Synthesis and chemical and biological comparison of nitroxyl- and nitric oxide-releasing diazeniumdiolate-based aspirin derivatives

Article abstract of DOI:10.1021/jm400196q

Structural modifications of nonsteroidal anti-inflammatory drugs (NSAIDs) have successfully reduced the side effect of gastrointestinal ulceration without affecting anti-inflammatory activity, but they may increase the risk of myocardial infarction with chronic use. The fact that nitroxyl (HNO) reduces platelet aggregation, preconditions against myocardial infarction, and enhances contractility led us to synthesize a diazeniumdiolate-based HNO-releasing aspirin and to compare it to an NO-releasing analogue. Here, the decomposition mechanisms are described for these compounds. In addition to protection against stomach ulceration, these prodrugs exhibited significantly enhanced cytotoxcity compared to either aspirin or the parent diazeniumdiolate toward nonsmall cell lung carcinoma cells (A549), but they were not appreciably toxic toward endothelial cells (HUVECs). The HNO-NSAID prodrug inhibited cylcooxgenase-2 and glyceraldehyde 3-phosphate dehydrogenase activity and triggered significant sarcomere shortening on murine ventricular myocytes compared to control. Together, these anti-inflammatory, antineoplasic, and contractile properties suggest the potential of HNO-NSAIDs in the treatment of inflammation, cancer, or heart failure.

Full text of DOI:10.1021/jm400196q

Subscriber access provided by NORTHEASTERN UNIV LIB
Article
Synthesis and Chemical and Biological Comparison of Nitroxyl and
Nitric Oxide Releasing Diazeniumdiolate-based Aspirin Derivatives
Debashree Basudhar, Gaurav Bharadwaj, Robert Y. Cheng, Sarthak Jain, Sa Shi, Julie L.
Heinecke, Ryan John Holland, Lisa A. Ridnour, Viviane M. Caceres, Regina C. Spadari-Bratfisch,
Nazareno Paolocci, Carlos Alberto Velazquez-Martinez, David A. Wink, and Katrina M. Miranda
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400196q • Publication Date (Web): 17 Sep 2013
Downloaded from http://pubs.acs.org on September 22, 2013
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis and Chemical and Biological Comparison of Nitroxyl and Nitric Oxide Releasing
Diazeniumdiolate-based Aspirin Derivatives
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1
2
3
4,5
Debashree Basudhar, Gaurav Bharadwaj, Robert Y. Cheng, Sarthak Jain, Sa Shi, Julie L.
2
6
2
4,7
Heinecke, Ryan J. Holland, Lisa A. Ridnour, Viviane M. Caceres, Regina C. Spadari-
7
4,8
3
2
Bratfisch, Nazareno Paolocci, Carlos A. Velázquez-Martínez, David A. Wink and Katrina M.
1
,*
Miranda
1
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721,
2
3
Radiation Biology Branch, National Institutes of Health, Bethesda, Maryland 20892, Faculty of
4
Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada, Division of
Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland
5
21287, Department of Pathophysiology, Harbin Medical University, Harbin, 150081, China;
6
Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, Frederick,
7
Maryland 21702,ꢀ Department of Biosciences, Federal University of São Paulo (UNIFESP),
8
Santos, SP, Brazil, Department of Clinical and Experimental Medicine, University of Perugia,
Perugia, Italy
Running title: Nitrogen oxide releasing prodrugs of aspirin
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
Structural modifications of non-steroidal anti-inflammatory drugs (NSAIDs) have
successfully reduced the side effect of gastrointestinal ulceration without affecting anti-
inflammatory activity, but may increase risk of myocardial infarction with chronic use. That
nitroxyl (HNO) reduces platelet aggregation, preconditions against myocardial infarction and
enhances contractility led us to synthesize a diazeniumdiolate-based HNO releasing aspirin and
to compare it to an NO-releasing analogue. Here, the decomposition mechanisms are described
for these compounds. In addition to protection against stomach ulceration, these prodrugs also
exhibited significantly enhanced cytotoxcity compared to either aspirin or the parent
diazeniumdiolate toward non-small cell lung carcinoma cells (A549) but were not appreciably
toxic toward endothelial cells (HUVECs). The HNO-NSAID prodrug inhibited cylcooxgenase-2
and glyceraldehyde 3-phosphate dehydrogenase activity and triggered significant sarcomere
shortening compared to control on murine ventricular myocytes. Together, these anti-
inflammatory, anti-neoplasic and contractile properties suggest the potential of HNO-NSAIDs in
the treatment of inflammation, cancer or heart failure.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Keywords: nitroxyl, nitric oxide, NONOate, diazeniumdiolate, IPA/NO, DEA/NO, NSAID,
aspirin, COX-2, GAPDH, anti-inflammatory, anticancer, cardioprotective
2
ACS Paragon Plus Environment

Page 3 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
Cyclooxygenase (COX)-mediated conversion of arachidonic acid to prostaglandins
1
affects various physiological functions, such as protection of stomach lining, contraction and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2, 3
4
5
6, 7
relaxation of smooth muscle, platelet aggregation, renal function, nerve and brain function,
8
9
bone metabolism, as well as pathological conditions, including pain, inflammation,
10-12
13
Alzheimer’s disease
and cancer. Use of non-steroidal anti-inflammatory drugs (NSAIDs) as
1
4
COX inhibitors is extensive to reduce pain, fever and inflammation. Given the diverse
physiological effects of prostaglandins, NSAID treatment can lead to serious side effects
15
including gastric ulcerogenicity and renal toxicity, due in part to a lack of specificity for COX-
1
, which is the constitutive isoform primarily responsible for baseline formation of
1
6
prostaglandins, and COX-2, which is induced during inflammation. More recent studies suggest
that this differentiation of the two isoforms is likely to be oversimplified, however, classic side
1
7
effects of NSAIDs remain attributed to non-specific inhibition of COX.
Development of later generation NSAIDs has relied on structural modification, for
example to induce COX-2 selectivity or to attach a nitric oxide (NO)-donating moiety. Selective
1
8
inhibitors of COX-2 (COXIBs) have successfully reduced ulcerogenicity, but chronic use leads
19
to an increased risk of heart attack and stroke. COX inhibiting NO donors, CINODs or NO-
2
0
NSAIDs , combine the anti-inflammatory properties of NSAIDs with the NO producing
capability of nitroglycerine, which has been used for decades to treat angina and chronic heart
2
1
failure. These organic nitrate derivatives reduce the side effects of NSAIDs and also have
2
2
efficacy as cancer therapeutics. However, prolonged use can lead to nitrate tolerance as
23
observed for nitroglycerine. In addition, the dependence on metabolic pathways to produce NO
2
4, 25
from organic nitrates
complicates measurement of NO release from CINODs. In fact, the
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
anticancer mechanism has been suggested to involve formation of electrophilic quinone
methides, which can deplete intracellular glutathione (GSH) levels, rather than production of
2
6
NO.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
More recently, NO releasing NSAIDs have been produced by derivatization of secondary
2
7, 28
29
amine diazeniumdiolates (NONOates),
which are widely used as NO donors. Such NONO-
NSAIDs are enzymatically cleaved to induce spontaneous release of NO from the
2
7, 28
diazeniumdiolate moiety,
thus removing dependence on the metabolic pathways required for
production of NO from organic nitrates. Another attractive attribute of secondary amine
diazeniumdiolates in terms of NO donation is the ability to tune the rate of decomposition based
3
0
on amine identity.
Decomposition of diazeniumdiolates can also lead to nitroxyl (HNO) production.
HNO has emerged as an important pharmacological agent with beneficial effects in overcoming
3
1-33
3
4
35
36
heart failure, preconditioning against myocardial infarction, decreasing tumor growth and
37
treating alcohol abuse. We have recently analyzed the pH-dependent mechanisms of production
of NO and HNO during decomposition of Angeli’s salt (Na N O ), which is the most commonly
2
2
3
3
3
used donor of HNO, and isopropylamine diazeniumdiolate (IPA/NO). For these related
compounds, protonation of the terminal nitroso nitrogen, by tautomerization of the amine proton
for example (Scheme 1), induces cleavage of the diazenium bond to produce HNO. In contrast,
protonation of the atom attached to the diazeniumdiolate moiety leads to separation of the NO
dimer from the nucleophile. Primary amine based diazeniumdiolates can function as dual donors
of NO and HNO in the pH range where both pathways are kinetically competitive. For instance,
IPA/NO is an NO donor at pH ≤ 5, an HNO donor at pH ≥ 8 and a dual donor at intermediate
pH.
4
ACS Paragon Plus Environment

Page 5 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
_
O
_
O
_
O
R
H
R
R
N N
_
N N
H
_
N N
+ HNO
N O
N O
H+
-H+
+
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
_
O
_
O
R
H
R
N N
H N N
H
_
RNH2 + 2NO
N OH
N O
Scheme 1. Dual decomposition mechanisms available for primary amine diazeniumdiolates
leading to release of HNO or NO.
Derivatization of amine-based diazeniumdiolates facilitates purification and increases
3
8,39
stability in neutral and acidic conditions.
There are now numerous examples of esterified
secondary amine diazeniumdiolates, and these compounds can typically be readily deprotected
3
0
by esterases either within cells or upon addition of purified protein or blood serum. Our recent
examination of the acetoxymethyl derivative of IPA/NO (IPA/NO-AcOM) demonstrated that
39
porcine live esterase catalyzed dissociation of free IPA/NO. However, spontaneous hydrolysis
of IPA/NO-AcOM unexpectedly increased HNO production over the parent diazeniumdiolate via
removal of the amine proton to produce a unique intermediate (see Scheme 3 below).
Here, two new NONO-NSAIDs were prepared by derivatizing both a primary and
2
secondary diazeniumdiolate with aspirin to produce O -(acetylsalicyloyloxymethyl)-1-(N-
2
isopropylamino)-diazen-1-ium-1,2-diolate
(IPA/NO-aspirin)
and
O -
(
acetylsalicyloyloxymethyl)-1-(N,N-diethylamino)-diazen-1-ium-1,2-diolate (DEA/NO-aspirin).
Their chemical and biological properties are compared to determine the efficacy of these
compounds as analgesic and anti-inflammatory agents. Additionally, the ability to induce HNO-
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mediated effects such as inhibition of thiol-containing proteins and enhancement of contractility
was investigated.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Results and Discussion
The half-life under physiological conditions of IPA/NO (5.7 min) is comparable to that of
29
DEA/NO (2.5 min), thus facilitating comparison of these related respective HNO and NO
2
38-40
donors. O -Modification of both IPA/NO and DEA/NO has previously been described,
as
Here,
IPA/NO-aspirin and DEA/NO-aspirin were newly synthesized by minor modifications of
2
7, 28
have the syntheses of NSAID derivatives of secondary amine diazeniumdiolates.
2
7, 41
published procedures (Scheme 2).
Briefly, each amine was first exposed to a high pressure of
2
NO to obtain the corresponding diazeniumdiolate. The desired products were produced by O -
2
methylthiomethyl derivatization followed by reaction with sulfuryl chloride to produce the O -
chloromethyl protected diazeniumdiolate, and finally by coupling with aspirin.
R
R
R
NO, 40 psi
N
ClCH SCH
N
CH3
2
3
NH
N N
O
O
N N
O
O
S
ether, NaOMe
DMF, K CO , 3 h
R'
R'
2
3
R'
Na+
1
4
; R = isopropyl, R' = H
; R = R' = Et
O
O
O
O
HO
O
O
R
R
SO Cl
N
N
2
2
N N
O
O
Cl
N N
O
O
O
o
CH Cl , 0 C, 3 h
DMSO, Et N
3
2
2
R'
R'
2
5
; R = isopropyl, R' = H
; R = R' = Et
3; R = isopropyl, R' = H
6; R = R' = Et
Scheme 2. Synthesis of NO or HNO releasing aspirin prodrugs.
6
ACS Paragon Plus Environment

Page 7 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Decomposition mechanism
Previously, examination of the decomposition of IPA/NO-AcOM established two
3
9
independent pathways for spontaneous and esterase-mediated hydrolysis. Assuming these
pathways are also accessible for the related aspirin derivative, spontaneous decomposition of
IPA/NO-aspirin (3) may be initiated by deprotonation of the amine proton (3a), which leads to 1-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
acyl migration via a cyclic intermediate (3b) and expulsion of formaldehyde (Scheme 3). The
resulting intermediate (3c) should then be susceptible to fragmentation by N-N bond cleavage to
produce a reactive diazoate ion (10) and an acyl nitroso derivative. The acetyl group of aspirin
provides a second hydrolysis site such that the acyl nitroso intermediate, which is likely to
further hydrolyze to produce HNO and a carboxylate, may be either the salicylate (pathway A;
9
a) or acetylsalicylate (pathway B; 9b) derivative. In either case, the products would be
salicylate, HNO and isopropanol (from decomposition of 10).
Alternatively, IPA/NO-aspirin (3) decomposition may be initiated by hydrolysis of one of
the two ester bonds (Scheme 4). Cleavage of the acetyl bond (pathway A) would produce
2
IPA/NO-salicylate (7), which could undergo further decomposition to form salicylate and the O -
hydroxymethyl-diazeniumdiolate. Spontaneous release of formaldehyde would give the free
diazeniumdiolate, which can then decompose by Scheme 1. Conversely, initial cleavage of the
2
prodrug (pathway B) would produce aspirin and the O -hydroxymethyl-diazeniumdiolate. Again,
regardless of the pathway, the products will include salicylate, HNO and isopropanol, but
isopropylamine and NO may also be formed. Scheme 3 is not accessible for the secondary amine
prodrug DEA/NO-aspirin nor is the HNO donating pathway in Scheme 1, thus the expected
products are salicylate, NO and diethylamine.
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
O
O
N
– H+
O
N
O
O
N
H
N
O
N
N
O
N
N
O
O
O
O
O
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
3
3a
3b
O
N
O
N
N
CH O
2
O
O
c
O
pathway A
pathway B
3
CH COOH
O
3
O
N
N
O
O
N
N
O
+
N
N
O
O
1
0
HO
O
3
d
9b
N2
O
N
O
O
O
O
+
N
N
OH
HO
O
HO
N2
HNO +
9
a
10
O
HO
HNO +
O
O
O
HO
slow
O
O
O
HO
CH COOH
3
8
ACS Paragon Plus Environment

Page 9 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 3. Possible pathways leading to HNO and salicylate production from spontaneous
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
decomposition of IPA/NO-aspirin via initial amine deprotonation.
R1
O
R2
R1
O
R2
N
N
pathway A
N
N
R1
O
R2
N
O
N
O
O
O
N
N
k1
k2
OH
+
N
O
O
O
O
O
CH COOH
3
O
OH
OH
O
3
or 6
7 or 8
k3
k'2
CH O
2
pathway B
k'1
CH COOH
3
O
O
R1
O
R2
R1
O
R2
N
N
N
N
k3
O
+
N
O
N
O
O
OH
CH2O
Scheme 4. Possible pathways leading to salicylate and free diazeniumdiolate production from
spontaneous decomposition of NONO-aspirin prodrugs via initial ester hydrolysis.
Of the many intermediates proposed in Schemes 3 and 4, those that may be both
diagnostic and long-lived enough to be detected are aspirin in pathway B of both Schemes 3 and
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
4
, NONO-salicylate (7 or 8) in pathway A of Scheme 4 and the free diazeniumdiolate in Scheme
. Spectrophotometric, NMR and HPLC analyses were thus performed to assist with mechanistic
assignment.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
O -Derivatization stabilizes diazeniumdiolates, allowing for purification by column
3
9
chromatography and increasing stability during storage, especially for primary amine adducts.
2
O -Protection also leads to a shift in the diazeniumdiolate absorption maximum from 250 to
~
240 nm. For the new aspirin prodrugs, the rates of spontaneous hydrolysis as determined by
loss of this peak/shoulder (Figures 1A and B) are significantly longer (t of 7.5 and ~36 h for
½
IPA/NO-aspirin and DEA/NO-aspirin, respectively, at pH 7.4 and 37°C) than for the parent
diazeniumdiolates or acetoxymethyl derivatives (compare to 5.7 and 2.5 min for IPA/NO and
2
9
39
DEA/NO , and 41 min for IPA/NO-AcOM ).
The final spectrum in Figure 1A corresponds to that of salicylate (λmax of 238 and 295
nm). Importantly, the presence of a clear isosbestic point indicates negligible accumulation of
aspirin (λmax at 267 nm). Spontaneous hydrolysis of aspirin to salicylate (Figure 1C) at pH 7.4
occurs with a nearly threefold slower rate constant than IPA/NO-aspirin decomposition. Elevated
pH accelerates the hydrolysis of both aspirin and IPA/NO-aspirin without leading to significant
spectral changes from those in Figures 1A and C (data not shown). The differences in the rates of
-
4
-1
-3 -
these processes however are even more pronounced ((1.6 ± 0.5) × 10 s and (3.5 ± 0.8) × 10 s
1
,
respectively at pH 10). These data therefore suggest that pathways 3-B and 4-B can be
eliminated as major components of the decomposition mechanism of IPA/NO-aspirin, due to
lower stability of the acetyl bond compared to the prodrug ester linker. Furthermore, that
accumulation of free IPA/NO was not observed at pH 10, where the rate of decomposition (4.1 ×
-
4
-1
10 s ) is slower than that of IPA/NO-aspirin, also eliminates pathway 4-A. Thus, as with the
1
0
ACS Paragon Plus Environment

Page 11 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
acetoxymethyl derivative, IPA/NO-aspirin is suggested to decompose by amine deprotonation
followed by pathway 3-A.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
A
0.8
0.6
0
0
.4
.2
0
2
20
240
260
280
300
320
340
Wavelength (nm)
1
B
0
0
0
0
.8
.6
.4
.2
0
220
240
260
280
300
320
340
Wavelength (nm)
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1.5
1
C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.5
0
2
20
240
260
280
300
320
340
Wavelength (nm)
2.5
2
D
1
.5
1
0
.5
0
2
40
260
280
300
320
340
Wavelength (nm)
1
2
ACS Paragon Plus Environment

Page 13 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
.5
1
E
1
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
.5
0
2
40
260
280
300
320
340
Wavelength (nm)
Figure 1. Spontaneous hydrolysis of (A) IPA/NO-aspirin, (B) DEA/NO-aspirin or (C) aspirin at
pH 7.4 and (D, E) DEA/NO-aspirin at pH 10 in PBS containing 50 µM DTPA at 37°C. Scans are
plotted at 0, 90, 210, 390, 570, 930, 1410 min in (A), 60, 570, 930, 1290, 1770, 2370, 2970, 3690
min in (B), 60, 210, 570, 1170, 1770, 2970, 5730 min in (C), 0, 10, 20, 30, 40, 50 min in (D) and
8
0, 110, 150, 190, 230, 270, 320, 360, 420, 460, 540, 660, 820 min in (E). The respective first-
order rate constants of IPA/NO-aspirin and DEA/NO-aspirin decomposition at 241 nm are (2.6 ±
-
5
-1
-6 -1
0.2) × 10 s (n > 3) and 5.3 × 10 s (n = 1 given the slow rate) while that of aspirin at 295 nm
-6 -1
2
is 9.8 × 10 s (n = 1 given the slow rate); all R > 0.997.
Analysis by NMR of the hydrolysis products of IPA/NO-aspirin at pH 10 (Figure S1)
revealed significant accumulation of isopropanol (δ 1.18 [d, (CH ) ]), acetate (δ 1.91 [s, CH ]),
3 2
3
formaldehyde (δ 4.86 [s, CH ]) and salicylate (δ 6.89-6.92, 7.39-7.42, 7.72-7.74). Notably, the
2
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
acetyl group of aspirin was not observed (δ 2.33 (s, CH )). Isopropylamine (δ 1.07 [d, (CH ) ])
3
3 2
was also not detected, indicating that under these conditions, the NO-donating pathway in
Scheme 1 was not appreciable, as would be expected even if pathways 4-A or 4-B were
significant.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The spectral changes observed during decomposition of IPA/NO-aspirin and DEA/NO-
aspirin are similar (Figures 1A,B), but a more pronounced initial shoulder near 275 nm may
suggest early formation of aspirin from decomposition of DEA/NO-aspirin. Since the rate of
hydrolysis of DEA/NO-aspirin is slower than that of aspirin by twofold, both pathways in
Scheme 4 are kinetically viable.
Given the potential for NONO-salicylate (7 or 8) formation by pathway 4-A, the
IPA/NO- and DEA/NO-salicylate derivatives were synthesized in a similar manner as shown in
Scheme 3 and were found to have unique maxima at 306 nm. Their faster rates of decomposition
-
5
-5 -1
(7.0 × 10 and 3.1 × 10 s ) indicate that early rate-limiting steps inhibit NONO-salicylate
accumulation as useful to discriminate between potential pathways for the aspirin derivatives.
At pH 10, unlike for IPA/NO-aspirin (data not shown), decomposition of DEA/NO-
aspirin produces observable intermediates. The peak near 275 nm in the initial spectrum suggests
the presence of aspirin (Figure 1D). With time, this peak shifts to ~295 nm, indicating cleavage
to salicylate (Figure 1E). Additionally, an initial growth of intensity near 250 nm suggests
-5 -1
production of DEA/NO ((4.5 ± 0.6) × 10 s ) (Figure 1D). Together, these data support
spontaneous hydrolysis of DEA/NO-aspirin through pathway 4-B.
DEA/NO-aspirin hydrolysis products were examined by NMR spectroscopy at pH 10 in a
similar manner as described for IPA/NO-aspirin. Formation of DEA/NO (2.99 and 0.94 ppm)
was evident as early as 10 min (Figure S2). With time, DEA/NO aspirin decreased while that of
1
4
ACS Paragon Plus Environment

Page 15 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a product (4.17, 3.72, 1.39, 1.27, and 1.12 ppm) increased, further reinforcing the generation of
free DEA/NO. Hydrolysis of DEA/NO-aspirin was not complete as late as 92 h, evident from
small observable peaks of the linker OCH O (6.13 ppm) and the acetyl group (2.40 ppm).
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Moreover, formation of DEA/NO-salicylate, free aspirin or the end product salicylate could not
be firmly established due to overlapping aromatic peaks, which complicated assignment.
Scheme 4 can also be considered for both prodrugs in the presence of esterases. Addition
of 2% guinea pig serum reduced the half-lives to 18 and 13 min, respectively for IPA/NO-aspirin
and DEA/NO-aspirin (Figure 2). Additionally, the spectra differed significantly from those in
Figure 1, with two processes evident. A new peak rapidly appeared at 306 nm with first-order
-
1
rate constants of 0.035 ± 0.009 and 0.027 ± 0.006 s , respectively (Figure 2A,B), and the slow
-4
-3
first-order loss of the characteristic peak at 241 nm (k = (6.5 ± 0.12) × 10 and (1.1 ± 0.6) × 10
2
-
1
s , respectively) was accompanied by shifting of the 306 nm peak to 295 nm (Figure 2C,D).
The rate of hydrolysis of aspirin in the presence of guinea pig serum (Figure 2E) was
-
4
-1
found to be slower than either observed process ((4.7 ± 0.17) × 10 s ), suggesting that aspirin
should accumulate if formed. Similarly, if produced during the faster first step, free
diazeniumdiolate should be observable spectrally, but a peak shift from 241 to 250 nm was not
apparent. Furthermore, the intermediate spectra corresponded well to that of the NONO-
salicylate derivatives, providing convincing evidence that esterase-mediated decomposition of
the NONO-aspirin prodrugs proceeds primarily by pathway 4-A. Additionally, modeling using
the kinetic data from Figure 2 and the rate constants for free diazeniumdiolate decomposition
verified that accumulation of IPA/NO or DEA/NO by pathway 4-A would be insufficient at any
time point to be observed spectrophotometrically (data not shown).
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
.4
A
1.2
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
0
0
.8
.6
.4
0.2
0
2
40
260
280
300
320
340
Wavelength (nm)
B
1
0.8
0.6
0
0
.4
.2
0
2
40
260
280
300
320
340
Wavelength (nm)
1
6
ACS Paragon Plus Environment

Page 17 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1.6
C
1
0
0
.2
.8
.4
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
40
260
280
300
320
340
Wavelength (nm)
D
0
0
0
.8
.6
.4
0.2
0
2
40
260
280
300
320
340
Wavelength (nm)
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
E
0.5
0.4
0.3
0.2
0.1
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
40
260
280
300
320
340
Wavelength (nm)
1
.4
F
1.2
1
0
0
0
.8
.6
.4
0.2
0
2
20
240
260
280
300
320
340
Wavelength (nm)
Figure 2. Guinea pig serum induced hydrolysis of (A, C) IPA/NO-aspirin, (B, D) DEA/NO-
aspirin, or (E) aspirin in PBS containing 50 µM DTPA at pH 7.4 and 37°C. Scans are plotted at
0
, 5, 10, 15, 20, 25, 40 s in (A), 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 s in (B), 2, 14, 26, 38, 62
min in (C), 3, 6, 9, 12, 15, 18, 39 min in (D) and 1, 7, 19, 31, 43, 59, 79, 99, 119 min in (E).
1
8
ACS Paragon Plus Environment

Page 19 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Panel (F) is as in (A) with the substitution of fetal bovine serum; scans are plotted at 0, 15, 30,
6
0, 150, 255, 555 and 885 min. All kinetic data fit to a first order equation (n ≥ 3 except for n = 1
2
for F) with R > 0.983. The presence of serum proteins impacts the blank-corrected spectra in the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
low 200 nm region in Figure 2 compared to Figure 1.
As additional confirmation, the species produced in the presence of guinea pig serum
were monitored temporally by HPLC with UV detection (Figure 3). The initial chromatograms
showed primarily the NONO-aspirin prodrugs (3 and 6), with traces of both aspirin and the
NONO-salicylate derivatives (7 and 8). At 5 min, complete conversion to 7 or 8 was apparent. At
4
0 min, significant accumulation of free salicylate occurred from cleavage of 8, while 7 provided
to be more stable toward hydrolysis. The products and timing are consistent with the
spectrophotometric analysis in Figure 2, thus providing further support of esterase-mediated
decomposition by pathway 4-A.
The effect of FBS on the decomposition of IPA/NO-aspirin (Figure 2F) was also
-
examined as it is commonly added to cell culture media. Interestingly, the rate constant (2.7 × 10
5
-1
s ) was comparable to that in the absence of serum, but the slow appearance of a 306 nm peak
and the subsequent shift to 295 nm suggested that esterase-mediated cleavage was occurring.
This suggests that effective cleavage of the ester bonds of NONO-aspirin derivatives may require
specific esterases.
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
0
ACS Paragon Plus Environment

Page 21 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 3. HPLC analysis of guinea pig serum induced hydrolysis of (A) DEA/NO-aspirin or (B)
IPA/NO-aspirin (100 µM) in 100 mM phosphate buffer containing 50 µM DTPA at pH 7.4 and
3
7°C. Authentic standards eluted from a Phenomenex Luna C18 column, 3 µm, 150 × 2.1 mm,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
using a water and acetonitrile gradient containing 0.1% formic acid as follows: aspirin (19.6
min), salicylate (20.5 min), IPA/NO-aspirin (23.8 min), IPA/NO-salicylate (24.6 min),
DEA/NO-aspirin (24.6 min) and DEA/NO-salicylate (25.7 min).
NO and HNO release from NONO-aspirin prodrugs
Although there are many methods to directly or indirectly measure NO, detection of
HNO is typically indirect and qualitative, in part due to metastability through an irreversible
6
-1 -1 42
dimerization pathway (producing N O and water (8 × 10 M s ) .We have designed and used a
2
4
3
39
multi-step protocol in our prior analysis of IPA/NO and IPA/NO-AcOM and utilized similar
methods here.
Reductive nitrosylation of ferric compounds such as metMb to the corresponding ferrous
nitrosyl complex (MbNO) is often used to provide spectral verification of HNO production. For
minute scale donors such as Angeli’s salt and IPA/NO, aerated conditions allow this assay to
discriminate between NO and HNO production due to the relative instability of the ferric
compared to the ferrous nitrosyl complex of myoglobin. For longer-lived donors of HNO, the
-4 -1 44
rate of autoxidation of MbNO back to metMb (2 × 10 s ) reduces the utility of this assay.
Since performing this assay under deaerated conditions introduces other complicating factors,
7
-1 -1 45, 46
here, we substituted oxymyoglobin (MbO ) for metMb. Although both NO (4 × 10 M s )
2
7
-1 -1 47
and HNO (1 × 10 M s ) react with MbO to produce metMb, addition of GSH (1 mM), which
2
6
-1 -1 47
48
reacts facilely (2 × 10 M s ) with HNO but not appreciably with NO, provides specificity to
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-5
-1
the assay. Since autoxidation of MbO (k = 1.2 × 10 s ; data not shown) can become
2
obs
significant during the long decomposition times of the prodrugs in the absence of esterases, an
excess of each prodrug was used to achieve higher reaction rates.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Both IPA/NO-aspirin and DEA/NO-aspirin converted MbO to metMb (Figure 4A-D),
2
and the calculated rate constants were similar to those determined for prodrug decomposition
-5
(
respectively for IPA/NO-aspirin and DEA/NO-aspirin mediated formation of metMb, 9.8 × 10
-
5
-1
-4
-4 -1
and 3.3 × 10 s in the absence of serum and 4.8 × 10 and 7.4 × 10 s in the presence of
serum). Addition of GSH only quenched metMb formation for IPA/NO-aspirin. In the absence of
-5 -1
serum (Figure 4A), formation of metMb (k = 4.3 × 10 s ) was reduced to a similar rate as that
obs
4
9
-5 -1
mediated by GSH alone (k = 4.8 × 10 s , data now shown), suggesting that HNO is the
obs
primary product under these conditions. In contrast, less efficient scavenging by GSH was
observed in the presence of serum (Figure 4C), indicating that NO production may be significant.
These results are consistent with the higher yield of HNO expected from spontaneous hydrolysis
via amine deprotonation (pathway 3-A) compared to ester cleavage to release free IPA/NO
(
pathway 4-A), which is a dual donor of HNO and NO at pH 7.4.
2
2
ACS Paragon Plus Environment

Page 23 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A
0
0
0
0
.8
.6
.4
.2
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
50
500
550
600
650
700
Wavelength (nm)
B
0
0
.8
.6
0.4
0
.2
0
4
50
500
550
600
650
700
Wavelength (nm)
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C
0
0
0
0
.8
.6
.4
.2
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
50
500
550
600
650
700
Wavelength (nm)
D
0
0
.8
.6
0.4
0
.2
0
4
50
500
550
600
650
700
Wavelength (nm)
Figure 4. Representative spectral changes (n ≥ 3) indicating trapping of NO and HNO by MbO2
(50 μM) during decomposition at 37°C of IPA/NO-aspirin or DEA/NO-aspirin in assay buffer ±
GSH (1 mM): (A) 250 µM IPA/NO-aspirin, (B) 250 µM DEA/NO-aspirin, (C) 100 µM IPA/NO-
aspirin + 2% guinea pig serum and (D) 50 µM DEA/NO-aspirin (given production of 2 eqs of
2
4
ACS Paragon Plus Environment

Page 25 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
NO) + 2% guinea pig serum. Initial scans are in red (MbO ), final scans without GSH are in blue
2
(155 min for A; 350 min for B; 1170 s for C; and 600 s for D) and final scans with GSH are in
green (145 min for A; 210 min for B; 1170 s for C; and 690 s for D).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In the presence of an oxidant such as ferricyanide, detection methods for NO can be used
50
to indirectly indicate formation of HNO. The slow rate of spontaneous hydrolysis impedes such
analysis, and therefore only the maximum current intensities observed with an NO-specific
electrode during decomposition of the prodrugs in the presence of guinea pig serum are shown.
For IPA/NO-aspirin (Figure 5A), the decrease in maximum signal intensity as the pH is elevated
from 6 is rescued by addition of ferricyanide, indicating a successive increase in the relative
3
3
yield of HNO at the expense of NO. That this profile is similar to that for IPA/NO further
supports cleavage as in Scheme 4. In contrast, the signal maxima from DEA/NO-aspirin are
relatively constant, as expected for an NO only donor. The decrease in maximum signal for both
IPA/NO-aspirin and DEA/NO-aspirin below pH 6 indicates reduced serum esterase activity.
Given the dependence of IPA/NO-aspirin decomposition on the presence of serum and
the sensitivity to serum type, the question arises as to the mechanism of decomposition within
human cells. Intracellular detection of NO and HNO is often achieved with fluorescent dyes such
5
1
as diaminofluorescein (DAF). Although typically non-specific toward HNO and NO, a higher
5
2
relative signal is produced by HNO compared to NO. Figure 6 shows the temporal profiles of
fluorescent signal upon exposure of A549 cells loaded with the cell permeable dye DAF-FM-
2DA to IPA/NO-aspirin, DEA/NO-aspirin and their constituent components.
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Maximum current intensity from an NO-specific electrode as a measure of NO and
HNO release from 100 µM of (A) IPA/NO-aspirin or (B) DEA/NO-aspirin in assay buffer
containing 2% guinea pig serum (in A, blue bars in assay buffer alone and green bars with
addition of 1 mM ferricyanide. The data are expressed as mean ± SD (n ≥ 3).
Although the ionic diazeniumdiolate precursors decompose relatively rapidly, HNO and
NO must then diffuse into the cells to react with the fluorophore. The higher signal intensities for
2
6
ACS Paragon Plus Environment

Page 27 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the prodrugs thus indicate significant cellular uptake, allowing HNO and NO to be produced in
close proximity to the reporter molecule, which will reduce cellular scavenging. Although NO
may be produced directly from IPA/NO-based donors or HNO may be converted to NO by
intracellular oxidants such as Cu,Zn SOD, the higher signals for IPA/NO and IPA/NO-aspirin
relative to DEA/NO and DEA/NO-aspirin respectively indicate significant reactivity with HNO
in the former cases. The only slightly slower rates of the signal increase for the prodrugs
compared to the parent donors suggests that esterase-mediated cleavage of the prodrugs was
significant within the cells.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
1
1
.4 10
.2 10
1 10
DMSO
5
5
4
4
4
4
IPANO-aspirin
DEANO-aspirin
IPA/NO
DEA/NO
aspirin
8
6
4
2
10
10
10
10
untreated
0
0
100
200
300
400
time (min)
Figure 6. NO and HNO release measured in A549 cells. The cells were exposed 100 µL of 10
µM DAF-2DA in PBS pH 7.4 for 75 min at 37°C and washed three times with PBS pH 7.4 to
remove excess dye. Upon addition of 10 µM of DEA/NO-aspirin, IPA/NO-aspirin or aspirin in
DMSO (<0.1%), 10 µM IPA/NO or DEA/NO in 10 mM NaOH or DMSO (n = 2, six replicates
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
per plate), the increase in fluorescence intensity at 535 nm was measured as a function of time at
3
7°C following excitation at 485 nm. The data are expressed as mean ± SD.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Effect of NONO-aspirin prodrugs on ulcerogenicity.
One of the most common side effects of aspirin use is gastrointestinal ulceration. NO
27
releasing NSAIDs have previously been shown to protect the stomach lining, but the effect of
HNO releasing NSAIDs is not known. To establish the safety profile of the new prodrugs, the
ulcerogenicity was measured by scoring the number and size of ulcerations. Both IPA/NO-
aspirin (3.3 ± 3) and DEA/NO-aspirin (0) (Figure 7) showed a substantially lower ulcer index as
compared to aspirin (57.4 ± 3.1; see for example reference 27 for images of aspirin-mediated
ulcerations).
Control
IPA/NO-aspirin
DEA/NO-aspirin
Figure 7. Examination of the stomach ulcerogenicity of IPA/NO-aspirin and DEA/NO-aspirin
(
1.38 mmol/kg). Ulcerogenicity was not significantly increased compared to control.
At highly acidic pHs such as that of the stomach, ionic diazeniumdiolates decompose
rapidly, and potentially accessible HNO-producing pathways are suppressed in favor of NO
2
production (Scheme 1). That O -derivatized diazeniumdiolates are stable under acidic conditions
2
8
ACS Paragon Plus Environment

Page 29 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
data not shown for the aspirin derivatives) may allow for significant absorption by the stomach
lining before cleavage. The reduced ulcerogenicity of IPA/NO-aspirin and DEA/NO-aspirin may
then be due to inhibition of an aspirin-mediated effect either by structural derivatization, by
decomposition to salicylate, by enhanced clearance or may be due to the prodrug itself
functioning as a selective inhibitor of COX-2. Ulcerogenicity may also be affected by the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
presence of NO or HNO, if cleavage does occur in the gastrointestinal system. O -Derivatization
may thus facilitate intracellular delivery and bioactivation as well as preserve pathways that lead
to HNO rather than NO production.
Anti-inflammatory properties of NONO-aspirin prodrugs compared to aspirin.
To verify whether IPA/NO-aspirin and DEA/NO-aspirin retain the anti-inflammatory
properties of the parent NSAID, they were orally administered to rats with paw edema induced
2
7
by carrageenan. Aspirin (ID = 129 mg/kg) and IPA/NO-aspirin (ID = 121 mg/kg) were
5
0
50
found to be comparably potent while DEA/NO-aspirin (ID = 169 mg/kg) had slightly lower
5
0
efficacy.
Administration of aspirin has previously been shown to increase COX-2 expression,
5
3
likely as a compensatory response to reduced prostaglandin levels due to irreversible inhibition.
Here, the effects of the prodrugs on COX-2 activity were examined in A549 cells, which are
5
4
known to express high levels of COX-2. PGE levels, which are a direct measure of COX-2
2
9
, 55
activity,
were decreased most significantly by IPA/NO-aspirin (Figure 8). These data support
the conclusion from Figure 6 that the prodrugs induce more potent effects, likely due to
improved cellular uptake and delivery.
2
9
ACS Paragon Plus Environment