NO-NSAIDs. Part 3: Nitric oxide-releasing prodrugs of non-steroidal anti-inflammatory drugs

Article abstract of DOI:10.1248/cpb.60.465

In continuation of our efforts to discover novel nitric oxide-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) as potentially "Safe NSAIDs," we report herein the design, synthesis and evaluation of 21 new NO-NSAIDs of commonly used NSAIDs such as aspirin, diclofenac, naproxen, flurbiprofen, ketoprofen, sulindac, ibuprofen and indomethacin. These prodrugs have NO-releasing disulfide linker attached to a parent NSAID via linkages such as an ester (compounds 9-16), a double ester (compounds 17-24), an imide (compounds 25-30) or an amide (compounds 31-33). Among these NO-NSAIDs, the ester-containing NO-aspirin (9), NO-diclofenac (10), NO-naproxen (11), and the imide-containing NO-aspirin (25), NO-flurbiprofen (27) and NO-ketoprofen (28) have shown promising oral absorption, anti-inflammatory activity and NO-releasing property, and also protected rats from NSAID-induced gastric damage. NO-aspirin compound 25, on further co-evaluation with aspirin at equimolar doses, exhibited comparable dose-dependent pharmacokinetics, inhibition of gastric mucosal prostaglandin E₂ (PGE₂) synthesis and analgesic properties to those of aspirin, but retained its gastric-sparing properties even after doubling its oral dose. These promising NO-NSAIDs could therefore represent a new class of potentially "Safe NSAIDs" for the treatment of arthritic pain and inflammation.

Full text of DOI:10.1248/cpb.60.465

April 2012
Regular Article
Chem. Pharm. Bull. 60(4) 465–481 (2012)
465
NO-NSAIDs. Part 3: Nitric Oxide-Releasing Prodrugs of Non-steroidal
Anti-inflammatory Drugs
a
b
a
b
Namdev Borhade, Asif Rahimkhan Pathan, Somnath Halder, Manoj Karwa,
c
b
a
b
Mini Dhiman, Venu Pamidiboina, Machhindra Gund, Jagannath Janardhan Deshattiwar,
Sunil Vasantrao Mali, Nitin Janardanrao Deshmukh, Subrayan Palanisamy Senthilkumar,
Parikshit Gaikwad, Santhosh Goud Tipparam, Jayesh Mudgal, Milan Chandra Dutta,
Aslam Usmangani Burhan, Gajanan Thakre, Ankur Sharma, Shubhada Deshpande,
Dattatraya Chandrakant Desai, Nauzer Pervez Dubash, Arun Kumar Jain, Somesh Sharma,
Kumar Venkata Subrahmanya Nemmani, and Apparao Satyam*
a
b
a
b
a
b
a
b
a
b
a
c
a
a
b
b
,a
a
b
Medicinal Chemistry Division, Piramal Life Sciences Limited; Pharmacology Division, Piramal Life Sciences
c
Limited: and Analytical Chemistry Division, Piramal Life Sciences Limited; Nirlon Complex, Goregaon East,
Mumbai–400 063, India.
Received October 19, 2011; accepted December 18, 2011; published online January 12, 2012
In continuation of our efforts to discover novel nitric oxide-releasing non-steroidal anti-inflammatory
drugs (NO-NSAIDs) as potentially “Safe NSAIDs,” we report herein the design, synthesis and evaluation
of 21 new NO-NSAIDs of commonly used NSAIDs such as aspirin, diclofenac, naproxen, flurbiprofen, keto-
profen, sulindac, ibuprofen and indomethacin. These prodrugs have NO-releasing disulfide linker attached
to a parent NSAID via linkages such as an ester (compounds 9–16), a double ester (compounds 17–24), an
imide (compounds 25–30) or an amide (compounds 31–33). Among these NO-NSAIDs, the ester-containing
NO-aspirin (9), NO-diclofenac (10), NO-naproxen (11), and the imide-containing NO-aspirin (25), NO-
flurbiprofen (27) and NO-ketoprofen (28) have shown promising oral absorption, anti-inflammatory activity
and NO-releasing property, and also protected rats from NSAID-induced gastric damage. NO-aspirin com-
pound 25, on further co-evaluation with aspirin at equimolar doses, exhibited comparable dose-dependent
pharmacokinetics, inhibition of gastric mucosal prostaglandin E (PGE ) synthesis and analgesic properties
2
2
to those of aspirin, but retained its gastric-sparing properties even after doubling its oral dose. These prom-
ising NO-NSAIDs could therefore represent a new class of potentially “Safe NSAIDs” for the treatment of
arthritic pain and inflammation.
Key words nitric oxide-aspirin; nitric oxide-diclofenac; nitric oxide-naproxen; nitric oxide-flurbiprofen; ni-
tric oxide-ketoprofen
Non-steroidal anti-inflammatory drugs (NSAIDs) are wide- activity comparable to those of diclofenac (2) in rat models.
ly used for the treatment of osteoarthritis, rheumatoid arthritis However, in case of NO-aspirin prodrugs (Fig. 2), the bio-
1,2)
and musculoskeletal pain. However, chronic use of NSAIDs availability and anti-inflammatory activity of the mono ester
has been associated with an increased risk of gastrointestinal compound 9, although better than those of double ester com-
3,4)
(
GI) complications. Lately, a new class of nitric oxide (NO)- pound 17, were not comparable to those of aspirin (1). We had
releasable prodrugs of NSAIDs (NO-NSAIDs) has been devel- also witnessed zero bioavailability for the amide-containing
oped with an objective to overcome the risks associated with aspirin compound 31. The promising compounds 9 and 10 had
5
)
NSAIDs. Naproxcinod (Fig. 1) is one such compound at late also shown NO-releasing properties and protected rats from
6
)
stage development.
NSAID-induced gastric damage which could be attributable
In this perspective, during the past six years, we have been to the beneficial effects of nitric oxide released from these
engaged in the design of novel NO-NSAIDs as potentially prodrugs.
gastric-sparing “Safe NSAIDs” by the application of our di-
We have further evaluated the promising NO-diclofenac
7
)
sulfide linker and prodrug technology. In a recent publication prodrug 10 for dose-dependent pharmacokinetics and anti-in-
of preliminary results from this study, we reported NO-aspirin flammatory activity in three different models of inflammation
and NO-diclofenac as potentially “safe” prodrugs of aspirin (i.e., acute, chronic and systemic), and published those results
8)
9)
(
1) and diclofenac (2), respectively. Among the reported NO- elsewhere. In those studies, the NO-diclofenac prodrug 10,
diclofenac prodrugs (Fig. 2), the mono ester prodrug 10 had rather uniquely, inhibited the production of pro-inflammatory
shown excellent oral bioavailability and anti-inflammatory cytokines such as tumor necrosis factor (TNF)-α (38.1%
inhibition), interleukin (IL)-1β (42.8% inhibition) and IL-6
(59.3% inhibition). In contrast, diclofenac (2) did not inhibit
the lipopolysaccharide (LPS)-induced production of IL-1β
and IL-6 and also, rather surprisingly, it increased the levels
of TNF-α (79.6% enhancement). The interaction between
NO and cytokines is still ambiguous within the complex in-
10)
terplay among inflammatory mediators. Depending on the
type and phase of inflammatory condition, NO can exert both
Fig. 1. Structure of Naproxcinod (I)
To whom correspondence should be addressed. e-mail: apparaosvgk@hotmail.com
*
© 2012 The Pharmaceutical Society of Japan

4
66
Vol. 60, No. 4
Encouraged by the successful identification of a potentially
safe NO-diclofenac” prodrug 10, we have further extended
“
the application of the “SS-nitrate” strategy to explore the pos-
sibility of discovering potentially “safe NO-NSAID” prodrugs
of other widely used NSAIDs such as naproxen (3), flurbipro-
fen (4), ketoprofen (5), sulindac (6), ibuprofen (7) and indo-
methacin (8) (Fig. 2) and the results from those studies are
reported in this article.
NO-NSAID Design The most important design feature
in our NO-NSAIDs is the presence of a disulfide linkage at
β-position to the nitrate ester group. Organic nitrates con-
taining a sulfur atom at β-position to a nitrate group were
originally termed as “SS-nitrates,”2 and they were designed
to prove the hypothesized sulfhydryl-dependent mechanism
of NO release from organic nitrates such as glyceryl trinitrate
0)
21,22)
(GTN).
Such ‘SS-nitrates’ exhibited good stability in neu-
tral aqueous solution but released significant amounts of NO
in aqueous medium at biologically relevant pH on addition
2
0)
of sulfhydryl-containing compounds. By partly adopting
this “SS-nitrate” feature into our disulfide-linker containing
7
)
prodrug strategy, we have designed, synthesized and evalu-
ated NO-NSAID prodrugs that contain a NO-releasing nitrate
moiety at β-position to the disulfide group, which is, in turn,
covalently bonded through “CH CH ” chain to the carboxyl
2
2
group of parent NSAIDs via linkages such as an ester (com-
pounds 9–16), a double ester (compounds 17–24), an imide
(compounds 25–30) or an amide (compounds 31–33) as shown
in Fig. 2. As proposed earlier, the disulfide group plays a
prominent role in the mechanism of release of NO from these
8
,23)
compounds.
NO-NSAID Synthesis General methods used for the
synthesis of NO-NSAIDs 9–33 are outlined in Chart 1. In
general, the desired NO-NSAIDs were synthesized from the
parent NSAIDs by activation of their carboxylic acid function
followed by appendage of appropriate linker intermediates
3
4–38a and 38b (Fig. 3) in the presence of a suitable base and
a suitable solvent. Thus, the ester-linked NO-NSAIDs 9–16
were synthesized by employing one of the four standard meth-
ods of carboxylic acid activation followed by their reaction
with linker intermediate 36 (35 in the case of NO-diclofenac
10) as depicted in Chart 2. The nitro linker intermediate 36
was prepared from the mono-bromide intermediate 34 as
reported earlier. NO-aspirin 9 and NO-ibuprofen 15 were
synthesized in 45−54% yields by first converting the parent
drugs aspirin (1) and ibuprofen (7) into their respective acid
The chlorides using oxalyl chloride and N,N-dimethylformamide
Fig. 2. Structures of NSAIDs (1–8) and Synthesized NO-NSAIDs
9–33)
8)
(
10–13)
14–17)
pro-inflammatory
and anti-inflammatory effects.
small amount of NO generated from constitutive nitric oxide (DMF) method, followed by their reaction with intermediate
synthase (cNOS), which includes endothelial (eNOS) and 36 in the presence of triethylamine. In case of naproxen (3),
neuronal (nNOS), is important for regulation of physiological flurbiprofen (4), ketoprofen (5) and indomethacin (8), they
homeostasis, whereas the large amount of NO produced from were first converted to their respective imidazolide species
inducible nitric oxide synthase (iNOS), which can remain ac- using N,N′-carbonyldiimidazole (CDI), followed by their in
tive for several hours or even days after expression, has been situ reaction with intermediate 36 to afford 28–67% of the
implicated in pathophysiology of several diseases and chronic corresponding NO-NSAIDs 11–13 and 16. NO-sulindac 14
18)
inflammatory conditions. However, NO-releasing derivatives was prepared in 53% yield by coupling sulindac (6) directly
of cyclooxygenase inhibitors are generally known to exhibit with intermediate 36 in presence of N,N′-dicyclohexyl car-
enhanced anti-inflammatory activity and the improved effi- bodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP).
cacy of these derivatives are most likely caused by its ability Previously, we synthesized NO-diclofenac 10 by reacting
19)
to suppress leukocyte infiltration and scavenge peroxynitrite.
diclofenac (2) with intermediate 36 in presence of DCC and
We therefore believe that the NO released from NO-diclofenac DMAP. However, we noticed formation of significant amounts
prodrug 10 could be responsible for the inhibition of pro- (ca. 30%) of diclofenac lactam 39 (Fig. 3). This lactam forma-
inflammatory cytokines.
tion was more pronounced when we attempted to synthesize

April 2012
467
Reagents and conditions: a) see Chart 2 for details; b) 1. Cs CO , MeOH/THF or K CO , DMF, 2. 37, THF/DMF, 15−80%; c) see Chart 3 for details; d) 1. 38a, TFA/
2
3
2
3
CH Cl (1:1), 30min; 2. oxalyl chloride, CH Cl or N,N′-carbonyldiimidazole, THF; 3. 38b, Et N, CH Cl , 17–34%.
2
2
2
2
3
2
2
Chart 1. Synthesis of NO-NSAIDs (9–33)
Fig. 3. Structures of Linker Intermediates (34–38a, 38b and 39)
Reagents and conditions: Route 1: a) aspirin (1) or ibuprofen (7), oxalyl
chloride, DMF (cat. amt.), benzene; b) 36, Et N, CH Cl , 45–54%; Route 2:
3
2
2
naproxen (3) or flurbiprofen (4) or ketoprofen (5) or indomethacin (8), N,N′-
carbonyldiimidazole, CH Cl or THF or CHCl , followed by 36, 28–67%;
2
2
3
Route 3: sulindac (6), 36, DCC, DMAP, CH Cl , 53%; Route 4: a) diclofenac
2
2
Reagents and conditions: (a) oxalyl chloride, DMF (cat. amt.)/thionyl chloride,
sodium (2a), 35, DMF, 50%; b) AgNO , acetonitrile, 49%.
benzene; (b) ammonia gas, CH Cl , 0 C, 0.5h, 90–100%; (c) oxalyl chloride,
°
2 2
24) 25)
3
CH Cl , reflux, 16h ; (d) silver cyanate, benzene, reflux, 0.5h ; (e) 36, ben-
Chart 2. Synthesis of NO-NSAIDs (9–16) with Ester Linkage
2
2
zene, 16h, 10–36%.
Chart 3. Synthesis of NO-NSAIDs (25–30) with Imide Linkage
NO-diclofenac 10 by the reaction of intermediate 36 with
diclofenac acid chloride in presence of a base such as tri-
ethylamine. We have therefore used an alternative two step were prepared in 15–80% yields by first converting the par-
procedure for the synthesis of NO-diclofenac 10 as shown in ent NSAIDs into their respective metal carboxylate salts and
route 4 of Chart 2. Thus, diclofenac was first converted to its then treating those salts with linker intermediate 37 (Chart 1,
sodium salt, which was then coupled with the dibromo linker Fig. 3). The intermediate 37 was prepared from intermediate
8)
intermediate 35 to give the bromide intermediate 40 in 50% 36 as reported earlier. Thus, NSAIDs 1–7 were converted to
yield, which was further treated with silver nitrate to afford their respective cesium carboxylate salts by treatment with ce-
the target compound 10 in 49% yield.
sium carbonate, which on reaction with linker intermediate 37
The double ester linkage-containing NO-NSAIDs 17–24 yielded NO-NSAIDs 17–23. The NO-indomethacin prodrug

4
68
Vol. 60, No. 4
2
4 could not be made by the above method. However, it was concentration and anti-inflammatory activity, some of the re-
made (15% yield) by the reaction of potassium carboxylate ported NO-NSAIDs were screened for either pharmacokinet-
salt of indomethacin with intermediate 37. Synthesis of imide ics or anti-inflammatory activity, depending on availability of
linkage-containing NO-NSAIDs 25–30 was achieved by first resources.
2
6)
converting the NSAIDs into their respective acid chlorides
1 by oxalyl chloride and DMF method (Chart 3). Bubbling (P1853) showed maximum anti-inflammatory activity, fol-
of ammonia gas into the solution containing acid chlorides lowed by the double ester prodrug 19. The imide prodrug 26
1 at 0°C afforded the corresponding NSAID-amides 42 and the amide prodrug 32 were inactive. In pharmacokinetic
Chart 3) in 90–100% yields. The said amides 42 were then (PK) studies, as anticipated, the ester prodrug 11 showed sig-
Among naproxen series (Table 1), the ester prodrug 11
4
4
(
refluxed with oxalyl chloride in dichloromethane to yield their nificant oral bioavailability (AUC value), which is only slightly
respective acyl isocyanates 43. Addition of linker intermedi- less than the AUC value of naproxen. However, the ester
ate 36 to these highly reactive acyl isocyanates 43 afforded prodrug 11 exhibited nearly comparable oral bioavailability,
2
4)
NO-NSAIDs 26–30 (via Route 1) in 10–36% yields, but anti-inflammatory activity and NO release profile to those of
2
7)
the NO-aspirin 25 could not be made via Route 1. However, naproxcinod (I) (Table 1), which is an investigational drug
2
5)
6)
the NO-aspirin 25 could be made via Route 2 in 35% yield in advanced clinical development. Thus, the ester group is
by the reaction of aspirin acid chloride with silver cyanate to found to be the best linkage for generation of a promising pro-
yield the desired aspirin acyl isocyanate intermediate 43 fol- drug in naproxen series using the specified linkers.
lowed by its reaction with linker intermediate 36.
While none of the listed NO-NSAID prodrugs of flurbi-
NO-NSAIDs 31–33 containing amide linkages were pre- profen, ketoprofen, sulindac, ibuprofen and indomethacin
pared in three steps as shown in Chart 1 by converting the showed comparable bioavailability to that of their respec-
parent NSAID into either its acid chloride or imidazolide, fol- tive parent NSAID, the ester prodrugs of all the reported
lowed by reaction with linker intermediate 38b in presence of NSAIDs, with the lone exception of indomethacin series,
triethylamine. Thus, in the first step, linker intermediate 38b showed superior bioavailability compared to the correspond-
was prepared by the deprotection of the Boc group of linker ing double ester, imide and amide prodrug counterparts in
8)
intermediate 38a using trifluoroacetic acid. In the second their respective series. In the indomethacin series, the double
step, the NSAIDs were converted to their respective acid chlo- ester prodrug exhibited better bioavailability than the ester
rides by treatment with thionyl chloride or oxalyl chloride/ and the imide prodrugs. Thus, the following is the order of
DMF (or imidazolide by the reaction with CDI). In the last bioavailability in each of the series of NO-NSAIDs (Table
2
6)
step, reaction of the amine intermediate (trifluoroacetic acid 1) : NO-flurbiprofen: ester prodrug 12>double ester prodrug
TFA) salt) 38b with the respective NSAID acid chlorides in 20>imide prodrug 27>amide prodrug 33; NO-ketoprofen:
(
the presence of a suitable base and solvent yielded the amide- ester prodrug 13>double ester prodrug 21>imide prodrug 28;
containing NO-NSAIDs 31–33 in 17–34% yields.
NO-sulindac: ester prodrug 14>double ester prodrug 22; NO-
ibuprofen: ester prodrug 15>double ester prodrug 23>imide
prodrug 29; NO-indomethacin: double ester prodrug 24>ester
Results and Discussion
Biological Evaluation The NO-NSAIDs 9–33 were eval- prodrug 16>imide prodrug 30.
uated in vivo to establish their pharmacokinetics and/or anti-
We have also evaluated anti-inflammatory activities of all
inflammatory activity using rat as animal model. Compounds the reported prodrugs of NO-flurbiprofen and only the ester
with more promising bioavailability and/or anti-inflammatory and imide prodrugs of NO-ketoprofen. In contrast to the trend
activity were selected and evaluated further for their NO- that was seen in other series of NO-NSAIDs, the imide pro-
releasing capabilities as well as their propensity for sparing/ drug 27 exhibited better anti-inflammatory activity than that
inducing gastric lesions vis-à-vis their parent NSAIDs.
of ester prodrug 12 and double ester prodrug 20, although
The bioavailability (area under curve (AUC)) data presented its bioavailability is less than those of ester prodrug 12 and
for NO-NSAIDs correspond to the released respective parent double ester prodrug 20. In the case of NO-ketoprofen series
NSAID, except in the case of aspirin and NO-aspirins, where too, the imide prodrug 28 showed better anti-inflammatory
the AUC data corresponds to plasma salicylate concentrations, activity than that of ester prodrug 13, although its bioavail-
2
6)
since aspirin and NO-aspirin get converted rapidly to salicylic ability is less than that of 13 (Table 1). It is rather surpris-
acid by enzymatic hydrolysis in vivo. All NSAIDs showed ing to see the enhanced anti-inflammatory activities for the
faster absorption with Tmax between 15 and 30min, whereas imide-containing flurbiprofen prodrug 27 and ketoprofen
NO-NSAIDs showed delayed Tmax with significant release of prodrug 28, in spite of their low bioavailability. However, as
parent NSAID over a longer period of time.
As mentioned earlier, the previously reported NO-aspirin enhanced anti-inflammatory activity which supports our be-
compounds 9 and 17 did not show comparable bioavailabil- lief that the enhanced anti-inflammatory effects shown by our
ity and anti-inflammatory activity to those of aspirin. We compounds 10, 25, 27 and 28 could also be partly due to the
now report a new “imide-containing” NO-aspirin prodrug NO released from these NO-NSAIDs. We have measured in
mentioned earlier, NO-releasing compounds generally exhibit
19)
8)
2
5 (P1539) that exhibited nearly comparable bioavailability vivo NO-release of the promising NO-NSAIDs 10, 11, 25, 27
2
6)
and anti-inflammatory activity to those of aspirin (Table 1). and 28 in rats. As anticipated, all tested NO-NSAIDs released
Thus, the imide group is found to be the best linkage for cre- significant amounts of NO. However, the NO release profile
ating a promising prodrug in aspirin series using the specified (rate, extent and fate) of these NO-NSAIDS is varied from one
linkers.
compound to another which may be due to differences in their
Since the data obtained on NO-aspirin and NO-diclofenac physicochemical properties. The corresponding plasma nitrate/
2
6)
prodrugs exhibited a strong correlation between plasma drug nitrite AUC values are shown in Table 1.

April 2012
469
a)
Table 1. Oral Bioavailability (AUC), Anti-inflammatory Activity and NO Release Data of NO-NSAIDs
c,d)
k)
Activity (% inhibition)
AUC of NO (nitrite/nitrate) [μM·h]
x
a)
b)
Compd.
AUC (μg/mL·h)
l)
4
h
6h
Compd. (Z mg/kgeq)
Vehicle (1mL/kg)
Aspirin (1)
4051± 312
1234± 93**
892± 140**
42± 9
20± 12
Inactive
48± 15
—
31± 8
17± 7
Inactive
28± 16
—
—
—
—
e)
e)
9
(P1538)
—
1
2
3
7 (P1911)
5 (P1539)
1 (P1537)
—
3681 (Z=150)
—
—
f)
3836± 164
330.4
—
e)
0
e)
Diclofenac (2)
65± 2
60± 5
55± 9
53± 7
34± 10
61± 6
45± 4
44± 9
Inactive
Inactive
21± 12
20± 13
Inactive
50± 6
35± 5
14± 6
e)
f)
1
1
0 (P2026)
8 (P1912)
612 (Z=5)
305
—
e)
49± 2*
238± 8
187± 12*
—
—
Naproxen (3)
—
—
1
1
2
3
1 (P1853)
9 (P1945)
6 (P1882)
2 (P1854)
1082 (Z=10)
433
—
—
—
Inactive
—
—
—
Inactive
—
—
g)
h)
Naproxcinod (I)
Flurbiprofen (4)
181± 5*
180± 15
88± 11**
53± 5**
27± 7**
4± 0.2**
90± 11
53± 10
68± 8
49± 8
842.3 (Z=10)
449
—
47± 5
—
i)
i)
12 (P1887)
20 (P1873)
27 (P1888)
33 (P1889)
46± 13
34± 10
—
—
i)
i)
57± 9
46± 6
—
—
i)
i)
61± 17
48± 2
1122 (Z=10)
418.7
—
i)
i)
23± 5
16± 5
—
Ketoprofen (5)
58± 6
47± 7
40± 10
20± 12
—
—
—
f)
j)
j)
1
2
2
3 (P1897)
1 (P1973)
8 (P1898)
58± 8
—
—
38± 8**
23± 6**
2498± 139
850± 108**
329± 44**
82± 3
36± 2**
28± 7**
26± 4**
323± 7
—
—
—
j)
j)
68± 1
46± 7
707 (Z=30)
363.5
—
Sulindac (6)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1
2
4 (P1995)
2 (P2025)
—
—
Ibuprofen (7)
—
1
2
2
5 (P1970)
3 (P1972)
9 (P1971)
—
—
—
Indomethacin (8)
—
1
2
3
6 (P1975)
4 (P5058)
0 (P5057)
57± 13**
96± 14**
22± 6**
—
—
—
a) All prodrugs were administered orally to rats at equimolar dose to their respective parent drugs (10mg/kg for diclofenac, naproxen, flurbiprofen, ibuprofen and indo-
26,27)
methacin; 150mg/kg for aspirin, 15mg/kg for ketoprofen and 100mg/kg for sulindac).
b) For aspirin and its prodrugs, plasma salicylate levels were estimated to calculate
bioavailability. c) Anti-inflammatory activity is expressed as % inhibition of paw edema volume over control rats. d) All values are expressed as mean± S.E.M. (n=3–4 for
8)
pharmacokinetic study; n=5–8 for anti-inflammatory activity). e) Reported earlier ; included here for comparison. f) Statistically not significant (i.e., p>0.05 versus parent
drug). g) Anti-inflammatory activity at 3h for naproxcinod. h) Anti-inflammatory activity at 5h for naproxcinod. i) 20mg/kg equivalent dose. j) 30mg/kg equivalent dose. k)
Pooled data (n=3–5). l) 100% polyethylene glycol 400 (PEG400) was used as vehicle for NO estimation study. *p<0.05, **p<0.01 versus parent drug; —=Not evaluated.
x
2
8)
Fig. 4.
Gastric Lesion Index of Selected NSAIDs versus Corresponding NO-NSAIDs (i.e., 25, 10, 11, 27 and 28) Measured as Gastric Lesion Area
2
(mm )
All NSAIDS (except aspirin) were dosed at 100mg/kg and all NO-NSAIDs (except NO-aspirin) were dosed at 100mg/kg equimolar to their respective parent NSAIDs;
.5% (w/v) carboxymethyl-cellulose (CMC) containing 1% (v/v) Tween80 was used as vehicle. a) Used aspirin oral dose of 150mg/kg. b) Used NO-Aspirin oral dose of
50mg/kg equimolar to aspirin.
0
1

470
Vol. 60, No. 4
Fig. 5. Pharmacokinetic and Pharmcodynamic Effects of Oral Aspirin and NO-Aspirin Compound 25 (P1539) in Rat
(
A) Dose dependent pharmacokinetics of aspirin and 25; (B) analgesic activity of aspirin and 25; (C) inhibition of gastric mucosal PGE2 production by aspirin and 25;
2
(
D) effect of aspirin and 25 (at equal and double doses) on gastric mucosa (gastric lesion index in mm ).
Since all the prodrugs in NO-sulindac, NO-ibuprofen and Table 2. Stability of Aspirin (1), Flurbiprofen (4), Ester Prodrugs 9 and
a,b)
NO-indomethacin series exhibited poor bioavailability when 12, and the Imide Prodrugs 25 and 27 in SGF
compared to those of their respective parent NSAIDs (Table
Prodrug 25
2
6)
1
), we did not carry out further evaluation of these com-
pounds.
We also selected the promising NO-NSAIDs 10, 11, 25, 27
Incubation time
c)
% 25
% SA
Half-life (t1/2)
0
5
min
min
96
59
33
15
0
0
34
57
72
90
90
and 28 and studied their gastric tolerance compared to their
respective parent NSAIDs in rats and the results of those ex-
10min
15min
30min
7min
2
8)
periments are presented graphically in Fig. 4.
While all parent NSAIDs caused significant gastric damage,
none of the tested NO-NSAIDs at equimolar doses caused any
gastric lesions. We believe that the observed gastric-sparing
60min
0
a) Values given are average of triplicate experiments. b) Aspirin (1), flurbiprofen
effect could be attributable to the physiological actions of NO (4), ester prodrugs 9 and 12, and the imide prodrug 27 remained stable for 60min
(
study time) in SGF at 37°C. c) SA=salicylic acid; other expected metabolites such
released from these NO-NSAIDs. However, additional factors
as aspirin, aspirin amide and salicylamide are not formed (for structures of metabo-
such as masking of free carboxylic acid group of NSAID as lites, see Fig. 6).
an ester also might be partly contributing to the gastric-spar-
2
9–31)
ing effect of these compounds.
Based on its promising oral bioavailability, anti-inflamma- carrageenan-induced hind paw model of hyperalgesia in rat
tory and gastric-sparing properties, we have selected the NO- (Fig. 5B). Similar to aspirin, NO-aspirin 25 also caused sig-
aspirin compound 25 for further evaluation and determined nificant reduction in PGE synthesis (Fig. 5C), which is an
2
its: A) dose-dependent pharmacokinetics; B) analgesic activity indirect measure of COX-1 inhibition. By inhibiting COX-1,
in carrageenan-induced hind paw model of inflammatory pain; which led to reduction in gastroprotective PGE levels, aspirin
2
C) effect on reducing mucosal prostaglandin E (PGE ) levels caused significant gastric lesions. Although compound 25 also
2
2
via inhibition of cyclooxygenase-1 (COX-1); and D) gastric- caused significant reduction in PGE levels in gastric mucosa,
2
sparing effects at equal as well as at double the doses and it did not lead to any gastric damage even after doubling its
compared the results with those of aspirin at equimolar doses dose (Fig. 5D). This result further confirms our belief that
(
Fig. 5).
Thus, as shown in Fig. 5A, compound 25 exhibited dose- 25 could be attributable to NO released from this promising
dependent oral absorption and its overall bioavailability was molecule.
the unique gastric-sparing property exhibited by compound
nearly comparable to that of aspirin at equimolar doses.
Metabolic Stability of NO-NSAIDs in Biological Fluids
As anticipated, compound 25 as well as aspirin at equimo- We have checked metabolic stability of a few representative
lar doses exhibited comparable anti-hyperalgesic effects in NO-NSAIDs such as ester prodrugs 9 and 12 and imide pro-

April 2012
471
drugs 25 and 27 along with their respective parent drugs aspi- flurbiprofen (4) and flurbiprofen amide (44) (Fig. 6). Thus,
rin (1) and flurbiprofen (4), in biological fluids such as simu- at 4h incubation, there still remained 32% of intact imide
lated gastric fluid (SGF) (Table 2) and human plasma (Table prodrug 27 along with metabolites 4 (21%) and 44 (23%).
3) and identified their metabolite(s) (Fig. 6). Additionally, the Interestingly, this flurbiprofen imide prodrug 27 degraded
stability of imide prodrug 27 was also tested in rat plasma faster (t =85min) and released more of flurbiprofen amide
1/2
(
Table 3).
(44) in rat plasma. Thus, when prodrug 27 was incubated in
While aspirin (1), the ester prodrugs 9 and 12, and the im- rat plasma at 37°C for 4h, there remained only, as shown in
ide prodrug 27 remained stable on incubation in SGF at 37°C parentheses in Table 3, 8% of intact imide prodrug 27 along
up to 60min (study period), the imide prodrug 25 degraded with the metabolites 4 (6%) and 44 (78%). Thus, although
completely within 30min [half-life (t )=7min] under similar both the aspirin prodrug 25 and the flurbiprofen prodrug 27
1/2
conditions to give salicylic acid (SA) as the sole metabolite contain the imide linkages in their structures, they exhibit
Table 2). However, as expected, all the tested NO-NSAIDs different metabolic stability, and also metabolize differently
degraded at varied rates [i.e., t =between 9, 150min] on in biological fluids. However, in PK study in rats, the flurbi-
(
1/2
incubation in human plasma at 37°C for up to 2h (up to 4h profen imide prodrug 27 released flurbiprofen (4) as the sole
in case of flurbiprofen prodrugs 12, 27) (Table 3). Here also, metabolite. We could not detect flurbiprofen amide (44) even
only the imide prodrug 25 degraded completely within 30min at early time points of PK study. It seems that the metabolite
(
t =9min) to give SA as the sole metabolite.
44 is either not formed or might be getting hydrolyzed to 4 as
Degradation of the remaining prodrugs (including aspirin) soon as it is formed under in vivo conditions.
1/2
was not complete even after 2h (4h in case of flurbiprofen
Since NO-aspirin 25 is expected to be hydrolyzed immedi-
prodrugs 12, 27) of incubation in human plasma. Thus, at ately after oral administration, the anti-inflammatory effects
h incubation time, there remained about 18% of intact ester shown by the compound 25 could be caused by the released
prodrug 9 (t =55min) and 82% of released SA metabolite. salicylate. It is also possible that both salicylic acid and NO
2
1/2
Surprisingly, degradation of flurbiprofen ester prodrug 12 could be contributing to the anti-inflammatory effect of com-
was very slow (t =200min) and even after 4h incubation pound 25. In order to understand the possible role of NO in
1/2
time, there still remained 40% of intact prodrug and 60% of this process, we have evaluated anti-inflammatory effects of
32)
released flurbiprofen (4). The flurbiprofen imide prodrug 27 the nitro linker 36 in vivo in rats. For comparison, we have
also degraded very slowly (t =150min), but rather interest- also simultaneously evaluated anti-inflammatory effects of the
1/2
ingly, this imide prodrug released the expected metabolites, non-nitro linker (HOCH CH SSCH CH OH), salicylic acid
2
2
2
2
(SA) and 1:1 mixture of nitro linker 36 and SA and the re-
sults are presented in Table 4.
Surprisingly, both non-nitro and nitro linkers exhibited sig-
nificant anti-inflammatory activity over control. The slightly
enhanced activity of nitro linker 36 over non-nitro linker,
although not significant, could be attributable to NO released
from the linker. As anticipated, an equimolar mixture of SA
and nitro linker 36 exhibited a slightly better anti-inflamma-
tory activity than either of constituents individually. However,
the difference in activity is not significant when compared
with salicylate, but it reached significance when compared
with nitro linker 36. We therefore can not attribute this en-
hanced activity solely to NO as the nitro linker 36 and/or the
non-nitro linker (after de-nitration) could as well contribute to
Fig. 6. Structures of Some of the Expected Metabolites from Prodrugs
, 12, 25 and 27
9
a)
Table 3. Stability of Aspirin (1), Ester Prodrugs 9 and 12, and Imide Prodrugs 25 and 27 in Human Plasma
Aspirin (1)
% SA
Prodrug 9
Prodrug 25
Prodrug 12
Prodrug 27
% 4
Incubation
time
b)
%
1
% 9
% SA
% 25
% SA
% 12
98
% 4
% 27
% 44
0
5
min
min
100
89
78
69
68
58
38
0
11
20
31
32
42
62
99
95
87
76
67
46
18
0
5
96
58
40
8
0.5
42
60
92
98
98
98
—
—
0
—
3
97
—
0
0
—
—
0
— (1)
—
3
10min
15min
30min
60min
10
17
33
54
82
—
—
96
94
— (95)
c)
c)
c)
c)
—
—
12
17
43
45
60
— (3)
c)
c)
0
83
82
57
55
40
86 (90)
76 (82)
56 (32)
48
0 (1)
6 (8)
c)
c)
c)
c)
0
4 (1)
8 (15)
14 (56)
17
c)
c)
120min
180min
240min
0
9 (6)
—
—
—
—
14
c)
c)
c)
—
—
—
—
32 (8)
21 (6)
23 (78)
c)
Half-lives
t1/2
70min
55min
9min
200min
150min (85min)
(
)
a) Values given are average of triplicate experiments. b) Other expected metabolites such as aspirin, aspirin amide and salicylamide are not formed (for structures, see Fig.
). c) Values in parentheses are for % of indicated prodrugs or metabolites in rat plasma at the specified incubation times; SA=salicylic acid; 44=flurbiprofen amide (for struc-
6
ture, see Fig. 6); —=not done.

472
Vol. 60, No. 4
the observed enhancement of activity of the mixture of salicy- mechanistic studies. Thus, in case of aspirin imide prodrug
late and nitro linker 36. 25, hydrolysis by plasma esterases would lead to a transient
Mechanism of Drug Release A plausible mechanism for intermediate o-phenoxybenzoyl isocyanate (TI1), which can
the release of NO and NSAID (native drug) from NO-NSAIDs break down via o-phenoxy-assisted cleavage to yield hydrogen
containing ester linkage has been proposed in our earlier pa- cyanate (which in turn can hydrolyze to ammonia and carbon
8
,23)
33)
per.
We now propose a plausible mechanism for the meta- dioxide as shown) and o-oxoketene (TI2A) via pathway a
33,34)
bolic degradation of NO-NSAIDs containing imide linkages and/or the lactone form TI2B
via pathway b. The geo-
by taking aspirin imide prodrug 25 and flurbiprofen imide metrically restricted o-oxoketene intermediate TI2A, being a
prodrug 27 as representative examples as depicted in Chart highly reactive species, would rapidly undergo hydration to
4. We believe that these imide prodrugs 25 and 27, because give a six-membered cyclic transition state TS1, which could
of their distinct differences in metabolic stability (t =9min quickly achieve aromatization via rearrangement to yield SA
1/2
3
4)
versus 150min) and degradation pattern in rat and/or hu- as the sole metabolite. The transient intermediate TI2B [i.e.,
man plasma, qualify themselves as ideal candidates for such benzodioxetane] could either equilibrate with o-oxoketene
33,34)
TI2A or directly undergo hydration to yield SA.
The
stability of such lactone form (i.e., TI2B) was reported to be
Table 4. Anti-inflammatory Activity of Non-nitro Linker, Nitro Linker nearly equal to o-oxoketene (TI2A) and it could be attributed
3
3
6, Salicylic Acid (SA) and an Equimolar Mixture of SA and Nitro Linker
6 in Rats
to aromatic stabilization, which might be partially offset by its
a)
ring strain. The photochemical inter-conversion of TI2A and
35)
TI2B has also been reported earlier.
Anti-inflammatory activity
b)
Compd.
c)
(% inhibition)
Metabolic degradation of flurbiprofen imide prodrug 27
is expected to proceed via two different pathways to yield
the metabolites flurbiprofen (4) and flurbiprofen amide (44),
respectively (Chart 4). Thus, the prodrug 27 is expected to
be hydrolyzed by plasma esterases to yield a transient acyl
isocyanate intermediate TI3. This reactive intermediate TI3
can break down into hydrogen cyanate (which can further hy-
(
Non-nitro) linker
41.99± 25.85*
59.26± 12.00*
74.10± 19.01*
Nitro linker 36
SA
,
SA+36 (1:1)
87.64± 9.34* **
a) Notably, the current batch of rats exhibited better sensitivity to all the test
compounds. b) All the compounds were dosed at 832.60μM/kg, which is equivalent to
1
50mg/kg dose of aspirin. c) Anti-inflammatory activity is expressed as % inhibition drolyze to ammonia and carbon dioxide as shown) and highly
of paw edema volume over control rats at 6h after dosing. All values are expressed
as mean± S.E.M. (n=5–6). *Statistically significant (i.e., p<0.05 versus control).
*
unstable transient intermediates TI4A and TI4B (pathway a),
which can rapidly undergo hydration to yield flurbiprofen (4).
*Statistically significant (i.e., p<0.05 versus nitro linker 36).
Chart 4. Plausible Mechanisms for the Metabolic Degradation of Imide Prodrugs 25 and 27 in Rat and/or Human Plasma

April 2012
473
Table 5. Determination of Sulfhydryl-Assisted Nitric Oxide (NO) Release Profiles of a Disulfide-Containing Compound 11 (NO-Naproxen) and a Non-
disulfide-Containing Compound I (Naproxcinod)
a)
Nitrate/nitrite concentration (μM)
Compd.
1
00μM
50μM
20μM
10μM
b)
c)
c)
c)
c)
b)
c)
c)
c)
c)
b)
c)
c)
c)
c)
b)
c)
c)
c)
c)
Standard (NaNO3)
99.88± 4.12 (<16.00)
48.90± 0.30 (<16.00)
21.87± 0.16 (<16.00)
<16.00 (<16.00)
b)
b)
b)
b)
I (Naproxcinod)
20.67± 0.44 (<16.00)
<16.00 (<16.00)
<16.00 (<16.00)
<16.00 (<16.00)
b)
b)
b)
b)
1
1 (P1853)
100.0± 4.00 (<16.00)
50.9± 3.60 (<16.00)
22.80± 2.80 (<16.00)
16.50± 0.60 (<16.00)
b)
b)
b)
b)
Blank
<16.00 (<16.00)
<16.00 (<16.00)
<16.00 (<16.00)
<16.00 (<16.00)
a) Used Griess Nitrate/Nitrite Assay Kit from Sigma-Aldrich; Values are expressed as mean± S.E.M. (n=3 experiments) and a value of <16.00 represents a minimum detec-
tion level. b) Values are obtained after treatment with nitrate reductase. c) Values in parentheses are obtained before treatment with nitrate reductase.
Alternatively, the highly reactive acyl isocyanate intermediate compound such as dithiothreitol (DTT) at biological relevant
TI3 can rapidly undergo hydrolysis to yield yet another unsta- temperature (ca. 37°C) and pH (ca. 7.4), only prodrug 11 ex-
ble transient intermediate TI5, which can rapidly lose carbon hibited nearly quantitative release of NO (Table 5). This result
dioxide to yield flurbiprofen amide (44).
confirms that our disulfide-containing NO-NSAIDs (9–33)
Thus, we have discovered a novel and unique aspirin imide indeed show characteristic properties of SS-nitrates. We also
prodrug 25, that: (1) metabolized quickly in biological fluids suspect that our NO-NSAIDs (9–33) and other organic nitrates
such as SGF or human plasma and released SA as the sole such as naproxcinod (I) metabolize and release NO in vivo by
metabolite; in contrast, the imide prodrugs of other NSAIDs, different mechanisms and may therefore show different phar-
such as flurbiprofen imide prodrug 27, exhibited good stabil- macological outcome.
ity in SGF and metabolized slowly and differently in human
plasma and rat plasma by releasing the expected metabolites, Conclusion
flurbiprofen (4) and flurbiprofen amide (44); (2) exhibited com-
In summary, we have designed, synthesized, and evaluated
parable and dose-dependent pharmacokinetics, anti-inflamma- a few novel NO-NSAID prodrugs of commonly used NSAIDs
tory and analgesic properties to those of aspirin; (3) exhibited such as aspirin, diclofenac, naproxen, flurbiprofen, ketoprofen,
comparable inhibition of mucosal PGE levels to that of aspi- sulindac, ibuprofen and indomethacin. These prodrugs have
2
rin; and (4) exhibited gastric-sparing properties. NO-releasing disulfide linker attached to the parent NSAID
Mechanistic Studies on NO Release By design, our NO- via linkages such as an ester (compounds 9–16), a double
7
)
NSAIDs carry salient features of both our disulfide linker
ester (compounds 17–24), an imide (compounds 25–30), or an
2
0)
and the known SS-nitrates. We have proposed a plausible amide (compounds 31–33). These NO-NSAIDs were evalu-
sulfhydryl-dependent mechanism of NO release from our ated for their oral bioavailability, anti-inflammatory activity,
8
,23)
NO-NSAIDs in our earlier paper.
In light of FDA’s recent NO-releasing capability and gastric-sparing effects in rats.
negative decision on Nicox’s naproxcinod (I),6) we wanted to Among the tested NO-NSAIDs, the ester containing NO-
determine the similarities as well as differences in NO release aspirin (9), NO-diclofenac (10), NO-naproxen (11), and the
profiles of naproxcinod (I) and our NO-NSAIDs. We have imide-containing NO-aspirin (25), NO-flurbiprofen (27) and
selected an appropriate ester containing NO-naproxen prodrug NO-ketoprofen (28) have shown promising pharmacokinetic
11 as it is the closest structural analog of naproxcinod (I) but and/or anti-inflammatory activities. However, only the ester
differs only by having a disulfide group in its linker. As dis- prodrugs of diclofenac (10) and naproxen (11), and the imide
cussed above, both these compounds exhibited nearly similar prodrug of aspirin (25) have shown significant oral absorption
anti-inflammatory activity and oral bioavailability (Table 1) and proportional anti-inflammatory activities that are nearly
(
i.e., AUC values are comparable although they showed dif- comparable to those of their respective parent NSAIDs. These
2
6,27)
ferent Tmax as well as Cmax).
In principle, prodrug 11 is a promising NO-NSAIDs have also shown NO-releasing proper-
simple organic nitrate like naproxcinod (I), but by having a ties and protected rats from NSAID-induced gastric damage
disulfide group at β-position to the nitrate group in its struc- which could be attributable to the physiological actions of NO
ture, it also falls into the category of ‘SS-nitrates.’ When these released from these prodrugs. NO-aspirin compound 25, on
two compounds were evaluated in vivo in rats at equimolar further co-evaluation with aspirin at equimolar doses, exhib-
oral doses, we observed significantly different NO release ited comparable dose-dependent pharmacokinetics, inhibition
profiles [i.e., AUC of NOx (nitrate/nitrite): 1082μM·h versus of gastric mucosal PGE2 synthesis and analgesic properties
8
1
42μM·h; Tmax: 2h versus 4h; Cmax: ca. 200μM versus ca. to those of aspirin, but retained its gastric-sparing properties
2
6,27)
25μM] for prodrug 11 and naproxcinod (I), respectively
even after doubling its oral dose. This prodrug 25 also showed
(
Table 1). Since prodrug 11 possesses SS-nitrate features, we less stability and metabolized differently (when compared
expected it to release NO (i.e., nitrate/nitrite) on treatment with imide prodrugs of other NSAIDs such as flurbiprofen im-
2
0)
with a sulfhydryl-containing compound. Based on known ide prodrug 27) on incubation in biological fluids such as SGF
hypothesis of sulfhydryl-dependent mechanism of NO release and human plasma. However, in pharmacokinetics studies, all
36,37)
from glyceryl nitrate (GTN),
mine whether a simple organic nitrate such as naproxcinod pounds, which release only salicylate) released their respective
I) can release NO on treatment with a sulfhydryl-containing NSAIDs as sole metabolites. Based on the above data, we
compound. Accordingly, when prodrug 11 and naproxcinod anticipate that these promising NO-NSAIDs could represent
I) were individually treated with sulfhydryl-containing a new class of potentially “Safe NSAIDs” for the treatment of
we also wanted to deter- the reported NO-NSAIDs (except aspirin or NO-aspirin com-
(
(

474
Vol. 60, No. 4
arthritic pain and inflammation. However, further studies are 34.77 (–SCH –), 37.16 (–SCH –), 45.01 (–CHCH ), 45.61
2
2
3
required to establish cardiovascular safety of these novel NO- (–ArCH –), 62.22 (–CO CH –), 70.57 (–CH ONO ), 127.17
2
2
2
2
2
NSAIDs over conventional NSAIDs.
(Ar-C), 129.39 (Ar-C), 137.40 (Ar-C), 140.71 (Ar-C), 174.13
−1
(–CO–); IR (NaCl) cm : 2955 (C–H), 1737 (C=O), 1634 (Ar-
Experimental
C=C), 1279 (–NO ), 1160 (C–O); HR-MS-electrospray ioniza-
2
+
Chemistry. General Methods Melting points were deter- tion (ESI) (m/z) [M+Na] Calcd for C H NNaO S : 410.1066;
17
25
5 2
mined using a capillary melting point apparatus and were un- Found: 410.1055 (mass accuracy: 2.68ppm); Anal. Calcd for
corrected. All solvents and reagents were of laboratory reagent C H NO S : C 52.69, H 6.50, N 3.61; Found: C 53.20, H,
17
25
5 2
grade and were used without purification. Dry solvents were 6.85, N, 3.56.
prepared by standard methods. Analytical TLC was performed
General Procedure for the Synthesis of NO-NSAIDs
on 0.25mm silica gel 60 F254 plates and visualized under UV (11–13, 16) with Ester Linkage To a solution of the ap-
light (254nm). IR spectra were recorded on Perkin Elmer propriate NSAID (5.0mmol) in dichloromethane (20mL) was
Spectrum One FTIR spectrometer; samples were prepared added N,N′-carbonyldiimidazole (7.5mmol). After stirring for
using either the KBR pellet method (for solids) or were run 2h, a solution of 36 (5.0mmol) in dichloromethane (15mL)
1
13
neat using NaCl disc (for oils). H- and C-NMR spectra were was added, and the mixture was stirred at room temperature
acquired using Bruker AVANCE 300 and Bruker AVANCE for 16h. Reaction mixture was washed successively with sat.
5
00 spectrometers. Chemical shifts are reported as δ values in NaHCO solution and brine, dried (Na SO ) and concentrated.
3
2
4
ppm using tetramethylsilane as an internal standard, and cou- The crude product was purified by silica gel column chroma-
pling constants (J) are given in Hertz. Signal multiplicities are tography (50–70% petroleum ether in dichloromethane) to af-
represented by: s=singlet, d=doublet, dd=doublet of doublet, ford the target compounds 11–13 and 16.
t=triplet, q=quartet, m=multiplet, br s=broad singlet. MS and
(S)-2-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-2-(6-me-
high resolution (HR)-MS data were obtained using a Bruker- thoxynaphthalen-2-yl)propanoate (11) The title compound
1
Micro-Q-time-of-flight (TOF) instrument. Reverse phase was obtained as a light yellow oil (46%). H-NMR (300MHz,
HPLC was performed on Waters HPLC using Waters X-terra CDCl ) δ: 1.60 (d, J=6.9Hz, 3H, –CHCH ), 2.84 (t, J=6.9Hz,
3
3
RP-18 analytical column (Dimension: 150mm×3.9mm, 5 2H, –SCH –), 2.90 (t, J=6.3Hz, 2H, –SCH –), 3.86–3.90 (m,
2
2
micron). Silica gel column chromatography was carried 1H, –CHCH ), 3.93 (s, 3H, –OCH ), 4.34 (dt, J=1.2, 6.3Hz,
3
3
out using (150–300 mesh) silica gel. Acetylsalicylic acid 2H, –CO CH –), 4.56 (t, J=6.9Hz, 2H, –CH ONO ), 7.10–7.18
2
2
2
2
(
aspirin) was purchased from Dr. Reddy’s Laboratories, (m, 2H, Ar-H), 7.42 (dd, J=1.1, 8.4Hz, 1H, Ar-H), 7.71 (m,
13
Hyderabad; Diclofenac sodium was purchased from Aarti 3H, Ar-H); C-NMR (75MHz, CDCl ) δ: 18.49 (–CHCH ),
3
3
Drugs Ltd., Mumbai. Naproxen was purchased from Divi’s 34.79 (–SCH –), 37.15 (–SCH –), 45.39 (–CHCH ), 55.33
2
2
3
Labs, Hyderabad. Flurbiprofen was purchased from Sun (–OCH ), 62.30 (–CO CH –), 70.53 (–CH ONO ), 105.56
3
2
2
2
2
Pharmaceutical Industries Ltd., Ahmednagar; Ketoprofen was (Ar-C), 119.14 (Ar-C), 126.01 (Ar-C), 126.19 (Ar-C), 127.22
purchased from BEC Chemicals, Roha; Ibuprofen, Sulindac (Ar-C), 128.89 (Ar-C), 129.26 (Ar-C), 133.73 (Ar-C), 135.33
−1
and Indomethacin were purchased from Sigma-Aldrich Co., (Ar-C), 157.71 (Ar-C), 174.44 (–CO–). IR (NaCl) cm : 2934
U.S.A. (C–H), 1735 (C=O), 1635 (Ar-C=C), 1277 (–NO ); HR-MS-
2
General Procedure for the Synthesis of NO-NSAIDs (9 ESI (m/z) [M+Na]+ Calcd for C H NNaO S : 434.0702,
18
21
6 2
and 15) with Ester Linkage Oxalyl chloride (45mmol) was Found: 434.0688 (mass accuracy: 5.4ppm); Anal. Calcd for
added to a solution of the appropriate NSAID (30mmol) in C H NO S : C 52.54, H 5.14, N 3.40; Found: C 53.36, H 5.21,
18
21
6 2
benzene (40mL). The reaction mixture was heated at 50°C for N 3.50.
1
h, concentrated, re-dissolved in benzene (40mL) and treated
2-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-2-(2-fluoro-[1,1′-
with a solution of 36 (30mmol) and triethylamine (45mmol) biphenyl]-4-yl)propanoate (12) The title compound was
in dichloromethane (20mL) at 0°C. After stirring for 16h, the obtained as a light yellow oil (43%). H-NMR (300MHz,
1
reaction mixture was concentrated and partitioned between CDCl ) δ: 1.55 (d, J=7.20Hz, 3H, –CH ), 2.86–2.95 (m, 4H,
3
3
ethyl acetate and 0.5N HCl. The organic layer was washed –CH S CH –), 3.78 (q, J=7.0Hz, 1H, –CH–), 4.38 (t, J=4.9,
2
2
2
with brine, dried (Na SO ) and concentrated. The crude com- 7.2Hz, 2H, –CO CH –), 4.66 (t, J=6.7Hz, 2H, –CH ONO ),
2
4
2
2
2
2
13
pound was purified by column chromatography (silica gel, 7.11–7.66 (m, 2H, Ar-H), 7.30–7.50 (m, 6H, Ar-H); C-NMR
–10% ethyl acetate in petroleum ether) to afford the target (75MHz, CDCl3) δ: 17.84 (–CH3), 34.39 (–SCH –),
5
2
compounds 9 and 15.
36.65 (–SCH –), 44.45 (–CH–), 62.03 (–CO CH –), 70.08
2
2
2
2
-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-2-acetoxybenzoate (–CH ONO ), 114.63 (Ar-C), 114.94 (Ar-C), 123.08 (Ar-C),
2
2
(
9) The synthesis of this compound, along with spectral and 127.22 (Ar-C), 127.54 (Ar-C), 128.43 (Ar-C), 130.35 (Ar-C),
8)
analytical data, has been described in our earlier publication.
134.92 (Ar-C), 140.93 (Ar-C), 141.03 (Ar-C), 157.55 (Ar-C),
−1
2
-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-2-(4-isobutylphe- 160.84 (Ar-C), 173.23 (–CO–); IR (NaCl) cm : 2936 (C–H),
nyl)propanoate (15) The title compound was obtained 1737 (C=O), 1634 (Ar-C=C), 1278 (–NO ), 1171 (C–O); HR-
2
1
+
as a light yellow oil (45%). H-NMR (300MHz, CDCl ) δ: MS-ESI (m/z) [M+Na] Calcd for C H FNNaO S : 448.0659;
3
19 20
5 2
0
–
2
.89 (d, J=6.6Hz, 6H, –CH(CH ) ), 1.49 (d, J=7.2Hz, 3H, Found 448.0655 (mass accuracy: 0.89ppm); Anal. Calcd for
3 2
CHCH ), 1.79–1.88 (m, 1H, –CH(CH ) ), 2.44 (d, J=7.2Hz, C H FNO S : C 53.63, H 4.74, N 3.29, S 15.07; Found: C
3
3 2
19 20
5 2
H, –ArCH –), 2.81–2.91 (m, 4H, –CH S CH –), 3.70 (q, 53.98, H 4.81, N 3.63, S 15.46.
2
2
2
2
J=7.2Hz, 1H, –CHCH ), 4.33 (t, J=6.9Hz, 2H, –CO CH –),
2-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-2-(3-benzoylphe-
3
2
2
4
.63 (t, J=6.9Hz, 2H, –CH ONO ), 7.10 (d, J=8.1Hz, 2H, Ar- nyl)propanoate (13) The title compound was obtained as a
2
2
13
1
H), 7.20 (d, J=7.8Hz, 2H, Ar-H); C-NMR (75MHz, CDCl3) light yellow oil (28%). H-NMR (300MHz, CDCl ) δ: 1.54 (d,
3
δ: 18.51 (–CHCH ), 22.38 (–CH(CH ) ), 30.19 (–CH(CH ) ), J=7.2Hz, 3H, –CH ), 2.91–2.93 (m, 4H, –CH S CH –), 3.82
3
3 2
3 2
3
2
2
2

April 2012
475
(
q, J=6.6Hz, 1H, –CH–), 4.34 (t, J=6.1Hz, 2H, –CO CH –), characterization data (spectral and analytical) obtained for the
2
2
8)
4
.64 (t, J=6.5Hz, 2H, –CH ONO ), 7.42–8.0 (m, 9H, Ar-H); compound 10 is identical to that reported earlier.
2 2
13
C-NMR (75MHz, CDCl ) δ: 17.89 (–CH ), 34.38 (–SCH –),
(Z)-2-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-2-(5-fluoro-
3
3
2
3
6.63 (–SCH –), 44.82 (–CH–), 61.97 (–CO CH –), 70.06 2-methyl-1-(4-(methylsulfinyl)benzylidene)-1H-inden-3-yl)-
2
2
2
(
–CH ONO ), 127.82 (Ar-C), 128.07 (Ar-C), 128.65 (Ar-C), acetate (14) To a solution of sulindac (1.00g, 2.80mmol)
2
2
129.56 (Ar-C), 130.99 (Ar-C), 132.04 (Ar-C), 136.96 (Ar- and linker 36 (0.78g, 3.91mmol) in dichloromethane (20mL)
C), 137.50 (Ar-C), 140.05 (Ar-C), 173.30 (–CO –), 195.94 at RT were added DCC (0.69g, 3.36mmol) and DMAP (0.41g,
2
−
1
(
ArCO–); IR (NaCl) cm : 2978 (C–H), 1736 (C=O), 1634 (Ar- 3.36mmol), and the mixture was stirred for 10h. The reac-
C=C), 1280 (–NO ), 1164 (C–O); HR-MS-ESI (m/z) [M+Na]
Calcd for C H NNaO S : 458.0702; Found 458.0698 (mass purified by silica gel column chromatography (5% acetone
+
tion mixture was filtered and concentrated, and the residue
2
2
0
21
6 2
accuracy: 0.87ppm); Anal. Calcd for C H NO S : C 55.16, H in dichloromethane) to afford 14 as a yellow solid (0.80g,
2
0
21
6 2
.86, N 3.22, S 14.73; Found: C 55.14, H 4.81, N 3.48, S 15.09. 53%). mp 171–172°C; 1H-NMR (300MHz, CDCl ) δ: 2.21
4
3
2
-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-2-(1-(4 - (s, 3H, Ar-CH ), 2.81 (s, 3H, –S(O)CH ), 2.90–2.96 (m, 4H,
3 3
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetate –CH S CH –), 3.59 (s, 2H, ArCH –), 4.38 (t, J=6.6Hz, 2H,
2
2
2
2
(
16) The title compound was obtained as an off-white –CO CH –), 4.64 (t, J=6.6Hz, 2H, –CH ONO ), 6.53–6.60
2 2 2 2
1
solid (67%). mp 91–93°C; H-NMR (300MHz, CDCl ) δ: (m, 1H, Ar-H), 6.88 (dd, J=2.4, 9.0Hz, 1H, –C=CH–),
2
3
.40 (s, 3H, Ar-CH ), 2.92–2.94 (m, 4H, –CH S CH –), 3.71 7.13–7.17 (m, 1H, Ar-H), 7.16 (s, 1H, Ar-H), 7.65–7.73 (m, 4H,
3 2 2 2
13
(
2
s, 2H, Ar-CH –), 3.86 (s, 3H, –OCH ), 4.39 (t, J=6.4Hz, Ar-H); C-NMR (75MHz, CDCl ) δ: 10.0 (Ar-CH ), 31.1 (Ar-
2
3
3
3
H, –CO CH –), 4.64 (t, J=4.7Hz, 2H, –CH ONO ), 6.71 (d, CH –), 34.4 (–SCH –), 36.7 (–SCH –), 43.3 (–S(O)CH ), 62.1
2 2 2 2 2 2 2 3
J=2.7Hz, 1H, Ar-H), 6.89 (d, J=9.0Hz, 1H, Ar-H), 6.98 (d, (–CO CH –), 70.0 (–CH ONO ), 105.4 (Ar-C), 105.7 (Ar-C),
2
2
2
2
J=2.7Hz, 1H, Ar-H), 7.49 (d, J=8.4Hz, 2H, Ar-H), 7.68 (d, 110.1 (Ar-C), 110.4 (Ar-C), 123.1 (Ar-C), 123.3 (Ar-C), 127.8
1
3
J=8.4Hz, 2H, Ar-H); C-NMR (75MHz, CDCl ) δ: 13.86 (Ar-C), 128.9 (Ar-C), 129.7 (Ar-C), 130.9 (Ar=C–), 137.9
3
(
Ar-CH ), 30.72 (Ar-CH –), 35.44 (–SCH –), 37.75 (–SCH –), (Ar-C), 139.1 (Ar-C), 141.0 (Ar-C), 145.0 (Ar-C), 146.0 (Ar-
3
2
2
2
−1
5
6.24 (–OCH ), 63.00 (–CO CH –), 71.04 (–CH ONO ), 101.85 C), 146.1 (Ar-C), 169.4 (–CO–); IR (KBr) cm : 2922 (C–H),
3 2 2 2 2
(
(
Ar-C), 112.11 (Ar-C), 112.69 (Ar-C), 115.45 (Ar-C), 129.62 1727 (C=O), 1625 (Ar-C=C), 1278 (–NO ), 1169 (C–O), 1049
2
Ar-C), 131.04 (Ar-C), 131.31 (Ar-C), 131.68 (Ar-C), 134.34 (S=O); HR-MS-ESI (m/z) [M+H]+ Calcd for C H FNO S :
24 25 6 3
(Ar-C), 136.54 (Ar-C), 139.80 (Ar-C), 156.56 (Ar-C), 168.78 538.0823; Found: 538.0806 (mass accuracy: 3.16ppm); Anal.
−1
(–N–CO–), 172.07 (ArCH CO–). IR (KBr) cm : 2930 (C–H), Calcd for C H FNO S : C 53.61, H 4.50, N 2.61; Found: C
2 24 24 6 3
1
722 (C=O), 1641 (Ar-C=C), 1278 (–NO ), 1133 (C–O); 54.70, H 4.84, N 2.56.
2
+
HR-MS-ESI (m/z) [M+Na] Calcd for C H ClN NaO S :
General Procedure for the Synthesis of NO-NSAIDs
61.0527; Found: 561.0511 (mass accuracy: 2.85ppm); Anal. (17, 19–23) with Double Ester Linkage A solution of the
Calcd for C H ClN O S : C 51.25, H 4.30, N 5.20; Found: C desired NSAID (13mmol) and cesium carbonate (13mmol) in
2
3
23
2
7 2
5
2
3
23
2
7 2
51.41, H 4.36, N 5.36.
methanol (40mL) was stirred for 24h at room temperature.
2
-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-2-(2-((2,6-dichlo- Methanol was removed under vacuum. The residual cesium
rophenyl)amino)phenyl)acetate (10) We have reported salt of the NSAID was suspended in a 1:1 mixture of tetra-
the synthesis of this compound by a different method in our hydrofuran (THF):DMF (40mL) and treated with a solution
8)
earlier publication. Here, we report an alternative method: To of linker intermediate 37 (13mmol) in THF (10mL). After 2h,
a solution of diclofenac sodium (2.80g, 8.80mmol) in DMF the reaction mixture was concentrated, re-dissolved in ethyl
(
20mL) at 0°C, was added a solution of dibromide 35 (2.46g, acetate, washed with water, dried (Na SO ) and concentrated.
2
4
8.80mmol) in DMF (10mL) over a period of 15min. The The crude material was purified by silica gel column chroma-
resulting mixture was stirred overnight at room temperature. tography (5–10% ethyl acetate in petroleum ether) to afford
The reaction mixture was quenched with water, extracted the target compounds 17 and 19–23.
with dichloromethane, washed with water and brine, dried
2-(2-((2-(Nitrooxy)ethyl)disulfanyl)ethoxy)-2-oxoethyl-
(
Na SO ) and concentrated. The crude material was purified 2-acetoxybenzoate (17) The synthesis of this compound,
2
4
by silica gel column chromatography (5% ethyl acetate in pe- along with spectral and analytical data, has been described in
8
)
troleum ether) to give the intermediate 40 as a pale yellow vis- our earlier publication.
1
cous liquid (2.18g, 50%). H-NMR (300MHz, CDCl ) δ: 2.95
(S)-2-(2-((2-(Nitrooxy)ethyl)disulfanyl)ethoxy)-2-oxoeth-
3
(
3
t, J=6.6Hz, 2H, –SCH –), 3.05 (t, J=8.4Hz, 2H, –SCH –), yl-2-(6-methoxy naphthalen-2-yl)propanoate (19) The title
2
2
1
.58 (t, J=8.4Hz, 2H, –CH Br), 3.84 (s, 2H, –ArCH –), 4.40 compound was obtained as a pale yellow gum (44%). H-NMR
2
2
(
6
t, J=6.6Hz, 2H, –CO CH –), 6.55 (d, J=8.1Hz, 1H, Ar-H), (300MHz, CDCl ) δ: 1.62 (d, J=7.2Hz, 3H, –CHCH ), 2.80 (t,
2
2
3
3
.82 (br s, 1H, –NH–), 6.94–7.01 (m, 2H, Ar-H), 7.10–7.16 (m, J=6.3Hz, 2H, –SCH –), 2.90 (t, J=6.6Hz, 2H, –SCH –), 3.91
2
2
1
H, Ar-H), 7.22–7.28 (m, 1H, Ar-H), 7.34 (d, J=8.1Hz, 2H, (s, 3H, –OCH ), 3.96 (q, J=7.2Hz, 1H), 4.35 (t, J=6.6Hz, 2H,
3
+
Ar-H); MS m/z 494 [M+H] . To a solution of intermediate 40 –CO CH –), 4.61–4.68 (m, 4H, –CO CH CO –, –CH ONO ),
2
2
2
2
2
2
2
(2.00g, 4.05mmol) in acetonitrile (20mL) was added a solu- 7.12–7.16 (m, 2H, Ar-H), 7.42 (dd, J=1.5, 8.4Hz, 1H, Ar-H),
13
tion of silver nitrate (0.86g, 5.07mmol) in acetonitrile (10mL). 7.71 (d, J=8.4Hz, 3H, Ar-H); C-NMR (75MHz, CDCl3)
The resulting mixture was stirred at room temperature for 4h. δ: 17.97 (–CHCH3), 34.32 (–SCH –), 36.19 (–SCH –), 44.56
2
2
The reaction mixture was diluted with ethyl acetate (50mL) (–CHCH ), 54.81 (–OCH ), 60.34 (–CO CH CH S–), 62.28
3
3
2
2
2
and filtered through celite. The filtrate was concentrated, dried (–CO CH2CO –), 70.06 (–CH ONO ), 105.12 (Ar-C), 118.54
2
2
2
2
(
Na SO ) and purified by silica gel column chromatography (Ar-C), 125.68 (Ar-C), 125.77 (Ar-C), 126.67 (Ar-C), 128.40
2 4
(5–10% ethyl acetate in petroleum ether) to afford the com- (Ar-C), 128.78 (Ar-C), 133.29 (Ar-C), 134.46 (Ar-C), 157.20
pound 10 as a pale yellow viscous liquid (0.95g, 49%). The (Ar-C), 167.01 (OCH CO–), 173.49 (CH CHCO–); IR (NaCl)
2
3

476
Vol. 60, No. 4
−
1
cm : 2950 (C–H), 1747 (C=O), 1633 (Ar-C=C), 1278 1630 (Ar-C=C), 1278 (–NO ), 1166 (C–O), 1049 (S=O); HR-
2
+
(
–NO ), 1166 (C–O); HR-MS-ESI (m/z) [M+K] Calcd for MS-ESI (m/z) [M+H]+ Calcd for C H FNO S : 596.0871;
2
26 27
8 3
C H KNO S : 508.0497; Found: 508.0504 (mass accuracy: Found: 596.0877 (mass accuracy: 1.01ppm); Anal. Calcd for
2
0
23
8 2
−
1.38ppm); Anal. Calcd for C H NO S : C 51.16, H 4.94, N C H FNO S : C 52.42, H 4.40, N 2.35; Found: C 52.17, H
20 23 8 2 26 26 8 3
2
.98; Found: C 51.28, H 5.05, N 3.17.
-(2-((2-(Nitrooxy)ethyl)disulfanyl)ethoxy)-2-oxoethyl-
-(2-fluoro-[1,1′-biphenyl]-4-yl)propanoate (20) The title 2-(4-isobutylphenyl)propanoate (23) The title compound
4.31, N, 2.39.
2
2-(2-((2-(Nitrooxy)ethyl)disulfanyl)ethoxy)-2-oxoethyl-
2
1
1
compound was obtained as a pale yellow gum (80%). H-NMR was obtained as a pale yellow oil (36%). H-NMR (300MHz,
500MHz, CDCl ) δ: 1.58 (d, J=7.5Hz, 3H, –CH ), 2.80–2.94 CDCl ) δ: 0.89 (d, J=6.6Hz, 6H, –CH(CH ) ), 1.54 (d,
(
3
3
3
3 2
(m, 4H, –CH S CH –), 3.88 (q, J=7.0Hz, 1H, –CH–), 4.40 (t, J=7.2Hz, 3H, –CHCH ), 1.80–1.87 (m, 1H, –CH(CH ) ),
2
2
2
3
3
2
J=6.5Hz, 2H, –CO CH –), 4.64–4.68 (m, 4H, –CO CH CO –, 2.44 (d, J=7.2Hz, 2H, –ArCH –), 2.88 (t, J=6.6Hz, 2H,
2
2
2
2
2
2
–
CH ONO ), 7.14–7.19 (m, 2H, Ar-H), 7.34–7.45 (m, 4H, Ar- –SCH –), 2.95 (t, J=6.9Hz, 2H, –SCH –), 3.82 (q, J=7.2Hz,
2 2 2 2
13
H), 7.52–7.54 (m, 2H, Ar-H); C-NMR (125MHz, CDCl3) 1H, –CHCH ), 4.38 (t, J=6.6Hz, 2H, –CO CH –), 4.62–4.69
3
2
2
δ: 18.4 (–CH ), 34.8 (–SCH –), 36.7 (–SCH –), 44.6 (–CH–), (m, 4H, –CO CH CO –, –CH ONO ), 7.11 (d, J=7.8Hz,
3
2
2
2
2
2
2
2
13
6
1
0.9 (–CO CH –), 62.8 (–CO CH CO –), 70.5 (–CH ONO ), 2H), 7.23 (d, J=7.8Hz, 2H); C-NMR (75MHz, CDCl ) δ:
2
2
2
2
2
2
2
3
15.3 (Ar-C), 115.5 (Ar-C), 123.7 (Ar-C), 127.7 (Ar-C), 128.4 18.56 (–CHCH ), 22.41 (–CH(CH ) ), 30.18 (–CH(CH ) ),
3 3 2 3 2
(
Ar-C), 128.9 (Ar-C), 130.8 (Ar-C), 135.4 (Ar-C), 140.1 (Ar- 34.85 (–SCH –), 36.76 (–SCH –), 44.74 (–CHCH ), 45.00
2 2 3
C), 159.0 (Ar-C), 167.4 (–OCH CO–), 173.3 (CH CHCO–); IR (–ArCH2–), 60.82 (–CO CH –), 62.78 (–CO CH CO –), 70.60
2
3
2
2
2
2
2
−1
(
(
NaCl) cm : 2950 (C–H), 1746 (C=O), 1634 (Ar-C=C), 1279 (–CH ONO ), 127.31 (Ar-C), 129.39 (Ar-C), 137.06 (Ar-C),
2 2
+
–NO ), 1164 (C–O); HR-MS-ESI (m/z) [M+Na] Calcd for 140.79 (Ar-C), 167.61 (–OCH CO –), 174.13 (CH CHCO–); IR
2 2 2 3
1
−
C H FNNaO S : 506.0719; Found: 506.0707 (mass accuracy: (NaCl) cm : 2955 (C–H), 1746 (C=O), 1634 (Ar-C=C), 1278
21
22
7 2
+
−
2
2.5ppm); Anal. Calcd for C H FNO S : C 52.16, H 4.59, N (–NO ), 1151 (C–O); HR-MS-ESI (m/z) [M+K] Calcd for
.90; Found: C 52.83, H 4.45, N 2.46.
21
22
7
2
2
C H KNO S : 484.0861; Found: 484.0864 (mass accuracy:
19
27
7 2
2
-(2-((2-(Nitrooxy)ethyl)disulfanyl)ethoxy)-2-oxoethyl- −0.62ppm); Anal. Calcd for C H NO S : C 51.22, H 6.11, N
19 27 7 2
2
-(3-benzoylphenyl)propanoate (21) The title compound 3.14; Found: C 51.94, H 6.08, N 3.34.
was obtained as a pale yellow oil (40%). H-NMR (300MHz,
1
2-(2-((2-(Nitrooxy)ethyl)disulfanyl)ethoxy)-2-oxoethyl-
CDCl ) δ: 1.59 (d, J=7.2Hz, 3H, –CH ), 2.89 (t, J=6.6Hz, 2H, 2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate (18) The
3
3
–
1
SCH –), 2.95 (t, J=6.6Hz, 2H, –SCH –), 3.92 (q, J=6.9Hz, synthesis of this compound, along with spectral and analytical
2 2
8)
H, –CH–), 4.39 (t, J=6.6Hz, 2H, –CO CH ), 4.62–4.68 data, has been described in our earlier publication.
2
2
(m, 4H, –CO CH CO –, –CH ONO ), 7.48 (t, J=7.8Hz, 3H,
2-(2-((2-(Nitrooxy)ethyl)disulfanyl)ethoxy)-2-oxoethyl-2-
2
2
2
2
2
Ar-H), 7.59 (d, J=7.5Hz, 2H, Ar-H), 7.69 (d, J=7.8Hz, 1H, (1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-
13
Ar-H), 7.80 (d, J=8.7Hz, 3H, Ar-H); C-NMR (75MHz, acetate (24) To a solution of indomethacin (2.5g, 7.0mmol)
CDCl ) δ: 16.40 (–CH ), 34.82 (–SCH –), 36.72 (–SCH –), in DMF (40mL) were added potassium carbonate (2.4g,
3
3
2
2
4
5.00 (–CH–), 60.91 (–CO CH –), 62.84 (–CO CH CO –), 17.5mmol) and the intermediate 37 (1.9g, 7.0mmol) and
2 2 2 2 2
7
0.58 (–CH ONO ), 128.33 (Ar-C), 128.58 (Ar-C), 129.23 the mixture was stirred for 16h. DMF was removed under
2
2
(Ar-C), 129.30 (Ar-C), 130.10 (Ar-C), 131.69 (Ar-C), 132.55 vacuum. The residue was partitioned between water and
(Ar-C), 137.43 (Ar-C), 137.94 (Ar-C), 140.18 (Ar-C), 167.38 ethyl acetate. The organic layer was washed with water
(
(
–OCH CO–), 173.44 (CH CHCO–), 196.44 (ArCO–); IR and brine, dried (Na SO ), concentrated and purified by
2
3
2
4
−1
NaCl) cm : 2950 (C–H), 1746 (C=O), 1634 (Ar-C=C), 1280 silica gel column chromatography (10% ethyl acetate in
+
(
–NO ), 1156 (C–O); HR-MS-ESI (m/z) [M+Na] Calcd for petroleum ether) to afford 24 as a light green gum (0.60g,
2
1
C H NNaO S : 516.0757; Found: 516.0767 (mass accuracy: 15%). H-NMR (300MHz, CDCl ) δ: 2.41 (s, 3H, Ar-CH ),
2
2
23
8
2
3
3
−
1.94ppm); Anal. Calcd for C H NO S : C 53.54, H 4.70, N 2.94 (m, 4H, –CH S CH –), 3.81 (s, 2H, Ar-CH –), 3.86 (s,
22 23 8 2 2 2 2 2
2
.84; Found: C 52.75, H 4.80, N 2.99.
(
3H, –OCH ), 4.42 (t, J=5.7Hz, 2H, –CO CH –), 4.62–4.67 (m,
3 2 2
Z)-2-(2-((2-(Nitrooxy)ethyl)disulfanyl)ethoxy)-2-oxoeth- 4H, –CO CH CO –, –CH ONO ), 6.69 (d, J=8.4Hz, 1H, Ar-
2
2
2
2
2
yl-2-(5-fluoro-2-methyl-1-(4-(methylsulfinyl)benzylidene)- H), 6.90 (d, J=8.7Hz, 1H, Ar-H), 7.00 (s, 1H, Ar-H), 7.49 (d,
1
3
1
H-inden-3-yl)acetate (22) The title compound was ob- J=7.8Hz, 2H, Ar-H), 7.68 (d, J=7.8Hz, 2H, Ar-H); C-NMR
1
tained as a light yellow solid (56%). mp 74–75°C; H-NMR (75MHz, CDCl3) δ: 13.85 (Ar-CH3), 30.24 (ArCH –),
2
(
300MHz, CDCl ) δ: 2.22 (s, 3H, Ar-CH ), 2.81 (s, 3H, –S(O) 35.36 (–SCH –), 37.25 (–SCH –), 56.23 (–OCH ), 61.46
3
3
2
2
3
CH ), 2.86–2.97 (m, 4H, –CH S CH –), 3.68 (s, 2H, Ar- (–CO CH CO –), 63.34 (–CO CH –), 71.06 (–CH ONO ),
3
2
2
2
2
2
2
2
2
2
2
CH –), 4.41 (t, J=6.6Hz, 2H, –CO CH –), 4.66 (t, J=6.6Hz, 101.82 (Ar-C), 112.22 (Ar-C), 112.37 (Ar-C), 115.44 (Ar-C),
2
2
2
2
H, –CH ONO ), 4.67 (s, 2H, –CO CH CO –), 6.54–6.60 (m, 129.63 (Ar-C), 131.01 (Ar-C), 131.30 (Ar-C), 131.70 (Ar-C),
2 2 2 2 2
1
H, Ar-H), 6.90 (dd, J=2.4, 9.0Hz, 1H, –C=CH–), 7.13–7.16 134.33 (Ar-C), 136.67 (Ar-C), 139.81 (Ar-C), 156.58 (Ar-C),
(
m, 1H, Ar-H), 7.17 (s, 1H, Ar-H), 7.65–7.73 (m, 4H, Ar-H); 167.87 (–N–CO–), 168.79 (ArCH CO–), 170.71 (–OCH CO–);
2
2
1
3
−1
C-NMR (75MHz, CDCl ) δ: 10.0 (Ar-CH ), 30.6 (Ar- IR (NaCl) cm : 2927 (C–H), 1735 (C=O), 1636 (Ar-C=C),
3
3
+
CH –), 34.3 (–SCH –), 36.2 (–SCH –), 43.4 (–S(O)CH ), 60.5 1278 (–NO ), 1149 (C–O); MS m/z: 597.1 [M+H] ; HR-MS-
2
2
2
3
2
+
(
–CO CH CO –), 62.3 (–CO CH –), 70.0 (–CH ONO ), 105.5 ESI (m/z) [M+H] Calcd for C H ClN O S : 597.0763;
2 2 2 2 2 2 2 25 26 2 9 2
(Ar-C), 105.8 (Ar-C), 110.1 (Ar-C), 110.4 (Ar-C), 123.1 (Ar-C), Found: 597.0739 (mass accuracy: 4.02ppm); Anal. Calcd for
1
23.3 (Ar-C), 127.9 (Ar-C), 128.9 (Ar-C), 129.7 (–C=CH–), C H ClN O S : C 50.29, H 4.22, N 4.69; Found: C 51.19, H
2
5
25
2
9 2
1
30.6 (Ar-C), 138.1 (Ar-C), 139.0 (Ar-C), 141.0 (Ar-C), 145.1 4.31, N 4.38.
Ar-C), 145.9 (Ar-C), 146.0 (Ar-C), 166.8 (ArCH CO–), 169.0 Synthesis of NO-NSAID (25) with Imide Linkage
–OCH CO–); IR (KBr) cm : 2958 (C–H), 1741 (C=O), 2-(((2-((2-(Nitrooxy)ethyl)disulfanyl)ethoxy)carbonyl)car-
(
(
2
−1
2

April 2012
477
bamoyl)phenylacetate (25) Thionyl chloride (0.81mL, Ar-H), 7.37–7.47 (m, 1H, Ar-H), 7.68–7.63 (m, 3H, Ar-H); MS
+
1
5
1.10mmol) was added to a solution of aspirin (1, 1.00g, m/z 429.27 [M+H] . Using this amide, the title compound
1
.55mmol) in benzene (8mL) at 0°C. The reaction mixture was obtained as a white solid (36%). mp 145–147°C; H-NMR
was heated at 70°C for 1.5h. Silver cyanate (0.99g, 6.66mmol) (300MHz, CDCl ) δ: 1.56 (d, J=6.9Hz, 3H, –CHCH ), 2.86
3
3
was added to the mixture containing aspirin acid chloride (m, 4H, –CH S CH –), 3.91 (s, 3H, –OCH ), 4.31–4.33 (m, 3H,
2
2
2
3
and the mixture was refluxed for 0.5h to get the correspond- –CO CH –, –CHCH ), 4.59 (t, J=6.6Hz, 2H, –CH ONO ),
2
2
3
2
2
2
5)
ing acyl isocyanate 43. The mixture was allowed to cool to 7.11–7.16 (m, 2H, Ar-H), 7.37–7.47 (m, 1H, Ar-H), 7.68–
room temperature. A solution of 36 (0.70g, 3.51mmol) in ben- 7.63 (m, 3H, Ar-H); C-NMR (75MHz, CDCl ) δ: 18.26
13
3
zene (4mL) was added to the mixture containing the interme- (–CHCH ), 34.05 (–SCH –), 36.15 (–SCH –), 45.14 (–CHCH ),
3
2
2
3
diate aspirin acyl isocyanate 43 and the mixture was stirred 55.12 (–OCH3), 62.44 (–CO CH –), 71.07 (–CH ONO ),
2
2
2
2
for 8h to generate the imide prodrug 25. The reaction mixture 105.66 (Ar-C), 118.69 (Ar-C), 125.72 (Ar-C), 126.27 (Ar-C),
was concentrated and the residue was partitioned between 126.85 (Ar-C), 128.30 (Ar-C), 129.10 (Ar-C), 133.27 (Ar-C),
ethyl acetate (40mL) and water (40mL). The organic layer 135.78 (Ar-C), 150.87 (Ar-C), 157.14 (–OCONH–), 173.17
−1
was washed with brine (40mL), dried (Na SO ) and concen- (–CO NHCO–); IR (KBr) cm : 3248 (N–H), 2960 (C–H),
2
4
2
trated. The crude material was purified by silica gel column 1749 (C=O), 1635 (Ar-C=C), 1278 (–NO ), 1180 (C–O); HR-
2
chromatography (5–10% ethyl acetate in hexane) to afford 25 MS-ESI (m/z) [M+Na]+ Calcd for C H N NaO S : 477.0761;
1
9
22
2
7 2
1
as a colourless oil (0.50g, 35%). H-NMR (300MHz, CDCl ) Found: 477.0766 (mass accuracy: −1.05ppm); Anal. Calcd for
δ: 2.22 (s, 3H, –COCH ), 2.87–2.95 (m, 4H, –CH S CH –), C H N O S : C 50.22, H 4.88, N 6.16; Found: C 50.92, H
3
3
2
2
2
19 22
2
7 2
4
–
.31 (t, J=6.0Hz, 2H, –CO CH –), 4.67 (t, J=7.5Hz, 2H, 4.92, N 6.28.
2 2
CH ONO ), 5.73 (br s, 1H, –NH–), 7.05 (d, J=9.0Hz, 1H,
2-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-(2-(2-fluoro-[1,1′-
2
2
Ar-H), 7.19 (t, J=7.5Hz, 1H, Ar-H), 7.61 (t, J=6.0Hz, 1H, biphenyl]-4-yl)propanoyl)carbamate (27) The intermedi-
13
Ar-H), 7.97 (d, J=6.0Hz, 1H, Ar-H); C-NMR (125MHz, ate flurbiprofen amide was obtained as a white solid (90%).
1
CDCl ) δ: 25.0 (–CH ), 34.7 (–SCH –), 37.2 (–SCH –), 62.9
(
H-NMR (300MHz, CDCl ) δ: 1.55 (d, J=6.9Hz, 3H, –CH ),
3
3
2
2
3
3
–CO CH CH –), 70.6 (–CH ONO ), 107.0 (Ar-C), 113.1 (Ar- 3.66 (q, J=7.2Hz, 1H, –CH–), 5.63 (s, 1H, –NH ), 5.95 (s,
2
2
2
2
2
2
C), 117.4 (Ar-C), 123.6 (Ar-C), 129.6 (Ar-C), 136.9 (Ar-C), 1H, –NH ), 7.1–7.20 (m, 2H, Ar-H), 7.38–7.56 (m, 6H, Ar-H);
2
13
1
(
52.6 (–CONH–), 154.4 (Ar-CO –), 159.6 (CH CO –); IR
C-NMR (75MHz, CDCl ) δ: 18.32 (–CH ), 46.00 (–CH–),
2
3
2
3
3
−1
NaCl) cm : 3305 (N–H), 2954 (C–H), 1731 (C=O), 1633 (Ar- 115.15 (Ar-C), 115.46 (Ar-C), 123.59 (Ar-C), 123.63 (Ar-C),
127.79 (Ar-C), 128.50 (Ar-C), 128.91 (Ar-C), 128.94 (Ar-C),
Calcd for C H N NaO S : 427.0246; Found: 427.0229 (mass 131.14 (Ar-C), 131.20 (Ar-C), 135.29 (Ar-C), 142.32 (Ar-C),
accuracy: −3.8ppm); Anal. Calcd for C H N O S : C 41.58, 142.42 (–CO–); MS m/z 244.14 [M+H] . Using this amide,
H 3.99, N 6.93, S 15.86; Found: C 41.85, H 3.97, N 6.65, S the title compound was obtained as a white solid (10%). mp:
+
C=C), 1278 (–NO ), 1156 (C–O); HR-MS-ESI (m/z) [M+Na]
2
14
16
2
8 2
+
14
16
2
8 2
1
15.78.
58–60°C; H-NMR (300MHz, CDCl ) δ: 1.54 (d, J=6.9Hz,
General Procedure for the Synthesis of NO-NSAIDs 3H, –CH ), 2.95 (m, 4H, –CH S CH –), 4.40–4.43 (m, 3H,
3
3
2
2
2
(
26–30) with Imide Linkage To a solution of the appro- –CO CH –, –CHCH ), 4.68 (t, J=6.6Hz, 2H, –CH ONO ),
2 2 3 2 2
13
priate NSAID (3–5, 7, 8) (30mmol) in benzene (70mL) was 7.13–7.19 (m, 2H, Ar-H), 7.35–7.55 (m, 6H, Ar-H); C-NMR
added DMF (2 drops) followed by oxalyl chloride (36mmol) (75MHz, CDCl3) δ: 18.28 (–CH3), 34.36 (–SCH –),
2
and stirred at room temperature for 3h. The solvent was re- 36.35 (–SCH –), 44.59 (–CH–), 63.17 (–CO CH –), 70.01
2
2
2
moved under vacuum. The residue (containing acid chlorides (–CH ONO ), 115.03 (Ar-C), 115.33 (Ar-C), 123.40 (Ar-C),
2
2
of respective NSAIDs) was dissolved in dichloromethane 127.27 (Ar-C), 127.98 (Ar-C), 128.42 (Ar-C), 130.54 (Ar-C),
100mL) and cooled to 0°C. Ammonia gas was passed slowly 134.82 (Ar-C), 140.65 (Ar-C), 140.79 (Ar-C), 150.38 (Ar-C),
through the solution for 30min. The reaction mixture was 157.60 (Ar-C), 160.89 (–NHCO –), 173.32 (CH CHCO–); IR
(
2
3
−1
washed successively with sat. NaHCO solution and brine. The (KBr) cm : 3259 (N–H), 2978 (C–H), 1752 (C=O), 1646 (Ar-
organic layer was dried (Na SO ) and concentrated to afford C=C), 1272 (–NO ), 1175 (C–O); HR-MS-ESI (m/z) [M+Na]
the respective NSAID amides. To a stirred suspension of the Calcd for C H FN NaO S : 491.0717; Found: 491.0718 (mass
3
+
2
4
2
2
0
21
2
6 2
NSAID amide (30mmol) in 1,2-dichloroethane (100mL) was accuracy: −0.2ppm); Anal. Calcd for C H FN O S : C 51.27,
2
0
21
2
6 2
added oxalyl chloride (36mmol) and refluxed for 16h to get H 4.52, N 5.98, S 13.69; Found: C 51.24, H 4.48, N 5.97, S
2
4)
the corresponding acyl isocyanate 43. The reaction mixture 13.71.
was cooled to room temperature. A solution of 36 (30mmol)
2-((2-(Nitrooxy)ethyl)disulfanyl)ethyl(2-(3-benzoylphe-
in 1,2-dichloroethane (10mL) was added to the above mixture nyl)propanoyl)carbamate (28) The intermediate ketopro-
1
and the mixture was stirred for 16h at room temperature to fen amide was obtained as a white solid (100%). H-NMR
generate the corresponding imide containing prodrugs 26–30. (300MHz, CD OD) δ: 1.55 (d, J=7.2Hz, 3H, –CH ), 3.68 (q,
3
3
The reaction mixture was concentrated; the residue was taken J=6.9Hz, 1H, –CH–), 5.54–5.69 (m, 2H, –NH ), 7.43–7.80 (m,
2
+
up in ethyl acetate, washed with sat. NaHCO solution, brine, 9H, Ar-H); MS m/z 453.97 [M+H] . Using this amide, the title
3
1
dried (Na SO ), concentrated and purified by silica gel column compound was obtained as a pale yellow oil (18%) H-NMR
2
4
chromatography (1% methanol in dichloromethane) to afford (300MHz, CDCl ) δ: 1.46 (d, J=7.1Hz, 3H, –CH ), 2.96–3.0
3
3
the target compounds 26–30.
(m, 4H, –CH S CH –), 4.00 (q, J=6.8Hz, 1H, –CH–),
2 2 2
(S)-2-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-(2-(6-methoxy- 4.37 (t, J=6.2Hz, 2H, –CO CH –), 4.68 (t, J=6.4Hz, 2H,
2
2
13
naphthalen-2-yl)propanoyl)carbamate (26) The interme- –CH ONO ), 7.47–4.77 (m, 9H, Ar-H); C-NMR (75MHz,
2
2
diate naproxen amide was obtained as a white solid (100%). CDCl ) δ: 18.40 (–CH ), 34.35 (–SCH –), 36.30 (–SCH –),
3
3
2
2
1
H-NMR (300MHz, CD OD) δ: 1.53 (d, J=6.9Hz, 3H, 45.05 (–CH–), 63.13 (–CO CH –), 70.09 (–CH ONO ),
CHCH ), 3.68 (q, J=7.2Hz, 1H, –CHCH ), 7.0–7.16 (m, 2H, 127.84 (Ar-C), 128.28 (Ar-C), 128.77 (Ar-C), 129.05 (Ar-
3
2
2
2
2
–
3
3

478
Vol. 60, No. 4
C), 129.60 (Ar-C), 131.29 (Ar-C), 132.12 (Ar-C), 136.84
General Procedure for the Synthesis of NO-NSAIDs
(
1
Ar-C), 137.62 (Ar-C), 139.84 (Ar-C), 150.26 (–NHCO –), (31–32) with Amide Linkage To an ice-cold solution of 38a
2
−1
73.27 (CH CHCO–), 195.90 (ArCO–); IR (NaCl) cm : (7mmol) in dichloromethane (10mL) was added trifluoroacetic
3
3
281 (N–H), 2977 (C–H), 1770 (C=O), 1634 (Ar-C=C), 1279 acid (TFA, 28mmol) and stirred for 1h. Excess TFA and sol-
+
(–NO ), 1199 (C–O); HR-MS-ESI (m/z) [M+Na] Calcd for vent was distilled off and the residue was dried in vacuum to
2
C H N NaO S : 501.0761; Found: 501.0770 (mass accuracy: afford the TFA salt 38b. Separately, to an ice-cold solution of
21
22
2
7 2
−
1.8ppm); Anal. Calcd for C H N O S : C 52.71, H 4.63, N the appropriate NSAID (7mmol) in dichloromethane (10mL)
21 22 2 7 2
5
.85, S 13.40; Found: C 52.62, H 4.63, N 6.15, S 13.73.
was added oxalyl chloride (9mmol), followed by 2 drops of
2
-((2-(Nitrooxy)ethyl)disulfanyl)ethyl(2-(4-isobutylphe- DMF and stirred for 1.5h. The intermediate NSAID acid chlo-
nyl)propanoyl)carbamate (29) The intermediate ibupro- ride was concentrated, dried and dissolved in dichloromethane
1
fen amide was obtained as a white solid (100%). H-NMR (10mL). A solution of the above TFA salt (38b) in dichloro-
(
300MHz, CD OD) δ: 0.88 (d, J=6.3Hz, 6H, –CH(CH ) ), methane (10mL) was added at 0°C to a solution of the NSAID
3
3
2
1
–
.41 (d, J=7.2Hz, 3H, –CHCH3), 1.78–1.87 (m, 1H, acid chloride, followed by drop-wise addition of triethylamine
CH(CH ) ), 2.41 (d, J=7.2Hz, 2H, –ArCH –), 3.62 (q, (21mmol) and stirred for 2h. The reaction mixture was
3
2
2
J=7.2Hz, 1H, –CHCH ), 7.00 (d, J=8.1Hz, 2H, Ar-H), 7.24 washed with water, brine, dried (Na SO ), concentrated and
3
2
4
13
(
(
d, J=7.8Hz, 2H, Ar-H); C-NMR (75MHz, CDCl ) δ: 17.45 purified by silica gel column chromatography (10–30% ethyl
3
–CHCH ), 21.30 (–CH(CH ) ), 30.06 (–CH(CH ) ), 44.64 acetate in petroleum ether) to afford the target compounds 31
3 3 2 3 2
(
–CHCH ), 45.34 (–ArCH –), 126.77 (Ar-C), 128.87 (Ar-C), and 32.
3
2
129.46 (Ar-C), 138.98 (Ar-C), 140.11 (Ar-C), 178.82 (–CO–).
2-((2-((2-(Nitrooxy)ethyl)disulfanyl)ethyl)carbamoyl)-
Using this amide, the title compound was obtained as a yel- phenylacetate (31) The synthesis of this compound, along
1
low solid (32%) mp 62-64°C; H-NMR (300MHz, CDCl ) δ: with spectral and analytical data, has been described in our
3
8)
0
–
2
.89 (d, J=6.6Hz, 6H, –CH(CH ) ), 1.49 (d, J=7.2Hz, 3H, earlier publication.
CHCH ), 1.80–1.86 (m, 1H, –CH(CH ) ), 2.45 (d, J=6.9Hz,
H, –ArCH –), 2.92 (q, J=6.9Hz, 4H, –CH S CH –), 4.18 (q, ethyl)disulfanyl)ethylnitrate (32) The title compound
J=6.9Hz, 1H, –CHCH ), 4.36 (t, J=6.3Hz, 2H, –CO CH –), was obtained as a colorless gum (17%). H-NMR (300MHz,
3 2
(S)-2-((2-(2-(6-Methoxynaphthalen-2-yl)propanamido)-
3
3 2
2
2
2
2
1
3
2
2
4
.67 (t, J=6.9Hz, 2H, –CH ONO ), 7.11 (d, J=8.1Hz, 2H, Ar- CDCl ) δ: 1.63 (d, J=2.4Hz, 3H, –CHCH ), 2.76–2.83 (m,
2 2 3 3
13
H), 7.20 (d, J=8.1Hz, 2H, Ar-H); C-NMR (75MHz, CDCl3) 4H, –CH S CH –), 3.53 (q, J=3.6Hz, 2H), 3.93 (q, J=4.5Hz,
2
2
2
δ: 18.65 (–CHCH ), 22.37 (–CH(CH ) ), 30.15 (–CH(CH ) ), 1H, –CHCH ), 3.94 (s, 3H, –OCH ), 4.58 (t, J=3.9Hz, 2H,
3
3 2
3 2
3
3
3
4.79 (–SCH –), 36.83 (–SCH –), 45.01 (–CHCH ), 45.93 –CH ONO ), 5.7 (br s, 1H), 7.14–7.19 (m, 2H, Ar-H), 7.39–7.39
2 2 3 2 2
13
(–ArCH –), 63.52 (–CO CH –), 70.58 (–CH ONO ), 127.52 (m, 1H, Ar-H), 7.69–7.76 (m, 3H, Ar-H); C-NMR (75MHz,
2 2 2 2 2
(
Ar-C), 129.73 (Ar-C), 136.99 (Ar-C), 141.15 (Ar-C), 150.66 CDCl ) δ: 18.71 (–CHCH ), 34.89 (–SCH –), 38.14 (–SCH –),
3 3 2 2
−1
(–NHCO –), 173.00 (CH CHCO–); IR (KBr) cm : 3248 38.91 (–NHCH –), 47.41 (–CHCH ), 55.71 (–OCH ), 70.87
2
3
2
3
3
(
N–H), 2951 (C–H), 1752 (C=O), 1635 (Ar-C=C), 1279 (–CH ONO ), 105.98 (Ar-C), 119.65 (Ar-C), 126.62 (Ar-C),
2
2
+
(
–NO ), 1170 (C–O); HR-MS-ESI (m/z) [M+Na] Calcd for 128.01 (Ar-C), 129.34 (Ar-C), 129.55 (Ar-C), 134.15 (Ar-C),
2
C H N NaO S : 453.1124; Found: 453.1110 (mass accuracy: 136.51 (Ar-C), 158.18 (Ar-C), 174.93 (–CO–); IR (NaCl)
1
8
26
2
6 2
−
1
2
6
.43ppm); Anal. Calcd for C H N O S : C 50.21, H 6.09, N cm : 3299 (N–H), 2936 (C–H), 1645 (C=O), 1546 (Ar-
.51; Found: C 50.54, H 6.14, N 6.47.
18
26
2
6 2
+
C=C), 1278 (–NO ), 1216 (C–O); MS m/z 410.99 [M+H] ;
2
+
2
-((2-(Nitrooxy)ethyl)disulfanyl)ethyl-(2-(1-(4- HR-MS-ESI (m/z) [M+H] Calcd for C H N O S : 411.1043;
18
23
2
5 2
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetyl)- Found: 411.1031 (mass accuracy: 2.79ppm); Anal. Calcd for
carbamate (30) The intermediate indomethacin amide C H N O S : C 52.66, H 5.40, N 6.82; Found: C 52.20, H
18
22
2
5 2
was obtained as a white solid (100%), from which the 5.98, N 6.43.
title compound was obtained as a pale yellow solid (10%)
2-((2-(2-(2-Fluoro-[1,1′-biphenyl]-4-yl)propanamido)eth-
1
mp 122–124°C; H-NMR (300MHz, CDCl ) δ: 2.39 (s, 3H, yl)disulfanyl)ethylnitrate (33) To an ice-cold solution of
3
Ar-CH ), 2.98 (d, J=6.5Hz, 2H, –SCH –), 3.00 (d, J=6.5Hz, 38a (3.00g, 10.24mmol) in dichloromethane (50mL) was add-
3
2
2
4
–
2
2
H, –SCH –), 3.84 (s, 3H, –OCH ), 4.00 (s, 2H, Ar-CH –), ed trifluoroacetic acid (3.00mL, 40.96mmol) and stirred for
2
3
2
.47 (t, J=6.5Hz, 2H, –CO CH –), 4.71 (t, J=6.5Hz, 2H, 1h. Excess TFA and solvent was distilled off and the residue
2
2
CH ONO ), 6.70 (dd, J=2.0, 9.0Hz, 1H, Ar-H), 6.89 (m, was dried in vacuum to afford the TFA salt 38b. Separately,
2
2
H, Ar-H), 7.49 (d, J=8.5Hz, 2H, Ar-H), 7.69 (d, J=8.0Hz, a solution of flurbiprofen (2.50g, 10.24mmol) and N,N′-
13
H, Ar-H), 7.73 (br s, 1H, –NH–); C-NMR (75MHz, carbonyldiimidazole (3.31g, 20.48mmol) in THF (35mL) was
CDCl ) δ: 13.94 (Ar-CH ), 32.71 (Ar-CH –), 35.39 (–SCH –), stirred for 4h, then treated with the above TFA salt 38b and
3
3
2
2
3
7.33 (–SCH –), 56.25 (–OCH ), 64.31 (–CO CH –), 71.11 triethylamine (4.30mL, 30.72mmol). This mixture was stirred
2 3 2 2
(
–CH ONO ), 101.64 (Ar-C), 112.04 (Ar-C), 112.33 (Ar-C), for 16h and concentrated under vacuum. The residue was
2 2
1
15.55 (Ar-C), 129.66 (Ar-C), 131.01 (Ar-C), 131.38 (Ar-C), dissolved in ethyl acetate, washed with sat. NaHCO solution
3
1
31.71 (Ar-C), 134.23 (Ar-C), 137.14 (Ar-C), 139.91 (Ar-C), and brine, dried (Na SO ), concentrated and purified by silica
2 4
1
51.73 (Ar-C), 156.65 (–N–CO–), 168.79 (–NHCO –), 170.83 gel column chromatography (25% dichloromethane in petro-
2
−1
(ArCH CO–); IR (KBr) cm : 3241 (N–H), 2959 (C–H), leum ether) to afford 33 as a white solid (1.50g, 34%). mp
2
1
1
756 (C=O), 1690 (Ar-C=C), 1281 (–NO ), 1217 (C–O); MS 48–50°C; H-NMR (300MHz, CDCl ) δ: 1.55 (d, J=6.9Hz,
2
3
+
+
m/z 582.1 [M+H] ; HR-MS-ESI (m/z) [M+Na] Calcd for 3H, –CH ), 2.82 (t, J=6.3Hz, 2H, –SCH –), 2.92 (t, J=6.9Hz,
3
2
C H ClN NaO S : 604.0586; Found: 604.0599 (mass accu- 2H, –SCH –), 3.58 (m, 2H, –NHCH –), 4.67 (t, J=6.6Hz,
2
4
24
3
8
2
2
2
racy: 2.15ppm); Anal. Calcd for C H ClN NaO S : C 47.64, 2H, –CH ONO ), 5.8 (br s, 1H, –NH–), 7.14 (t, J=6.9Hz, 2H,
2
4
24
3
8
2
2
2
13
H 4.00, N 6.95; Found: C 47.00, H 3.88, N 7.07.
Ar-H), 7.35–7.55 (m, 6H, Ar-H); C-NMR (75MHz, CDCl3)

April 2012
479
δ: 17.94 (–CH ), 34.17 (–SCH –), 37.31 (–SCH –), 37.98 ment cylinder of plethysmometer (Letika, Model No. LE7500)
3
2
2
(
–NHCH –), 46.13 (–CH–), 70.06 (–CH ONO ), 114.66 (Ar-C), and basal paw volume was recorded. Aspirin (150mg/kg),
2
2
2
1
1
14.97 (Ar-C), 123.14 (Ar-C), 127.28 (Ar-C), 128.00 (Ar-C), diclofenac, naproxen, flurbiprofen (each at 10mg/kg) and
28.41 (Ar-C), 130.64 (Ar-C), 134.80 (Ar-C), 141.90 (Ar-C), ketoprofen (15mg/kg) or equimolar doses of respective NO-
1
42.01 (Ar-C), 159.22 (Ar-C), 157.12 (Ar-C), 173.17 (–CO–); NSAIDs (exception: NO-flurbiprofen at 20mg/kg equivalent
−1
IR (KBr) cm : 3267 (N–H), 2969 (C–H), 1752 (C=O), 1627 (eq.) and NO-ketoprofen at 30mg/kgeq.) were administered
Ar-C=C), 1281 (–NO ), 1231 (C–O); HR-MS-ESI (m/z) orally to overnight fasted rats. One hour after compound ad-
(
2
+
[
M+H] Calcd for C H FN O S : 425.1000; Found: 425.0994 ministration, carrageenan (100μL, 1% w/v) suspension was in-
19 22 2 4 2
(mass accuracy: 1.41ppm); Anal. Calcd for C H FN O S : C jected intra-plantarly in the right hind paw. Paw volume were
19 21 2 4 2
53.76, H 4.99, N 6.60, S 15.11; Found: C 53.89, H 5.18, N 6.03, measured at 4 and 6h post injection. Change in paw volume
S 15.35.
was obtained by subtracting basal paw volume from the post-
Biological Methods. Animals For all our animal stud- injection paw volumes at specified time points and the percent
ies, male Sprague-Dawley (SD) and Wistar rats (175–325g) inhibition of paw edema was calculated.
were procured from the animal facility, Piramal Life Sciences
Analgesic Assay Analgesic activity was evaluated in
Limited, Mumbai, India. The animals were fed with normal carrageenan-induced hind paw model of inflammatory pain
pellet diet and water ad libitum, and maintained in standard where threshold response to a thermal noxious stimulus was
environmental conditions (temperature 22± 2°C, humidity determined using Hargreaves’s test.4 Briefly, SD rats were
0)
6
0± 5%, and 12h light/12h dark cycle). Rats were housed in tested for paw withdrawal latency in response to noxious
a group of three per cage and acclimatized to the laboratory thermal stimuli using a Plantar Analgesia Meter (IITC Life
condition one week prior to experiments. All the study proto- Sciences Inc., CA, U.S.A.). Animals were placed in Plexiglas
cols were approved by Institutional Animal Ethics Committee cages with a glass floor. After 5min of habituation, a radiant
(
IAEC), and performed in accordance with the guidelines of heat light source was focused onto the plantar surface of right
Committee for the Purpose of Control and Supervision on hind paw until the animal lifted the paw away from the heat
Experiments on Animals (CPCSEA), Government of India. source. The basal paw withdrawal latency (in seconds) was
Materials Carrageenan (λ-Carrageenan, Type IV) was recorded for each animal. A cut-off latency of 20s was used
purchased from Sigma Chemicals, St. Louis, MO, U.S.A. to avoid tissue damage. Aspirin (150mg/kg) or NO-aspirin
Dose Preparation NSAIDs and equivalent doses of NO- compound 25 (150mg/kg eq.) was administered orally. One
NSAIDs were weighed and solution formulations were pre- hour after compound administration, carrageenan (100μL, 1%
pared in 100% polyethylene glycol (PEG400). These solution w/v) suspension was injected intra-plantarly in the right hind
formulations were used for the pharmacokinetics and pharm- paw. The paw withdrawal latency of the ipsilateral paw was
codynamic studies. Since PEG400 is reported to possess measured at various time points (i.e., 1, 2, 4, 6, 8, 10h) after
38)
gastro-protective effect, suspension formulations of drugs carrageenan injection.
were prepared in 0.5% (w/v) carboxymethylcellulose (CMC)
Acute Gastric Ulcerogenesis Study The acute ulcero-
containing 1% (v/v) Tween80 for gastric ulcerogenic studies genic potential of compounds was evaluated in SD rats ac-
41)
and for estimation of gastric mucosal PGE levels. The drug cording to the reported procedure. Vehicle (1mL/kg), aspirin
2
formulations were prepared freshly just before dosing and (150mg/kg), diclofenac, naproxen, flurbiprofen and ketoprofen
were vortexed thoroughly before administration to each ani- (each at 100mg/kg) or equimolar doses of respective NO-
mal. Carrageenan (1%) was suspended in physiological saline NSAIDs were administered orally to overnight fasted rats.
and a uniform solution was achieved by warming (58°C) with Three hours (aspirin groups) or five hours (for other NSAID
constant agitation for 1h.
groups) later, the rats were euthanized and their stomachs
Biological Studies. Oral Bioavailability Study The were removed and opened along the greater curvature. The
pharmacokinetics studies of NO-NSAIDs were carried out in stomach were rinsed with physiological saline and then fixed
SD rats. Aspirin (150mg/kg), sulindac (100mg/kg), diclofenac, with 2% formalin saline for 5min, spread and pinned on
naproxen, flurbiprofen, ketoprofen and indomethacin (each at paper. The images were captured using a stereomicroscope
10mg/kg) or equimolar doses of respective NO-NSAIDs were (at 0.5× magnification) attached to a digital camera (Stemi
administered orally to overnight fasted rats. Blood was col- 2000, Zeiss, Germany). An observer unaware of the treatment
lected in heparinized micro-centrifuge tubes from retro-orbit- regimen quantified the hemorrhagic ulcer lesions from the im-
al plexus under isoflurane anesthesia (Animal Anesthesia Unit, ages using Image Pro Plus 5.1 software (Media Cybernetics,
Surgivet Inc. Waukesha, WI, U.S.A.) at different time points Bethesda, MD, U.S.A.). The total area of ulcer lesions for each
2
and plasma was obtained by centrifugation at 10000rpm for stomach were measured and expressed as mm and the aver-
1
0min at 4°C. The plasma samples were processed with ace- age total lesion area for each treatment group was calculated.
tonitrile and were further centrifuged at 10000rpm for 10min Estimation of Gastric Mucosal PGE2 Levels Vehicle,
at 4°C to remove plasma proteins. The supernatant samples aspirin (150mg/kg) or compound 25 (150mg/kgeq.) was
obtained were analyzed by HPLC (Waters 2695 Separations administered orally to the overnight fasted SD rats. Three
Module with 2996 Photodiode Array Detector; Waters Corp., hours later, rats were euthanized, stomachs were removed
Milford, MA, U.S.A.).
and mucosa (ca. 100mg) was obtained by scraping the gastric
Anti-inflammatory Assay To assess the anti-inflammato- tissue with the help of ice-cold glass slide. The mucosa was
ry activity of compounds, the rat paw edema model was used, weighed and transferred to a tube containing 100% ethanol
39)
as previously reported. Briefly, right hind paw of SD rats and 0.1M indomethacin. The samples were then homogenized
was marked with an indelible ink marker at the lateral malleo- and centrifuged for 10min at 12000rpm at 4°C. The superna-
lus of the ankle and dipped up to the mark in water displace- tant was collected and used for estimation of PGE by EIA kit
2

4
80
Vol. 60, No. 4
42)
(Cayman Chemical Co., Ann Arbor, MI, U.S.A.).
ture was centrifuged at 13000rpm for 15min at 4°C, and the
Estimation of Plasma NOx Plasma NOx (nitrate/nitrite) supernatant analyzed by HPLC. The amounts (area percent-
was estimated as per the Griess method described previ- ages) of the remaining intact prodrug (if any) and the released
42)
ously, with minor modifications. Briefly, aspirin (150mg/ metabolite(s) were estimated by HPLC.
kg), diclofenac, naproxen, flurbiprofen (each drug at 10mg/
In Vitro Studies on DTT-Mediated NO Release from
kg) and ketoprofen (15mg/kg) or equimolar doses of respec- NO-NSAIDs 11 and Naproxcinod (I). Preparation of
tive NO-NSAIDs (exceptions: NO-diclofenac compound 10 Solutions Dithiothreitol: Five milligrams of DTT was dis-
at 5mg/kgeq and NO-ketoprofen compound 28 at 30mg/ solved in 2mL of acetonitrile/water (4:1).
kgeq) were administered orally to overnight fasted Wister
rats and blood was collected in heparinized micro-centrifuge pounds were prepared in acetonitrile/water (4:1).
tubes from the retro-orbital plexus at various time points Procedure: One hundred microliters of DTT solution was
Samples: One hundred micromolars solutions of test com-
under light isoflurane anesthesia (Animal Anesthesia Unit, added to 1mL of 100μM test compound solution and the pH of
Surgivet Inc. Waukesha, WI, U.S.A.). Samples were centri- the mixture was adjusted to ca. 7.5 by adding 0.1N NaHCO3.
fuged at 10000rpm for 10min at 4°C to obtain plasma that The mixture was stirred at RT under nitrogen and the DTT
was passed through Multi-Screen filter plate with Ultracel-10 mediated decomposition of test compound was monitored
membrane (Millipore Corp. Billerica, MA, U.S.A.) for further by HPLC. After 2h, about 2/3 of compound 11 decomposed
purification. The filtrate was collected and stored at −20°C. (Note: Naproxcinod (I) remained stable). At this stage, ad-
Nitrate/nitrite, an indirect measure for plasma NO release, ditional 50mL of DTT solution was added followed by 0.1N
was estimated in aliquots of the samples by the Griess method NaHCO to adjust pH to ca. 7.5 and the mixture was stirred
3
using commercially available nitrate/nitrite assay kit (Sigma at RT under nitrogen for additional 1h. HPLC analysis of
Chemicals, St. Louis, MO, U.S.A.).
the mixture at this stage indicated >95% decomposition of
Statistical Analysis Statistical analyses of data consist- compound 11 (Note: Naproxcinod (I) remained stable). During
ing of three or more groups were performed using one-way this reaction period (ca. 3h), while naproxcinod (I) remained
analysis of variance (one-way ANOVA) followed by post-hoc stable, compound 11 (t =9.37min) was converted into another
R
Dunnett’s multiple comparison test, and values of p<0.05 unidentified compound (t =10.38min). The resulting mixture
R
were considered as significant. For data consisting of two was freeze dried. HPLC analysis of the freeze dried residue
groups, analyses were performed using student’s t-test and indicated mainly two unidentified peaks at tR 9.85min (52%)
values of p<0.05 were considered as significant. All analy- and tR 10.33min (48%) with no trace of compound 11 peak
ses were carried out using GraphPad Prism version 4.00 for (tR 9.37min). Freeze dried naproxcinod containing mixture
Windows (GraphPad Software, San Diego, CA, U.S.A.). For showed only the naproxcinod peak with no signs of DTT-
data consisting of only pooled/mean values, the statistical mediated decomposition. The residue obtained after freeze
analysis could not be performed.
drying was dissolved in 1mL of PBS buffer and the liberated
In Vitro Metabolic Stability Studies in Biological Fluids. NO (i.e., nitrate/nitrite levels) was estimated by Griess method
Preparation of Biological Fluids Simulated Gastric Fluid as described above. Blank experiments (i.e., without adding
(
SGF): SGF was prepared according to a procedure described the test compounds) were simultaneously performed as con-
in Test Solution-USP. Thus, 0.20g of sodium chloride and trol.
.32g of purified pepsin (Sigma), which is derived from por- Analysis of Metabolites: This was performed by using
0
cine stomach mucosa with an activity of 800 to 2500 units per HPLC instrument (Waters alliance), pump 2695, and PDA
mg of protein, were dissolved in 0.70mL of hydrochloric acid detector 2996 with the following chromatographic param-
and sufficient water to make 100mL. This test solution has a eters: wavelength-210nm; column, Waters X-Terra RP-18,
pH of about 1.2 and it was utilized in further in vitro studies.
150×3.9mm, 5μm; injection volume, 25μL; run time, 13min.
Rat Plasma: Rat plasma was harvested from in-house rats. Mode of operation was linear gradient with mobile phase A:
Fresh blood was collected from the male rat using a retro- Acetonitrile and B: 0.1% TFA in water (filtered and degassed).
orbital bleeding method into a tube containing heparin (50IU/ Flow rate was 1.0mL/min at 25°C.
mL blood). After collection of blood, plasma was separated
Analysis of Naproxen: Compounds were analyzed at room
from the blood by centrifugation at 9000rpm for 5min at 4°C. temperature on a Waters X-Terra RP 18 column (150×3.5mm,
The supernatant plasma was separated and utilized for the in particle size 5μm; Waters Corp., Milford, MA, U.S.A.) and
vitro experiments.
eluted with a gradient of acetonitrile (A) and 0.1% v/v TFA
Human Plasma: Human plasma was similarly obtained in water (B) at a flow rate of 1mL/min as per the following
by processing the blood taken from healthy human male schedule: 0–10min, 20%A, 10–14min, 100%A, 14–18min,
volunteers (age group 25–35 years) who have not consumed 20%A. UV absorbance was monitored at PDA 3D Max plot
any NSAIDS one week prior to the collection of blood. This wavelength.
plasma was utilized for the in vitro experiments.
Analysis of Flurbiprofen: Compounds were analyzed
In Vitro Metabolic Stability of Aspirin (1), Ester Prodrugs at room temperature on a Waters X-Terra RP 18 column
and 12, and Imide Prodrugs 25 and 27 in SGF, Human (150×3.5mm, particle size 5μm; Waters Corp., Milford, MA,
9
Plasma and Rat Plasma: The test compound solution in ace- U.S.A.) and eluted with a gradient of acetonitrile (A) and 0.1%
tonitrile (10μL of 100μM solution) was dissolved in SGF or v/v TFA (B) in water at a flow rate of 1mL/min as per the
Human Plasma or Rat Plasma (990μL). The resulting reaction following schedule: 0–10min, 30%A, 10–13min, 100%A,
mixtures were incubated at 37°C. At specified time intervals, 13–18min, 30%A. UV absorbance was monitored at PDA 3D
aliquots (60μL) were withdrawn and added to acetonitrile Max plot wavelength.
(200μL) and mixed well by vortexing for 2min. The mix-
Analysis of Ketoprofen, Ibuprofen and Indomethacin:

April 2012
481
1
5) Kosonen O., Kankaanranta H., Malo-Ranta U., Ristimäki A.,
Compounds were analyzed at room temperature on a Waters
X-Terra RP 18 column (150×3.5mm, particle size 5μm;
Waters Corp., Milford, MA, U.S.A.) and eluted with a gradi-
ent of acetonitrile (A) and 0.1% v/v TFA (B) in water at a
flow rate of 1mL/min as per the following schedule: 0–4min,
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1
was monitored at PDA 3D Max plot wavelength.
3
Analysis of Sulindac: Compounds were separated at room
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1
K., McKnight W., del Soldato P., Morelli A., Fiorucci S.,
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sented as Chart S1 in the supporting information.
(
B) in water at a flow rate of 1mL/min as per the follow-
ing schedule: 0–4min, 15%A, 4–14min, 80%A, 14–18min,
5%A. UV absorbance was monitored at PDA 3D Max plot
2
1
2
2
wavelength.
Analysis of Aspirin and Diclofenac: Analytical methods of
8)
aspirin and diclofenac were carried out as reported earlier.
Acknowledgments We sincerely thank Dr.
H. 24) Speziale A. J., Smith L. R., J. Org. Chem., 27, 3742–3743 (1962).
Sivaramakrishnan and Dr. Meenakshi Sivakumar for the 25) Hill A. J., Degnan W. M., J. Am. Chem. Soc., 62, 1595–1596 (1940).
bulk synthesis of NO-diclofenac and NO-aspirin for pre- 26) Graphical representation of oral bioavailability, anti-inflammatory
activity and NO release data for NO-NSAIDs and their correspond-
ing NSAIDs or control are presented as Fig. S1 in the supporting
information.
clinical studies. Our special thanks are also due to Analytical
Department for providing spectral and analytical data. We
also thank our patents department for their timely professional
help.
2
7) Graphical representation of oral bioavailability, anti-inflammatory
activity and NO release data for naproxcinod (I) and their corre-
sponding NSAIDs or control are presented as Fig. S1 in the sup-
porting information.
8) Representative images of rat stomachs showing gastric lesion induc-
tion/sparing effects following oral administration of NSAIDs or
NO-NSAIDs equimolar to NSAIDs are presented as Fig. S3 in the
supporting information.
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Med. Chem., 54, 1191–1201 (2011).
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