Second-generation aspirin and indomethacin prodrugs possessing an O 2-(acetoxymethyl)-1-(2-carboxypyrrolidin-1-yl)diazenium-1,2-diolate nitric oxide donor moiety: Design, synthesis, biological evaluation, and nitric oxide release studies

Article abstract of DOI:10.1021/jm701450q

The carboxylic acid group of the anti-inflammatory (AI) drugs aspirin and indomethacin was covalently linked to the 1-(2-carboxypyrrolidin-1-yl)diazen-1- ium-1,2-diolate ion via a one-carbon methylene spacer to obtain two new hybrid prodrugs. The aspirin prodrug (23) was a 2.2-fold more potent AI agent than aspirin, whereas the indomethacin prodrug (26) was about 1.6-fold less potent than indomethacin. Prodrugs 23 and 26 slowly released nitric oxide (NO) upon dissolution in phosphate buffer at pH 7.4 (1.1 mol of NO/mol of compound after 43 h), but the rate and the extent of NO release were higher (1.9 mol of NO/mol of compound in 3 min or less) when the compounds were incubated in the presence of porcine liver esterase. In vivo ulcer index (UI) studies showed that the aspirin prodrug 23 (UI = 0.7) and indomethacin prodrug 26 (UI = 0) were substantially less ulcerogenic than the parent drugs aspirin (UI = 51) and indomethacin (UI = 64).

Full text of DOI:10.1021/jm701450q

1954
J. Med. Chem. 2008, 51, 1954–1961
Second-Generation Aspirin and Indomethacin Prodrugs Possessing an
O2-(Acetoxymethyl)-1-(2-carboxypyrrolidin-1-yl)diazenium-1,2-diolate Nitric Oxide Donor
Moiety: Design, Synthesis, Biological Evaluation, and Nitric Oxide Release Studies
Carlos A. Velázquez,† Qiao-Hong Chen,‡ Michael L. Citro,§ Larry K. Keefer,† and Edward E. Knaus*,‡
Chemistry Section, Laboratory of ComparatiVe Carcinogenesis and Basic Research Program, SAIC-Frederick Inc., National Cancer Institute at
Frederick, Frederick, Maryland 21702, and Faculty of Pharmacy and Pharmaceutical Sciences, UniVersity of Alberta,
Edmonton Alberta, T6G 2N8, Canada
ReceiVed NoVember 16, 2007
The carboxylic acid group of the anti-inflammatory (AI) drugs aspirin and indomethacin was covalently
linked to the 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate ion via a one-carbon methylene spacer
to obtain two new hybrid prodrugs. The aspirin prodrug (23) was a 2.2-fold more potent AI agent than
aspirin, whereas the indomethacin prodrug (26) was about 1.6-fold less potent than indomethacin. Prodrugs
23 and 26 slowly released nitric oxide (NO) upon dissolution in phosphate buffer at pH 7.4 (1.1 mol of
NO/mol of compound after 43 h), but the rate and the extent of NO release were higher (1.9 mol of NO/mol
of compound in 3 min or less) when the compounds were incubated in the presence of porcine liver esterase.
In vivo ulcer index (UI) studies showed that the aspirin prodrug 23 (UI ) 0.7) and indomethacin prodrug
26 (UI ) 0) were substantially less ulcerogenic than the parent drugs aspirin (UI ) 51) and indomethacin
(UI ) 64).
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs)a are one of
the most frequently used medications worldwide to treat pain,
fever, and inflammation. NSAIDs exert their therapeutic activity
by inhibiting cyclooxygenase-derived prostaglandin synthesis,
but this mechanism of action is inherently responsible for the
gastrointestinal (GI),1–5 renal,6–8 and hepatic9 side effects
observed in patients undergoing a long-term treatment. The most
common side effects associated with NSAID therapy are upper
GI irritation, ulceration, dyspepsia, bleeding, and in some cases
death.10 There are numerous reports describing the incidence
of NSAID-induced GI side effects, but the estimates vary greatly
depending upon the study design, patient population, drug(s)
used, dosage, and duration of treatment; nevertheless, it is clear
that NSAID-induced toxicity is a serious public-health problem
contributing significantly to morbidity and mortality.
Three different strategies have emerged to improve the safety
profile of NSAIDs: (a) the development of selective cyclooxy-
genase-2 (COX-2) inhibitors; (b) the coadministration of a
Figure 1. Chemical structures of 1 (rofecoxib) and some representative
* Corresponding author. Phone: (780)-492-5993. Fax: (780)-492-1217.
E-mail: eknaus@pharmacy.ualberta.ca.
NO-NSAIDs (organic nitrates): 2–4 and 5 (NO-diclofenac).
†
Laboratory of Comparative Carcinogenesis, National Cancer Institute.
University of Alberta.
SAIC-Frederick, Inc., National Cancer Institute.
proton pump inhibitor with the NSAID; and (c) the linkage of
a nitrate-based nitric oxide (NO)-releasing moiety to classical
NSAIDs (NO-NSAIDs). Each strategy presents a different set
of advantages and limitations. For example, despite the relatively
safe profile of COX-2 inhibitors in the GI tract, their role with
respect to the adverse cardiovascular effects reported in some
patients undergoing chronic treatment has attracted considerable
attention.11 In this regard, the adverse hypertensive effect
induced by rofecoxib (1) was the primary factor that prompted
its withdrawal from the market.12 Organic nitrate-based NO-
NSAIDs (Figure 1) such as NCX-4016 (2),13 AZD-3835 (3),14
NCX-2216 (4),15,16 and NO-diclofenac (5)17 suppress prosta-
glandin synthesis as effectively as the parent NSAIDs18–20 but
have been shown to spare the GI tract in animals and humans.
However, an important drawback to this design is the fact that
production of NO from nitrate esters requires a three-electron
‡
§
a Abbreviations: AUC, area under the curve; COX-2, cyclooxygenase-
2; DCM, dichloromethane; DMA/NO, 1-(N,N-dimethylamino)diazen-1-ium-
1,2-diolate nitric oxide donor; DMF, dimethylformamide; DMSO, dimethyl
sulfoxide; GI, gastrointestinal; cGMP, cyclic guanosine monophosphate;
2-HEMA/NO, 1-[2-(hydroxyethyl)methylamino]diazen-1-ium-1,2-diolate
ntric oxide donor; NO, nitric oxide; NOA, nitric oxide analyzer; NONO-
aspirin,O2-acetoxymethyl1-[2-(acetylsalicyloyloxymethyloxycarbonyl)pyrrolidin-
1-yl]diazen-1-ium-1,2-diolate; NONOate, N-diazen-1-ium-1,2-diolate moiety
that can theoretically release two molecules of nitric oxide; NONO-
indomethacin, O2-acetoxymethyl 1-{2-[2-(1-(4-chlorobenzoyl)-5-methoxy-
2-methyl-1H-indol-3-yl)acetoxy methoxycarbonyl]pyrrolidin-1-yl}diazen-
1-ium-1,2-diolate; NO-NSAIDs and NONO-NSAIDs, nonsteroidal anti-
inflammatory drugs that can theoretically release one and two molecules
of nitric oxide, respectively; NSAIDs, nonsteroidal anti-inflammatory drugs;
PLE, porcine liver esterase; PROLI/NO, 1-(2-carboxypyrrolidin-1-yl)diazen-
1-ium-1,2-diolate ntric oxide donor; PYRRO/NO, 1-(pyrrolidin-1-yl)diazen-
1-ium-1,2-diolate nitric oxide donor; UI, ulcer index.
10.1021/jm701450q CCC: $40.75
2008 American Chemical Society
Published on Web 02/29/2008

Second Generation Aspirin and Indomethacin Prodrugs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1955
Figure 2. Chemical structures of the 1-(pyrrolidin-1-yl)diazen-1-ium-
1,2-diolate (PYRRO/NO, 6), 1-(N,N-dimethylamino)diazen-1-ium-1,2-
diolate (DMA/NO, 7), and 1-[2-(hydroxyethyl)methylamino]diazen-1-
ium-1,2-diolate (2-HEMA/NO, 8).
Figure 4. Chemical structures of N-nitrosopyrrolidine (12), N-
nitrosodimethylamine (13), N-nitroso-N-(2-hydroxyethyl)methylamine
(14), N-nitrosoproline (15), and 1-[2-(hydroxycarbonyl)pyrrolidin-1-
yl]diazen-1-ium-1,2-diolate (PROLI/NO, 16).
Scheme 1. Synthesis of 20a
a Reagents and conditions: (a) nitric oxide (40 psi), NaOCH3/CH3OH,
THF/ether, 25 °C, 24 h; (b) CH3CO2CH2Br, K2CO3, DMSO, 25 °C, 15 h;
and (c) NaIO4, RuCl3 ·H2O, MeCN, EtOAc, H2O, 25 °C, 2 h.
Figure 3. Chemical structures of representative NONO-NSAIDs
(9-11).
fore, NONO-NSAIDs possessing a N-diazeniumdiolate would
release (upon esterase-mediated hydrolysis) not only the active
components (NSAID, NO, and/or HNO) but also 1 equiv of
the corresponding amine. Considering the toxicity of N-
nitrosopyrrolidine (12), N-nitrosodimethylamine (13), and N-
nitroso-N-(2-hydroxyethyl)methylamine (14, possible decom-
position products of the NONO-NSAIDs shown in Figure 4),30
we wanted to address this issue by developing strategies to
minimize or eliminate the risk of exposure to toxic nitrosamines
in patients who may be treated with NONO-NSAIDs. One way
to deal with this concern is to use a diazeniumdiolate ion
obtained from an amine, the N-nitroso derivative of which is
considered to be nontoxic. One such secondary amine is
L-proline. Its N-nitroso derivative (15) has been the subject of
numerous reports examining the effects of its long-term
administration to animals, but none of these studies showed it
to be tumorigenic.31–36
reduction, and this metabolic activation can decrease in ef-
ficiency on continued use of the drugs, contributing to nitrate
tolerance.21–23
To address safety and efficacy issues related to classical
NSAIDs, COX-2 selective inhibitors, and organic nitrate-based
NO-NSAIDs, our group has introduced and developed the
concept of NONO-NSAIDs, which is based on the linkage of a
N-diazen-1-ium-1,2-diolate (NONOate, ions 6-8, Figure 2)
functional group to the structure of a classical NSAID.24,25
Diazen-1-ium-1,2-diolate ions release up to 2 equiv of NO
without metabolic activation (first-order kinetics), they are
structurally diverse, and they possess a rich derivatization
chemistry that facilitates delivery of NO to specific organ and/
or tissue sites.26 These features distinguish NONO-NSAIDs
9-11 (Figure 3) from currently available nitrate-based NO-
NSAIDs which require redox activation before NO is released.
As part of our ongoing research program targeted toward the
development of improved anti-inflammatory agents with a
greater safety profile, we now report the synthesis, in vivo
analgesic and anti-inflammatory activities, nitric oxide release
data, and results from ulcerogenicity studies for two new
The spontaneous decomposition reaction of N-diazeniumdi-
olates in physiological medium releases up to 2 equiv of NO
(for secondary amine-based NONOates) or different ratios of
NO/nitroxyl (for primary amine-based NONOates);27–29 there-

1956 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Scheme 2. Synthesis of 23 and 26a
Velázquez et al.
a Reagents and conditions: (a) ClSO3CH2Cl, CH2Cl2, H2O, NaHCO3, (Bu)4NSO4H, 25 °C, 30 min; (b) TEA, DMSO, 20, 25 °C, 24 h; and (c) TEA, DMF,
17, 40-45 °C, 17 h.
compounds, O2-acetoxymethyl 1-[2-(acetylsalicyloyloxymethyl-
oxycarbonyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (23, NONO-
aspirin) and O2-acetoxymethyl 1-{2-[2-(1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl)acetoxy methoxycarbonyl]-
pyrrolidin-1-yl}diazen-1-ium-1,2-diolate (26, NONO-indometha-
cin) possessing a 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-
diolate (PROLI/NO) moiety (16). Hydrolysis of PROLI/NO
under aerobic conditions yields NO, L-proline (a naturally
occurring amino acid), nitrite/nitrate ions, and N-nitrosoproline
(15).37
sulfoxide (DMSO) to afford O2-acetoxymethyl 1-[2-(hydroxy-
methyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (19), which was
subsequently oxidized with sodium periodate and ruthenium
chloride in a mixture of water, ethyl acetate, and acetonitrile to
afford compound 20 in 83% yield.
Aspirin (21) and indomethacin (24) were reacted with
chloromethyl chlorosulfate in dichloromethane at 25 °C to
afford chloromethyl 2-(acetoxy)benzoate (22) and chloro-
methyl 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl]acetate (25), respectively, on the basis of a procedure
reported by Binderup et al.39 The target ester prodrugs 23
and 26 were synthesized in moderate to good yields (90 and
56%, respectively) by condensation of chloromethyl esters
22 or 25 with intermediate 20 by using polar aprotic solvents
such as DMSO or dimethylformamide (DMF) (Scheme 2).
Chemistry
The synthesis of O2-(acetoxymethyl) 1-[2-(hydroxycarbonyl)-
pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (20) was carried out by
a procedure patterned after that of Chakrapani et al.,38 as
illustrated in Scheme 1. Thus, reaction of L-prolinol (17) with
nitric oxide gas (40 psi) at room temperature in the presence of
sodium methoxide afforded sodium 1-[2-(hydroxymethyl)pyr-
rolidin-1-yl]diazen-1-ium-1,2-diolate (18) in 86% yield. The
sodium salt was alkylated with bromomethyl acetate in dimethyl
Results and Discussion
Two novel diazeniumdiolate-based nonsteroidal anti-inflam-
matory prodrugs (NONO-NSAIDs) possessing an O2-acetoxy-
methyl-protected PROLI/NO moiety were obtained. When

Second Generation Aspirin and Indomethacin Prodrugs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1957
Table 1. Anti-inflammatory and Analgesic Activities of Prodrugs
23, 26, and the Reference Compounds Acetylsalicylic Acid (21) and
Indomethacin (24)
Table 2. Nitric Oxide from NONO-NSAIDs 23, 26, and PROLI/NO
(16)
nitric oxide releaseda
analgesic activityb
compound
buffer pH 7.4b
t (h)c
PLEd
t (min)e
AI activity
dose % inhibition % inhibition
23
26
1.1f
1.1h
2.0j
43
45
1.9g
2.4
3.2
0.03
compound
ED50 (µmol/kg)a (µmol/kg) at 30 min at 60 min
1.8i
23
314
710
18.5
11.8
68
277
48
24.6 ( 3.8 65.8 ( 5.5
56.5 ( 9.8 64.3 ( 13.7
36.3 ( 8.7 61.5 ( 13.7
51.8 ( 3.5 67.6 ( 9.4
PROLI/NO (16)
aspirin (21)
26
indomethacin (24)
a Moles of NO/mol of test compound. b Incubated in 0.1 M phosphate-
buffered solution at pH 7.4 and 37 °C. c Time required to detect about 50%
of the theoretical total amount of NO/mol of test compound. Calculated
graphically from the corresponding time vs mol NO/min curves. d Moles
of NO released at 37 °C in the presence of porcine liver esterase (PLE, 29
units, 10 µL of a suspension in 3.2 M (NH4)2SO4, Sigma). e Time required
to detect more than 90% of the theoretical total amount of NO/mol of test
compound. Calculated graphically from the corresponding time vs mol NO/
min curves. f 10 µL of a 37 µM solution (in DMSO) injected into a flask
containing 3 mL of phosphate buffer pH 7.4. g 10 µL of a 37 µM solution
(in DMSO) injected into a flask containing 3 mL of phosphate buffer pH
8.0. h 10 µL of a 46 µM solution (in DMSO) injected into a flask containing
3 mL of phosphate buffer pH 7.4. i 10 µL of a 46 µM solution (in DMSO)
injected into a flask containing 3 mL of phosphate buffer pH 8.0. j Moles
of NO released, with a calculated half-life of 1.8 s (0.03 min) reported by
Saavedra et al.49
84
a Anti-inflammatory (AI) activity in a carrageenan-induced rat paw edema
assay. The results are expressed as the effective dose of the test compound
necessary to decrease by 50% the inflammatory response in the group of
animals (n ) 4). b Inhibitory activity in the 4% NaCl-induced rat abdominal
constriction assay. The results are expressed as mean ( SEM (n ) 4).
administered orally to rats, both prodrugs 23 and 26 produced
a significant anti-inflammatory effect in vivo. The carrageenan-
induced rat paw edema assay data (Table 1) showed a 2.2-fold
increase in potency for prodrug 23 (ED50 ) 314 µmol/kg)
compared with the reference drug aspirin (ED50 ) 710 µmol/
kg), whereas NONO-indomethacin 26 (ED50 ) 18.5 µmol/kg)
was about 1.6-fold less potent relative to indomethacin (ED50
) 11.8 µmol/kg). Plausible explanations for the observed
difference between the potency ratio for the prodrugs 23 and
26 relative to the respective aspirin and indomethacin parent
compounds include different absorption, bioconversion to the
parent compound, and biodistribution and/or pharmacodynamic
profiles. These results are consistent with previous observations
reported by our group, describing the anti-inflammatory proper-
ties of diazeniumdiolate-based NO-NSAIDs (NONO-NSAIDs)
possessing a PYRRO/NO (6, Figure 2), a DMA/NO (7, moieties
attached via a one-carbon methylene spacer to the carboxylic
acid group of traditional NSAIDs),24 or an O2-acetoxymethyl-
protected 2-HEMA/NO (8, moiety attached via a two-carbon
ethylene spacer to the carboxylic acid group of the NSAID).25
In vivo analgesic activity was determined by using a 4%
NaCl-induced rat abdominal constriction assay, and the results
are shown in Table 1. Prodrugs 23 and 26 exhibited moderate
analgesic activities (24.6 and 36.3% inhibition of writhing,
respectively) 30 min after oral administration of a 30 mg/kg
dose, compared to the reference drugs aspirin (56.5% inhibition,
50 mg/kg) and indomethacin (51.8% inhibition, 30 mg/kg);
however, at 60 min post-administration, the analgesic activity
observed for both prodrugs (23, 65.8% inhibition and 26, 61.5%
inhibition) was equipotent to that of the parent compounds
(aspirin, 64.3% inhibition and indomethacin, 67.6% inhibition).
Nevertheless, when we analyzed the correspondent doses on a
molar basis, it was evident that NONO-aspirin (68 µmol/kg
dose) and NONO-indomethacin (48 µmol/kg dose) showed an
increased potency relative to the parent NSAIDs aspirin (277
µmol/kg dose) and indomethacin (84 µmol/kg dose). On the
basis of these results, NONO-aspirin was 4-fold more potent
than aspirin, and NONO-indomethacin was about 1.7-fold more
potent than indomethacin.
(95% of NO released in 2.4-3.2 min, see Table 2). NONO-
NSAID ester prodrugs 23 and 26 were designed to possess three
different ester groups (four in the case of NONO-aspirin). Two
carboxyl groups (the one from NSAID and the one from PROLI/
NO) are linked to each other by a one-carbon methylene spacer,
forming two ester groups which bind the active components of
the prodrug (the NSAID and the NO-releasing group). The third
ester group protects the PROLI/NO moiety from releasing NO
spontaneously because of the presence of a one-carbon meth-
ylene spacer between the acetoxy group of the acetoxymethyl
protecting group and the diazen-1-ium-1,2-diolate O2-atom.
Following enzymatic hydrolysis of the acetate moiety, the O2-
(hydroxymethyl)diazen-1-ium-1,2-diolate moieties of 27 or 28
thus produced would spontaneously eliminate formaldehyde to
form the free NONOate moiety (16 or 29), which would
subsequently fragment to release two molecules of NO (Scheme
3A or B).
The most common side effects associated with the long-term
administration of NSAIDs are gastric erosions, ulcer formation,
and sometimes severe bleeding. Therefore, the potential ulcero-
genic side effects of prodrugs 23 and 26 were determined and
compared to those produced by the parent NSAIDs (Table 3).
Both NONO-NSAIDs showed an improved safety profile
relative to their NSAID counterparts. Unlike acetylsalicylic acid
(ulcer index (UI) ) 51), the NONO-aspirin 23 (UI ) 0.75)
produced only a few detectable lesions on the gastric mucosa
in the group of animals examined, and NONO-indomethacin
26 showed no visible lesions. This represents a remarkable
improvement considering that indomethacin (UI ) 64) was the
most irritant compound on a molar basis. The gastric-sparing
effect observed for NONO-NSAIDs is believed to be due (at
least in part) to the nitric oxide-releasing properties of these
compounds. Future studies will be conducted to measure COX-
derived prostaglandin levels at target tissues, cyclic guanosine
monophosphate (cGMP) concentration (guanylate cyclase activ-
ity), leukocyte recruitment, and other biochemical mechanisms
associated with cytoprotective effects.
Previously reported O2-acetoxymethyl diazen-1-ium-1,2-
diolates are relatively stable compounds that hydrolyze slowly
at pH 7.4.40 Consistent with these observations, when prodrugs
23 and 26 were incubated in phosphate buffer at pH 7.4, the
time required to detect about 1 mol of NO/mol of test compound
(50% of the total amount of NO typically obtained from
diazeniumdiolate ions) was 43-45 h, which is indicative of slow
NO release under these conditions. However, both ester prodrugs
are hydrolyzed more extensively (1.9 mol of NO/mol of
prodrug) in the presence of porcine liver esterase (PLE) with a
considerable increase in their corresponding rates of hydrolysis
Conclusions
We demonstrated that design of N-diazeniumdiolate-based
nitric oxide-releasing nonsteroidal anti-inflammatory prodrugs
(NONO-NSAIDs) represents a rational approach to improved
anti-inflammatory and analgesic agents, with markedly reduced

1958 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Velázquez et al.
Scheme 3. (A and B) Theoretical Metabolic Activation Mechanisms (Esterase-Based Hydrolysis) of Prodrug 26 (NO-indomethacin,
Shown as a Representative Example)
GI side effects. NONO-NSAIDs invoke metabolic activation
by esterase-mediated hydrolysis. The use of a second-generation
1-[2-(hydroxycarbonyl)pyrrolidin-1-yl]diazen-1-ium-1,2-di-
olate group (PROLI/NO) derived from a naturally occurring
amino acid (L-proline) proved to be an advantageous alternative
to the use of previously reported N-diazeniumdiolates. Finally,
NONO-aspirins obtained with the use of this technology could
represent a suitable alternative to the use of aspirin for the
prophylactic prevention of a wide variety of disorders where
the pharmacological inhibition of cyclooxygenase enzymes is
recommended or required, including (but not limited to)
inflammation, stroke, myocardial infarction, or colon cancer
prevention.
Experimental Section
General. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra
were acquired by using a Varian Unity Inova spectrometer. UV
spectra were recorded by using an Agilent 8453 spectrophotometer
(Agilent Technologies). IR spectra were acquired by using a Buck
Scientific M-500 spectrometer. Optical rotations were measured by
using a JASCO P-1010 polarimeter. Microanalyses were performed

Second Generation Aspirin and Indomethacin Prodrugs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1959
Table 3. Ulcer Index Assay for Compounds 23, 26, Aspirin (21),
and Indomethacin (24)
The white solid suspended in the reaction media was removed by
filtration, and the clear solution was diluted with ethyl acetate (30
mL) and water (30 mL). After shaking, the water layer was
separated and acidified to pH 2 with conc. HCl solution. One
additional extraction with ethyl acetate (20 mL) was carried out.
Combined organic phases were dried (Na2SO4), and the solvent
was evaporated. The residue (dark oil) was purified by flash
chromatography by using DCM/MeOH (95:5, v/v) as eluent to
furnish 20 (0.8 g, 83%). UV (2-propanol) λmax (ꢀ) 247 nm (7.9
mM-1 cm-1); 1H NMR (CDCl3) δ 2.02-2.17 (m, 2H, pryrrolidin-
1-yl H-4), 2.12 (s, 3H, COCH3), 2.21-2.38 (m, 2H, pyrrolidin-1-
yl H-3), 3.63-3.77 (m, 1H, pyrrolidin-1-yl H-5), 3.82-3.88 (m,
1H, pyrrolidin-1-yl H′-5), 4.61 (dd, J ) 8.9, 4.1 Hz, 1H, pyrrolidin-
1-yl H-2), 5.75 (d, J ) 7.2 Hz, 1H, -OCHH′O-), 5.78 (d, J ) 7.2,
1H, -OCHH′O-); 13C NMR (CDCl3) δ 20.8, 22.5, 27.5, 51.1, 61.8,
87.2, 169.5, 175.0.
Chloromethyl 2-(Acetoxy)benzoate (22). Acetylsalicylic acid
(21, 2.0 g, 11 mmol), dichloromethane (DCM, 10 mL), water (10
mL), sodium bicarbonate (3.5 g, 42 mmol), and tetrabutylammo-
nium hydrogen sulfate (0.4 g, 1.1 mmol) were stirred at 25 °C for
2 min. A solution of chloromethyl chlorosulfate (2.14 g, 13 mmol)
in DCM (3 mL) was added dropwise. This biphasic system was
stirred vigorously at 25 °C for 30 min; the organic phase was
separated and dried (MgSO4). After evaporation of the solvent, the
residue (2.2 g of a colorless oil) was purified by flash chromatog-
raphy by using EtOAc/hexane (1:4, v/v) as eluent to give 22 (2.1
g, 86%). 1H NMR (CDCl3) δ 3.37 (s, 3H, -CH3), 5.89 (s, 2H,
-OCH2Cl), 7.14 (dd, J ) 8.1, 1.1 Hz, phenyl H-3), 7.34 (td, J )
7.8, 1.1 Hz, 1H, phenyl H-5), 7.61 (td, J ) 7.8, 1.4 Hz, 1H, phenyl
H-4), 8.04 (dd, J ) 7.8, 1.4 Hz, 1H, phenyl H-6); 13C NMR (CDCl3)
δ 20.9, 69.0, 121.7, 124.0, 126.1, 131.9, 134.8, 151.1, 162.2, 169.5.
Anal. (C10H9ClO4) C, H.
Chloromethyl 2-[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-
1H-indol-3-yl]acetate (25). Indomethacin (24, 2.0 g, 5.6 mmol),
DCM (5 mL), water (5 mL), sodium bicarbonate (1.8 g, 21.0 mmol),
and tetrabutylammonium hydrogen sulfate (0.2 g, 0.5 mmol) were
stirred at 25 °C for 2 min. A solution of chloromethyl chlorosulfate
(1.7 g, 10.3 mmol) in DCM (3 mL) was added dropwise. This
biphasic system was stirred vigorously at 25 °C for 3 h; the organic
phase was separated and dried (MgSO4). After evaporation of the
solvent, the residue was purified by flash chromatography by using
EtOAc/hexane (1:4, v/v) as eluent to give 25 (1.7 g, 74%). 1H NMR
(CDCl3) δ 2.38 (s, 3H, CH3), 3.74 (s, 2H, CH2CO2), 3.83 (s, 3H,
OCH3), 5.71 (s, 2H, OCH2Cl), 6.68 (dd, J ) 8.9, 2.5 Hz, 1H, indolyl
H-6), 6.87 (d, J ) 8.9 Hz, 1H, indolyl H-7), 6.92 (d, J ) 2.5 Hz,
1H, indolyl H-4), 7.47 (ddd, J ) 8.6, 2.2, 1.9 Hz, 2H, benzoyl
H-3, H-5), 7.65 (ddd, J ) 8.6, 2.2, 1.9 Hz, 2H, benzoyl H-2, H-6);
13C NMR (CDCl3) δ 13.3, 30.0, 55.7, 69.0, 101.0, 111.2, 111.8,
114.9, 129.1, 130.2, 130.7, 131.1, 133.7, 136.2, 139.3, 156.0, 168.2,
168.8. Anal. (C20H17Cl2NO4) C, H, N.
compound
dose (mmol/kg)
ulcer indexa
23
1.38
1.38
0.08
0.08
0.75 ( 0.87
aspirin (21)
26
51.4 ( 9.0
0
indomethacin (24)
64.5 ( 10.5
0
control groupb
a Calculated by adding the total length (in mm) of individual ulcers in
each stomach and averaging over the number of animals (n ) 4) in each
group. Data are presented as mean total length ( SEM at 6 h after oral
administration of the test compound. b 1.0% methylcellulose solution.
by Midwest Analytic (Indianapolis, IN) and were within (0.4%
of theoretical values for all elements listed. Flash column chroma-
tography was performed by using Versapak 23 × 53 mm or 23 ×
110 mm cartridges (silica gel 20-45 µm). Nitric oxide gas was
purchased from Matheson Gas Products (Montgomeryville, PA).
Quantification of NO by chemiluminescence was determined by
using a Sievers nitric oxide analyzer (NOA) model 280i, as
previously described.41 Sodium 1-[2-(hydroxymethyl)pyrrolidin-
1-yl]diazen-1-ium-1,2-diolate (18),38 chloromethyl chlorosulfate,39
and bromomethyl acetate42 were prepared according to reported
procedures, but all other reagents (including the PLE, suspended
in 3.2 M ammonium sulfate) were purchased from Sigma-Aldrich
Chemical Co. (Milwaukee, WI) and used without further purifica-
tion. The in vivo anti-inflammatory,43 analgesic,44 and ulcer index
assays45–48 were carried out by using protocols approved by the
Health Sciences Animal Welfare Committee at the University of
Alberta.
Sodium 1-[2-(Hydroxymethyl)pyrrolidin-1-yl]diazen-1-ium-
1,2-diolate (18). (S)-(+)-2-(Hydroxymethyl)pyrrolidine (17, 10.0 g,
98 mmol) was dissolved in a 1:1 mixture of THF/diethyl ether (200
mL) and mixed with sodium methoxide (118 mmol, 22 mL of a
30% w/v solution in methanol) while being stirred at 25 °C for 5
min. This mixture was flushed with dry nitrogen for 5 min, and
the reaction was allowed to proceed under an atmosphere of nitric
oxide (40 psi internal pressure) with stirring at 25 °C for 24 h. The
product, which precipitated as a fine white powder, was isolated
by filtration and then suspended in diethyl ether (100 mL) while
being stirred for 5 min. The suspension was filtered, and the
collected solid was dried at 25 °C under reduced pressure for 4 h
to afford 18 as a fine white powder (15.5 g, 86%). mp 122-125
°C (dec.), UV (2-propanol) λmax (ꢀ) 232 nm (23.0 mM-1 cm-1);
1H NMR (NaOD-D2O) δ 1.67-2.10 (m, 4H, pyrrolidin-1-yl H-3,
H-4), 3.15-3.62 (m, 5H, CH2OH, pyrrolidin-1-yl H-2, H-5).
Intermediate 18 was used immediately after drying without further
purification.
O2-(Acetoxymethyl) 1-[2-(Hydroxymethyl)pyrrolidin-1-yl]dia-
zen-1-ium-1,2-diolate (19). The sodium diazeniumdiolate 18 (5.0
g, 27 mmol) was added to a suspension of potassium carbonate
(1.8 g, 13 mmol) in DMSO (70 mL) at 25 °C while being stirred
for 5 min. Freshly distilled bromomethyl acetate (4.1 g, 27 mmol)
was added dropwise, and the reaction was allowed to proceed at
25 °C for 15 h with stirring. Ethyl acetate (200 mL) was added to
dilute the reaction, the solids were filtered off, and the organic phase
was washed with water (5 × 80 mL) and dried (Na2SO4). The
solvent was removed in vacuo to give a liquid residue which was
purified by flash column chromatography by using EtOAc/hexane
(1:1, v/v) as eluent. Compound 19 (1.1 g, 18%) was obtained as a
pale yellow liquid. UV (2-propanol) λmax (ꢀ) 220 nm (16.7 mM-1
cm-1), 1H NMR (CDCl3) δ 1.80-2.11 (m, 4H, pyrrolidin-1-yl H-3,
H-4), 2.12 (s, 3H, OCOCH3), 3.57-3.72 (m, 3H, HOCHH′,
pyrrolidin-1-yl H-5), 3.79 (dd, J ) 11.3, 3.8 Hz, 1H, HOCHH′),
O2-Acetoxymethyl 1-[2-(Acetylsalicyloyloxymethyloxycar-
bonyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (23). Compound
20 (0.8 g, 3.2 mmol) was dissolved in DMSO (3 mL) and stirred
with triethylamine (0.4 mL, 3.2 mmol) at 25 °C for 5 min. Then,
it was added to a solution of compound 22 (0.7 g, 3.2 mmol) in
DMSO (3 mL). After the solution was stirred for 24 h at 25 °C,
ethyl acetate (100 mL) was added to dilute the reaction. The organic
phase was washed with water (5 × 30 mL), dried (MgSO4), and
evaporated under vacuum. The residue (pale yellow oil) was purified
by flash chromatography by using EtOAc/hexane (1:2, v/v) as eluent
to furnish 23 (1.2 g, 90%). UV (2-propanol) λmax (ꢀ) 208 nm (17.2
mM-1 cm-1); [R]18.8 ) -39.7 (0.0433, CHCl3); IR (neat) 2982
D
(C-H aliphatic), 1760 (CdO); 1H NMR (CDCl3) δ 1.96-2.15 (m,
3H, pyrrolidin-1-yl H-3, H-4, H′-4), 2.09 (s, 3H, COCH3),
2.27-2.34 (m, 1H, pyrrolidin-1-yl H′-3), 2.35 (s, 3H, COCH3),
3.66-3.72 (m, 1H, pyrrolidin-1-yl H-5), 3.85-3.91 (m, 1H,
pyrrolidin-1-yl H′-5), 4.61 (dd, J ) 8.8, 4.0 Hz, 1H, pyrrolidin-1-
yl H-2), 5.68 (d, J ) 7.2 Hz, 1H, -OCHH′OAc), 5.72 (d, J ) 7.2,
1H, OCHH′OAc), 5.94 (d, J ) 5.6 Hz, 1H, -CO2CHH′O2C-), 6.05
(d, J ) 5.6 Hz, 1H, -CO2CHH′O2C-), 7.12 (dd, J ) 8.0, 1.2 Hz,
1H, phenyl H-3), 7.34 (td, J ) 7.6, 1.2 Hz, 1H, phenyl H-5), 7.61
4.11-4.17 (m, 1H, pyrrolidin-1-yl H-2), 5.77 (s, 2H, OCH2O); 13
C
NMR (CDCl3) δ 20.8, 23.0, 26.9, 52.7, 63.9, 65.2, 87.24, 169.5.
O2-(Acetoxymethyl) 1-[2-(Hydroxycarbonyl)pyrrolidin-1-yl]-
diazen-1-ium-1,2-diolate (20). A solution of compound 19 (1.0 g,
4.2 mmol) in acetonitrile (8 mL), water (12 mL), and ethyl acetate
(8 mL) was stirred with sodium periodate (3.76 g, 17.6 mmol) and
ruthenium chloride monohydrate (cat. amount) at 25 °C for 2 h.

1960 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Velázquez et al.
(td, J ) 7.6, 1.6 Hz, 1H, phenyl H-4), 8.07 (dd, J ) 8.0, 1.6 Hz,
1H, phenyl H-6); 13C NMR (CDCl3) δ 20.8, 20.97, 22.2, 27.7, 50.7,
61.5, 79.6, 87.2, 121.8, 124.0, 126.1, 132.2, 134.8, 151.1, 162.9,
169.4, 169.6, 169.9. Anal. (C18H21N3O10) C, H, N.
the greater curvature of the stomach, gently rinsed with water, and
placed on ice. The number and the length of ulcers observed in
each stomach were determined by using magnifier lenses. The
severity of each gastric lesion was measured along its greatest length
(1 mm, rating of 1; 1-2 mm, rating of 2; and >2 mm, rating
according to their length in millimeter). The UI for each test
compound was calculated by adding the total length (L, in mm) of
individual ulcers in each stomach and averaging over the number
of animals in each group (n ) 4): UI ) (L1 + L2 + L3 + L4)/4.
O2-Acetoxymethyl 1-{2-[2-(1-(4-Chlorobenzoyl)-5-methoxy-
2-methyl-1H-indol-3-yl)acetoxy methoxycarbonyl]pyrrolidin-1-
yl}diazen-1-ium-1,2-diolate (26). A solution of compound 20 (1.0
g, 4.0 mmol) in DMF (4 mL) and triethylamine (0.6 mL, 4.4 mmol)
was stirred at 25 °C for 10 min, and a solution of compound 25
(1.6 g, 4.0 mmol) in DMSO (3 mL) was added. After the solution
was stirred for 17 h at 40-45 °C, the reaction mixture was diluted
with ethyl acetate (100 mL). The organic phase was washed with
water (5 × 30 mL), dried (MgSO4), and evaporated under vacuum.
The residue (yellow oil) was purified by column chromatography
by using EtOAc/hexane (1:1, v/v) as eluent to furnish 26 (1.4 g,
56%) as a clear yellow oil. UV (2-propanol) λmax (ꢀ) 247 nm (14.8
Acknowledgment. This work was supported by federal
funds from the National Cancer Institute, National Institutes of
Health (NIH) under contract N01-CO-12400 with SAIC-
Frederick Inc., as well as by the Intramural Research Program
of the NIH, Center for Cancer Research. We are also grateful
to the Canadian Institutes of Health Research (Grant no. MOP-
14712).
mM-1 cm-1); [R]18.8 ) -23.8 (0.0068, CHCl3); IR (neat) 2978
D
(C-H aliphatic), 1749 (CdO); 1H NMR (CDCl3) δ 1.70-1.78 (m,
1H, pyrrolidin-1-yl H-3), 1.90-1.97 (m, 2H, pyrrolidin-1-yl H-4),
2.10 (s, 3H, COCH3), 2.12-2.22 (m, 1H, pyrrolidin-1-yl H′-3), 2.36
(s, 3H, CH3), 3.61-3.67 (m, 1H, pyrrolidin-1-yl H-5), 3.72 (s, 2H,
CH2CO2), 3.76-3.82 (m, 1H, pyrrolidin-1-yl H′-5), 3.84 (s, 3H,
OCH3), 4.51 (dd, J ) 9.2, 4.4 Hz, 1H, pyrrolidin-1-yl H-2), 5.69
(d, J ) 7.2 Hz, 1H, -CO2CHH′O2C-), 5.72 (d, J ) 7.2 Hz, 1H,
-CO2CHH′O2C-), 5.75 (d, J ) 5.6 Hz, 1H, -OCHH′OAc), 5.85 (d,
J ) 5.6 Hz, 1H, -OCHH′OAc), 6.67 (dd, J ) 8.8, 2.4 Hz, 1H,
indolyl H-6), 6.90 (d, J ) 9.2 Hz, 1H, indolyl H-7), 6.95 (d, J )
2.4 Hz, indolyl H-4), 7.47 (ddd, J ) 8.8, 2.4, 2.0 Hz, 2H, benzoyl
H-2, H-6), 7.66 (ddd, J ) 8.4, 2.4, 1.6 Hz, 2H, benzoyl H-3, H-5);
13C NMR (CDCl3) δ 13.3, 20.8, 22.2, 27.4, 30.0, 50.7, 55.7, 61.3,
79.6, 87.1, 101.1, 111.4, 111.8, 114.9, 129.1, 130.3, 130.7, 131.1,
131.4, 133.7, 136.2, 139.3, 156.1, 168.2, 169.3, 169.4, 169.7. Anal.
(C28H29ClN4O10) C, H, N.
Anti-inflammatory Assay. The test compounds 23 and 26, as
well as the reference drugs aspirin and indomethacin were evaluated
by using the in vivo rat carrageenan-induced foot paw edema model
reported previously.43
Analgesic Activity. Analgesic activity was determined by using
a 4% sodium chloride-induced writhing (abdominal constriction)
assay previously reported.44
Nitric Oxide Release Assay. NO gas measurements were
performed by using a Sievers NOA, model 280i, by using a method
described earlier.41 The instruments were calibrated before each
experiment with nitrogen as the zero gas. Mixtures of NO/He
(certified standards, MG Industries, Morrisville, PA) at different
concentrations were injected into the reaction chamber, recording
the area under the curve (AUC) for each peak and plotting µmol
of NO vs peak area. Linear regression analysis of resultant graphs
showed correlation coefficients of 0.999 or better. Measurement
of NO released from the test compounds was performed by injecting
the prodrug dissolved in DMSO into a clean, dry, NOA measure-
ment cell (sealed with a septum) containing deoxygenated 0.1 M
phosphate-buffered solution (3 mL, pH 7.4) and 50 µM diethyl-
enetriaminepentaacetic acid. The NO generated from the samples
was carried from the phosphate-buffered solution to the NOA via
a constant nitrogen purge. Integration of the resulting AUC was
used to calculate the amount of NO released from each test
compound on the basis of the calibration curve. The experiments
with PLE (10 µL of a suspension in 3.2 M ammonium sulfate)
were carried out in phosphate-buffered solution (3 mL) at pH 8.0.
Acute Ulcerogenesis Assay. The ability to produce gastric
damage was evaluated according to reported procedures.45–48
Ulcerogenic activity was evaluated after oral administration of
aspirin (1.38 mmol/kg), indomethacin (0.08 mmol/kg), or an
equimolar amount of the corresponding test compound (23 or 26).
All drugs were suspended and administered in 1.7 mL of a 1%
methylcellulose solution. Control rats received oral administration
of vehicle (1.7 mL of 1% methylcellulose solution). Food, but not
water, was removed 24 h before administration of test compounds.
Six hours after oral administration of the drug, rats were euthanized
in a CO2 chamber, and their stomachs were removed, cut out along
Supporting Information Available: A table of combustion data
is presented for compounds 23, 26, 22, and 25. This material is
available free of charge via the Internet at http://pubs.acs.org.
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