Evaluation of nitrate-substituted pseudocholine esters of aspirin as potential nitro-aspirins

Article abstract of DOI:10.1016/j.bmcl.2007.03.009

Herein we explore some designs for nitro-aspirins, compounds potentially capable of releasing both aspirin and nitric oxide in vivo. A series of nitrate-bearing alkyl esters of aspirin were prepared based on the choline ester template preferred by human plasma butyrylcholinesterase. The degradation kinetics of the compounds were followed in human plasma solution. All compounds underwent hydrolysis rapidly (t_1/2 ～ 1 min) but generating exclusively the corresponding nitro-salicylate. The one exception, an N-propyl, N-nitroxyethyl aminoethanol ester produced 9.2% aspirin in molar terms indicating that the nitro-aspirin objective is probably achievable if due cognisance can be paid to the demands of the activating enzyme. Even at this low level of aspirin release, this compound is the most successful nitro-aspirin reported to date in the key human plasma model.

Full text of DOI:10.1016/j.bmcl.2007.03.009

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3217–3220
Evaluation of nitrate-substituted pseudocholine esters of
aspirin as potential nitro-aspirins
John F. Gilmer,* Louise M. Moriarty and John M. Clancy
School of Pharmacy and Pharmaceutical Sciences, Trinity College, Dublin 2, Ireland
Received 12 February 2007; accepted 5 March 2007
Available online 12 March 2007
Abstract—Herein we explore some designs for nitro-aspirins, compounds potentially capable of releasing both aspirin and nitric
oxide in vivo. A series of nitrate-bearing alkyl esters of aspirin were prepared based on the choline ester template preferred by human
plasma butyrylcholinesterase. The degradation kinetics of the compounds were followed in human plasma solution. All compounds
underwent hydrolysis rapidly (t1/2 ꢀ 1 min) but generating exclusively the corresponding nitro-salicylate. The one exception, an
N-propyl, N-nitroxyethyl aminoethanol ester produced 9.2% aspirin in molar terms indicating that the nitro-aspirin objective is
probably achievable if due cognisance can be paid to the demands of the activating enzyme. Even at this low level of aspirin release,
this compound is the most successful nitro-aspirin reported to date in the key human plasma model.
ꢀ 2007 Elsevier Ltd. All rights reserved.
NO-NSAIDs are compounds in which a non-steroidal
anti-inflammatory drug is coupled to a nitric oxide
donating moiety through a spacer group, usually an
ester.1 They are also referred to as cyclo-oxygenase
(COX) inhibitory NO donors (CINODs).2 The original
pharmacological rationale for these compounds was
that nitric oxide has gastro-protective effects which
would ameliorate the gastrointestinal toxicity of the
NSAID. A general observation is that the nitric oxide
and COX-inhibitory partners exhibit synergy especially
in models of carcinogenesis.3,4
nificant aspirin release in human tissue.9,11 In the case of
NCX-4016 irreversible cyclooxygenase inhibition is
reported to be mediated by the intact drug.12
The difficulty in designing aspirin ester prodrugs (with
or without an NO-donating capability) is that aspirin
already has an ester, its acetate, which becomes highly
labile in plasma when the carboxylic acid is converted
to an ester.13 The dominant esterase in human plasma,
butyrylcholinesterase (BuChE), is not efficient in the
hydrolysis of negatively charged substrates, but pro-
cesses neutral phenylacetates extremely rapidly.14 In
order to release aspirin in vivo, the carrier group
(bearing the NO-donor) must be detached at a faster
rate than the acetyl ester, whose hydrolysis the carrier
ester promotes. This conundrum was the focus of
numerous investigations at the simple prodrug level,
long before the prospect of an NO-aspirin emerged.
One of the very few successful solutions was the
design of glycolamide esters, which act as pseudo-
choline substrates for BuChE.13 The N,N-diethylglyco-
lamide of aspirin and its hydroxy-analogue (3) were
reported to undergo hydrolysis in dilute human
plasma releasing around 50% aspirin on a molar basis.
There have been reports of glycolamide esters of
NSAIDs,15,16 including one recent nitrate-bearing
glycolamide of naproxen,17 but the obvious promise
of this approach to the design of NO-aspirins does
not appear to have been evaluated.
Aspirin is an obvious candidate for the NO-hybrid ap-
proach. It is gastro-toxic even at the low doses used in
cardio-protection while its antiplatelet effects are com-
plementary to those of NO.5 NCX-4016, the prototypi-
cal NO-aspirin, has shown promising effects in a variety
of cardiovascular disease models and in cancer.6,7 Alter-
native NO-aspirin hybrid designs include 1-(pyrrolidin-
1-yl) diazen-1-ium-1,2-diolates8 (e.g., 2), furoxans9,
and aspirin derivatives of isosorbide mononitrate.10
The actions of these compounds appear to be complex
and not easily explicable in simple terms of aspirin
and nitric oxide release. Indeed there is no evidence that
any of the compounds reported so far are capable of sig-
Keyword: Nitroaspirin.
*
Corresponding author. Tel.: +353 1 6082795; fax: +353 1 6082793;
e-mail: gilmerjf@tcd.ie
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.009

3218
J. F. Gilmer et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3217–3220
O
O
O
O
O
O
O
O
O
N
N
O
N+
O
O
O
OH
2
ONO2
1
NCX-4016
The purpose of this study was to determine if the pro-
ductive hydrolysis characteristics of the aspirin glycola-
mides could be maintained when the carrier group is
further substituted with an NO-donor, for example, 4.
Some related ethanolamine esters (5) as well as simple
nitrooxyalkyl esters were also tested as true aspirin
prodrugs, also potentially capable of releasing nitric
oxide.
Four ethanolamine carrier groups were prepared
(Scheme 3). The appropriate N-alkyl amino ethanol
was nitrated using fuming nitric acid in DCM.19 The
resulting nitroxyalkylammonium salts were dissolved
in 7 M NaOH and alkylated using bromoethanol.20
Esterification of 10 with commercially available acetyl-
salicoyl chloride in the presence of triethylamine affor-
ded the mono- or di-nitrate esters (5a–d).
R
R
N
OAc
OAc
O
OAc
O
O
N
N
ONO2
O
ONO2
O
O
OH
O
O
5
4
3
Simple nitroxyalkyl esters (8a–c) were prepared by stir-
ring acetylsalicoylchloride with the appropriate
The various nitrate-esters were incubated in dilute hu-
man or rat plasma solution (pH 7.4, 37 ꢁC) or in the
presence of horse serum BuChE in order to predict their
potential to act as prodrugs for aspirin in vivo. The
product profile was determined using an RPHPLC/
PDA method capable of discriminating between aspirin,
salicylic acid, the parent ester and its deacetylated
analogue-salicylate esters elute after aspirin esters in
RPHPLC and possess a distinctive absorbance at
296 nm. The disappearance of test compounds followed
pseudo first-order kinetics with half-lives (0.693/kobs) in
the range 20 s–5 min (Table 1). Vmax and Km values were
obtained by non-linear regression of the disappearance
6
bromoalcohol in the presence of triethylamine followed
by Br-displacement using AgNO3 in acetonitrile.18 In
preparing the nitroglycolamide esters, the linking gly-
colic acid unit was introduced by esterification with ben-
zyl protected glycolic acid (12). The resulting ester 13
was deprotected and treated with oxalyl chloride to give
the acid chloride. The glycolamides 4a–d were generated
in the presence of the appropriate alkylamino nitrate.
The N-ethyl, N-hydroxyethylglycolamide ester of aspirin
(3), reported to release aspirin in human plasma solu-
tion,13 was prepared as a reference (Schemes 1 and 2).
OAc
O
OAc
O
OAc
AgNO3
CH3CN
O
Et3N
8a: R=CH2CH2
8b: R=CH(CH3)CH2
8c: R=(CH2)3
R
R
Cl
O
ONO2
O
Br
R
6
HO
Br
7
Scheme 1. Synthesis of simple nitroxyalkyl aspirin esters.
O
O
O
a,b
OH
c
O
O
O
HO
11
HO
O
O
O
12
13
d,e
O
O
O
4a:R=Me
4b:R=Et,
4c:R=Pr
f
Cl
O
14
O
4d:R=(CH2)2NO2
Scheme 2. Synthesis of nitroxyglycolamides. Reagents: (a) CsCO3, MeOH/H2O; (b) BnBr, DMF; (c) 6, Et3N, DCM; (d) H2, Pd/C; (e) (COCl)2,
DCM; (f) RN(CH2)2ONO2, Et3N.

J. F. Gilmer et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3217–3220
3219
R
5a:R=Me
5b:R=Et
5c:R=Pr
a,b
c
NHR
N
HO
ONO2
HO
9
5d:R=(CH2)2ONO2
10
Scheme 3. Synthesis of ethanolamine nitrate substituted esters. Reagents and conditions: (a) HNO3, DCM, 0 ꢁC then acetic anhydride;
(b) bromoethanol, 7 M NaOH, rt; (c) 6, Et3N, DCM.
The hydrolysis pathways of the glycolamide esters (4a–
d) were both complex and unproductive. None of the
compounds produced more than 0.5% aspirin on a
molar basis in any of the solutions tested-aqueous pH
7.4 (37 ꢁC), human or rat plasma solution or in solutions
containing purified horse BuChE. Meanwhile, the
unsubstituted N,N-diethylglycolamide ester liberated
around 60% aspirin in agreement with the report of
Bundgaard et al. Nitroxy-substitution of the N-alkyl
group therefore has a deleterious effect on hydrolysis
characteristics, promoting the usual acetyl group hydro-
lysis over aspirin liberation. This seems likely to be due
to differences in interaction orientation between the
carrier group and the BuChE active site. In human plas-
ma solution and in the presence of horse BuChE, initial
acetyl group hydrolysis was unexpectedly followed by
cleavage at the glycolamide bond, generating the gly-
colic acid ester of salicylic acid. In aqueous solution
(pH 7.4, 37 ꢁC) the glycolamides underwent quite rapid
hydrolysis at the amide bond and acetyl groups in
parallel (t1/2 ꢀ 90 min). Since the unsubstituted glycola-
mides were reported to enjoy reasonable aqueous stabil-
ity by Bundgaard (and do not look especially unstable),
we are forced to conclude that the nitrate group pro-
motes hydrolysis of the neighbouring glycolamide bond.
Hydrolysis in rat plasma solution occurred rapidly and
exclusively at the acetyl site generating the glycolamide
esters of salicylic acid, with no evidence of glycolamide
hydrolysis over the time course of the experiment, indi-
cating that BuChE might have contributed to the glyco-
lamide hydrolysis in the other experiments.
Table 1. Kinetic parameters for nitroaspirin hydrolysis in plasma
solution
0.1
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
0
1
2
3
4
Time (minutes)
Progress curve for the hydrolysis of ester 5c in
10% buffered human plasma (pH 7.4) at 37ºC:
5c ( ), salicylate ester (x), aspirin ( ) and
salicylic acid (o).
Compound
Km,
·104 M
Vmax
,
kobs
,
t1/2
,
Aspirin
release (%)
·104 M
min
s
8a
8b
8c
4a
4b
4c
4d
5a
5b
5c
5d
4.62
7.17
2.060
4.24
1.26
3.68
2.59
3.57
2.23
2.64
2.51
4.62
7.8
0.44
0.47
0.34
0.6
96
88
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
9.2
2.904
5.13
3.06
134
70
0.68
1.13
6.12
2.62
0.66
1.33
0.63
62
69
4.83
5.405
1.235
3.352
3.073
12.82
367
21
The design of a NO-aspirin mutual prodrug or hybrid is
challenging. Our experiments indicate that ethanol-
amine esters hold some promise as a platform. The
design process needs to take account of plasma BuChE
because even if it that is not the intended vector for drug
release, it will make a very effective and destructive com-
petitor. A structure-based design approach employing
one of the excellent models of human BuChE22,23 might
be useful in that regard.
63
39
67
<0.5
data to the integrated Michaelis–Menten equation.21 In
the cases of all but one of the esters, deacetylation pre-
dominated over carrier group detachment with no aspi-
rin production. The hydrolysis was most rapid in the
case of the ethanolamine esters (5) with half-lives of a
minute or less. The most promising compound, the N-
propyl, N-nitroxy-ethyl aminoethyl ester 5c liberated
9–10% aspirin on a molar basis. Interestingly, this was
not the most rapidly hydrolysed ester, underlining the
fact that the relative rates of aspirin ester and acetyl ester
hydrolysis is what determines the extent of aspirin re-
lease. The experiment was repeated with similar results
when human plasma was replaced by purified horse ser-
um butyrylcholinesterase at about the same concentra-
tion as the enzyme is found in plasma. The hydrolysis
rate was also found to be dependent on esterase concen-
tration without any change in hydrolysis direction.
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