Nitric oxide release is not required to decrease the ulcerogenic profile of nonsteroidal anti-inflammatory drugs

Article abstract of DOI:10.1021/jm200973j

The objective of this work was to evaluate the biological properties of a new series of nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs) possessing a tyrosol linker between the NSAID and the NO-releasing moiety (PROLI/NO); however,

Full text of DOI:10.1021/jm200973j

Article
pubs.acs.org/jmc
Nitric Oxide Release Is Not Required to Decrease the Ulcerogenic
Profile of Nonsteroidal Anti-inflammatory Drugs
Sarthak Jain,† Susan Tran,† Mohamed A. M. El Gendy,† Khosrow Kashfi,‡ Paul Jurasz,†
and Carlos A. Velazquez-Martínez*,†
́
†Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8 Canada
‡Department of Physiology and Pharmacology, City University of New York Medical School, New York, NY, United States
ABSTRACT: The objective of this work was to evaluate the biological
properties of a new series of nitric oxide-releasing nonsteroidal anti-
inflammatory drugs (NO-NSAIDs) possessing a tyrosol linker between the
NSAID and the NO-releasing moiety (PROLI/NO); however, initial
screening of ester intermediates without the PROLI/NO group showed
the required (desirable) efficacy/safety ratio, which questioned the need for
NO in the design. In this regard, NSAID ester intermediates were potent and
selective COX-2 inhibitors in vitro, showed equipotent anti-inflammatory
activity compared to the corresponding parent NSAID, but showed a
markedly reduced gastric toxicity when administered orally. These results provide complementary evidence to challenge the
currently accepted notion that hybrid NO-NSAIDs exert their cytoprotective effects by releasing NO. Results obtained in this
work constitute a good body of evidence to initiate a debate about the future replacement of NSAID prodrugs for unprotected
NSAIDs (possessing a free carboxylic acid group) currently in clinical use.
intestine,9 and 2−4% of these individuals present clinically
INTRODUCTION
■
significant GI ulcers and bleeding, sometimes leading to
Nonsteroidal anti-inflammatory drugs (NSAIDs) is one of the
death.10 Consequently, the development of new anti-inflam-
most widely used classes of medications worldwide. NSAIDs
matory drugs is still a strong clinical need, especially after the
are employed predominantly to treat pain, fever, and
withdrawal of some selective COX-2 inhibitors such as
inflammation; however, new studies support an expanded
rofecoxib and valdecoxib11,12 and current concerns about the
repertoire for NSAIDs and, more importantly, their chemically
potential dose-dependent hypertensive effect of other NSAIDs
modified derivatives. Expanded medical applications include the
which is not easily predicted by COX selectivity alone.13
prophylactic treatment of a wide variety of human diseases such
as atherosclerosis,1 thrombosis,2 cancer,3,4 Alzheimer’s disease,5
The discovery that nitric oxide (NO) and hydrogen sulfide
(H2S) exert protective effects in the GI tract, modulate many
and other disorders for which chronic inflammation is an
aspects of GI mucosal defense, and accelerate the healing of
etiological factor. The major pharmacological mechanism of
pre-existing ulcers, led to the development of chemically
action of NSAIDs is inhibiting production of prostaglandins
modified NSAIDs possessing “donor” groups which release NO
(PGs) and thromboxanes (TXs), molecular species that are
or H2S upon metabolic activation in vivo. In this regard, the
derived from the enzymatic transformation of arachidonic acid
most extensively studied hybrid prodrugs are the NO-NSAIDs
(AA) by cyclooxygenase (COX)-1 and COX-2 enzymes. COX-
(1, Figure 1) and the first NO-releasing group employed in the
1 is generally regarded as a constitutive enzyme that is present
in most tissues; it is involved in the physiological production of
PGs and provides maintenance functions such as cytoprotec-
design of NO-NSAIDs was the organic nitrate (−ONO2),14
which invariably necessitates the presence of a “linker” between
the NO-donor group and the NSAID.
A few years ago, we proposed the use of NONOates (2, N-
tion in the stomach. In contrast, COX-2 has been regarded as
an inducible enzyme and is expressed in inflammatory cells.6
diazen-1-ium-1,2-diolates) as a suitable replacement to organic
However, recent reports have challenged these “traditional”
nitrates.15 Three attributes distinguished NONOates from
roles for COX-1 and COX-2 enzymes, emphasizing the
organic nitrates, namely structural diversity, dependable rates of
importance of re-evaluating their roles not only in the
inflammatory process,7 but their contribution in the underlying
NO-release, and rich derivatization chemistry that facilitates
targeting of NO to specific target organ and/or tissue sites.16
mechanisms of NSAID-induced side effects.
Gastrointestinal (GI) erosions and bleeding are two of the
The first generation of NONOate-containing NSAIDs (3,
Figure 1) were obtained using secondary amines as the source
most common toxic side effects associated with the
of NONOates; however, concerns about the generation of
administration of NSAIDs, which have been observed even
with low prophylactic doses of aspirin (81 mg/day).8 It is
estimated that approximately 50% of patients taking NSAIDs
on a long-term basis develop mucosal damage in the small
Received: July 20, 2011
Published: December 7, 2011
© 2011 American Chemical Society
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Figure 1. Chemical structures of organic nitrate-containing NO-aspirin (1); representative NONOate ion (2, where R1 and R2 groups represent
different alkyl moieties); first-generation NONO-aspirin (3); second-generation NONO-indomethacin (4); naturally occurring phenols tyrosol (5)
and hydroxytyrosol (6); and the NONOate ion obtained from L-proline (PROLI/NO).
potentially carcinogenic N-nitrosamines as metabolic products
in vivo prompted us to modify this strategy. Subsequent
NONO-NSAIDs (4) were designed to possess a PROLI/NO
moiety (NONOate obtained from L-proline), which were
synthesized and evaluated in vivo (Figure 1).17 Nevertheless,
even though NONO-NSAIDs possessing a PROLI/NO moiety
were effective anti-inflammatory agents and their design
addressed the safety concerns related to the generation of
carcinogenic nitrosamines,18 they released one or two
equivalents of formaldehyde (HCHO) per mol of drug upon
metabolism, which may be considered favorable if the goal is to
kill cancer cells (chemotherapeutic), but it might be a potential
safety concern in routine anti-inflammatory treatments.
Scheme 1. Design of New NONO-NSAIDs Possessing
PROLI/NO and a Simple Phenol Linker (Tyrosol)
a
As part of our ongoing research program aimed to develop
new anti-inflammatory agents with a suitable efficacy/safety
profile, we now propose the design and biological evaluation of
new NONO-NSAIDs possessing PROLI/NO and a naturally
occurring diol linker which replaced formaldehyde-generating
acetal linkers (−OCH2O−) used in our previous work. We
considered the use of a wide variety of diols including sugars
and polyphenols. However, sugar chemistry represented a
challenging approach and the use of polyphenols would require
a cumbersome protection−deprotection synthetic strategy, so
we decided to start with a relatively simple phenol such as 4-(2-
hydroxyethyl)phenol (5, also called tyrosol); tyrosol is
structurally related to hydroxytyrosol (6, Figure 1), a well
studied natural anti-inflammatory and antioxidant compound
found in olives.19,20
The rationale behind the design of new NONO-NSAIDs
possessing a PROLI/NO moiety as the source of NO and a
simple phenol such as tyrosol as linker (Scheme 1) is based on
the assumption that, upon metabolic activation, these ester
prodrugs would release the anti-inflammatory NSAID,
cytoprotective NO, and innocuous metabolites such as L-
proline, tyrosol, and a β-D-glucopyranosyl moiety (Scheme 2)
containing 0−4 acetate ester groups. We now report the
synthesis, in vitro COX inhibitory activity, in vivo anti-
a
R2 = 2,3,4,6-tetra-O-acetyl-D-glucopyranosyl protecting group.
inflammatory potency, and the unexpected low ulcerogenicity
of four NSAID ester prodrugs possessing a tyrosol moiety.
RESULTS AND DISCUSSION
■
Determination of Molecular Length, Surface, and
Volume. Considering that the COX-1 active site consists of a
long narrow channel about 8 Å × 25 Å (total volume 316 Å3),
and the COX-2 binding site is about 25% larger (394 Å3),21
bulkier molecules normally fit better into the bigger active site
of COX-2 and, consequently, larger compounds generally show
a higher selectivity for this enzyme.22 This is a well established
concept reported in the literature which has been employed in
the development of potent and selective COX-2 inhibitors by
several groups.23−25 Therefore, the first step was the
determination of molecular length (Å), surface (Å2), and
volume (Å3) of prodrugs to assess if they would possess the
minimum essential structural requirements to enter the COX
binding site. In this regard, after a standard energy
minimization procedure using Alchemy 2000 (Version 2.0,
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a
Scheme 2. Theoretical Metabolic Activation of NONO-Ibuprofen in Vivo
a
Compounds marked with a solid rectangle represent the two active components (ibuprofen and NO) released by metabolic activation; compounds
marked with a broken-line rectangle represent innocuous metabolites produced after ester and glycosidase hydrolysis. R2 represents a protecting
2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl moiety. An alternative ester hydrolysis would involve the hydrolysis of the L-proline (ester hydrolysis-1),
and then the NSAID carboxylate (ester hydrolysis-2).
1997, Tripos Inc.), we determined these parameters for
NONO-aspirin (7), NONO-ibuprofen (8), and their corre-
sponding ester intermediates without the PROLI/NO group (9
and 10, see Scheme 3).
aliphatic alcohol, which was in agreement with reports
describing similar esterification reactions using a structurally
related 3-hydroxybenzyl alcohol.26 The second step involved
the preparation of O2-protected PROLI/NO derivatives (16,
Scheme 3B); in this regard, it has been reported in the
literature that it is not possible to obtain O2-alkylation products
by simple nucleophilic displacement (SN2) using PROLI/NO
(O2-sodium salt of 16) and electrophiles; therefore, the
preparation of intermediate (16) would require an indirect
route involving the synthesis of O2-sodium PROLINOL/NO
(14), O2-alkylation with 1-bromo-2,3,4,6-tetraacetoxyglucose to
obtain 15, and subsequent oxidation of the alcohol group in L-
prolinol to obtain the target carboxylic acid 16.27 The last step
involved the esterification reaction between intermediates 9 or
10 with 16 to yield the corresponding NONO-aspirin (7) or
NONO-ibuprofen (8, Scheme 3C). However, on the basis of
the preliminary screening of intermediate esters 9 and 10, we
decided not to carry out the synthesis of NONO-NSAIDs (see
Results and Discussion).
The predicted physicochemical parameters for ester inter-
mediates possessing a tyrosol moiety, suggested that the aspirin
tyrosol ester (9) has a suitable molecular volume (264.7 Å3) to
fit into the active site of both COX-1 and COX-2 enzymes,
whereas the bulkier ibuprofen tyrosol ester (10, volume =
322.6 Å3) would probably fit better into the larger COX-2 bind-
ing site (Table 1). As expected, the corresponding NONO-
aspirin (7, volume = 411.9 Å3) and NONO-ibuprofen (8,
volume = 469.6 Å3) are considerably bulkier than their corres-
ponding ester intermediates. Considering that molecular
volume and length are the initial determinant features control-
ling the access to the active site of COX enzymes, it is unlikely
that large NONO-NSAIDs would enter the long narrow channel,
and therefore, they will require bioactivation (ester hydrolysis,
see Scheme 2) to release either the tyrosol esters (7 and 8) or
the active NSAIDs. Nevertheless, we recognized that even
though tyrosol esters possessed proper volumes and lengths, they
may or may not interact favorably with essential residues within
the binding site of either enzyme (this would be better assessed
by specific molecular modeling (docking) studies).
In Vitro Cyclooxygenase Inhibition Assay. We observed
that esterification of ibuprofen with a tyrosol moiety increased
both the inhibitory potency and COX-2 selectivity, whereas
esterification of aspirin increased its potency but maintained the
COX-1 selective inhibitory profile of the parent NSAID. In this
Chemistry. The chemical synthesis of new NONO-NSAID
prodrugs possessing a tyrosol moiety involved three steps; the
first one was the preparation of the corresponding ester
intermediates (7 and 8) by reacting aspirin (11) or ibuprofen
(12) acid chlorides with tyrosol (Scheme 3A). Under the
experimental conditions used for these reactions (THF/TEA,
at 25 °C), the phenol group reacted much faster than the
regard, aspirin ester intermediate 9 showed a COX-1 IC50 =
0.03 μM, and COX-2 IC50 = 0.38 μM, which compared to aspirin
(COX-1 = 0.30 μM, COX-2 IC50 = 2.40 μM) represents a 10-
fold increase in COX-1 inhibitory activity and about 6-fold
increase on that of COX-2 (Table 2). This effect was also
observed for the ibuprofen ester (10), which showed a
remarkable inhibitory potency on COX-2 (IC50 = 0.2 nM) and
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a
Scheme 3. Chemical Synthesis of NONO-NSAIDs Possessing PROLI/NO and a Tyrosol Linker
a
Reagents and conditions: (a) THF, TEA, 25 °C, 5 h; (b) NO (40−60 psi), NaOCH3/CH3OH, ether, 25 °C, 6 h; (c) 1-bromo-2,3,4,6-
tetraacetoxyglucose, 5% aq NaHCO3/acetone; (d) NaIO4, RuCl3 (cat.), CH3CN, EtOAc, H2O, 25 °C, 2 h; (e) DCC, THF, TEA, 25 °C. (A)
Chemical synthesis of tyrosol intermediates 9 and 10; (B) proposed synthesis of intermediate O2-protected PROLI/NO (R2 = 2,3,4,6-tetra-O-acetyl-
β-D-glucopyranosyl); (C) Proposed synthesis of NONO-aspirin (7) and NONO-ibuprofen (8). Reactions represented by arrows with a broken line
were not actually carried out (see Results and Discussion Ulcer Index Assay).
Table 1. Prediction of Physicochemical Parameters for
NONO-NSAIDs (7, 8) and the Corresponding
Intermediates without the PROLI/NO Group (9 and 10)
Table 2. In Vitro COX-1/COX-2 Enzyme Inhibition,
Selectivity Index, and in Vivo Anti-Inflammatory Activity
Data for NSAID Tyrosol Esters (9 and 10)
a
a
a
b
compd
volume
264.7
surface
314.7
length
14.1
Log P
COX-1
COX-2
IC
IC
50a
50a
b
c
d
9
7
2.0 0.4
3.3 0.6
1.2 0.2
compd
(μM)
(μM)
SI
AI activity
58
UI
c
c
c
c
c
c
411.9
153.7
507.2
195.9
17.2
9
0.03
0.38
2.40
0.08
0.14
32220
2.63
2.6 1.2
57.4 3.7
3.5 0.8
45.8 2.9
e
e
f
aspirin
8.1
aspirin
10
ibuprofen
0.30
6.44
2.90
50
0.0002
63
50
e
e
f
1.10
10
322.6
469.6
209.5
394.6
574.9
260.4
16.6
4.6 0.2
5.9 0.6
3.7 0.2
c
c
a
8
20.7
The in vitro test compound concentration required to produce 50%
ibuprofen
10.1
inhibition of COX-1 or COX-2. The result (IC50, μM) is the mean of
two determinations acquired using an ovine COX-1 and human
recombinant COX-2 inhibitor screening assay kit (cat. no. 560131,
Cayman Chemicals Inc., Ann Arbor MI), and the deviation from the
a
Calculated using Alchemy 2000 (version 2.0, 1997, Tripos Inc.) after
energy minimization; molecular volume is expressed in cubic
angstroms (Å3), molecular surface is expressed in squared angstroms
b
b
(Å2), length is expressed in angstroms (Å). Negative logarithm of the
mean is <10% of the mean value. Selectivity index (SI) = COX-1
c
IC50/COX-2 IC50. Results are expressed as % decrease in the
distribution coefficient of each compound between n-octanol/water
(theoretical value, expressed as Log P, calculated using ACD/
ChemSketch version 12.01 freeware, 2010, ACD Laboratories Inc.)
inflammatory response compared to control group receiving vehicle at
3 h after oral administration of the test compounds (equimolar dose to
the corresponding reference drugs). The average overall length (in
d
c
Calculated for the corresponding NO-NSAIDs possessing an O2-
mm) of individual ulcers in each stomach SEM, n = 4, 6 h after oral
administration of the test compound (aspirin = 1.4 mmol/kg, ibuprofen
= 1.2 mmol/kg, 9 and 10 = equimolar amounts to their corresponding
methyl PROLI/NO moiety.
a significantly increased selectivity (SI = 32200) compared to the
parent drug ibuprofen (COX-2 IC50 = 1.1 μM, SI = 2.63). As it
was anticipated, the calculated size and volume for the smaller
aspirin ester 9 allowed it to fit into the active sites of both COX
enzymes but it maintained the preferential selectivity for COX-1
e
f
parent NSAIDs). Reported literature values.15 ID50 values reported
for aspirin (0.7 mmol/kg) and ibuprofen (0.3 mmol/kg).15
of aspirin, whereas the bulkier ibuprofen ester 10 seems to fit
better into the active site of the bigger COX-2 active site.
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These results are in agreement with previous reports
published by other groups, describing an improved potency
and COX-2 selectivity of different NSAID derivatives by
“masking” the carboxylic acid present in classical anti-
inflammatory drugs of this type. For example, Kalgutkar et al.
reported the increased potency and improved COX-2
selectivity of 28 “normal” indomethacin esters possessing a
wide variety of aliphatic and aromatic moieties,28 and 20
“reversed” indomethacin amides and esters showing the same
biological profile in vitro.29
respectively) than the parent NSAIDs (aspirin UI = 57.4,
ibuprofen UI = 45.8; Table 2). This observation was not in
accordance with our original hypothesis because prodrugs 9
and 10 were supposed to be intermediate compounds to which
the NO-releasing moiety was to be linked. In other words, the
two compounds that were expected to be devoid of ulcerogenic
side effects were the corresponding NONO-NSAIDs 7 and 8
(not synthesized), possessing a PROLI/NO moiety, and not
prodrugs 9 and 10. These results suggested that the NO-
releasing moiety was not essential to counteract the ulcerogenic
effects of aspirin or ibuprofen, and it changed our approach
because it seemed that the synthesis of NONO-NSAIDs 7 and
8 was not required (Scheme 3B,C).
One plausible explanation for the lack of acute gastric toxicity
of compound 9, despite its higher selectivity for COX-1
enzyme, could be its increased lipophilic character. Ester 9 has a
Log P = 2.0, whereas aspirin Log P = 1.2. As it was discussed for
the modest increase in anti-inflammatory activity observed with
tyrosol esters, we hypothesize that prodrugs are being absorbed
intact and at a higher rate than their corresponding acid
counterparts; faster rates of absroption might decrease the time
at which prodrugs are available in gastric tissues to exert local
toxicity. This observation is supported by previous reports
describing significantly higher bioavailability for lipophilic
sulindac sulfide amides compared to sulindac in mice.37
Evidence collected with prodrugs 9 and 10 suggested that
NO-releasing moieties are not essential to protect the gastric
mucosal layer from the ulcerogenic effects of an acute dose of
aspirin or ibuprofen. The implications of these observations are
considerable because it is possible to obtain simple ester COX
inhibitors with significant anti-inflammatory activity in vivo but
devoid of ulcerogenic side-effects without incorporating donors
of bioactive mediators. This statement supports the discussion
and conclusions described by Halen et al. in a recent review
paper.31
In 1994, Wallace et al. proposed the design of NO-releasing
NSAIDs as a promising approach to decrease the severity and
the relatively high incidence of gastric ulcers associated with the
long-term administration of NSAIDs.38 This approach was
based on the well documented bioregulatory properties of NO
in the stomach (gastroprotection and modulation of GI
mucosal defense), and it is still considered valid today. The
design of hybrid NO-NSAIDs continues to be described in the
literature as a suitable strategy to protect the gastric mucosa
from detrimental effects of NSAIDs.31,39
To obtain additional/complementary evidence in support of
this hypothesis, we synthesized and screened the corresponding
tyrosol ester of indomethacin (18, Scheme 4). Indomethacin is
a potent anti-inflammatory agent, but it is associated with a
high incidence of gastric toxicity, and unlike aspirin or
ibuprofen, indomethacin is available to patients by prescription
only. In this regard, esterification of indomethacin with a
tyrosol moiety did not change its inhibitory potency on COX-2
because prodrug 18 showed a similar COX-2 IC50 value to that
obtained with indomethacin (4.6 and 5.7 μM, respectively, see
Table 3); however, prodrug 18 showed a markedly reduced
inhibitory potency on COX-1 (IC50 > 100 μM) compared to
indomethacin (IC50 = 0.1 μM). This shift in COX selectivity
was similar to that obtained for the ibuprofen tyrosol prodrug
(10). When administered orally to rats, compound 18 showed
an improved anti-inflammatory activity in vivo (61% decrease
in the inflammatory response), which represents a 1.6-fold
increase in potency compared to indomethacin (38% inhibition
In Vivo Anti-inflammatory Activity. After evaluating the
in vitro inhibitory profile of tyrosol esters 9 and 10, both
NSAID prodrugs were screened in vivo using the carageenan-
induced rat paw edema assay.30 Experimental drugs were
administered orally (1% methylcellulose as vehicle). Tyrosol
esters 9 and 10 showed an equipotent anti-inflammatory pro-
file compared to aspirin and ibuprofen; the administration of
these prodrugs resulted in 58% and 63% decrease in the
inflammatory response, respectively, when compared to control
animals receiving vehicle only. Equimolar amounts of aspirin
and ibuprofen produced about 50% decrease in inflammation
(Table 2). It is reasonable to assume that the modest
improvement in anti-inflammatory potency observed for tyrosol
esters 9 and 10 may be due to the increased lipophilic character
of esters (Log P = 2.0 and 4.6 respectively) compared to the
parent NSAIDs (aspirin Log P = 1.2, ibuprofen Log P = 3.7).
Increased lipophilicity increases cell membrane permeability (to
a certain extent) and, ultimately, improves bioavailability.31
Nevertheless, this possibility does not necessarily mean that the
intact ester prodrugs reach the target tissues before they are
metabolized by nonspecific esterases in blood and other organs,
it would only increase their bioavailability compared to the
parent NSAID. In this regard, another plausible explanation to
support the increased anti-inflammatory activity of tyrosol
esters involves the cleavage (via ester hydrolysis) of the prodrug
before they interact with the active site of COX enzymes,
releasing one equivalent of NSAID + one equivalent of tyrosol;
this assumption would implicate complementary mechanisms
of action, other than, or in addition to, COX inhibition, exerted
by the NSAID and tyrosol. In this regard, there are several
reports in the literature describing the antioxidant and anti-
inflammatory properties of naturally occurring phenols such as
hydroxytyrosol, tyrosol, and 4-hydroxybenzyl alcohol, three
common phenols present in olive fruits.32 Hydroxytyrosol
has been reported to reduce serum TNFα in a LPS treated
BALB mice model33 and inhibits 5-LOX activity in vitro;34 4-
hydroxybenzyl alcohol (4-HBA) released from a biodegradable
polymer induced a dose-dependent down-regulation of iNOS
and significantly reduced the expression of TNFα in RAW
264.7 cells.35 Additionally, Lim et al. reported the anti-
inflammatory effect of 4-HBA on carageenan-induced pouch
model in rats by preventing the release of inflammatory media-
tors and reducing vascular permeability.36 These mechanisms
of anti-inflammatory activity might play a role in the improved
anti-inflammatory profile of prodrugs 9 and 10 and are the
subject of current research in our group.
Ulcer Index Assay. To evaluate the potential (unwanted)
ulcerogenic side-effects of tyrosol prodrugs, we conducted an
acute toxicity assay by administering aspirin (1.4 mmol/kg),
ibuprofen (1.2 mmol/kg), 9, or 10 (equimolar doses, po) to
rats. All drugs were suspended in 1% methylcellulose solution.
Surprisingly, aspirin tyrosol and ibuprofen tyrosol prodrugs
were considerably less ulcerogenic (UI = 2.6 and 3.5,
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a
Scheme 4. Chemical Synthesis of Indomethacin Tyrosol Ester (18)
a
Reagents and conditions: (a) THF, TEA, 25 °C, 5 h.
Table 3. In Vitro COX-1/COX-2 Enzyme Inhibition, in Vivo
Anti-inflammatory Activity, and Ulcer Index Data for
Indomethacin Tyrosol Ester (9)
appears that gastroprotection provided by both hybrid prodrugs
was not entirely dependent on the amount of NO released
from them”. This observation suggested that either NO was not
involved in reducing the ulcerogenic profile of NO-NSAIDs, or
the required threshold of NO to obtain gastroprotection is ob-
tained with only one mol of NO/mol of drug. Results provided
in our current work offer additional evidence to assume that
NO might not be required at all.
Experimental prodrugs (9, 10, 18) tested in vivo did not
possess a nitric oxide-releasing moiety, and in spite of this, all
showed a significantly reduced ulcerogenic profile compared to
their parent NSAID counterparts. In this regard, we provide
complementary evidence to support the observation that
esterification may be good enough to minimize the ulcerogenic
toxicity of NSAIDs.
COX-1 IC50
COX-2 IC50
AI
activity
a
a
b
c
compd
(μM)
(μM)
UI
18
Indomethacin
>100
4.6
61.3
9.6 2.5
d
d
d
d
0.1
5.7
38.3
34.4 4.2
a
The in vitro test compound concentration required to produce 50%
inhibition of COX-1 or COX-2. The result (IC50, μM) is the mean of
two determinations acquired using an ovine COX-1 and human
recombinant COX-2 inhibitor screening assay kit (cat. no. 560131,
Cayman Chemicals Inc., Ann Arbor MI), and the deviation from the
b
mean is <10% of the mean value. Results are expressed as % decrease
in the inflammatory response compared to control group receiving
vehicle at 3 h after oral administration of the test compound
c
(equimolar dose to indomethacin [0.01 mmol/kg]). The average
Several other groups have reported similar results, even
though they did not correlate their findings to the presence (or
absence) of NO-releasing groups, for example, nonulcerogenic
indomethacin amide derivatives,41 aminoalkyl esters of
ibuprofen and naproxen,42,43 sulindac sulfide amides,37 and
D-galactose esters of ketorolac.44 In a recent publication, Jiang
et al. reported a series of new conjugates of aspirin having one
“phenolic acid antioxidant” group (p-coumaric acid, ferulic acid,
or caffeic acid) connected through a diol linker to the car-
boxylic acid group present in aspirin. These prodrugs showed
considerable anti-inflammatory activity (croton oil-induced
mice-ear swelling) without significant GI side-effects;45 one of
the diol linkers used by Jiang’s group was tyrosol, which is the
protecting group we used in this work to form NSAID esters,
meaning that it is probably not essential to have the second
(additional) phenolic acid antioxidants linked to tyrosol to
maintain the anti-inflammatory profile of the corresponding
NSAID or to decrease its ulcerogenic effects.
overall length (in mm) of individual ulcers in each stomach SEM, n
= 4, 6 h after oral administration of the test compound (0.08 mmol/
d
kg). Reported literature values.15
at equimolar dose). Nevertheless, indomethacin tyrosol pro-
drug (18) was significantly less ulcerogenic (UI = 9.6) in rats
when administered orally (0.08 mmol/kg) compared to
indomethacin at an equimolar dose (UI = 34.4, Table 3).
The biological evaluation of compounds 9, 10, and 18
showed a common trend. All prodrugs inhibited cyclo-
oxygenase activity in vitro; ibuprofen (10) and indomethacin
(18) prodrugs showed selective inhibition of COX-2 isoform;
all prodrugs showed comparable anti-inflammatory profile in
vivo compared to their corresponding parent NSAID but
exerted a considerably lower ulcerogenic effect. This provides
complementary evidence to bring into question the cytopro-
tective mechanisms of action exerted by NO-donors present in
hybrid NO-NSAIDs.
In a recent publication, Chattopadhyay et al. conducted a
head-to-head comparison of two NO-NSAIDs, namely 1
(NCX-4016, possessing an organic nitrate) and 3 (CVM-01,
possessing a NONOate group).40 The main difference between
these two compounds (Figure 1) is the amount of NO released
from them; unlike organic nitrates, NONOates release two
equivalents of NO per mol of drug and do not require meta-
bolic activation (3-electron reduction) to release it. The as-
sumption that 3 would release twice as much NO as compared
to 1 led to the expectation that NONOate-containing drugs
would show an improved biological profile and lower degree of
ulcerogenicity; however, there was no statistically significant
difference between groups of animals receiving these two
drugs. Both NO-NSAIDs were equipotent analgesic and anti-
inflammatory agents (they reduced stomach PGE2 levels to the
same extent), and they were devoid of major gastric side effects.
In this regard, Chattopadhyay’s group concluded that “it
It is worth mentioning that the improved anti-inflammatory
profile exerted by NO-NSAIDs constitutes only one of the
several potential applications for these molecules, and the
presence of NO-releasing moieties may have a significant effect
in other conditions (for example, the use of NO-aspirin for the
prophylactic treatment of cardiovascular disorders and cancer2).
Therefore, the scope of the current work is only limited to
discussing the role of simple ester groups in decreasing
the gastric toxicity of NSAIDs while maintaining their anti-
inflammatory efficacy in vivo. In this regard, our work makes an
important distinction between the potential applications of
NO-NSAIDs and NSAID esters. If the intended application is
to treat pain and inflammation only (as it is currently the case
for most patients using NSAIDs), the use of simple NSAID esters
might be good enough to decrease the severity and high incidence
of gastric ulcers; on the other hand, if the intended application
of NSAIDs is prophylactic or the patient is considered at high
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Journal of Medicinal Chemistry
Article
molecular properties were calculated [molecular volume was expressed
in cubic angstroms (Å3), molecular surface was expressed in squared
angstroms (Å2), and length was expressed in angstroms (Å)]; mole-
cular lengths were calculated by measuring the linear distance of two
selected atoms in the molecule with the highest separation between
them. Log P values (negative logarithm of the distribution coefficient
of drugs between n-octanol/water) were calculated using ACD/
ChemSketch version 12.01 (2010) freeware, ACD Laboratories Inc.
Chemistry. Melting points were determined with an Electro-
thermal Mel-Temp melting point apparatus (Dubuque, IA, USA) and
are uncorrected. Infrared (IR) spectra were recorded as films
(chloroform solutions or neat compounds) on NaCl plates using a
risk of developing thrombotic events, the use of NO-NSAIDs
(particularly NO-aspirins) would offer additional advantages com-
pared to the use of original NSAIDs. In this regard, NO-releasing
moieties may counteract the cardiovascular toxicity of selective
COX-2 inhibitors (particularly COXIBs) and the release of NO
from NO-aspirins would potentiate the antithrombotic effect of
aspirin. Furthermore, another interesting setting on which simple
esters could be compared to NO-NSAIDs in future experiments
is on chronic animal models designed to measure the effect of
drugs on healing of pre-existing ulcers.46 Our study used relatively
high oral doses of NSAIDs rather than the administration of
lower amounts over a long period of time, which would provide
data regarding the effects of NSAID ester prodrugs when admin-
istered on a long-term basis. This constitutes one of the current
topics of research in our group.
To provide a possible explanation to the observation that
simple NSAID esters exert similar efficacy/safety ratios than
those observed with NO-NSAIDs, we hypothesize that after
oral administration, most of the NSAID ester prodrug
(regardless if it has a NO-donor or not) is absorbed mostly
intact and at a relatively faster rate than the corresponding free
NSAID. Once the ester is absorbed it may interact with and
inhibit COX enzymes, either as the tyrosol ester (active in
vitro) or as the parent NSAID. This will depend on the rate,
extent, and location of ester hydrolysis in vivo. Future research
work in this area will need to address gaps in knowledge related
to the rate, extent, and site of cleavage of esterified NSAIDs,
and such analysis should also include the investigation of the
impact of NO on NSAID-induced ulcerogenicity.
1
Nicolet 550 series II Magna FTIR spectrometer. H and 13C NMR
spectra were measured on a Bruker AM-300 spectrometer with TMS
as internal standard, where coupling constants (J) are estimated in
hertz (Hz). Mass spectra (MS) were recorded on a Water’s micromass
ZQ 4000 mass spectrometer using the ESI mode. Microanalyses were
within 0.4% of theoretical values for all elements listed (Faculty of
Chemistry, University of Alberta). Compounds 9, 10, and 18 showed a
single spot on RediSep silica gel glass plates (UV254, 0.2 mm) using a
high, medium, and low polarity solvent mixture, and no residue
reminded after combustion, indicating a purity higher than 95%.
Column chromatography was performed on a CombiFlash Retrieve
system using RediSep Rf silica gel (40−60 μM) cartridges.
Indomethacin acid chloride (17),47 ibuprofen acid chloride (12,
racemic),47 and 148 were synthesized according to reported literature
procedures. Acetylsalicyloyl chloride (11) was obtained from TCI
America (Portland, OR); all other reagents were purchased from
Aldrich Chemical Company (Milwaukee, WI) and 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.
4-(2-Hydroxyethyl)phenyl Acetylsalicyloate (9). A solution of
tyrosol (1.4 g, 7.2 mmol) and TEA (0.2 g, 1.9 mmol) in dry THF
(10 mL) was stirred for 10 min under nitrogen atmosphere before
adding (dropwise) a solution of acetylsalicyloyl chloride (1.0 g, 7.2
mmol) previously dissolved in dry THF (5 mL). This reaction mixture
was stirred 25 °C for 5 h; all solids (triethylammonium chloride) were
filtered out and the solvent was evaporated under vacuum. The residue
was purified by silica gel column chromatography using EtOAc/hexane
(2:8) as eluent to give (9) as white solid (1.1 g, 50.6% yield); mp: 66−
69 °C. IR (NaCl) 3370 (OH), 2936 (C−H aromatic), 2867 (C−H
CONCLUSIONS
■
Esterification of aspirin, ibuprofen, and indomethacin with
tyrosol yields potent and selective COX-2 inhibitors in vitro,
equivalent or slightly improved anti-inflammatory activity in
vivo, and significantly decreased gastric ulcerogenicity. Even
though this design is similar to others reported earlier, our work
offers essential evidence suggesting that NO-releasing groups
are not required to decrease the ulcerogenic profile of classical
NSAIDs, regardless if they are available over-the-counter
(aspirin, ibuprofen) or by prescription only (indomethacin).
Results obtained in this work, along with those reported
previously by other groups, constitute a good body of evidence
to question why despite the reliable efficacy/safety profile of
simple NSAID prodrugs, there is not a single NSAID oral
prodrug commercially available in North America. In this
regard, the vast majority of NSAID esters commercially
available are used topically (i.e., methyl salicylate). Therefore,
we believe it is essential to re-evaluate the potential use of new
and/or existing NSAID prodrugs as a safer alternative to the
use of classical (unprotected) NSAIDs and to start a debate
about the future replacement of NSAID prodrugs for
unprotected NSAIDs currently in clinical use. The significance
of this statement is evident considering that (i) NSAIDs are
one of the most highly used drugs worldwide, and (ii) the
relatively high incidence of gastrointestinal side effects
associated with their long-term use.
1
aliphatic), 1740 (CO) cm−1. H NMR (300 MHz, CDCl3): δ = 1.51
(bs, 1H, OH), 2.31 (s, 3H, CH3), 2.89 (t, J = 6.7 Hz, 2H, PhCH2),
3.88 (t, J = 6.7 Hz, 2H, CH2OH), 7.12 (d, J = 8.5 Hz, 2H, phenyl H-2,
H-6), 7.17 (dd, J = 7.9 Hz, 1.2 Hz, salicyloate H-3), 7.28 (d, J = 8.5
Hz, 2H, phenyl H-3, H-5), 7.39 (td, J = 7.3, 1.2 Hz, salicyloate H-5),
7.64 (td, J = 7.3, 1.8 Hz, salicyloate H-4), 8.22 (dd, J = 7.3, 1.2 Hz,
salicyloate H-6). 13C NMR (300 MHz, CDCl3) δ = 21.0, 38.6, 63.5,
115.4, 121.7, 122.5, 124.0, 126.1, 130.1, 132.2, 134.5, 136.4, 149.1,
151.1, 163.0. MS: 301.0 [M + 1]+. Anal. Calcd for C17H16O5: C, 67.99;
H, 5.37. Found C, 67.74; H, 5.15.
4-(2-Hydroxyethyl)phenyl 2-(4-isobutylphenyl)propanoate (10).
A solution of tyrosol (1.5, 11.1 mmol) and TEA (3.3 g, 13.3 mmol) in
dry THF (15 mL) was stirred for 10 min under nitrogen atmosphere
before adding (dropwise) a solution of ibuprofen acid chloride (1b, 2.5
g, 11.1 mmol) in dry THF (10 mL). This reaction mixture was stirred
at 25 °C for 5 h; all solids (triethylammonium chloride) were filtered
out and the solvent was evaporated under vacuum. The residue was
purified by silica gel column chromatography using EtOAc/hexane
(2:8) as eluent to give (10) as white solid (1.9 g, 53.2% yield); mp:
47−50 °C. IR (NaCl) 3383 (OH), 2955 (C−H aromatic), 2867 (C−
H aliphatic), 1740 (CO) cm−1. 1H NMR (300 MHz, CDCl3) δ = 0.91
(d, J = 6.6 Hz, 6H, CH(CH3)2), 1.60 (d, J = 7.3 Hz, 3H, PhCHCH3),
1.85 (nonet, J = 6.7 Hz, 1H, CH2CH(CH3)2), 2.47 (d, J = 7.3 Hz, 2H,
PhCH2), 2.83 (t, J = 6.7 Hz, 2H, PhCH2), 3.82 (t, J = 6.7 Hz, 2H,
CH2OH), 3.93 (q, J = 7.3 Hz, 1H, PhCH), 6.93 (d, J = 8.5 Hz, 2H,
phenyl H-2, H-6), 7.14 (d, J = 7.9 Hz, 2H, tyrosol phenyl H-2, H-6),
7.18 (d, J = 8.5 Hz, 2H, phenyl H-3, H-5), 7.29 (d, J = 7.9 Hz, 2H,
tyrosol phenyl H-3, H-5). 13C NMR (300 MHz, CDCl3) δ = 18.5,
EXPERIMENTAL SECTION
■
Determination of Molecular Length, Surface, and Volume.
Test compounds were drawn using BioDraw Ultra (version 11.0,
Cambridge Soft USA); the structures were copied and pasted to
Alchemy 2000 (version 2.0, 1997, Tripos Inc.) and their steric energies
were minimized. Immediately after energy minimization, their
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Journal of Medicinal Chemistry
Article
22.3, 30.1, 38.5, 45.0, 45.2, 63.5, 121.2, 121.4, 127.1, 129.2, 129.3,
129.4, 129.8, 135.9, 137.1, 140.7, 149.4, 173.3. MS 327 [M + 1]+. Anal.
Calcd for C21H26O3: C, 77.27; H, 8.03. Found C, 77.33; H, 8.07.
4-(2-Hydroxyethyl)phenyl 2-(1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indol-3-yl)acetate (18). A solution of tyrosol (0.18 g,
1.3 mmol) and TEA (0.16 g, 1.5 mmol) in dry THF (10 mL) was
stirred under nitrogen atmosphere at −80 °C for 10 min before adding
(dropwise) a solution of indomethacin acid chloride (17, 0.50 g, 1.3
mmol) in THF (5 mL). This reaction mixture was stirred at −80 °C
for 5 h; the precipitated salts (triethylammonium chloride) were
filtered out and the solvent was evaporated under vacuum. The residue
was purified by silica gel column chromatography using EtOAc/hexane
(3:7) as eluent to give 18 as a dark-green viscous liquid (0.12 g,
19.46% yield). IR (NaCl): 3458 (OH), 2999 (C−H aromatic), 2904
(C−H aliphatic). 1H NMR (300 MHz, CDCl3) δ = 2.29 (s, 3H, CH3),
2.81 (t, J = 6.7 Hz, 2H, PhCH2), 3.62 (s, 2H, CH2CO2), 3.82 (s, 3H,
OCH3), 4.27 (t, J = 6.7 Hz, 2H, CH2OH), 6.64 (dd, J = 9.1 Hz, 2.4
Hz, 2H, indolyl H-6), 6.65 (d, J = 8.5 Hz, 2H, phenyl H-2, H-6), 6.84
(d, J = 9.1 Hz, 1H, indolyl H-7), 6.91 (d, J = 8.5 Hz, 2H, phenyl H-3,
H-5), 6.94 (d, J = 2.4 Hz, 1H, indolyl H-4), 7.43 (dd, J = 8.5 Hz, 2.4
Hz, 2H, benzoyl H-3, H-5), 7.63 (dd, J = 8.5 Hz, 2.4 Hz, 2H, benzoyl
H-2, H-6). 13C NMR (300 MHz, CDCl3) δ = 13.3, 30.4, 34.1, 55.7,
65.5, 101.4, 111.6, 112.5, 114.9, 115.3, 129.1, 129.6, 129.8, 130.6,
130.8, 131.1, 133.9, 135.7, 139.2, 154.1, 156.0, 168.3, 170.7. MS: 477
[M + 1]+. Anal. Calcd for C27H24ClNO5: C, 67.85; H, 5.06; N, 2.93.
Found C, 67.61; H, 5.10; N, 2.62.
In Vitro Cyclooxygenase Inhibition Assay. Experimental
compounds 9, 10, and 18 were evaluated for their ability to inhibit
human recombinant COX-2 and ovine COX-1 using a cyclooxygenase
inhibitor screening assay kit (catalogue no. 560131, Cayman Chemical,
Ann Arbor, MI, USA) following the procedure suggested by the
manufacturer.
In Vivo Anti-inflammatory Assay. NSAID prodrugs (9, 10, and
18) were evaluated using the carrageenan-induced rat foot paw edema
model reported previously.30 All experimental compounds were
suspended in 1.2 mL of 1% methylcellulose solution and administered
orally by gavage at the following doses: aspirin and aspirin deriva-
tive (9) = 0.71 mmol/kg; ibuprofen and ibuprofen derivative (10) =
0.32 mmol/kg; indomethacin and indomethacin derivative (18) =
0.01 mmol/kg; animals in the control group received an equivalent
volume of 1% methylcellulose solution. This assay was carried out
using protocols approved by the Health Sciences Animal Welfare
Committee at the University of Alberta.
Ulcer Index Assay. The ability to produce gastric damage was
evaluated according to a reported procedure.49 Ulcerogenic activity
was evaluated after oral administration of aspirin (1.4 mmol/kg),
ibuprofen (1.4 mmol/kg), indomethacin (0.08 mmol/kg), or an
equivalent amount of the correspondent test compounds (9, 10, or
18). All drugs were suspended and administered in 1.2 mL of a 1%
methylcellulose solution. Control rats received oral administration of
vehicle (1.2 mL of 1.0% 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 the
greater curvature of the stomach, gently rinsed with water, and placed
on ice. The number and the length of ulcers were determined using a
magnifier lense. The severity of the gastric lesion was measured along
its greatest length (1 mm = rating of 1, 1−2 mm = rating of 2, >2 mm
= rating according to their length in mm). The “ulcer index” (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.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support provided by
the Faculty of Pharmacy and Pharmaceutical Sciences at the
University of Alberta for providing start up funds. We thank
Ying Dong for carrying out the anti-inflammatory assay in vivo.
ABBREVIATION USED
■
AA, arachidonic acid; COX, cyclooxygenase enzyme; GI,
gastrointestinal; NO, nitric oxide; NONOate, N-diazen-1-ium-
1,2-diolate; NSAID, nonsteroidal anti-inflammatory drug; NO-
NSAID, nitric oxide-releasing nonsteroidal anti-inflammatory
drug; PG, prostaglandin; Tx, thromboxane; PROLI/NO,
NONOate ion derived from L-proline; SI, selectivity index;
UI, ulcer index
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: (780)-248-1557. Fax: (780)-492-1217. E-mail:
cvelazquez@pharmacy.ualberta.ca. Full address: 2142-L Katz
Centre for Pharmacy and Health Research, University of
Alberta, Edmonton, Alberta T6G 2E9, Canada.
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