Hydrolysis mechanisms for indomethacin and acemethacin in perchloric acid

Article abstract of DOI:10.1021/jo052561k

The acid-catalyzed hydrolysis reactions of the antiinflammatory drugs indomethacin and acemethacin were investigated at 25.0 °C in a number of strongly concentrated perchloric acid media. The reaction rates were evaluated by UV measurements, and the intermediate species were detected by UV-vis, ¹H NMR, ¹³C NMR, and mass spectroscopy measurements. A switchover from an A-2 to an A-1 mechanism as a function of the medium acidity is reported for the acid-catalyzed hydrolyses of the amide group of both indomethacin and acemethacin. In the A-2 hydrolysis, two water molecules are involved in the rate-determining step. An analysis of the kinetic data collected for acemethacin by the different techniques used reveals a complex mechanism, indomethacin being a metabolite intermediate species in the hydrolysis of acemethacin. The rate constants for the hydrolysis of the acemethacin ester group were considerably larger compared to those of the amide group.

Full text of DOI:10.1021/jo052561k

Hydrolysis Mechanisms for Indomethacin and Acemethacin in
Perchloric Acid
B. Garc´ıa,* F. J. Hoyuelos, S. Ibeas, and J. M. Leal
UniVersidad de Burgos, Departamento de Qu´ımica, 09001 Burgos, Spain
begar@ubu.es
ReceiVed December 13, 2005
The acid-catalyzed hydrolysis reactions of the antiinflammatory drugs indomethacin and acemethacin
were investigated at 25.0 °C in a number of strongly concentrated perchloric acid media. The reaction
1
rates were evaluated by UV measurements, and the intermediate species were detected by UV-vis, H
NMR, 13C NMR, and mass spectroscopy measurements. A switchover from an A-2 to an A-1 mechanism
as a function of the medium acidity is reported for the acid-catalyzed hydrolyses of the amide group of
both indomethacin and acemethacin. In the A-2 hydrolysis, two water molecules are involved in the
rate-determining step. An analysis of the kinetic data collected for acemethacin by the different techniques
used reveals a complex mechanism, indomethacin being a metabolite intermediate species in the hydrolysis
of acemethacin. The rate constants for the hydrolysis of the acemethacin ester group were considerably
larger compared to those of the amide group.
SCHEME 1. Indomethacin (1) and Acemethacin (2)
Introduction
The indole moiety is a fundamental constituent for a number
of both natural and synthetic compounds with biological activity.
This functional group represents probably one of the most
important structural types in drug development;1 pharmaceutical
compounds such as nonsteroidal antiinflammatories (NSAIDs)
or analgesic and antipyretic drugs such as indomethacin (1) and
acemethacin (2) (Scheme 1) contain the indole ring. These
compounds are widely used in the treatment of pain, arthritis,2
cardiovascular3 and Alzheimer4 diseases, and cancer prevention;5
however, despite the adverse side effects reported, especially
those related to the gastrointestinal tract,6 so far the use of
NSAIDs drugs has become widespread.
Acemethacin can be synthesized by hydroxyacetic acid
esterification of indomethacin (1-(4-chlorobenzoyl)-5-methoxy-
(1) Smith, A. L.; Stevenson, G. I.; Swain, C. J.; Castro, J. L. Tetrahedron
Lett. 1998, 39, 8317.
(2) Schuna, A. A. J. Am. Pharm. Assoc. 1998, 38, 728.
(3) (a) Goodnight, S. H. Curr. Opin. Hematol. 1996, 3, 355. (b)
Rodriguez, L. A.; Varas, C.; Patrono, C. Epidemiology 2000, 11, 382.
(4) (a) Sloane, P. D. Am. Fam. Physician 1998, 58, 1577. (b) Flynn, B.
L.; Theesen, K. A. Ann. Pharmacother. 1999, 33, 840.
(5) (a) Shiff, S. J.; Rigas, B. Gastroenterology 1997, 113, 1992. (b) Jones,
M. K.; Wang, H.; Peskar, B. M.; Levin, E.; Itani, R. M.; Sarfels, I. J.;
Tarnawski, A. S. Nat. Med. 1999, 5, 1418.
(6) Matos, C.; Lima, J. L. C.; Reis, S.; Lopes, A.; Bastos, M. Biophys.
J. 2004, 2, 946.
10.1021/jo052561k CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/14/2006
3718
J. Org. Chem. 2006, 71, 3718-3726

Hydrolysis Mechanisms for Indomethacin and Acemethacin
SCHEME 2
evolves in the rate-determining unimolecular step to some
intermediate species A+, which subsequently reacts speedily to
products. The criteria employed to elucidate the A-1 mechanism
are described next.
(a) The Zucker-Hammett criterion.10 The reaction rate
defined as the disappearance of the total substrate concentration
can be expressed in the form
2-methyl-1H-indole-3-acetic acid). Despite the close similarity
of their UV-vis absorption curves, which prevent the spectral
features from a clear distinction between the two compounds,
in a previous paper we have evaluated the protonation constant
of the indomethacin amide group in perchloric acid medium
d(cs + cSH
dt
)
fSH
fq
+
+
υ ) -
) kobs(cs + cSH+) ) k0,1 cSH
(4)
+
+
where fSH and fq represent the activity coefficients of the
+
(pKInH ) -4.0) and the two constants for the protonation of
protonated substrate and transition state, respectively. From the
+
+
the acemethacin ester (pKAcH ) -2.0) and amide (pKAcH
)
+
2
mathematical definition of the KSH constant, eq 1, and the
-4.2) groups;7 although N-protonation has been suggested for
the amide group,8 it is generally agreed that protonation occurs
at the oxygen site.7 In addition to basic hydrolyses of in-
domethacin and acemethacin,9 acidic hydrolyses are also
feasible; to properly elucidate the latter mechanisms, the kinetic
study of the acid-catalyzed hydrolyses of the two drugs has been
undertaken by monitoring spectrophotometrically the reaction
rates in different perchloric acid concentrations, the intermediates
Hammett acidity function H0,14 eq 5 is arrived at when cSH
>
+
cS, that is, when the predominantly protonated substrate prevails
at high acidity levels.
fSH
+
log kobs ) log k0,1 + log
(5)
(
)
fq
Therefore, if, for an A-1 mechanism, the hydrolysis follows
1
and reaction products both being analyzed by UV-vis, H
+
the reaction in Scheme 2 and the ratio fSH /fq ≈ 1 for all acid
NMR, 13C NMR, and mass spectroscopy.
concentrations, then the kobs values become independent of the
medium acidity and will remain constant.
A number of different methods are available to deal with the
treatment of kinetic data at medium and high acidity levels; to
reliably determine the reaction mechanisms, the following
methods have been used: Zucker-Hammett,10 Bunnett,11
LFER,12 and Cox-Yates13 (or excess acidity) methods; the three
first treatments introduce the medium acidity in terms of the
Hammett acidity function H0,14 whereas the last one does so in
terms of the excess acidity function, X.15,16 To discern the
reaction order on the basis of reacting species different from
the proton, Ingold referred to the acid-catalyzed hydrolysis
reactions as A-1 and A-2 mechanisms.17 Over the last years, a
number of methods have been put forward to differentiate
between the A-1 and A-2 mechanisms; these are applicable at
high and low acidity levels, respectively, and will be reflected
hereafter. Depending on the relative concentrations of the
protonated (SH+) and nonprotonated (S) substrate forms, two
different situations can be differentiated in each of the A-1 and
A-2 mechanisms.
(b) Linear Free Energy Relationship (LFER). Bunnett and
Olsen11,18 extended the linear free energy relationships to acid-
catalyzed reactions,19 obtaining the equation
k0,1
log kobs + H0 ) φ(H0 + log cH+) + log
(6)
KSH
+
where the solvation parameter φ represents the response of the
equilibrium to the change in the medium, that is, increase in
cH and subsequent decrease in aH 0, and measures the effects
+
2
brought about by the changes of hydration. If primary anilines
are taken as reference bases (φ ) 0),19 then positive φ values
are expected for bases with higher solvation requirements and
negative when the hydration of SH+ is comparatively low.
Hence, the parameter φ fluctuates between -1 and +1 for the
A-1 mechanism and is evaluated as the sum of (1) the φe
contribution, related to the protonation equilibrium, and (2) the
φq contribution, more sensitive to changes in the mechanism,
related to the rate-determining step. Lucchini et al.20 have
detailed the equations that correlate the rate constants with the
medium acidity through the free-energy relationships, arriving
at the following general equation:
A-1 Mechanism. If the starting material is the S species, that
is, the predominantly unprotonated substrate form, then the
mechanism shown in Scheme 2 applies.
The reaction Scheme 2 involves a fast proton-transfer
preequilibrium of the substrate S to give SH+; this species
(7) Hoyuelos, F. J.; Garc´ıa, B.; Ibeas, S.; Mun˜oz, M. S.; Navarro, A.
M.; Pen˜acoba, I.; Leal, J. M. Eur. J. Org. Chem. 2005, 1161.
(8) Liler, M. Reaction mechanisms in sulphuric acid; Academic Press:
London, 1971; p 189.
(9) a)Archontaki, H. A. Analyst 1995, 120, 2627. (b) Hajratwala, B. R,
Dawson, J. E. J. Pharm. Sci. 1977, 66, 27. (c) Cipiciani A.; Ebert, C.; Linda,
P.; Rubessa, F.; Savelli, G. J. Pharm. Sci. 1983, 72, 1075-1076. (d) Arcos,
J.; Lopez-Palacios, J.; Leal, J. M.; Sanchez Batanero, P.; Mata. F. Bull.
Soc. Chim. Fr. 1991, 128, 314. (e) Alibrandi, G.; Coppolino, S.; D’Aliberti,
S.; Ficarra, P.; Micali, N.; Villari, A. J. Pharm. Sci. 2003, 92 (8), 1730.
(10) Zucker, L.; Hammett, L. P. J. Am. Chem. Soc. 1939, 61, 2791.
(11) Bunnett, J. F. J. Am. Chem. Soc. 1961, 83, 4956; 4968.
(12) Bunnett, J. F.; Olsen, F. P. Chem. Commun. 1965, 601.
(13) Cox, R. A.; Yates, K. Can. J. Chem. 1983, 61, 2225.
cSH
+
log kobs - log
)
(
)
cS + cSH
+
log k0,1 + (φq - φe)(H0 + log cH+) (7)
+
At high acidities, when cSH > cS, the predominantly
protonated substrate prevails, then eq 7 converts to
log kobs ) log k0,1 + (φq - φe)(H0 + log cH
)
+
(8)
(14) Hammett, L. P.; Deyrup, A. J. J. Am. Chem. Soc. 1932, 54, 2721.
(15) Cox, R. A.; Yates, K. J. Am. Chem. Soc. 1978, 100, 3861.
(16) Cox, R. A.; Yates, K. Can. J. Chem. 1981, 59, 2116.
(17) Ingold, C. K. Structure and Mechanism in Organic Chemistry, 2nd
Ed.; Cornell University Press: Ithaca, NY, 1969; Chapter XIV.
(18) Bunnett, J. F.; Olsen, F. P. Can. J. Chem. 1966, 44, 1917.
(19) Bunnett, J. F.; Olsen, F. P. (a) Can. J. Chem. 1966, 44, 1899; (b)
J. Chem. Soc., Chem. Commun. 1965, 601.
(20) Lucchini, V.; Modena, G.; Scorrano, G.; Tonellato, U. J. Am. Chem.
Soc. 1977, 99, 3387.
J. Org. Chem, Vol. 71, No. 10, 2006 3719

Garc´ıa et al.
SCHEME 3
If eq 8 is fulfilled, then the process obeys the A-1 pattern
+
and the log kobs vs (H0 + log cH ) plot gives a straight line, the
slope (φq - φe) being in the range -1 to +1.
(c) The Excess Acidity Method.13 Combination of eq 4 and
+
KSH leads to
cS
log kobs - log
- log cH )
number of molecules covalently bound to the protonated
substrate, forming the transition state.30 The A-2 hydrolysis
involves a rapid preequilibrium to form the SH+ form which,
in turn, is attacked by the nucleophile Nu (water) in the rate-
determining step.
+
(
)
cS + cSH
+
k0,1
fSfH
fq
+
log
+ log
(9)
(
)
(
)
KSH
+
(a) The Zucker-Hammett Criterion.10 The reaction rate
for the A-2 mechanism, Scheme 3, can be expressed as
where the last term, the activity coefficients ratio term, is related
to the excess acidity function X,15,16 in the form
n
fSfH
fq
fSfH
fSH
fBfH
fBH
a
f
+
+
+
+
d(cS + cSH
dt
)
H2O SH
) mq log
) mqm* log
) mqm*X
+
log
υ ) -
) k0,2cSH
(15)
(
)
(
)
(
)
+
+
+
fq
(10)
Reasoning analogous to that applied to A-1 leads to the
following expression:
where B is a hypothetical reference base on which the acidity
function X is defined; the excess acidity, defined as the
+
+
log(fBfH /fBH ) term, represents the extra medium acidity brought
about by the solvent nonideal behavior, that is, the difference
between the actual solution acidity and the stoichiometric
acidity. The excess acidity displays the very useful feature of
being 0 in the standard state of activity coefficients unity. When
fSfH+an
cS + cSH
k0,2
H2O
+
kobs
)
cH
+
(16)
(
)
cS
KSH
fq
+
+
If cSH < cS, then eq 16 converts to
+
cSH > cS, that is, for substrates predominantly protonated, then
eq 10 leads to
fSfH+an
k0,2
H2O
log kobs ) log cH+ + log
+ log
(17)
(
)
(
)
KSH
fq
+
cSH
+
log kobs - log
) log k0,1 + (mq - 1)m*X (11)
(
)
cS + cSH
If the Zucker-Hammett assumption is correct,10 according
to which the activity coefficients ratio term becomes unity, then
the kobs value for A-2 must be directly related to proton
concentration in the form
+
To find out the suitable mq value and thereby establish the
reaction mechanism, the proper m* value corresponding to the
protonation equilibrium in high acidity media must be previously
determined; the parameter m* measures the ability of the set of
bases to become stabilized by solvation and reflects the
equilibrium sensitivity to the large changes in the medium
required to complete the protonation; therefore, the value m*
) 0 for H3O+ in water denotes the highest solvation require-
ments, m* ) 1 corresponds to solvation of primary anilines,
and higher values denote weaker solvation. A suitable plot of
the left-hand side of eq 11 vs X should give a straight line, the
intercept providing the log k0,1 value. For A-1 the activation
parameter should be mq > 1, and in fact, it normally fluctuates
between 2 and 3.21,22 This treatment was applied to deduce the
mechanism of a variety of hydrolyses, including esters,23
substituted benzaldehydes,24-27 and the chromic acid oxidation
of propanal28 and glycolic acid.29
log kobs ) log cH+ + constant
(18)
(b) Linear Free Energy Relationship (LFER). Bunnett11a
observed that the linear (log kobs + H0) vs log aH O plot for a
2
variety of reactions enabled the equation
log kobs + H0 ) w log a + constant
(19)
H2O
A number of empirical mechanistic criteria have been detailed
for hydrolysis reactions that follow the A-2 pattern, according
to which the w parameter must vary between +1.2 and +3.3
for O- and N-protonated substrates, the nucleophile playing a
critical role in the rate-determining step;12 the parameter w bears
relation to the hydration change between the transition state and
the reactants. In this treatment, the assumption was made that
the activity coefficients ratio term is solvent-independent for
species with the same charge. Application of Lucchini et al.’s20
correlation to eq 16 leads to eq 20, representative of an A-2
mechanism.
A-2 Mechanism. The A-2 mechanism is similar to A-1, but
the protonated substrate reacts with a nucleophile species in
the rate-determining step (Scheme 3) where n represents the
(21) Cox, R. A.; Yates, K. Can. J. Chem. 1979, 57, 2944.
(22) Lajunen, M.; Uotila, R. Acta Chem. Scand. 1992, 46, 968.
(23) Cox, R. A.; Goldman, M. F.; Yates, K. Can. J. Chem. 1979, 57,
2960.
(24) Cox, R. A.; Yates, K. J. Org. Chem. 1986, 51, 3619.
(25) Ali, M.; Satchell, D. P. N. J. Chem. Soc., Perkin Trans. 2 1991,
575; 1993, 1825.
(26) Satchell, D. P. N.; Wassef, W. N. J. Chem. Soc., Perkin Trans. 2
1992, 1855.
(27) Motie, R. E.; Satchell, D. P. N.; Wassef, W. N. J. Chem. Soc., Perkin
Trans. 2 1993, 1087.
cSH
+
log kobs - log
- log aH O )
2
(
)
cS + cSH
+
log k0,2 + (φq - φe)(H0 + log cH+) (20)
+
The left-hand side vs (H0 + log cH ) plot should lead to a
straight line.
(28) Alvarez, M. P.; Montequi, M. I. An. Quim. 1992, 88, 149.
(29) Montequi, M. I.; Alvarez, M. P. An. Quim. 1989, 85, 331.
(30) Yates, K.; McClelland, R. A. J. Am. Chem. Soc. 1967, 89, 2686.
3720 J. Org. Chem., Vol. 71, No. 10, 2006

Hydrolysis Mechanisms for Indomethacin and Acemethacin
(c) Excess Acidity Method. Starting from eqs 10 and 16, a
kinetic analysis similar to that performed for the A-1 mechanism
+
can be established. If cSH < cS, that is, if the substrate
predominantly unprotonated prevails, then the relevant rate
equation derived for this mechanism is as follows21
cS
log kobs - log
- log cH+ - n log aNu )
(
)
cS + cSH
+
k0,2
log
+ mqm*X (21)
(
)
KSH
+
where n stands for the number of water molecules in the
transition state and the parameter mq must be close to unity; if
the KSH constant is already known, then the left-hand side vs
+
X plot gives the k0,2 value as the intercept. This equation has
been applied successfully to hydrolyses of benzoic acid deriva-
tives31,32 and azopyridines.33,34
FIGURE 1. UV-vis absorption spectral curves corresponding to
hydrolysis of (a) indomethacin in 9.22 M HClO4, time interval 600 s,
and (b) magnified view in the 245-270 nm range.
In summary, both the LFER and excess acidity methods are
reliable for the A-1 and A-2 hydrolyses. The slope parameters
m*, mq, φe, and φq are related to each other as φq ≈ 1 - mqm*
and m* ≈ (1 - φq)/(1 - φe); that is, m* ≈ 1 - φe.20 Once the
mechanism is deduced, the excess acidity method provides
reliable solvent-independent k0,i (i ) 1, 2); the values deduced
compare favorably with those determined in buffered media,
leaving out the disadvantages inherent to the behavior of the
activity coefficients or the different H0 acidity functions.25
Results and Discussion
The very weak bases indomethacin and acemethacin undergo
acid-base equilibria in the acidic range 1.7-11.6 M HClO4.
Acemethacin displays two protonation equilibria corresponding
+
2+
to the ester (pKAcH ) and amide (pKAcH ) groups, whereas
2
indomethacin undergoes only a single protonation of the amide
7
+
group (pKInH ). To rationalize the kinetics of the respective
hydrolyses, the overall acidity interval was split into region 1
(1.7-7.5 M HClO4), region 2 (8.0-10.0 M HClO4), and region
3 (10.4-11.6 M HClO4). In region 1, the amide group was found
to be predominantly unprotonated, and the reaction was rather
slow; in region 3, the substrate was found to be predominantly
protonated and hydrolyzed fast. In region 2, the hydrolysis
proceeded at an intermediate speed. In regions 1 and 3, the
indomethacin and acemethacin UV spectral curves were quite
similar, with well-defined isosbestic points, whereas in region
2 only acemethacin displayed UV spectral curves without a clear
isosbestic point, which denotes the existence of a complex
reaction. As an example, Figures 1 and 2 show sets of spectral
curves for both indomethacin and acemethacin in 9.22 M HClO4.
In regions 1 and 3, for acemethacin, and in regions 1-3 for
indomethacin, the kobs rate constants were calculated
by nonlinear least-squares fitting, using the single-exponential
equation35
FIGURE 2. UV-vis absorption curves corresponding to hydrolysis
of (a) acemethacin in 9.22 M HClO4, time interval 600 s, and (b)
magnified view in the 245-270 nm range.
double-exponential equation
obst
At - A∞ ) Re-k t + âe-k′
(23)
obs
where A0 and A∞ stand for the initial and final absorbance
readings, respectively, and At represents that at different reaction
times.
The rate constants kobs and k′obs for the disappearance of
acemethacin and kobs for indomethacin at different perchloric
acid concentrations are listed in Table 1. The acidity effect on
k
obs, plotted in Figure 3, reveals that the kinetic behavior for
the two drugs are nearly identical, consistent with the observa-
tion that the A-2 hydrolysis switched over to an A-1 with
increasing medium acidity, being 8.0 M HClO4 (roughly) the
acid concentration at which the change in mechanism occurs,
that is, on the border between regions 1 and 2.
obst
At ) A∞ - (A0 - A∞)e-k
(22)
whereas in region 2 the data for acemethacin were fitted to the
For acemethacin the values for kobs deduced in region 2 on
the basis of eq 23 (Figure 3) matched perfectly well with those
obtained in the regions 1 and 3 on the basis of eq 22. The rate
of hydrolysis, kobs, increased progressively with increasing acid
(31) Cox, R. A.; Smith, C. R.; Yates, K. Can. J. Chem. 1979, 57, 2952.
(32) Cox, R. A.; Yates, K. Can. J. Chem. 1982, 60, 3061.
(33) Cox, R. A.; Onyido, I.; Buncel, E. J. Am. Chem. Soc. 1992, 114,
1358.
(34) Ghosh, K. K.; Rajput, S. K.; Krihnani, K. K. J. Phys. Org. Chem.
1992, 5, 39.
(35) Moore, P. J. Chem. Soc., Faraday Trans. 1972, 68, 1890.
(36) von Dell, H. D.; Doersing, M.; Fischer W.; Jacobi, H.; Kamp, R.;
Ko¨hler G.; Scho¨llnhammer G. Arzneim. Forsch. 1980, 30-II-8a, 1391.
J. Org. Chem, Vol. 71, No. 10, 2006 3721

Garc´ıa et al.
TABLE 1. Rate Constants for Indomethacin and Acemethacin as a Function of HClO4 Concentration (cHClO ), Hammett Acidity Function, H0,
4
Excess Acidity Function, X, and Water Activity aH O
2
indomethacin
acemethacin
cHClO (M)
H0
X
log aH O
kobs × 105 (s-1
)
kobs × 105 (s-1
)
k′obs × 105 (s-1
)
4
2
0.922
1.38
1.73
1.84
2.30
2.77
2.88
3.23
3.46
3.69
4.03
4.15
4.61
5.07
5.18
5.53
5.76
5.99
6.34
6.45
6.91
7.37
7.49
7.83
8.06
8.29
8.30
8.64
8.76
8.87
8.99
9.1
-0.28745208
-0.53706339
-0.70744830
-0.76074035
-0.96957324
-1.17755975
-1.22914696
-1.38616260
-1.49464458
-1.60222114
-1.76999344
-1.82933105
-2.07048089
-2.32504421
-2.38895687
-2.59662524
-2.73821895
-2.88454035
-3.11341513
-3.18817181
-3.50727983
-3.84528504
-3.93323576
-4.19771576
-4.37624522
-4.56181914
-4.56589694
-4.85176223
-4.94988044
-5.04729564
-5.15090293
-5.24684207
-5.35256434
-5.55832838
-5.76642607
-5.87017832
-6.08645722
-6.19912319
-6.42407854
-6.65169672
-6.88401655
-7.12115943
-7.36324572
-7.61039087
0.181
0.301
0.403
0.439
0.594
0.770
0.816
0.960
1.061
1.163
1.323
1.379
1.606
1.841
1.899
2.086
2.212
2.340
2.538
2.602
2.873
3.157
3.230
3.451
3.599
3.753
3.757
3.993
4.075
4.155
4.241
4.320
4.407
4.576
4.747
4.831
5.006
5.097
5.275
5.454
5.632
5.810
5.988
6.163
1.662
1.619
1.587
1.577
1.535
1.492
1.482
1.448
1.425
1.401
1.364
1.351
1.295
1.235
1.219
1.167
1.131
1.092
1.030
1.009
0.916
0.812
0.784
0.697
0.636
0.571
0.570
0.466
0.429
0.392
0.352
0.315
0.273
0.190
0.104
0.060
-0.033
-0.082
-0.183
-0.287
-0.395
-0.508
-0.626
-0.749
0.283
0.417
0.683
0.823
1.029
1.418
1.510
1.896
2.093
2.236
2.638
2.630
2.838
0.600
0.800
0.933
1.050
1.233
1.467
1.617
1.833
2.033
2.200
2.319
2.417
2.567
2.700
3.031
3.283
34.01
3.265
4.083
36.41
31.64
3.380
3.400
4.055
4.400
5.305
6.709
7.071
7.250
13.35
19.35
4.621
33.52
5.381
6.876
7.697
10.10
13.94
33.88
33.68
35.26
35.99
37.38
9.22
9.45
9.68
9.79
10.02
10.14
10.37
10.60
10.83
11.06
11.29
11.52
31.73
41.24
34.17
43.32
40.83
96.83
213.3
700.0
71.63
123.5
175.5
262.3
354.0
533.5
8.0 M HClO4, the reaction proceeded through a slow nucleo-
philic attack of the protonated substrate by two water molecules
(A-2 mechanism). At the highest acidity, above 8.0 M HClO4,
the reaction proceeded by rate-determining decomposition of
the protonated substrate (A-1 mechanism).
Acemethacin: Hydrolysis Mechanisms of the Amide
Group. A-2 Mechanism. The above kinetic criteria were
applied to the data pairs collected for the hydrolysis of
+
acemethacin below 8.0 M HClO4, where cSH < cS, and they
all pointed to an A-2 mechanism. The kinetic criterion by
+
Zucker-Hammett, eq 18, is fulfilled; the log kobs vs log cH
plot (Figure 4) led to a straight line (r ) 0.995) with slope 1.04,
close to unity. The requirements by the Bunnett method, eq 19,
were also met; the (log kobs + H0) vs aH O led to a straight line
2
(Figure 5) with slope parameter w ) 3.3 (r ) 0.999). The LFER
Lucchini correlation was also fulfilled (eq 20); the plot [log
+
+
+
kobs - log(cSH /(cS + cSH )) - log aH O) vs (H0 + log cH ) led
2
FIGURE 3. kobs vs X and kobs vs cHClO plots for indomethacin (2)
and acemethacin (O) at 25 °C.
4
to a straight line (Figure 6) with slope (φq - φe) ) 0.363 (r )
0.995). Finally, the excess acidity method was found to be very
useful in the obtaining of mechanistic information. The [log
concentration, whereas k′obs remained (roughly) constant, the
average value being 3.5 × 10-4 s-1. At acidity levels below
+
+
kobs - log(cS/(cS + cSH )) - log cH - n log aNu) vs X plot, eq
3722 J. Org. Chem., Vol. 71, No. 10, 2006

Hydrolysis Mechanisms for Indomethacin and Acemethacin
+
FIGURE 4. log kobs vs log cH plot for acemethacin. A-2 Zucker-
+
+
FIGURE 7. (log kobs - log(cS/(cS + cSH )) - log cH - 2‚log aH O
)
2
Hammett criterion (eq 18).
vs X plot (eq 21) for an A-2 mechanism for acemethacin in HClO4,
showing the involvement of two water molecules. T ) 25 °C.
+
FIGURE 5. (log kobs + H0) vs log cH plot for acemethacin. A-2
Bunnett criterion (eq 19).
+
FIGURE 8. log kobs vs (H0 + log cH ) plot for acemethacin. A-1 LFER
analysis (eq 8).
+
+
FIGURE 6. log kobs - log(cSH /(cS + cSH )) - log aH O vs (H0 + log
2
+
+
+
cH ) plot for acemethacin. A-2 LFER Lucchini correlation (eq 20).
FIGURE 9. log kobs - log(cSH /(cS + cSH )) vs X plot (eq 11) for
acemethacin in HClO4 for an A-1 mechanism. T ) 25 °C.
21, attained linearity when n ) 2 (Figure 7), n being the number
of water molecules in the transition state; the ordinate yielded
the rate constant kA0,2 ) 2.04 × 10-5 M-1 s-1. Therefore, the
rate-determining step involves the nucleophilic attack by two
water molecules to the protonated substrate. If one recalls that
m* ) 0.60,7 then the value mq ) 0.98 close to unity is deduced,
in good agreement with the criterion by Cox-Yates for an A-2
mechanism (Scheme 4, eqs 12-14).
A-1 Mechanism. In the region above 8.0 M HClO4, where
+
cSH > cS, all the mechanistic criteria analyzed point to an A-1
hydrolysis. According to the LFER method, the linear log kobs
+
vs (H0 + log cH ) plot (eq 8, Figure 8, r ) 0.998), yield the
slope (φ - φe) ) -0.82, a value in the range -1 and +1 that
*
meets the mechanistic requirements; hence, the constant value
of the rate-determining step kA0,1 ) 1.17 × 10-6 s-1 is deduced
J. Org. Chem, Vol. 71, No. 10, 2006 3723

Garc´ıa et al.
SCHEME 4. A-2 Mechanism for the Hydrolysis of the Amide Group of Acemethacin (R ) CH2COOCH2COOH) and
Indomethacin (R ) CH2COOH)
SCHEME 5. A-1 Mechanism for the Hydrolysis of the Amide Group of Acemethacin (R ) CH2COOCH2COOH) and
Indomethacin (R ) CH2COOH)
from the ordinate. The Excess Acidity criterion for an A-1
mechanism, eq 11, is also fulfilled, as shown in Figure 9 (r )
0.998), with slope (mq - 1)m* ) 0.60 and the ordinate yielding
the rate-determining step kA0,1 ) 1.17 × 10-6 s-1. Introduction
of the value m* ) 0.60 reported earlier7 yields the value mq )
2.0, concurrent with the Cox-Yates requirement for an A-1
mechanism (Scheme 5, eqs 1-3).
Indomethacin. To deduce the hydrolysis mechanism for
indomethacin, the same equations and mechanistic criteria were
applied and revealed a switchover from an A-2 to an A-1
mechanism as a function of the medium acidity.
linearity when n ) 2, n being the number of water molecules
in the transition state, and the ordinate yielding the rate constant
kI0,2 ) 1.05 × 10-5 M-1 s-1. Therefore, the rate-determining
step is the nucleophilic attack of the protonated substrate by
two water molecules. If one recalls that m* ) 0.60,7 then the
value close to unity mq ) 1.04 is deduced, in accordance with
the criterion by Cox-Yates for an A-2 mechanism, (Scheme
4).
A-1 Mechanism. In the acidity region above 8.0 M HClO4,
+
where cSH > cS, the criteria tested pointed to an A-1
mechanism. According to the LFER analysis, the log kobs vs
+
(H0 + log cH ) plot (eq 8) leads to a straight line, r ) 0.998).
A-2 Mechanism. The Zucker-Hammett criterion, eq 18, was
The slope (φq - φe) ) -0.87, in the range -1 to +1, concurrent
with the mechanistic requirements. The excess acidity criterion
for an A-1 mechanism, eq 11, is also fulfilled (r ) 0.997), with
an slope (mq - 1)m* ) 0.63; introduction of the m* ) 0.60
value reported earlier,7 yields the value mq ) 2.05, concurrent
with the Cox-Yates requirement for an A-1 mechanism
(Scheme 5), and provides the rate-determining step kI0,1 ) 3.94
fulfilled within the acidity region below 8.0 M HClO4, where
+
+
cSH < cS: the log kobs vs log cH plot was linear (r ) 0.999)
with slope 1.12, close to unity. The LFER analysis, eq 19, was
also fulfilled: the (log kobs + H0) vs log aH O plot yielded a
2
straight line (r ) 0.999) with slope w ) 3.08. Furthermore, the
LFER Lucchini correlation, eq 20, was also fulfilled: the [log
+
+
+
kobs - log(cSH /(cS + cSH )) - log a ) vs (H0 + log cH ) plot
H2O
× 10-6 s-1
.
led to a straight line, and yielded a slope (φq - φe) ) 0.285 (r
) 0.978). The excess acidity method was found to be very useful
in the obtaining of mechanistic information; the [log kobs - log-
The above results confirm that the two antiinflammatory drugs
follow the same hydrolysis pattern for the amide group, with
rate constants of the same order of magnitude. In both cases,
+
+
(cS/(cS + cSH )) - log cH - nlog aNu) vs X plot, eq 21, attained
3724 J. Org. Chem., Vol. 71, No. 10, 2006

Hydrolysis Mechanisms for Indomethacin and Acemethacin
SCHEME 6. Indomethacin (3), (2-Methyl-5-methoxy-1H-indol-3-yl)acetic Acid (4),
(2-Methyl-5-methoxy-1H-indol-3-yl)ethoxycarbonylmethylacetic Acid (5), and p-Chlorobenzoic Acid (6)
both at the ester and amide sites, InH+ represents indomethacin
SCHEME 7. Mechanism for Hydrolysis of Acemethacin
protonated at the amide site, P2+, is the indole protonated at
the ester site resulting from hydrolysis of the amide group of
acemethacin (acid form 5, Scheme 6), P1 is the indole in the
acid form 4, X is the p-chlorobenzyl, acid form 6, kA0,i and k0I ,i
(i ) 1, 2) represent the step-determining rate constants for
hydrolysis of the amide group for AcH22+ and InH+ according
to an A-1 (higher acidity) or A-2 (lower acidity) mechanism,
respectively. Schemes 4 and 5 show the A-2 and A-1 hydrolysis
mechanisms for acemethacin and indomethacin; k3 refers to
hydrolysis of the acemethacin ester group at acidity levels above
2+
7 M HClO4. Assuming that above 8.0 M HClO4 the AcH2
and InH+ species follow A-1 hydrolyses, InH+ being a reaction
intermediate, then application of the kinetic formalism to the
hydrolysis of acemethacin (Scheme 7) leads to the equations
A+k3)t
cAcH 2+ ) (c
2+) e-(k
(24)
0,1
the NMR and GC data have enabled the identification of the
reaction intermediates; for indomethacin, the intermediates and
reaction products were those that resulted from the hydrolysis
of the amide group (4 and 6 forms, Scheme 6), whereas for
acemethacin, the intermediates were the 3, 4, 5, and 6 forms;
this feature indicates that the ester and amide groups hydrolyze
concurrently. On the other hand, in the second acidity range
acemethacin undergoes both the ester and amide hydrolyses,
and displays a more complex kinetic behavior compared to
indomethacin (Figure 2); this feature tells the difference between
the two substrates. Finally, Figure 10 shows that the two
antiinflammatory drugs yield the same reaction products. In
accordance with these results, the hydrolysis mechanism put
forward for acemethacin is as follows:
AcH2
0
2
kI0,1(cInH
(kA0,1 + k3 - kI0,1)
)
+
A
0,1It
0
0,1
3
cInH
)
(e-(k +k )t - e-k
)
(25)
+
which are consistent with the fitting of the experimental data
(eqs 22 and 23). Therefore, for acemethacin (Table 1), k′obs
equals (kA0,1 + k3) ) 3.5 × 10-4 s-1, and kobs ) kI ) 3.94 ×
0,1
10-6 s-1. In view that k0A,1 ) 1.17 × 10-6 s-1, it follows that k3
. kA0,1, and hence, above 8.0 M HClO4, the hydrolysis of the
acemethacin ester group to produce InH+ prevails, the acidity-
independent value k′obs ≈ k3 being consistent with an A-1
mechanism (eq 5). The noncatalyzed acemethacin hydrolysis
of the ester group (k3 roughly constant) can be justified because
In Scheme 7 AcH+ represents the acemethacin form proto-
nated at the ester site, AcH22+ represents acemethacin protonated
+
above 8.0 M HClO4 the ester group is fully protonated (pKAcH
) -2.0), the AcH+ concentration being constant over the whole
acidity range. Table 2 summarizes the equilibria and rate
constants referred to in Scheme 7.
Conclusions
Indomethacin and acemethacin hydrolyze in perchloric acid
above 1.7 M. The reaction products for both indomethacin and
acemethacin are the same. The reaction mechanism, which
depends on the medium acidity, switched from a bimolecular
addition A-2 mechanism involving two water molecules, to a
unimolecular addition A-1 mechanism.The hydrolysis of acem-
ethacin is a complex reaction and involves indomethacin as an
intermediate reaction; hence, the hydrolyses of indomethacin
and acemethacin are concurrent. The reaction rate of hydrolysis
of the ester group is two orders higher compared to that of the
amide group.
FIGURE 10. UV-vis absorption spectral curves of the hydrolysis
products of indomethacin (dashed line), and acemethacin (solid line)
in 8.64 M HClO4, at 99.9% completion 25 °C. Initial indomethacin
and acemethacin concentrations, 5 × 10-5 M.
Experimental Section
Acemethacin and indomethacin were purchased. All other
materials were of the highest commercially available grade and used
J. Org. Chem, Vol. 71, No. 10, 2006 3725

Garc´ıa et al.
TABLE 2. Dissociation Equilibrium (pK) and Rate Constants of the Determining Step (k) at 298 K for the Hydrolyses of Acemethacin and
Indomethacin in HClO4
+
pKAcH (acemethacin ester protonation)
-2.07
-4.27
-4.07
2+
pKAcH (acemethacinium amide protonation)
2
+
pKInH (indomethacin amide protonation)
kA0,2 (amide acemethacin hydrolysis rate constant, A-2 mechanism)
kA0,1 (amide acemethacin hydrolysis rate constant, A-1 mechanism)
kI0,2 (indomethacin hydrolysis rate constant, A-2 mechanism)
kI0,1 (indomethacin hydrolysis rate constant, A-1 mechanism)
k3 (ester acemethacin hydrolysis rate constant)
2.04 × 10-5 M-1 s-1
1.17 × 10-6 s-1
1.05 × 10-5 M-1 s-1
3.94 × 10-6 s-1
3.5 × 10-4 s-1
without further purification. All solutions were prepared using
deionized water. Perchloric acid was used to attain the required
medium acidity. The acid solutions of the proper concentration were
prepared by careful addition of appropriate weights of commercial
HClO4 to a 100 mL reagent bottle by dilution with doubly distilled
deionized water. Since the substrates are not very soluble in water,
the substrate stock solutions were prepared in 5 mL volumetric
flasks by accurately weighing the mass samples (9-10 mg) and
dissolving in freshly distilled acetonitrile, giving concentrations of
5 × 10-3 M. To rule out the occurrence of photochemical
decomposition, the flasks were wrapped up in aluminum foil.
Aliquots (20 µL) of the relevant stock solution were syringed into
a thermostated cell (25.0 ( 0.1 °C) containing 2 mL of the
appropriate aqueous HClO4 solution, the final substrate concentra-
tion being ∼5 × 10-5 M in 1% v/v acetonitrile/H2O.
checked at all relevant wavelengths within the working acidity
range; the concentration effect on the rate constant was negligible
over the acidity range. Full protonation of indomethacin and
acemethacin was attained only at about 74 wt % HClO4, therefore
the two substrates remained unprotonated in dilute acid media. The
excess acidities values, X, for perchloric acid were taken from Cox-
Yates15 and the proton concentrations cH regarded as the acid
+
molarities.16
Intermediate Analyses. The analyses of the intermediates and
reaction products were performed using a gas chromatograph (GC)
1
equipped with a mass selective detector. The H and 13C NMR
spectra were recorded with a 200 MHz instrument. A solution of
indomethacin (0.25 g, 0.0007 mol) or acemethacin (0.25 g, 0.0006
mol) (Scheme 1) in acetonitrile (8 mL) was stirred magnetically
for 2 h with 8 mL HClO4 70 wt % aq. The resulting solution was
then slowly added to 8 mL ethanol and stirred for 12 h at 293 K;
in view of the high volatility of the acidic form, esterification was
needed for the proper GC detection. Samples of the crude product
were pulled out for GC-MS analyses; the isolated products of
Kinetics. The spectrophotometer used to monitor the course of
the reaction was fit out with a diode array detection system and a
temperature cell-holder adapter, electrically regulated and controlled
by computer. The kinetics were investigated by repetitive scans in
the 200-450 nm range. The rate constants were evaluated by
monitoring the disappearance with time of the substrate absorption
At at the different maxima recorded in the λ ) 260-280 nm range.
The course of the reaction was monitored at least up to three half-
lives, the absorbance value at infinity (A∞) being attained after four
half-lives. The computer program used in the fitting of kinetic data
is based on the Marquardt-Levenberg method; after introducing
the initial estimated values (A0, A∞, and kobs), the process is iterated
until the convergence is achieved for the minimum value of the ø2
parameter;35 the average rate constants deviation of the repetitive
scans was <5%. The fulfillment of the Lambert-Beer Law was
1
acemethacin hydrolysis, identified by H and 13C NMR and mass
spectroscopy (MS) analyses, are shown in Scheme 6.
Acknowledgment. The financial support by Junta de Castilla
y Leo´n, project BU26-02, and Ministerio de Ciencia y Tecno-
log´ıa, project BQU2002-01061, Spain, is gratefully acknowl-
edged. Thanks are due to Dr. R. D´ıez and Dr. J. Delgado
(Burgos University) for characterization of the compounds.
JO052561K
3726 J. Org. Chem., Vol. 71, No. 10, 2006