Discovery of a "true" aspirin prodrug

Article abstract of DOI:10.1021/jm801094c

Aspirin prodrugs formed by derivatization at the benzoic acid group are very difficult to obtain because the promoiety accelerates the rate of hydrolysis by plasma esterases at the neighboring acetyl group, generating salicylic acid derivatives. By tracing the hydrolysis pattern of the aspirin prodrug isosorbide-2,5-diaspirinate (ISDA) in human plasma solution, we were able to identify a metabolite, isosorbide-2-aspirinate-5-salicylate, that undergoes almost complete conversion to aspirin by human plasma butyrylcholinesterase, making it the most successful aspirin prodrug discovered to date.

Full text of DOI:10.1021/jm801094c

J. Med. Chem. 2008, 51, 7991–7999
7991
Discovery of a “True” Aspirin Prodrug
Louise M. Moriarty, Maeve N. Lally, Ciaran G. Carolan, Michael Jones, John M. Clancy, and John F. Gilmer*
School of Pharmacy and Pharmaceutical Sciences, Trinity College, Dublin 2, Ireland
ReceiVed September 2, 2008
Aspirin prodrugs formed by derivatization at the benzoic acid group are very difficult to obtain because the
promoiety accelerates the rate of hydrolysis by plasma esterases at the neighboring acetyl group, generating
salicylic acid derivatives. By tracing the hydrolysis pattern of the aspirin prodrug isosorbide-2,5-diaspirinate
(ISDA) in human plasma solution, we were able to identify a metabolite, isosorbide-2-aspirinate-5-salicylate,
that undergoes almost complete conversion to aspirin by human plasma butyrylcholinesterase, making it
the most successful aspirin prodrug discovered to date.
Introduction
The design of an aspirin ester prodrug capable of enzyme-
mediated activation is one of the classical problems in prodrug
chemistry. Aspirin derivatives, such as esters and amides, are
expected to be less toxic to the gastrointestinal tract (GIT)a
following oral administration.1 They usually have better aqueous
stability than aspirin, making them potentially more suitable
for a variety of pharmaceutical presentations.2 There has
accordingly been a lot interest in the area in the past both as a
drug development opportunity and because the design of a
successful prodrug represents a significant and interesting
scientific conundrum.3,4 Reported aspirin derivatives include
aspirin anhydride,5 benzodioxinone derivatives,6 acylal deriva-
tives,7 N-(hydroxyalkyl) amides,8 2-formylphenyl derivatives,9
straightforward alkyl and aryl esters,10 triglycerides for lipase
targeting,11 acyloxyalkyl esters,12 sulfinyl and sulfonyl esters,13
phenylalanine amides,14 amino acid derivatives,15 indolediones
as hypotoxic tissue targeting agents,16 and dual release mech-
anism prodrugs.17 Ideally, an aspirin prodrug should be stable
under aqueous conditions and only undergo activation during
the absorption and distribution process.18
The obstacle to aspirin prodrug development is that aspirin
esters (1) are hydrolyzed rapidly by butyrylcholinesterase
([BuChE, EC3.1.1.8]) present in human plasma to the corre-
sponding salicylate ester (2) and then, more slowly, to salicylic
acid (3) [Figure 1a].18 Aspirin (4) is comparatively stable in
plasma, being a poor substrate for BuChE because of low
affinity, a phenomenon observed for that enzyme with negatively
charged substrates.19 Neutral aspirin esters behave more like
phenyl acetate (5), a “supersubstrate” for BuChE. For example,
the half-life for the hydrolysis of phenylaspirinate (6) in 10%
human plasma is 1.3 min, and it occurs exclusively at the acetate
group. Aspirin under similar conditions undergoes hydrolysis
at its acetate group, with a half-life of about 2 h.18 As pointed
out by Bundgaard and Nielsen, for a prodrug to release aspirin
in plasma, the benzoic ester must undergo hydrolysis at a greater
Figure 1. (a) Routes of hydrolysis of aspirin esters. (b) Phenyl acetate
(5), some aspirin esters, and prodrugs.
rate than at the acetyl group, whose hydrolysis is significantly
accelerated by the presence of the benzoic ester. The implication
is that a successful prodrug candidate in the plasma model
requires a carrier group of even greater compatibility than the
phenyl acetate or better still one which at the same time
depresses the phenylacetate hydrolysis. The pseudo-choline
glycolamide esters (Figure 1b, 7) vindicated this strategy with
hydrolysis, in the best case, to aspirin and the corresponding
salicylate esters in a ratio of around 1:1.18
Interest in the aspirin prodrug area has been renewed with
the advent of the so-called nitro-aspirins and related com-
pounds.20 These are mutual prodrugs, in which aspirin is
connected via an ester group to a nitric oxide releasing moiety,
such as a nitrate ester, e.g., NCX4016 (8) and the aspirin
diazenium diolates.21,22 However, to exert both actions of its
* To whom correspondence should be addressed: School of Pharmacy
and Pharmaceutical Sciences, Trinity College, Dublin 2, Ireland. Telephone:
+353-1-896-2795. Fax: +353-1-896-2793. E-mail: gilmerjf@tcd.ie.
a Abbreviations: BNPP, bis-p-nitrophenylphosphate; BuChE, butyryl-
cholinesterase; BW254c51, 1:5-bis(4-allyl-dimethyl)ammoniumphenyl-pen-
tan-3-one; DCC, dicyclohexylcarbodiimide; GIT, gastrointestinal tract;
HPLC, high-performance liquid chromatography; ISDA, isosorbide-2,5-
diaspirinate; EDTA, ethylenediaminetetraacetic acid; ISMN, isosorbide
mononitrate; ISMNA, isosorbide mononitrate aspirinate; iso-OMPA, tet-
raisopropylpyrophosphoramide; PDA, photodiode array detector; TXB2,
thromboxane B2.
10.1021/jm801094c CCC: $40.75
2008 American Chemical Society
Published on Web 12/02/2008

7992 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Scheme 1. ISDA Potential Hydrolysis Routesa
Moriarty et al.
a All of the possible hydrolysis metabolites are illustrated, but some potential pathways are omitted for clarity; e.g., 16 may be generated from 10 and 14
from 11. Processes leading directly to aspirin (4) are highlighted in bold. All processes lead ultimately to salicylic acid (3) and isosorbide (17).
component parts, a nitro-aspirin must be able to release aspirin.
None of the compounds reported thus far in this category appear
able to satisfy this requirement.23
network of parallel competing and consecutive pathways, with
four initial routes characterized by the rate constants k1, k2, k3,
and k4 (Scheme 1). Aspirin production could arise because of a
direct hydrolysis on ISDA at either the isosorbide 2 or 5
positions, leading to primary monoaspirinate products 10 or 11
(k1 and k4 in Scheme 1), or because of hydrolysis on one of
these monoaspirinates (k5 and k6 in Scheme 1), leading to
isosorbide(17).Hydrolysisoftheprimarymixedsalicylate-aspirinate
compounds 12 or 13 along the k7 and k8 pathways would also
lead to aspirin liberation. Altogether there are six possibilities
for aspirin production from ISDA in the presence of plasma
esterases. However, given the highly specific requirements for
enzyme-mediated aspirin release from an aspirin ester, it seemed
more likely that a smaller number of processes, possibly only
one, leads to aspirin evolution from ISDA. We sought to
characterize the distribution of ISDA metabolites in human
plasma solution and to identify the productive pathway. Ac-
cordingly, the potential hydrolysis metabolites appearing in
Scheme 1 were independently synthesized for use as standards
to monitor hydrolysis progress and to evaluate their ability to
generate aspirin separately.
We reported some interesting characteristics of aspirinate
esters of ISMN (ISMNA)24 and then on the diaspirinate ester
of isosorbide (ISDA, 9, Figure 1). This latter diester produces
significant amounts of aspirin when incubated in human
plasma.25 In a single oral dose study in dogs, ISDA administra-
tion caused sustained inhibition of ex ViVo platelet function and
TXB2 production up to 24 h.26 For an aspirin ester to produce
aspirin in Vitro and exhibit aspirin-like effects ex ViVo is unusual
and potentially valuable. The aim of the present study was to
investigate the hydrolysis pathways of ISDA in human plasma
and to explain its ability to produce aspirin. The potential
metabolites of ISDA were synthesized, and their evolution and
decay were monitored chromatographically following incubation
in the presence of human plasma. We were surprised to discover
that aspirin is produced from a hydrolysis product of ISDA along
a pathway involving the first “true” aspirin prodrug. The
compound behaves as an aspirin prodrug because of a highly
specific and efficient interaction with human BuChE.
Synthesis of ISDA Hydrolysis Products. Isosorbide mono-
nitrate aspirinate (ISMNA,24 19) was identified as a key
intermediate in the synthesis of potential metabolites, and it was
prepared from ISMN (18). To access the 2-monoaspirinate (10),
the 5-nitrate was removed using H2 in the presence of Pd/C in
methanol/ethyl acetate (Scheme 2). The isomeric isosorbide-5-
aspirinate (11) was prepared by a similar strategy (Scheme 3),
employing isosorbide-2-mononitrate (20) instead.27 The monosal-
icylates (14 and 16) were prepared by coupling of the appropri-
ate mononitrates (18 and 20) to salicylic acid (DCC/DMAP)
and removal of the nitrate group using Pd/C under an atmo-
sphere of H2. The disalicylate (15) and mixed 2/5-aspirinate-
Results and Discussion
Approach toward Characterizing the Hydrolysis of
ISDA. ISDA hydrolysis in human plasma solution is rapid (t1/2
) 1.1 min in 30% plasma solution), leading to a complex
mixture of aspirinate and salicylate metabolites as identified by
HPLC/PDA (the salicylates have a characteristic UV λmax at
around 305 nm).25 Around 60% aspirin is produced on the basis
of starting ester concentration of the diester. ISDA has four ester
bonds, one to each of the aspirin moieties (at the isosorbide 2
and 5 positions), as well as one acetyl group on each of the
aspirin moieties. Hydrolysis of ISDA can occur through a

DiscoVery of a “True” Aspirin Prodrug
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7993
Scheme 2. Synthesis of Isosorbide-2- and 5-Aspirinates 10 and
consumption of the aspirinate-salicylate pair 12 and 13,
implying that one or both of these acts as an aspirin prodrug. A
second curious feature of the progress curve was the predomi-
nance of the monosalicylate 14 over salicylic acid. Compound
14 can be generated from 11, 12, or 15. However, if compound
14 was formed from 15, an equimolar amount of salicylic acid
would be generated, suggesting that it was formed from either
11 or 12. Only small amounts of the 5-aspirinate (11) were
detected (∼10%) at several early time points, suggesting that it
was not the source of 14, although it remained possible that 11
wasprocessedmorerapidlythanitformed.Thesalicylate-aspirinate
pair 12 and 13 were consistently produced under a variety of
conditions in a ratio of 70:30 independent of the plasma
concentration (and therefore independent of the rate of disap-
pearance of 9). Because aspirin is a significant product following
ISDA incubation in plasma, we hypothesized that the more
significant salicylate-aspirinate metabolite, 12, was acting as
an aspirin prodrug. The substantial amount of 14 was consistent
with this observation. To investigate this possibility, 12 and 13
were incubated separately in human plasma. Kinetic data for
the disappearance of these and other potential aspirin-producing
metabolites (10-11) are presented in Table 1. The disappearance
profile in each case had the appearance of first-order kinetics,
which is typically the case for Michaelis-Menten processes at
11a
a (i) Acetylsalicoyl chloride, Et3N, DCM, RT, 8 h; (ii) H2, Pd/C, EtOAc/
MeOH, RT, 8 h.
2/5-salicylates (12 and 13) could not be prepared by this directed
acylation with salicylic acid because of competition between
the salicylic acid-OH group and the isosorbide-OH group.
Instead, salicylic acid was modified first by perbenzylation using
benzylbromide (Scheme 3). The benzylester was removed by
hydrolysis, leaving the phenolic benzylether intact. The disali-
cylate 15 was prepared by diesterification of isosorbide (17)
with 2 equiv of 24 and removal of both benzyl groups, again
by treatment under reductive conditions. Finally, the mixed
2-aspirinate-5-salicylate (12) and isomeric 5-aspirinate-2-sali-
cylate (13) were obtained by esterification of the corresponding
monoaspirinates (10 and 11) with 24 and removal of the benzyl
groups as before. All of the potential metabolites were charac-
terized appropriately.
Incubation Experiments with ISDA: Identification of
Prodrug Metabolite. ISDA was incubated in human plasma
solution in the range of 10-50% (pH 7.4, 37 °C), and the
evolution and decay of its primary and secondary hydrolysis
products monitored by HPLC using the synthetic external
reference standards (Table 1). ISDA undergoes rapid hydrolysis
at a high plasma concentration (50%) with apparently instan-
taneous evolution of aspirin. The apparent first-order rate
constant (kT) for the disappearance represents the combined rate
constants for the primary k1, k2, k3, and k4 processes (Scheme
1). We were unable to detect any isosorbide-2-aspirinate (10)
or the corresponding salicylate (16) at any time point, suggesting
that the k1 pathway does not play a significant role in the plasma-
mediated hydrolysis of ISDA or in aspirin production. This
interpretation, which was later vindicated, also ruled out the
hypothetical k5 pathway, which requires the availability of 10.
Hydrolysis of isosorbide-2-esters is very rapid, especially where
the 5 position is substituted, and the failure to detect 2-aspirinate
(10) or the corresponding salicylate ester (16) was not that
surprising.
low substrate concentration (and the rate constant relates to Vmax
/
Km). Compound 12 disappeared rapidly, with a half-life of <1
min in human plasma (50%), serum (50%) and whole blood,
producing 70-90% aspirin based on the initial concentration,
along with a corresponding amount of the monosalicylate
“carrier” 14 (Figure 4). The disalicylate compound (15) was
produced in small amounts (<15%), and it decayed rapidly
afterward. The carrier (14) and disalicylate were hydrolyzed
eventually to isosorbide and salicylic acid substances with well-
characterized safety profiles and good tolerability. It is likely
that this pattern would be followed in ViVo.
Compound 12, isosorbide-2-aspirinate-5-salicylate, is the most
successful aspirin prodrug discovered to date in the important
human plasma model. The isomeric 5-aspirinate-2-salicylate 13
did not produce aspirin at all, being exclusively hydrolyzed to
isosorbide disalicylate (15) along the k10 pathway. The other
prodrug candidates, the monoaspirinates 10 and11, were also
unproductive, being rapidly hydrolyzed to the corresponding
salicylates (a progress curve for the disappearance of 10 in
human plasma solution is presented in Figure 5). The apparent
Michaelis-Menten parameters in Table 1 were estimated by
nonlinear regression of the disappearance data for each com-
pound to the integrated form of the Michaelis-Menten equa-
tion.28 The KaMpp values for the disappearance of these esters is
difficult to interpret. In the case of 9, for example, the KMapp
represents a mean value for three parallel processes involving
three different productive presentations of the same compound
at the BuChE active site. Equally, whereas the concentration
of substrate decays below its KaMpp value, it generates an
equimolar set of species that undergo processing with similar
efficiency, so that active site occupancy remains constant over
the initial phase of the reaction. Nevertheless, the KaMpp values
provide us with a crude indication of the catalytic efficiency
for the hydrolysis processes and the relative affinities of the
The complex pattern of the hydrolysis progress for ISDA in
10% human plasma solution is illustrated in the progress curve
appearing in Figure 2. The principal initial hydrolysis products
were the isosorbide-2/5-aspirinate-salicylate isomeric pair 12
and 13, then the disalicylate 15, the monosalicylate 14, and later,
salicylic acid. We were surprised to find that aspirin production
from ISDA accelerated as the parent disappeared, with an
apparent burst late in the decay profile. This pulsative behavior,
which was reproducible in 10% plasma solution was not evident
at higher plasma concentration (30%), where the disappearance
of ISDA is extremely rapid (Figure 3). The appearance of aspirin
in the 10% plasma solutions seemed to correlate with the
substrate. The failure to detect any relationship between KMapp
app
max
or V
values and aspirin production for compounds 9-13
illustrates that one kinetic process is only marginally preferred
over the other even where aspirin production dominates.

7994 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Scheme 3. Synthesis of Salicylates 12-16a
Moriarty et al.
a (i) Acetylsalicoyl chloride, Et3N, DCM, RT, 8 h; (ii) H2, Pd/C, EtOAc/MeOH, RT, 8 h; (iii) DCC, DMAP, DCM, RT, 4 h; (iv) Cs2CO3, acetone,
evacuation, and then BnBr; (v) NaOH, H2O/MeOH, RT, 4 h.
Table 1. Kinetic Data for the Hydrolysis of ISDA (9) and Potential Aspirinate Metabolites 10-13 in Human Plasma Solution and for 12 in Other
Relevant Mediaa
compound
Km,app (M, ×104)
Vmax,app (M min-1
)
kobs (min-1
)
t1/2 (min)
aspirin yield (%)
9 (ISDA), 10% human plasma
9, 30% human plasma
9, 50% human plasma
10,10% human plasma
11, 10% human plasma
13, 10% human plasma
12, 10% human plasma
12, 50% human plasma
12, 50% human serum
12, whole blood
1.59
0.3
0.20 ( 0.04
0.59 ( 0.15
1.2 ( 0.35
0.21 ( 0.04
0.15 ( 0.04
0.16 ( 0.04
0.17 ( 0.05
1.39 ( 0.13
1.22 ( 0.29
0.79 ( 0.25
1.5 ( 0.56
3.35
1.17
0.57
3.25
4.73
4.21
4.9
0.49
0.56
0.96
0.47
51 ( 10
61 ( 4
0.61
1.11
0.91
0.81
0.69
0.93
1.41
1.34
0.22
0.25
0.23
0.17
0.36
0.85
1.49
1.75
3
nd
nd
74 ( 8
81 ( 11
73 ( 5
72 ( 13
nd
12, 10% rat plasma
a All measurements were performed in triplicate. The kobs range is the standard deviation for three independent measurements sometimes on different days
or with different donor plasma, and they reflect biological variation in BuChE activity and specificity. The r2 values for individual decay determinations
were >0.995 in all cases.
Identification of Human BuChE as Responsible for
Activation of Compound 12. There are a number of proteins
in human plasma with esterase activity, including BuChE,
paraoxonase (EC 3.1.8.1), trace amounts of acetylcholinesterase
(EC 3.1.1.7), and albumin, which, although not highly efficient,
is present at high concentrations (50-60 g/L).29,30 Human
plasma does not contain carboxylesterase.31 When compound
12 was incubated in rat plasma solution (10%), the exclusive
hydrolysis product was the disalicylate (15); there was no aspirin
production (Table 1). Rat plasma esterase activity is largely due
to carboxylesterases rather than cholinesterases.31 ISDA had
already been established as a substrate for human BuChE;
therefore, it seemed likely that its aspirin-producing metabolite
would be too. Compound 12 was nevertheless incubated in
human plasma solution in the presence of esterase subtype
inhibitors to confirm the identity of BuChE as the activating
enzyme (Table 2). The presence of EDTA (arylesterase), BNPP
(serine protease), or BW254c51 (0.1 µM, AChE) had little or
no effect on aspirin production. There was a significant decrease
in the rate of disappearance and aspirin production when the
latter experiment was repeated with BW254c51 (100 µM), at
which concentration it inhibits BuChE and AChE. Co-incubation
with the selective BuChE inhibitor iso-OMPA (10 µM) and
unselective cholinesterase inhibitor eserine (20 µM) caused a
complete blockade of aspirin production and a significant drop
in the hydrolysis rate. There was a significant drop in the
hydrolysis rate and aspirin production following incubation in
the presence of dibucaine in normal plasma but little difference
in plasma from a donor previously classified as having the
BuChE mutation conferring dibucaine resistance. Compound
12 was incubated in solution containing purified BuChE from
human serum and in the presence of horse serum BuChE.
Hydrolysis in the presence of the horse enzyme occurred at the
acetyl and benzoic acid sites in a ratio of around 60:40. The
purified human serum enzyme catalyzed hydrolysis almost
exclusively toward aspirin, with only around 5% salicylate
formation (Figure 6). The exceptionally high specificity of the
purified BuChE suggests that there were competing nonproduc-
tive processes occurring in the plasma and serum experiments.
Indeed, in the presence of the arylesterase inhibitors, there was

DiscoVery of a “True” Aspirin Prodrug
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7995
Figure 2. Progress curve for the disappearance of ISDA (9) (b) in
10% human plasma at 37 °C, showing the in Vitro metabolites: aspirin
(9), isosorbide-5-aspirinate (11) (×), isosorbide-2-aspirinate-5-salicylate
12 (2), isosorbide-5-aspirinate-2-salicylate 13 (1), salicylic acid, 3 (O),
isosorbide-5-salicylate, 14, (4), and isosorbide disalicylate, 15 (]). The
solid line for ISDA disappearance was calculated from exponential
decay, using the rate constant appearing in Table 1. The data represent
the average from three separate experiments using plasma from three
different donors.
Figure 4. Progress curve for the disappearance of 12 (b) in 50%
human plasma buffered at pH 7.4 (37 °C), showing the in Vitro
metabolites: aspirin, 4 (9), isosorbide-5-salicylate, 14 (4), salicylic acid,
3 (O), and isosorbide disalicylate, 15 (]). Note the stability of 14
relative to 12. Solid lines were constructed using the appropriate rate
constants appearing in Table 3 and eqs 4-6.
act as a prodrug, without having to generate 12? This was not
a straightforward question because 12 is generated from 9 and
consumed rapidly, producing aspirin. As already stated, small
amounts (<10%) of the 5-monoaspirinate 11 were found at
initial time points in 10% plasma solution, indicating some direct
hydrolysis of 9 to aspirin. The only other way that 11 could be
generated is from the mixed salicylate 13, but when 13 was
incubated separately in human plasma solution, it did not
produce 11, indicating that, in plasma solution, 11 is produced
directly from ISDA, with an equimolar amount of aspirin
(<10%). Another way of investigating the production of aspirin
from ISDA would be to determine how much of its hydrolysis
could be accounted for by alternative processes to salicylate
production.
Figure 3. Progress curve for the disappearance of ISDA, 9, (b) in
30% human plasma at 37 °C, showing the in Vitro metabolites: aspirin
(9), the pair 12 + 13 (0), isosorbide-2-aspirinate-5-salicylate, 12 (2),
isosorbide-5-aspirinate-2-salicylate, 13 (1), salicylic acid, 3 (O),
isosorbide-5-salicylate, 14, (4), and isosorbide disalicylate, 15 (]).
Solid lines were constructed using rate constants appearing in Table 3
and eqs 1-3.
ISDA decays through three pathways, k2, k3, and k4, and
therefore
-d[9]/dt ) kT[9] ) k2[9] + k3[9] + k4[9]
(1)
Because 12 decays through the k7 and k9 pathways, leading to
aspirin and disalicylate (15)
marginally greater aspirin release, suggesting that an ary-
lesterase, probably paraoxonase, contributes to nonproductive
hydrolysis in human plasma. The hydrolysis pathways of 12
were similar in human plasma, serum, and whole blood, with
the esterase activity of the latter being substantially a result of
the presence of BuChE. It is hard to see how 12 could be further
optimized for aspirin release in human plasma using BuChE as
the vector because BuChE is almost completely specific for the
benzoic ester of 12. On the other hand, designs that target other
plasma esterases, such as paraoxonase, must still overcome the
high efficiency with which acetyl group detachment is catalyzed
by BuChE.
Is ISDA (9) Intrinsically an Aspirin Prodrug? Compound
12 acts as an aspirin prodrug because its interaction with BuChE
overrides the normal preference of the enzyme for the pheny-
lacetate group. This interaction is connected with a specific set
of structural attributes of the prodrug; for example, the isomeric
2-salicylate-5-aspirinate (13) is not an aspirin prodrug. We were
also aware that isosorbide-2-aspirinate (10) (unsubstituted at the
5 position) was not an aspirin prodrug being hydrolyzed in
plasma solution along the salicylate pathway (Figure 5). This
indicated to us that the 5-salicylate ester of 12 is critical for
productive binding, leading to aspirin evolution. ISDA is
hydrolyzed first at the acetyl group of the 5-aspirinate, producing
12, which acts as a prodrug in human plasma. Does ISDA itself
d[12]/dt ) k2[9] - k7[12] - k9[12]
(2)
Similarly, 13 decays exclusively through the k10 pathway and
therefore
d[13]/dt ) k3[9] - k10[13]
(3)
By estimating k2 and k3 from nonlinear regression to integrated
forms of eqs 2 and 3, it was possible to estimate k4, the rate
constant for aspirin production directly from ISDA. In calculat-
ing these kinetic parameters, kT was estimated from the
disappearance of 9 in 30% plasma solution (Table 3). Initial
values for the disappearance of esters 12 and 13 were estimated
from the rate of hydrolysis independent of 9. The difference
between the sum of the rate constants for the appearance of 12
and 13 and the disappearance rate for ISDA yielded a small
value for k4, indicating that around 7% of 9 undergoes hydrolysis
directly to aspirin (Table 3). ISDA is an intrinsic aspirin prodrug
in plasma but a poor one. It probably fits the enzyme quite well
considering its similarity to 12, but it bears two acetates and
therefore has two competing processes rather than one, as is
the case for 12.
Interpretation of hydrolysis data for 12 is kinetically simpler
because it has one less ester group than 9 and its principal
product, aspirin, is not consumed as rapidly (Scheme 4). In
addition, there was no detectable hydrolysis at the 5-ester over

7996 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Moriarty et al.
Table 2. Data for the Hydrolysis of 12 in 30% Human Plasma (pH 7.4, 37 °C) in the Presence of Esterase Subtype Inhibitorsa
inhibitor
concentration
mM
10 µM
20 µM
100 µM
0.1 µM
20 µM
target enzyme
kobs (min-1
)
t1/2 (min)
% aspirin
EDTA32
2
arylesterase
carboxylesterase
cholinesterase
AChE
0.49 ( 0.08
1.41
0.95
5.8
3.9
1.1
85
84
BNPP33
0.73 ( 0.006
0.12 ( 0.001
0.18 ( 0.001
0.66 ( 0.064
0.15 ( 0.013
0.35 ( 0.07
eserine34
not detected
30
74
BW284c5135
dibucaine36
BuChE subtype
BuChE
4.6
6
2.0b
7.04
69b
iso-OMPA37
10 µM
0.098( 0.003
not detected
a Average of three determinations ( standard deviation (SD). b Plasma from a donor with dubucaine-insensitive BuChE.
Table 3. Rate Constants for the Hydrolysis of 9 and 12 in Human Plasma (pH 7.4, 37°C)a
compound
kobs
k2
k3
k4
k2+ k3/kobs
k7′
k9′b
0.36 ( 0.06
k7′/kobs
9c
0.591( 0.05
1.39 (0.11
0.39 ( 0.02
0.16 (0.02
0.028 ( 0.01
0.93
12d
nr
nr
nr
1.0( 0.06
0.72
a Data are from a single representative progress curve. r2 > 0.999. b r2 > 0.95. c A 30% plasma solution. d A 50% plasma solution.
Figure 5. Progress curve for the disappearance of isosorbide-2-
Figure 6. Progress curve for the disappearance of 12 (b) in the
presence of BuChE purified from human serum buffered at pH 7.4 (37
°C), showing the in Vitro metabolites: aspirin (9), isosorbide-5-
salicylate, 14 (4), salicylic acid, 3 (O), and isosorbide disalicylate, 15
(]). The data are for one representative run. The ratio of aspirin/
disalicylate (15) at Cmax for each was 95:5.
aspirinate (10) (b) in 10% human plasma solution at pH 7.4 (37 °C),
showing the in Vitro metabolites: aspirin (0), isosorbide-2-salicylate,
16 (1), and salicylic acid, 3 (O). The solid line was constructed using
rate constants appearing in Table 1.
Table 4. IC50 Values for Inhibition of Platelet Aggregation in Whole
Blood Stimulated by Arachidonic Acid (0.5 mM), n ) 3
Scheme 4. Actual Hydrolysis Routes of ISDAa
compound
IC50, 95% CL (µM)
9
12
aspirin
34.1 (28.2-44.3)
17.3 (15.9-18.8)
25.5 (20.1-31.7)
the time course of the experiment; it does not contribute to the
disappearance of 12. The kobs for the consumption of 12 (kT′)
corresponds to the sum of the rate constants for deacetylation
to 15, k9′ and the larger rate constant for hydrolysis to aspirin,
k7′ (where kT′, k7′, and k9′ are rate constants determined for the
hydrolysis of 12 independent of 9).
a The 5-acetyl group is removed first (65%), generating an effective
prodrug (12). The other primary route (k3) involving 2-acetyl group
hydrolysis does not lead to aspirin generation because 13 is not an aspirin
prodrug.
-d[12]/dt ) kT′[12] ) k9′[12] + k7′[12]
(4)
Similarly, the disappearance of the disalicylate (15), following
incubation of 12, occurs along the k12′ and to a small extent the
k13′ pathway
constant for 12 disappearance, in accordance with the estimate
from extrapolation of maximum aspirin during the disappearance
of 12. The data appearing in Scheme 4 is based on the
assumption that the disappearance kinetics for 12 are similar in
the presence of ISDA.
Platelet Aggregation Studies. One of the hallmark pharma-
cological effects of aspirin is its ability to interfere with platelet
function through acetylation of platelet COX-1. Compound 12,
aspirin, and ISDA (9) were evaluated as inhibitors of platelet
aggregation stimulated by arachidonic acid (0.5 mM) using the
whole blood impedance method.38 Compound 12 was signifi-
cantly more potent than ISDA in accordance with its enhanced
d[15]/dt ) k9′[12] - k12′[15] - k13′[15]
(5)
Aspirin evolution and decay are through the k7 pathway and
the slow decay to salicylic acid, k14
d[4]/dt ) k7′[12] - k14′[4]
(6)
The value for k7′ obtained in this way was in good agreement
with the product of kT′ and the maximum mole percent of aspirin
extrapolated from the progress curve for 12 (Figure 4).
Meanwhile, the k7′ value accounts for 72% of the total rate

DiscoVery of a “True” Aspirin Prodrug
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7997
m), 4.64 (1H, d), 5.04 (1H, t), 5.45 (2H, m), 6.92 (1H, t), 7.02
(1H, d), 7.12 (1H, d), 7.32 (1H, t), 7.49 (1H, m), 7.58 (1H, m),
7.89 (1H, dd, J ) 1.5 and 1.5 Hz), 8.01 (1H, dd, J ) 1.5 and 1.5
Hz). Anal. Calcd (C22H20O9): C, H.
ability to generate aspirin (Table 4). Puzzling, 12 was also
marginally (but significantly) more potent than aspirin. Com-
pound 10, which did not release aspirin in the plasma hydrolysis
experiment was not active in this assay (<30% inhibition at
100 µM), indicating that platelet inhibition by 12 and ISDA
(9) is due to aspirin release.
Isosorbide-2-salicylate-5-aspirinate (13). A solution of isos-
orbide-5-aspirinate-2-benzyloxy benzoate (27) (0.13 g, 0.25 mmol)
in a mixture of methanol and ethyl acetate (5 mL, 3:1) was stirred
overnight over palladium on charcoal under an atmosphere of
hydrogen. The reaction mixture was filtered through a bed of silica,
and the filtrate was removed in Vacuo to yield 0.05 g of crude
product. Purification by column chromatography using hexane and
ethyl acetate (2:1) as the eluant afforded 0.02 g of product as a
colorless crystalline material (18.69%). mp 84-86 °C. HRMS [M
Conclusions
Compound 12 is the first “true” aspirin prodrug in BuChE
solutions, and it produces more aspirin in human plasma than
any other prodrug discovered to date. It does so because of an
unusually favorable interaction with BuChE in human plasma.
These observations remind us that seemingly insurmountable
esterase preferences can be overcome with appropriate substrate
complementarity. A pseudo-sugar is probably the last place one
would start in pursuit of a cholinesterase substrate type; however,
BuChE does not have an established endogenous substrate or
ligand, and the esters reported here are more successful
substrates than the ostensibly more promising pseudo-choline
esters reported by Nielsen and Bundgaard.18 Compound 12 will
be tested in appropriate preclinical models of platelet inhibition
and then gastric toxicity. One of the challenges will be to
develop and validate an animal model that mimics the ability
of human blood to convert 12 to aspirin (which excludes, for
example, the rat). Without aspirin generation, an ester can be
expected to be safe but not efficacious.
1
+ Na]+: requires, 451.1005; found, 451.1006. H NMR (CDCl3)
δ: 2.36 (3H, s), 4.03 (4H, m), 4.67 (1H, d), 5.02 (1H, t), 5.38 (1H,
m), 5.49 (1H, d), 6.87 (1H, t), 6.98 (1H, d), 7.12 (1H, d), 7.34
(1H, t), 7.47 (1H, m), 7.58 (1H, m), 7.83 (1H, dd, J ) 1.5 and 1.5
Hz), 8.08 (1H, dd, J ) 2.0 and 1.5 Hz), 10.50 (1H, s). Anal. Calcd
(C22H20O9): C, H.
Hydrolysis and Enzyme Studies. High-performance liquid
chromatography (HPLC) was performed using a system consisting
of a Waters 600E pump and controller, a Waters 717 autosampler,
and a Waters (2)996 PDA, all controlled by Millennium or Alliance
Chromatography Manager. A Waters Spherisorb (4 µm) C18 3.9
× 250 mm column was used for the plasma and enzyme study
samples. The enzyme and plasma study samples were analyzed
using a gradient method, employing a mobile phase consisting of
pH 3.19 phosphate buffer/acetonitrile at 90:10, grading to 10:90
over the first 10 min, then back to 65:35 to 12 min, and grading to
90:10 to 17 min, at which it was held to 30 min. The eluent in
both methods was monitored at 230 nm, and peak identity and
homogeneity were confirmed by PDA analysis. Quantitation was
performed by a comparison of peak areas with external standards
run under the same conditions at about the same concentration.
The order of elution in this system was salicylic acid (3), aspirin
(4), isosorbide-2-aspirinate (10), isosorbide-5-aspirinate (11), isos-
orbide-2-salicylate (16), isosorbide-5-salicylate (14), isosorbide-2/
5-aspirinate-2/5-salicylate (12/13), and isosorbide-disalicylate (15).
The flow rate was 1 mL/min. ISDA incubation samples were re-
analysed using an alternative isocratic method, with a mobile phase
consisting of 60% phosphate buffer (pH 2.4)/40% acetonitrile. This
permitted the separation of 12 and 13. Both methods (gradient and
isocratic) were validated for linearity, precision (repeatability),
specificity, and sensitivity in accordance with ICH guidelines on
analytical validation Q2A and Q2B. A linear response was observed
for each analyte (r > 0.999) in the range of 1-100 µg/mL. The
RSD on multiple injection of each analyte at 10 and 100 µg/mL
was <0.75%. The limit of quantitation for the relevant analytes in
the gradient method was 1 µg/mL. The limit of quantitation for
aspirin and salicylic acid in the isocratic method used in the aqueous
hydrolysis study samples was 5 µg/mL, while the prodrug 12 was
quantifiable at 0.5 µg/mL using this method.
For the hydrolysis experiments, pooled plasma or serum solutions
(4 mL) were prepared by centrifugation of citrated human venous
blood and dilution with phosphate buffer at pH 7.4, as appropriate.
Whole blood was used undiluted. The test compounds in acetonitrile
(100 µL) were incubated in 4 mL of the preheated solution (37 (
0.5 °C) at a concentration of 5 × 10-5 M, and 250 µL aliquots
were withdrawn at appropriate intervals. Samples were transferred
to 1.5 mL Eppendorf tubes containing 500 µL of a 2% ZnSO4 ·7H2O
in MeCN/H2O (1:1) solution, vortexed, and then centrifuged for 4
min at 10 000 rpm. Aliquots (20 µL) of the supernatant were
analyzed by HPLC. Hydrolysis was monitored until consumption
of the parent ester. The hydrolysis experiments were repeated in
the presence of the following inhibitors, which were incubated in
the buffered plasma solution for 5 min before addition of the ester
solution: EDTA (2 mM), BNPP (10 µM), eserine (20 µM),
BW254c51 (0.1-100 µM), dibucaine (20 µM), iso-OMPA (10 µM).
The samples were then processed as above. All experiments were
performed in triplicate. Cholinesterase activity of plasma, serum,
and whole blood was evaluated using the Ellman approach, with
Experimental Section
Chemistry. Infrared (IR) spectra were obtained using a Perkin-
Elmer 205 FT Infrared Paragon 1000 spectrometer. Band positions
are given in cm-1. Solid samples were obtained by KBr discs, and
oils were analyzed as neat films on NaCl plates. 1H and 13C spectra
were recorded at 27 °C on a Brucker DPX 400 MHz FT NMR
1
spectrometer (400.13 MHz, H; 100.61 MHz, 13C) or a Brucker
AV600 (600.13 MHz, 1H; 150.6 MHz, 13C) in either CDCl3 or
(CD3)2CO with TMS as an internal standard. In CDCl3, 1H spectra
were assigned relative to the TMS peak at 0.0 ppm and 13C spectra
were assigned relative to the middle CDCl3 triplet at 77.00 ppm.
1
In (CD3)2CO, H spectra were assigned relative to the (CD3)2CO
peak at 2.05 ppm and 13C spectra were assigned relative to the
(CD3)2CO at 29.5 ppm. Coupling constants were reported in hertz.
High-resolution mass spectrometry (HRMS) was performed using
a micromass mass spectrophotometer with electrospray ionization
at the School of Chemistry, Trinity College Dublin, Ireland.
Elemental analyses were performed at the Microanalytical Labora-
tory, Department of Chemistry, University College Dublin, Ireland.
Flash chromatography was performed on Merck Kieselgel (particle
size of 60 mm). Thin-layer chromatography (TLC) was performed
on silica gel Merck F-254 plates. Compounds were visually detected
by UV absorbance at 254 nm. ISMN was obtained as a gift from
Shwartz Pharma, Shannon Co Clare. Compound 24 was obtained
according to ref 39.
Isosorbide-2-aspirinate-5-salicylate (12). Compound 24 (1.6
mmol) was dissolved in dry DCM (20 mL) and stirred. Isosorbide-
2-aspirinate 10 (1.6 mmol) and DMAP (0.1 equiv) were added.
The flask was cooled to 0 °C, and DCC (1.6 mmol) was added.
Stirring was continued for 5 min, and the temperature was allowed
to come to room temperature, where it was stirred overnight. The
reaction was filtered, and the filtrate was washed with 0.1 M HCl,
5% NaHCO3, and water, dried over sodium sulfate and evaporated
to an oil. This was purified by column chromatography hexane/
ethyl acetate (2:1) to give a white product (Rf ) 0.4, 228 mg).
This was dissolved in methanol/ethyl acetate (1:1). Pd/C was added,
and the reaction was stirred under hydrogen overnight. The reaction
was filtered and concentrated. Oil was purified by column chro-
matography using hexane/ethyl acetate (1:1) to yield a white solid.
mp 82-84 °C. LRMS: requires, 451.0984 (M+ + 23); found,
451.0971 (M+ + 23). 1H NMR (CDCl3) δ: 2.37 (1H, s), 4.10 (4H,

7998 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Moriarty et al.
(6) (a) Ankersen, M.; Nielsen, K. K.; Senning, A. Synthesis, properties
and prodrug potential of 2-methyl-2-oxy- and 2-methyl-2-thio-4H-
1,3-benzodioxinones. Acta Chem. Scand. 1989, 34, 213–221. (b)
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prodrug potential. Acta Chem. Scand. 1990, 44, 952–956.
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of acylals of aspirin: Hydrolysis of (1′-ethoxy)ethyl 2-acetoxybenzoate.
J. Pharm. Sci. 1974, 63, 627–628. (b) Hussain, A.; Truelove, J. E.;
Kostenbauder, H. B. Kinetics and mechanism of hydrolysis of 1-(2′-
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J. Pharm. Sci. 1979, 68, 299–301.
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butyryl thiocholine as the substrate. Values were typically between
2200 and 4000 nmol (mL of plasma)-1 min-1. The hydrolysis of
12 was evaluated in the presence of purified horse serum BuChE
(Sigma) at a concentration of 0.1 mg/mL (1000 units/mg of protein)
in phosphate buffer (pH 7.4) and in the presence of purified human
serum BuChE at 0.1 mg/mL (14 units/mg of protein) (Sigma). The
activity of these preparations was confirmed using the Ellman assay,
with butyrylthiocholine as the substrate.40
app
max
The parameters KaMpp (Michaelis constant) and V (maximum
rate of substrate consumption) for the hydrolysis of 12 were
estimated by fitting depletion data to the integrated form of the
Michaelis-Menten equation28 by multiple nonlinear least-squares
regression using Scientist, MicroMath (Salt Lake City, UT)
Vmaxt ) So - S + KM ln(So/S)
(7)
Rate constants for the hydrolysis of esters were obtained by
nonlinear regression of the concentration of remaining ester against
(10) (a) Rainsford, K. D.; Whitehouse, M. W. Anti-inflammatory/anti-
pyretic salicylic acid esters with low gastric ulcerogenic activity. Agents
Actions 1980, 10, 451–456. (b) Rainsford, K. D.; Whitehouse, M. W.
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time to exponential decay. Half-lives were calculated from t1/2
)
0.693/kobs. Integrated forms of eqs 1-6 were used to determine
the rate constants appearing in Table 3 and to generate the solid
lines appearing in Figure 3 for ISDA hydrolysis41
(11) (a) Kumar, R.; Billimoria, J. D. Gastric ulceration and the concentration
of salicylate in plasma in rats after administration of 14C labelled
aspirin and its synthetic triglyceride, 1,3-dipalmitoyl-(2′-acetoxy-
k2[9]0
[12] )
(e-(k +k )t - e-k t
)
(8)
(9)
7
9
T
[
14C]carboxylbenzoyl) glycerol. J. Pharm. Pharmacol. 1978, 30, 754–
kT - (k7 + k9)
k3[9]0
758. (b) Paris, G. Y.; Garmaise, D. L.; Cimon, D. G. Synthesis and
anti-inflammatory activity of 1,3-bis(alkanoyl)-2-(O-acetylsalicyloyl)g-
lycerides (aspirin triglycerides). J. Med. Chem. 1979, 22, 683–687.
(c) Paris, G. Y.; Garmaise, D. L.; Cimon, D. G.; Swett, L.; Carter,
G. W.; Young, P. Glycerides as prodrugs. 2. 1,3-Dialkanoyl-2-(2-
methyl-4-oxo-1,3-benzodioxan-2-yl) glycerides (cyclic aspirin trig-
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82.
[13] )
(e-k t - e-k t
)
10
T
kT - k10
The following were used to generate the solid lines in Figure 4,
for hydrolysis of 12, separately:
k9′[12]0
(12) Los, M.; Piccinali, C. A.; Tosti, E. L.; Torriani, H. Sintesis de nuevos
derivados de los acidos 2-hidroxi (y) 2-acetiloxibenzoico. Boll. Chim.
Farm. 1982, 121, 285–302.
12′+k13
(e-(k
′)t - e-(k ′t)
)
(10)
(11)
T
[15] )
kT′ - (k12′ + k13′)
k7′[12]0
(13) (a) Lofttson, T.; Bodor, N. Improved delivery through biological
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Carboxypeptidase A hydrolysis of aspirin derivatives. J. Pharm. Sci.
1981, 70, 1307–1309. (b) Banerjee, P. K.; Amidon, G. L. Physico-
chemical property modification strategies based on enzyme substrate
specificities II: R-Chymotrypsin hydrolysis of aspirin derivatives.
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G. L. Physicochemical property modification strategies based on
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(e-k ′t - e-k ′t
)
14
T
[4] )
kT′ - k14′
Nonlinear regression to eqs 8-11 was performed using GraphPad
Prism5 (La Jolla, CA).
Whole Blood Aggregation. A 500 µL aliquot of whole blood
was mixed with 500 µL of physiological saline and allowed to
incubate at 37 °C for 10 min in the incubation well of a Chrono-
Log Whole Blood Aggregometer model 591/592. Aggregation was
initiated with arachidonic acid (0.5 mM), and impedance was
monitored over 6 min. During inhibition studies, the diluted blood
sample was pre-incubated with appropriate concentrations of
inhibitor at 37 °C for 10 min, with stirring before initiating
aggregation. Test compounds (10 µL) were introduced in DMSO
(a DMSO level shown to have no effect on platelet function).
Inhibition of aggregation was monitored in the range of 5-100
µM, and results were analyzed by nonlinear regression using
GraphPad Prism5.
Supporting Information Available: Characterization and purity
data for compounds 10, 11, 14, 15, 16, 20, 21, 23, and 25 and
platelet aggregation data for 9, 12, and aspirin. This material is
available free of charge via the Internet at http://pubs.acs.org.
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