Gastric-sparing nitric oxide-releasable 'true' prodrugs of aspirin and naproxen

Article abstract of DOI:10.1016/j.bmcl.2014.10.096

Nitric oxide-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) are gaining attention as potentially gastric-sparing NSAIDs. Herein, we report a novel class of '1-(nitrooxy)ethyl ester' group-containing NSAIDS as efficient NO releasing 'true' pro

Full text of DOI:10.1016/j.bmcl.2014.10.096

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5587–5592
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Gastric-sparing nitric oxide-releasable ‘true’ prodrugs of aspirin
and naproxen
Machhindra Gund a, Parikshit Gaikwad b, Namdev Borhade a, Aslam Burhan b, Dattatraya C. Desai c,
Ankur Sharma b, Mini Dhiman c, Mohan Patil b, Javed Sheikh a, Gajanan Thakre a, Santhosh G. Tipparam a,
Somesh Sharma b, Kumar V. S. Nemmani b, Apparao Satyam a,
⇑
a Medicinal Chemistry Division, Piramal Life Sciences, Piramal Enterprises Limited, 1-Nirlon Complex, Goregaon East, Mumbai 400063, India
b Pharmacology Division, Piramal Life Sciences, Piramal Enterprises Limited, 1-Nirlon Complex, Goregaon East, Mumbai 400063, India
c Analytical Chemistry-Medicinal Chemistry Division, Piramal Life Sciences, Piramal Enterprises Limited, 1-Nirlon Complex, Goregaon East, Mumbai 400063, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2014
Revised 18 October 2014
Accepted 30 October 2014
Available online 5 November 2014
Nitric oxide-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) are gaining attention as
potentially gastric-sparing NSAIDs. Herein, we report a novel class of ‘1-(nitrooxy)ethyl ester’ group-con-
taining NSAIDS as efficient NO releasing ‘true’ prodrugs of aspirin and naproxen. While an aspirin prodrug
exhibited comparable oral bioavailability and antiplatelet activity (i.e., TXB2 inhibition) to those of aspi-
rin, a naproxen prodrug exhibited better bioavailability than naproxen. These promising NO-NSAIDs pro-
tected experimental rats from gastric damage. We therefore believe that these promising NO-NSAIDs
could represent a new class of potentially ‘Safe NSAIDs’ for the treatment of arthritic pain, inflammation
and cardiovascular disorders in the case of NO-aspirin.
Keywords:
NO-aspirin
NO-naproxen
NO-NSAIDs
Ó 2014 Elsevier Ltd. All rights reserved.
Gastric-sparing NSAIDs
Nitric oxide donors
Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin
and naproxen are widely used to reduce pain, arthritis and inflam-
mation. However, NSAIDs can cause undesirable gastrointestinal
(GI) toxic effects such as bleeding, dyspepsia and peptic ulcers.
The NSAID-induced GI toxicities could be of either systemic or local
origin.1,2 While the systemic effects are thought to be due to the
inhibition of COX-1 isozyme, the local effects are largely due to
the presence of free carboxylic acid group, which can cause local
irritation upon oral administration. A widely used approach to
reduce local GI effects is to temporarily mask the free carboxyl
group in the form of an ester prodrug.3–7 It was for the first time,
Bayer introduced aspirin (acetylsalicylic acid) as a less gastric irri-
tating derivative of the NSAID sodium salicylate in 1899. Based on
known definitions of prodrugs, aspirin can be considered as a pro-
drug as it is hydrolyzed by plasma esterases to release its parent
drug salicylate in vivo. However, aspirin also exhibits intrinsic
pharmacological activity such as inhibition of COX isozymes.8
Aspirin (O-acetylsalicylic acid) is the most versatile pharmaceu-
tical agent known with antipyretic, analgesic, anti-inflammatory,
and antiplatelet properties. Aspirin can inhibit platelet cyclooxy-
genase, thromboxane production and protect against arterial
thrombosis at low doses of 75–100 mg per a day.9 However, similar
to other NSAIDs, chronic treatment with aspirin even at smaller
doses can increase the risk of major bleeding complications. Unfor-
tunately, the ester prodrug strategy cannot be applied easily to
aspirin to reduce the local GI toxicity by temporarily masking of
its free carboxyl acid group. This is due to the fact that aspirin exits
in carboxylate form at biological pH and the carboxylate anion
seems to stabilize the adjacent O-acetyl group from hydrolysis.
However, esterification of carboxyl group in aspirin, which
amounts to neutralizing the negative group of the aspirin, renders
the adjacent O-acetyl group extremely susceptible to enzymatic
cleavage.10 In order to act as a ‘true’ prodrug of aspirin, its carboxyl
masking group must be cleaved faster than its O-acetyl group. It is
therefore a challenging task to design prodrugs of aspirin. Hence,
there are only a few reported examples of true prodrugs of aspirin
in the literature.11–13,10,14–19
Another potentially promising approach to reduce NSAID-
induced gastrotoxicity is to attach a nitric oxide (NO)-releasing
moiety to standard NSAIDs to yield a new class of compounds called
NO-releasing NSAIDs (NO-NSAIDs), which are also sometimes
known as cyclooxygenase inhibitory NO-donors (CINODs). The
rationale behind this strategy is to harness the beneficial effects
of NO, which is recognized as a critical mediator of GI mucosal
defense, influencing factors such as mucus secretion, mucosal blood
⇑
Corresponding author. Tel.: +91 93238 02658; fax: +91 22 3081 8334.
E-mail address: apparaosvgk@hotmail.com (A. Satyam).
http://dx.doi.org/10.1016/j.bmcl.2014.10.096
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

5588
M. Gund et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5587–5592
represented by ‘AC(@O)AOAC(H)(R)AOANO2’, which can undergo
H
R
O
facile hydrolysis (either by acid-catalyzed or esterase-catalyzed) to
release the constituent NSAID as shown by plausible mechanisms
in Figure 3. In fact, these prodrugs can be structurally considered
as double esters of an organic carboxylic acid such as aspirin or
naproxen and an inorganic acid such as nitric acid. However, these
prodrugs also possess a unique acid-labile acetal function, which
can force these unique prodrugs to degrade only in a particular
way to release nitrate species under acidic aqueous conditions.
Thus, under acidic aqueous conditions such as in simulated gastric
fluid (SGF, pH 1.2), initial protonation on the ester/acetal oxygen
atom of either inorganic acid (i.e., Pathway A) or carboxylic acid
(i.e., Pathway B) would trigger cleavage of the prodrug to release
free carboxylic acid (i.e., aspirin or naproxen), the corresponding
aldehyde (i.e., RCHO) and nitrate group (NOÀ3 ) as shown in Figure 3.
In simulated intestinal fluid (SIF, pH 6.8) or human plasma, ester-
ases can initiate cleavage of carboxyl ester group, which can trigger
further cleavage to release the same species (i.e., aspirin or
naproxen, the corresponding aldehyde and nitrate) as shown in
Figure 3. Thus, as per the proposed mechanisms of cleavage of
these prodrugs, an aldehyde (RCHO) and nitrate (NOÀ3 ) species
NO2
Dn
1A, Dn = D1, R = CH3;
O
O
1B, Dn = D1, R = CH2CH3;
1C, Dn = D1, R = CH2CH2CH3;
2A, Dn = D2, R = CH3;
2B, Dn = D2, R = CH2CH3;
2C, Dn = D2, R = CH2CH2CH3;
2D, Dn = D2, R = CH2CH2CH2CH3;
Where, Dn = D1, D2, D3 and so on.
AcO
O
O
=
O
D1
O
Aspirin
CH3
O
O
=
D2
O
O
MeO
Naproxen
Figure 2. Structures of NO-aspirin prodrugs (1A–C) and NO-naproxen prodrugs
(2A–D).
ALDH
R-CHO
R-CO2H
O
H
O
R
NO2
flow, ulcer repair, down-regulation of inflammatory responses,
scavenging of various free radical species and protection of the GI
mucosa from injury induced by many topical irritants.20–22 As
expected, NO-NSAIDs have been shown to cause reduced GI dam-
age, while exhibiting comparable analgesic and anti-inflammatory
activity to those of their respective parent NSAIDs.23 Except
NO-aspirin compounds, all other reported NO-NSAIDs acted as true
prodrugs by releasing nitric oxide and their respective parent
NSAIDs in vivo or in plasma. Even a widely studied NO-aspirin com-
pound such as NCX-4016 did not act as true prodrug of aspirin and
released only NO and salicylic acid in vivo or in plasma.24 Here
again, it is a very challenging task to design a true NO releasing pro-
drug of aspirin. Hence, there are only a few examples of NO-aspirin
prodrugs which can release NO and some amount (9–71%) of aspi-
rin along with salicylic acid in human plasma or serum. Structures
of known NO-releasing true prodrugs of aspirin are shown in
Figure 1 (see Supplementary material).25–29
In our quest for finding a potentially gastric-sparing ‘Safe NSAID’,
we have earlier designed, synthesized and evaluated a number of
novel NO-NSAIDs containing ‘SS-nitrates’ as NO-releasing moieties
and we were successful in identifying a few promising NO-NSAIDs
such as NO-aspirin (P1539), NO-diclofenac (P2026) and NO-
naproxen (P1853), which showed significant oral bioavailability,
anti-inflammatory activity and protected experimental rats from
NSAID-induced gastric damage, which could be attributable to the
physiological actions of NO released from these promising pro-
drugs.30–32 Unfortunately, like NCX-4016, P1539 also failed to act
as a true prodrug of aspirin (i.e., it released only salicylic acid in
human plasma). We now report in this preliminary communication,
our design, synthesis and evaluation of a novel class of 1-(nitro-
oxy)alkyl esters of NSAIDs as efficient NO-releasing ‘true’ prodrugs
of aspirin and naproxen (Fig. 2). To our knowledge, this is the first
report of such unique NO-releasing true prodrugs of aspirin and
naproxen. We also believe that it is the first report of a prodrug of
naproxen which exhibits significantly superior oral bioavailability
than its parent drug naproxen.
Dn
O
O
Dn
OH
H
Released Aspirin
or Naproxen
NO2
H
O
H
H+ -
O
Pathway B
(or)
H
(Nitrate)
O
R
H
O
H2O
NO2
Dn
O
O
Dn
O
R
H
Pathway A
NO2
H+ -
O
O
H
H2O
H3O
(Nitrate)
37 oC
H
O
SGF (pH 1.2)
R
H
Dn
O
OH
NO2
Released Drug
Dn
O
O
[Aspirin (when Dn = D1) or
Naproxen (when Dn = D2)]
NO-NSAIDs
(1A-C or 2A-D)
(when R = Me)
SIF (pH 6.8) or
Human Plasma
37 oC
Esterase
CH3
H
O
H
O
R
Acetaldehyde
NO2
O
Dn
O
O
ALDH
Esterase
H2O
Dn
OH
CH3
HO
Released Aspirina
or Naproxen
R-CHO
ALDH
O
Acetic acid
Bacterial
nitrate
reductase
H+
NO2
NO
R-CO2H
-O
Nitrate
-O
Nitrite
NO
Nitric oxide
Where, R = CH3, CH3CH2, CH3CH2CH2 or CH3CH2CH2CH2;
ALDH = Aldehyde dehydrogenase; SGF = Simulated Gastric
Fluid at pH 1.2; SIF = Simulated Intestinal Fluid, pH 6.8.
Figure 3. Proposed plausible mechanisms of drug release from the reported NO-
NSAIDs either by acid-catalyzed (shown by red arrows) or by enzyme-catalyzed
(shown by blue arrows) hydrolysis. aIn human plasma, NO-aspirin prodrug 1A
released only 8–9% of aspirin and the rest as salicylic acid. Where R = CH3, CH3CH2,
CH3CH2CH2 or CH3CH2CH2CH2; ALDH = aldehyde dehydrogenase; SGF = simulated
gastric fluid at pH 1.2; SIF = simulated intestinal fluid, pH 6.8.
Design of NO-NSAIDs
A salient feature in our design of these novel NO-NSAIDs is the
introduction of a unique ‘1-(nitrooxy)alkyl ester–acetal linkage’

M. Gund et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5587–5592
5589
Thus, in the first step, aspirin (D1-C(@O)OH) or naproxen
(D2-C(@O)OH) was converted to their respective acid chlorides
(D1-1 or D2-1) by a known procedure such as treating with oxalyl
chloride–DMF or thionyl chloride. In the second step, the acid chlo-
ride was reacted with an appropriate aldehyde [i.e., acetaldehyde
(Aa) or propionaldehyde (Ab) or butyraldehyde (Ac) or pentanal-
dehyde (Ad)] in presence of zinc chloride to yield the respective
O
O
a
Dn
OH
Dn
D1-1 D2-1
Cl
Step 1
Aspirin (when Dn = D1) or
Naproxen (when Dn = D2)
or
b
Step 2
a
-chloroalkyl ester (D1-2a–c or D2-2a–d). In the third step, the
H
R
O
c
a
-chloroalkyl ester (D1-2a or D1-2b or D1-2c or D2-2a
1A-C or 2A-D
resulting
O
Step 3
Dn
D1-2a-c or D2-2a-d
Cl
or D2-2b or D2-2c or D2-2d) was treated with silver nitrate in
acetonitrile35 to afford the respective NO-aspirin prodrugs 1A–C
and NO-naproxen prodrugs 2A–D.
Scheme 1. Synthetic method for NO-aspirin prodrugs (1A–C) and NO-naproxen
prodrugs (2A–D). Reagents and conditions: (a) oxalyl chloride, DMF (1 or 2 drops),
DCM, rt, 3 h or thionyl chloride, rt or reflux; (b) ZnCl2, DCM, À15 °C to rt, R-CHO
(Aa-d), 23% (D1-2a), 30% (D1-2b), 43% (D1-2c), 80% (D2-2a), 30% (D2-2b), 65%
(D2-2c), 23% (D2-2d); (c) AgNO3, acetonitrile, rt or reflux, 81% (1A), 65% (1B), 47%
(1C), 74% (2A), 31% (2B), 57% (2C), 35% (2D).
Metabolic studies in biologically relevant fluids
The NO-aspirin prodrug 1A and the NO-naproxen prodrugs
2A–D were incubated in biologically relevant simulated gastric
fluid (SGF at pH 1.2) at 37 °C, which is close to acidic stomach envi-
ronment, and estimated their half-lives (t1/2) and the amount (%) of
drug release over 1–3 h (Table 1). The aspirin prodrug 1A was also
incubated in simulated intestinal fluid (SIF) and 100% human
plasma and estimated its drug release profile (Table 1).
are also generated along with the free Aspirin or Naproxen. Under
normal physiological conditions, the aldehyde species (RCHO, pos-
sibly toxic) would be quickly oxidized further by the enzyme alde-
hyde dehydrogenase (ALDH) (EC 1.2.1.3) to their respective acids
that are likely to be less toxic and can be excreted from the body
easily. However, it is preferable to select methyl as R group so that
the liberated aldehyde would be a less toxic acetaldehyde, which is
the usual metabolite formed in our body via oxidation of ethyl
alcohol by the enzyme alcohol dehydrogenase (ADH) (EC 1.1.1.1)
and the acetaldehyde thus generated can be oxidized further to
an innocuous acetic acid by the enzyme ALDH. The liberated
nitrate group can be reduced by oral bacterial nitrate reductase
to nitrite ion, which in turn can get reduced to NO in the acidic
conditions of the stomach.33,34 Thus, after oral administration,
these prodrugs are expected to be hydrolyzed quickly in the acidic
stomach region to release NO, acetaldehyde (i.e., when R = CH3)
and the free NSAID. Notably, prodrugs derived from aspirin acted
as true prodrugs of aspirin by releasing quantitative amounts of
aspirin in biologically relevant fluids. In fact, this platform prodrug
technology can be applied to any therapeutic agent containing at
least one derivatizable carboxylic acid group.
Thus, all the prodrugs released their respective parent drug
[either aspirin (D1-C(@O)OH) or naproxen (D2-C(@O)OH)], but the
rate of drug release was found to be dependent on the nature of their
R group. Thus, both the NO-aspirin prodrug 1A (R is methyl) and the
NO-naproxen prodrug 2A (R is methyl) decomposed in SGF with
half-lives of <10 min and <30 min, respectively, and released quan-
titative amounts (i.e., 99–100%) of their parent drugs aspirin and
naproxen, respectively, within 60 min of incubation in SGF. How-
ever, the NO-naproxen prodrugs 2B–D exhibited half-lives (t1/2) of
ꢀ60 min (when R is ethyl), <3 h (when R is n-propyl) and >3 h (when
R is n-butyl) and released 93%, 61% and 29% of naproxen, respec-
tively, after 3 h of incubation is SGF. This is expected because the
amount of drug release from these prodrugs is proportional to their
hydrophobicity, which is expected to increase proportionally with
increase in the size of R group. In SIF, the aspirin prodrug 1A decom-
posed with a half-life of <20 min and released 100% of aspirin in
about 2 h of incubation. However, in 100% human plasma, this aspi-
rin prodrug 1A decomposed with a half-life of <5 min. It seems that
this prodrug has acted more like a prodrug of salicylic acid by releas-
ing 79% of salicylic acid and only 8–9% of aspirin within 5 min of
incubation is human plasma (Table 1).
Synthesis of NO-NSAIDs
Synthesis of NO-aspirin prodrugs (1A–C) and NO-naproxen pro-
drugs (2A–D) was performed in 3 steps as shown in Scheme 1.
Table 1
Stability of NO-aspirin prodrug (1A) and NO-naproxen prodrugs (2A–D) in SGF, SIF and human plasma and percentage (%) of drug {aspirin [D1-C(@O)OH]} or {naproxen
[D2-C(@O)OH]} or salicylic acid (SA) release
Time (min)
SGF
SIF
100% HP
(pH 1.2)
(pH 6.8)
1A
2A
2B
2C
2D
1A
1A
1Aa
Aspb
2Aa
Npb
2Ba
Npb
2Ca
Npb
2Da
Npb
1Aa
Aspb
Aspb
SAc
0
2
5
10
15
20
30
40
100
78
65
45
—
14
—
12
0
0
100
—
85
75
65
—
33
—
0
0
—
100
—
94
90
74
—
62
—
51
39
7
ꢀ60 min
0
—
6
100
—
96
94
83
—
83
—
75
58
39
<3 h
0
—
4
6
17
—
17
—
25
42
61
100
—
98
96
87
—
87
—
87
80
71
>3 h
0
—
2
4
13
—
13
—
13
20
29
100
91
82
52
—
32
—
—
0
9
0
—
8
9
—
8
—
6
0
—
22
35
55
—
86
—
88
99
—
15
25
35
—
67
—
100
—
—
18
48
—
68
—
79
86
—
90
—
90
95
100
—
10
26
—
38
—
49
61
93
—
60
120
180
Half-life (t1/2
4
0
—
96
100
—
0
0
—
—
—
—
—
—
<5 min
)
<10 min
<30 min
<15 min
SGF = simulated gastric fluid; SIF = simulated intestinal fluid; HP = human plasma; — = not determined.
a
Percentage (%) of NO-NSAID (i.e., 1A or 2A or 2B or 2C or 2D) remaining.
b
Percentage (%) of Asp = aspirin [D1-C(@O)OH] or Np = naproxen [D2-C(@O)OH] released.
c
Percentage (%) of SA released.

5590
M. Gund et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5587–5592
centration in naproxen treated animals, although showed a Cmax
of above 55 g/mL at 15 min, quickly reached to just above
30 g/ml in 2 h and to just above 20 g/mL in a period of 4 h and
it further dropped to below 15 g/ml in a period of 8 h. So, the pro-
drug 2A has exhibited controlled release of higher amounts of
naproxen over a longer period of time (over 30
6 h duration) when compared to naproxen at equimolar doses. This
prodrug is therefore expected to offer better pain relief for a longer
period of time than the parent drug naproxen although the parent
drug is expected to offer quicker relief from pain than its prodrug
due to its faster absorption within 15 min of administration of the
drug. The remaining prodrugs in the naproxen series (i.e., 2B, 2C
and 2D) exhibited either comparable (2B with an AUC value of
Biological evaluation
l
l
l
The NO-aspirin prodrugs 1A–C and NO-naproxen prodrugs
2A–D were evaluated in vivo to establish their bioavailability.
Based on their promising bioavailability data, the NO-aspirin
prodrug 1A and the NO-naproxen prodrug 2A were selected and
evaluated further for their nitric oxide release capabilities and their
gastric-sparing/damaging effects in comparison to those of their
respective parent drugs. The NO-aspirin prodrug 1A was also
evaluated for its ability to inhibit thromboxane B2 (TXB2) and
compared the result with that of aspirin at equimolar dose.
l
l
g/mL⁄⁄⁄ up to
Bioavailability studies
182.70 8.10
values of 178.60 8.10
respectively) bioavailability when compared to that of naproxen
with an AUC value of 207.80 18.20 g*h/mL and also showed
some decreasing trend, although not significant, in bioavailability
with increasing chain length of ‘R’ group. Based on the above bio-
availability data and on the in vitro drug release profile (Table 1),
we have selected the best NO-naproxen prodrug 2A for further
evaluation. To our knowledge, this is the first report of a naproxen
prodrug, which has shown significantly superior oral bioavailabil-
ity than its parent drug naproxen.
l
g*h/mL) or slightly less (i.e., 2C and 2D with AUC
All the reported NO-NSAIDs 1A–C and 2A–D were subjected to
pharmacokinetics studies using rat as animal model and deter-
mined their bioavailabilities. For NO-naproxen prodrugs 2A–D
and naproxen, the presented bioavailability [area under curve
(AUC)] data correspond to the plasma concentration of the released
parent drug naproxen. However, in the case of NO-aspirin prodrugs
1A–C, the reported AUC data corresponds to plasma concentration
of the released salicylic acid, since aspirin and NO-aspirin rapidly
undergo enzymatic hydrolysis in vivo to generate salicylic acid.
Among the NO-aspirin series, as shown in Figure 4 (see Supplemen-
tary material), the prodrug 1A showed nearly comparable bioavail-
ability to that of aspirin (i.e., AUCs: 91.13 12.2 vs
l
g*h/mL and 177.40 4.10 g*h/mL,
l
l
Anti-inflammatory efficacy
89.78 10.2 lg*h/ml). The other two prodrugs 1B and 1C exhibited
lower bioavailabilities when compared to that of aspirin. Thus, the
prodrug 1A (with methyl as R group) is the best among three NO-
aspirin prodrugs. When R group is ethyl, the corresponding prodrug
It is known that the anti-inflammatory activity of an NSAID is
directly proportional to the plasma concentration of the drug.30,31
Thus, based on their comparable oral bioavailability data, the
promising NO-aspirin prodrug 1A is expected to show comparable
anti-inflammatory activity to that of the parent drug aspirin.
Similarly, based on its superior and improved bioavailability, the
NO-naproxen prodrug 2A is expected to show superior or at least
comparable anti-inflammatory activity to that of naproxen in the
carrageenan-induced rat paw edema model. We have chosen to
defer these in vivo experiments to save experimental animals
and resources but the anti-inflammatory activity of these promis-
ing prodrugs can be readily assessed in carrageenan-induced rat
paw edema model according to the reported procedure.36
1B
53.56 15.6
ing prodrug 1C showed slightly better bioavailability (AUC:
73.54 4.9 g*h/ml) than that of 1B. Although the AUC values are
showed
significantly
less
bioavailability
(AUC:
lg*h/ml). When R group is n-propyl, the correspond-
l
nearly comparable for aspirin and NO-aspirin prodrug 1A, they
exhibited significantly different Tmax values (15 min and 2 h,
respectively) and Cmax values (26.12 2.11
18.57 1.60 g/ml, respectively).
lg/ml and
l
In order to assess the species-specific differences in oral bioavail-
ability of these prodrugs, we have carried out PK studies on the most
promising NO-aspirin prodrug 1A and aspirin in Wistar rats and the
corresponding results are presented in Figure 5 (see Supplementary
material). Interestingly, both aspirin and its prodrug 1A have shown
Estimation of NO release from the promising NO-NSAIDs
comparable bioavailability (AUCs: 436.8 26.2
lg*h/mL vs
We have evaluated the NO releasing capability of the promising
NO-NSAIDs 1A and 2A in SD rats. The NO release profile in the blood
plasma which is an indirect measure of the NO (i.e., nitrate/nitrite)
released in the blood was estimated by using Griess method37 and
the data obtained from these experiments is presented in Figure 7
(see Supplementary material) and Table 2. As shown in Table 2,
the NO-aspirin prodrug 1A released more NO than the
NO-naproxen prodrug 2A (AUCs: 1481.00 lM*h vs 686.80 lM*h).
However, their NO release profiles were different in that the
NO-aspirin prodrug 1A treated rats showed plasma NOx
397.6 28.0 g*h/mL) in Wistar rats also. However, both aspirin
l
and its prodrug 1A have shown strikingly improved oral absorption
in Wistar rats as compared to that in Sprague–Dawley (SD) rats
(AUCs for Aspirin: 436.8 26.2 vs 91.13 12.20 at 30 mg/kg equi-
molar dose; AUCs for prodrug 1A: 397.6 28.0
lg*h/mL vs
89.78 10.20 g*h/mL at 44.83 mg/kg, which is equimolar to
l
30 mg/kg dose of aspirin). Based on the above bioavailability data
and on the in vitro drug release profile (Table 1), we have selected
the best NO-aspirin prodrug 1A for further evaluation.
Among the NO-naproxen series also, as shown in Figure 6 (see
Supplementary material), the prodrug 2A, which contains methyl
as R group, exhibited superior and statistically significant increase
concentration of ꢀ180
lM over a period of 8 h, where as the
Table 2
l
g*h/mL, **p <0.01) over that
Estimation of NO release (i.e., plasma nitrate/nitrite concentration) from the most
promising NO-NSAIDs 1A and 2A in SD rats
in bioavailability (AUC: 272.60 8.50
of naproxen (AUC: 207.80 18.20 g*h/mL) in SD rats. However, it
l
Prodruga
Plasma nitrate/nitrite
AUC ( M*h)
is interesting to see their important PK parameters: while their
Tmax values are different (i.e., 15 min for naproxen vs 1 h for the
prodrug 2A), their Cmax values are not significantly different (i.e.,
l
Vehicle
1Ab
371.10
1481.00
686.80
54.97 2.42
lg/ml and 49.37 5.61 lg/ml, respectively). It is also
2Ac
interesting to see that the plasma drug concentration in prodrug
a
b
c
treated animals was found to be between 30 and 35
the period from 0.5 h to 6.0 h (between 40 and 55
the period between 1 h and 4 h). However, the plasma drug con-
l
g/mL during
All the compounds were administered orally.
At a dose equimolar to 10 mg/kg dose of aspirin.
At a dose equimolar to 30 mg/kg dose of naproxen.
lg/mL during

M. Gund et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5587–5592
5591
NO-naproxen prodrug 2A treated rats have shown ꢀ150
plasma NOx concentration during first 1 h, which fell to ꢀ120
l
l
M
M
800
600
400
200
0
580 80
by 2 h and to ꢀ60
lM by 4 h, which finally reached to basal level
by 6 h.
Gastric tolerance study
Since the reported prodrugs release their parent NSAIDs quan-
titatively in the stomach region following their oral administration,
it is important see whether the released NSAIDs could cause the
usual NSAID-induced gastric bleeding, lesions and ulcers in exper-
imental animals. We have therefore evaluated the most promising
aspirin prodrug 1A and the naproxen prodrug 2A, after acute oral
dosing of rats with 298.8 mg/kg of 1A, which is equivalent to
200 mg/kg of aspirin and 138.67 mg/kg of 2A, which is equivalent
to 100 mg/kg of naproxen, to determine their gastric tolerance
capability compared to gastric ulcer-causing potential of their
respective parent drugs, aspirin and naproxen (both at doses of
100 mg/kg). The results associated with these experiments are pre-
sented graphically in Figures 8 and 9, respectively. The actual
stomach images from these experiments are presented in Figures
S1 and S2, respectively, (see Supplementary material). These
results clearly establish that none of the animals treated with the
prodrugs 1A and 2A showed any significant development of gastric
lesions or ulcers. However, severe hemorrhagic lesions and ulcers
were developed in rats administered with parent drugs, aspirin
and naproxen (both at doses of 100 mg/kg). We believe that the
observed gastric-sparing effects of these promising prodrugs 1A
and 2A could be partly attributable to the physiological functions
of NO released from these novel prodrugs. However, it is possible
that the masking of carboxylic acid groups of NSAIDs as ester pro-
drugs might have partly contributed to the gastric-sparing effects
of these prodrugs.38–40
39 20
***
Naproxen
(100 mg/kg, p.o.)
2A
(138.67 mg/kg, p.o.)
***p < 0.001 versus Naproxen
Figure 9. Gastric lesion area (mm2) of rat stomachs after acute oral dosing of rats
with naproxen sodium (109.52 mg/kg, which is a dose equimolar to 100 mg/kg dose
of naproxen) and its prodrug 2A (138.67 mg/kg, which is a dose equimolar to
100 mg/kg of naproxen).
300
200
72.59%
100
0
75.97%
**
**
Vehicle
(1 ml/kg)
Aspirin
1A
(30 mg/kg) (30 mg/kg eq.)
** p < 0.01 versus vehicle
Figure 10. In vivo inhibition of TXB2 (i.e., indicated by the reduction in serum TXB2
levels) after oral dosing of rats with aspirin (30 mg/kg) and its promising prodrug
1A (44.82 mg/kg, which is equimolar to 30 mg/kg dose of aspirin).
TXB2 inhibition assay
Aspirin shows its antiplatelet activity via inhibition of platelet
cyclooxygenase (COX), which is responsible for generation of a
potent platelet activator thromboxane A2 (TXA2), which in turn indi-
rectly inhibits the formation of its stable metabolite serum TXB2.41 It
is therefore possible to achieve complete suppression of platelet
TXA2 (and the TXB2) formation via chronic administration of aspirin
at a dose of 30 mg/daily.42 The antiplatelet activity of aspirin
(30 mg/kg) and its prodrug 1A (at a dose equimolar to 30 mg/kg
dose of aspirin) was evaluated in SD rats through estimation of
serum TXB2 levels.43 As expected, aspirin (30 mg/kg, p.o., o.d.,
7 days) and its prodrug 1A (44.82 mg/kg, equivalent to 30 mg/kg
of aspirin, p.o., o.d., 7 days) exhibited nearly comparable inhibition
of platelet TXB2 formation (75.97% vs 72.59%) at equimolar doses as
shown in Figure 10. This result unequivocally establishes that the
compound 1A, which exhibits significant antiplatelet activity (a
unique property of the wonder drug aspirin), is indeed a true pro-
drug of aspirin.
Stability studies
150
An investigational drug must show sufficient stability at room
temperature (rt) so that it can have an acceptable shelf life when
it is developed as a drug. This stability issue is even more critical
for aspirin prodrugs as they tend to be less stable by design. We
have therefore tested stability of the promising NO-aspirin prodrug
1A and the NO-naproxen prodrug 2A at room temperature and at
50 °C over a period of 25–30 days and the results are presented
in Table S1 (see Supplementary material).
93 32
100
50
As anticipated, the aspirin prodrug 1A was found to be very stable
at RT up to 1 month. However, when it was incubated at 50 °C, it
degraded slightly (ꢀ1%) after 5 days and about 11% after 1 month.
After 1 month of incubation at 50 °C, about 2.8% of aspirin and
0.6% of salicylic acid were generated. In the case of naproxen prodrug
2A, the prodrug remained stable both at rt and at 50 °C for up to
25 days (period of study) and released only negligible amounts
(ꢀ0.20% at rt and ꢀ0.33% at 50 °C) of naproxen after 25 days.
Although we have disclosed only the NO releasing prodrugs of
NSAIDs aspirin and naproxen here, the linker technology is not lim-
1
***
1A
1
0
Aspirin
(200 mg/kg, p.o.) (298.8 mg/kg, p.o.)
*** p < 0.0001 versus Aspirin
Figure 8. Gastric lesion & ulcer area (mm2) of rat stomachs after acute oral dosing
of rats with aspirin (100 mg/kg) and its prodrug 1A (298.85 mg/kg, which is a dose
equimolar to 200 mg/kg of aspirin).

5592
M. Gund et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5587–5592
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Cl
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Tomlinson, E., Eds.; Plenum: New York and London, 1986. pp 49.
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CH3
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O
O
NO2
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agent containing at least one derivatizable carboxylic acid group.
Thus, we have made an example using an anti-cancer drug, chlor-
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Acknowledgments
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We are grateful to the Piramal Enterprises management for
their support and encouragement. We also express our thanks to
our chemistry colleagues Dnyaneshwar Pacharne, Sunil Mali, Milan
Dutta, Somnath Halder, Nauzer Dubash, Arun K. Jain and Vijaya
Nadar for their technical help in the development of linker technol-
ogy. We are also thankful to our pharmacology colleague Vivek
Khatanglekar for his technical help in pharmacology studies. Help
given by our colleagues at the animal care facility, patents depart-
ment and analytical chemistry department is gratefully
acknowledged.
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Supplementary data
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Supplementary data (Figs. 1, 4–7, S1 and S2, Table S1, experi-
mental details for the synthesis and characterization of all
reported compounds and procedures for biological experiments)
associated with this article can be found, in the online version,
at http://dx.doi.org/10.1016/j.bmcl.2014.10.096.
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