Dinitroglyceryl and diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs of aspirin, indomethacin and ibuprofen: Synthesis, biological evaluation and nitric oxide release studies

Article abstract of DOI:10.1016/j.bmcl.2009.04.059

A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) ester prodrugs (NONO-NSAIDs) wherein a 1,3-dinitrooxy-2-propyl (12a-c), or O²-acetoxymethyl-1-[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-1, 2-diolate (14a-c), NO-donor moiety is directly attached to the carboxylic acid group of aspirin, indomethacin or ibuprofen were synthesized. NO release from the dinitrooxypropyl, or diazen-1-ium-1,2-diolate, ester prodrugs was increased substantially upon incubation in the presence of l-cysteine (12a-c) or rat serum (14a-c). The ester prodrugs (12a-c, 14a-c), which did not inhibit the COX-1 isozyme, exhibited modest inhibitory activity against the COX-2 isozyme. The NONO-NSAIDs 12a-c and 14a-c exhibited in vivo AI activity that was similar to that exhibited by the parent drug aspirin, indomethacin or ibuprofen when the same oral dose (μmol/kg) was administered. These similarities in oral potency profiles suggest these NONO-NSAIDs act as classical prodrugs that require metabolic activation by esterase-mediated hydrolysis. Hybrid NO-donor/anti-inflammatory prodrugs of this type (NONO-NSAIDs) offer a potential drug design concept targeted toward the development of anti-inflammatory drugs with reduced adverse gastrointestinal effects.

Full text of DOI:10.1016/j.bmcl.2009.04.059

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3014–3018
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dinitroglyceryl and diazen-1-ium-1,2-diolated nitric oxide donor ester
prodrugs of aspirin, indomethacin and ibuprofen: Synthesis, biological
evaluation and nitric oxide release studies
Khaled R. A. Abdellatif, Morshed Alam Chowdhury, Ying Dong, Dipankar Das, Gang Yu,
*
Carlos A. Velázquez, Mavanur R. Suresh, Edward E. Knaus
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alta, Canada T6G 2N8
a r t i c l e i n f o
a b s t r a c t
Article history:
A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) ester prodrugs (NONO-NSAIDs)
wherein a 1,3-dinitrooxy-2-propyl (12a–c), or O2-acetoxymethyl-1-[2-(methyl)pyrrolidin-1-yl]diazen-1-
ium-1,2-diolate (14a–c), NO-donor moiety is directly attached to the carboxylic acid group of aspirin,
indomethacin or ibuprofen were synthesized. NO release from the dinitrooxypropyl, or diazen-1-ium-
Received 24 March 2009
Revised 6 April 2009
Accepted 7 April 2009
Available online 20 April 2009
1,2-diolate, ester prodrugs was increased substantially upon incubation in the presence of L-cysteine
(12a–c) or rat serum (14a–c). The ester prodrugs (12a–c, 14a–c), which did not inhibit the COX-1 iso-
zyme, exhibited modest inhibitory activity against the COX-2 isozyme. The NONO-NSAIDs 12a–c and
14a–c exhibited in vivo AI activity that was similar to that exhibited by the parent drug aspirin, indo-
Keywords:
NSAIDs
Nitrate and diazen-1-ium-1,2-diolate nitric
oxide donor ester prodrugs
Cyclooxygenase inhibition
Anti-inflammatory activity
methacin or ibuprofen when the same oral dose (lmol/kg) was administered. These similarities in oral
potency profiles suggest these NONO-NSAIDs act as classical prodrugs that require metabolic activation
by esterase-mediated hydrolysis. Hybrid NO-donor/anti-inflammatory prodrugs of this type (NONO-NSA-
IDs) offer a potential drug design concept targeted toward the development of anti-inflammatory drugs
with reduced adverse gastrointestinal effects.
Ó 2009 Elsevier Ltd. All rights reserved.
Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspir-
in, indomethacin and ibuprofen are widely used for the treatment
of pain, fever and inflammation. The major mechanism by which
NSAIDs exert their ant-inflammatory activity, inhibition of cyclo-
oxygenase-derived prostaglandin synthesis, is also responsible for
the gastrointestinal (GI),1–5 renal,6–8 and hepatic9 side effects ob-
served in patients undergoing long-term treatment. The most
common side effect is the propensity of NSAIDs to induce gastric
or intestinal ulceration. Thus, the patients who use NSAIDs on a
chronic basis have about three times greater relative risk for
serious adverse gastrointestinal events compared to the general
population of non-users.10 A variety of approaches have been
investigated to prevent and/or treat NSAID-induced gastroenter-
opathy. Among these diverse strategies, prodrugs wherein a nitric
oxide (NO)-donating moiety is covalently attached to NSAIDs
have been designed with the aim of reducing gastrointestinal
toxicity by exploiting the protective effect of locally released
NO on the gastric mucosa.11 Accordingly, we showed that a novel
group of hybrid NO-releasing NONO-NSAID ester prodrugs pos-
sessing a 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate or 1-(N,N-
dimethylamino) diazen-1-ium-1,2-diolate moiety attached via a
one-carbon spacer, or an O2-acetoxymethyl-1-(N-ethyl-N-methyl-
amino)diazen-1-ium-1,2-diolate moiety attached directly to the
carboxylic acid group of aspirin (1a–c), ibuprofen (2a–c) and
indomethacin (3a–c) showed approximately equipotent anti-
inflammatory activity to the parent NSAID, without significant
gastric toxicity when administered orally.12,13 (see structures in
Fig. 1). Studies carried out by others have shown that nitrate-
based NO-NSAIDs such as NO-aspirin (4, 5),10,11 NO-indomethacin
(6, 7),14,15 and NO-ibuprofen (8),15 are gastrointestinal sparing
while simultaneously suppressing prostaglandin synthesis similar
to the parent drugs. As part of our ongoing research program to
develop anti-inflammatory agents devoid of side effects, we
now report the synthesis, in vitro COX-1/COX-2 inhibitory activ-
ity, in vivo antiinflammatory activity and nitric oxide release data
for a group of ester prodrugs of aspirin, indomethacin or ibupro-
fen that possess alternative dinitroglyceryl (12a–c) or diazen-1-
ium-1,2-diolate (14a–c) NONO-donor moieties.
A group of ester prodrugs possessing 1,3-dinitrooxy-2-propyl
12a–c and O2-acetoxymethyl-1-[2-(methyl)pyrrolidin-1-yl]dia-
zen-1-ium-1,2-diolate 14a–c ester substituents were synthesized
using the reaction sequence illustrated in Scheme 1. Accordingly,
aspirin (9a), indomethacin (9b) and ibuprofen (9c) were converted
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.059

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3014–3018
3015
OAc
OR
Cl
O
O
O
N
Me
-CH2O
N
+
;
-CH2O
N
+
a, R =
c, R =
b, R =
CH3
OR
N
N
O
N
N
MeO
1 (NONO-Aspirin)
Me
CH3
O
O
OR
N
+
O
O
Me
3 (NONO-Indomethacin)
N
N
O
Me
O
i-Bu
2 (NONO-Ibuprofen)
Cl
O
O
O
O
ONO2
OAc
N
CH3
OR
OR
O
MeO
ONO2
R =
O
CH3
O
4 (NCX 4016, NO-Aspirin)
6 (NO-Indomethacin)
5 (NCX 4215, NO-Asprin)
Cl
O
CH3
OR
OH
N
CH3
ONO2
R =
MeO
OR
O
i-Bu
O
8 (NO-Ibuprofen)
7 (NO-Indomethacin)
Figure 1. Chemical structures of some diazen-1-ium-1,2-diolate ester prodrugs of aspirin (1a–c), ibuprofen (2a–c) and indomethacin (3a–c), the 3-(nitrooxymethylphenyl)
ester of aspirin (4), some nitrooxybutyl ester prodrugs of aspirin (5) and indomethacin (6), and the nitroglyceryl esters of indomethacin (7) and ibuprofen (8).
to the respective acid chloride 10a–c upon treatment with either
thionyl chloride or oxalyl chloride. Reaction of each acid chloride
10a–c with 1,3-dinitrooxy-2-propanol (11) in dry THF, in the pres-
ence of the non-nucleophilic base triethylamine, furnished the
respective dinitrate ester 12a–c in 19–35% yield. The correspond-
ing O2-acetoxymethyl-1-[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-1,
2-diolate esters 14a–c were prepared in higher yields (51–78%)
using the same method employing O2-acetoxymethyl 1-[2-
(hydroxymethyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (13) in
place of 1,3-dinitrooxy-2-propanol (11).
A group of new hybrid nonsteroidal anti-inflammatory prodrugs,
derived from aspirin (12a, 14a), indomethacin (12b, 14b) and
ibuprofen (12c, 14c) possessing a 1,3-dinitrooxy-2-propyl, or
O2-acetoxymethyl-1-[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-
1,2-diolate, NO-donor moiety were synthesized. The 1,3-dinitrooxy-
2-propyl esters 12a-c were designed (i) to have two NO-donor
nitrooxygroups compared tooneNO-donormoietyinthepreviously
reported NO-NSAIDs 4-8 (see structures in Fig. 1),10,11,14,15 and (ii)
following complete release of NO from the 1,3-dinitrooxy-2-propyl
group in conjunction with cleavage of the ester group would release
the parent NSAID 9a–c and the non-toxic glycerol (11) as illustrated
in Figure 2A. The diazen-1-ium-1,2-diolates 14a–c were designed (i)
such that the NSAID (9a–c) carboxyl group is covalently attached di-
rectly to the alcohol substituent of the diazenium-1,2-diolate (15),
and (ii) subsequent cleavage of the ester groups and release of NO
would furnish the parent NSAID (9a–c) and the natural amino alco-
the COX-1 isozyme at the highest test compound concentration
used (100 M). In contrast, this same group of compounds exhib-
ited COX-2 selectivity (COX-2 S.I. = 13.0–166.7 range) with COX-2
inhibitory activities (IC50 = 0.6–9.3 M range) that were generally
lower than that of the parent drugs aspirin, indomethacin and ibu-
profen showing COX-2 IC50 values ( M) of 2.4, 5.7 and 1.1, respec-
l
l
l
tively. These COX isozyme inhibition data indicate that attachment
of a NO-releasing 1,3-dinitrooxy-2-propyl, or O2-acetoxymethyl-
1-[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate, moiety to
the parent NSAID (9a–c) completely abolished COX-1 inhibitory
activity and decreased COX-2 inhibitory activity. The only amino
acid residue lining the COX binding site that is not identical be-
tween COX-1 and COX-2 is at position 523 where COX-2 has Val
and COX-1 has Ile. This difference changes the size and shape of
the NSAID binding site for COX-1 and COX-2. In this regard, the
COX-2 enzyme has a second internal pocket extending off the
NSAID binding site. In COX-2, the volume of the primary inhibitor
binding site and the secondary pocket (394 Å3) is about 25% larger
than the COX-1 binding site (316 Å3) due to the presence of Val523
in COX-2 relative to Ile523 in COX-1. It is plausible that these dif-
ferences in size, shape and conformation of the COX isozyme bind-
ing sites may explain the differences in COX isozyme inhibition
observed.16,17 However, when the hybrid compounds 12a–c and
14a–c were administered orally to rats, data acquired using the
carrageenan-induced rat paw edema assay to determine anti-
inflammatory activity (see data in Table 1) showed that the % inhi-
bition of anti-inflammatory activity was similar for the same oral
hol
In vitro COX-1/COX-2 enzyme inhibition studies (Table 1)
showed that none of the NONO-NSAIDs (12a–c, 14a–c) inhibited
L-prolinol (Fig. 2B).
dose (lmol/kg) of the parent drug aspirin, indomethacin or ibupro-
fen (9a–c) and the respective NONO-NSAID (12a–c, 14a–c). These

3016
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3014–3018
zen-1-ium-1,2-diolates are stable compounds that hydrolyze
O
C
O
C
O
C
CH2ONO2
CH2ONO2
b, 11
a
slowly even in acidic solution.19 The percent of NO released from
the hybrid O2-acetoxymethyl-1-[2-(methyl)pyrrolidin-1-yl]dia-
zen-1-ium-1,2-diolate ester prodrugs (14a–c) upon incubation in
phosphate-buffered-saline (PBS at pH 7.4), varied over a narrow
range (4.4–7.9%) which is indicative of slow NO release.20 In con-
trast, the effect of non-specific esterases present in rat serum on
the NO release properties of NONO-NSAIDs 14a–c was substan-
tially higher (50.6–80.7% range). These data indicate the non-spe-
cific serum esterases present in rat serum cleave these hybrid
prodrug esters more effectively than PBS at pH 7.4. From a mech-
anistic perspective, the hybrid ester prodrugs 14a–c cannot release
NO prior to cleavage of the terminal O2-acetoxymethyl ester group.
This requirement is consistent with the observation that O2-so-
dium 1-[2-(hydroxymethyl)pyrrolidin-1-yl]diazen-1-ium-1,2-dio-
late (15), which does not possess an ester group that requires
prior ester cleavage, releases 84.5 and 84.8% of the theoretical
maximal release of two molecules of NO/molecule of the parent
NO donor in PBS and serum, respectively. Comparative studies
showed that the extent of NO release upon incubation of the hy-
brid 1,3-dinitrooxy-2-propyl ester prodrugs (12a–c) in PBS at a
pH of 7.4 was lower (3.4–3.8% range) than the corresponding
O2-acetoxymethyl-1-[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-1,
2-diolate ester prodrugs (14a–c, 4.4–7.9% range). The percentage of
NO released from the 1,3-dinitrooxy-2-propyl compounds 12a–c
R
OH
R
Cl
R
O
10a-c
9a-c
12a-c
b, 13
O
N
N
O
R
N
O
CH3
O
O
O
14a-c
H3C
CH3
O
N
OCOCH3
CH3
CH3
a
Cl
b
c
O
was about twofold higher in the presence of 5 mM
L-cysteine
ONO2
ONO2
N
N
+
(6.8–7.8% range) than in the absence of -cysteine. This latter
L
N
CH3
O
O
observation is consistent with reports that NO release from organic
nitrates is facilitated by thiols.21,22 The lower NO release from the
nitrate esters 12a–c is attributed to the fact that production of NO
from nitrate esters requires a demanding three-electron reduction
(redox activation).23 Theoretical metabolic activation pathways for
the release of the parent NSAID 9a-c, and the release of NO, from
1,3-dinitrooxy-2-propyl esters (12a–c, Path A) and the O2-acetoxy-
methyl-1-[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate es-
ters (14a–c, Path B) are illustrated in Figure 2.
In conclusion, a group of hybrid NO releasing ester prodrugs
possessing a 1,3-dinitrooxy-2-propyl (12a–c), or O2-acetoxy-
methyl-1-[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate
(14a–c), moiety attached directly to the carboxylic acid group of
aspirin, indomethacin and ibuprofen were synthesized24 for evalu-
O
HO
11
HO
13
Scheme 1. Reagents and conditions: (a) 9a, 9c: SOCl2, benzene, reflux 3–6 h; 9b:
(COCl)2, CH2Cl2, 25 °C, 12 h; (b) Et3N, THF, 25 °C, 24–48 h.
similarities in oral potency profiles between the parent NSAIDs
9a–c, and the respective NONO-NSAID ester, suggest that 12a–c
and 14a–c act as classical prodrugs that require metabolic activa-
tion by esterase-mediated hydrolysis.
The rate of NO release from diazen-1-ium-1,2-diolates can be
decreased by chemical modification involving the attachment of
alkyl substituents to the O2-position.18 Thus, O2-substituted-dia-
2 NO
CH2OH
HO CH
CH2OH
Glycerol
CH2ONO2
HO CH
CH2ONO2
CH2ONO2
CH
O
C
O
C
A
B
esterase
+
R
OH
R
O
Parent
NSAID (9a-c)
CH2ONO2
12a-c
O
esterase
N
N
+
N
N
+
O
O
+
CH3CO2H
R
R
N
CH3
N
O
O
R
O
O
H
O
O
O
O
14a-c
CH2O
O
C
R
OH
Parent
2 NO
NSAID (9a-c)
+
N
+
N
esterase
O
N
O
R
N
O
H
O
O
O
N
HO
H
L-Prolinol
Figure 2. Theoretical metabolic activation (esterase hydrolysis) and nitric oxide release from the 1,3-dinitrooxy-2-propyl esters (12a–c, Path A), and the O2-acetoxymethyl-1-
[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate esters (14a–c, Path B). The sequence of esterase cleavage and nitric oxide release may also occur in the reverse order.

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3014–3018
3017
Table 1
In vitro COX-1 and COX-2 inhibition, percent (%) nitric oxide release, and anti-inflammatory (AI) data for dinitroglyceryl esters (12a–c), diazeniumdiolate esters (14a–c), O2-
sodium 1-[2-(hydroxymethyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (15), glycerol trinitrate (GTN, 16) and the parent NSAIDs, aspirin, indomethacin and ibuprofen
O
CH2ONO2
CHONO2
CH2ONO2
O
C
CH2ONO2
CH2ONO2
N
N
O
R
N
N
HO
N
O
CH3
R
O
O
O
N
O
ONa
O
12a-c
16 (GTN)
14a-c
15
Compound
IC50
(l
M)a
COX-2 S.I.b
% NO releasedc
Serume
AI activity % inhibition (umol/kg)g
COX-1
COX-2
PBSd
L
-Cysteinef
12a
12b
12c
14a
14b
14c
15
16 (GTN)
Aspirin
Indomethacin
Ibuprofen
>100
> 100
> 100
> 100
> 100
> 100
—
—
0.3
0.1
2.9
7.7
0.6
1.7
2.2
9.3
6.4
—
> 13
> 166
> 58
> 45
> 10
> 15
—
—
2.4
0.02
2.4
3.6
3.8
3.4
7.4
7.9
4.4
84.5
2.8
—
—
—
—
50.6
80.7
79.5
84.8
—
7.8
7.4
6.8
—
—
—
—
10.1
—
—
—
30.5 (357)
42.2 (11.7)
59.1 (327)
23.7 (357)
53.6 (11.7)
57.1 (327)
—
—
—
2.4h
5.7h
1.1h
—
—
—
50 (714)
50 (11.7)
50 (327)
—
—
a
The in vitro test compound concentration required to produce 50% inhibition of ovine COX-1 or human recombinant COX-2. The result (IC50, lM) is the mean of two
determinations acquired using the enzyme immuno assay kit (Catalog No. 560131, Cayman Chemicals Inc., Ann Arbor, MI, USA) and the deviation from the mean is <10% of
the mean value.
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
Percent of nitric oxide released based on a theoretical maximum release of (i) 2 mol of NO/mol of the dinitroglyceryl test compounds (12a–c), (ii) 2 mol of NO/mol of the
c
diazen-1-ium-1,2-diolate test compounds (14a–c) and (iii) 3 mol of NO/mol of glycerol trinitrate (16). The result is the mean value of three measurements (n = 3) where
variation from the mean% value was 60.2%.
d
A solution of the test compound (2.4 mL of a 1.0 ꢀ 10ꢁ2 mM solution in phosphate buffer at pH 7.4, 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 at pH 7.4 to which 90
lL rat serum had been added), was incubated at 37 °C for
1.5 h.
f
A solution of the test compound (2.4 mL of a 1.0 ꢀ 10ꢁ2 mM solution in phosphate buffer at pH 7.4 which contained 5.0 mM
L-cysteine), was incubated at 37 °C for 1.5 h.
g
Inhibitory activity in a carrageenan-induced rat paw edema assay. The results are expressed as the% inhibition of inflammation at 3 h after oral administration of the test
compound at the specified dose (lmol/kg).
h
Data acquired using ovine COX-2 (Catalog No. 56101, Cayman Chemicals Inc.).
ation as COX-1/COX-2 isozyme inhibitors,25 NO donors,26 and as
anti-inflammatory agents.27 Structure–activity and biological sta-
bility studies showed that (i) the dinitroglyceryl (12a–c), and
diazen-1-ium-1,2-diolate (14a–c), ester prodrugs do not inhibit
COX-1 but that they exhibit weak COX-2 inhibitory activity, (ii)
all prodrugs 12a–c and 14a–c show approximately equipotent
in vivo anti-inflammatory activity to the parent drugs, (iii) both
classes of prodrugs (12a–c, 14a–c) are relatively stable in phos-
phate-buffered saline at pH 7.4 where NO release is in the 3.4–
7.9% range, and specifically (iv) the O2-acetoxymethyl-1-[2-
(methyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates (14a–c) under-
go extensive ester cleavage by rat serum esterase(s) that is fol-
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Acknowledgment
We are grateful to the Canadian Institutes of Health Research
(CIHR) (MOP-14712) for financial support of this research.
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23. Csont, T.; Ferdinandy, P. Pharmacol. Ther. 2005, 105, 57.
24. Experimental procedures and spectral data for compounds 10a–c, 12a–c,
14a–c.General: Melting points were determined on a Thomas–Hoover capillary
apparatus and are uncorrected. Unless otherwise noted, infrared (IR) spectra
References
1. Cryer, B. Am. J. Gastroenterol. 2005, 100, 1694.
2. Go, M. F. Gastrointest. Endosc. Clin. N. Am. 2006, 16, 83.

3018
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3014–3018
were recorded as films on NaCl plates using a Nicolet 550 Series II Magna FT-IR
spectrometer. 1H NMR spectra were measured on
Bruker AM-300
1,3-Dinitrooxy-2-propyl 2-(4-(isobutyl)phenyl)propionate (12c): Yield, 19%; pale
yellow oil; IR (film) 2959 (C–H aromatic), 2870 (C–H aliphatic), 1753 (CO2),
1656, 1280 (ONO2) cmꢁ1 1H NMR (CDCl3) d 0.89 [d, J = 6.7 Hz, 6H, CH(CH3)2],
a
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). 1,3-Dinitrooxy-2-
propanol (11)22 and O2-acetoxymethyl 1-[2-(hydroxymethyl)pyrrolidin-1-
yl]diazen-1-ium-1,2-diolate (13)28 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.
Aspirin acid chloride (10a): Aspirin (0.72 g, 4 mmol) was added to a solution of
thionyl chloride (0.952 g, 8 mmol) in benzene (100 mL) and the mixture was
refluxed for 6 h. The solvent and excess thionyl chloride were removed under
reduced pressure. Upon cooling, the residue crystallized as white crystals
(0.73 g, 92%); mp 45–47 °C; 1H NMR (CDCl3) d 2.27 (s, 3H, CH3), 7.07 (dd, J = 8.0,
1.9 Hz, 1H, phenyl H-3), 7.31 (ddd, J = 8.1, 8.0, 1.9 Hz, 1H, phenyl H-5), 7.59
(ddd, J = 8.1, 8.0, 1.9 Hz, 1H, phenyl H-4), 8.15 (dd, J = 8.0, 1.9 Hz, 1H, phenyl H-
6).
Indomethacin acid chloride (10b): Oxalyl chloride (0.6 mL, 6.7 mmol) was added
drop wise to a solution of indomethacin (2.0 g, 5.6 mmol) in dry CH2Cl2 (20 mL)
under argon at 25 °C. The reaction mixture was stirred at 25 °C for 12 h, and the
solvent was removed in vacuo. The crude product was washed with hexane
(3 ꢀ 25 mL) and dried under vacuum to give 10b (2.05 g, 95% yield) as a pale
gray solid; mp 125–127 °C; 1H NMR (CDCl3) d 2.42 (s, 3H, CH3), 3.85 (s, 3H,
OCH3), 4.18 (s, 2H, CH2), 6.70 (dd, J = 9.1, 2.4 Hz, 1H, indolyl H-6), 6.84 (d,
J = 9.1 Hz, 1H, indolyl H-7), 6.89 (d, J = 2.4 Hz, 1H, indolyl H-4), 7.50 (dd, J = 6.9,
1.8 Hz, 2H, benzoyl H-3, H-5), 7.68 (dd, J = 6.9, 1.8 Hz, 2H, benzoyl H-2, H-6).
General procedure for the synthesis of the dinitrate esters (12a–c) and diazen-1-
ium-1,2-diolate esters (14a–c): The respective acid chloride 10a–c (1 mmol),
either 1,3-dinitrooxy-2-propanol (11, 1 mmol) or O2-acetoxymethyl 1-[2-
(hydroxymethyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (13, 1 mmol), and
triethylamine (1 mmol) were dissolved in dry tetrahydrofuran (15 mL), and the
resulting mixture was stirred at 25 °C for 24 h (12a, 12b, 14a), or for 48 h (12c,
14b, 14c). The precipitate (triethylammonium chloride) was filtered off and the
solvent was removed in vacuo. The residue was dissolved in dichloromethane,
washed with water, the organic layer was dried (Na2SO4), and the solvent was
removed in vacuo. The product was purified by silica gel column
chromatography using EtOAc/hexane (1:3, v/v) as eluent for compounds 12a,
12b, 14c, EtOAc/hexane (1:10, v/v) for compound 12c, and EtOAc/hexane (1:1,
v/v) for compounds 14a, 14b. Physical and spectral data for 12a–c and 14a–c
are listed below.
;
1.51 (d, J = 7.3 Hz, 3H, CHCH3), 1.85 [m, 1H, CH(CH3)2], 2.45 (d, J = 7.3 Hz, 2H,
CH2), 3.73 (q, J = 7.3 Hz, 1H, CHCH3), 4.46 (dd, J = 12.8, 5.8 Hz, 1H, CHH0ONO2),
4.55 (dd, J = 12.8, 5.8 Hz, 1H, CHH0ONO2), 4.61 (dd, J = 12.4, 4.0 Hz, 1H,
CHH0ONO2), 4.73 (dd, J = 12.4, 4.0 Hz, 1H, CHH0ONO2), 5.35 (m, 1H, CO2CH),
7.10 (d, J = 8.0 Hz, 2H, phenyl H-3, H-5), 7.16 (d, J = 8.0 Hz, 2H, phenyl H-2, H-
6); 13C NMR (CDCl3) d 18.1, 22.3, 30.2, 44.9, 45.0, 66.4, 69.3, 69.4, 126.9, 129.5,
136.5, 141.0, 173.5; MS 371.02 (M+1), 393.1 (M+Na); Anal. Calcd for
C16H22N2O8: C, 51.89; H, 5.99; N, 7.56. Found: C, 52.40; H, 5.99; N, 7.21.
O2-Acetoxymethyl 1-[2-(acetylsalicyloyloxymethyl)pyrrolidin-1-yl]diazen-1-ium-
1,2-diolate (14a): Yield, 51%; white gum; ½a D21:0
ꢁ47.2 (1.0200, CHCl3); IR
ꢂ
(film) 2964 (C–H aromatic), 2881 (C–H aliphatic), 1755, 1728 (CO2), 1225, 1077
(N@N–O) cmꢁ1
;
1H NMR (CDCl3) d 1.95ꢁ2.12 (m, 4H, pyrrolidin-1-yl H-3, H-4),
2.10 (s, 3H, COCH3), 2.35 (s, 3H, COCH3), 3.57 (m, 1H, pyrrolidin-1-yl H-5), 3.60
(m, 1H, pyrrolidin-1-yl H0-5), 4.42–4.46 (m, 3H, pyrrolidin-1-yl H-2, COOCH2),
5.72 (d, J = 6.6 Hz, 1H, –OCHH0OAc), 5.76 (d, J = 6.6, 1H, OCHH0OAc), 7.11 (dd,
J = 7.9, 1.2 Hz, 1H, phenyl H-3), 7.33 (ddd, J = 8.0, 7.9, 1.2 Hz, 1H, phenyl H-5),
7.58 (ddd, J = 8.0, 7.9, 1.2 Hz, 1H, phenyl H-4), 8.00 (dd, J = 8.0, 1.2 Hz, 1H,
phenyl H-6); MS 396.14 (M+1), 417.94 (M+Na). Anal. Calcd for C17H21N3O8: C,
51.64; H, 5.35; N, 10.63. Found: C, 51.73; H, 5.58; N, 10.29.
O2-Acetoxymethyl 1-{2-[2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetoxymethyl]pyrrolidin-1-yl}diazen-1-ium-1,2-diolate (14b): Yield, 78%;
yellow gum; ½a D21:0
ꢂ
ꢁ37.0 (1.0300, CHCl3); IR (film) 2979 (C–H aromatic), 2882
(C–H aliphatic), 1733, 1717 (CO2), 1684 (CO), 1225, 1075 (N@N–O) cmꢁ1
;
1H
NMR (CDCl3) d 1.65ꢁ2.04 (m, 4H, pyrrolidin-1-yl H-3, H-4), 2.11 (s, 3H, COCH3),
2.39 (s, 3H, CH3), 3.49-3.54 (m, 2H, pyrrolidin-1-yl H-5), 3.70 (s, 2H, CH2), 3.83
(s, 3H, OCH3), 4.19-4.33 (m, 3H, pyrrolidin-1-yl H-2, COOCH2), 5.73 (d,
J = 6.6 Hz, 1H, –OCHH0OAc), 5.76 (d, J = 6.6, 1H, OCHH0OAc), 6.67 (dd, J = 8.5,
2.4 Hz, 1H, indolyl H-6), 6.89 (d, J = 8.5 Hz, 1H, indolyl H-7), 6.96 (d, J = 2.4 Hz,
1H, indolyl H-4), 7.48 (dd, J = 6.7, 1.9 Hz, 2H, benzoyl H-3, H-5), 7.67 (dd,
J = 6.7, 1.9 Hz, 2H, benzoyl H-2, H-6); MS 594.96 (M+Na), 596.97 (M+2+Na).
Anal. Calcd for C27H29ClN4O8: C, 56.60; H, 5.10; N, 9.76. Found: C, 56.45; H,
5.12; N, 9.60.
O2-Acetoxymethyl 1-{2-[2-(4-isobutylphenyl)propionoyloxymethyl]pyrrolidin-1-
yl}diazen-1-ium-1,2-diolate (14c): Yield, 69%; pale yellow oil; ½a D21:0
ꢁ65.9
ꢂ
(1.0300, CHCl3); IR (film) 2961 (C–H aromatic), 2870 (C–H aliphatic), 1734,
1717 (CO2), 1222, 1073 (N@N–O) cmꢁ1 1H NMR (CDCl3) d 0.89 [d, = 6.7 Hz, 6H,
;
CH(CH3)2], 1.50 (d, J = 7.3 Hz, 3H, CHCH3), 1.53ꢁ1.88 [m, 5H, pyrrolidin-1-yl H-
3, H-4, CH(CH3)2], 2.11 (s, 3H, COCH3), 2.45 (d, J = 7.3 Hz, 2H, CH2), 3.46–3.48
(m, 2H, pyrrolidin-1-yl H-5), 3.71 (q, J = 7.3 Hz, 3H, CHCH3), 4.20–4.28 (m, 3H,
pyrrolidin-1-yl H-2, CO2CH2), 5.73 (d, J = 6.6 Hz, 1H, –OCHH0OAc), 5.77 (d,
J = 6.6, 1H, OCHH0OAc), 7.09 (d, J = 8.0 Hz, 2H, phenyl H-3, H-5), 7.19 (d,
J = 8.0 Hz, 2H, phenyl H-2, H-6); MS 422.23 (M+1), 444.08 (M+Na); Anal. Calcd
for C21H31N3O6ꢃ1/9 H2O: C, 59.56; H, 7.43; N, 9.92. Found: C, 59.92; H, 7.20; N,
9.53.
Ibuprofen acid chloride (10c): Ibuprofen (2.0 g, 9.7 mmol) was dissolved in
thionyl chloride (5 mL) and the mixture was refluxed for 3 h. Removal of the
thionyl chloride under vacuum, yielded 10c as a yellow liquid (2.07 g, 95%); 1H
NMR (CDCl3) d 0.92 [d, J = 6.7 Hz, 6H, CH(CH3)2], 1.60 (d, J = 7.3 Hz, 3H, CHCH3),
1.87 (m, 1H, CH(CH3)2), 2.49 (d, J = 7.3 Hz, 2H, CH2), 3.77 (q, J = 7.3 Hz, 1H,
CHCH3), 7.15-7.22 (m, 4H, phenyl hydrogens).
25. Cyclooxygenase inhibition assays: The ability of the test compounds listed in
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).
1,3-Dinitrooxy-2-propyl 2-acetoxybenzoate (12a): Yield, 23%; white crystals; mp
92–94 °C; IR (film) 2963 (C–H aromatic), 2904 (C–H aliphatic), 1755, 1685
(CO2), 1633, 1287 (ONO2) cmꢁ1
;
1H NMR (CDCl3) d 1.83 (s, 3H, CH3), 4.63 (m,
26. Nitric oxide release assays: In vitro nitric oxide release, upon incubation of the
4H, CH2ONO2), 4.67 (m, 1H, COOCH), 7.05 (dd, J = 8.5, 1.9 Hz, 1H, phenyl H-3),
7.22 (ddd, J = 8.5, 8.0, 1.9 Hz, 1H, phenyl H-5), 7.63 (ddd, J = 8.5, 8.0, 1.9 Hz, 1H,
phenyl H-4), 8.05 (dd, J = 8.0, 1.9 Hz, 1H, phenyl H-6); MS 345.06 (M+1), 366.87
(M+Na). Anal. Calcd for C12H12N2O10: C, 41.87; H, 3.51; N, 8.14. Found: C,
41.87; H, 3.57; N, 7.76.
test compound at 37 °C for 1.5 h 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 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 (12a–c, 14a–c, 15–16) using the
reported procedures (Velázquez, C.; Vo, D.; Knaus, E. E. Drug Dev. Res. 2003, 60,
204).
1,3-Dinitrooxy-2-propyl 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetate (12b): Yield, 35%; yellow gum; IR (film) 2962 (C–H aromatic), 2931
(C–H aliphatic), 1751 (CO2), 1647 (CO), 1615, 1285 (ONO2) cmꢁ1 1H NMR
;
(CDCl3) d 2.39 (s, 3H, CH3), 3.73 (s, 2H, CH2CO2), 3.85 (s, 3H, OCH3), 4.56 (dd,
J = 12.4, 4.4 Hz, 2H, CHH’ONO2), 4.76 (dd, J = 12.4, 4.4 Hz, 2H, CHH’ONO2), 5.39
(m, 1H, CO2CH), 6.68 (dd, J = 8.5, 2.4 Hz, 1H, indolyl H-6), 6.85 (d, J = 8.5 Hz, 1H,
indolyl H-7), 6.91 (d, J = 2.4 Hz, 1H, indolyl H-4), 7.47 (dd, J = 6.7, 1.9 Hz, 2H,
benzoyl H-3, H-5), 7.68 (dd, J = 6.7, 1.9 Hz, 2H, benzoyl H-2, H-6); MS 544.07
(M+Na). Anal. Calcd for C22H20ClN3O10: C, 50.63; H, 3.86; N, 8.05. Found: C,
50.81; H, 4.08; N, 8.11.
27. In vivo anti-inflammatory assay: The test compounds 12a–c, 14a–c, and the
reference drugs aspirin, indomethacin 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).
28. Velázquez, C. A.; Chen, Q.-H.; Citro, M. L.; Keefer, L. K.; Knaus, E. E. J. Med. Chem.
2008, 51, 1954.