Nitrooxymethyl-substituted analogues of celecoxib: Synthesis and pharmacological characterization

Article abstract of DOI:10.1002/cbdv.200800307

Nitrooxymethyl-substituted analogues of celecoxib were synthesized and tested for their cyclooxygenase (COX)-inhibiting, vasodilator, and anti-aggregatory activities, as well as for their metabolic stability in human serum and whole blood. The results showed their potency and selectivity in inhibiting the COX isoforms, evaluated in whole human blood, as well as their anti-aggregatory activity to depend closely on the position at which the NO-donor moiety is introduced. All products dilated rat aorta strips precontracted with phenylephrine in a dose-dependent manner through a cGMP-dependent mechanism. They were stable in human serum while, in blood, they were metabolically transformed, principally to the related alcohols.

Full text of DOI:10.1002/cbdv.200800307

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
369
Nitrooxymethyl-Substituted Analogues of Celecoxib: Synthesis and
Pharmacological Characterization
by Donatella Boschi, Loretta Lazzarato, Barbara Rolando, Andrea Filieri, Clara Cena, Antonella Di
Stilo, Roberta Fruttero, and Alberto Gasco*
`
Dipartimento di Scienza e Tecnologia del Farmaco, Universita degli Studi di Torino, Via Pietro Giuria 9,
I-10125 Torino (phone: þ390116707670; fax: þ390116707286; e-mail: alberto.gasco@unito.it)
Nitrooxymethyl-substituted analogues of celecoxib were synthesized and tested for their cyclo-
oxygenase (COX)-inhibiting, vasodilator, and anti-aggregatory activities, as well as for their metabolic
stability in human serum and whole blood. The results showed their potency and selectivity in inhibiting
the COX isoforms, evaluated in whole human blood, as well as their anti-aggregatory activity to depend
closely on the position at which the NO-donor moiety is introduced. All products dilated rat aorta strips
precontracted with phenylephrine in a dose-dependent manner through a cGMP-dependent mechanism.
They were stable in human serum while, in blood, they were metabolically transformed, principally to the
related alcohols.
Introduction. – Cyclooxygenase (COX) is one of the key enzymes implicated in the
transformation of arachidonic acid into prostanoids. It exists in at least two isoforms,
COX-1 and COX-2. The former is prevalently a constitutive and the latter an inducible
isoform. Selective blocking of these isoforms induces specific pharmacological effects
which can be exploited in therapy [1]. COX-2-Selective inhibitors (Coxibs) are a new
class of drugs recently introduced into the market [2]; they induce anti-inflammatory
effects without the gastrotoxic side-effects typical of the classical non-steroidal anti-
inflammatory drugs (NSAIDs), which are nonselective COX inhibitors. An interesting
aspect of these drugs is their potential use in treating Alzheimerꢀs disease and certain
forms of cancer [3][4]. The drawback in their use is that they increase the risk of heart
attack and stroke [5][6]. Blood vessels and platelets are the major targets of
prostanoids such as prostacycline PGI2, prostaglandin PGE2, and thromboxane TxA2
in the cardiovascular system. The proaggregatory and vasoconstrictor TxA2 is mainly
synthesized via COX-1 in the platelets, while vasodilator (PGI2, PGE2) and antiplatelet
(PGI2) compounds are synthesized mainly in the vascular endothelium. A strategy to
improve the benefit–risk profiles of these drugs is to design a multi-target drug by
combining COX-2-selective inhibition with nitric oxide (NO)-dependent activities. NO
displays a variety of effects in the cardiovascular system, including vasodilation,
inhibition of platelet aggregation, modulation of platelet and leukocytes adherence to
vessels, and inhibition of smooth muscle cell proliferation [7]. Examples of this type of
approach have already been reported [8][9] including by our group [10][11]. As a
development of our work in this field, we now describe the synthesis and structural
characterization of a new series of NO-donor COX-2 inhibitors obtained by
introducing NO-donor nitrooxy functions into the well-known selective COX-2
ꢁ 2009 Verlag Helvetica Chimica Acta AG, Zꢂrich

370
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
inhibitor celecoxib (1) [12] that has recently been placed under surveillance by FDA,
following its suspected cardiotoxicity.
The results of a preliminary pharmacological screening of these new hybrid
products and of their alcoholic metabolites show that their potency and selectivity in
inhibiting the COX isoforms, evaluated in whole human blood, is closely dependent on
the position at which the NO-donor moiety is introduced. This aspect is discussed, as
well as the NO-dependent ability of the products to relax rat aorta strips precontracted
with phenylephrine and, for selected compounds, their capacity to inhibit collagen-
induced platelet aggregation of human platelet rich plasma (PRP).
Results and Discussion. – 1. Chemistry. The general strategy for the synthesis of the
nitrooxy substituted celecoxib derivatives 7, 11, and 15 is outlined in Schemes 1 and 2.
The trifluoromethyl b-diketone 4 was obtained by refluxing 4-(hydroxymethyl)aceto-
phenone (2) and ethyl trifluoroacetate in MeOH in the presence of MeONa. The
intermediate 4 was isolated but not purified, since it was immediately condensed in
refluxing EtOH with the hydrochloride of the phenylhydrazine derivative 5 to give,
with a fair yield, the 1,5-diphenyl-1H-pyrazole derivative 6, accompanied by a small
amount of the 1,3-diphenyl isomer. This is in keeping with reports that 1,5-diaryl-
pyrazole derivatives are almost exclusively produced, when the hydrochloride salt of
the phenylhydrazine is condensed with trifluoromethyl b-diketones in refluxing EtOH
or MeOH [13]. Treatment of 6 dissolved in MeCN with N-bromosuccinimide (NBS) in
the presence of Ph3P and AgNO3 afforded a crude product that was purified by flash
chromatography to give the expected final compound 7, mononitrooxymethyl-
substituted at the 5-phenyl group. When 4 underwent reaction with the hydrochloride
salt of phenylhydrazine derivative 8, it produced the pyrazolylbenzoate 9 in a highly
regiospecific manner. Reduction of the ester function of 9 with LiAlH4 yielded the
dimethanol 10, which was transformed into the final dinitrooxy derivative 11 according
to the same procedure used to prepare 7 from 6. This reaction was accompanied by the
formation of small amounts of a mixture of the two isomeric mononitrooxy alcohols 11a
and 11b in a ratio of ca. 1:1 (NMR detection). When 8 was allowed to react in refluxing
MeOH with the trifluoromethyl b-diketone 12, obtained from the commercially
available 4-methylacetophenone (3), the 1,5-diphenylpyrazole derivative 13 was
yielded in a moderately regiospecific manner. This product was separated by flash
chromatography from a minor amount of 1,3-diphenyl isomer and transformed into the
final mononitrooxy derivative 15, through the intermediate formation of the alcohol 14,
following the sequence of reactions used to prepare 11 from 9.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
371
Scheme 1
i) MeONa, CF3COOEt, MeOH, reflux. ii) Refluxing in MeOH (in EtOH for 6). iii) LiAlH4, THF.
2. Stability in Human Serum and in Human Whole Blood. It is known that nitrates
can undergo chemical and enzymatic metabolism [14]. Celecoxib (1) and its derivatives
were found to be stable following 24 h incubation in human serum. By contrast,
derivatives 7, 11, and 15 were extensively metabolized when incubated in whole human
blood. This transformation was monitored by RP-HPLC. The half-lives (t1/2) are
reported in Table 1. Products 11 and 15 behaved similarly, while 7 surprisingly did not:
its t1/2 is ca. 1 h, vs. 25–30 h of the other compounds, and after 24 h only 4.5% of the
original product remained unchanged. Compounds 11 and 15 were principally

372
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Scheme 2
i) Ph3P, AgNO3, N-bromosuccinimide (NBS), ꢀ158!r.t.
transformed into the corresponding alcohols 10, 11a/11b and 14, respectively (Table 1).
These metabolites were identified by comparison of their retention times with those of
alcohols 10 and 14, which were used as intermediates to prepare the final compounds.
Similarly, the mixture 11a/11b was used as reference to identify the two isomeric
mononitrooxy alcohols that derive from the biotransformation of 11. The behavior of
Table 1. Whole Human Blood Stability
Compound
% of unchanged compound
% of alcoholic metabolites
t1/2 [h]
after 24 h (ꢁ SEM)
after 24 h (ꢁ SEM)
1
100
7
11
4.5ꢁ1.1
4.5ꢁ0.5 (6)
0.8
30.0
59.4ꢁ4.4
16.2ꢁ0.7 of 11a/11b
11.7ꢁ1.0 of 10
41.2ꢁ1.7 (14)
15
54.4ꢁ2.3
25.4

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
373
compound 7 again differed: HPLC analysis, using the alcohol 6 as standard, revealed
only 4.5% of this product as detectable metabolite. This indicates that the other
metabolites formed are not free in the cytosol, but they are sequestrated by
components of the blood compartment.
3. COX Inhibition. The ability of the products and of their alcoholic metabolites to
inhibit the COX enzymes was evaluated in human whole blood according to an
established procedure [15]. To assay the extent of COX-2 isoform inhibition, human
heparinized whole blood was incubated with lipopolysaccharide (LPS) overnight in the
presence of the inhibitors, and plasma reduction of PGE2 production in comparison
with control samples was assayed as a function of COX-2 inhibition. For COX-1
inhibition, whole blood samples without any anticoagulant were incubated with the
inhibitors for 1 h; serum was then collected and analyzed for TxB2 production. The
potencies of the products are expressed as IC50 (Table 2); when this could not be
calculated because inhibition did not reach 50%, inhibition at the maximum
concentration tested (100 mm) is reported.
Table 2. COX-1 and COX-2 Inhibition Data for Compounds and Celecoxib (1) Taken as Reference
Compound R1
R2
COX-1
COX-2
IC50 ꢁSE [mm] % InhibitionꢁSE (100 mma)) IC50 ꢁSE [mm]
1
6
7
10
11
14
15
SO2NH2
Me
14ꢁ2
–
1.3ꢁ0.4
b
SO2NH2
SO2NH2
CH2OH
CH2ONO2 CH2ONO2
CH2OH Me
CH2ONO2 Me
CH2OH
CH2ONO2
CH2OH
)
20ꢁ13
52ꢁ3
b
)
6.7ꢁ3.8
67ꢁ19
12ꢁ3
0.51ꢁ0.14
–
b
)
15ꢁ7
19ꢁ1
0.41ꢁ0.08
–
–
11ꢁ3
4.6ꢁ1.6
31 ꢁ10
a) Maximum concentration tested. b) Inhibition of control TxB2 production did not reach 50%.
The data show that product 7, derived from substituting the nitrooxymethyl function
for the methyl group of celecoxib (1), is a feeble COX-2 inhibitor, being ca. 50 times
less potent than the lead, but that it retains a good degree of COX-2 selectivity: it
displayed negligible COX-1 activity when tested at 100 mm concentration. Its alcoholic
metabolite 6 behaves similarly. When it is the aminosulfonyl group that is substituted,
which is an important determinant for the COX-2 selectivity of 1 [16], 15 is obtained,
which is a more potent COX-1 inhibitor than 1, and which displays a rather good COX-
1 selectivity. COX-1 Inhibition potency and selectivity are even more evident in its
alcoholic metabolite 14. Finally, the simultaneous substitution of both the Me and the

374
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
aminosulfonyl moiety in 1 provides 11, which is a fairly potent COX-2 inhibitor, with
negligible COX-1 activity when tested at a concentration of 100 mm. By contrast, its
dihydroxy metabolite 10 shows a COX-inhibitory profile very close to that of 14. All
compounds were also tested for their ability to inhibit collagen-induced platelet
aggregation of human platelet rich plasma (PRP). The products 10 and 14 display
antiplatelet action in the mm range (Table 3) in keeping with their ability to inhibit the
COX-1 isoform. Also the activity of the NO donor 15 is essentially COX-1- and not
NO-dependent, in agreement with the reduced ability of platelets to effect NO release
from organic nitrates [17]. Indeed, the anti-aggregatory potency of this compound was
not modified by the presence of ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one),
a well-known inhibitor of the soluble guanylate cyclase (sGC).
Table 3. Anti-Aggregatory and Vasodilating Properties of Derivatives
Compound R1
R2
Anti-aggregatory activity
Vasodilating activity
IC50 (95% CL) % inhibitionꢁSE EC50 ꢁSE [mm]
[mm]
(100 mma))
1
6
7
SO2NH2
SO2NH2
SO2NH2
Me
CH2OH
CH2ONO2
31 (25–39)
–
–
–
b
)
)
5.8ꢁ2.1
b
3.4ꢁ2.6
0.26ꢁ0.05
7.0ꢁ1.3c)
–
0.10ꢁ0.03
26ꢁ9c)
–
0.12ꢁ0.01
>100c)
4.7ꢁ0.6
>100c)
10
11
CH2OH
CH2ONO2 CH2ONO2
CH2OH
8.4 (7.0–10)
)
–
b
7.4ꢁ4.3
14
15
CH2OH
CH2ONO2 Me
Me
11 (10–12)
–
–
48 (44–52)d)
ISDNe)
a) Maximum concentration tested. b) Inhibition of control aggregation effect did not reach 50%. c) In the
presence of 1 mm ODQ. d) Unchanged in the presence of 100 mm ODQ. e) Isosorbide dinitrate.
4. Vasodilation. The in vitro vasodilating activities of this new series of COX
inhibitors was assessed on rat aorta strips precontracted with phenylephrine. All
products relaxed the contracted tissues in a concentration-dependent manner. Their
potencies, expressed as EC50, are shown in Table 3. All act as potent vasodilators, their
potencies being closely distributed in the submicromolar range. Vasodilating potencies
markedly decreased, when the experiments were repeated in the presence of 1 mm

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
375
ODQ. This is typical of the classical nitrooxy-containing compounds, such as isosorbide
dinitrate (ISDN), which was used as reference.
Conclusions. – Introduction at the para-position of the phenyl rings present in
celecoxib (1) of either the nitrooxymethyl group or the hydroxymethyl group, deriving
from its metabolic transformation, gives rise to a complex behavior. When these
moieties are linked to the N-phenyl ring, the selective and potent COX-1 inhibitors 14
and 15, endowed with anti-aggregatory activity, are obtained. By contrast, when they
are attached to the C-phenyl group, the selective but weak COX-2 inhibitors 6 and 7 are
generated. Interesting results occurred following the simultaneous introduction of
either two nitrooxy or two hydroxymethyl functions on the two phenyl groups. In the
former case, the fairly potent and selective COX-2 inhibitor 11, endowed with a feeble
anti-aggregatory activity, was obtained, while, in the latter case, compound 10 was
obtained, which is a potent and selective COX-1 inhibitor endowed with a high anti-
aggregatory activity. All of the products containing nitrooxy moieties displayed NO-
dependent vasodilatory activity. Compound 11 emerges as the most interesting product
arising from these chemical manipulations of compound 1, in terms of the possibility of
producing a COX-2-selective inhibitor potentially possessing low cardiotoxicity.
This work was supported by a MIUR grant (COFIN 2005).
Experimental Part
General. Compounds 2 [18], 5 [19], 8 [20], and 12 [12] were synthesized according to the methods
described in the literature. THF was distilled immediately before use from Na and benzophenone. Anh.
MgSO4 was used as drying agent for the org. phases. Flash column chromatography (FC): silica gel
(Merck Kieselgel 60, 230–400 mesh ASTM) with the eluents indicated. TLC: 5ꢂ20 cm plates with 0.25-
mm layer thickness. M.p.: cap. apparatus (Bꢀchi 540); m.p. with decomposition were determined after
placing the sample in a bath at a temp. 108 below the m.p.; a heating rate of 18 minꢀ1 was used. FT-IR:
1
Perkin-Elmer SPECTRUM BXII. H- and 13C-NMR: Bruker Avance-300. MS: Finnigan-Mat TSQ-700.
Analysis (C, H, N) of the new compounds was performed by REDOX (Monza) and the results are within
ꢁ0.4% of the theoretical values.
4,4,4-Trifluoro-1-[4-(hydroxymethyl)phenyl]butane-1,3-dione (4). 1.5m MeONa in MeOH (5 ml,
7.5 mmol) was added to a soln. of 4-(hydroxymethyl)acetophenone (2) (0.72 g, 4.8 mmol) in dry MeOH
(4 ml). The mixture was stirred for 5 min, after which ethyl trifluoroacetate (0.7 ml, 7.0 mmol) was added.
After refluxing for 24 h, the mixture was cooled to r.t. and concentrated in vacuo; 10% HCl (10 ml) was
added, and the mixture was extracted with AcOEt. The extracts were dried and concentrated to afford 4
as a brown oil (0.99 g), which was used without further purification.
4-{5-[4-(Hydroxymethyl)phenyl]-3-(trifluoromethyl)-1H-pyrazol-1-yl}benzenesulfonamide (6). (4-
Sulfamoylphenyl)hydrazine hydrochloride (5; 0.93 g, 4.2 mmol) was added to a stirred soln. of 4 (0.99 g)
in EtOH (50 ml). The mixture was heated to reflux for 24 h. After cooling to r.t., the mixture was
concentrated in vacuo, the residue was taken up in AcOEt, washed with H2O and brine, dried, and
concentrated in vacuo to give an oil that was purified by FC (petroleum ether (PE)/i-PrOH 9 :1) to give 6
1
(0.95 g, 57%). Pale yellow solid. M.p. 157–1588 (from benzene). H-NMR (CDCl3): 7.84 (d, J¼8.6, 2
arom. H); 7.41 (d, J¼8.6, 2 arom. H); 7.34 (d, J¼8.1, 2 arom. H); 7.20 (d, J¼8.1, 2 arom. H); 6.76 (s, CH
(pyrazole)); 5.31 (s, NH2); 4.69 (s, CH2); 2.06 (br. s, OH). 13C-NMR (CDCl3): 144.9; 144.2 (q, 2J¼38);
1
142.4; 142.3; 141.5; 129.0; 127.7; 127.5; 127.4; 125.6; 121.0 (q, J¼268); 106.6 (q, 3J¼2); 64.5. CI-MS (i-
Bu): 398 ([Mþ1]þ ). Anal. calc. for C17H14F3N3O3S (397.38): C 51.38, H 3.55, N 10.57; found C 51.68, H
3.66, N 10.52.

376
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Methyl 4-{5-[4-(Hydroxymethyl)phenyl]-3-(trifluoromethyl)-1H-pyrazol-1-yl}benzoate (9). [4-(Me-
thoxycarbonyl)phenyl]hydrazine hydrochloride (8; 0.84 g, 4.2 mmol) was added to a stirred soln. of 4
(0.99 g) in MeOH (40 ml). The mixture was heated to reflux for 3 h. After cooling to r.t., the mixture was
concentrated in vacuo, the residue was taken up in AcOEt, washed with H2O and brine, dried, and
concentrated in vacuo to give an oil that was purified by FC (CH2Cl2/AcOEt 99 :1) to give 9 (0.73 g,
46%). Yellow solid. M.p. 95–978 (dec.; i-Pr2O). 1H-NMR (CDCl3): 8.02 (d, J¼8.6, 2 arom. H); 7.39 (d,
J¼8.6, 2 arom. H); 7.34 (d, J¼8.1, 2 arom. H); 7.21 (d, J¼8.1, 2 arom. H); 6.76 (s, CH (pyrazole)); 4.71 (s,
2
CH2); 3.92 (s, Me); 2.15 (br. s, OH). 13C-NMR (CDCl3): 166.1; 144.8; 143.9 (q, J¼38); 142.7; 142.3;
130.6; 130.0; 129.8; 128.0; 127.2; 125.0; 121.1 (q, 1J¼267); 106.3 (q, 3J¼2); 64.5; 52.4. CI-MS (i-Bu): 377
([Mþ1]þ ). Anal. calc. for C19H15F3N2O3 (376.34): C 60.64, H 4.02, N 7.44; found: C 60.87, H 4.40, N 7.40.
Methyl 4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzoate (13). Compound 8
(3.41 g, 17.0 mmol) was added to a stirred soln. of 12 (3.87 g, 17.0 mmol) in MeOH (100 ml). The
mixture was heated to reflux for 20 h. After cooling to r.t., the mixture was concentrated in vacuo, the
residue was taken up in AcOEt, washed with H2O and brine, dried, and concentrated in vacuo to give an
oil that was purified by FC (PE/AcOEt 97:3) to give 13 (2.06 g, 34%). White solid. M.p. 78.5–79.58
1
(hexane). H-NMR (CDCl3): 8.03 (d, J¼8.6, 2 arom. H); 7.39 (d, J¼8.6, 2 arom. H); 7.16–7.08 (m, 4
arom. H); 6.73 (s, CH (pyrazole)); 3.92 (s, MeO); 2.36 (br. s, Me). 13C-NMR (CDCl3): 166.2; 145.2; 143.9
(q, 2J¼38); 142.8; 139.6; 130.6; 129.8; 129.7; 128.8; 126.1; 125.1, 121.3 (q, 1J¼267); 106.1 (q, 3J¼2); 52.5;
21.4. EI-MS: 360 (100, Mþ ), 329 (50). Anal. calc. for C19H15F3N2O2 (360.34): C 63.33, H 4.20, N 7.77;
found: C 63.95, H 4.13, N 7.81.
4-{1-[4-(Hydroxymethyl)phenyl]-3-(trifluoromethyl)-1H-pyrazol-1-yl}phenylmethanol (10). A soln.
of 9 (1.26 g, 3.4 mmol) in dry THF (10 ml) was added dropwise to a stirred mixture of LiAlH4 (0.13 g,
3.4 mmol) in dry THF (10 ml) under N2. After 30 min, H2O (1 ml), NaOH 15% (1 ml), and H2O (1 ml)
were added sequentially to the mixture, after which the solid was filtered and washed with MeOH. The
filtrate was concentrated in vacuo, and the residue was taken up in AcOEt, washed with H2O and brine,
dried, and concentrated in vacuo to give an oil that was purified by FC (CH2Cl2/AcOEt 8 :2) to give 10
(0.70 g, 59%). White solid. M.p. 107–1118 (i-Pr2O). 1H-NMR (CDCl3): 7.35–7.26 (m, 6 arom. H); 7.21 (d,
J¼8.1, 2 arom. H); 6.74 (s, CH (pyrazole)); 4.70 (s, 2 CH2); 1.98 (br. s, OH); 1.90 (br. s, OH). 13C-NMR
(CDCl3): 144.5; 143.2 (q, 2J¼38), 141.8; 141.3; 138.3; 128.9; 128.3; 127.4; 127.1; 125.6; 121.2 (q, 1J¼267);
105.6 (q, 3J¼2); 64.6; 64.4. CI-MS (i-Bu): 349 (Mþ1)þ . Anal. calc. for C18H15F3N2O2 (348.32): C 62.07, H
4.34, N 8.04; found: C 62.45, H 4.42, N 7.94.
4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenylmethanol (14). A soln. of 13
(2.18 g, 6.0 mmol) in dry THF (25 ml) was added dropwise to a stirred mixture of LiAlH4 (0.23 g,
6.0 mmol) in dry THF (25 ml) under N2. After 30 min, H2O (1 ml), NaOH 15% (1 ml) and H2O (1 ml)
were added sequentially to the mixture, then the solid was filtered and washed with MeOH. The filtered
soln. was concentrated in vacuo, and the residue was taken up in AcOEt, washed with H2O and brine,
dried, and concentrated in vacuo to give a solid (1.91 g, 96%) that was purified by crystallization from
1
EtOH/H2O to give 14. White solid. M.p. 109–1118 (EtOH/H2O). H-NMR (CDCl3): 7.35–7.27 (m, 4
arom. H); 7.11–7.09 (m, 4 arom. H); 6.71 (s, CH (pyrazole)); 4.70 (s, CH2); 2.34 (s, Me); 1.93 (br. s, OH).
13C-NMR (CDCl3): 144.8; 143.2 (q, 2J¼38); 141.2; 139.1; 138.5; 129.4; 128.7; 127.3; 126.2; 125.5; 121.3 (q,
3
1J¼267); 105.3 (q, J¼2); 64.4, 21.3. EI-MS: 332 (100, Mþ ). Anal. calc. for C18H15F3N2O (332.32): C
65.06, H 4.55, N 8.43; found: C 65.12, H 4.53, N 8.33.
4-{1-[4-(Aminosulfonyl)phenyl]-3-(trifluoromethyl)-1H-pyrazol-5-yl}benzyl Nitrate (7). AgNO3
(1.00 g, 5.8 mmol) and Ph3P (0.75 g, 2.9 mmol) were added to a soln. of 6 (1.00 g, 2.5 mmol) in MeCN
(30 ml) under N2. The mixture was cooled to 08, and NBS (0.50 g, 2.8 mmol) was added portionwise.
Stirring was continued at 08 for 1 h and then at r.t. for 3 h. AcOEt was added to the mixture, and the solid
was filtered; the filtrate was washed with H2O, brine, dried, and concentrated in vacuo to give a solid that
was purified by FC (PE/AcOEt 7:3) to give 7 (0.76 g, 70%). White solid. 1H-NMR ((D6)DMSO): 7.89 (d,
J¼8.6, 2 arom. H); 7.58–7.51 (m, 4 arom. H, NH2); 7.39 (d, J¼8.3, 2 arom. H); 7.30 (s, CH of pyrazol);
5.60 (s, CH2). 13C-NMR ((D6)DMSO): 144.5; 144.5; 142.2 (q, 2J¼37); 140.9; 133.4; 129.5; 129.2; 129.0;
126.8; 126.0; 121.2 (q, 1J¼267); 106.7 (q, 3J¼2); 74.3. EI-MS: 442 (40, Mþ ), 397 (40), 302 (100). Anal.
calc. for C17H13F3N4O5S (442.38): C 46.16, H 2.96, N 12.66; found: C 45.96, H 2.89, N 12.29.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
377
4-{1-[4-(Nitrooxymethyl)phenyl]-3-(trifluoromethyl)-1H-pyrazol-5-yl}benzyl Nitrate (11). AgNO3
(1.03 g, 6 mmol) and Ph3P (1.31 g, 5 mmol) were added to a soln. of 10 (0.7 g, 2 mmol) in MeCN
(20 ml) under N2. The mixture was cooled to ꢀ158 under N2, and NBS (0.89 g, 5 mmol) was added
portionwise. Stirring was continued at ꢀ158 for 1 h and then at r.t. for 12 h. AcOEt was added to the
mixture, and the solid was filtered; the filtrate was washed with H2O, brine and dried, and concentrated in
vacuo to give an oil which was purified by FC (hexane/AcOEt 9 :1). The first eluted compound was 11
(0.42 g, 48%). White solid. M.p. 52–538 (i-Pr2O/PE). 1H-NMR (CDCl3): 7.43–7.25 (m, 8 arom. H); 6.78
(s, CH (pyrazole)); 5.44, 5.43 (2s, 2 CH2ONO2). 13C-NMR (CDCl3): 143.9; 143.7 (q, 2J¼38); 139.8;
133.4; 132.7; 130.0; 129.8; 129.4; 129.4; 125.7; 121.1 (q, 1J¼268); 106.3 (q, 3J¼2); 73.9; 73.6. EI-MS: 438
(30, Mþ ), 345 (48), 316 (100). Anal. calc. for C18H13F3N4O6 (438.32): C 49.32, H 2.99, N 12.78; found: C
49.38, H 2.95, N 12.56.
The second eluted fraction was the mixture of the two mononitrate products 11a and 11b, obtained as
1
a transparent oil. H-NMR (CDCl3): 7.41–7.20 (m, 16 arom. H); 6.75, 6.77 (2s, 2 CH (pyrazole)); 5.42,
5.43 (2s, 2 CH2ONO2); 4.73, 4.72 (2s, 2 CH2OH); 1.79 (br. s, 2 OH). 13C-NMR (CDCl3): 144.6; 143.8;
2
143.4 (q, J¼38); 142.1; 141.6; 140.0; 138.2; 133.1; 132.5; 130.3; 129.7; 129.3; 129.2; 129.0; 128.1; 127.5;
127.2; 125.7; 125.6; 121.4 (q, 1J¼268); 106.0 (q, 3J¼2); 105.9 (q, 3J¼2); 73.9; 73.7; 64.6; 64.4. EI-MS: 393
(97, Mþ ), 347 (100), 317 (99). Anal. calc. for C18H14F3N3O4 (393.32): C 54.97, H 3.59, N 10.68; found: C
55.44, H 3.94, N 10.30.
4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzyl Nitrate (15). AgNO3 (0.68 g,
4 mmol) and Ph3P (0.92 g, 3.5 mmol) were added to a soln. of 14 (0.66 g, 2 mmol) in MeCN (30 ml)
under N2. The mixture was cooled to ꢀ158 under N2, and NBS (0.62 g, 3.5 mmol) was added portionwise.
Stirring was continued at ꢀ158 for 1 h and then at r. t. for 4 h. AcOEt was added to the mixture, and the
solid was filtered; the filtrate was washed with H2O, brine, dried, and concentrated in vacuo to give a
residue that was purified by FC (PE/AcOEt 9.5 :0.5) to give 15 (0.52 g, 68%). White solid. M.p. 106–1078
(MeOH). 1H-NMR (CDCl3): 7.41–7.34 (m, 4 arom. H); 7.16–7.09 (m, 4 arom. H); 6.72 (s, CH
(pyrazole)); 5.43 (s, CH2); 2.37 (s, Me). 13C-NMR (CDCl3): 144.9; 143.5 (q, 2J¼38); 140.2; 139.4; 132.3;
130.0; 129.7; 128.7; 126.1; 125.7; 121.2 (q, 1J¼267); 105.8 (q, 3J¼2); 73.8; 21.3. EI-MS: 377 (100, Mþ ), 331
(85), 300 (90). Anal. calc. for C18H14F3N3O3 (377.32): C 57.30, H 3.74, N 11.14; found: C 57.30, H 3.91, N
10.82.
Evaluation of Stability in Human Serum and in Whole Human Blood. Blood samples for both
stability, COX selectivity, and platelet aggregation assays were obtained from healthy volunteers who had
not taken any drug for at least two weeks. Volunteers, who were treated according to the Helsinki
protocol for biomedical experimentation, gave their informed consent to the use of blood samples for
research purposes.
Compounds 7, 11, and 15 were incubated in human serum (sterile-filtered from human male AB
plasma, Sigma-Aldrich) and in heparinized human blood at 100 mm concentration. The resulting solns.
were maintained at 37ꢁ0.58, and, at appropriate time intervals, the mixtures were diluted 1:2 with
MeCN containing 0.1% CF3COOH in order to deproteinize and precipitate cells, proteins, and enzymes.
Samples were sonicated, vortexed, and then centrifuged for 15 min at 2150g. The clear supernatant was
filtered by 0.45 mm PTFE filters (Alltech) and analyzed by RP-HPLC.
The RP-HPLC procedure allowed separation and quantitation of remaining compounds and
metabolites. HPLC Analyses were performed with an HP1100 chromatograph system (Agilent
Technologies, Palo Alto, CA, USA) equipped with a quaternary pump (model G1311A), a membrane
degasser (model G1379A), a diode-array detector (DAD; model G1315B) integrated in the HP1100
system. Data were analyzed using an HP ChemStation system (Agilent Technologies). The anal. column
was a ZORBAX Eclipse XDB-C8 (4.6ꢂ150 mm, 5 mm; Agilent). The mobile phase consisted of MeCN/
H2O (70 :30) with 0.1% CF3COOH, and the flow-rate was 1.2 ml/min. The injection volume was 20 ml
(Rheodyne, Cotati, CA). The column effluent was monitored at 226 and 254 nm. Quantitation was
achieved by comparison of peak areas with standards chromatographed under the same conditions.
Cyclooxygenase Inhibition Studies. Awhole blood assay [15] was performed to evaluate the ability of
synthesised compounds to inhibit COX-1 and COX-2.
Blood samples were divided in two aliquots to test COX-1 and COX-2 inhibition. The COX-2
aliquots were treated with 10 IU/ml of sodium heparine, 10 mg/ml acetylsalicylic acid, and 10 mg/ml

378
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
lipopolysaccaride (LPS) from E. coli. Methanolic solns. of the tested compounds at different
concentrations were prepared, 10-ml aliquots were distributed in incubation tubes, and the solvent was
evaporated. The residues were dissolved by vortexing either in 1 ml of heparinized blood to test COX-2
inhibition, or in 1 ml of untreated blood to test COX-1 inhibition. The final concentrations of the tested
compounds were, therefore, diluted 100 times in the incubation tubes. The COX-1 aliquots were
incubated in glass tubes for 1 h at 378, which is sufficient to complete coagulation, then centrifuged at
2000g for 10 min, after which the serum was ready to be tested for platelet TxB2 production. % Inhibition
in samples treated with the test compounds was evaluated in comparison with control samples with basal
TxB2 production.
The COX-2 aliquots were incubated in polyethylene tubes for 24 h at 378 to allow COX-2 expression
in monocytes and maximal PGE2 production. They were then centrifuged at 2000g for 10 min after which
the plasma was ready to be tested for PGE2 production. Basal PGE2 production in blood untreated with
LPS was subtracted from values for each sample, and % inhibition in samples incubated with tested
compounds was calculated vs. control samples with maximal PGE2 production.
Prostanoid production was evaluated by enzyme immunoassay, following the specific instructions
provided by Cayman Chemical, based on a competitive reaction, for COX-1, between TxB2 and a TxB2-
acetylcholinesterase conjugate (TxB2 tracer) for a specific TxB2 antiserum, and, for COX-2, between
PGE2 and PGE2-acetylcholinesterase (PGE2-tracer) for a specific PGE2 antiserum. Standard curves with
known concentrations of TxB2 and of PGE2 were used to determine prostanoid concentrations in the
sample wells. % Inhibition in compound-treated samples was calculated by comparison with untreated
controls. The concentration of the tested compounds causing 50% inhibition (IC50) was calculated from
the concentration–inhibition response curve (5–6 experiments).
Inhibition of Platelet Aggregation in vitro. Platelet rich plasma (PRP) was prepared by centrifugation
of citrated blood at 200g for 20 min. Aliquots (500 ml) of PRP were added into aggregometer (Chrono-
log 4902D) cuvettes, and aggregation was recorded as increased light transmission under continuous
stirring (1000 rpm) at 378 for 10 min after addition of the stimulus. Collagen (1.0 mg/ml) was used as
platelet activator in PRP. The inhibitory activity of the compounds was tested by addition of drug to PRP
10 min before addition of the stimulus (collagen). Drug vehicle (ꢃ0.5% DMSO) added to PRP did not
affect platelet function in control samples. At least, five experiments were performed for each compound.
The potential role of NO and sGC in the inhibitory effect of 15 was verified using the sGC inhibitor,
ODQ (100 mm).
The anti-aggregatory activity of the test compounds was evaluated as % inhibition of platelet
aggregation compared to controls (5–6 experiments), and IC50 values were calculated by nonlinear
regression analysis.
Vasodilating Activity Assay. Thoracic aortas were isolated from male Wistar rats weighing 180–200 g.
The endothelium was removed, and the vessels were helically cut: three strips were obtained from each
aorta. The tissue was placed in organ baths containing 30 ml of Krebs-bicarbonate buffer of the following
composition (mm): NaCl 111.2, KCl 5.0, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.0, NaHCO3 12, glucose 11.1
maintained at 378 and continuously gassed with 95% O2/5% CO2 (pH 7.4). The aortic strips were allowed
to equilibrate for 120 min and then contracted with 1 mm (ꢀ)-phenylephrine. When the response to the
agonist reached a plateau, cumulative concentration–response curves to compounds 7, 11, and 15 were
determined. The effect of 1 mm ODQ was evaluated in a separate series of experiments in which the ODQ
was added 5 min before contraction. EC50 Values are means of 5–9 determinations. Responses were
recorded by an isometric transducer connected to the MacLab System PowerLabꢃ.
REFERENCES
[1] A. L. Blobaum, L. J. Marnett, J. Med. Chem. 2007, 50, 1425.
[2] G. A. FitzGerald, C. Patrono, N. Engl. J. Med. 2001, 345, 433.
[3] L. Gasparini, E. Ongini, G. Wenk, J. Neurochem. 2004, 91, 521.
[4] Y. S. Bakhle, Br. J. Pharmacol. 2001, 134, 1137.
[5] E. M. Antman, D. DeMets, J. Loscalzo, Circulation 2005, 112, 759.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
379
´
[6] J.-M. Dogne, C. T. Supuran, D. Pratico, J. Med. Chem. 2005, 48, 2251.
[7] S. Moncada, E. A. Higgs, Br. J. Pharmacol. 2006, 147, S193.
[8] R. R. Ranatunge, M. Augustyniak, U. K. Bandarage, R. A. Earl, J. L. Ellis, D. S. Garvey, D. R.
Janero, L. G. Letts, A. M. Martino, M. G. Murty, S. K. Richardson, J. D. Schroeder, M. J. Shumway,
S. W. Tam, A. M. Trocha, D. V. Young, J. Med. Chem. 2004, 47, 2180.
´
[9] C. Velazquez, P. N. P. Rao, R. McDonald, E. E. Knaus, Bioorg. Med. Chem. 2005, 13, 2749.
[10] E. Del Grosso, D. Boschi, L. Lazzarato, C. Cena, A. Di Stilo, R. Fruttero, S. Moro, A. Gasco, Chem.
Biodiversity 2005, 2, 886.
[11] K. Chegaev, L. Lazzarato, P. Tosco, C. Cena, E. Marini, B. Rolando, P.-A. Carrupt, R. Fruttero, A.
Gasco, J. Med. Chem. 2007, 50, 1449.
[12] T. D. Penning, J. J. Talley, S. R. Bertenshaw, J. S. Carter, P. W. Collins, S. Docter, M. J. Graneto, L. F.
Lee, J. W. Malecha, J. M. Miyashiro, R. S. Rogers, D. J. Rogier, S. S. Yu, G. D. Anderson, E. G.
Burton, J. N. Cogburn, S. A. Gregory, C. M. Koboldt, W. E. Perkins, K. Seibert, A. W. Veenhuizen,
Y. Y. Zhang, P. C. Isakson, J. Med. Chem. 1997, 40, 1347.
[13] S. K. Singh, M. S. Reddy, S. Shivaramakrishna, D. Kavitha, R. Vasudev, J. M. Babu, A.
Sivalakshmidevi, Y. K. Rao, Tetrahedron Lett. 2004, 45, 7679.
[14] B. Testa, J. M. Mayer, ꢄ Hydrolysis in Drug and Prodrug Metabolism – Chemistry, Biochemistry, and
Enzymologyꢀ, Verlag Helvetica Chimica Acta, Zꢂrich, Wiley-VCH, Weinheim, 2003, p. 534.
[15] P. Patrignani, M. R. Panara, A. Greco, O. Fusco, C. Natoli, S. Iacobelli, F. Cipollone, A. Ganci, C.
´
Creminon, J. Maclouf, J. Pharmacol. Exp. Ther. 1994, 271, 1705.
[16] J. J. Talley, Prog. Med. Chem. 1999, 36, 201.
[17] A.-A. Weber, T. Neuhaus, C. Seul, R. Dꢂsing, K. Schrçr, A. Sachinidis, H. Vetter, Eur. J. Pharmacol.
1996, 309, 209.
[18] S. Ganapathy, B. B. V. Soma Sekhar, S. M. Cairns, K. Akutagawa, W. G. Bentrude, J. Am. Chem.
Soc. 1999, 121, 2085.
[19] R. Soliman, J. Med. Chem. 1979, 22, 321.
[20] R. B. Kelly, E. G. Daniels, J. W. Hinman, J. Org. Chem. 1962, 27, 3229.
Received October 10, 2008