are incubated in an hybridisation oven. At
each time point (0, 30, 60, and 120 min), a 100 L aliquot is taken
and quenched immediately with 300 L of 100% ice cold acetoni-
lL of 1 mM solution of each test compound is added to each
l
l
trile (containing 60 ng/mL of internal standard, i.e. phenacetine)
and stored at À80 °C. All the sample then are centrifuged at
14,000 rpm for 5 min. The supernatant is transferred into an HPLC
vial and analysed on LC–MS/MS (Shimadzu-API4000).
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4.2.10. Pharmacokinetic study
Male Han Wistar rats, three animals per dosage route, were
treated with each compound solution (5% DMSO, 5% CHREMO-
PHOR EL, 0.1% Tween 80 in phosphate buffer pH 7 (150 mM) at
the dose of 10 mg/kg/5 mL intravenously and orally. Approxi-
mately 0.3 mL of blood per time point was taken from left jugular
vein using a syringe. The samples where then centrifuged at
12,000 rpm for 5 min at 4 °C. Plasma was separated, placed in
pre-labeled Eppendorf tubes, and stored at À80 °C pending the as-
say. LC–MS/MS analysis was conducted on Applied Biosystem API
4000 Qtrap mass spectrometer operating in ESI positive ion mode
with the LLOQ of 1 ng/mL for 1c and 4c and LLOQ of 15 ng/mL for
3a and 5 ng/mL for 6a. Data were elaborated using Analyst 1.5.1
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(2.1 Â 50 mm, 3.5
l
m) 3.5
l
m and Zorbax Sb C18, 2.1 Â 50 mm
columns, heated at 45 °C. Compounds 1c and 4c and 3a and 6a sep-
aration was achieved in gradient conditions at a flow rate of
0.4 mL. Mobile phase composition in both cases was 0.1% HCOOH
in H2O/CH3CN, 10/90 (v/v). Compounds quantification was done
at following MRM transitions: 1c: m/z 476 > 342, 4c: m/z
431 > 342 and 3a: m/z 491 > 369, 6a: m/z 446 > 368.
Extraction method consisted in plasma protein precipitation of
rat plasma samples with CH3OH into 96-deep well plates 1 mL
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15 min at 3700 rpm at 6 °C and 5 lL of the supernatant was in-
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Acknowledgments
We are thankful to Rottapharm Madaus S.p.A. for its technical,
scientific and financial support.
We thank Francesca Lodovichetti and Mario De Miranda, Rot-
tapharm Madaus Analytical Labs for analytical method develop-
ment and analytical support. We wish to thank Paola Anzellotti,
Ph.D, for her valuable input and assistance.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.008.
36. Biava, M.; Porretta, G. C.; Poce, G.; Supino, S.; Manetti, F.; Botta, M.; Sautebin,
L.; Rossi, A.; Pergola, C.; Ghelardini, C.; Norcini, M.; Makovec, F.; Anzellotti, P.;
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