Novel analgesic/anti-inflammatory agents: Diarylpyrrole acetic esters endowed with nitric oxide releasing properties

Article abstract of DOI:10.1021/jm200715n

The design of compounds that are able to inhibit cyclooxygenase (COX) and to release nitric oxide (NO) should give rise to drugs endowed with an overall safer profile for the gastrointestinal and cardiovascular systems. Herein we report a new class of pyrrole-derived nitrooxy esters (11a-j), cyclooxygenase-2 (COX-2) selective inhibitors endowed with NO releasing properties, with the goal of generating new molecules able to both strongly inhibit this isoform and reduce the related adverse side effects. Taking into account the metabolic conversion of nitrooxy esters into corresponding alcohols, we also studied derivatives 12a-j. All compounds proved to be very potent and selective COX-2 inhibitors; nitrooxy derivatives displayed interesting ex vivo NO-dependent vasorelaxing properties. Compounds 11c, 11d, 12c, and 12d were selected for further in vivo studies that highlited good anti-inflammatory and antinociceptive activities. Finally, two selected compounds (11c and 12c) tested in human whole blood (HWB) assay proved to be preferential inhibitors of COX-2.

Full text of DOI:10.1021/jm200715n

Article
pubs.acs.org/jmc
Novel Analgesic/Anti-Inflammatory Agents: Diarylpyrrole Acetic
Esters Endowed with Nitric Oxide Releasing Properties
Mariangela Biava,*,† Giulio Cesare Porretta,† Giovanna Poce,† Claudio Battilocchio,† Salvatore Alfonso,†
Michele Rovini,‡ Salvatore Valenti,‡ Gianluca Giorgi,§ Vincenzo Calderone,∥ Alma Martelli,∥ Lara Testai,∥
Lidia Sautebin,⊥ Antonietta Rossi,⊥ Giuseppina Papa,⊥ Carla Ghelardini,# Lorenzo Di Cesare Mannelli,#
Antonio Giordani,× Paola Anzellotti,● Annalisa Bruno,● Paola Patrignani,● and Maurizio Anzini‡
†Dipartimento di Studi di Chimica e Tecnologie del Farmaco, Universita
̀
“Sapienza”, Piazzale Aldo Moro 5, I-00185 Roma, Italy
§
‡Dipartimento Farmaco Chimico Tecnologico and Dipartimento di Chimica, Universita
̀
degli Studi di Siena, Via A. de Gasperi 2,
I-53100 Siena, Italy
∥Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita
̀
di Pisa, Via Bonanno 6, I-56126 Pisa, Italy
di Napoli “Federico II”, Via D. Montesano 49, I-80131 Napoli, Italy
̀
di Firenze, Viale G. Pieraccini 6, I-50139 Firenze, Italy
⊥Dipartimento di Farmacologia Sperimentale, Universita
̀
#Dipartimento di Farmacologia, Universita
×Rottapharm Madaus, Via Valosa di Sopra 7, I-20052 Monza, Italy
●Dipartimento di Medicina e Scienze dell’Invecchiamento, Universita
dell’Invecchiamento (CeSI), Via dei Vestini 31, 66100 Chieti, Italy
̀
degli Studi “G. D’Annunzio” di Chieti e Centro Scienze
S
* Supporting Information
ABSTRACT: The design of compounds that are able to
inhibit cyclooxygenase (COX) and to release nitric oxide
(NO) should give rise to drugs endowed with an overall safer
profile for the gastrointestinal and cardiovascular systems.
Herein we report a new class of pyrrole-derived nitrooxy esters
(11a−j), cyclooxygenase-2 (COX-2) selective inhibitors endowed with NO releasing properties, with the goal of generating new
molecules able to both strongly inhibit this isoform and reduce the related adverse side effects. Taking into account the metabolic
conversion of nitrooxy esters into corresponding alcohols, we also studied derivatives 12a−j. All compounds proved to be very
potent and selective COX-2 inhibitors; nitrooxy derivatives displayed interesting ex vivo NO-dependent vasorelaxing properties.
Compounds 11c, 11d, 12c, and 12d were selected for further in vivo studies that highlited good anti-inflammatory and
antinociceptive activities. Finally, two selected compounds (11c and 12c) tested in human whole blood (HWB) assay proved to
be preferential inhibitors of COX-2.
inhibition of endothelial COX-2-dependent prostacyclin
associated with incomplete inhibition of antithrombotic factors
deriving from platelet COX-1, resulting in an impaired platelet
function and increased risk of thromboembolic accidents.7 The
COX-inhibiting nitric oxide (NO) donors (CINODs)
represent a new class of agents that may offer an improved
safety profile having a dual mechanism of action involving COX
inhibition and NO donation.
NO is a pleyotropic radicalic molecule involved in vascular
smooth muscle tone modulation, angiogenesis, thrombosis,
endothelium permeability, and neuronal transmission. This
endogenous autacoid exerts its physiological roles mainly
through the activation of guanylate cyclase (GC), which gives
rise to increasing levels of c-GMP and, as a consequence, to
smooth muscle tone relaxation, platelet aggregation inhibition,
and chemokine and cytokine down-regulation (which accounts
for the inhibitory activity on leukocyte adhesion), finally
INTRODUCTION
■
Nonsteroidal anti-inflammatory drugs (NSAIDs) selective for
cyclooxygenase-2 (COX-2), named coxibs, were developed to
overcome the gastrointestinal (GI) side effects associated with
the use of traditional NSAIDs (t-NSAIDs).1,2 Large random-
ized clinical trials highlighted how almost all highly selective
COX-2 inhibitors were endowed with safer GI profiles
compared to t-NSAIDs.1−3 The withdrawal of rofecoxib from
the marketplace has been related to a small but significant
increase of thrombotic events associated with the chronic use of
rofecoxib in patients with a history of colorectal adenoma.4
Numerous studies of clinical pharmacology and pharmacoepi-
demiology as well as studies performed in animal models
showed quite convincingly that selective inhibition of COX-2
by coxibs translates into a cardiovascular (CV) hazard. The
most plausible hypothesis is that they cause the inhibition of
the vasorelaxing and antithrombotic prostacyclin which is
generated mainly by the activity of constitutively expressed
COX-2, in endothelial cells, in vivo.5,6 This CV hazard is shared
with t-NSAIDs through the same mechanism, i.e., profound
Received: June 6, 2011
Published: October 12, 2011
© 2011 American Chemical Society
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Figure 1. New class of pyrrole-containing antiflogistic/analgesic agents (1−4) as selective COX-2 inhibitors.
leading to an antithrombotic mode of action.8 It is also worth
noting that NO is endowed with a cardioprotective function,
and gene knock-out studies demonstrated that NO depletion
leads to an increased incidence of myocardial infarction. It is
well-known and understood that organic nitrates as well as
some other compounds (nitrous thiols, furoxan N-oxides)9 are
endowed with releasing property as exogenous “NO donors”,
mimicking its actions in such a way to be employed as
therapeutic agents.9,10 On these grounds, the synthesis of
hybrid molecules, which conjugate a COX-2 inhibiting feature
with a NO releasing moiety, could bring about new anti-
inflammatory and analgesic agents, with an improved safety
profile for the CV system versus NSAIDs (both t-NSAIDs and
coxibs) which are associated with renal and occlusive vascular
events.7,11 Some CINODs, such as nitronaproxen,12−14 are
known in literature and underwent clinical development
studies. Nitronaproxen has been reported have reduced renal
and gastrointestinal toxicity compared to naproxen.11,15,16
Though the approach of combining an NO-donor moiety to
t-NSAIDs was widely applied, only few examples can be found
in the design of COX-2 inhibiting nitric oxide donors.17 An
interesting prodrug of rofecoxib, able to release NO, was
reported as well as nitroxy-substituted 1,5-diarylimidazoles and
nitroxy substituted analogues of celecoxib.18−24
In the past, we reported several investigations concerning the
development of a new class of pyrrole-containing antiflogistic/
analgesic agents (compounds 1−4, Figure 1) as selective COX-
2 inhibitors.25−31 Structure−activity relationships of these
compounds were examined in depth. In addition to the impact
by the substitution pattern on the phenyl ring at N1 (activity
decreasing in the order H > 3-F = 4-F > 3,4-diF > 4-OMe >
4-CF3 > 4-CH3) and, of course, to the presence of the
p-methylsulfonyl substituent in the C5 pyrrole ring, the side
chain at C3 appeared to be crucial for the COX-2 inhibition
potency. Only aliphatic side chains were previously explored,
and no information was available about the effect exerted by the
introduction into the side chain of other substituents (Figure 1).
Thus, starting on these grounds, we decided to investigate the
effects exerted on COX-2 inhibitory activity by the introduction
of a NO donor moiety on this scaffold. Manipulation of the
3-position of the scaffold allowed us to retain COX-2 inhibitory
properties in terms of both potency and selectivity and to
introduce a NO-releasing moiety with appropriate character-
istics. Conversely, manipulation of the scaffold in other
positions led to the loss of COX-2 inhibition. We report
herein the syntheses and the biological activities of new
derivatives 11a−j (Scheme 1), obtained by the combination of
selective COX-2 inhibitors and nitrooxyalkyl chains in order to
obtain effective analgesics and anti-inflammatory agents CV-
sparing. These compounds have been designed by combining
some 1,5-diarylpyrroles that were selected on the basis of their
COX-2 inhibitory activity31 and NO-donor alkyl chains with
different length. Furthermore, considering that the nitro-
oxyalkyl groups are subjected to in vivo metabolism, we also
synthesized the corresponding hydroxyl derivatives (com-
pounds 12a−j) of compounds 11a−j. All the target compounds
were evaluated in vitro for their ability to inhibit COX-1 and
COX-2 in a J774 murine macrophage cell line. In order to
evaluate their NO-releasing properties, the vasorelaxing
activities of compounds 11a−j were evaluated ex vivo on
isolated rat aortic rings. The in vivo anti-inflammatory and
analgesic activities for selected compounds were also evaluated
in several animal models. Among all compounds, derivatives
11c and 12c, a nitro ester and its hydroxy-related derivative,
were selected to evaluate their selectivity ex vivo, performing a
human whole blood (HWB) assay.
CHEMISTRY
■
The synthesis of the target compounds is described in Scheme 1.
Briefly, the reaction of methyl vinyl ketone with 4-(methylthio)-
benzaldehyde in a microwave apparatus gave intermediate 5,30
which was transformed into the corresponding 4-methylsul-
fonyl derivative 6 by means of Oxone oxidation.32 Intermediate
6 cyclized to yield the expected 1,5-diarylpyrroles 7a−e in the
presence of the appropriate arylamine (45 min by employing a
microwave apparatus).30 Regioselective acylation of 7a−e with
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a
Scheme 1. Synthesis of Compounds 11a−j and 12a−j
a
Compounds: 7a, R = H; 7b, R = 3-F; 7c, R = 4-F; 7d, R = 4-SCH3; 7e, R = 4-OCH3; 8a, R = H; 8b, R =3-F; 8c, R = 4-F; 8d, R = 4-SCH3; 8e, R =
4-OCH3; 9a, R = H; 9b, R = 3-F; 9c, R = 4-F; 9d, R = 4-SCH3; 9e, R = 4-OCH3; 10a, R = H; 10b, R = 3-F; 10c, R = 4-F; 10d, R = 4- SCH3; 10e,
R = 4-OCH3; 11a, R = H, n = 2; 11b, R = H, n = 3; 11c, R = 3-F, n = 2; 11d, R = 3-F, n = 3; 11e, R = 4-F, n = 2; 11f, R = 4-F, n = 3; 11g, R =
4-SCH3, n = 2; 11h, R = 4-SCH3, n = 3; 11i, R = 4-OCH3, n = 2; 11j, R = 4-OCH3, n = 3; 12a, R = H, n = 2; 12b, R = H, n = 3; 12c, R = 3-F, n = 2;
12d, R = 3-F, n = 3; 12e, R = 4-F, n = 2; 12f, R = 4-F, n = 3; 12g, R = 4-SCH3, n = 2; 12h, R = 4-SCH3, n = 3; 12i, R = 4-OCH3, n = 2; 12j, R =
4-OCH3, n = 3; 13a, n = 2; 13b, n = 3; 14a, n = 2; 14b, n = 3. Reagents and conditions: (i) CH2CHCOMe, TEA, 3-ethyl-5-(2-hydroxyethyl)-
4-methylthiazolium bromide, microwave, 15 min; (ii) Oxone, MeOH/H2O, room temp, 2 h; (iii) RPhNH2, p-toluensulfonic acid, EtOH, microwave,
45 min; (iv) EtOCOCOCl, TiCl4, CH2Cl2, room temp, 4 h; (v) Et3SiH, TFA, room temp, 2 h; (vi) 1 N NaOH, MeOH, 1 h; (vii) 14a,b,
EDCI, DMAP, CH2Cl2, room temp, 3h; (viii) 13a,b, EDCI, DMAP, CH2Cl2, room temp, 3 h; (ix) HNO3, CH3COOH, (CH3CO)2O, EtOAc,
room temp, 20 h.
ethoxalyl chloride in the presence of titanium tetrachloride
gave the α-keto ester derivatives 8a-e, which were in turn
reduced by means of triethylsilane in trifluoroacetic acid to
give the pyrrole ethyl esters 9a−e.30 Treatment of 9a−e with
1 N NaOH in MeOH for 1 h gave the corresponding acetic
acids 10a−e. Target compounds 12a−j and 11a−j were
finally obtained by reacting acetic acids 10a−e with the
appropriate diols 13a,b or hydroxyalkyl nitrates 14a,b
(previously prepared by reacting 13a,b with nitric acid,
acetic acid, and acetic anhydride overnight)33 in the presence
of EDCI and DMAP for 3 h.
channel of the enzyme. Nonetheless, it is noteworthy that
especially in the meta-fluorine substituted (11c, 11d) and para-
methoxy substituted (11i, 11j) subclasses, the activity seems to
be related to the side chain length; an inverse relationship is
found between side chain length and inhibitory concentration.
Compound 11c, which is characterized by a two-carbon side
chain, showed a 19 nM activity on COX-2 isoenzyme, while
compound 11d (three-carbon side chain) displayed a 3-fold
higher activity with a 7.3 nM inhibitory concentration. This
trend is also found in increased form comparing compounds
11i (0.17 μM) and 11j (2.4 nM). On the other hand, nitrated
compounds bearing a para-fluorine, a para-methylsulfonyl, or an
unsubstituted phenyl ring at N1 of the central core displayed
activities that are not influenced by length of the side chain
(Table 1). The results obtained for the hydroxylated derivatives
(12a−j) showed a similar trend concerning the inhibition of
COX-2 isozyme; the only exception is found in compound 12b
which is devoid of any activity. No correlation, however,
between the length of the hydroxyl compound side chain and
the activity can be postulated. All compounds, either in the
form of nitroester or corresponding hydroxyl or acid
derivatives, showed outstanding selectivity toward COX-2.
Compounds 11a−j were further evaluated to assess their
RESULTS AND DISCUSSION
■
All compounds were tested in the J774 murine macrophage
assay to evaluate their inhibitory potency and selectivity on
both COX isoforms (Table 1). All compounds inhibited COX-
2 activity at concentrations ranging from micromolar to
nanomolar values. In particular, nitrated compounds (11a−j)
displayed much better biological profiles than those of the
corresponding alcohol compounds (12a−j). Similar trends
have been previously observed by other authors.19,20 This result
could be due to an electronic interaction of the nitrate group or
to other interactions of this moiety at the inner hydrophobic
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Table 1. In Vitro COX-1 and COX-2 Inhibitory Activity of 11a−j, 12a−j, and Celecoxib
a
a
b
compd
R
n
concn (μM) COX-1 inhibition (%)
COX-1 IC50 (μM)
COX-2 inhibition (%) COX-2 IC50 (μM)
selectivity index
>10
c
10a
H
0.01
0.1
1
>10
13
17
43
100
40
60
89
96
25
60
100
25
47
73
94
98
47
62
90
22
68
91
100
13
76
91
90
43
67
92
96
55
83
97
97
31
72
100
100
25
71
90
91
38
84
100
100
40
61
90
99
34
89
97
39
67
90
90
51
92
100
1
10
0
4
d
10b
10c
10d
0.01
0.1
>10
>10
0.028
0.5
>357.1
>20
1
10
0.1
1
10
0.1
>10
>10
0.5
>20
0.01
0.1
0.013
>769.2
1
10
45
0
d
10e
11a
0.1
>10
>10
0.17
>58.8
1
10
H
2
3
2
3
2
3
2
3
0.01
0.10
1.00
10.00
0.01
0.10
1.00
10.00
0.01
0.10
1.00
10.00
0.01
0.10
1.00
10.00
0.01
0.10
1.00
10.00
0.01
0.10
1.00
10.00
0.01
0.10
1.00
10.00
0.01
0.10
1.00
10.00
0.100
1.00
10.00
0.001
0.01
0.1
0.0430
>232.6
19
27
36
11b
11c
11d
11e
11f
H
>10
>10
1.1
0.0420
0.0190
0.0073
0.0290
0.0372
0.0170
0.0270
>238.1
>526.3
150.7
3-F
3-F
4-F
4-F
20
50
75
>10
>10
>10
>10
>344.8
>268.8
>588.2
>370.4
0
28
0
11g
11h
4-SCH3
4-SCH3
0
11i
11j
4-OCH3
4-OCH3
2
3
12
30
55
6.4
0.1700
0.0024
37.6
>10
>4166.7
1
0
12a
H
2
0.10
1.00
10.00
>10
0.0960
>104.2
22
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Table 1. continued
a
a
b
compd
12b
R
n
concn (μM) COX-1 inhibition (%)
COX-1 IC50 (μM)
COX-2 inhibition (%) COX-2 IC50 (μM)
selectivity index
H
3
0.100
1.00
0
0
10.00
0.01
100
12c
12d
12e
12f
3-F
2
3
2
3
2
>10
>10
>10
>10
3.18
30
50
78
80
40
66
80
85
25
68
92
100
22
60
90
199
24
50
85
87
34
74
85
2
0.0850
0.0230
0.0390
0.0560
0.0850
>117.6
>434.8
>256.4
>178.6
37.4
0.100
1.00
10.00
0.01
0.10
1.00
10.00
0.01
0.10
1.00
10.00
0.01
0.10
1.00
10.00
0.01
0.10
1.00
10.00
0.10
1.00
10.00
0.10
1.00
10.00
0.10
1.00
10.00
0.01
0.10
1.00
10.00
33
0
3-F
4-F
4-F
21
0
12g
4-SCH3
11
40
62
12h
4-SCH3
4-OCH3
4-OCH3
3
2
3
>10
>10
>10
3.84
0.2600
1.1000
0.7000
0.0610
>38.5
>9.1
36
5
12i
52
84
3
12j
>14.3
>63.0
64
93
32
52
80
100
27
0
celecoxib
4
31
64.7
a
Results are expressed as the mean (n = 3) of the % inhibition of PGE2 production by the test compounds with respect to control samples.
IC50(COX-1)/IC50(COX-2). See ref 25. See ref 30.
b
c
d
Table 2. Evaluation of Efficacy and Potency in Determining
NO-Dependent Vasorelaxing Responses of 11a−j and GTN
efficacy and potency in determining NO-dependent vasorelaxing
responses in a suitable experimental model of vascular smooth
muscle and in particular in endothelium-denuded rat aortic rings
(Table 2). Again, the side chain length seems to be the crucial
feature for this activity: the two-carbon chain-bearing compounds
(11a, 11c, 11e, 11g, 11i) are always endowed with higher
vasorelaxing effects than the related three-carbon chain-bearing
derivatives (11b, 11d, 11f, 11h, 11j). In particular, all two-
carbon chain-bearing derivatives exhibited vasorelaxing efficacy
(Emax) higher than 50% and potency values (pIC50) ranging
between 5.47 (11e) and 6.75 (11i), while the efficacy parameters
of all three-carbon chain-bearing derivatives were always lower
than 50%, thus rendering the calculation of the parameters of
pIC50 impossible. The experiments carried out in the presence of
guanylate cyclase (GC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-
a]quinoxalin-1-one (ODQ) confirmed that the vasorelaxing
effects were due to the release of NO, since such a
pharmacological inhibition of GC significantly antagonized the
vasodilator responses evoked by the nitrooxy derivatives 11a−j
(Figure 2). These experiments also showed that the correspond-
ing alcohol derivatives 12a−j were devoid of significant
a
a
compd
R
n
R′
Emax
pIC50
11a
11b
11c
11d
11e
11f
H
2
3
2
3
2
3
2
3
2
3
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
65 ± 2
44 ± 8
69 ± 4
39 ± 1
58 ± 5
41 ± 2
65 ± 1
41 ± 8
77 ± 2
41 ± 11
73 ± 2
5.76 ± 0.08
<5
H
3-F
3-F
4-F
4-F
6.48 ± 0.06
<5
5.47 ± 0.07
<5
11g
11h
11i
4-SCH3
4-SCH3
4-OCH3
4-OCH3
6.34 ± 0.06
<5
6.75 ± 0.05
<5
11j
GTN
6.90 ± 0.07
a
Emax represents the vasorelaxing efficacy, while the parameter of
potency is expressed as pIC50.
vasorelaxing effects (data not shown), further emphasizing the
fact that the pharmacodynamic feature is only due to the
nitrooxy group and its biotransformation to NO. GTN, selected
as reference drug endowed with rapid NO-releasing properties,
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Table 3. Effect of 11c, 11d, 12c, 12d, Celecoxib, and Vehicle
(CMC) in the Mouse Abdominal Constriction Test (Acetic
Acid 0.6%)
no.
a
b
c
compd
CMC
mice
dose po, mg/kg (μmol/kg)
no. writhes
43
12
12
18
10
8
32.6 ± 2.1
11c
11c
11d
11d
12c
12c
12c
12c
12d
12d
20 (42)
40 (84)
20 (41)
40 (82)
3 (7)
17.1 ± 2.3*
9.4 ± 2.5*
25.7 ± 2.8∧
24.9 ± 2.2*
26.9 ± 3.0*
17.4 ± 2.9*
16.2 ± 2.8*
8.9 ± 2.1*
25.3 ± 2.8
15.6 ± 2.2*
8
10 (23)
20 (46)
30 (70)
20 (45)
40 (90)
10 (26)
8
8
10
10
10
celecoxib
13.4 ± 2.6*
a
b
All compounds were administered per os 30 min before test. The
doses are expressed in mg/kg. The equivalent doses in μmol/kg are
c
indicated in parentheses. ∗, P < 0.01 versus vehicle-treated mice. ∧,
P < 0.05.
hypothesize that its related hydroxyl derivative 12c is not the
main metabolite of 11c. Indeed, the complex metabolic
pathway that ensures the biotransformation of the nitrooxy
derivatives, leading to the production of NO and the related
hydroxyl derivatives, is widely considered as the most usual
metabolic destiny of organic nitrates.34 Nevertheless, recent
experimental evidence suggests that this is not a unique
pathway. For example, the pharmacokinetic feature of some
nitrooxy derivatives of aspirin indicates that alternative
pathways involving the formation of O-glutathionyl
metabolites can play a relevant role in the metabolism of
organic nitrates.35 A more detailed characterization of the
pharmacokinetic profile of 11c is currently being investigated.
Compounds 11d and 12d performed well but significantly less
well than 12c. The antiedemigenic activity of compounds is
remarkable albeit not comparable to celecoxib. The extent of
selective inhibition gained through the cell-based assay is,
however, not to be considered the most reliable one; indeed,
the use of exogenous AA in the assay for COX-1 activity in
vitro, in the murine monocyte/macrophage J774 cell line, might
have caused a loss in COX-1 affinity for an AA-dependent
allosteric activation of COX-1, which induces a conformation
change within the enzyme binding site.36 With the aim of
assessing COX-2 selectivity, the HWB assay was carried out
on selected compounds 11c and 12c (nitro ester and nitro-
free one, respectively). In particular, the assay was performed
to predict the actual extent of isozyme inhibition achievable
in vivo by circulating drug levels, since a number of variables
are potentially able to affect drug−enzyme interactions. As
shown in Figure 3 and in Table 5, compounds 11c and 12c
inhibited platelet COX-1 activity in clotting blood incubated
for 60 min at 37 °C in a concentration-dependent fashion,
with different IC50 values of 9.6 μM (95% CI of 6.3−14.6
μM) and 12.2 μM (95% CI of 7 −21 μM), respectively, the
effect of the compounds on inducible COX-2 activity in LPS-
stimulated HWB in vitro. 11c and 12c showed as well similar
inhibitions of monocyte COX-2 activity with IC50 values of
3.2 μM (95% CI of 2−5.5 μM) and 1.7 μM (95% CI of
0.94−3 μM), respectively. Hence, 11c was 3-fold and 12c
was 7.3-fold more potent toward COX-2 than toward COX-1
Figure 2. Representative concentration−response curves relative to
the vasorelaxing effects evoked by compounds 11c and 11i, recorded
on endothelium-denuded rat aortic rings in the absence (control, black
squares) or in the presence (white circles) of 1 μM ODQ (inhibitor of
guanylate cyclase). The vasorelaxing responses are expressed as a
percentage of the contractile effect induced by 30 mM KCl. The
vertical bars indicate the standard error.
showed strong vasorelaxing effects and higher levels of
vasorelaxing potency (Table 2). Experimental evidence suggests
that the compounds reported herein ensure a NO release that is
slower than that for GTN, thus acting as “modulated” NO
donors, which are preferred for hybrid drugs. The meta-fluorine
subclass of compounds (11c,d and 12c,d) was selected for
further in vivo studies. The results related to the writhing test
emphasized the strong dependence of the activity on the side
chain length (Table 3). Even though compounds bearing three-
carbon side chains (Table 1) proved to be more active than the
corresponding two-carbon side chain bearing ones in vitro,
compounds with two-carbon side chains, such as nitrated and
nitro-free derivatives 11c and 12c, showed better analgesic
profiles than those found for three-carbon side chain
derivatives. The 70% writhes reduction was associated with
the activities of both 11c and 12c at their maximum dosage (40
and 30 mg kg−1, respectively), while compounds 11d and 12d
showed a lower activity. Findings reported for carrageenan
induced hyperalgesia and edema show that all compounds are
endowed with good pharmacological profiles (Table 4). As
discussed for the writhing test, compounds bearing two-carbon
side chains were able to cut down carrageenan induced pain.
Compound 12c exerted a unique profile: 80% of hyperalgesia
reduction 30 min after the treatment, when administered at a
dose of 30 mg kg−1. A comparison of 12c with celecoxib clearly
emphasizes that their profiles nearly resemble each other until
60 min after treatment, with the only difference being that 12c
can be associated with a faster onset of the effect. As shown for
12c, the nitro-related compound 11c is characterized by the
same fast effect but its efficacy is abated after 60 min. The
shorter duration of the analgesic effect of 11c allows us to
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Table 4. Effect of 11c,d, 12c,d, Celecoxib, and Vehicle (CMC) on Hyperalgesia and Edema Induced by Carrageenan in the Rat
Paw Pressure Test
paw pressure (g)
pretreatment,
ipl
treatment,
po
before
treatment
a
b
b
dose mg/kg (μmol/kg)
after treatment
volume (mL)
30 min
60 min
120 min
62.7 ± 2.5
34.8 ± 2.3
ND
60 min
saline
CMC
CMC
11c
62.6 ± 2.1
61.9 ± 2.0
32.6 ± 2.7
29.8 ± 3.1
31.9 ± 3.0
62.5 ± 3.1
63.2 ± 3.3
31.8 ± 2.5
62.6 ± 3.1
61.3 ± 3.0
61.5 ± 3.4
62.5 ± 1.8
33.7 ± 2.6
40.1 ± 3.4∧
54.1 ± 3.7*
57.2 ± 3.5*
43.2 ± 3.2
45.5 ± 3.7*
55.7 ± 3.1*
41.2 ± 3.0
43.1 ± 3.6
54.3 ± 3.9*
63.4 ± 2.2
35.1 ± 2.3
35.3 ± 2.8
38.7 ± 3.1
36.5 ± 3.8
45.1 ± 3.7∧
44.9 ± 4.2∧
52.3 ± 2.8*
45.8 ± 3.6∧
48.8 ± 3.6*
1.33 ± 0.07
2.44 ± 0.08
2.52 ± 0.06
2.17 ± 0.08*
1.88 ± 0.08*
2.05 ± 0.08∧
1.96 ± 0.09*
2.51 ± 0.05
ND
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
20 (42)
40 (84)
100 (210)
20 (41)
40 (82)
30 (70)
20 (45)
40 (90)
10 (26)
11c
ND
11c
ND
11d
40.7 ± 2.6
36.8 ± 3.8
39.3 ± 3.5
36.4 ± 3.1
44.4 ± 3.3∧
54.9 ± 3.6*
11d
12c
12d
12d
ND
celecoxib
57.1 ± 4.0*
1.36 ± 0.07*
a
b
The doses are expressed in mg/kg. The equivalent doses in μmol/kg are indicated in parentheses. ∗, P < 0.01 versus vehicle-treated mice. ND, not
determined. ∧, P < 0.05.
oxygen atom as well as a simultaneous double hydrogen bond
interaction of Arg120 with alcoholic and carboxylic oxygen
atoms. Moreover, in the docked conformation of 12c, an
intramolecular hydrogen bond was observed between the OH
group and the carboxylic oxygen that induces the side chain to
adopt a pseudocyclic conformation (Figure 4a). This distinctive
feature is not conserved in the interaction of hydroxypropyl
chain-bearing derivative; that is, 12d (Figure 4b) is able to
inhibit COX-2 activity with higher potency (IC50 = 23 nM)
compared to compound 12c (IC50 = 85 nM). In fact, the
hydroxypropyl chain of 12d allows for an additional hydrogen
bond with Val116, while the hydrogen bond between Arg120
and the carboxylic oxygen atom is retained. In this way, the
lipophilic pocket defined by Val116, Leu93, and Leu359 allows
the hydroxypropyl ester side chain of 12d to be accommodated
in a more profitable manner compared to the case of
Figure 3. Effects of 11c and 12c on COX-1 and COX-2 activity in
hydroxyethyl chain of 12c. For the nitrooxyalkyl derivatives
vitro in human whole blood. Results are expressed as average percent
11c,d docking simulations demonstrated that the side chains of
of inhibition (N = 3, mean ± SD) from three separate experiments.
these compounds interact with Arg120, adopting a conforma-
tion similar to the hydroxypropyl chain of compound 12d.
Different from the hydroxypropyl ester side chain of 12d, the
nitrooxyethyl and nitrooxypropyl acetate moieties of the highly
active derivative 11d (IC50 = 7.3 nM) and 11c (IC50 = 19 nM)
interact with the COX-2 active site by means of a hydrogen
bond between Ser119 and one of the oxygen atom of the NO2
group (Figure 5), confirming what Wey et al. previously
suggested for polar-end tethers.20 Thus, independently from
the length of the spacer between the acetate moiety and the
nitroester, both residues Val116 and Ser119 seem to elicit the
same effect on compounds 11c,d and 12d, stretching the side
chains toward the solvent accessible surface.
Furthermore, the substituted phenyl ring in position 1 of the
pyrrole core is located in the “hydrophobic pocket” defined by
hydrophobic (Leu384) and aromatic (Tyr385, Trp387, and
Phe518) residues; this finding corresponds to those observed
for compounds 3 and 4. Finally, the p-methylsulfonylphenyl
moiety at position 5 of the diarylpyrrole scaffold of compounds
11c,d and 12c,d occupies the same space as for compounds 3
and 4, being accommodated within the so-called “selectivity
site”. In fact, the entire p-methylsulfonylphenyl group of the
inhibitor undregoes profitable interactions with COX-2
(Figure 3 and Table 5); however, a dramatic change in COX-
2 selectivity in HWB assay versus J774 models was observed.
The drop of selectivity in the HWB assay in relation to that
by the cell-based assay can be potentially explained by
different sensitivities of mice and human COXs enzymes, as
adequately described in the literature for other COX-2
inhibitors.26,37
MOLECULAR MODELING SIMULATIONS
■
The COX-2 inhibition data for selected compounds 11c,d and
12c,d were rationalized through docking simulations using a
crystallographic model of the COX-2 active site [complex
between COX-2 and SC-558, refined at 3 Å resolution
(Brookhaven Protein Data Bank entry: 6cox)]25 by means of
AutoDock 4.0 (for details see the Supporting Information).
Those docking simulations suggest that the binding modes
observed for hydroxylalkyl esters 12c,d and nitro diesters 11c,d
are very similar to the ones previously reported for 1,5-
diarylpyrrole-3-alkoxyethyl ether 327 and 1,5-diarylpyrrole-3-
acetic ester 4.25 In particular, the hydroxyethyl ester moiety of
compound 12c establishes multiple interactions with the active
site: a simple hydrogen bond between Tyr355 and the alcoholic
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a
Table 5. In vitro Inhibition (HWB) of COX-1 and COX-2 by 11c, 12c, and Celecoxib
compd
11c
R
n
R′
COX-1 IC50 (μM)
COX-2 IC50 (μM)
SI
3-F
3-F
2
NO2
OH
9.6 (6.3−14.6)
12.2 (7−21)
12.47 (8.6−17.9)
3.2 (2−5.5)
1.7 (0.94−3)
0.54 (0.29−0.52)
3
12c
2
7.3
23
celecoxib
a
IC50 values of COX-1 and COX-2 are reported as the mean of three different experiments performed in duplicate. In parentheses, the 95%
confidence intervals (CI) are shown.
Figure 4. (a) Hydroxyethyl ester 12c and (b) hydroxypropyl ester 12d docked into the COX-2 cyclooxygenase site. The main residues and the
inhibitors are represented as licorice. Hydrogen bonds are represented as blue dotted lines. For the sake of clarity, only a few amino acids of the
binding site are displayed.
Figure 5. (a) Nitrooxypropyl ester 11d and (b) nitrooxyethyl ester 11c docked into the COX-2 cyclooxygenase site. The main residues and the inhibitors are
represented as licorice. Hydrogen bonds are represented as blue dotted lines. For the sake of clarity, only a few amino acids of the binding site are displayed.
residues. The oxygen atoms of the sulfone groups show
hydrogen bond contacts with the terminal NH2 group of
Gln192 and with the backbone NH group of Phe518, while the
aromatic ring interacts with the side chain of hydrophobic
amino acids, such as Leu352 and Val523.
Not surprisingly, the docking calculations reported herein
were not able to explain the different COX-2 inhibitory activity
of compounds 11c and 11d. More detailed and time-
consuming metadynamics simulations could potentially reveal
a possible alternative binding mode of these inhibitors within
COX-2, as it was very recently reported by Limongelli et al.38
who by means of a metadynamic-based approach illuminated
the highly dynamical character of selective 1,5-diarylpyrrole
derivatives/COX-2 recognition process.
X-ray diffraction (see Supporting Information.) provides
further support to the binding mode herein proposed.
CONCLUSIONS
■
The preclinical evaluation of a new class of pyrrole-derived
compounds displayed satisfactory profiles. Nitrooxy derivatives,
acting as hybrid molecules, conjugate a COX-2 inhibiting feature
with a NO releasing moiety. In vitro tests highlighted a profound
inhibition of COX-2 isozyme both in rats and in humans. Their
vasorelaxing properties are dependent on NO release. Moreover,
it was shown that compounds 11c, 11d, 12c, and 12d are
endowed with good anti-inflammatory and antinociceptive
activities. Nevertheless, it still remains to be addressed whether
the NO-releasing property of this new class of compounds would
translate into inhibition of platelet function, thus counteracting
the possible CV hazard related to COX-2-dependent prostacyclin
inhibition.
In addition, as for compound 11c, the energy difference (ΔE
= −0.4059 hartree) between the docked conformation (see
Figure 5b) and the conformation resulting from single crystal
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1-[(4-Methylthio)phenyl]-2-methyl-5-[4-(methylsulfonyl)-
phenyl]-1H-pyrrole (7d). Yellowish needles, mp 105−110 °C (yield
79%). 1H NMR (400 MHz, CDCl3) δ (ppm): 7.60 (d, 2H, J =
8.6 Hz), 7.26 (d, 2H, J = 8.5 Hz), 7.17 (d, 2H, J = 8.6 Hz), 6.98 (d,
2H, J = 8.5 Hz), 6.39 (m, 1H), 6.11 (m, 1H), 3.01 (s, 3H), 2.51 (s,
3H), 2.06 (s, 3H). MS-ESI: m/z 380 (M + Na+).
General Procedure for the Preparation of 1,5-Diarylpyrrol-3-
glyoxylic Esters (8a−e). To a solution of the appropriate pyrrole
[7a−e (4.82 mmol)] in dry dichloromethane (5 mL) at 0 °C under
nitrogen atmosphere, ethoxalyl chloride (4.82 mmol) and TiCl4 (4.82
mmol) were added in sequence. The solution was stirred for 4 h at
room temperature and then quenched with water. The mixture was
extracted with chloroform, and the organic layer was washed with
brine, dried, and evaporated in vacuo. Purification of the residue by
chromatography on silica gel employing petroleum ether/chloroform/
ethyl acetate, 4:3:1 (v/v/v), as the eluent gave a solid which, after
recrystallization from hexane, afforded the expected product.
EXPERIMENTAL SECTION
■
Chemistry. All chemicals used were of reagent grade. Yields refer
to purified products and are not optimized. A CEM Discovery
microwave system apparatus was used for the Stetter and Paal−Knorr
reactions. Melting points were determined in open capillaries on a
Gallenkamp apparatus and are uncorrected. Microanalyses were
performed on a Perkin-Elmer 240C or a Perkin-Elmer series II
CHNS/O analyzer 2400 instrument. Sigma-Aldrich silica gel 60 (230−
400 mesh) was used for column chromatography. Merck TLC plates
(silica gel 60 F254) were used for thin-layer chromatography (TLC).
Sigma-Aldrich aluminum oxide (activity II−III, according to
Brockmann) 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 standard).
The values of the chemical shifts are expressed in ppm and the
coupling constants (J) in hertz.
HPLC Analysis. The purity of the title compounds was assessed by
means of a Waters Alliance 2695 instrument equipped with an UV−vis
Waters PDA 996 as the detector and working at 333 nm. Millennium
Empower with Windows XP was used. A Phenomenex LUNA C8,
5 μm (150 mm × 4.6 mm) column (code 00F-4249-E0) at 40 °C was
used as the chromatographic column at a flow rate of 1.0 mL/min. The
eluent was (A) a 65/15/20 (v/v/v) 10 mM (pH 2.5) KH2PO4/
MeOH/acetonitrile mixture or (B) a 15/75/10 (v/v/v) 10 mM (pH
2.5) KH2PO4/MeOH/acetonitrile mixture. A gradient from 100% A to
100% B was run in 25 min, and then isocratic conditions at 100% B
were maintained for an additional 10 min. Using the analytical
conditions reported above, the retention times of the compounds
range between 7.44 and 17.25 min. All the synthesized compounds
were generally more than 95% pure.
1-[4-(Methylthio)phenyl]pentane-1,4-dione (5). 4-Methyl-
thiobenzaldehyde (11.97 mL, 90 mmol), triethylamine (19.5 mL,
600 mmol), methyl vinyl ketone (5.8 mL, 90 mmol), and 3-ethyl-5-(2-
hydroxyethyl)-4-methylthiazolium bromide (14 mmol) were put into a
round-bottomed flask equipped with a stir bar. The flask was inserted
into the cavity of the microwave system apparatus, and the reaction
was run for 15 min (150 W, 70 °C, 170 psi).27 The residue obtained
was treated with 2 N HCl (10 mL) until pH 2 was reached. After the
extraction with ethyl acetate, the organic layer was washed with
aqueous sodium bicarbonate and water. The organic fractions were
dried over Na2SO4, filtered, and concentrated to give a crude orange
liquid. After crystallization from cyclohexane, intermediate 5 was
isolated as white needles (78% yield). Physicochemical, spectroscopic,
and analytical data were consistent with those reported in the
literature.25−31
1-[4-(Methylsulfonyl)phenyl]pentane-1,4-dione (6). To a
solution of 5 (35 mmol) in methanol (250 mL), a solution of
Oxone (61.4 mmol) in water (150 mL) was added over a period
15 min. The reaction mixture was stirred for 2 h at room temperature,
then diluted with water (400 mL) and extracted with dichloromethane.
The organic layer was washed with brine (200 mL), water (200 mL)
and dried over Na2SO4. After filtration and concentration under
reduced pressure, compound 6 was obtained as a white solid (>92%
yield). Physicochemical, spectroscopic, and analytical data were
consistent with those reported in the literature.25−31
Ethyl 2-[1-[(4-Methylthio)phenyl]-2-methyl-5-[4-
(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]glyoxylate (8d). Yel-
1
lowish needles, mp 105−110 °C (yield 77%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.60 (d, 2H, J = 8.6 Hz), 7.25 (d, 2H, J = 8.5 Hz),
7.17 (d, 2H, J = 8.6 Hz), 7.03−6.99 (m, 3H,), 4.10 (q, 2H, J = 7.0 Hz),
3.00 (s, 3H), 2.54 (s, 3H), 2.09 (s, 3H), 1.55 (t, 3H, J = 7.0 Hz). MS-
ESI: m/z 480 (M + Na+).
General Procedure for the Preparation of Ethyl 1,5-
Diarylpyrrole-3-acetic Esters (9a−e). To a solution of the suitable
1,5-diarylpyrrol-3-glyoxylic ester (8a−e) (2.3 mmol) in TFA (9 mL,
0.12 mol), stirred at 0 °C under nitrogen atmosphere, triethylsilane
(0.75 mL, 4.7 mmol) was slowly added, and the mixture was reacted
for 2 h at room temperature. Then the mixture was made alkaline (pH
12) with 40% aqueous ammonia and extracted with chloroform. The
organic solution was dried over Na2SO4, filtered, and evaporated in
vacuo. The resulting residue was purified by chromatography on silica
gel using petroleum ether/chloroform/ethyl acetate, 4:3:1 (v/v/v), as
the eluent to give a solid which, after recrystallization from ethanol,
afforded the desired product.
Ethyl 2-[1-[(4-Methylthio)phenyl]-2-methyl-5-[4-
(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate (9d). Yellowish
needles, mp 123−127 °C (yield 79%). 1H NMR (400 MHz, CDCl3) δ
(ppm): 7.60 (d, 2H, J = 8.6 Hz), 7.25 (d, 2H, J = 8.5 Hz), 7.17 (d, 2H,
J = 8.6 Hz), 6.99 (d, 2H, J = 8.5 Hz), 6.46 (s, 1H), 4.10 (q, 2H, J = 7.0
Hz), 3.51 (s, 2H), 3.00 (s, 3H), 2.54 (s, 3H), 2.09 (s, 3H), 1.55 (t, 3H,
J = 7.0 Hz). MS-ESI: m/z 466 (M + Na+).
General Procedure for the Preparation of 1,5-Diarylpyrrole-
3-acetic Acids (10a−e). To a solution of the appropriate ethyl 1,5-
diarylpyrrole-3-acetic ester (9a−e) (0.67 mmol) in methanol (4.83
mL) was added 4.83 mL of 1 N NaOH. The resulting mixture was
refluxed for 1.5 h, cooled, and concentrated in vacuo. The residue was
solubilized in water and then acidified with concentrated HCl. The
precipitate was filtered off to give the expected acid as a white solid.
2-[1-[(4-Methylthio)phenyl]-2-methyl-5-[4-(methylsulfonyl)-
phenyl]-1H-pyrrol-3yl]acetic Acid (10d). White needles, mp
1
147−152 °C (yield >95%). H NMR (400 MHz, CDCl3) δ (ppm):
11.00 (br s, 1H), 7.60 (d, 2H, J = 8.6 Hz), 7.25 (d, 2H, J = 8.5 Hz),
7.17 (d, 2H, J = 8.6 Hz), 7.00 (d, 2H, J = 8.5 Hz), 6.49 (s, 1H), 3.02
(s, 3H), 2.55 (s, 3H), 2.10 (s, 3H). MS-ESI: m/z 416 (M + H+).
General Procedure for the Preparation of 1,5-Diarylpyrrole-
3-acetic Nitroxyalkyl and Hydroxylalkyl Esters (11a−j, 12a−j). To
a solution of the opportune 1,5-diarylpyrrole-3-acetic acid 10a−e
(0.1 mmol) in dichloromethane (5 mL), under nitrogen atmosphere,
an excess of alcohol [(13a−c, 14a−c) 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 N HCl, NaHCO3 saturated solution,
brine and dried over Na2SO4. 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.
General Procedure for the Preparation of 1,5-Diarylpyrroles
(7a−e). Following the Paal−Knorr condensation conditions, com-
pound 6 (2.28 mmol) was dissolved in ethanol (2 mL) in a round-
bottomed flask equipped with a stir bar. Corresponding aniline (2.28
mmol) and p-toluenesulfonic acid (0.17 mmol) were added. The flask
was inserted into the cavity of the microwave system apparatus and
heated (150 W for 45 min, temperature 160 °C, pressure 150 psi).27
The reaction mixture was cooled to room temperature and
subsequently concentrated. The crude material was purified by
chromatography on aluminum oxide using cyclohexane/ethyl acetate,
3:1 (v/v), as the eluent to give the expected 1,5-diarylpyrroles 7a−e as
solids in satisfactory yields.
2-(Nitroxy)ethyl 2-[1-[(4-Methylthio)phenyl]-2-methyl-5-[4-
(methylsulfonyl)phenyl]-1H-pyrrol-3yl]acetate (11g). Yellow
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powder, mp 118−123 °C (yield 70%). FT-IR: 1738 cm−1 (νC−O),
1280 cm−1 (νasC−O−C), 1144 cm−1 (νsC−O−C), 1625 cm−1 (νasO-
NO2), 843 cm−1 (νsO-NO2). 1H NMR (400 MHz, CDCl3) δ (ppm):
7.80 (d, 2H, J = 8.6 Hz), 7.24 (d, 2H, J = 8.5 Hz), 7.17 (d, 2H, J = 8.6
Hz), 6.99 (d, 2H, J = 8.5 Hz), 6.51 (s, 1H), 4.73 (t, 2H, J = 6.3 Hz),
4.27 (m, 2H, J = 6.3 Hz), 3.50 (s, 2H), 3.00 (s, 3H), 2.55 (s, 2H), 2.10
(s, 3H). 13C NMR (400 MHz, CDCl3) δ (ppm): 10.89 (CH3
pyrrolic), 29.79 (CH3−S), 32.21 (CH2), 44.60 (CH3 sulfonyl),
60.41 (OCH2), 69.90 (CH2ONO2), 112.96, 113.50, 115.89, 120.67,
125.39, 127.00, 130.59, 130.70, 131.30, 131.80, 136.10, 138.00,
177.03 (COO). MS-ESI: m/z 505 (M + H+). HPLC: >98% pure
(tR = 14.19 min).
2-Hydroxyethyl 2-[1-(4-Methylthiophenyl)-2-methyl-5-[4-
(methylsulfonyl)phenyl]-1H-pyrrol-3-yl]acetate (12g). Yellow
powder, mp 110−112 °C (yield 55%). 1H NMR (400 MHz,
CDCl3) δ (ppm): 7.80 (d, 2H, J = 8.6 Hz), 7.24 (d, 2H, J = 8.5
Hz), 7.17 (d, 2H, J = 8.6 Hz), 7.00 (d, 2H, J = 8.5 Hz), 6.48 (s, 1H),
4.25 (t, 2H, J = 6.8 Hz), 3.89 (t, 2H, J = 6.8 Hz), 3.52 (s, 2H), 2.55 (s,
3H), 2.07 (s, 3H), 2.01 (br s, 1H). 13C NMR (400 MHz, CDCl3) δ
(ppm): 10.89 (CH3 pyrrolic), 29.79 (CH3−S), 32.20 (CH2), 44.54
(CH3 sulfonyl), 60.40 (OCH2), 66.29 (CH2OH), 112.96, 113.50,
115.90, 120.67, 125.39, 127.01, 130.63, 130.71, 131.27, 131.80, 136.10,
138.00, 177.00 (COO). MS-ESI: m/z 460 (M + H+). HPLC: >97%
pure (tR = 9.32 min.)
General Procedure for the Preparation of Hydroxylalkyl
Nitrates (14a,b). To a solution of diol (13a,b, 20 mmol) in ethyl
acetate (100 mL) were added nitric acid (65% w/v, 16 mmol), glacial
acetic acid (97 mmol), and acetic anhydride (97% w/v, 59 mmol). The
mixture was allowed to react overnight. The reaction was quenched
with 20% KOH solution. The pH was adjusted to 12−13, and the
acqueous phase was extracted with ethyl acetate (3 × 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.
performed using the sigmoidal dose−response equation (variable
slope) (GraphPad).
Ex Vivo Vasorelaxing Activity. 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 anesthesia, 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 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)
thermostated 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 to a computerized
system (Biopac). After an equilibration period of 60 min, the
endothelium removal was confirmed by the administration of
acetylcholine (ACh) (10 μM) 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 ≥10% (i.e., significant
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 increasing concentrations
of compounds (1nM to 10 μM) 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 μM
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 concentration of 10 μM (the highest concentration that could be
administered) 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 logarithm 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 6−10 experiments. Two-way ANOVA
was used for statistical analysis, and P < 0.05 was considered
representative of significant statistical differences. Experimental data
were analyzed by a computer fitting procedure (software: GraphPad
Prism, version 4.0).
In Vitro Human Whole Blood (HWB) Assay. Compounds 11c and
12c were also evaluated for COX-1 versus COX-2 selectivity in HWB
assay. Three healthy volunteers (two females and one male, aged 29 ±
3 years) were enrolled to participate in the study after its approval by
the Ethical Committee of the University of Chieti, Italy. Informed
consent was obtained from each subject. Compounds 11c (0.05−50
mM) and 12c (0.05−50 mM) were dissolved in DMSO. Aliquots of
the solutions (2 μL) or vehicle were pipetted directly into test tubes to
give final concentrations of 0.1−100 μM in whole blood samples. To
evaluate COX-2 activity, 1 mL aliquots of peripheral venous blood
samples containing 10 IU of sodium heparin were incubated in the
presence of LPS (10 μg/mL) or saline for 24 h at 37 °C, as previously
described.40 The contribution of platelet COX-1 was suppressed by
pretreating the subjects with aspirin (300 mg, 48 h) before sampling.
Plasma was separated by centrifugation (10 min at 2000 rpm) and
kept at −80 °C until assayed for PGE2 as an index of monocyte COX-
2 activity. Moreover, peripheral venous blood samples were drawn
from the same donors when they had not taken any NSAID during the
2 weeks preceding the study. Aliquots (1 mL) of whole blood were
immediately transferred into glass tubes and allowed to clot at 37 °C
2-Nitroxyethanol (14a). Yellowish oil (yield 70%). FT-IR: 1623
cm−1 (νasO-NO2), 850 cm−1 (νsO-NO2).1H NMR: 4.67 (t, 2H, J =
6.9 Hz), 4.20 (t, 2H, J = 6.9 Hz), 2.10 (br s, 1H). MS-ESI: m/z 108
(M + H+).
Pharmacology. In Vitro Anti-Inflammatory Study. The in vitro
profiles of compounds 11a−j and 12a−j, related to their inhibitory
activity toward 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, 25 mM HEPES, 100 units/mL penicillin, 100 μg/mL
streptomycin, 10% fetal bovine serum (FBS), and 1.2% sodium
pyruvate. Cells were plated in 24-well culture plates at a density of 2.5
× 105 cells/mL or in 60 mm diameter culture dishes (3 × 106 cells per
3 mL per dish) and allowed to adhere at 37 °C in 5% CO2 for 2 h.
Immediately before the experiments, the culture medium was replaced
with fresh medium and cells were stimulated as described previously.39
The evaluation of COX-1 inhibitory activity was achieved by
pretreating cells with test compounds (10 μM) for 15 min and then
incubating them at 37 °C for 30 min with 15 μM arachidonic acid to
activate the constitutive COX. For the compounds with COX-1
inhibition higher than 50% (at 10 μM), the cells were also treated with
lower concentrations (0.01−1 μM). At the end of the incubation, 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 Escherichia coli
lipopolysaccharide (LPS, 10 μg/mL) to induce COX-2, in the absence
or presence of test compounds (0.01−10 μM). Celecoxib was utilized
as a reference compound 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 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
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Article
for 1 h. Serum was separated by centrifugation (10 min at 3000 rpm)
and kept at −80 °C until assayed for TXB2. Whole blood TXB2
production was measured as a reflection of maximally platelet COX-1
activity in response to endogenously formed thrombin.41
Analysis of PGE2 and TXB2. PGE2 and TXB2 concentrations were
measured by previously described and validated radioimmuno-
assays.40,41 Unextracted plasma and serum samples were diluted in
the standard diluent of the assay (0.02 M phosphate buffer, pH 7.4)
and assayed in a volume of 1.5 mL at a final dilution of 1:50 to
1:30000. [3H]PGE2 or [3H]TBX2 (3000 cpm, specific activity of >100
Ci/mmol, 1:100000 dilution) and anti-TBX2 (1:120000 dilution) sera
were used. The least detectable concentration was 1−2 pg/mL for
both prostanoids.40,41
compound 11c. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 06 4991 3812. Fax: +39 06 4991 3133. E-mail:
mariangela.biava@uniroma1.it.
■
ACKNOWLEDGMENTS
We thank Rottapharm Madaus (Monza, Italy) for financial
support of this research.
■
In Vivo Analgesic and Anti-Inflammatory Study. In vivo anti-
inflammatory activity of the new compounds was also assessed. Male
Swiss albino mice (23−25 g) and Sprague−Dawley 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. The carrageenan-
induced paw edema 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 movements was counted for 10 min, starting 5 min after
administration.
Computational Details. Inhibitor Setup. The structures of the
inhibitors used in the docking simulations were generated by means of
Extensible Computational Chemistry Environment (ECCE) soft-
ware42 and then geometry optimized by means of NWChem (100
steps of RHF, 6-31G*). Partial atomic charges RESP were calculated
by means of the NWChem and then used in the following docking
simulations.43 All the relevant torsion angles were treated as rotatable
during the docking process, thus allowing a search of the conforma-
tional space.
Enzyme Setup. The COX-2 protein was set up for docking as
follows: polar hydrogens were added by means of ECCE software, and
Kollman united-atom partial charges were assigned.44 The ADDSOL
utility of AutoDock was used to add solvation parameters to the
protein structures, and the grid maps representing the proteins in the
docking process were calculated using AutoGrid. The grids, one for
each atom type in the inhibitor plus one for the electrostatic
interactions, were chosen to be large enough to include not only the
cyclooxygenase sites but also a significant part of the protein around it.
As a consequence, the dimensions of grid maps were 59 × 45 × 59
points with a grid point spacing of 0.375 Å for COX-2 for all docking
calculations.
Docking Calculations. Compounds 11c,d and 12c,d were docked
into the enzymes using AutoDock, version 4.0. Docking simulations of
the compounds were carried out using the Lamarckian genetic
algorithm and through a protocol with an initial population of 300
randomly placed individuals, a maximum number of 25 million energy
evaluations, a mutation rate of 0.02, a crossover rate of 0.80, and an
elitism value of 1. The pseudo Solis and Wets algorithm with a
maximum of 300 interactions was applied for the local search. Two-
hundred independent docking runs were carried out for each inhibitor,
and the resulting conformations that differed by less than 2.0 Å in
positional root-mean-square deviation (rmsd) were clustered together.
Cluster analysis was performed by selecting the most populated
cluster, which in all cases coincided with the one endowed with the
best energy.
ABBREVIATIONS USED
■
t-NSAID, traditional nonsteroidal anti-inflammatory drug;
COXIB, cyclooxygenase-2 inhibitor; COX, cyclooxygenase;
GI, gastrointestinal; NO, nitric oxide; AA, arachidonic acid;
GC, guanylate cyclase; c-GMP, cyclic guanosine mono-
phosphate; CV, cardiovascular; CINOD, cyclooxygenase-
inhibiting nitric oxide donor; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; DMAP, dimethylami-
nopyridine; Ach, acetylcholine; DMEM, Dulbecco’s modi-
fied Eagle’s medium; PGE2, prostaglandin E2; FBS, fetal
bovine serum; LPS, lipopolysaccharide; RIA, radioimmuno-
assay; TX, thromboxane; CMC, carboxymethylcellulose;
ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ipl, in-
traplantar:; po, per os; ip, intraperitoneal; GTN, glyceryl
trinitrate; Emax, maximal vasorelaxing response
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