A diazen-1-ium-1,2-diolated nitric oxide donor ester prodrug of 3-(4-hydroxymethylphenyl)-4-(4-methanesulfonylphenyl)-5H-furan-2-one: Synthesis, biological evaluation and nitric oxide release studies

Article abstract of DOI:10.1016/j.bmcl.2011.05.017

A novel hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrug (NONO-coxib 14) wherein an O²-acetoxymethyl 1-(2-carboxypyrrolidin-1- yl)diazen-1-ium-1,2-diolate (O²-acetoxymethyl PROLI/NO) NO-donor moiety was covalently c

Full text of DOI:10.1016/j.bmcl.2011.05.017

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3951–3956
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A diazen-1-ium-1,2-diolated nitric oxide donor ester prodrug of 3-(4-hydrox-
ymethylphenyl)-4-(4-methanesulfonylphenyl)-5H-furan-2-one: Synthesis,
biological evaluation and nitric oxide release studies
Khaled R. A. Abdellatif a, Zhangjian Huang a, Morshed A. Chowdhury a, Susan Kaufman b, Edward E. Knaus a,
⇑
a Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
b Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrug (NONO-coxib 14) wherein an
O2-acetoxymethyl 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate (O2-acetoxymethyl PROLI/NO)
NO-donor moiety was covalently coupled to the CH2OH group of 3-(4-hydroxymethylphenyl)-4-(4-meth-
ylsulfonylphenyl)-5H-furan-2-one (12), was synthesized. The prodrug 14 released a low amount of NO
(4.2%) upon incubation with phosphate buffer (PBS) at pH 7.4 which was significantly higher (34.8% of
the theoretical maximal release of two molecules of NO/molecule of the parent hybrid ester prodrug)
upon incubation in the presence of rat serum. These incubation studies suggest that both NO and the
parent compound 12 would be released from the prodrug 14 upon in vivo cleavage by non-specific serum
esterases. The prodrug ester 14 is a selective COX-2 inhibitor that exhibited AI activity (ED50 = 72.2
Received 23 April 2011
Revised 5 May 2011
Accepted 6 May 2011
Available online 14 May 2011
Keywords:
Rofecoxib
Diazen-1-ium-1-2-diolate
Ester prodrug
Nitric oxide donor
Cyclooxygenase inhibition
Vascular effects
mmol/kg po) between that of the reference drugs celecoxib (ED50 = 30.9
lmol/kg po) and ibuprofen
(ED50 = 327 mol/kg po). The NO donor compound 14 exhibited enhanced inhibition of phenylephrine-
l
induced vasoconstriction of isolated mesenteric arteries compared with that observed under control con-
ditions. These studies indicate hybrid ester AI/NO donor prodrugs (NONO-coxibs) constitutes a plausible
drug design concept targeted toward the development of selective COX-2 inhibitory AI drugs that are
devoid of adverse cardiovascular effects.
Anti-inflammatory activity
Ó 2011 Elsevier Ltd. All rights reserved.
The original drug design concept that selective cyclooxygenase-2
(COX-2) inhibitors would be effective anti-inflammatory (AI) agents
showing minimal gastrointestinal (GI) toxicity1–4 was confirmed
with the discovery of celecoxib (1),5 rofecoxib (2),6 and valdecoxib
(3, see structures in Fig. 1).7 Accordingly, these drugs preferentially
inhibit the inducible COX-2 isozyme that causes inflammation in
the periphery rather than the constitutive COX-1 isozyme that
provides gastroprotection and maintains vascular homeostasis.
However, this apparently safe pharmacological profile shown by
selective COX-2 inhibitors was relatively short-lived. Evidence be-
gan to accumulate suggesting that highly selective COX-2 inhibitors
alter the balance in the COX pathway causing a decrease in the level
of the desirable vasodilatory and anti-aggregatory prostacyclin
(PGI2) in conjunction with an increase in the level of the undesirable
prothrombotic thromboxane A2 (TxA2). This alteration provided a
rational explanation for the observed elevation in blood pressure
and increased incidences of an adverse cardiovascular thrombotic
event such as myocardial infarction.8 Accordingly, the clinical use
of rofecoxib and valdecoxib were subsequently terminated due to
adverse cardiovascular effects associated with their use.9
Nitric oxide (NO) exhibits a number of biological actions that are
similar to those for PGI2 that encompass a cytoprotective role in GI
homeostasis by facilitating mucosal blood flow, and inhibition of
platelet aggregation and inflammatory-cell activation.10–13 It is
plausible that these beneficial actions of NO could enhance the gas-
tro-sparing features of selective COX-2 inhibitors and potentially in-
duce peripheral vasodilation to circumvent the elevation in blood
pressure exhibited by selective COX-2 inhibitors that decrease the
physiological level of PGI2. In this regard, hybrid selective COX-2
inhibitors possessing a NO-donor moiety (NO-coxibs) have been
investigated as a method to increase the clinical safety of COX-2
inhibitors. Examples of NO-coxibs (see Fig. 1) having a nitrate ester
NO-donor moiety include the oxazole (4) which exhibits anti-
inflammatory activity similar to that of valdecoxib with antithrom-
botic action at higher doses,14 and the imidazoles (5a–b) that
exhibit a NO-dependent vasodilator activity.15 In earlier studies,
we described second generation celecoxib (6)16 and indomethacin
(7)17 prodrugs having a NONO-donor O2-(acetoxymethyl)-1-(2-
carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate moiety that are
effectively cleaved by esterases to release the parent COX-2 inhibi-
tory AI agent and NO. We now report the synthesis and biological
evaluation of a novel hybrid ester prodrug derivative of rofecoxib
(14) in which the second generation NONO-donor moiety indicated
⇑
Corresponding author. Tel.: +1 780 492 5993; fax: +1 780 492 1217.
E-mail address: eknaus@pharmacy.ualberta.ca (E.E. Knaus).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.017

3952
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3951–3956
Me
Me
O
O
S
O
O
O
NH
O NO
2
2
NH
2
S
Me
S
S
O
O
N
N
O
O
O
O
N
N
O
Me
F C
3
Valdecoxib (3)
Rofecoxib (2)
Celecoxib (1)
NMI-1093 (4)
O
N
N
Me
O
O
O
N
O
CH
3
O
O
1
S
Cl
R O
O
O
N
F
N
CH
3
N
+
N
O
CH
O
O
3
MeO
Cl
5a, R = CH CH ONO
2
O
N
N
SO NH
2 2
N
O
O
N
O
1
O
2
2
F C
3
6
7
1
5b, R = CH CH(ONO )CH ONO
2
2
2
2
Figure 1. Chemical structures of the selective cyclooxygenase-2 (COX-2) inhibitors celecoxib (1), rofecoxib (2), valdecoxib (3), selective COX-2 inhibitors that possess a nitric
oxide donor nitrate (4–5) or diazen-1-ium-1,2-diolate (6) moiety, and the indomethacin prodrug (7) that unlike indomethacin is completely devoid of ulcerogenicity.
above16,17 is attached directly to the hydroxyl group of 3-(4-hydrox-
ymethylphenyl)-4-(4-methanesulfonylphenyl)-5H-furan-2-one (12).
This hybrid ester prodrug of rofecoxib was designed with the expec-
tation that the anti-platelet aggregation and hypotensive actions of
NO that is released from the diazen-1-ium-1,2-diolate moiety
would circumvent the adverse thrombotic and hypertensive effects
that led to the clinical withdrawal of rofecoxib (VioxxÒ).18
The 3-(4-hydroxymethylphenyl)-4-(4-methanesulfonylphenyl)
-5H-furan-2-one (12), and the O2-acetoxymethyl PROLI/NO prodrug
(14), were synthesized using the reaction sequence illustrated in
Scheme 1. Accordingly, reaction of the ketone 8 with para-tolylace-
tic acid (9) in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) afforded the furan-2-one product 10 (63%). Subsequent pho-
tochemical promoted bromination of the tolyl methyl group present
in compound 10 furnished the benzyl bromide 11 in 41% yield. The
bromomethyl compound 11 was converted to the respective
hydroxymethyl product 12 in moderate yield (36%) upon heating
under reflux in an acetone-water solvent system (13:1, v/v) for
110 h. The target O2-acetoxymethyl PROLI/NO prodrug ester 14
was synthesized in 30% yield by condensation of the bromomethyl
compound 11 with O2-acetoxymethyl 1-(2-carboxypyrrolidin-1-
yl)diazen-1-ium-1,2-diolate (13) in the presence of triethylamine
in dimethyl sulfoxide.
Three positions on the structure of rofecoxib (2) were considered
for attachment of an O2-acetoxymethyl-1-(2-methylpyrrolidin-1-
yl)diazen-1-ium-1,2-diolate (PROLI/NO) NO donor moiety via an es-
ter linkage. The MeSO2 COX-2 pharmacophore must be present at
the para-position on the C-4 phenyl ring since the corresponding
C-3 regioisomer is inactive. A substituent (halogen, methyl, meth-
oxy) at the para-position of the C-3 phenyl generally has little effect
on COX-2 potency although it may result in a moderate increase in
COX-1 potency thereby reducing the COX-2 selectivity index.6
Although the C-5 position of the furan-2-one central ring tolerates
small alkyl and/or hydroxyl substituents,19,20 it was anticipated that
a large PROLI/NO moiety and opening of the lactone ring from a syn-
thetic perspective may be deterrents. Accordingly, we decided based
on this structure-activity information to couple the PROLI/NO donor
moiety (13) to the C-3 para-C6H4-CH2OH moiety present in the
MeO2S
MeO2S
CH2Br
Me
SO2Me
Me
9
+
b
a
O
Br
O
O
11
O
10
O
HOOC
8
O
d, 13
c
CH3
N
O
O
O
N
O
N
MeO2S
CH2OH
O
MeO2S
CH2
O
N
CH3
O
O
O
N
O
O
O
N
12
O
HO
O
14
13 (PROLI/NO)
Scheme 1. Reagents and conditions: (a) CH3CN, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 0 °C, 4 h; (b) N-bromosuccinimide, benzoyl peroxide, light from a 100-W sun
beam lamp, benzene, reflux, 5 h; (c) acetone, H2O, reflux, 110 h; (d) Et3N, DMSO, 25 °C, 36 h.

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3951–3956
3953
benzyl alcohol 12 via an ester moiety to prepare the target PROLI/NO
hybrid ester prodrug 14.
PBS and serum. Two plausible pathways for the ester hydrolysis of
hybrid O2-(acetoxymethyl)-1-(2-carboxypyrrolidin-1-yl)diazen-1-
ium-1,2-diolate ester prodrugs and the subsequent release of acetic
acid, formaldehyde, two molecules of NO, and the natural amino
In vitro COX enzyme inhibition studies (Table 1) showed that
the hydroxymethyl (CH2OH) compound 12 (COX-1 IC50 = 33.1
COX-2 IC50 = 4.2 M) showed weaker COX-2 inhibitory activity
compared to the reference drugs rofecoxib (IC50 = 0.5 M) and
celecoxib (IC50 = 0.07 M). The observation that the hydroxy-
lM;
l
acid L
-proline were described in an earlier publication.17 Similar
l
esterase cleavage and NO release pathways for the ester prodrug
14 are illustrated in Figure 2. The prodrug 14 was designed (i) such
that the –CH2OH group of the parent compound 12 is covalently at-
tached directly to the CO2H substituent of the diazenium-1,2-diolate
(13), and (ii) subsequent cleavage of the ester groups and release of
NO would furnish the parent COX-2 inhibitor (12) and the non-toxic
l
methyl compound 12 is a weak COX-1 inhibitor is consistent with
literature data indicating that incorporation of a small substituent
at the para-position of the C-3 phenyl ring in rofecoxib (2, COX-1
IC50 > 100 l
M) increases COX-1 inhibition.6 In comparison to the
parent hydroxymethyl compound 12, which showed a COX-2
selectivity index (COX-1 IC50/COX-2 IC50) of 7.9, the PROLI/NO hy-
brid ester prodrug 14 was a highly selective COX-2 inhibitior
natural amino acid L-proline.
Vascular studies showed that none of the COX-2 inhibitors
(rofecoxib, compound 12, and compound 14) altered the potency
(IC50) of the vasoconstrictive response to phenylephrine (PE) com-
pared with the control vessels (Table 2). On the other hand, the
slope of the PE concentration-response curve for the NO donor
compound 14 was very much steeper than that of the other drugs
(Figure 3), such that the IC20 of this inhibitor was significantly
higher than that of the hydroxymethyl compound 12, or indeed
of rofecoxib (Table 2). The maximum responses of the COX-2 inhib-
itors were all significantly lower than those of the control vessels
and, although the maximum response of the NO donor compound
14 tended to be the lowest of the groups, this failed to reach signif-
icance compared with the two inhibitors. The IC50’s for ACh-
induced relaxation of pre-constricted vessels was not altered by
either rofecoxib or the hydroxymethyl compound 12 although
maximal relaxation was enhanced since the COX-2 inhibitor-
treated vessels relaxed to a greater extent than did the control ves-
sels (Table 3).
(COX-1 IC50 > 100 lM; COX-2 IC50 = 4.9 lM).
The percent NO released from the PROLI/NO hybrid ester prodrug
14 upon incubation in phosphate-buffered-saline (PBS at pH 7.4),
and in the presence of rat serum, was determined (see data in Table
1). The rate of NO release from diazen-1-ium-1,2-diolates can be
controlled by chemical manipulation such as attachment of an alkyl
substituent to the O2-position21 that furnishes stable O2-substi-
tuted-diazen-1-ium-1,2-diolates that hydrolyze slowly even in
acidic solution.22 Consistent with these observations, when the PRO-
LI/NO prodrug 14 was incubated for 1.5 h in PBS at pH 7.4, the per-
centage of NO released was 4.2% which is indicative of slow NO
release.23 On the other hand, the effect of non-specific esterases
present in rat serum with respect to NO release from 14 was sub-
stantiallyhigher (38.4%). In thisregard, non-specificserumesterases
present in rat serum cleave the hybrid prodrug ester more effec-
tively than PBS at pH 7.4. From a mechanistic perspective, it is not
possible for the hybrid ester prodrug 14 to release NO prior to cleav-
age of the terminal O2-acetoxymethyl ester group. This requirement
is consistent with the observation that O2-sodium 1-[2-(hydroxy-
methyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (15), which does
not possess an ester group that requires prior ester cleavage, re-
leased 84.5 to 85% of the theoretical maximal release of two mole-
cules of NO/molecule of the parent NO-donor compound in both
As anticipated, the novel COX-2 inhibitor NO donor compound
14 exhibited enhanced inhibition of PE-induced vasoconstriction
of isolated mesenteric arteries compared with that observed under
control conditions, after incubations with rofecoxib, and after incu-
bation with its parent hydroxymethyl compound 12. This was
manifest, not as a change in IC50, but rather as a marked inhibition
of PE-induced vasoconstriction at the lower doses of PE (IC20). This
Table 1
In vitro COX-1/COX-2 enzyme inhibition, in vivo anti-inflammatory activity, and in vitro nitric oxide release data for the 3-(4-hydroxymethylphenyl)-4-(4-methanesulfonyl-
phenyl)-5H-furan-2-one (12), the diazen-1-ium-1,2-diolate prodrug ester (14), the reference drugs rofecoxib, celecoxib, ibuprofen, and the diazen-1-ium-1,2-diolate (15)
12, R1 = OH
MeO2S
CH2R1
O
O
N
ONa
N
N
N
O
O
N
N
O
CH3
O
HO
14, R1 =
O
15
O
O
AI activityb ED50
(l
mol/kg)
NO releasedc (%)
a
a
Compound
COX-1 IC50
(lM)
COX-2 IC50
(l
M)
PBSd
Serume
12
14
15
Rofecoxib
Celecoxib
Ibuprofen
33.1
>100
—
>100
7.7
4.2
2.4
—
0.5
0.07
1.1
112.9
72.2
—
—
—
4.2
84.5
—
—
—
38.4
85.0
—
—
—
4.8f
30.9
326.7
2.9
a
The in vitro test compound concentration required to produce 50% inhibition of COX-1 or COX-2. The result (IC50
, lM) is the mean of two determinations acquired using
an ovine COX-1/COX-2 assay kit (Catalog No. 560101, Cayman Chemicals Inc., Ann Arbor, MI, USA) and the deviation from the mean is <10% of the mean value.
b
Inhibitory activity in a carrageenan-induced rat paw edema assay. The results are expressed as the ED50 value (lmol/kg) at 3 h after oral administration of the test
compound.
c
Percent of nitric oxide released based on a theoretical maximum release of 2 mol of nitric oxide/mole of the diazen-1-ium-1,2-diolate test compounds (14, 15). The result
is the mean value of 3 measurements (n = 3) where variation from the mean% value was 60.5%.
d
A solution of the test compound (2.4 mL of a 1.0 ꢀ 10ꢁ2 mM solution in phosphate buffer containing a small amount of DMSO at pH 7.4),17 was incubated at 37 °C for
1.5 h.
e
A solution of the test compound (2.4 mL of a 1.0 ꢀ 10ꢁ2 mM solution in phosphate buffer containing a small amount of DMSO at pH 7.4 to which 90
lL rat serum had been
added), was incubated at 37 °C for 1.5 h.
f
Literature data (J. Pharmacol. Exp. Ther. 1999, 290, 551).

3954
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3951–3956
O
R1
O
esterase
+
N
N
R1
O
+
N
N
+
CH3CO2H
N
O
O H
N
CH3
O
O
O
O
O
O
14
CH2O
Parent
compound
(12, R = OH)
1
2 NO
MeO2S
R1 =
CH2
esterase
R1
O
R1
O
+
N
N
N
H
N
+
O
O
O
O
O
O
N
HO
H
O
L-Proline
Figure 2. Theoretical metabolic activation (esterase hydrolysis) and nitric oxide release from the O2-(acetoxymethyl)-1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate
ester prodrug (14). The sequence in which ester cleavage and nitric oxide release may also occur in a reverse order.
Table 2
There have been very few studies of the effects of COX-2 inhibi-
Effect of rofecoxib, compound 12 and compound 14 on PE-induced constriction of
isolated small mesenteric arteries
tors on vascular reactivity of isolated resistance blood vessels. Bru-
eggeman et al24 found that celecoxib, but not rofecoxib, dilated
Compound
IC50 (M)
IC20 (M)
Maximum
response (g/mm)
preconstricted small mesenteric arteries. They attributed the vasod-
ilatory activity of celecoxib to enhancement of KCNQ potassium
currents and suppression of L-type voltage-sensitive calcium cur-
rents. There were however significant methodological differences
between that study and our own in that their vessels were pre-con-
stricted with vasopressin and studied by pressure myography,
whereas ours were preconstricted with PE and evaluated by isomet-
ric wire myography. Other studies have also reported enhanced
vasorelaxation in the presence of celecoxib: in KCl-pre-constricted
vessels from celecoxib-fed animals hypertensive rats,25 and in cor-
onary vessels of celecoxib-infused guinea pig Langendorff hearts.26
In neither of these two aforementioned studies did the authors find
rofecoxib altered vascular tone. We, on the other hand, found that
rofecoxib reduced maximal PE-induced vasoconstriction and en-
hanced maximal ACh-induced vasorelaxation. We suggest that the
unique conditions under which we conducted our studies allowed
this activity to be demonstrated. Furthermore, our in vitro studies
demonstrate that coupling of the parent hydroxymethyl drug 12
to an NO-donor moiety produces a prodrug 14 which, when prein-
cubated with isolated small arteries, significantly impairs PE-
induced vasoconstriction.
Control (n = 11)
Rofecoxib (n = 13)
12 (n = 11)
1.48 0.10 ꢀ 10ꢁ6
1.66 0.14 ꢀ 10ꢁ6
1.68 0.15 ꢀ 10ꢁ6
1.75 0.06 ꢀ 10ꢁ6
0.91 0.08
0.95 0.07
1.01 0.07
1.37 0.04#
2.13 0.23
1.32 0.14*
1.51 0.14*
1.17 0.05*
14 (n = 9)
*
Significant difference from control group (P < 0.05).
Significant difference from compound 12 (P < 0.05).
#
100
80
60
40
20
0
The AI activities exhibited by the parent rofecoxib CH2OH deriv-
ative 12, and the O2-acetoxymethyl-protected PROLI/NO prodrug
14, were determined using a carrageenan-induced rat foot paw
edema model (see data in Table 1). Upon oral administration to rats,
both compounds produced a significant AI activity with the prodrug
1e-7
1e-6
PE (Log M)
1e-5
1e-4
Figure 3. Effect of COX-2 inhibitors on concentration-response curve of isolated
mesenteric arteries to PE. Control: filled circles (n = 11); Rofecoxib: open circles
(n = 13); Compound 12: filled triangles (n = 11); Compound 14: open triangles
(n = 14). Vertical bars delineate standard error of mean.
14 (ED50 = 72.2
lmol/kg po) showing a greater AI potency than the
parent compound 12 (ED50 = 112.9
l
mol/kg po). Plausible explana-
tions for the greater potency shown by the prodrug 14 include differ-
ences in their absorption, biodistribution and/or pharmacodynamic,
profiles. In comparison, both compounds 12 and 14 were more po-
tent than the reference drug ibuprofen (ED50 = 326.7 lmol/kg po),
but less potent than the reference drug celecoxib (ED50 = 30.9 l-
Table 3
Effect of rofecoxib and compound 12 on ACh-induced relaxation of pre-constricted
(PE EC80) isolated small mesenteric arteries
Compound
IC50 (M)
Maximum response (g/mm)
mol/kg po).
In conclusion, the hitherto-unknown ester prodrug O2-acetoxy-
methyl 1-[2-[4-(4-(4-methanesulfonylphenyl)-5H-furan-2-on-3-yl)
phenylmethoxy-carbonyl]pyrrolidin-1-yl]diazen-1-ium-1,2-dio-
late (14) was synthesized27 for evaluation as a selective COX-2
inhibitor,28 NO donor,29 vascular relaxant,30 and AI31 agent. Struc-
ture-activity and biological stability studies showed that the NO do-
nor prodrug 14 (i) like rofecoxib is a selective COX-2 inhibitor, (ii) is
relatively stable in phosphate-buffered saline at pH 7 where NO re-
lease is low (4.2%), (iii) undergoes extensive cleavage of the terminal
acetoxy group by rat serum esterase(s) that is followed by a
Control (n = 11)
Rofecoxib (n = 13)
12 (n = 11)
5.9 1.6 ꢀ 10ꢁ8
8.8 1.6 ꢀ 10ꢁ8
9.5 1.6 ꢀ 10ꢁ8
0.38 0.01
0.23 0.02*
0.30 0.04*
*
Significant difference from control group (P < 0.05).
observation is consistent with the fact that the amount of NO
released from the NO donor prodrug 14 in this assay will be low
since the mesenteric artery preparation does not contain the
required esterase enzyme to release the vasorelaxant NO.

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3951–3956
3955
26. Klein, T.; Eltze, M.; Grebe, T.; Hatzelmann, A.; Kömhoff, M. Cardiovasc. Res.
2007, 75, 390.
27. Experimental procedures and spectral data for compounds 10–14.
significant release of NO (38.4%), (iv) upon preincubation with iso-
lated small arteries, significantly impairs PE-induced vasoconstric-
tion, and (v) the relatively potent AI activity exhibited by this ester
prodrug 14 support the drug design concept that covalent attach-
ment of the NO donor moiety directly to a suitably positioned
CH2OH group present in a selective COX-2 inhibitor such as rofecox-
ib offers a rational drug design approach to circumvent adverse
thrombotic and hypertensive effects associated with chronic use of
selective COX-2 inhibitors.
General: Melting points were determined on
a Thomas-Hoover capillary
apparatus and are uncorrected. Unless otherwise noted, infrared (IR) spectra
were recorded as films on NaCl plates using a Nicolet 550 Series II Magna FT-IR
spectrometer. 1H NMR spectra were measured on
a Bruker AM-300
spectrometer in CDCl3, DMSO-d6, or CDCl3 + DMSO-d6 with TMS as the
internal standard. Microanalyses were performed for C, H, N (MicroAnalytical
Service Laboratory, Department of Chemistry, University of Alberta). Nominal
mass, positive polarity, electrospray, spectra were acquired using a Water’s
Micromass ZQ 4000 mass spectrometer. Silica gel column chromatography was
performed using Merck silica gel 60 ASTM (70–230 mesh). 2-Bromo-40-
(methylsulphonyl)acetophenone
(8)32
and
O2-acetoxymethyl
1-(2-
Acknowledgements
carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate (13)17 were prepared
according to literature procedures. All other reagents, purchased from the
Aldrich Chemical Company (Milwaukee, WI), were used without further
purification. The in vivo anti-inflammatory assay was carried out using a
protocol approved by the Health Sciences Animal Welfare Committee at the
University of Alberta.
We (EEK and SK) are grateful to the Canadian Institutes of
Health Research (CIHR) for financial support of this research. The
skilled technical support of Sareh Panah in performing the vascular
relaxation studies is gratefully appreciated.
3-(para-Tolyl)-4-(4-methanesulfonylphenyl)-5H-furan-2-one
(10):
Triethyl
amine (0.7 mL, 5 mmol) was added drop wise to a mixture of 2-bromo-40-
(methylsulphonyl)acetophenone (8, 0.61 g, 2.25 mmol) and para-tolylacetic
acid (9, 0.3 g, 2 mmol) in acetonitrile (8 mL) under argon. The resulting mixture
was maintained at 25 °C for 1 hour with stirring prior to cooling to 0 °C. DBU
(0.58 mL, 3.88 mmol) was added, the mixture was stirred for 2 h at 0 °C, a
solution of 1 N HCl (7 mL) was added, and the mixture was extracted with
EtOAc (3 ꢀ 50 mL). The organic extract was dried (Na2SO4), the solvent was
removed in vacuo, and the residue was purified by elution from a silica gel
column using EtOAc-hexane (2:1, v/v) as eluant to furnish 10 (0.41 g, 63%) as a
yellow powder: mp 174–176 °C; IR (film) 3030 (C–H aromatic), 2929 (C–H
References and notes
1. Bombardier, C.; Laine, L.; Reicin, A.; Shapiro, D.; Burgos-Vargas, R.; Davies, B.;
Day, R.; Ferraz, M. B.; Hawkey, C. J.; Hochberg, M. C.; Kvien, T. K.; Schnitzer, T. J.
N. Engl. J. Med. 2000, 343, 1520.
2. Silverstein, F. E.; Faich, G.; Goldstein, J. L.; Simon, L. S.; Pincus, T.; Whelton, A.;
Makuch, R.; Eisen, G.; Agrawa, N. M.; Stenson, F. W.; Burr, A. M.; Zhao, W. W.;
Kent, J. D.; Lefkwith, J. B.; Verburg, K. M.; Geis, G. S. JAMA 2000, 284, 1247.
3. Turini, M. E.; DuBois, R. N. Ann. Rev. Med. 2002, 53, 35.
4. Rodrigues, C. R.; Veloso, M. P.; Verli, H.; Fraga, C. A.; Miranda, A. L.; Barreiro, E. J.
Curr. Med. Chem. 2002, 9, 849.
5. Penning, T. D.; Tally, J. J.; Bertenshaw, S. R.; Carter, J. S.; Collins, P. W.; Doctor, S.;
Graneto, M. J.; Lee, L. F.; Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier, D.
J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.; Koboldt,
C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.; Isakson, P. C. J.
Med. Chem. 1997, 40, 1347.
6. Prasit, P.; Wang, Z.; Brideau, C.; Chan, C. C.; Charleson, S.; Cromlish, W.; Ethier,
D.; Evans, J. F.; Ford-Hutchinson, A. W.; Gauthier, J. Y.; Gordon, R.; Guay, J.;
Gresser, M.; Kargman, S.; Kennedy, B.; Leblanc, Y.; Leger, S.; Mancini, J.; O’Neill,
G. P.; Quellet, M.; Percival, M. D.; Perrier, H.; Riendeau, D.; Rodger, I.; Tagari, P.;
Therien, M.; Vickers, P.; Wong, E.; Xu, L. J.; Young, R. N.; Zamboni, R.; Boyce, S.;
Rupniak, N.; Forrest, M.; Visco, D.; Patrick, D. Bioorg. Med. Chem. Lett. 1999, 9,
1773.
7. Talley, J. A.; Brown, D. L.; Carter, J. S.; Masferrer, M. J.; Perkins, W. E.; Rogers, R.
S.; Shaffer, A. F.; Zhang, Y. Y.; Zweifel, B. S.; Seibert, K. J. Med. Chem. 2000, 43,
775.
8. Charlier, C.; Michaux, C. Eur. J. Med. Chem. 2003, 38, 645.
9. Marnett, L. J. Can. Prev. Res. 2009, 2, 288.
10. Wallace, J. L.; Reuter, B.; Cicala, C.; McKnight, W.; Cirino, G.; Gisham, M. B.
Gastroenterology 1994, 107, 173.
11. Wallace, J. L.; Reuter, B.; Cicala, C.; McKnight, W.; Grisham, M. B.; Cirino, J.
Pharmacol. 1994, 257, 249.
12. Elliott, S. N.; McKnight, W.; Cirino, G.; Wallace, J. L. Gastroenterology 1995, 109,
524–530.
13. Muscara, M. N.; McNight, W.; Del Soldato, P.; Wallace, J. L. Pharmacol. Lett.
1998, 62, 235.
14. Dhawan, V.; Schwalb, D. J.; Shumway, M. J.; Warren, M. C.; Wexler, R. S.;
Zemtseva, I. S.; Zifcak, B. M.; Janero, D. R. Free Radical Biol. Med. 2005, 39, 1191.
15. Chegaev, K.; Lazzarato, L.; Tosco, P.; Cena, C.; Marini, E.; Rolando, B.; Carrupt,
P.-A.; Fruttero, R.; Gasco, A. J. Med. Chem. 2007, 50, 1449.
16. Abdellatif, K. R. A.; Chowdhury, M. A.; Dong, Y.; Das, D.; Yu, G.; Velázquez, C.;
Suresh, M. R.; Knaus, E. E. Bioorg. Med. Chem. 2009, 17, 5182.
17. Velázquez, C. A.; Chen, Q.-H.; Citro, M. L.; Keefer, L. K.; Knaus, E. E. J. Med. Chem.
2008, 51, 1954.
18. http://www.merckfrosst.com/mfcl/en/corporate/products/vioxx_withdrawal/
vioxx_release_Voluntary_Worldwide_Withdrawal.html
19. Leblanc, Y.; Roy, P.; Wang, Z.; Li, C. S.; Chauret, N.; Nicoll-Griffith, D. A.; Silva,
J. M.; Aubin, Y.; Yergey, J. A.; Chan, C. C.; Riendearu, D.; Brideau, C.; Gordon,
R.; Zu, L.; Webb, J.; Visco, D. M.; Prasit, P. Bioorg. Med. Chem. Lett. 2002, 12,
3317.
20. Black, W. C.; Brideau, C.; Chan, C. C.; Charleson, S.; Cromlish, W.; Gordon, R.;
Grimm, E. L.; Hughes, G.; Leger, S.; Li, C. S.; Riendeau, D.; Therien, M.; Wang, Z.;
Xu, L. J.; Prasit, P. Bioorg. Med. Chem. Lett. 2003, 13, 1195.
21. Saavedra, J. E.; Dunams, T. M.; Flippen-Anderson, J. L.; Keefer, L. K. J. Org. Chem.
1992, 57, 6134.
aliphatic), 1750 (CO), 1315, 1150 (SO2) cmꢁ1 1H NMR (CDCl3) d 2.39 (s, 3H,
;
CH3), 3.08 (s, 3H, SO2CH3), 5.19 (s, 2H, furanone CH2), 7.21 (d, J = 7.9 Hz, 2H,
tolyl H-3, H-5), 7.30 (d, J = 7.9 Hz, 2H, tolyl H-2, H-6), 7.54 (dd, J = 6.7, 1.8 Hz,
2H, methanesulfonylphenyl H-2, H-6), 7.94 (d, J = 6.7, 1.8 Hz, 2H,
methanesulfonylphenyl H-3, H-5); MS m/z (ES+) 329.08, C18H17O4S (M+H)
requires 329.38; 350.96, C18H16O4SNa (M+Na) requires 351.08.
3-(4-Bromomethylphenyl)-4-(4-methanesulfonylphenyl)-5H-furan-2-one (11): N-
Bromosuccinimide (0.641 g, 3.6 mmol) was added to a stirred solution of 10
(0.985 g, 3.0 mmol) in benzene (40 mL). Benzoyl peroxide (0.73 g, 0.3 mmol)
was added and the reaction mixture was then irradiated with light from a 100-
watt sun beam lamp for 5 h. After cooling to 25 °C, the reaction mixture was
filtered, the filtrate was washed with water and then brine, the filtrate was
dried (Na2SO4), filtered, and the solvent was removed from the filtrate in
vacuo. The residue was purified by silica gel column chromatography using a
gradient of EtOAc-hexane (1:8, v/v) to EtOAc/hexane (1:1, v/v) as eluent to give
11 (0.501 g, 41%) as a yellow powder: mp 160–163 °C; IR (film) 3030 (C–H
aromatic), 2929 (C-H aliphatic), 1755 (CO), 1310, 1150 (SO2) cmꢁ1 1H NMR
;
(CDCl3) d 3.08 (s, 3H, SO2CH3), 4.50 (s, 2H, CH2Br), 5.21 (s, 2H, furanone CH2),
7.42 (m, 4H, bromomethylphenyl H-2, H-3, H-5, H-6), 7.53 (dd, J = 6.7, 1.8 Hz,
2H, methanesulfonylphenyl H-2, H-6), 7.95 (d, J = 6.7, 1.8 Hz, 2H,
methanesulfonylphenyl H-3, H-5); MS m/z 406.96,
requires 407.28; 408.93,
18H1681BrO4S (M+H) requires 409.28; 428.91,
18H1579BrO4SNa (M+Na) requires 429.28; 430.88, C18H1581BrO4SNa (M+Na)
requires 431.28.
3-(4-Hydroxymethylphenyl)-4-(4-methanesulfonylphenyl)-5H-furan-2-one (12):
solution of the bromomethyl compound 11 (102 mg, 0.25 mmol) in
C
18H1679BrO4S (M+H)
C
C
A
acetone (4 mL) and water (0.3 mL) was refluxed for 110 h. After removal of
the solvents in vacuo, the residue was dissolved in EtOAc (20 mL), the EtOAc
fraction was dried (MgSO4), and the solvent was removed in vacuo. The residue
was purified by silica gel column chromatography using EtOAc-hexane (3:1, v/
v) as eluent to afford the alcohol 12 as a yellow powder (31 mg, 36%); mp 68–
70 °C; IR (film) 3623–3184 (OH), 3030 (C-H aromatic), 2925 (C-H aliphatic),
1750 (CO), 1305, 1148 (SO2) cmꢁ1 1H NMR (CDCl3) d 1.76 (br s, 1H, OH,
;
exchangeable with D2O), 3.08 (s, 3H, SO2CH3), 4.75 (s, 2H, CH2OH), 5.20 (s, 2H,
furanone CH2), 7.39–7.43 (m, 4H, hydroxymethylphenyl H-2, H-3, H-5, H-6),
7.53 (d, J = 8.5 Hz, 2H, methanesulfonylphenyl H-2, H-6), 7.93 (d, J = 8.5 Hz, 2H,
methanesulfonylphenyl H-3, H-5); MS m/z 344.98, C18H17O5S (M + H) requires
345.38; 366.99, C18H16O5SNa (M + Na) requires 367.38.
O2-Acetoxymethyl
1-[2-[4-(4-(4-methanesulfonylphenyl)-5H-furan-2-on-3-
yl)phenylmethoxy-carbonyl]pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (14):
A
solution of the bromomethyl compound 11 (273 mg, 0.67 mmol) in DMSO
(1 mL) and Et3N (0.08 mL, 0.67 mmol) was stirred at 25 °C for 5 minutes. A
solution of compound 13 (166 mg, 0.67 mmol) in DMSO (1 mL) was added and
the reaction was allowed to proceed for 36 h at 25 °C with stirring. Ethyl
acetate (30 mL) was added to dilute the reaction mixture, the organic phase
was washed with water (5 ꢀ 10 mL), dried (MgSO4), and the solvent from the
organic fraction was removed in vacuo. The residue was purified by silica gel
column chromatography using EtOAc-hexane (2:1, v/v) as eluent to give 14 as
a white powder (115 mg, 30%): mp 75–77 °C; IR (film) 3030 (C-H aromatic),
2920 (C-H aliphatic), 1755 (CO), 1311, 1148 (SO2), 1221, 1086 (N = N-O) cm1;
1H NMR (CDCl3) d 2.06ꢁ2.10 (m, 3H, pyrrolidin-1-yl H-3, H-4, H0-4), 2.08 (s, 3H,
COCH3), 2.30ꢁ2.39 (m, 1H, pyrrolidin-1-yl H0-3), 3.10 (s, 3H, SO2CH3),
3.73ꢁ3.81 (m, 1H, pyrrolidin-1-yl H-5), 3.85ꢁ3.93 (m, 1H, pyrrolidin-1-yl H0-
5), 4.66 (dd, J = 8.5, 3.2 Hz, 1H, pyrrolidin-1-yl H-2), 5.19 (d, J = 12.8 Hz, 1H, -
CHH0OCO), 5.21 (s, 2H, furanone CH2), 5.26 (d, J = 12.8, 1H, CHH0OCO), 5.70 (d,
22. Hrabie, J. A.; Klose, J. R.; Wink, D. A.; Keefer, L. K. J. Org. Chem. 1993, 58, 1472.
23. Saavedra, J. E.; Shami, P. J.; Wang, L. Y.; Davies, K. M.; Booth, M. N.; Citro, M. L.;
Keefer, L. K. J. Med. Chem. 2000, 43, 261.
24. Brueggermann, L. I.; Mackie, A. R.; Mani, B. K.; Cribbs, L. L.; Byron, K. L. Mol.
Pharm. 2009, 76, 1053.
25. Hermann, M.; Camici, G.; Fratton, A.; Hurlimann, D.; Tanner, F. C.; Hellermann,
J. P.; Fiedler, M.; Thiery, J.; Neidhart, M.; Gay, R. E.; Gay, S.; Lüscher, T. F.;
Ruschitzka, F. Circulation 2003, 108, 2308.

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K. R. A. Abdellatif et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3951–3956
J = 7.4, 1H, OCHH0O), 5.73 (d, J = 7.4, 1H, OCHH0O), 7.41 (m, 4H, benzyl H-2, H-3,
H-5, H-6), 7.52 (d, J = 8.5 Hz, 2H, methanesulfonylphenyl H-2, H-6), 7.96 (d,
J = 8.5 Hz, 2H, methanesulfonylphenyl H-3, H-5); MS m/z 595.93,
buffered phosphate saline (Concentration in mmol Lꢁ1: 142 NaCl, 4.7 KCl, 1.17
MgSO4, 1.56 CaCl2, 1.18 K2PO4, 10 HEPES and 5.5 glucose, @ pH 7.4).
Vascular reactivity: Isolated mesenteric arteries were mounted on an isometric
wire myograph system (Kent Scientific, Litchfield, CA, USA) and pretreated
C26H27N3O10SNa (M + Na) requires 596.14. Anal. Calcd for C26H27N3O10S.1/
7H2O: C, 54.24; H, 4.78; N, 7.30. Found: C, 54.70; H, 5.23; N, 6.84.
28. Cyclooxygenase inhibition assays: The ability of the test compounds listed in
with rofecoxib (1 lM), Compound 12 (1 lM), compound 14 (1 lM) or DMSO
for 45 min. Constrictive responses to phenylephrine (PE, 10ꢁ7ꢁ5 ꢀ 10ꢁ5 M)
were then measured as previously described (Andrew, P. S.; Kaufman, S. Am. J.
Physiol. Regul. Integr. Comp. Physiol. 2003, 284, R567). Separate arterial
segments were pretreated with the inhibitors, and constricted with PE at a
Table 1 to inhibit ovine COX-1 and human recombinant COX-2 (IC50 value, lM)
was determined using an enzyme immuno assay (EIA) kit (catalog no. 560131,
Cayman Chemical, Ann Arbor, MI, USA) according to our previously reported
method (Rao, P. N. P.; Amini, M.; Li, H.; Habeeb, A.; Knaus, E. E. J. Med. Chem.
2003, 46, 4872).
submaximal dose of
1 lM (EC80). After reaching a plateau contraction,
cumulative concentration response curves to acetylcholine (ACh, 10ꢁ10 to
10ꢁ4 M) were obtained to evaluate endothelium-dependent relaxation (Tawfik,
H. E.; Cena, J.; Schulz, R.; Kaufman, S. Am. J. Physiol. Heart Circ. Physiol. 2008,
295, H1736).
29. Nitric oxide release assays: In vitro nitric oxide release, upon incubation of the
test compound at 37 °C for 1.5 hour with either 2.4 mL of a 1.0 ꢀ 10ꢁ2 mM
solution in phosphate buffer at pH 7.4, or with 2.4 mL of a 1.0 ꢀ 10ꢁ2 mM
solution in phosphate buffer at pH 7.4 to which 90
lL
rat serum had
Statistical analysis: Between groups variation was assessed using one-way
ANOVA, followed by post hoc analysis with the Student-Newman-Keuls test..
31. In vivo anti-inflammatory assay: The test compounds 12, 14, and the reference
drugs celecoxib and ibuprofen were evaluated using the in vivo carrageenan-
induced rat foot paw edema model reported previously (Winter, C. A.; Risley, E.
A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544).
been added, was determined by quantification of nitrite produced by the
reaction of nitric oxide with oxygen and water using the Griess reaction. Nitric
oxide release data were acquired for test compounds (14–15) using the
reported procedures (Velázquez, C.; Vo, D.; Knaus, E. E. Drug Dev. Res. 2003, 60,
204).
30. Preparation of isolated vessels: Adult male rats (Long Evans) were decapitated
and a segment of the small intestine and attached mesentery was isolated
(ꢂ10 cm from the ileal-cecal junction). Second order vessels, <250 mm in
diameter and ꢂ2 mm in length, were dissected out in cold (0–4 °C) HEPES-
32. Huang, H. C.; Li, J. J.; Garland, D. J.; Chamberlain, T. S.; Reinhard, E. J.; Manning,
R. E.; Seibert, K.; Koboldt, C. M.; Gregory, S. A.; Anderson, G. D.; Veenhuizen, A.
W.; Zhang, Y.; Perkins,W. E.; Burton,E. G.; Cogburn, J. N.; Isakson, P. C.; Reitz D.
B. .J. Med. Chem. 1996, 39, 253.