Ethanesulfohydroxamic acid ester prodrugs of nonsteroidal anti-inflammatory drugs (NSAIDs): Synthesis, nitric oxide and nitroxyl release, cyclooxygenase inhibition, anti-inflammatory, and ulcerogenicity index studies

Article abstract of DOI:10.1021/jm101403g

The carboxylic acid group of the anti-inflammatory (AI) drugs indo-methacin, (S)-naproxen and ibuprofen was covalently linked via a two-carbon ethyl spacer to a sulfohydroxamic acid moiety (CH₂CH ₂SO₂NHOH) to furnish a group of hybrid ester prodrugs that release nitric oxide (NO) and nitroxyl (HNO). Biological data acquired for this hitherto unknown class of ethanesulfohydroxamic acid ester prodrugs showed (i) all compounds exhibited superior NO, but similar HNO, release properties relative to arylsulfohydroxamic acids, (ii) the (S)-naproxen and ibuprofen prodrug esters are more potent AI agents than their parent NSAID, (iii) the indomethacin prodrug ester, in contrast to indomethacin which is highly ulcerogenic, showed no visible stomach lesions [ulcer index (UI) = 0 for a 80 μmol/kg oral dose] while retaining potent AI activity, and iv) that the indomethacin prodrug ester, unlike indomethacin which is an ulcerogenic selective COX-1 inhibitor, is a selective COX-2 inhibitor (COX-2 selectivity index = 184) devoid of ulcerogenicity that is attributed to its high COX-2 SI and/or ability to release cytoprotective NO.

Full text of DOI:10.1021/jm101403g

ARTICLE
pubs.acs.org/jmc
Ethanesulfohydroxamic Acid Ester Prodrugs of Nonsteroidal Anti-
inflammatory Drugs (NSAIDs): Synthesis, Nitric oxide and Nitroxyl
Release, Cyclooxygenase Inhibition, Anti-inflammatory, and
Ulcerogenicity Index Studies
Zhangjian Huang,† Carlos A. Velꢀazquez,† Khaled R. A. Abdellatif,† Morshed A. Chowdhury,† Julie A. Reisz,‡
Jenna F. DuMond,‡ S. Bruce King,‡ and Edward E. Knaus*,†
†Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton Alberta T6G 2N8, Canada
‡Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109, United States
ABSTRACT: The carboxylic acid group of the anti-inflammatory (AI) drugs indo-
methacin, (S)-naproxen and ibuprofen was covalently linked via a two-carbon ethyl
spacer to a sulfohydroxamic acid moiety (CH2CH2SO2NHOH) to furnish a group of
hybrid ester prodrugs that release nitric oxide (NO) and nitroxyl (HNO). Biological
data acquired for this hitherto unknown class of ethanesulfohydroxamic acid ester
prodrugs showed (i) all compounds exhibited superior NO, but similar HNO, release properties relative to arylsulfohydroxamic
acids, (ii) the (S)-naproxen and ibuprofen prodrug esters are more potent AI agents than their parent NSAID, (iii) the indomethacin
prodrug ester, in contrast to indomethacin which is highly ulcerogenic, showed no visible stomach lesions [ulcer index (UI) = 0 for a
80 μmol/kg oral dose] while retaining potent AI activity, and iv) that the indomethacin prodrug ester, unlike indomethacin which is
an ulcerogenic selective COX-1 inhibitor, is a selective COX-2 inhibitor (COX-2 selectivity index = 184) devoid of ulcerogenicity
that is attributed to its high COX-2 SI and/or ability to release cytoprotective NO.
’ INTRODUCTION
COX-1 and COX-2 inhibitory, and in vivo anti-inflammatory
(AI), activities relative to the reference drugs celecoxib,
ibuprofen, and aspirin. The arylsulfohydroxamic acids 5 acted
as NO donors at physiogical pH that was increased in the
presence of a strong oxidant, but a nonphysiological alkaline
pH was required to release HNO.
Nonsteroidal anti-inflammatory drugs (NSAIDs) of the aryl-
(heteroaryl)acetic acid class (1-3, see Figure 1) exert their
therapeutic effects by inhibiting cyclooxygenase-derived prosta-
glandin synthesis. However, inhibition of the constitutive
cyclooxygenase-1 (COX-1) isozyme is responsible for gastro-
intestinal (GI),1,2 renal,3,4 and hepatic5 side effects often ob-
served in patients undergoing long-term treatment. Selective
cyclooxygenase-2 (COX-2) inhibitors, despite their safe profile
in the GI tract, may cause an increased risk of adverse cardio-
vascular effects such as thrombosis and stroke in some patients
undergoing chronic treatment.6
Nitric oxide (NO) is an effective vasodilation agent, an
inhibitor of platelet aggregation and adhesion,7 and it provides
a promising concept to suppress vascular side effects observed
with NSAID use.8,9 Nitroxyl (HNO) is (i) the reduced form of
nitric oxide (NO), (ii) an effective vasodilation agent and
inhibitor of platelet aggregation and adhesion like NO,10 (iii) a
positive inotropic cardiac agent,11-13 (iv) a protector against
cardiac ischemia-reperfusion injury,14 and (v) it is resistant to
superoxide radical anion.11 Benzenesulfohydroxamic acid
(PhSO2NHOH), common name of Piloty’s acid (PA), can serve
as a HNO and/or NO donor.15 In this regard, we recently
reported a novel group of nonselective COX inhibitors (5)
wherein the MeSO2 COX-2 pharmacophore present in 1,1-
diphenyl-2-(4-methanesulfonylphenyl)hex-1-ene (4) was re-
placed by a SO2NHOH NO/HNO donor prodrug moiety.16
These arylSO2NHOH compounds 5 showed moderate in vitro
The simplest alkylsulfohydroxamic acid CH3SO2NHOH is
reported to release HNO at a rate comparable to the well-
known HNO donor Angeli’s salt (sodium trioxodinitrate) at
physiological pH.17,18 Accordingly, it was of interest to in-
vestigate whether coupling a short-chain alkylsulfohydroxa-
mic acid to the CO2H group of NSAIDs would provide a
hitherto-unknown class of ester compounds that act as dual
NO and HNO donors having good NO/HNO release proper-
ties at physiological pH. In continuation of our ongoing
studies to design novel AI drugs that are devoid of adverse
ulcerogenic and/or cardiovascular side effects, we now de-
scribe the synthesis of a group of CH2CH2SO2NHOH and
CH2CH2SO2NHOMe ester prodrugs (9a-e) of the NSAIDs
indomethacin, ibuprofen, (S)-naproxen, and the reference
compound phenylacetic acid (Scheme 1), in vitro NO
and HNO release, in vitro COX-1/COX-2 inhibition, and in
vivo ulcerogenic index studies, and their evaluation as AI
agents.
Received: October 28, 2010
Published: January 31, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
ARTICLE
Nitric Oxide Release. The % NO released from the N-
hydroxy (methoxy) ethanesulfonamides 9a-e and ethanesulfo-
hydroxamic acid (EA) upon incubation in phosphate-buffered
saline (PBS at pH 7.4) or PBS containing rat serum were
measured by quantitation of nitrite using the Griess reaction
(see data in Table 1). The % NO released from the ethanesulfo-
hydroxamic acids esters 9a-d in PBS at pH 7.4 varied over a
44.5-54.3% range, which is indicative of much higher NO
release relative to that previously reported for a group of
arylsulfohydroxamic acids 5 (4.3-11.4% range).16 The % NO
release from 9a-d and EA was suppressed (1.3-4.8% range) in
PBS containing rat serum. One plausible explanation for this
reduction in NO release because NO is not expected to react with
serum thiols,11 could be due to the likely probability that the
highly lipophilic (log P = 2.1-3.4 range; see data in Table 2)
sulfohydroxamic acids 9a-b, d-e undergo strong protein bind-
ing to rat serum which results in suppressed NO release. This
observation is consistent with a similar serum effect for arylsul-
fohydroxamic acids 5.16 These data suggest that the sulfohy-
droxamic acid moiety present in the prodrugs 9a-d should
possess some degree of stability in the circulatory system. In
contrast, the N-methoxyl ethanesulfonamide 9e showed a much
lower % NO release in PBS and PBS containing rat serum (4.3%
and 0.9%, respectively) relative to the N-hydroxyl ethanesulfon-
amides (9a-d).
Indirect Assay of Nitroxyl (HNO) Release As Nitrous Oxide
(N2O). Quantitative gas chromatographic analysis of N2O,
which arises from HNO dimerization and dehydration under
anaerobic conditions (HNO þ HNO f [HONNOH] f N2O þ
H2O), was employed because direct HNO detection continues
to be difficult.22 HNO release from 9a-e and ethanesulfohy-
droxamic acid (EA) was measured using three MeOH-based
solvent mixtures (see data in Table 1) based on the fact that
methanesulfohydroxamic acid releases HNO at a rate compar-
able to Angeli’s salt at physiological pH,17,18 an alkaline pH can
enhance the amounts of HNO released from the sulfohydroxa-
mic acid moiety,23 and HNO reacts rapidly with thiols to form
disulfides and hydroxylamine or sulfinamides.24 The percentage
of N2O arising from 9a-d and ethanesulfohydroxamic acid in
MeOH/TBS was very low (<1%). In contrast, in the presence of
the base NaOH that produces a nonphysiological alkaline pH,
the % N2O produced was substantially larger (20-25% range).
In comparison, the % N2O produced in the MeOH/NaOH/
GSH solvent system was much smaller (e2% and e7% for 2 and
24 h incubations, respectively) because addition of the thiol
glutathione (GSH) reacts with HNO, thereby resulting in the
expected decrease in N2O production observed. This latter group
of experiments in which the incubation solvent system contains
GSH provides strong evidence for the release of HNO from
9a-d and the subsequent dimerization of the released HNO to
N2O.
Figure 1. Chemical structures of some representative NSAIDs (1-3), a
representative selective COX-2 inhibitory triaryl olefin (4), and an
acyclic triaryl olefin possessing a sulfohydroxamic acid dual NO/HNO
donor moiety (5).
’ RESULTS AND DISCUSSION
Chemistry. Reaction of indomethacin, (S)-naproxen, phenyl-
acetic acid, or ibuprofen with oxalyl chloride in dry CH2Cl2 and a
catalytic amount of DMF furnished the respective acid chloride
(6a, 6b, 6c, or 6d) as previously reported.19 Because indometha-
cin acid chloride (6a) is unstable at high temperature (>130 °C),
condensation of 6a with 2-hydroxyethanesulfonic acid sodium
salt was carried out using dry pyridine as solvent at 60 °C to
prepare the carbonyloxyethanesulfonic acid sodium salt (7a).20
In contrast, reaction of the (S)-naproxen, phenylacetic, and
ibuprofen acid chlorides with 2-hydroxyethanesulfonic acid
sodium salt at 130 °C in the absence of a solvent afforded the
respective carbonyloxyethanesulfonic acid sodium salt (7b, 7c, or
7d).21 Subsequent reaction of each sulfonic acid sodium salt
7a-d with thionyl chloride in DMF provided the respective
sulfonyl chloride 8a-d. Reaction of each sulfonyl chloride 8a-d
with hydroxylamine hydrochloride in the presence of potassium
carbonate in dry THF afforded the respective target products
9a-d. A similar reaction of the ethanesulfonyl chloride 8d with
methoxylamine hydrochloride in the presence of sodium bicar-
bonate in dry THF furnished the target CH2CH2SO2NHOMe
product 9e (Scheme 1).
Drug Design Rationale. In a recent study, we reported a
novel group of acyclic triaryl olefins possessing an arylsulfo-
hydroxamic acid moiety (5) which act as dual NO/HNO
donors.16 The ethanesulfohydroxamic acid ester prodrugs
9a-d described in the current investigation were designed
based on the expectation that (i) an ethanesulfohydroxamic
acid prodrug moiety would possess similar HNO and/or NO
release properties to that of methanesulfohydroxamic acid
(MeSO2NHOH), thereby providing release of HNO at phy-
siological pH, attempts to synthesize the -CH2SO2NHOH-
(OMe) analogues of the -CH2CH2SO2NHOH(OMe) com-
pounds 9a-e have to date not been successful, and (ii)
esterase induced-cleavage of the ester group (see Figure 2)
would furnish the parent NSAID indomethacin, (S)-naproxen,
or ibuprofen along with the putative hydroxyethanesulfohy-
droxamic acid (HOCH2CH2SO2NHOH).
Cyclooxygenase-1 (COX-1) and -2 Enzyme Inhibition. In
vitro COX-1/COX-2 isozyme inhibition studies (Table 2)
showed that the ethanesulfohydroxamic acids (9a-b, 9d) and
the N-methoxyl ethanesulfonamide (9e) ester prodrugs were
more potent inhibitors of COX-2 (IC50 = 0.4-15.8 μM range)
than COX-1 (IC50 = 1.1-77.7 μM range). Within this group of
compounds, the N-methoxyl ethanesulfonamide ester of ibupro-
fen 9e showed a higher COX-2 selectivity index than the parent
NSAID ibuprofen where the SI’s are 23.3 and 2.9, respectively.
The observation that the ethanesulfohydroxamic acid ester of
ibuprofen 9d showed a much lower COX-2 SI (0.07) compared
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Journal of Medicinal Chemistry
ARTICLE
Scheme 1a
a Reagents and conditions: (a) oxalyl chloride, dry CH2Cl2, DMF, 25 °C, 12 h; (b) 2-hydroxyethanesulfonic acid sodium salt, dry
pyridine, 60 °C, 24 h for 7a, 2-hydroxyethanesulfonic acid sodium salt, 130 °C, 4 h for 7b-d; (c) SOCl2, DMF, 25 °C, 1 h; (d) dry THF,
K2CO3, HO-NH2 HCl, 25 °C, 2 h for 9a-d, dry THF, NaHCO3, MeO-NH2 HCl, 25 °C, 2 h, for compound 9e.
3
3
highest test compound concentration tested (100 μM). Simi-
larly, EA did not inhibit COX-1 or COX-2 at the highest
concentration (100 μM) tested.
Anti-inflammatory Activity. AI ID50 values acquired for the
ethanesulfohydroxamic acid esters of indomethacin (9a) and
(S)-naproxen (9b), % inhibition of inflammation induced by the
phenylacetic acid ester 9c for a 100 mg/kg oral dose, and %
inhibition shown by the ibuprofen prodrug esters 9d and 9e for a
327 μmol/kg oral dose were determined using a carrageenan-
induced rat foot paw edema model (see data in Table 2). Within
this group of compounds, all ester prodrugs of NSAIDs 9a-b
and 9d-e showed potent AI activities. In this regard, the (S)-
Figure 2. Putative esterase induced cleavage of the ester group to
release the parent NSAID and 3-hydroxyethanesulfohydroxamic acid.
to the N-methoxyl analogue 9e (23.3) indicates that the N-
methoxyl group must enhance binding to the COX-2 enzyme. It
is most noteworthy that the COX-2 SI for the ethanesulfohy-
droxamic acid ester of indomethacin 9a (COX-2 SI = 184) is
completely reversed relative to the parent NSAID indomethacin,
which is a highly ulcerogenic selective COX-1 inhibitor (COX-2
SI = 0.02). The lipophilicity of 9a (log P = 3.4) and indomethacin
(log P = 3.6) are relatively similar. The molecular volume of the
primary COX-2 binding site and its associated secondary pocket
is about 25% larger (394 Å3) than the volume of the smaller
(316 Å3) COX-1 binding site.25 Accordingly, it is highly probable
that the larger molecular volume of 9a (381 Å3) compared to that
of the much smaller molecular volume of indomethacin (298 Å3)
is an important contributor to the COX-2 selectivity observed for
9a (see data in Table 2). The phenylacetic acid analogue 9c, as
expected, did not inhibit the COX-1 or COX-2 isozymes at the
naproxen analogue 9b showed a more potent AI activity (ID50
=
11.8 μmol/kg po) than (S)-naproxen (ID50 = 29.7 μmol/kg
po).26 On the other hand, the COX-2 selective indomethacin
analogue 9a is a less potent AI agent (ID50 = 19.1 μmol/kg po)
relative to indomethacin (ID50 = 11.7 μmol/kg po). Despite the
fact that the ethanesulfohydroxamic acid ester of ibuprofen 9d
(COX-2 SI = 0.07) and the N-methoxyl ethanesulfonamide ester
of ibuprofen 9e (COX-2 SI = 23.3) showed distinctly different
COX-2 selectivity indexes, 9d and 9e both exhibited more
potent, yet similar, AI activities (79.5 and 78.9% inhibition of
inflammation for a 327 μmol/kg oral dose) relative to the parent
NSAID ibuprofen (AI ID50 = 327 μmol/kg po). One plausible
explanation for this AI data, because 9e released a low 4.3% of
NO and a negligible amount of HNO (Table 1), could be due to
the likely probability that in vivo cleavage of the ester group
present in both 9d and 9e by an esterase releases a similar
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Table 1. Percent (%) Nitric Oxide and Nitrous Oxide Release from 9a-e and the Reference Compound Ethanesulfohydroxamic
Acid (EA)
% N2O release a
% NO releaseb
MeOH/TBS c
24 h
MeOH/base d
24 h
MeOH/base/GSH e
24 h
PBS f
PBS þ serum g
2 h
2 h
2 h
9a
9b
9c
9d
9e
EA
44.5
52.9
54.3
46.3
4.3
3.2
1.3
4.8
2.1
0.9
1.4
0
0
0
0
0
0
0
0
0
0
0
0
20
24
23
25
0
1
23
21
24
0
1
2
0
0
0
0
1
4
7
7
0
0
54.5
24
7
a Percent of N2O released, based on the condensation of 2 mol of HNO f 1 mol N2O þ H2O. The result is the mean value of three measurements (n =
3). The HNO donor test compound (9a-e, EA) concentration is 50 mM in each experiment unless otherwise noted. b Percent NO released based on a
theoretical maximum release of 1 mol of NO/mol of the sulfohydroxamic acid test compound (9a-e) and the reference agent ethanesulfohydroxamic
acid (EA). The result is the mean value of three measurements (n = 3) where variation from the mean % value was e0.2%. c MeOH/TBS solvent is
comprised of 0.6 mL MeOH and 0.2 mL of 700 mM Tris buffer solution (TBS) at pH 7.00. d MeOH/base solvent is comprised of 0.6 mL MeOH and 0.2
mL of 1 M NaOH. e MeOH/base/GSH experiments are 100 mM in glutathione (GSH) and 250 mM in NaOH except for compounds 9b and EA which
were 25 mM in NaOH. f A solution of the test compound (2.4 mL of a 5.0 ꢀ 10-2 mM) in phosphate buffer at pH 7.4 was incubated at 37 °C for 1.5 h. g A
solution of the test compound (2.4 mL of a 5.0 ꢀ 10-2 mM) in phosphate buffer at pH 7.4, to which 90 μL rat serum had been added, was incubated
at 37 °C for 1.5 h.
Table 2. In Vitro COX-1 and COX-2 Inhibition, Anti-inflammatory (AI), Ulcer Index (UI), log P, and Molecular Volume (Å3)
Data
IC50 (μM) a
AI activity:
ID50 (μmol/kg) c
compd
COX-1
COX-2
COX-2 SI b
ulcer index d
0
log P e
volume (Å3) f
9a
9b
9c
9d
9e
77.7
4.5
0.42
2.3
184
1.9
19.1
3.4
2.1
0.65
2.9
3.4
3.6
3.0
3.8
381
297
212
295
311
298
214
211
11.8
>100
1.1
>100
15.8
0.63
6.9
weak g
potent h
potent i
11.7
0.07
23.3
0.02
0.01
2.9
14.6
0.13
0.18
4.0
indomethacin
(S)-naproxen
ibuprofen
EA
64.5 ( 10.5
12.4
1.4
29.7
327
>100
>100
a The in vitro test compound concentration required to produce 50% inhibition of ovine COX-1 or human recombinant COX-2. The result (IC50, μM)
is the mean of two determinations acquired using the enzyme immuno assay kit (catalogue 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). c Inhibitory activity in a
carrageenan-induced rat paw edema assay. The results are expressed as the inhibitory dose (ID50 value in μmol/kg, determined from a dose-response
curve, required to decrease inflammation by 50%) at 3 h after oral administration of the test compound. d Calculated by adding the total length (in mm)
of individual ulcers in each stomach and averaging over the number of animals (n = 3) in each group. Data are presented as mean total length ( SEM at 6
h after oral administration of the test compound. e The log P value was calculated using the ChemDraw Ultra program, version 11.0, CambridgeSoft
company. f The volume of the molecule, after minimization using the molecular mechanics geometry optimization module, was calculated using the
Alchemy 2000 program, Tripos Inc. g A 12.4% (n = 3, SEM = 4.6) inhibition of inflammation was observed for a 100 mg/kg oral dose. h A 79.5% (n = 3,
SEM = 2.4) inhibition of inflammation was observed for a 327 μmol/kg oral dose. i A 78.9% (n = 3, SEM = 9.2) inhibition of inflammation was observed
for a 327 μmol/kg oral dose.
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quantity of ibuprofen thereby providing similar AI activities. The
phenylacetic acid ester 9c, as expected, exhibited weak AI activity
(12.4% inhibition of inflammation for a 100 mg/kg po dose).
Ulcer Index (UI) Assay. The most common side effects
associated with the chronic use of NSAIDs such as indomethacin
include the development of gastric erosions, ulcer formation, and
sometimes severe bleeding that is attributed to inhibition of the
constitutive COX-1 isozyme. It was therefore anticipated that the
ethanesulfohydroxamic acid ester of indometachin 9a, in view of
its COX-2 selectivity and ability to release NO which is a
beneficial mediator of GI mucosal protection that induces many
actions similar to prostaglandins in the GI tract,27 would be free
of contraindicated GI effects. Accordingly, the UI for compound
9a was determined for comparison to the parent NSAID
indomethacin (Table 2). Unlike indomethacin (UI = 64 for a
80 μmol/kg po dose), 9a showed no visible lesions (UI = 0 for a
80 μmol/kg po dose). This important gastric-sparing property
observed for 9a is attributed to its good COX-2 selectivity and
nitric oxide-releasing properties.
recorded on a Water’s Micromass ZQ 4000 mass spectrometer using the
ESI ionization mode. Microanalyses were performed for C, H, N by the
Microanalytical Service Laboratory, Department of Chemistry, Univer-
sity of Alberta. Compounds 7a-d and 8a-d showed a single spot on
Macherey-Nagel Polygram Sil G/UV254 silica gel plates (0.2 mm) using
a low, medium, and highly polar solvent system, and no residue
remained after combustion, indicating a purity >95%. Column chroma-
tography was performed on a Combiflash Rf system using either a gold
silica (8a-d) or a C18 (7a-d) column. All other reagents, purchased
from the Aldrich Chemical Co. (Milwaukee, WI), were used without
further purification. The in vivo anti-inflammatory and ulcer index assays
were carried out using protocols approved by the Health Sciences
Animal Welfare Committee at the University of Alberta.
Indomethacin Acid Chloride (6a). Oxalyl chloride (0.52 mL, 6
mmol) was added dropwise to a solution of indomethacin (1.07 g, 3
mmol) and two drops of dry DMF in dry CH2Cl2 (20 mL) at 0-5 °C.
The reaction mixture was stirred at 25 °C for 12 h, and the solvent was
removed in vacuo to give 6a as a pale-gray solid; yield 94%; mp 124-
126 °C (lit. mp 125-127 °C).19 1H NMR (300 MHz, CDCl3): δ 2.42
(s, 3H, CH3), 3.85 (s, 3H, OCH3), 4.18 (s, 2H, CH2COCl), 6.70 (dd, J =
9.2, 2.4 Hz, 1H, indolyl H-6), 6.85-6.90 (m, 2H, indolyl H-7 and H-4),
7.48 (dd, J = 6.7, 1.8 Hz, 2H, benzoyl H-3 and H-5), 7.68 (dd, J = 6.7,
1.8 Hz, 2H, benzoyl H-2 and H-6).
’ CONCLUSIONS
Hybrid NSAIDs that possess a nitrate28,29 or diazeniumdiolate30
NO-releasing moiety provided an attractive drug design strategy to
improve the safety profile of traditional NSAIDs. However, produc-
tion of NO from nitrate esters requires a demanding three-electron
reduction, and this metabolic activation can lead to nitrate tolerance
upon continued use.31,32 Diazen-1-ium-1,2-diolates, after release of
two molecules of NO, also give rise to the secondary amine from
which they are derived. This secondary amine can subsequently
undergo biotransformation to a potentially toxic and carcinogenic
nitrosoamine.33 To-date no NO-NSAID or NONO-NSAID has
achieved clinical approval. The sulfohydroxamic acid moiety is a
desirable drug design pharmacophore because it can act as a dual
NO/HNO donor. The difficult challenge of utilizing the unique
biological and pharmacological properties of HNO, particularly
upon the cardiovascular system, has not been adequately
investigated.11-14 In this regard, a synthetic methodology to prepare
ethanesulfohydroxamic acid prodrug esters of indomethacin, (S)-
naproxen, and ibuprofen was developed. Biological studies indicated
that this hitherto-unknown group of NSAID-CO2CH2CH2SO2N-
HOH compounds showed (i) a much higher NO release at
physiological pH relative to a group of arylsulfohydroxamic acid
compounds (5) previously reported,16 (ii) that the ethanesulfohy-
droxamic acid ester of indomethacin 9a is a selective COX-2
inhibitor, (iii) that the (S)-naproxen and ibuprofen prodrug esters
9b and 9d-e showed more potent AI activities than their respective
parent NSAID (S)-naproxen or ibuprofen, (iv) that the ethane-
SO2NHOMe compound 9e, which did not act as a NO/HNO
donor, exhibited a more potent AI activity than ibuprofen that is
attributed to an esterase induced (Figure 2) ester cleavage to release
ibuprofen, and (v) that the indomethacin prodrug ester 9a, which
exhibited potent AI activity, unlike indomethacin was completely
devoid of adverse ulcerogenic effects.
(S)-Naproxen Acid Chloride (6b). Compound 6b was prepared,
using the procedure described for the synthesis of 6a, except that (S)-
naproxen was used in place of indomethacin, in 97% yield as a pale-gray
solid, mp 94-96 °C (lit. mp 90-95 °C).34 1H NMR (CDCl3): δ 1.68
(d, J = 6.7 Hz, 3H, CH3), 3.93 (s, 3H, OCH3), 4.26 (q, J = 6.7 Hz, 1H,
CHMe), 7.15 (dd, J = 7.9 Hz, 2.5 Hz, 1H, naphthyl H-3), 7.20 (d, J = 2.5
Hz, 1H, naphthyl H-1), 7.36 (dd, J = 8.5 Hz, 1.8 Hz, 1H, naphthyl H-7),
7.69-7.78 (m, 3H, naphthyl H-4, H-5 and H-8).
Phenylacetyl Chloride (6c). Compound 6c was prepared, using
the procedure described for the synthesis of 6a, except that phenylacetic
acid was used in place of indomethacin, in 95% yield as a yellow oil. 1H
NMR (CDCl3): δ 4.15 (s, 2H, ArCH2), 7.27-7.42 (m, 5H, ArH).
Ibuprofen Acid Chloride (6d). Compound 6d was prepared
using the procedure described for the synthesis of 6a, except that
ibuprofen was used in place of indomethacin, in 94% yield as a yellow
oil. 1H NMR (CDCl3): δ 0.91 (d, J = 6.7 Hz, 6H, (CH3)2CHCH2), 1.59
(d, J = 7.4 Hz, 3H, CHCH3), 1.87 (heptet, J = 6.7 Hz, 1H, (CH3)2-
CHCH2), 2.48 (d, J = 7.4 Hz, 2H, (CH3)2CHCH2), 4.10 (q, J = 7.4 Hz,
1H, CHMe), 7.14-7.22 (m, 4H, phenyl hydrogens).
2-{2-[1-(4-Chlorobenzoyl)-2-methyl-5-methoxyl-1H-indoyl-
3-yl)]acetoxy}ethanesulfonic Acid Sodium Salt (7a). 2-Hydro-
xyethanesulfonic acid sodium salt (488 mg, 3.3 mmol), indomethacin acid
chloride (1.13 g, 3.0 mmol), and anhydrous pyridine (8 mL) were stirred
under argon at 50-60 °C for 24 h. After removal of pyridine, H2O (10 mL)
was added to the residue, and the mixture was washed with EtOAc (2 ꢀ 10
mL). The water phase was concentrated under vacuum to give a brown
product. Purification using a C18 column with H2O-acetonitrile (95:5, v/
v) as eluent afforded the title compound (313 mg, 22.1%) as a yellow syrup.
IR (KBr): 2955, 2894, 1685, 1234, 1159, 1043 cm-1. ESI-MS: 464 [M -
Na]-. 1H NMR (DMSO-d6): δ 2.19 (s, 3H, CH3), 2.75 (t, J = 7.9 Hz, 2H,
CH2SO3Na), 3.74 (s, 2H, CH2CO), 3.76 (s, 3H, OCH3), 4.24 (t, J = 7.9
Hz, 2H, COOCH2CH2 SO3Na), 6.70(dd, J= 9.2, 2.5 Hz, 1H, indolyl H-6),
6.94 (d, J = 9.2 Hz, 1H, indolyl H-7), 7.04 (d, J = 2.5 Hz, 1H, indolyl H-4),
7.0-7.62 (m, 4H, benzoyl H-2, H-3, H-5 and H-6). 13C NMR (DMSO-
d6): δ 13.1, 50.0, 55.4, 57.6, 61.2, 101.7, 111.5, 112.7, 114.5, 127.2, 129.0,
130.1, 130.5, 134.1, 135.3, 137.5, 155.5, 167.8, 170.3.
’ EXPERIMENTAL SECTION
General. Melting points were determined on a Thomas-Hoover
capillary apparatus and are uncorrected. 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 with TMS as the internal standard, where J (coupling
constant) values are estimated in hertz (Hz). Mass spectra (MS) were
(S)-2-[2-(6-Methoxynaphthyl-2-yl)propionoyloxy]ethane-
sulfonic Acid Sodium Salt (7b). An intimate mixture of anhydrous
2-hydroxyethanesulfonic acid sodium salt (400 mg, 2.7 mmol) and (S)-
naproxen acid chloride (6b, 740 mg, 3.0 mmol) was heated at 130 °C
until no further hydrogen chloride was evolved (about 4 h). The dark
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Journal of Medicinal Chemistry
ARTICLE
solid was dissolved in boiling water (20 mL), filtered through a charcoal
pad, and extracted with ethyl ether (30 mL) to remove excess (S)-
naproxen. The aqueous liquor was adjusted to pH 7 with sodium
hydroxide and evaporated under reduced pressure. The residue obtained
was purified on a C18 column using H2O-acetonitrile (95:5, v/v) as
eluent to furnish 7b (700 mg, 72.2%) as a white solid; mp 232-235 °C. IR
(KBr): 2977, 2941, 1732, 1185 cm-1. ESI-MS: 337 [M - Na]-.
1H NMR (DMSO-d6): δ 1.45 (d, J = 7.3 Hz, 3H, CHCH3), 2.68 (t,
J = 7.3 Hz, 2H, CH2SO3Na), 3.85 (s, 3H, OCH3), 3.91 (q, J = 7.3 Hz, 1H,
CHMe), 4.16-4.26 (m, 2H, COOCH2CH2), 7.14 (dd, J = 9.2 Hz, 2.5
Hz, 1H, naphthyl H-3), 7.28 (d, J = 2.5 Hz, 1H, naphthyl H-1), 7.38 (dd,
J = 8.0 Hz, 1.3 Hz, 1H, naphthyl H-7), 7.71-7.81 (m, 3H, naphthyl H-4,
H-5 and H-8). 13NMR (DMSO-d6): δ 18.4, 44.4, 49.9, 55.1, 61.2, 105.7,
118.6, 125.5, 126.1, 126.9, 128.3, 129.1, 133.2, 135.6, 157.1, 173.4.
2-(Phenylacetyloxy)ethanesulfonic Acid Sodium Salt
(7c). The title compound was prepared, using a similar procedure to
that described for the synthesis of 7b starting with phenylacetyl chloride,
in 54.6% yield as a white solid; mp 252-254 °C. IR (KBr): 2958, 1725,
1199 cm-1. ESI-MS: 243 [M - Na]-. 1H NMR (DMSO-d6): δ 2.75 (t,
J = 7.3 Hz, 2H, CH2SO3Na), 3.64 (s, 2H, ArCH2), 4.22 (t, J = 7.3 Hz, 2H,
COOCH2), 7.24-7.31 (m, 5H, ArH). 13NMR (DMSO-d6): δ 40.3,
49.9, 61.2, 126.7, 128.2, 129.2, 134.3, 171.0.
2-[2-(4-Isobutylphenyl)propionoyloxy]ethanesulfonic Acid
Sodium Salt (7d). The title compound was prepared, using a similar
procedure to that described for the synthesis of 7b starting with ibuprofen
acid chloride in 86.7% yield as a white solid; mp 179-181 °C. IR (KBr):
2962, 2835, 1737, 1656, 1198, 1036 cm-1. ESI-MS: 313 [M - Na]-. 1H
NMR (DMSO-d6): δ 0.84 (d, J = 6.8 Hz, 6H, (CH3)2CHCH2), 1.34 (d,
J = 7.3 Hz, 3H, CHCH3), 1.79 (heptet, J = 6.8 Hz, 1H, (CH3)2CHCH2),
2.40 (d, J = 6.7 Hz, 2H, (CH3)2CHCH2), 2.66 (t, J = 7.9 Hz, CH2SO3Na),
3.70 (q, J = 7.3 Hz, 1H, CHMe), 4.13-4.23 (m, 2H, COOCH2CH2), 7.09
(d, J = 8.0 Hz, 2H, phenyl H-3 and H-5), 7.16 (d, J = 8.0 Hz, phenyl H-2
and H-6). 13NMR (DMSO-d6): δ 18.5, 22.1, 29.5, 44.0, 44.1, 49.8, 61.1,
126.9, 128.9, 137.8, 139.6, 173.7.
2-{2-[1-(4-Chlorobenzoyl)-2-methyl-5-methoxyl-1H-indoyl-
3-yl)]acetoxy}ethanesulfonyl Chloride (8a). The sulfonic acid
sodium salt 7a (300 mg, 0.615 mmol) was dissolved in DMF (3 mL),
and SOCl2 (0.22 mL, 3.08 mmol) was added dropwise. The reaction
mixture was allowed to stir at 25 °Cfor1h, pouredintocoldwater(30mL),
and extracted with EtOAc (3 ꢀ 30 mL). The combined organic fractions
were washed with 2N HCl aqueous solution and brine and dried (MgSO4).
After concentration, the resulting brown syrup was purified using ethyl
acetate-hexane (1:2, v/v) as eluent to give the title compound (130 mg,
43.3%) as a yellow syrup. IR (film): 2966, 2930, 1749, 1693, 1480, 1378,
1322 cm-1. ESI-MS: 484 [M þ H]þ. 1H NMR (CDCl3): δ 2.40 (s, 3H,
CH3), 3.74 (s, 2H, CH2), 3.85 (s, 3H, OCH3), 4.00 (t, J = 5.4 Hz, 2H,
CH2SO2Cl), 4.68 (t, J = 5.4 Hz, 2H, COOCH2CH2), 6.69 (dd, J = 9.2 Hz,
2.5 Hz, 1H, indolyl H-6), 6.89 (d, J = 9.2 Hz, 1H, indolyl H-7), 6.94 (d, J =
2.5 Hz, 1H, indolyl H-4), 7.48 (dd, J = 6.7, 1.8 Hz, 2H, benzoyl H-3, H-5),
7.67 (dd, J = 6.7 Hz, 1.8 Hz, 2H, benzoyl H-2, H-6). 13C NMR (DMSO-
d6): δ 13.3, 55.7, 57.9, 63.6, 68.2, 101.1, 111.5, 111.8, 115.0, 129.1, 130.4,
130.8, 131.2, 133.7, 136.2, 129.4, 156.1, 168.2, 170.1.
2-(Phenylacetyloxy)ethanesulfonyl Chloride (8c). The sul-
fonic acid sodium salt 7c (1.6 g, 6.0 mmol) was dissolved in SOCl2
(10 mL), and a catalytic amount of DMF was added. The mixture was
allowed to stir at 25 °C for 3 h. The SOCl2 was removed under reduced
pressure at about 40 °C, ethyl acetate (30 mL) was added to the residue
that was removed by filtration, and the filtrate was concentrated under
vacuum to give a yellow residue which was purified using ethyl acetate-
hexane (1/3, v/v) as eluent to give the title compound (1.1 g, 66.6%) as a
yellow oil. IR (film): 2963, 1703, 1202 cm-1. ESI-MS: 263 [M þ H]þ.
1H NMR (CDCl3): δ 3.69 (s, 2H, ArCH2), 3.98 (t, J = 6.1 Hz, 2H,
CH2SO2Cl), 4.66 (t, J = 6.1 Hz, 2H, COOCH2CH2), 7.27-7.38 (m, 5H,
phenyl hydrogens). 13C NMR (DMSO-d6): δ 40.9, 57.8, 63.4, 127.4,
128.7, 129.3, 132.9, 170.9.
2-[2-(4-Isobutylphenyl)propionoyloxy]ethanesulfonyl
Chloride (8d). The title compound was synthesized using a method
similar to that used to prepare 8c starting from 7d, in 51.7% yield as a
yellowish solid; mp 39-40 °C. IR (film): 2966, 1752, 1379, 1167 cm-1
.
ESI-MS: 355 [M þ Na]þ. 1H NMR (CDCl3): δ 0.90 (d, J = 6.7 Hz, 6H,
(CH3)2CHCH2), 1.52 (d, J = 7.3 Hz, 3H, CHCH3), 1.85 (heptet, J = 7.3
Hz, 1H, (CH3)2CHCH2), 2.45 (d, J = 7.3 Hz, 2H, (CH3)2CHCH2), 3.74
(t, J = 7.3 Hz, CH2SO2Cl), 3.94 (q, J = 7.3 Hz, 2H, CHMe), 4.59-4.65
(m, 2H, COOCH2CH2), 7.11 (d, J = 7.9 Hz, 2H, phenyl H-3 and
H-5), 7.20 (d, J = 7.9 Hz, phenyl H-2 and H-6). 13C NMR (DMSO-d6):
δ 18.2, 22.4, 30.1, 44.8, 45.0, 57.7, 63.4, 127.1, 129.4, 136.7, 140.9,
174.0.
2-{2-[1-(4-Chlorobenzoyl)-2-methyl-5-methoxyl-1H-indoyl-
3-yl)]acetoxy}ethanesulfonyl Hydroxamic Acid (9a). The etha-
nesulfonyl chloride 8a (110 mg, 0.23 mmol) was dissolved in dry THF
(5 mL), and then hydroxylamine hydrochloride (32 mg, 0.46 mmol) and
potassium carbonate (127 mg, 0.92 mmol) were added. The reaction
mixture was vigorously stirred at 25 °C until the sulfonyl chloride had
completely disappeared (TLC; EtOAc-hexane, 1:1, v/v) in about 2 h. The
reaction mixture was filtered through a pad of Celite that provided a clear
filtrate which was added to ethyl acetate (20 mL), this mixture was washed
with water (20 mL) and brine (20 mL), and the organic fraction was dried
(MgSO4). Removal of the solvent from the organic fraction in vacuo gave a
residue that was purified by flash silica gel column chromatography using n-
hexane-EtOAc (2:1, v/v) as eluent to afford the title compound 9a (75
mg, 68.2%) as a yellow solid; mp 143-144 °C. IR (film): 3385, 3247, 2963,
1685, 1485, 1327 cm-1. ESI-MS: 481 [M þ H]þ. 1H NMR (DMSO-d6):
δ 2.20 (s, 3H, CH3), 3.49 (t, J = 6.1 Hz, 2H, CH2SO2NHOH), 3.76 (s, 3H,
OCH3), 3.79 (s, 2H, CH2), 4.40 (t, J = 6.1 Hz, 2H, COOCH2CH2), 6.71
(dd, J = 9.2 Hz, 2.4 Hz, 1H, indolyl H-6), 6.94 (d, J = 9.2 Hz, 1H, indolyl
H-7), 7.03 (d, J = 2.4 Hz, 1H, indolyl H-4), 7.62-7.69 (m, 4H, benzoyl
H-2, H-3, H-5 and H-6), 9.25 and 9.69 (two d, J = 3.1 Hz, 1H each,
HO-NH). 13C NMR (CDCl3): δ 12.8, 45.8, 55.2, 58.0, 59.5, 100.9, 111.5,
111.8, 114.3, 128.5, 130.0, 130.2, 130.6, 133.3, 135.3, 138.5, 155.5, 167.6,
169.8. Anal. Calcd for C21H21ClN2O7S: C, 52.45; H, 4.40; N, 5.82. Found:
C, 52.38; H, 4.52; N, 5.57.
(S)-(þ)-2-[2-(6-Methoxynaphthyl-2-yl)propionoyloxy]etha-
nesulfonylhydroxamic Acid (9b). The title compound was synthe-
sized, using a procedure similar to that used to prepare 9a starting from 8b,
in 75.7% yield as a white solid; mp 107-109 °C. IR (film): 3386, 3259,
2940, 1739, 1156 cm-1; [R]21.0D = þ22.7 (1.000, CHCl3). ESI-MS: 354
[M þ H]þ, 371 [M þ NH4]þ, 376 [M þ Na]þ. 1H NMR (DMSO-d6): δ
1.47 (d, J = 7.3 Hz, 3H, CH3), 3.44 (t, J = 6.7 Hz, 2H, CH2SO2NHOH),
3.85 (s, 3H, OCH3), 3.93 (q, J = 7.3 Hz, CHMe), 4.28-4.44 (m, 2H,
COOCH2CH2), 7.14 (dd, J = 9.1 Hz, 2.5 Hz, 1H, naphthyl H-3), 7.28 (d,
J = 2.5 Hz, 1H, naphthyl H-1), 7.40 (dd, J = 8.5 Hz, 1.2 Hz, 1H, naphthyl
H-7), 7.72-7.80 (m, 3H, naphthyl H-4, H-5 and H-8), 9.23 and 9.66 (two
d, J = 3.1 Hz, 1H each, HO-NH). 13C NMR (DMSO-d6): δ 18.1, 45.4,
46.0, 58.5, 105.7, 119.4, 125.8, 126.1, 127.6, 128.9, 129.2, 133.8, 134.7,
157.8, 174.1. Anal. Calcd for C16H19NO6S: C, 54.38; H, 5.42; N, 3.96.
Found: C, 54.18; H, 5.23; N, 3.93.
(S)-2-[2-(6-Methoxynaphthyl-2-yl)propionoyloxy]ethane-
sulfonyl Chloride (8b). The title compound was synthesized, using
a method similar to that used for the preparation of 8a starting from 7b
in 45.3% yield as a white solid; mp 76-78 °C. IR (film): 2999, 2939,
1
1743, 1607, 1376, 1160 cm-1. ESI-MS: 357 [M þ H]þ. H NMR
(CDCl3): δ 1.62 (d, J = 7.3 Hz, 3H, CH3), 3.92 (s, 3H, OCH3),
3.88-3.96 (m, 3H, CHMe and CH2SO2Cl), 4.56-4.68 (m, 2H,
COOCH2CH2), 7.14 (dd, J = 6.7 Hz, 2.4 Hz, 1H, naphthyl H-3), 7.28
(d, J = 2.4 Hz, 1H, naphthyl H-1), 7.39 (dd, J = 8.6 Hz, 1.9 Hz, 1H,
naphthyl H-7), 7.67-7.73 (m, 3H, naphthyl H-4, H-5 and H-8). 13C
NMR (DMSO-d6): δ 18.2, 45.2, 55.3, 57.7, 63.4, 105.6, 119.1, 126.0,
126.1, 127.3, 128.9, 129.2, 133.8, 134.6, 157.8, 174.0.
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ARTICLE
2-(Phenylacetyloxy)ethanesulfonylhydroxamic Acid
(9c). The title compound was synthesized using a procedure similar
to that used to prepare 9a starting from 8c, in 61.9% yield as a white
solid; mp 96-97 °C. IR (film): 3393, 3255, 1736, 1338, 1150 cm-1. ESI-
MS: 282 [M þ Na]þ. 1H NMR (DMSO-d6): δ 3.48 (t, J = 6.1 Hz, 2H,
CH2SO2NHOH), 3.68 (s, 2H, ArCH2), 4.37 (t, J = 6.1 Hz, 2H,
COOCH2), 7.25-7.34 (m, 5H, phenyl hydrogens), 9.25 and 9.67
(two d, J = 3.1 Hz, 1H each, HO-NH). 13C NMR (DMSO-d6): δ
40.7, 45.9, 58.1, 126.9, 128.7, 129.0, 133.2, 170.8. Anal. Calcd for
C10H13NO5S: C, 46.32; H, 5.05; N, 5.40. Found: C, 46.45; H, 5.05;
N, 5.32.
were identical, except that the vehicle did not contain a test compound.
The volume of the injected paw was measured at 0 and 3 h using a UGO
Basile 7141 Plethysmometer (series no. 43201). A dose response curve
was constructed which was used to determine the ID50 value.
Acute Ulcerogenesis Assay. The ability to produce gastric
damage was evaluated according to reported procedures.38 Ulcerogenic
activity was evaluated after oral administration of 9a (80 μmol/kg po
dose) that was suspended 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 12 h before
administration of the test compound. Six hours after oral administration
of the drug, rats were euthanized in a CO2 chamber and their stomachs
were removed, cut out along 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 millimeters). The UI for 9a and blank
control 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 = 3): UI) (L1 þ L2 þ L3)/3.
2-[2-(4-Isobutylphenyl)propionoyloxy]ethanesulfonyl-
hydroxamic Acid (9d). The title compound was synthesized, using a
method similar to that used to prepare 9a starting from 8d, in 71.4%
yield as a white solid; mp 68-70 °C. IR (film): 3398, 3252, 2959, 1748,
1
1340, 1158 cm-1. ESI-MS: 347 [M þ NH4]þ, 352 [M þ Na]þ. H
NMR (DMSO-d6): δ 0.84 (d, J = 6.7 Hz, 6H, (CH3)2CHCH2), 1.38 (d,
J = 6.7 Hz, 3H, CHCH3), 1.77 (heptet, J = 6.7 Hz, 1H, (CH3)2CHCH2),
2.40 (d, J = 7.4 Hz, 2H, (CH3)2CHCH2), 3.43 (t, J = 6.7 Hz,
CH2SO2NHOH), 3.76 (q, J = 6.7 Hz, 1H, ArCH), 4.26-4.43 (m, 2H,
COOCH2CH2), 7.10 (d, J = 7.9 Hz, 2H, phenyl H-3 and H-5), 7.19 (d,
J = 7.9 Hz, phenyl H-2 and H-6), 9.22 and 9.66 (two d, J = 2.5 Hz, 1H
each, HO-NH). 13C NMR (DMSO-d6): δ 18.1, 22.4, 30.1, 44.9, 45.0,
45.9, 58.5, 127.1, 129.7, 136.9, 141.2, 174.1. Anal. Calcd for
Nitric Oxide Release Assay. In vitro nitric oxide release, upon
incubation of the test compound (2.4 mL of 5.0 ꢀ 10-2 mM) with either
(i) phosphate buffer solution (PBS) at pH 7.4 and 37 °C for 1.5 h or (ii)
PBS at pH 7.4 and 37 °C to which 90 μL of 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 (9a-e) and the reference
compound ethanesulfonylhydroxamic acid (EA) using the reported
procedures.39
C15H23NO5S 0.2H2O: C, 54.10; H, 7.08; N, 4.21. Found: C, 54.08;
3
H, 7.00; N, 4.26.
N-Methoxyl 2-[2-(4-Isobutylphenyl)propionoyloxy]etha-
nesulfonylamide (9e). The sulfonyl chloride 8d (300 mg, 0.90
mmol) was reacted with methoxylamine hydrochloride (150 mg, 1.8
mmol) that was neutralized with an solution of NaHCO3 (300 mg, 3.57
mmol) in dry THF (14 mL). The reaction was completed using a
procedure similar to that described for the isolation of 9a, to yield the
title compound 9e as a colorless oil (65.1%). IR (film): 3260, 2963,
1745, 1344, 1156 cm-1. ESI-MS: 361 [M þ NH4]þ, 366 [M þ Na]þ.
1H NMR (DMSO-d6): δ 0.84 (d, J = 6.7 Hz, 6H, (CH3)2CHCH2), 1.37
(d, J = 7.3 Hz, 3H, CHCH3), 1.79 (heptet, J = 6.7 Hz, 1H,
(CH3)2CHCH2), 2.40 (d, J = 7.3 Hz, 2H, (CH3)2CHCH2), 3.46 (t,
J = 6.1 Hz, CH2SO2NHOMe), 3.64 (s, 3H, SO2NHOCH3), 3.75 (q, J =
7.4 Hz, 1H, ArCH), 4.24-4.44 (m, 2H, COOCH2CH2), 7.10 (d, J = 7.9
Hz, 2H, phenyl H-3 and H-5), 7.19 (d, J = 7.9 Hz, phenyl H-2 and H-6),
10.2 (s, 1H, SO2NHOCH3). 13C NMR (DMSO-d6): δ 18.1, 22.3, 30.3,
45.0, 45.1, 46.3, 58.5, 64.9, 127.1, 129.7, 138.9, 141.2, 173.8. Anal. Calcd
Gas Chromatographic N2O Analysis. For headspace analysis,
substrate (0.04 mmol) was placed in a 10 mL round-bottom flask, which
was sealed with a rubber septum and flushed with inert gas. Solvent
(0.8 mL) was added, and headspace aliquots (0.25 mL) from three
separate experiments were injected at 2 and 24 h onto a 7890A Agilent
Technologies Gas Chromatograph equipped with a thermal conductiv-
1
ity detector and a 6 ft ꢀ /8 in.0 Porapack Q column. The oven operated
at 40 °C for 5 min was then ramped to 150 °C over 4.5 min for a total run
time of 9.5 min. The purged packed inlet with a total flow (He as carrier
gas) of 18 mL/min and a septum purge flow of 3 mL/min was held at
140 °C. The back detector with a reference flow of 9 mL/min and a
makeup flow of 6 mL/min was held at 150 °C. The retention time of
nitrous oxide was 2.5 min, and yields (% of N2O produced) were
calculated based on a standard curve prepared using known amounts of
nitrous oxide gas (Matheson Tri-Gas).
for C16H25NO5S 0.25H2O: C, 55.23; H, 7.39; N, 4.03. Found: C, 54.91;
3
H, 7.27; N, 4.17.
Ethanesulfonylhydroxamic Acid (EA). The title compound
was prepared by reaction of ethanesulfonyl chloride with hydroxylamine
hydrochloride using a procedure similar to that used to prepare 9a, in
54.0% yield as a colorless oil according to a literature method.35 IR
(film): 3404, 3238, 1333, 1157 cm-1. 1H NMR (DMSO-d6): δ 1.19 (t,
J = 7.3 Hz, 3H, CH3CH2), 3.11 (q, J = 7.3 Hz, 2H, CH3CH2), 9.16 and
9.50 (two d, J = 3.1 Hz, 1H each, HO-NH).
Cyclooxygenase Inhibition Assays. The ability of the test
compounds 9a-e listed in Table 2 to inhibit ovine COX-1 and human
recombinant COX-2 (IC50 value, μM) was determined using an enzyme
immuno assay (EIA) kit (catalogue no. 560131, Cayman Chemical, Ann
Arbor, MI, USA) according to a previously reported method.36
Anti-inflammatory Assay. Anti-inflammatory activity was mea-
sured using a carrageenan-induced rat paw edema assay described by
Winter et al.37 Briefly, three male Sprague-Dawley rats weighing
160-180 g were used in each group. Test compounds suspended in
water containing 1% methyl cellulose were administered orally for a
minimum of three different doses (2.8-112 mg/kg range) 1 h prior to a
0.05 mL subcutaneous injection of 1% carrageenan in 0.9% NaCl
solution under the planter skin of the right hind paw. Control experiments
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (780)492-5993. Fax: (780)492-1217. E-mail: eknaus@
pharmacy.ualberta.ca.
’ ACKNOWLEDGMENT
We are also grateful to the Canadian Institutes of Health
Research (grant no. MOP-14712) for financial support of this
research.
’ ABBREVIATIONS USED
AI, anti-inflammatory; COX-1, cyclooxygenase-1; COX-2, cyclo-
oxygenase-2; EA, ethanesulfohydroxamic acid; GI, gastrointest-
inal; GSH, glutathione; NO, nitric oxide;HNO, nitroxyl; N2O,
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Journal of Medicinal Chemistry
ARTICLE
nitrous oxide; NSAIDs, nonsteroidal anti-inflammatory drugs;
PBS, phosphate buffer solution; PA, Piloty’s acid; SI, selectivity
index; TBS, tris buffer solution; UI, ulcer index.
(18) Shoeman, D. W.; Nagasawa, H. T. The reaction of nitroxyl
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