NO-NSAIDs: Gastric-sparing nitric oxide-releasable prodrugs of non-steroidal anti-inflammatory drugs

Article abstract of DOI:10.1016/j.bmcl.2009.07.142

Recently, a new class of nitric-oxide-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) is being studied as 'Safe NSAIDs' because of their gastric-sparing properties. As an extension of our novel disulfide linker technology, we have designed, sy

Full text of DOI:10.1016/j.bmcl.2009.07.142

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5297–5301
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
NO-NSAIDs: Gastric-sparing nitric oxide-releasable prodrugs
of non-steroidal anti-inflammatory drugs
Kumar V. S. Nemmani a, Sunil V. Mali b, Namdev Borhade b, Asif R. Pathan a, Manoj Karwa a,
Venu Pamidiboina a, S. P. Senthilkumar b, Machhindra Gund b, Arun K. Jain b, Naveen K. Mangu b,
Nauzer P. Dubash b, Dattatraya C. Desai b, Somesh Sharma a, Apparao Satyam b,
*
a Pharmacology Department, Piramal Life Sciences Limited, Nirlon Complex, Goregaon East, Mumbai 400 063, India
b R&D Special Projects Department, Medicinal Chemistry Division, Piramal Life Sciences Limited, Nirlon Complex, Goregaon East, Mumbai 400 063, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently, a new class of nitric-oxide-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) is
being studied as ‘Safe NSAIDs’ because of their gastric-sparing properties. As an extension of our novel
disulfide linker technology, we have designed, synthesized and evaluated novel NO-releasing NSAID pro-
drugs such as NO-Aspirin (1b–d) and NO-Diclofenac (2b–c). Although the amide-containing derivative 1d
did not show any bioavailability, the remaining compounds have shown fair to excellent pharmacoki-
netic, anti-inflammatory and gastric-sparing properties. Among them, however, the NO-Diclofenac (2b)
has shown the most promising pharmacokinetic, anti-inflammatory and NO-releasing properties and
protected rats from NSAID-induced gastric damage which could be attributable to the beneficial effects
of NO released from these prodrugs.
Received 3 June 2009
Revised 29 July 2009
Accepted 30 July 2009
Available online 3 August 2009
Keywords:
NO-NSAIDs
Nitric oxide donors
Prodrugs of NSAIDs
Gastric-sparing NSAIDs
NO-Aspirin
Ó 2009 Elsevier Ltd. All rights reserved.
NO-Diclofenac
Aspirin and diclofenac are among the most widely used non-
steroidal anti-inflammatory drugs (NSAIDs) for the treatment of
pain, fever, and inflammatory diseases such as arthritis.1 Because
of its anti-thrombotic properties, aspirin is now indicated for pa-
tients with stable angina, unstable angina, acute myocardial infarc-
tion, transient cerebral ischemia, thrombotic stroke, and peripheral
arterial disease. The use of aspirin for the prevention of arterial
thrombosis is likely to increase further as public awareness of this
wonder drug grows. It has been reported that more than 80 billion
aspirin tablets are consumed annually in the USA, and more than
37% of the individuals taking aspirin do so to prevent blood clots.2
Similarly, diclofenac or its sodium salt (VoltarenÒ) is the most
widely prescribed NSAID for the treatment of osteoarthritis, rheu-
matoid arthritis and ankylosing spondylitis.1 However, chronic use
of these NSAIDs can induce severe gastrointestinal (GI) toxicity
such as bleeding, ulceration and perforation, and also cardiorenal
complications which greatly limit their therapeutic usefulness.
According to an investigative report on ‘Toxic and Deadly NSAIDs’,3
conservative estimates of NSAID-related GI complications account
for more than 107,000 hospitalizations and 16,500 deaths annually
among arthritis patients in the United States alone. Therefore,
there exists an unmet medical need for ‘Safe NSAIDs’ that do not
cause adverse effects on GI tract and cardiorenal systems. As a po-
tential solution to this problem, a new class of nitric oxide (NO)-
releasable prodrugs of non-steroidal anti-inflammatory drugs
(NO-NSAIDs), also sometimes known as CINODs (cyclooxygenase
inhibitory NO donors), has been developed recently by the addition
of NO-releasing moiety to an existing NSAID. The rationale behind
this strategy is to harness the beneficial effects of NO that is re-
leased from these NO-NSAIDs. NO is now widely recognized as a
critical mediator of GI mucosal defense and greatly suppresses
NSAID-induced COX-1 inhibition related adverse events such as
suppression of prostanoid synthesis, reduction in mucosal blood
flow and over-expression of inflammatory mediators such as plas-
ma tumor necrosis factor alfa (TNF-a) and the leukocyte–endothe-
lial cell adherence.4 As anticipated, NO-NSAIDs such as NO-Aspirin
(NCX 4016), NO-Diclofenac (NITROFENAC),5,6 and NO-Naproxen
(NAPROXCINOD/HCT 3012/AZD 3582) (Fig. 1) have been shown
either pre-clinically or clinically to cause minimal or insignificant
gastric damage with comparable analgesic and anti-inflammatory
activity to their respective parent NSAIDs.7 These studies have pro-
vided a ‘Proof of concept’8 that is in agreement with their intended
design and application. Interestingly, naproxcinod (AZD 3582/HCT
3012), whose Phase III studies were completed recently with a very
favorable outcome, has been tipped by the media to be one of the
ten potential ‘Future Blockbusters’.9
With an intent to apply our disulfide-based linker and prodrug
technology10 to this emerging area of NO-NSAIDs, we have
designed and synthesized novel NO-releasing prodrugs of
* Corresponding author. Tel.: +91 93238 02658; fax: +91 22 3081 8334.
E-mail address: apparaosvgk@hotmail.com (A. Satyam).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.142

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K. V. S. Nemmani et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5297–5301
O
OAc
NSAID
O
S
NO2
O
Cl
S
O
NO2
O
O
RS-
NO-NSAID Prodrug
NCX 4016
NH
O
NO2
Cl
RSH At biological pH
O
O
CO2H
S
O
NITROFENAC
RS
NSAID
Released Drug
RSH
NO2
S
+
+
RS-
CH3
O
M2
RSH
M1
NO2
O
O
RSSR
RSSR
H3CO
H2O
O
O
N
NAPROXCINOD
H2O
O
O
N
S
H+
OH
Figure 1. Structures of some known NO-NSAIDs.
O
S
+
S
SR
M4
Transient Intermediate M3
anti-inflammatory agents, 1b–c (NO-Aspirin) and 2b–c (NO-Dic-
lofenac) by utilizing widely used anti-inflammatory drugs aspirin
(1a) and diclofenac (2a), respectively (Fig. 2), and evaluated their
oral bioavailability, anti-inflammatory activity, NO-releasing pro-
file and gastric-sparing properties in rats. We now report these
promising preliminary results in this Letter.
OH
Pathway 1
RSSR
Pathway 2
RSSR
S
RSH
RSH
SR
H2O
M5
OH
S
-
NO3
+
+
NO
OH
The most important feature in our design of these NO-NSAIDs is
the presence of an appropriately positioned disulfide linkage that is
at b-position to both the carboxyl ester group of NSAID and NO-
releasing nitrate ester group (Fig. 2) and their design was partly
based on previously proposed sulfhydryl-dependent metabolism
of organic nitrates such as glyceryl trinitrate (GTN) to NO.11,12
Although drug release from ester bearing prodrugs can very likely
occur via enzymatic hydrolysis, we have logically proposed a plau-
sible sulfhydryl-assisted cleavage of these novel prodrugs as
shown in Scheme 1. We thus suggest that a strategically placed
disulfide bond in our NO-NSAID prodrug can get reduced by an
intracellular sulfhydryl-containing species such as cysteine or glu-
tathione via the proposed mechanism of cleavage to release free
drug and intermediate metabolites, M1 and M2. The presumed
toxic episulfide (M1) can rapidly get metabolized further to a be-
nign glutathione or cysteine conjugate M5 as shown in the Scheme.
The other presumed metabolite M2 can undergo further break-
down in the presence of cysteine or glutathione as shown in the
scheme which is based on the mechanism proposed earlier for such
‘SS-nitrates’.13 The presumed transient intermediate M3 can break-
down further under biological conditions in two probable path-
ways as shown in the Scheme. While pathway 2 can lead to direct
generation of NO and a benign glutathione or cysteine conjugate
M6, pathway 1 may lead to the generation of a benign nitrate
group and a reactive episulfide M1 which can get converted further
S
Nitrate
Released
Nitric Oxide
M1
SR
Bacterial
nitrate
reductase
M6
H+
-
NO2
Nitrite
Wherein, RSH = Cellular Cysteine or Glutathione
Scheme 1. Plausible mechanism of drug and NO release.
to a benign glutathione or cysteine conjugate M5 as discussed
above. It was also reported that the nitrate ion thus generated
can get reduced by oral bacterial nitrate reductase to nitrite ion
which in turn can rapidly get reduced to NO via nitrous acid in
the acidic environment of the stomach.14,15
Synthesis of NO-NSAIDs (1b–c) and (2b–c) was performed as
depicted in Scheme 2.16 Syntheses of NO-Aspirin (1b) and NO-
Diclofenac (2b) containing ester linkages were carried out by
Method A and Method B, respectively. In Method A, selective
mono-bromination of 2-hydroxyethyl disulfide (3) using carbon
tetrabromide, triphenylphosphine and triethylamine afforded
2-((2-bromoethyl)disulfanyl)ethanol (4) in 52% yield. The mono-
bromide 4 was treated with silver nitrate in acetonitrile to afford
2-((2-hydroxyethyl)disulfanyl)ethyl nitrate (5) in ꢀ59% yield. The
nitrate ester 5 was acylated with freshly prepared aspirin acid
chloride in the presence of triethylamine to afford 2-((2-(nitro-
oxy)ethyl)disulfanyl)ethyl 2-acetoxybenzoate (NO-Aspirin, 1b) in
64% yield. In Method B, the intermediate nitrooxy alcohol 5 was
reacted with diclofenac in the presence of DCC and DMAP in 1:4
DMF/DCM to afford 2-((2-(nitrooxy)ethyl)disulfanyl)ethyl 2-(2-
(2,6-dichlorophenylamino)phenyl)acetate (NO-Diclofenac, 2b) in
50% yield. For the synthesis of diester-containing NO-NSAIDs (1c
and 2c), the intermediate nitrooxy alcohol 5 was first acylated with
chloroacetyl chloride in the presence of triethylamine to afford
2-((2-(nitrooxy)ethyl)disulfanyl)ethyl 2-chloroacetate (6) which
was further reacted with freshly prepared aspirin cesium salt in
THF or diclofenac sodium salt in DMF to afford the double ester-
containing 2-(2-((2-(nitrooxy)ethyl)disulfanyl)ethoxy)-2-oxoethyl
2-acetoxybenzoate (NO-Aspirin, 1c) or 2-(2-((2-(nitrooxy)ethyl)dis-
ulfanyl)ethoxy)-2-oxoethyl 2-(2-(2,6-dichlorophenylamino)phenyl)-
acetate (NO-Diclofenac, 2c), respectively.
O
O
O
H3C
O
Cl
R
NH
R
Cl
2a-c
1a-d
1a, R = OH (Aspirin)
2a, R = OH (Diclofenac)
S
O
1b & 2b, R =
1c & 2c, R =
O
S
NO2
NO2
O
S
O
O
S
O
1d , R =
NO2
HN
S
S
O
Similarly, synthesis of amide linkage-containing NO-Aspirin
(1d) was also performed by using appropriate reagents and reac-
tion conditions.16
Figure 2. Structures of aspirin (1a), diclofenac (2a), NO-Aspirin prodrugs (1b–d)
and NO-Diclofenac prodrugs (2b–c).

K. V. S. Nemmani et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5297–5301
5299
a
OAc
S
OH
S
Br
O
HO
S
HO
S
NO2
S
O
4
3
O
S
O
O
1c
b
S
O
HO
S
5
NO2
f
[Method A]
e
OAc
O
c
O
S
O
O
S
Cl
S
O
NO2
O
S
d
NO2
[Method B]
1b (P 1538)
6
Cl
Cl
NH
g
NH
O
NO2
Cl
Cl
O
O
S
O
S
S
O
S
O
NO2
O
O
2c
2b (P 2026)
Scheme 2. Reagents and conditions: (a) CBr4, PPh3, CH2Cl2, 30 min at 0 °C, then 10 h at 25 °C, 52%; (b) Silver nitrate, acetonitrile, 25 °C, 3 h, 59%; (c) aspirin acid chloride, Et3N,
benzene, 25 °C, 5 h (mixed reagents at 0 °C), 64%; (d) diclofenac, DCC, DMAP,1:4 DMF/CH2Cl2, 0–5 °C, 3 h, 50%; (e) chloroacetyl chloride, Et3N, CH2Cl2, 2 h, 49%; (f) aspirin
cesium, THF, 25 °C, 20 h, 35%; (g) diclofenac sodium, 1:3 DMF/THF, 24 h at 25 °C, then 8 h at 60 °C, 64%.
Biological studies on these novel NO-NSAIDs were carried out in
rats for establishing their bioavailability, anti-inflammatory activ-
ities, and gastric tolerance relative to their respective parent drugs
and the results of these experiments are presented in Table 1. The
data presented for aspirin is actually for plasma salicylate levels as
aspirin or its NO-Aspirin prodrugs quickly get hydrolyzed enzy-
matically in vivo to salicylic acid. We have not seen any intact
NO-Aspirin derivative in plasma even at initial time points of the
study. It is therefore obvious that none of the tested NO-Aspirin
compounds behaved like a true prodrug of aspirin, but they may
be termed as prodrugs of salicylic acid. Interestingly, only the ester
linkage-containing prodrug 1b showed appreciable bioavailability
when compared to that of aspirin. Although the double ester link-
age-containing prodrug 1c showed moderate bioavailability, the
amide linkage-containing compound 1d did not show even traces
of salicylate in plasma of the tested rats. It is therefore obvious that
the compound 1d did not act even as a prodrug of salicylic acid.
Interestingly, the bioavailability (AUC value) for the monoester-
containing NO-Diclofenac prodrug 2b is comparable to that of dic-
lofenac. The double ester-containing NO-Diclofenac prodrug (2c)
has also showed appreciable bioavailability. However, we have
not seen any of these intact NO-Diclofenac prodrugs in the plasma
at any time point and we therefore measured the plasma concen-
tration of the released diclofenac for calculation of AUC values of
NO-Diclofenac prodrugs (Table 1).
Although the NO-Aspirin prodrug 1b showed appreciable bio-
availability, it exhibited only moderate anti-inflammatory activity
when compared to that of aspirin at equimolar doses at all the
tested time points (Table 1).
As anticipated, the monoester-containing NO-Diclofenac pro-
drug 2b showed nearly equal anti-inflammatory activity to that
of diclofenac at equimolar doses at all the tested time points
(Fig. 3). However, the double ester-containing NO-Diclofenac pro-
drug 2c exhibited only moderate anti-inflammatory activity. These
results clearly establish that the extent of anti-inflammatory activ-
ity is proportional to the plasma concentration of diclofenac.
Based on their extent of bioavailability and/or anti-inflamma-
tory efficacy, we selected the NO-Aspirin prodrug 1b and NO-Dic-
lofenac prodrug 2b and studied their gastric tolerance compared to
their respective parent drugs in rats and the results of these exper-
iments are presented in Table 1 as well as in Figure 4.
While both the parent drugs caused significant gastric damage,
our NO-NSAIDs 1b and 2b at equimolar doses did not cause any GI
damage. This gastric-sparing effect could be attributable to the
beneficial actions of NO released from these promising NO-NSAIDs.
However, additional factors such as masking of free acid group as
an ester might be partly contributing to the gastric-sparing effect.
In order to experimentally determine the NO-releasing proper-
ties of these novel molecules, the most promising NO-Diclofenac
prodrug 2b was selected and studied in rats. The nitrate/nitrite
concentration of the plasma of tested rats was determined by using
Griess method.19 As shown in Figure 5, NO-Diclofenac 2b released
significant quantities of NO in a sustained fashion when compared
to that of control.
Table 1
Data for bioavailability, anti-inflammatory activity,17 and gastric tolerance18 studies
on NO-NSAIDs and their parent drugs
Compda
AUCd
(
lg/ml h)
Activityc (% inhibition)d
Gastric ulcer
aread (mm2)
4 h
6 h
Aspirinb
4051 312
1234 93
892 140
0
42 9*
20 12
Inactive
ND
31 8*
99 32
0
ND
ND
108 36
0
ND
1b (P1538)
1c (P1911)
1d (P1537)
Diclofenac
2b (P2026)
2c (P1912)
17
7
Inactive
ND
21 12
20 13
Inactive
65
60
49
2
5
2
55 9**
54 7**
34 10*
a
Aspirin (150 mg/kg) or NO-Aspirin (150 mg/kg equimolar to aspirin), diclofenac
(10 mg/kg) or NO-Diclofenac (10 mg/kg equimolar to diclofenac) was administered
orally to rats.
b
Based on this promising data, we selected the NO-Diclofenac
prodrug 2b and carried out additional in vivo pharmacology and
toxicology studies and those results will be described elsewhere.
This molecule is currently undergoing IND enabling preclinical tox-
icology studies.
For aspirin and its prodrugs, plasma salicylate levels were estimated to calcu-
late their bioavailability.
c
Activity is expressed as % inhibition of paw volume over control rats. ND = Not
determined.
d
All values are expressed as mean S.E.M. (n = 3–4 for pharmacokinetic study;
n = 5–8 for anti-inflammatory activity; n = 5 for gastric tolerance study).
*
P <0.05.
P <0.01 versus control.
Interestingly, the non-bioavailability exhibited by the amide
derivative 1d may support the view that an ester bond may be
**

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Vehicle
Control
2b (P2026)
120
1.5
1.2
0.9
0.6
0.3
0.0
Diclofenac
2c (P1912)
2b (P2026)
**
**
**
**
**
90
*
60
30
**
**
2
0
4
6
8
Time (h)
0
0
2
4
6
8
Figure 3. Anti-inflammatory activity of diclofenac (10 mg/kg) or NO-Diclofenac at
equimolar dose in carrageenan-induced paw odema in rat. *P <0.05, **P <0.01 versus
control.
Time (h)
Figure 5. Plasma NOx (nitrite/nitrate) following oral administration of NO-
Diclofenac (5 mg/kg equimolar diclofenac) in rat. **P <0.01 versus control.
Aspirin
1b (P1538)
the ‘Proof of concept’ in that our novel disulfide linker-containing
NO-NSAIDs are indeed acting as potentially gastric-sparing ‘Safe
NSAIDs’. Further application of this promising disulfide linker
and prodrug technology to other prominent NSAIDs such as na-
proxen, ibuprofen, indomethacin, flurbiprofen, and ketoprofen, to
identify a few more potentially gastric-sparing ‘Safe NSAIDs’, is in
progress.
Acknowledgments
We gratefully acknowledge the support and encouragement
provided by the Piramal Life Sciences management. We are thank-
ful to our colleagues Mini Dhiman, Milan Dutta, Lokesh Babu,
Shubhada Deshpande, Gajanan Thakre, Dnyaneshwar Pacharne,
and Vijaya Nadar for their technical help in the development of
NO-releasing disulfide linker and prodrug technology. We are also
thankful to our colleagues Parikshit Gaikwad, Jagannath J. Deshat-
tiwar, Jayesh Mudgal, Aslam U. Burhan, Nitin J. Deshmukh, Ankur
Sharma, and Thirumalaivelu T. Mariappan for their technical help
in pharmacology studies. We also thank Professor M. Himaja of
VIT University, Vellore, for her technical advice. Help given by
our colleagues at animal house, patents department and analytical
chemistry department is gratefully acknowledged.
Diclofenac
2b (P2026)
Figure 4. Representative images of rat stomachs showing gastric ulcer induction/
sparing following oral administration of NSAID (100 mg/kg) or NO-NSAID (100 mg/
kg equimolar to NSAID) in rats.
Supplementary data
Supplementary data (experimental details for the synthesis and
characterization of all reported compounds; procedures for biolog-
ical experiments and some additional biological data) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.07.142.
essential for this class of NO-NSAIDs to act as prodrugs. This struc-
tural requirement may also support the view that these ester pro-
drugs might be releasing free drugs via enzymatic hydrolysis.
However, the proposed sulfhydryl-dependent mechanism of NO
release from these novel NO-NSAIDs still looks very plausible.
In summary, we have designed, synthesized, and evaluated bio-
logical activity of novel NO-NSAID prodrugs such as NO-Aspirin
and NO-Diclofenac. They have shown promising pharmacokinetic,
anti-inflammatory and NO-releasing properties and also protected
rats from NSAID-induced gastric damage which could be most
likely due to their NO-releasing capability. Among the evaluated
prodrugs, the NO-Diclofenac prodrug 2b has shown excellent gas-
tro-protective and NO-releasing properties in addition to exhibit-
ing comparable bioavailability and anti-inflammatory activities to
those of diclofenac. These preliminary results clearly establish
References and notes
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his website http://www.healthsentinel.com and the relevant references cited
therein.
4. Lanas, A. Arthritis Res. Ther. 2008, 10, S4.
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Pharmacol. Res. 1994, 29, 89.
6. Wallace, J. L.; Reuter, B.; Cicala, C.; McKnight, W.; Grisham, M.; Cirino, G. A. Eur.
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7. Stefano, F.; Distrutti, E. Curr. Top. Med. Chem. 2007, 7, 277.
8. Wallace, J. L.; Soldato, P. D. Fundam. Clin. Pharmacol. 2003, 17, 11.

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9. Humphreys, A. Eighth Annual Report on Future Blockbusters: Naproxcinod.
MedAdNews, Feb 2008, USA.
10. Satyam, A. Bioorg. Med. Chem. Lett. 2008, 18, 3196.
11. Tian, Y.; Zhang, C.; Fang, Z.; Betts, W. H. Clin. Chim. Acta 2001, 313, 51. and
relevant references cited therein.
12. Thatcher, G. R. J.; Nicolescu, A. C.; Bennett, B. M.; Toader, V. Free Radical Biol.
Med. 2004, 37, 1122. and relevant references cited therein.
13. Zavorin, S. I.; Artz, J. D.; Dumitrascu, A.; Nicolescu, A.; Scutaru, D.; Smith, S. V.;
Thatcher, G. R. J. Org. Lett. 2001, 3, 1113.
14. Doel, J. J.; Benjamin, N.; Hector, M. P.; Rogers, M.; Allaker, R. P. Eur. J. Oral Sci.
2005, 113, 14.
compound 1d: 1H NMR (300 MHz, CDCl3): d 2.35 (s, 3H), 2.92 (t, J = 6.1 Hz, 2H),
2.98 (t, J = 6.81 Hz, 2H), 3.76 (q, J = 6.05 Hz, 2H), 4.71 (t, J = 6.73 Hz, 2H), 6.70
(br s, 1H), 6.90,7.12 (dd, J = 1.04 Hz, 1H), 7.31(t, J = 3 Hz, 1H), 7.48 (t, J = 3 Hz,
1H), 7.77, 7.79 (dd, J = 1.68 Hz, 1H). 13C NMR (100 MHz, CDCl3): d 21.3, 34.5,
37.6, 38.2, 70.5, 123.2, 126.3, 127.7, 129.8, 132.0, 148.0, 165.8, 169.1. HRMS
ESI (m/z): [M+Na]+ calcd for C13H16N2O6S2Na: 383.0347; found: 383.0342
(mass accuracy: 1.5 ppm); compound 2b: 1H NMR (CDCl3): d 2.93–3.03 (m,
4H), 3.86 (s, 2H), 4.44 (t, J = 6.6 Hz, 2H), 4.67 (t, J = 6.9 Hz, 2H), 6.57 (d,
J = 7.8 Hz, 1H), 6.83 (br s, 1H), 6.96–7.04 (m, 2H), 7.13–7.18 (m, 1H), 7.25–7.28
(m, 1H), 7.36–7.38 (m, 2H). 13C NMR (75 MHz, CDCl3): d 34.3, 36.5, 37.9, 62.4,
70.0, 117.8, 121.6, 123.6, 127.6, 128.4, 129.0, 130.4, 137.2, 142.1, 171.5. HRMS
ESI (m/z): [M+H]+ calcd for C18H19Cl2N2O5S2: 477.0107; found: 476.0114
(mass accuracy: À1.47 ppm); compound 2c: 1H NMR (300 MHz, CDCl3): d 2.86
(t, J = 6.6 Hz, 2H), 2.95 (t, J = 6.6 Hz, 2H), 3.96 (s, 2H), 4.41 (t, J = 6.6 Hz, 2H),
4.69 (t, J = 6.6 Hz, 2H), 4.72 (s, 2H), 6.56–6.59 (m, 1H), 6.72 (br s, 1H), 6.97–
7.04 (m, 2H), 7.13–7.19 (m, 1H), 7.27–7.30 (m, 1H), 7.36 (d, J = 8.1 Hz, 2H). 13C
NMR (75 MHz, CDCl3): d 34.3, 36.1, 37.5, 60.6, 62.4, 70, 117.9, 121.7, 123.3,
123.6, 127.7, 128.4, 129, 130.5, 137.3, 142.2, 166.7, 170.9. HRMS ESI
(m/z): [M+H]+ calcd for C20H21Cl2N2O7S2: 535.0162; found: 535.0181 (mass
accuracy: À3.55 ppm).
15. Weitzberg, E.; Lundberg, J. O. Nitric Oxide 1998, 2, 1.
16. Refer to Supplementary data for complete experimental details and compound
characterization data. Analytical and spectral data for final compounds are
given below: Compound 1b: 1H NMR (300 MHz, CDCl3): d 2.35 (s, 3H), 2.97 (t,
J = 7.5 Hz, 2H), 3.05 (t, J = 7.5 Hz, 2H), 4.53 (t, J = 7.5 Hz, 2H), 4.69 (t, J = 7.5 Hz,
2H), 7.11 (dd, J = 6 Hz, 1H), 7.33 (dt, J = 6 Hz, 1H), 7.58 (dt, J = 6 Hz, 1H), 8.02
(dd, J = 1.8, 1.5 Hz, 1H). 13C NMR (100 MHz, CDCl3): d 21.1, 34.8, 37.1, 62.6,
70.6, 122.7, 123.8, 126.1, 131.7, 134.2, 150.8, 164.0, 169.7. HRMS ESI (m/z):
[M+Na]+ calcd for C13H15NO7NaS2: 384.0188; found: 384.0193 (mass accuracy:
1.3 ppm); compound 1c: 1H NMR (500 MHz, CDCl3): d 2.35 (s, 3H), 2.80–3.03
(m, 4H), 4.46 (t, J = 9.5 Hz, 2H), 4.62 (t, J = 7 Hz, 2H), 4.90 (s, 2H), 7.14 (d,
J = 8 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 8.11 (d, J = 8 Hz, 1H).
13C NMR (125 MHz, CDCl3): d 21.0, 34.8, 36.7, 61.04, 62.7, 70.6, 122.2, 123.9,
126.1, 132.0, 134.1, 150.9, 163.6, 167.3, 169.6. HRMS ESI (m/z): [M+Na]+ calcd
for C15H17N1Na1O9S2: 442.0237; found: 442.0233 (mass accuracy: 0.90 ppm);
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