7998 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
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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.;
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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.
Gastric irritancy of aspirin and its congeners: Anti-inflammatory
activity without this side-effect. J. Pharm. Pharmacol. 1976, 28, 599–
601.
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-
lycerides) as anti-inflammatory agents. J. Med. Chem. 1980, 23, 79–
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
membranes XD: Percutaneous absorption and metabolism of meth-
ylsulfinylmethyl 2-acetoxybenzoate and related aspirin prodrugs.
J. Pharm. Sci. 1981, 70, 756–758. (b) Lofttson, T.; Bodor, N. Improved
delivery through biological membranes IX: Kinetics and mechanism
of hydrolysis of methylsulfinymethyl 2-acetoxybenzoate and related
aspirin prodrugs. J. Pharm. Sci. 1981, 70, 750–755. (c) Lofttson, T.;
Kaminski, J. J.; Bodor, N. Improved delivery through biological
membranes VIII: Design, synthesis, and in vivo testing of true prodrugs
of aspirin. J. Pharm. Sci. 1981, 70, 743–749.
(14) (a) Banerjee, P. K.; Amidon, G. L. Physicochemical property
modification strategies based on enzyme substrate specificities III:
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.
J. Pharm. Sci. 1981, 70, 1304–1306. (c) Banerjee, P. K.; Amidon,
G. L. Physicochemical property modification strategies based on
enzyme substrate specificities I: Rationale, synthesis, and pharmaceuti-
cal properties of aspirin derivatives. J. Pharm. Sci. 1981, 70, 1299–
1303. (d) Muhi-Eldeen, Z.; Kawahara, M.; Dakkuri, A.; Hussain, A.
Kinetics and mechanism of hydrolysis of aspirin phenylalanine ethyl
ester. Int. J. Pharm. 1985, 261, 15–23.
(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
(15) Tsunematsu, H.; Ishida, E.; Yoshida, S.; Yamamoto, M. Synthesis and
enzymatic hydrolysis of aspirin-basic amino acid ethyl esters. Int.
J. Pharm. 1991, 68, 77–86.
(16) Jaffar, M.; Everett, S. A.; Naylor, M. A.; Moore, S. G.; Ulhaq, S.;
Patel, K. B.; Stratford, M. R.; Nolan, J.; Wardman, P.; Stratford, I. J.
Prodrugs for targeting hypoxic tissues: regiospecific elimination of
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