Nitrooxymethyl-substituted analogues of Rofecoxib: Synthesis and pharmacological characterization

Article abstract of DOI:10.1002/cbdv.200900421

Nitrooxymethyl-substituted derivatives of Rofecoxib were synthesized and tested for their cyclooxygenase (COX)-inhibiting activity in whole human blood, vasodilator potency on rat aorta strips, and for their capacity of inhibiting platelet aggregation of

Full text of DOI:10.1002/cbdv.200900421

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1173
Nitrooxymethyl-Substituted Analogues of Rofecoxib: Synthesis and
Pharmacological Characterization
by Donatella Boschia), Clara Cenaa), Antonella Di Stiloa), Barbara Rolandoa), Paola Manzinib),
Roberta Frutteroa), and Alberto Gasco*a)
a
`
) 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)
b) SIMT Banca del Sangue, AOU S. Giovanni Battista, C.so Bramante 88, I-10126 Torino
Nitrooxymethyl-substituted derivatives of Rofecoxib were synthesized and tested for their cyclo-
oxygenase (COX)-inhibiting activity in whole human blood, vasodilator potency on rat aorta strips, and
for their capacity of inhibiting platelet aggregation of human platelet-rich plasma. The results show that
their potency and selectivity in inhibiting COX isoforms, as well as their anti-aggregatory properties, are
closely dependent on the position at which the NO-donor nitrooxymethyl function is introduced into the
Rofecoxib scaffold. All the products were capable of dilating rat aorta strips precontracted with
phenylephrine in a dose-dependent manner, through a cGMP-dependent mechanism. Compound 10
emerged as a quite potent COX-2-selective inhibitor endowed with good vasodilator activity.
Interestingly, compound 19 behaved as a potent selective COX-1 inhibitor, and displayed good
vasodilator and anti-aggregatory properties. The hydroxymethyl derivatives, potential metabolites of the
nitrooxymethyl analogues, were similarly studied for a comparison.
Introduction. – The principal pharmacological effects of the traditional non-
steroidal anti-inflammatory drugs (tNSAIDs) are related to their ability to inhibit the
cyclooxygenase isoforms COX-1 and COX-2, two enzymes involved in the production
of prostanoids from arachidonic acid [1]. A wide amount of experimental evidence
shows that the inhibition of the COX-2 isoform is principally associated with the anti-
inflammatory and analgesic properties of the NSAIDs, while the inhibition of the
COX-1 isoform is responsible for their anti-aggregatory and gastrotoxic effects [2][3].
On these bases, a new class of inhibitors endowed with a high selectivity for the COX-2
isoform was developed as anti-inflammatory drugs [4]. These products, called Coxibs,
are characterized by reducing prostaglandin (PG)-dependent inflammation, while
maintaining protective gastric PG synthesis [2][5][6]. A number of them, Rofecoxib
(1) [7], Celecoxib (2), Valdecoxib (3), Etoricoxib (4), and Lumiracoxib (5), were
introduced into the market [8].
During the clinical use of these Coxibs, it was found that they can increase the risk
of heart attack and stroke. This cardiotoxicity is due to their capacity of reducing the
biosynthesis of prostacycline (PGI2) in vivo, with consequent tipping of prostanoid
balance in favor of thrombogenic tromboxane [9–11]. Because of their adverse
cardiovascular effects, Rofecoxib and Valdecoxib were withdrawn from the market [8].
This removal decreased the interest in the selective COX-2 inhibitors and discouraged
the pharmaceutical companies to develop further this kind of drugs. Today, there is a
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

1174
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
renewed attention to Coxibs based on the finding that a meta-analysis of 140
randomized trials of five different Coxibs showed that their use was associated with a
relatively low incidence of major vascular events [12][13]. Therefore, these drugs could
be used with appropriate population of patients at low cardiovascular risk. In addition,
recent discoveries revealed new functions for COX-2 enzyme, including an important
role in tumorigenesis [14] with consequent possible applications of Coxibs in the
treatment and in the detection of cancer [15]. The use of these drugs has been proposed
also for the management of Alzheimerꢂs disease [16]. A possible way of attempting to
reduce cardiotoxicity of Coxibs, and consequently to improve their benefit–risk profile,
is to design nitric oxide (NO)-releasing coxibs (NO-Coxibs). Indeed, NO is an
endogenous messenger which displays very important roles in maintaining micro- and
macrovascular homeostasis, among them vasodilation, inhibition of platelet aggrega-
tion, modulation of platelet and leukocyte adherence to vessels, and inhibition of
smooth muscle-cell proliferation. In addition, it is essential for normal physiological
function in central nervous system (CNS), and it is one of the final effectors in the
immune system [17]. Consequently, NO donors are potential useful tools in cancer
therapy and in the management of Alzheimerꢂs disease [18][19]. There are a number of
NO-Coxibs reported in literature. They were obtained by using NO donor 3,4-
diphenylfuroxan system bearing appropriate substituents [20][21], or by linking
selective COX-2 inhibitors with either diazen-1-ium 1,2-diolate [22], or nitrooxy NO-
donor moieties [23–26]. As development of our work in this field, we now describe the
synthesis, COX-inhibition profile studied in human whole blood, and anti-aggregatory
and in vitro vasodilator activities of the compounds 10, 15, and 19, containing nitrooxy
function(s) inserted into the Rofecoxib scaffold. The inhibitory activity of the related
OH analogs 9, 14, and 18, used as intermediates for the preparation of the target
compounds, is also reported in view of their possible role of metabolites [27].

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1175
Results and Discussion. – 1. Chemistry. The synthetic pathway used to prepare the
nitrooxy-substituted Rofecoxib derivatives is depicted in the Scheme. The method used
`
to prepare furan-2(5H)-one derivatives is the same reported by Therien et al. for the
Scheme
a) HBr/Br2, CH2Cl2, 08. b) Et3N, MeCN. c) 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), MeCN, 608 (r.t.
for 9). d) Ph3P, AgNO3, N-Bromosuccinimide (NBS), ꢀ158!r.t. (608 for 19).

1176
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
synthesis of Rofecoxib [28]. The Br-substituted ketone 6 treated with the [4-
(hydroxymethyl)phenyl]acetic acid (7) afforded, in the presence of Et3N, esther 8.
This product underwent cyclization, under the action of 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU), to give the 4-hydroxymethyl-substituted Rofecoxib 9. Treatment of 9,
dissolved in MeCN, with N-bromosuccinimide (NBS) in the presence of Ph3P, and then
with AgNO3, afforded the expected mononitrooxy substituted final compound 10. This
is a recent method for the preparation of nitrates under mild conditions [29]. The
sequence of reactions aforementioned was used to prepare 13, 14, and the dinitrooxy-
substituted final compound 15, starting from 7 and 12. This latter intermediate was
obtained by bromination of the known 1-[4-(hydroxymethyl)phenyl]ethanone (11).
Similarly, the intermediates 17, 18, and the mononitrooxy-substituted Rofecoxib analog
19 were sequentially synthesized starting from 12 and 2-phenylacetic acid (16).
2. COX Inhibition and Platelet Anti-aggregatory Activity. The ability of the products
and of their alcoholic analogues to inhibit the COX enzymes was evaluated in human
whole blood according to a well-established procedure [30]. To assess 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 was
assayed for PGE2 production as a function of COX-2 inhibition. For COX-1 inhibition
activity, whole blood samples without any anticoagulant were incubated with the
inhibitors for 1 h. Then, plasma was collected and analyzed for TXB2 production. The
potencies of the products expressed as IC50 values are collected in Table 1. When these
values could not be derived because inhibition did not reach 50%, the inhibition at
maximal concentration tested (100 mm) was calculated. Analysis of the data shows that
the introduction of one nitrooxymethyl group in para-position of the 3-phenyl ring of
Rofecoxib gives rise to 10, which is a COX-2 inhibitor ca. 15 times less potent than the
lead, but it retains a quite good degree of COX-2/COX-1 selectivity. Its OH analogue 9
behaves similarly. By contrast, when in 1 the MeSO2 group, which is an important
determinant for the COX-2 selectivity [4], is replaced with the nitrooxymethyl moiety,
19 is obtained that is a COX-1 inhibitor ca. 100 times more potent and a COX-2
inhibitor about sixfold less potent than the lead. Inhibitory potencies and selectivity are
even more evident in its OH analog 18. Finally, the simultaneous presence of two
nitrooxymethyl groups in para-positions of the two phenyl rings of 1 provides 15, which
displays very feeble COX inhibition, when tested on both the two isoforms. By contrast,
its dihydroxy analog 14 is a quite potent and selective COX-1 inhibitor. The COX
inhibitory profiles of the nitrooxy-substituted Rofecoxib compounds and of their OH
analogs here described parallel very well those of the related Celecoxib derivatives
previously described [26]. The only difference is the definitively lower inhibitor
activities of the bis(hydroxymethyl)- and bis(nitrooxymethyl)-substituted compounds
14 and 15, respectively. All products were also tested for their ability to inhibit collagen-
induced platelet aggregation of human platelet-rich plasma (PRP). Only the nitro-
oxymethyl-substituted final compound 19 displayed potent anti-aggregatory activity in
the micromolar range, in keeping with its high ability to inhibit COX-1 isoform
(Table 2). A similar behavior was shown by the HOCH2-substituted intermediates 14
and 18.
3. Vasodilation. The in vitro vasodilator activity of the NO-donor analogs of
Rofecoxib 10, 15, and 19 was assessed on rat aorta strips precontracted with

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1177
Table 1. COX-1 and COX-2 inhibition data for compounds and Rofecoxib 1, taken as reference
Com- R1
pound
R2
COX-1
COX-2
IC50 ꢁSE [mm]a) % InhibitionꢁSEb) IC50 ꢁSE [mm]a) % InhibitionꢁSEb)
(100 mm)c)
(100 mm)c)
1
9
SO2Me
H
64ꢁ13
–
1.5ꢁ0.8
22ꢁ5
d
SO2Me
SO2Me
CH2OH
CH2ONO2 CH2ONO2
CH2OH
CH2OH
CH2ONO2
CH2OH
)
7.6ꢁ6.5
d
10
14
15
18
19
)
11ꢁ4
22ꢁ6
d
5.7ꢁ1.7
)
)
21ꢁ13
39ꢁ11
d
d
)
36ꢁ2
H
H
0.24ꢁ0.03
8.4ꢁ2.8
CH2ONO2
0.61ꢁ0.17
9.6ꢁ4.2
a) Values are expressed as IC50 ꢁstandard errors. b) Values are expressed as mean percentage of inhibitionꢁ
c
standard errors. ) Maximum concentration tested. d) Inhibition of control prostanoid production did not reach
50%.
Table 2. Anti-Aggregatory and Vasodilating Properties of Derivatives
Compound
R1
R2
Anti-aggregatory activity
Vasodilating activity
IC50 (95% CL)
[mm]a)
% InhibitionꢁSEb)
EC50 ꢁSE [mm]d)
(100 mm)c)
1
9
10
SO2Me
SO2Me
SO2Me
H
48 (44–51)
inactive
)
e
f
CH2OH
CH2ONO2
)
)
1.7ꢁ1.7
29ꢁ15
e
0.21ꢁ0.06
11ꢁ1.3g)
f
14
15
CH2OH
CH2ONO2
CH2OH
CH2ONO2
23 (19–28)
)
e
)
5.3ꢁ4.4
0.28ꢁ0.05
15ꢁ4g)
f
18
19
CH2OH
CH2ONO2
H
H
39 (35–42)
46 (42–50)
)
0.22ꢁ0.08
35ꢁ3g)
ISDNh)
)
)
4.7ꢁ0.6
f
f
>100g)
a) Values are expressed as IC50 with 95% confidential limits in brackets. b) Values are expressed as mean
c
percentage of inhibitionꢁstandard errors. ) Maximum concentration tested. d) Values are expressed as EC50
ꢁ
standard errors. e) Inhibition of control aggregation effect did not reach 50%. f) Not tested. g) In the presence of
1 mm 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). h) Isosorbide dinitrate (ISDN) was used as a
reference compound.

1178
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
phenylephrine. All the products were capable of relaxing the contracted tissues in a
concentration-dependent manner. Their potencies, expressed as EC50 values, are
collected in Table 2. All of them behave as potent vasodilators, more active than
isosorbide dinitrate (ISDN) taken as a reference. The vasodilator potencies of the
products were reduced, when the experiments were repeated in the presence of 1 mm
ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a well-known heme site inhibitor
of sGC. This is in keeping with the involvement of NO in the vasodilator action.
Conclusions. – Introduction of nitrooxymethyl and of hydroxymethyl groups on the
scaffold of Rofecoxib (1) gives rise to compounds with different COX-inhibitor
profiles. The two most interesting products obtained are the compounds 19 and 10. The
former is derived from replacement of methylsulfonyl group with nitrooxymethyl
moiety in 1. It is a rather potent and selective COX-1 inhibitor, endowed with
vasodilator and anti-aggregatory properties. This finding confirms the importance of
MeSO2 group for COX-2 selectivity of the lead. To our knowledge, it is the first
example of NO-donor selective COX-1 inhibitor which might be further studied as
cardioprotective, chemopreventive, and analgesic agent [31]. The latter arises from
introduction of nitrooxymethyl group in the para-position of the 3-phenyl ring of 1. It is
a selective and quite potent COX-2 inhibitor, which displays good vasodilator activity.
This product is certainly an interesting NO-Coxib, worthy of additional study owing to
its potential low cardiotoxicity.
This work was supported by a grant from Regione Piemonte Progetto Ricerca Sanitaria Finalizzata
2007.
Experimental Part
General. Anh. MgSO4 was used as drying agent for the org. phases. Flash column chromatography
(FC): silica gel (Merck Kieselgel 60, 230–400 mesh ASTM) using the reported eluents. TLC: 5ꢂ20 cm
plates with 0.25-mm layer thickness. M.p.: in capillaries (Bꢀchi 540 instrument); m.p. with decomposition
were determined after introduction of the sample in the bath at 108 below the m.p.; a heating rate of
18 minꢀ1 was used. All compounds were routinely checked by FT-IR (Perkin-Elmer SPECTRUM BXII),
1H- and 13C-NMR (Bruker Avance 300), and MS (Finnigan-Mat TSQ-700). Elemental analysis (C, H, N):
REDOX (Monza); the results were within ꢁ0.4% of the theoretical values. Compounds 6 [28] and 11
[32] were synthesized according to the methods described in the literature, 7 and 16 were supplied by
Sigma-Aldrich.
2-Bromo-1-[4-(hydroxymethyl)phenyl]ethanone (12). A soln. of Br2 (4.38 g, 27.3 mmol) in CH2Cl2
(50 ml) was added at 08, during 2 h, to a stirred soln. of 11 (4.11 g, 27.0 mmol) in CH2Cl2 (200 ml)
containing conc. HBr (4 drops). The mixture was washed with H2O and with a sat. soln. of NaHCO3, then
dried, and evaporated to give a crude compound that was purified by FC (CH2Cl2/AcOEt 98 :2) to give
1
12 (2.23 g, 36%). White solid. M.p. 68–698 (i-Pr2O). H-NMR (CDCl3): 7.98 (AA’BB’, J¼8.4, 2 arom.
H); 7.49 (AA’BB’, J¼8.4, 2 arom. H); 4.80 (s, CH2OH); 4.45 (s, CH2Br); 1.94 (s, OH). 13C-NMR
(CDCl3): 191.1; 147.3; 133.1; 129.2; 126.8; 64.5; 30.9. EI-MS: 230, 228 (Mþ ), 135 (100). Anal. calc. for
C9H9BrO2 (229.07): C 47.19, H 3.96; found: C 47.14, H 3.98.
2-[4-(Methylsulfonyl)phenyl]-2-oxoethyl [4-(Hydroxymethyl)phenyl]acetate (8). A soln. of 6 (1.66 g,
6.0 mmol) in MeCN (25 ml) was added dropwise to a soln. of 7 (1.00 g, 6.0 mmol) and Et3N (0.83 ml,
6.0 mmol) in MeCN (30 ml) at r.t. After 7 h, the mixture was concentrated in vacuo, and the residue was
dissolved in AcOEt. The combined org. phases were washed with H2O, 1n HCl, and brine, dried and
evaporated. FC (petroleum ether (PE)/i-PrOH 7:3) of the crude product gave 8 (1.85 g, 85%). White

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1179
solid. M.p. 107–1088 (MeOH). 1H-NMR (CDCl3): 7.98 (s, 4 arom. H); 7.34–7.26 (m, 4 arom. H); 5.28 (s,
CH2O); 4.69 (s, CH2OH); 3.81 (s, CH2CO); 3.07 (s, Me); 2.03 (s, OH). 13C-NMR (CDCl3): 191.9; 170.8;
144.6; 140.3; 138.1; 132.4; 129.5; 128.8; 127.9; 127.3; 66.7; 64.9; 44.3; 40.6. EI-MS: 362 (Mþ ), 165 (100).
Anal. calc. for C18H18O6S (362.40): C 59.66, H 5.01; found: C 59.85, H 5.05.
2-[4-(Hydroxymethyl)phenyl]-2-oxoethyl [4-(Hydroxymethyl)phenyl]acetate (13). Compound 13
was obtained by the above described procedure for 8, starting from 7 and 12. After 24 h, the mixture was
poured into ice/H2O, and the pure white precipitate was filtered and dried (yield 82%). M.p. 92–948 ((i-
1
Pr)2O/AcOEt 3 :1). H-NMR ((D6)DMSO): 7.92 (AA’BB’, J¼8.4, 2 arom. H); 7.48 (AA’BB’, J¼8.4,
2 arom. H); 7.28 (s, 4 arom. H); 5.49 (s, CH2O); 5.40 (t, J ¼ 5.7, OH); 5.16 (t, J¼5.7, OH); 4.59 (d, J¼5.7,
CH2OH); 4.48 (d, J¼5.7, CH2OH); 3.81 (s, CH2CO). 13C-NMR ((D6)DMSO): 193.3; 171.9; 149.5; 141.5;
133.1; 133.0; 129.9; 128.5; 127.4; 127.3; 67.4; 63.3; 63.0; 40.4. EI-MS: 296 ([MꢀH2O]þ ), 135 (100). Anal.
calc. for C18H18O5 (314.33): C 68.78, H 5.77; found: C 68.65, H 5.78.
2-[4-(Hydroxymethyl)phenyl]-2-oxoethyl 2-Phenylacetate (17). Compound 17 was obtained by the
above described procedure for 8, starting from 12 and 16. The mixture was kept at r.t. for 24 h. The
mixture was poured into ice/H2O, and the pure white precipitate was filtered and dried (yield 52%). M.p.
60–628 ((i-Pr)2O). 1H-NMR (CDCl3): 7.86 (AA’BB’, J¼8.4, 2 arom. H); 7.45 (AA’BB’, J¼8.4, 2 arom.
H); 7.26–7.36 (m, 5 arom. H); 5.33 (s, CH2O); 4.76 (s, CH2OH); 3.82 (s, CH2CO); 1.98 (br. s, OH).
13C-NMR (CDCl3): 192.2; 171.6; 147.7; 134.0; 133.7; 129.3; 128.5; 127.7; 127.3; 126.4; 66.9; 64.9; 41.3. EI-
MS: 284 (Mþ ), 135 (100). Anal. calc. for C17H16O4 (284.31): C 71.82, H 5.67; found: C 71.57, H 5.71.
3-[4-(Hydroxymethyl)phenyl]-4-[4-(methylsulfonyl)phenyl]furan-2(5H)-one (9). A soln. of DBU
(0.36 g, 2.4 mmol) in dry MeCN (15 ml) was added dropwise during 1 h to a soln. of 8 (1.70 g, 4.7 mmol)
in dry MeCN (50 ml) at r.t. under N2. Then, the mixture was poured into H2O, and the product was
extracted with CH2Cl2. The combined org. phase was treated with brine, dried, and evaporated. FC
(CH2Cl2/MeOH 98 :2) of the crude product gave 9 (1.15 g, 71%). White solid. M.p. 177–1808 (MeOH/
1
Et2O). H-NMR ((D6)DMSO): 8.00 (AA’BB’, J¼8.4, 2 arom. H); 7.63 (AA’BB’, J¼8.4, 2 arom. H);
7.38–7.29 (m, 4 arom. H); 5.40 (s, 1 CH2 of furan); 5.27 (t, J¼5.4, OH); 4.53 (d, J¼5.4, CH2OH); 3.26 (s,
Me). 13C-NMR ((D6)DMSO): 172.7; 155.7; 143.5; 142.0; 135.9; 128.9; 128.7; 128.0; 127.5; 126.9; 126.7;
70.9; 62.6; 43.2. EI-MS: 344 (Mþ, 100%). Anal. calc. for C18H16O5S (344.39): C 62.78, H 4.68; found: C
62.45, H 4.68.
3,4-Bis[4-(hydroxymethyl)phenyl]furan-2(5H)-one (14). A soln. of DBU (0.27 g, 1.7 mmol) in dry
MeCN (20 ml) was added dropwise during 10 min to a soln. of 13 (1.08 g, 3.4 mmol) in dry MeCN
(180 ml) kept at 608 under N2. After 45 min, the soln. was concentrated to 20 ml under vacuum, the
residue was poured into H2O, and the product was extracted with AcOEt. The combined org. phases were
treated with H2O and brine, and dried and evaporated. The crude product was purified by crystallization
from 1,2-dichloroethane to give the title product as a yellow solid (0.70 g, 69% yield). M.p. 161–1628.
1H-NMR ((D6)DMSO): 7.37–7.28 (m, 8 arom. H); 5.36 (s, CH2 of furan); 5.24–5.30 (m, 2 OH); 4.54–
4.49 (m, 2 CH2OH). 13C-NMR ((D6)DMSO): 173.1; 157.2; 145.2; 142.3; 128.9; 128.7; 127.3; 126.6; 126.5;
126.4; 124.1; 70.5; 62.5; 62.4. EI-MS: 296 (Mþ, 100%). Anal. calc. for C18H16O4 ·0.05 C2H4Cl2 (301.27): C
72.16, H 5.42; found: C 71.78, H 5.37.
4-[4-(Hydroxymethyl)phenyl]-3-phenylfuran-2(5H)-one (18). A soln. of DBU (0.41 g, 2.6 mmol) in
dry MeCN (10 ml) was added dropwise during 10 min to a soln. of 17 (1.50 g, 3.4 mmol) in dry MeCN
(20 ml) kept at 608 under N2. After 60 min, the mixture was concentrated to 20 ml under vacuum, and the
residue was poured into H2O. The product was extracted with AcOEt, and the combined org. phases were
treated with H2O and brine, and dried (MgSO4) and evaporated. The crude product obtained was
crystallized by H2O/EtOH to give 18 (1.05 g, 75%). White solid. M.p. 141–1428. 1H-NMR (CDCl3): 7.43–
7.26 (m, 9 arom. H); 5.16 (s, CH2 of furan); 4.69 (s, CH2OH); 2.21 (br. s, OH). 13C-NMR (CDCl3): 173.7;
156.0; 143.8; 130.2; 129.8; 129.3; 128.8; 128.7; 127.6; 127.2; 126.0; 70.6; 64.4. EI-MS: 266 (100, Mþ ). Anal.
calc. for C17H14O3 (266.30): C 76.68, H 5.30; found: C 76.47, H 5.26.
4-{2,5-Dihydro-4-[4-(methylsulfonyl)phenyl]-2-oxofuran-3-yl}benzyl Nitrate (10). AgNO3 (0.39 g,
2.3 mmol) and Ph3P (0.51 g, 1.9 mmol) were added to a soln. of 9 (0.53 g, 1.5 mmol) in dry MeCN (15 ml)
under N2. The mixture was cooled to ꢀ158, and NBS (1.69 g, 1.7 mmol) was added portionwise. Stirring
was continued at ꢀ158 for 1 h and then at r.t. for 24 h. CH2Cl2 was added to the mixture, and the
precipitate was removed by filtration. The filtrate was washed with H2O and brine, and dried and

1180
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
concentrated in vacuo. The residue was purified by FCC (CH2Cl2/AcOEt 95 :5) to give 10 (0.40 g, 67%).
White solid. M.p. 170–1728 (MeOH). 1H-NMR (CDCl3): 7.95 (AA’BB’, J¼8.4, 2 arom. H); 7.51
(AA’BB’, J¼8.4, 2 arom. H); 7.47–7.40 (m, 4 arom. H); 5.45 (s, CH2ONO2); 5.21 (s, CH2 of furan); 3.09
(s, Me). 13C-NMR (CDCl3): 172.2; 154.4; 142.2; 136.0; 133.6; 130.3; 129.7; 129.5; 128.5; 128.3; 128.2; 74.0;
70.5; 44.3. EI-MS: 389 (Mþ ), 343 (100). Anal. calc. for C18H15NO7S (389.38): C 55.52, H 3.88; found: C
55.44, H 3.94.
(2,5-Dihydro-2-oxofuran-3,4-diyl)bis(benzene-4,1-diylmethanediyl) Dinitrate (15). AgNO3 (1.71 g,
10.0 mmol) and Ph3P (2.19 g, 8.0 mmol) were added to a soln. of 14 (0.99 g, 3.0 mmol) in dry MeCN
(15 ml) under N2. The mixture was cooled to ꢀ158, and NBS (1.42 g, 8.0 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
precipitate was removed by filtration. The filtrate was washed with H2O and brine, and dried and
concentrated in vacuo. The residue was purified by FCC (CH2Cl2) to give 15 (0.50 g, 39%). White solid.
M.p. 100–1018 (MeOH). 1H-NMR (CDCl3): 7.50–7.34 (m, 8 arom. H); 5.46 (s, CH2ONO2); 5.44 (s,
CH2ONO2); 5.20 (s, CH2 of furan). 13C-NMR (CDCl3): 172.8; 155.8; 135.1; 133.1; 131.7; 131.1; 129.5;
129.4; 129.0; 128.8; 126.3; 74.2; 73.6; 70.6. EI-MS: 386 (Mþ ), 264 (100). Anal. calc. for C18H14N2O8
(386.31): C 55.96, H 3.65; found: C 55.89, H 3.67.
4-(2,5-Dihydro-5-oxo-4-phenylfuran-3-yl)benzyl Nitrate (19). AgNO3 (1.28 g, 7.5 mmol) and Ph3P
(1.23 g, 4.7 mmol) were added to a soln. of 18 (1.00 g, 3.8 mmol) in dry MeCN (50 ml) under N2. The
mixture was cooled to 58, and NBS (0.84 g, 4.7 mmol) was added portionwise. Stirring was continued for
1 h at r.t. and then for 2.5 h at 608. AcOEt was added to the mixture, and the precipitate was removed by
filtration. The filtrate was washed with H2O and brine, and dried and concentrated in vacuo to give a solid
that was purified by crystallization from MeOH to give 19 (0.64 g, 55%). White solid. M.p. 78–798
(MeOH). 1H-NMR (CDCl3): 7.40–7.26 (m, 9 arom. H); 5.41 (s, CH2ONO2); 5.17 (s, CH2 of furan).
13C-NMR (CDCl3): 173.1; 154.9; 134.8; 132.0; 129.8; 129.4; 129.2; 129.1; 128.8; 128.0; 127.2; 73.7; 70.5. EI-
MS: 311 (Mþ ), 178 (100). Anal. calc. for C17H13NO5 (311.29): C 65.59, H 4.21; found: C 65.68, H 4.28.
Cyclooxygenase Inhibition Studies. Awhole blood assay [30] was performed to evaluate the ability of
synthesized 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
lipopolysaccaride from E.coli (LPS). MeOH 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
TXB2ꢀacetylcholinesterase conjugate (TXB2 tracer) for a specific TXB2 antiserum, and, for COX-2,
between PGE2 and PGE2ꢀacetylcholinesterase (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. Percent 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 centrifu-
gation 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

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1181
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 anti-aggregatory activity of the tested 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 and 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
10, 15, and 19 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 4–10
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] D. E. Griswold, J. L. Adams, Med. Res. Rev. 1996, 16, 181.
[3] J. A. Mitchell, T. D. Warner, Br. J. Pharmacol. 1999, 128, 1121.
[4] J. J. Talley, Prog. Med. Chem. 1999, 36, 201.
[5] X. de Leval, F. Julemont, V. Benoit, M. Frederich, B. Pirotte, J.-M. Dogne, Mini-Rev. Med. Chem.
2004, 4, 597.
[6] P. Singh, A. Mittal, Mini-Rev. Med. Chem. 2008, 8, 73.
[7] P. Prasit, Z. Wang, C. Brideau, C.-C. Chan, S. Charleson, W. Cromlish, D. Ethier, J. F. Evans, A. W.
Ford-Hutchinson, J. Y. Gauthier, R. Gordon, J. Guay, M. Gresser, S. Kargman, B. Kennedy, Y.
´
Leblanc, S. Leger, J. Mancini, G. P. OꢂNeill, M. Ouellet, M. D. Percival, H. Perrier, D. Riendeau, I.
´
Rodger, P. Tagari, M. Therien, P. Vickers, E. Wong, L.-J. Xu, R. N. Young, R. Zamboni, S. Boyce, N.
Rupniak, N. Forrest, D. Visco, D. Patrick, Bioorg. Med. Chem. Lett. 1999, 9, 1773.
[8] S. Shi, U. Klotz, Eur. J. Clin. Pharmacol. 2008, 64, 233.
´
[9] J.-M. Dogne, C. T. Supuran, D. Pratico, J. Med. Chem. 2005, 48, 2251.
[10] E. M. Antman, D. DeMets, J. Loscalzo, Circulation 2005, 112, 759.
[11] F. Andersohn, S. Suissa, E. Garbe, Circulation 2006, 113, 1950.
[12] P. M. Kearney, C. Baigent, J. Godwin, H. Halls, J. R. Emberson, C. Patrono, Br. Med. J. 2006, 332,
1302.
[13] C. Patrono, B. Rocca, Pharmacol. Res. 2009, 59, 285.
[14] O. C. Trifan, T. Hla, J. Cell. Mol. Med. 2003, 7, 207.
[15] C. A. Rouzer, L. J. Marnett, J. Lipid Res. 2009, 50, S29.
[16] M. Nivsarkar, A. Banerjee, H. Padh, Pharmacol. Rep. 2008, 60, 692.
[17] J. F. Kerwin Jr., M. Heller, Med. Res. Rev. 1994, 14, 23.
[18] S. Huerta, S. Chilka, B. Bonavida, Int. J. Oncol. 2008, 33, 909.
[19] G. R. J. Thatcher, B. M. Bennett, J. N. Reynolds, Curr. Alzheimer Res. 2006, 3, 237.
[20] E. Del Grosso, D. Boschi, L. Lazzarato, C. Cena, A. Di Stilo, R. Fruttero, S. Moro, A. Gasco, Chem.
Biodiversity 2005, 2, 886.
´
[21] C. Velazquez, P. N. P. Rao, R. McDonald, E. E. Knaus, Bioorg. Med. Chem. 2005, 13, 2749.
´
[22] K. R. A. Abdellatif, M. A. Chowdhury, Y. Dong, D. Das, G. Yu, C. Velazquez, M. R. Suresh, E. E.
Knaus, Bioorg. Med. Chem. 2009, 17, 5182.
[23] F. C. Engelhardt, Y.-J. Shi, C. J. Cowden, D. A. Conlon, B. Pipik, G. Zhou, J. M. McNamara, U.-H.
Dolling, J. Org. Chem. 2006, 71, 480.

1182
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
[24] 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.
[25] 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.
[26] D. Boschi, L. Lazzarato, B. Rolando, A. Filieri, C. Cena, A. Di Stilo, R. Fruttero, A. Gasco, Chem.
Biodiversity 2009, 6, 369.
[27] B. Testa, J. M. Mayer, in ꢄ Hydrolysis in Drug and Prodrug Metabolism – Chemistry, Biochemistry
and Enzymologyꢂ, Eds. B. Testa, J. M. Mayer, VHCA/Wiley-VCH, Zurich, 2003, p. 535.
´
´
[28] M. Therien, J. Y. Gauthier, Y. Leblanc, S. Leger, H. Perrier, P. Prasit, Z. Wang, Synthesis 2001, 1778.
[29] P. Frøsyen, Phosphorus, Sulfur Silicon Relat. Elem. 1997, 129, 89.
[30] 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.
[31] N. Handler, G. Brunhofer, C. Studenik, K. Leisser, W. Jaeger, S. Parth, T. Erker, Bioorg. Med. Chem.
2007, 15, 6109.
[32] S. Ganapathy, B. B. V. Sekhar, S. M. Cairns, K. Akutagawa, W. G. Bentrude, J. Am. Chem. Soc. 1999,
121, 2085.
Received December 23, 2009