Cyclic amide derivatives as potential prodrugs II: N- hydroxymethylsuccinimide- / isatin esters of some NSAIDs as prodrugs with an improved therapeutic index

Article abstract of DOI:10.1016/S0223-5234(00)80025-2

Ester prodrugs of aspirin la, ibuprofen 1b, naproxen 1c and indomethacin 1d were synthesized using N-Hydroxymethylsuccinimide (HMSI) 3 and N- hydroxymethylisatin (HMIS) 4 as promoieties to reduce their gastrointestinal toxicity and improve bioavailability. Additionally, the kinetics of hydrolysis of the synthesized prodrugs 5a-d and 6a-d were studied at 37°C in non-enzymatic simulated gastric fluid (SGF; hydrochloric acid buffer pH = 1.2); 0.02 M phosphate buffer (pH = 7.4); 80% human plasma and 10% rat liver homogenate. The results indicate higher chemical stability of the ester prodrugs in non-enzymatic SGF (t(1/2) ? 6.5-18.6 h) and rapid conversion to the parent drugs in 80% human plasma (t(1/2) ? 11.4-235 min) as well as in 10% rat liver homogenates (t(1/2) ? 12.0-90.0 min). As a general pattern, the HMSI esters 5a-d revealed higher chemical stability than the corresponding HMIS analogues 6a-d. The pH-rate profile of 5c and 6a indicated maximum stability of the former at pH = 1.2-8.0 and of the latter at pH = 1.2-4.0. The distribution coefficient (D₇.₄) values of the prodrugs 5a-d, 6a-d and the parent drugs 1a-d in an n-octanol/phosphate buffer (pH =7.4) system indicated enhanced lipophilic properties of the prodrugs. Furthermore, the HMIS ester prodrugs 6a-d are more lipophilic than the corresponding HMSI derivatives 5a-d. In vivo ulcerogenicity studies using scanning electron microscopy on stomach specimens of rats treated with an oral dose for 4 d revealed that the synthesized ester prodrugs are significantly less irritating to gastric mucosa than the parent drugs. These results suggested HMSI and/or HMIS esters possess good potential as prodrugs with an improved therapeutic index for oral delivery of NSAIDs.

Full text of DOI:10.1016/S0223-5234(00)80025-2

Eur. J. Med. Chem. 34 (1999) 551−562
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
551
Original article
Cyclic amide derivatives as potential prodrugs II: N-hydroxymethylsuccinimide- /
isatin esters of some NSAIDs as prodrugs with an improved therapeutic index
Nadia M. Mahfouz, Farghaly A. Omar*, Tarek Aboul-Fadl
Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
(Received 15 October 1998; accepted 29 December 1998)
Abstract – Ester prodrugs of aspirin 1a, ibuprofen 1b, naproxen 1c and indomethacin 1d were synthesized using N-Hydroxy-
methylsuccinimide (HMSI) 3 and N-hydroxymethylisatin (HMIS) 4 as promoieties to reduce their gastrointestinal toxicity and improve
bioavailability. Additionally, the kinetics of hydrolysis of the synthesized prodrugs 5a–d and 6a–d were studied at 37 °C in non-enzymatic
simulated gastric fluid (SGF; hydrochloric acid buffer pH = 1.2); 0.02 M phosphate buffer (pH = 7.4); 80% human plasma and 10% rat liver
homogenate. The results indicate higher chemical stability of the ester prodrugs in non-enzymatic SGF (t1/2 6.5–18.6 h) and rapid conversion
to the parent drugs in 80% human plasma (t1/2 11.4–235 min) as well as in 10% rat liver homogenates (t1/2 12.0–90.0 min). As a general
pattern, the HMSI esters 5a–d revealed higher chemical stability than the corresponding HMIS analogues 6a–d. The pH-rate profile of 5c and
6a indicated maximum stability of the former at pH = 1.2–8.0 and of the latter at pH = 1.2–4.0. The distribution coefficient (D7.4) values of
the prodrugs 5a–d, 6a–d and the parent drugs 1a–d in an n-octanol/phosphate buffer (pH =7.4) system indicated enhanced lipophilic properties
of the prodrugs. Furthermore, the HMIS ester prodrugs 6a–d are more lipophilic than the corresponding HMSI derivatives 5a–d. In vivo
ulcerogenicity studies using scanning electron microscopy on stomach specimens of rats treated with an oral dose for 4 d revealed that the
synthesized ester prodrugs are significantly less irritating to gastric mucosa than the parent drugs. These results suggested HMSI and/or HMIS
esters possess good potential as prodrugs with an improved therapeutic index for oral delivery of NSAIDs. © 1999 Éditions scientifiques et
médicales Elsevier SAS
NSAI prodrugs / N-hydroxymethylsuccinimide (HMSI) / N-hydroxy methylisatin (HMIS) / in vitro stability / lipophilicity /
ulcerogenicity
1. Introduction
therapeutic properties of a wide variety of drugs through
development of prodrugs or soft drugs [5]. Accordingly,
the carboxylic group of the NSAID can thus be tempo-
rarily masked and its direct effect on the gastric mucosa
will be prohibited [6].
The gastrointestinal toxicity of the acidic NSAIDs is
one of the most challenging problems in medicinal
chemistry, since these side effects are usually related to
the intrinsic mechanism responsible for the desired activ-
ity [1, 2]. The direct contact mechanism appears to play
the major role in the production of the gastrointestinal
lesions [3]. It is probably caused by a combination of
local irritations produced by the free carboxylic group of
the NSAIDs and by local inhibition of the cytoprotective
action of prostaglandin on gastric mucosa [4]. One of the
recent approaches for overcoming such therapeutic prob-
lems is the concept of retrometabolic drug design, that
incorporates targeting and metabolic considerations into
the design process. This has been used for improving the
We have recently shown the N-hydroxymethyl-
phthalimide ester prodrugs of some NSAIDs to be almost
devoid of the gastric ulcerogenic activity of the parent
drugs [7]. In a trial for optimization of this prodrug
system, two additional analogous cyclic amides:
N-hydroxymethylsuccinimide (HMSI) 3 and N-hydroxy-
methyl isatin (HMIS) 4 were synthesized, to be used as
alternative promoieties for N-hydroxymethyl-phthalimide
(HMPhI). The new promoieties 3 and 4 having advan-
tages as derivatives of endogenous substances [8, 9].
Moreover, several related succinimide derivatives were
reported as promoieties for improving the physicochemi-
*Correspondence and reprints

552
cal and/or therapeutic properties of some drugs [10, 11].
This paper describes the synthesis, physicochemical
properties, kinetics of the chemical and enzymatic deg-
radation of HMSI and HMIS esters of aspirin, ibuprofen,
naproxen and indomethacin for potential use as prodrugs
with an improved therapeutic index.
2. Chemistry
The
promoieties
N-hydroxymethylsuccinimide
(HMSI) 3 and N-hydroxymethylisatin (HMIS) 4 were
prepared by reaction of formaldehyde (40% w/v) and
succinimide or isatin in boiling ethanol. The selected
NSAIs 1a–c were allowed to react with ethyl chlorofor-
mate in the presence of triethylamine in carefully dried
dichloromethane to give the mixed anhydrides 2a–c as
illustrated in figure 1. Treatment with HMSI 3 or HMIS 4
at ambient temperature gave the corresponding ester
prodrugs 5a–c and 6a–c respectively. Attempts to react
indomethacin 1d and ethyl chloroformate in the same
manner failed, since such treatment led to the formation
1
of indomethacin ethylester, as evidenced from the H-
NMR spectrum of the product. This is probably due to the
presence of a methyl group at C-2 of the indole nucleus
that provides sufficient steric hindrance to discourage the
attack of the bulky HMSI 3 or HMIS 4 at the carboxylic
carbonyl. The mixed anhydride undergoes simultaneous
rearrangement to afford the sterically favoured ethyl ester
of indomethacin. However, 1d could be esterified to 5d
and 6d (figure 1) by conversion to the corresponding acid
chloride 2d, that is then treated with the respective
promoiety in the presence of triethylamine. The physical
constants, yields and elemental analysis of the ester
prodrugs 5a–d and 6a–d are listed in table I.
Figure 1. Synthesis of the ester prodrugs 5a–d and 6a–d.
The assignment of the structures was established on the
also worth noting that the methylenic protons of the
HMIS-derived ester prodrugs 6a–d are downfield shifted
(δ 6.0 ppm) relative to their corresponding ones of the
HMSI-derived series 5a–d. This could also be attributed
to the possible conjugation of the lone pair of electrons of
the isatin-nitrogen atom with the adjacent aromatic sys-
tem resulting in a subsequent deshielding of the methyl-
ene group.
The mass spectral data (table II) revealed the parent
peaks, base peaks in addition to some other peaks of the
investigated ester prodrugs in different relative intensi-
ties. In the case of the HMSI-derived esters of aspirin 5a
and indomethacin 5d, the base peak at m/z = 112 could be
attributed to the ion fragment [C5H6NO2]+. A plausible
fragmentation pattern would be the cleavage of the
O-CH2- bond resulting in the formation of N-methylene-
succinimide, which corresponds to the aforementioned
ion fragment. A similar fragmentation pattern was also
1
basis of the characteristic H-NMR and Mass spectral
data and the purity was ascertained by elemental analy-
1
ses, TLC and HPLC. The H-NMR data of the promoi-
eties 3 and 4 as well as the prodrugs 5a–d and 6a–d are
summarized in table II. As evidenced from the table, the
promoieties HMSI 3 and HMIS 4 showed the character-
istic pattern of the methylene and hydroxyl protons along
with the signal of the aliphatic protons in the former and
the aromatic multiplet in the latter. The methylene pro-
tons appeared at nearly equal chemical shifts in both
compounds (δ 5.0 ppm), whereas the chemical shift of
the hydroxyl proton in HMSI (δ = 4.2 ppm) differ from
that in HMIS (δ = 6.3 ppm). The observed downfield shift
of the hydroxyl proton in the latter might be explained on
basis of its orientation relative to the deshielding aniso-
tropic effects of the neighbouring phenyl nucleus in
addition to the effect of d6-DMSO used as a solvent. It is

553
Table I. Physical constants; yields and elemental analysis of the
ester prodrugs 5a–d and 6a–d.
Compound
% Yield
M.p. °C
(solvent)
Mol. formula
(MW)
5a
5b
5c
5d
6a
6b
6c
6d
65
63
64
60
86
82
85
75
114–16*
a
66–68
b
148–50
c
120–23
d
165–67
e
100–103
d
161–63
e
92–94
f
C14H13NO6
(291.26)
C18H23NO4
(317.16)
C19H19NO5
(341.13)
C24H21ClN2O6
(468.11)
C18H13NO6
(339.30)
C22H23NO4
(365.43)
C23H19NO5
(389.41)
C28H21ClN2O6
(516.94)
a: ethyl acetate/petroleum ether; b: ether/petroleum ether; c:
acetone; d: methanol; e: methanol/ethyl acetate; f: acetone/
petroleum ether.
* reported [Lit. 11]: 117–118 °C.
Figure 2. Suggested fragmentation pattern for the ester pro-
drugs 5c and 6c.
observed in the HMIS-derived esters of aspirin 6a,
ibuprofen 6b and indomethacin 6d, leading to the ion
fragment at m/z = 160 for [C9H6NO2]+, which is best
represented by N-methylene-isatin, the promoiety of this
series. This latter ion fragment was further degraded in
the usual pathway reported for the isatin derivatives [12],
whereby loss of CO affords the most stable ion fragment
at m/z = 132. Moreover, the aspirin prodrugs 5a and 6a
are characterized also by rapid cleavage of the acetyl
moiety [CH2CO]+, as shown in table II. On the contrary,
the base peak in the case of naproxen prodrugs 5c and 6c
at m/z = 185 may be resulting from ion fragments of the
parent. The suggested fragmentation pattern, as illus-
trated in figure 2, involves also cleavage of the O-CH2
bond to give naproxen and the previously observed ion
fragments of the promoieties. The most stable ion frag-
ment was produced in this case by loss of a CO2 molecule
from [nap-COO]+, whereas the ion fragments of the
promoieties at m/z = 112 and 132 were found to be less
abundant.
free NSAIDs which would then exert their characteristic
pharmacological actions. Therefore, it was desirable to
determine the relative susceptibility of these compounds
at 37 °C in simulated gastric fluid (hydrochloric acid
buffer, pH 1.2), phosphate buffer of pH 7.4, 80% human
plasma and 10% rat-liver homogenate. The use of 10%
liver homogenate in hydrolysis studies instead of an
undiluted state was based on the fact that liver homoge-
nates are much more enriched with esterases than
plasma [13] and the need to obtain comparable data for
all the studied esters.
All prodrugs were found to be converted quantitatively
to the parent drugs in both chemical and enzymatic
hydrolysis as revealed by HPLC analysis of the reactions.
It is worthwhile mentioning that in case of the prodrugs
5a and 6a, the resulting aspirin was subjected to subse-
quent degradation into salicylic acid at the studied pH
values. Figure 3 illustrates a representative HPLC chro-
matogram for the hydrolysis of aspirin ester prodrug 6a
after 20 min incubation in citrate buffer of pH = 6 at
37 °C. The progress of both chemical and enzymatic
hydrolysis of the esters 5a–d and 6a–d followed pseudo
first-order kinetics over several half-lives. The rate con-
stants (kobs) for the individual reactions were calculated
3. Results and discussion
3.1. In vitro stability
It was expected that the synthesized ester prodrugs
5a–d and 6a–d would be hydrolysed in-vivo before,
during or after absorption to release the corresponding

554
Table II. 1H-NMR and MS data of the N-hydroxymethyl cyclic amides 3, 4 and the ester prodrugs 5a–d and 6a–d.
1H-NMR δ ppm(m; J Hz)
M+;
Mass
others(%)
(%)
100 %
3
2.9 (4H, s, succinim-H), 4.2 (1H, bs, -OH), 5.1 (2H, s, N-CH2-)
4*
5.0 (2H, d, J = 7 Hz, N-CH2), 6.3 (1H, t, J = 7 Hz, -OH),
6.8–7.8 (4H, m, isat-H)
5a
5b
2.3 (3H, s, -CH3), 2.8 (4H, s, succinim-H), 5.7 (2H, s, N-CH2-)
7.0–8.0 (4H, m, ph)
291;
(0.14)
112
–
249 (91):
M-C2H2O
0.9 [6H, d, J = 7 Hz, -(CH3)2], 1.4 (3H, d, J = 7 Hz, -CHCH3),
1.9 [1H, m, -CH(CH3)2], 2.5 (2H, d, J = 7 Hz, -CH2-CH-),
2.8 (4H, s, succinim-H), 3.6 (1H, q, J = 7 Hz,-CHCH3),
5.5 (2H, s, -N-CH2-), 7.1 (4H, bs, ph-H)
–
5c
5d
6a
1.7 (3H, d, J = 7 Hz, -CHCH3), 2.6 (4H, s, succinim-H),
3.8 (1H, q, J = 7 Hz, -CHCH3), 3.9 (3H, s, 3H, OCH3),
5.5 (2H, s, -N-CH2-), 7.0 - 7.8 (6H, m, naphthyl-H)
341;
(17.4)
185
112;
132
229 (8.8)
(M-112)
2.3 (3H, s, -CH3), 2.6 (4H, s, succinim-H), 3.6 (2H, s, CH2CO-),
3.9 (3H, s, OCH3), 5.5 (2H, s, -NCH2), 6.6–7.1 (3H, m,indol-H)
7.3–7.8 (4H, m, ph-H)
468;
(6.2)
249; 189;
139(51.6):
C7ClH4O
2.3 (3H, s, CH3), 6.0 (2H, s, -N-CH2-), 6.9–7.8(7H, m, isat-4H +
ph-H4,5,6),
8.0 (1H, dd, J3,4 = 8Hz, J3,5 = 3Hz, ph-H3)
339;
(4.3)
297 (5.5):
M-C2H2O
160 (17.8)
6b
0.9 [6H, d, J = 7 Hz, CH(CH3)2], 1.5 (3H, d, J = 7Hz, -CHCH3),
1.9 (1H, m, -CH(CH3)2), 2.3 (2H, d, J = 7 Hz, -CH2CH-),
3.8 (1H, q, J = 7 Hz, -CHCH3), 5.7 (2H, s -N-CH2-),
6.9–7.8 (4H, m, isat-H), 7.1 (4H, bs, ph-H)
365;
(6.0)
132
161 (82):
[CH3-isat]+
6c
1.8 (3H, d, J = 7 Hz, -CHCH3), 3.9 (1H, q, J = 7 Hz, -CHCH3),
3.9 (3H, s, -OCH3), 5.8 (2H, s -N-CH2-),
6.5–7.7 ( 10 H, m, naphthyl + isat-H)
389;
(11.2)
185
132
161 (16.5);
132 (80.2)
6d*
2.2 (3H, s, -CH3), 3.8 (2H, s, CH2COO-), 4.0 (3H, s, -OCH3)
5.9 (2H, s -N-CH2-), 6.6–7.4 (7H, m, indolyl-H + isatin-H),
7.7 (4H, bs, ph-H)
516;
(7.7)
161 (49.3)
139 (85.5):
[C7ClH4O]+
1H-NMR: solvent CDCl3; *compounds 4 and 6d in d6-DMSO; bs: broad singlet; dd: doublet of doublet; MS: M+ = molecular ion peak; base
peak (100%); [C7ClH4O]+: p-chlorobenzoyl.
from the linear regression equations correlating the re-
sidual ester concentrations vs. time. The corresponding
half-life for the respective prodrugs 5a–d and 6a–d were
then calculated. The results are summarized in table III.
The kinetic data revealed that HMSI and HMIS esters
5a–d and 6a–d of the selected NSAIDs were hydrolysed
more slowly at pH 1.2 (t1/2 ~ 6.5–18.6 h) than at pH 7.4
(t1/2 ~ 10.7–85 min). Moreover, it has been observed that,
at constant pH and temperature the HMIS-derived ester
prodrugs 6a–d are more susceptible to chemical hydroly-
sis than their respective analogues of HMSI series 5a–d.
The observed acid stability of the ester prodrugs 5a–d
and 6a–d fulfills the essential requirements for the oral
delivery system of NSAIDs [14], whereby the masking
group should be acid-stable to prevent the direct contact
effects with the stomach mucosa as well as the local
inhibition of the protective prostaglandins.
The stability of the ester prodrugs 5c and 6a as
representative examples for each series was further in-
vestigated over the pH range 1.2–10 at 37 °C to evaluate
the effect of pH on the degradation rate and to determine
the pH of maximum stability. The values of the pseudo
first-order rate constants (kobs), for hydrolysis of the two
compounds 5c and 6a, at the respective pH are given in
table IV. The pH-rate profiles are shown in figure 4 in
1
As previously evidenced in H-NMR spectra, the ester
linkage in the former group of compounds is relatively
activated due to the neighbouring phenyl moiety of isatin.

555
hydrolysis of the acetyl group along with the ester
function of the prodrug 6a. As seen from figure 4, the
ester 5c is much more stable than 6a and both of them
show minimal degradation rates at pH 1–2, which is the
normal pH range of the stomach.
The rate data for hydrolysis in 80% human plasma
(table III) indicated an increase in the observed rates of
hydrolysis of most prodrugs (prodrug 6d exempted) by
factors ranging from 2–7-fold (t1/2 ~ 11.4–35 min) rela-
tive to their respective values in aqueous buffer solution
of pH 7.4. In 10% rat liver homogenate (t1/2
12.0–110.0 min), the rates of hydrolysis are much more
accelerated, taking into account the low enzymatic con-
centration due to dilution. In addition, no appreciable
differences could be observed between the two series
5a–c and 6a–c with respect to their susceptibility to
enzymatic hydrolysis. The ester prodrugs of indometha-
cin 5d and 6d exhibit low rates of enzymatic hydrolysis
(t1/2 = 3.5 and 1.5 h respectively). As previously observed
for the N-hydroxyymethylphthalimide analogue [7], the
bulkiness of these ester prodrugs interfered with their
ability to bind to the esterase active sites.
3.2. Lipophilicity
Lipophilicity is well known as a prime physicochemi-
cal descriptor of drugs with relevance to their biological
properties. Consequently, there must be a valid and quick
procedure to quantify molecular lipophilicity. In this
context calculative approaches are superior to experimen-
tal procedures [16]. Validity studies of the most com-
monly used calculation methods confirm the superiority
of the fragmental methods over the atom-based ap-
proaches [16]. The partition coefficients of the synthe-
sized prodrugs 5a–d and 6a–d as well as those of the
parent drugs 1a–d were calculated using a PC-software
package, based on the fragmental method developed by
Leo [17] and the values are listed in table V.
Figure 3. HPLC chromatogram showing the degradation of the
prodrug 6a after 20 min in citrate buffer of pH 6 at 37 °C:
mobile phase, acetonitrile:water (45:55). a) aspirin, b) salicylic
acid, c) remaining ester prodrug 6a.
which the logarithms of the observed pseudo-first-order
rate constants (kobs) are plotted against pH. The ester 5c
exhibits a broad U-shape hydrolysis profile, indicative of
+
the presence of acid-catalysed (kH ), base-catalysed
–
(kOH ) and pH-independent (ko) processes according to
the following rate expression:
It is worthwhile mentioning that the parent drugs 1a–d
are weak acids and are partially ionized at pHs higher
than their pKa values. The effect of the pH on the
partitioning behaviour of such compounds has been
documented and must be taken into consideration [18].
The adopted calculation method cannot adequately deal
with differing contributions of their mesomeric forms at
the pH of the intestine (pH > 7). Consequently, the
distribution coefficients (D) of the synthesized prodrugs
5a–b and 6a–b, as well as their parent drugs 1a–d were
determined in an n-octanol/phosphate buffer (pH = 7.4)
system. Alternatively, a chloroform/phosphate buffer
(pH = 7.4) system was used for determination of the D7.4
values of the ester prodrugs 5c–d and 6c–d due to their
k
obs = k0 + kH+ H+ + kOH− OH−
͓ ͔ ͓ ͔
This profile is analogous to those reported for simple
acyloxymethylamides [15]. It could be observed that the
HMSI-derived ester prodrugs 5a–d represented by 5c are
significantly stable at the pH range 2–8.
The hydrolysis rate profile of 6a deviates from the
simple U-shape (figure 4) and shows two narrow pH-
independent regions, with different rates, between pH
2.2–4 as well as at pH 6–7.4. Maximal stability of this
ester prodrug could be attained at pH-values < 4. Signifi-
cant acid catalysis of hydrolysis has been observed at pH
4–6. This complex profile might be due to the possible

556
Table III. In vitro kinetic data for chemical and enzymatic degradation of the prodrugs 5a–d and 6a–d.
Kobs min–1; (t1/2
pH 1.2a
)
PD
pH 7.4b
80 % h. plasma
10 % rat liver
5a
1.4615 × 10-3
1.9457 × 10-2
4.6014 × 10-2
3.3340 × 10-2
(7.90 h)
(35.6 min)
(15.1 min)
(20.8 min)
5b
5c
5d
6a
6b
6c
6d
0.7689 × 10-3
(15.02 h)
1.5028 × 10-2
(46.1 min)
3.6093 × 10-2
(19.2 min)
1.3653 × 10-2
(50.8 min)
0.9324 × 10-3
(12.39 h)
0.8387 × 10-2
(1.37 h)
6.0897 × 10-2
(11.4 min)
3.0499 × 10-2
(22.7 min)
0.6197 × 10-3
(18.64 h)
1.6644 × 10-2
(59.4 min)
1.9866 × 10-2
(34.9 min)
3.0499 × 10-2
(22.7 min)
1.7645 × 10-3
(6.55 h)
2.884 × 10-2
(24.03 min)
n.d
5.7644 × 10-2
(12 min)
1.0995 × 10-3
(10.5 h)
6.4683 × 10-2
(10.71 min)
4.0635 × 10-2
(17.05 min)
2.7127 × 10-2
(25.55 min)
1.2757 × 10-3
(9.05h)
2.8838 × 10-2
(24.03 min)
5.5898 × 10-2
(12.4 min)
3.3240 × 10-2
(36.00 min)
1.4876 × 10-3
3.9817 × 10-2
3.2480 × 10-3
7.6769 × 10-3
(7.76 h)
(17.4 min)
(3.55 h)
(1.50 h)
a
b
PD: Prodrug; : hydrochoric acid buffer (0.2 M); : phosphate buffer (0.02 M).
poor solubility in n-octanol. The latter D-values were
The measured D7.4-values as well as the calculated
partition coefficients of the prodrugs 5a–d, 6a–d and the
parent drugs 1a–d are listed in table V.
corrected to the respective ones in the n-octanol system
on the basis of a reported regression equation correlating
the two solvent systems [19].
LogPchloroform = 1.10 ± 0.12 logP − 0.649 ± 0.18
͒
͑
͒
͑
oct
Table IV. Pseudo-first-order rate constants (kobs) and the corres-
ponding (t1/2) for the hydrolysis of the prodrugs 5c and 6a in
aqueous buffer solutions at 37oC.
5c
6a
PH*
kobs (min-1)
t1/2
kobs (min-1)
t1/2
1.2
2.2
3.0
4.0
5.0
6.0
7.4
8.0
9.0
10.0
0.932 × 10-3
0.996 × 10-3
2.553 × 10-3
3.037 × 10-3
2.815 × 10-3
3.978 × 10-3
0.8387 × 10-2
4.522 × 10-3
12.40 h
11.50 h
4.50 h
3.80 h
4.10 h
2.90 h
1.38 h
2.60 h
1.765 × 10-3
2.226 × 10-3
2.103 × 10-3
2.469 × 10-3
8.846 × 10-3
2.701 × 10-2
2.884 × 10-2
–
6.50 h
5.20 h
5.00 h
4.70 h
1.31 h
25.70 min
24.00 min
–
1.590 × 10-2 43.60 min
0.932 × 10-2 74.40 min
–
–
–
–
* pH =1.2 (hydrochloric acid buffer); pH = 2.2–6.0 and 8.0–9.0
(citrate buffer); pH = 7.4 (phosphate buffer); pH = 10.0 (borate
buffer).
Figure 4. pH rate profiles for the hydrolysis of prodrugs 5c (+)
and 6a (N) in aqueous buffers at 37 °C.

557
Table V. Chromatographic data for analytical HPLC and the lipohilicity parameters of the prodrugs: 5a–d; 6a–d and the parent drugs 1a–d.
PD
Eluent
Detection
Rt (min)
PD
Lipophilicity [D7.4; (Clog p)]
PD
λmax (nm)
D
D
5a
5b
5c
5d
6a
6b
6c
6d
ACN/H2O (45:55)
ACN/H2O (75:25)
ACN/H2O (70:30)
ACN/H2O (75:25)
ACN/H2O (65:35)
ACN/H2O (78:22)
ACN/H2O (65:35)
ACN/H2O (80:20)
230
228
232
257
230
254
232
257
6.7
5.3
5.7
5.9
5.3
6.6
7.0
6.3
4.5
3.7
3.3
4.8
3.2
3.4
4.3
4.5
1.01 (0.67)*
2.62 (3.37)
2.46 (2.51)
5.47 (4.64)
3.72 (1.81)
4.23 (4.51)
4.84 (3.65)
5.13 (5.78)
0.18 (1.02)
1.28 (3.50)
1.48 (3.34)
2.55 (4.27)
0.18 (1.02)
1.28 (3.50)
1.48 (3.34)
2.55 (4.27)
D
7.4: distribution coefficient in n-octanol/ phosphate buffer (pH = 7.4). Clog P: calculated using PC-software package based on Lit. 17; PD:
*
Prodrug; D: Drug; Reported Log P = 0.500 [11]
Unlike the calculated values, the results in table V
revealed that there are appreciable differences in the D7.4
values of the parent drugs and their prodrugs. This is
because the drug will be negatively charged, whereas, the
respective prodrug is still neutral at pH = 7.4. It is evident
that the prodrugs 5a–d and 6a–d are more lipophilic and
might be characterized by enhanced intestinal absorption
relative to the parent drugs 1a–d. Moreover, the
N-hydroxymethylisatin-derived (HMIS) ester prodrugs
6a–d are more lipophilic than their respective ones of the
HMSI series 5a–d. This could be attributed to the higher
CH index [20] of promoiety in the HMIS-derived esters
as compared with that of the corresponding ones of the
HMSI series.
Figures 5 and 6 illustrate scanning-electromicrographs
for stomach as well as duodenum specimens of rats
treated with a daily oral dose (4 d) of aspirin 1a
(figure 5A) and indomethacin 1d (figure 6A) and their
respective prodrugs 5a, 6a, 5d and 6d (figures 5B, 5C, 6B
and 6C) respectively. The figures revealed damage of the
protective mucous layer of the stomach in the case of the
drugs-treated groups (figure 5A and 6A). The bared sur-
face epithelial cells of the mucosa were then subjected to
the direct effect of the acid resulting in ulceration as can
be seen in the rats treated with indomethacin (figure 6A).
As previously observed [7, 24], the aforementioned toxic
effects are not detected in stomach specimens of the
prodrugs-treated groups (figures 5B, 5C, 6B and 6C) and
the scanning-electromicrographs are almost identical to
that of the control group (figure 5D). It is also worth
noting that neither of the parent drugs 1a, 1d nor the
prodrugs 5a, 6a, 5d and 6d exerted any ulcerogenic
activity on the upper intestine under the used dosing
schedule. The corresponding scanning-electron micro-
graphs of the duodenum specimen are illustrated in
figures 5D, 6D, 5E, 6E, 5F and 6F.
3.3. Ulcerogenic activity
Gastrointestinal ulceration is a serious side effect
associated with many acidic NSAIDs. The clinical uses of
many potent anti-inflammatory agents are strongly lim-
ited by its gastrointestinal toxicity. To diminish these
deleterious effects, ester prodrugs were synthesized and
ulcerogenic activities were tested after repeated dosing
(once daily for four days) of the prodrugs in comparison
to the parent carboxylic acid drugs. The ulcerogenic
activity was routinely determined through counting the
number of ulcers of the stomach and intestine by visual
examination using a binocular magnifier. The severity of
the mucosal damage was then assessed on the basis of the
frequency and size of the observed ulcers [21, 22].
Alternatively, we reported the application of the scanning
electron microscope for investigation of the ulcerogenic
activity of NSAI agents as well as their prodrugs [7, 23,
24]. This new technique represents a highly accurate and
self-explanatory description for the effects of the inves-
tigated compounds on the protective as well as the
mucosal layer of the gastrointestinal tract.
4. Conclusion
The results indicated that the suggested ester prodrugs
5a–d and 6a–d were sufficiently chemically stable in
simulated-gastric fluid and characterized by enhanced
lipophilicity at pHs simulating that of the intestine that
attain intact absorption at an appreciably higher rate than
the parent drugs. In vitro and in vivo studies indicated
that the prodrugs were rapidly bioconverted to the parent
drugs in 80% human plasma and were appreciably less
irritating to the stomach than the parent drugs.

558
Cs
D
A
B
E
C
F
Figure 5. Scanning electron micrographs following a daily oral dose for 4 d of aspirin 1a and its prodrugs 5a and 6a in rats. Stomach:
A. 1a; B. 5a; C. 6a; Cs. control. Intestine: D. 1a; E. 5a; F. 6a; Ci. control.

559
Ci
D
A
B
E
C
F
Figure 6. Scanning electron micrographs following a daily oral dose for 4 d of indomethacin 1d and its prodrugs 5d and 6d in rats.
Stomach: A. 1d; B. 5d; C. 6d. Intestine: D. 1d; E. 5d; F. 6d.

560
5. Experimental protocols
benzene (150 mL) and the mixture was refluxed for 3 h.
The solvent and excess thionyl chloride were then re-
moved under reduced pressure. The resulting acid chlo-
ride was dissolved in methylene chloride (50 mL) and
added dropwise to a cooled solution containing the
appropriate cyclic amide (0.01 mol), triethylamine
(1.1 mL, 0.01 mol) and 4-dimethylaminopyridine 0.01 g
in methylene chloride (50 mL). The reaction mixture was
stirred for 10 h at room temperature, and the precipitated
product was then filtered, dried and recrystallized from
acetone/petroleum ether.
5.1. Chemistry
Aspirin 1a, ibuprofen 1b, naproxen 1c and indometha-
cin 1d were kindly provided by several pharmaceutical
companies in Cairo and used as received. All other
chemicals and reagents used in the synthesis were of
reagent-grade and those for the kinetic studies were of
analytical grade. Fresh doubly distilled water was used in
the preparation of the solutions.
5.1.1. Synthesis of N-hydroxymethylsuccinimide 3
5.2. Analytical methods
A solution of succinimide 5.0 g (0.05 mol) and 3.0 mL
formaldehyde solution (40% w/v) in 100 mL ethanol
(90%) was refluxed for 1 h. The solvent was removed
under reduced pressure and the oily residue was crystal-
lized from ether/petroleum ether. Yield 4.0 g 60%. m.p.
55–58 °C (ether/petroleum ether). Lit. [25] no specific
Melting points were determined with an electrothermal
apparatus (Stuart Scientific, England) and were uncor-
rected. Precoated silica gel plates (Kieselgel 60G
F254 nm, Merck, Germany) were used for TLC. 1H-
NMR spectra [δ (ppm) J (Hz)] were obtained on a Varian
EM-360L (60 MHz) spectrometer (reference TMS). Mass
spectra were determined on a Shimadzu GC/MS QP-5000
(Shimadzu Co., Kyoto, Japan) at the Institute of Pharma-
ceutical Chemistry, Vienna University, Vienna, Austria.
Elemental analysis for C, H and N were obtained on a
Perkin-Elmer 240 °C analyser and were within ± 0.4% of
the theoretical values.
The HPLC system consisted of a pump [Knauer pump
64, Germany], a variable-wavelength detector [Knauer],
a reverse-phase C18 column (Eurospher 80 RP-18, 25 ×
0.5 cm i.d.) equipped with a cartridge guard column, a
Shimadzu C-R 6A chromatopac recording integrator, and
a 20 µL injection loop. Chromatographic separations
were achieved using a mobile phase of acetonitrile and
water containing 1% triethylamine at a flow rate of
1.0 mL/min. The relative ratio of acetonitrile and water
(table V) has been adjusted in each case to attain separa-
tion of the peaks of the prodrug, parent drug as well as the
expected degradation products (figure 3). The column
effluent was monitored at the convenient wavelengths
(table V).
1
constants were cited; H-NMR data are listed in table II.
5.1.2. Synthesis of N-hydroxymethylisatin 4
A suspension of isatin 7.5 g (0.05 mol) and 3.0 mL
formaldehyde solution (40% w/v) in water (100 mL) was
refluxed for 2 h. The hot solution was filtered, cooled
overnight and the separated crystalline product was
filtered and dried in air. Recrystallization from ethyl-
acetate affords a pure product. Yield 6.2 g (70%), m.p
150–52 °C (ethyl acetate). Lit. [26] m.p. 148–150 °C;
1H-NMR data are listed in table II.
5.1.3. General method for synthesis of the ester prodrugs
5a–c and 6a–c
To a cooled solution (0–5 °C) of the respective non-
steroidal anti-inflammatory carboxylic acid 1a–c
(0.01 mol) and triethylamine 1.02 g (0.01 mol) in meth-
ylene chloride (50 mL) was added dropwise ethyl chloro-
formate 1.1 g (0.01 mol) and the mixture was stirred for
a further 30 min. The respective cyclic amide (0.01 mol)
was then added portionwise over a period of 30 min and
stirring was continued overnight at room temperature.
The reaction mixture was then washed successively with
water and 5% sodium hydrogen carbonate solution, dried
over anhydrous sodium sulfate and filtered. The solvent
was then removed under reduced pressure and the resi-
dues recrystallized from the appropriate solvent. Physical
5.3. In vitro hydrolysis kinetics
5.3.1. Chemical hydrolysis
The hydrolysis of the studied HMSI (5a–d) and HMIS
(6a–d) ester prodrugs was studied at pH 1.2 using a buffer
solution of hydrochloric acid and potassium chloride and
in pH 7.4 phosphate buffer. A constant ionic strength of
0.5 µ at 37 °C was maintained in both buffers by adding
a calculated amount of potassium chloride. Buffer solu-
tions containing 0.02% w/v tween 80 and the ester
prodrug (1 × 10–4 M) were kept at a constant temperature
of 37 °C in a water bath. At appropriate time intervals,
1
constants and yields are in table I. H-NMR and mass
spectral data are summerized in table II.
5.1.4. Synthesis of indomethacin ester prodrugs 5d and
6d
Thionyl chloride (3.84 g, 0.03 mol) was added to a
suspension of indomethacin (3.57 g, 0.01 mol) in dried

561
samples of 20 µL were taken and analysed immediately
by HPLC for the respective NSAID and the remaining
ester prodrug. Least-square equations, derived by corre-
lating peak areas in HPLC chromatograms to known
concentrations of each compound were used for calcula-
tion of the residual ester concentrations in the studied
samples. The correlation coefficients of the standard
curves were 0.999.
5.4. Determination of distribution coeffıcients
The distribution coefficient (D) of the prodrugs 5a–d,
6a–d and the parent drugs 1a–d were determined by
dissolving 10 mg of the respective compound in 10 mL of
n-octanol or chloroform, in the case of 5c–d and 6c–d,
2 mL aliquots were then taken and added to an equal
volume of phosphate buffer (pH = 7.4) in screw-capped
test tubes. The mixtures were then vortexed for 15 min.
and centrifuged at 1 × 104 rpm for 5 min. The layers were
separated and aliquots of 100 µl were diluted to 10 mL in
volumetric flasks. The absorption of the diluted solutions
were then measured at the convenient λ-max. The con-
centration in each of the organic and aqueous layers were
then calculated for each compound by virtue of calibra-
tion curves correlating absorbances and known concen-
trations. All experiments were conducted in triplicate and
the mean values were taken. The values of D7.4 for the
respective compounds were then calculated by the usual
method and the results are listed in table V.
The pH profile: the hydrolysis of the ester prodrugs 5c
and 6a was studied in aqueous buffer solutions of pH
1.2–10 at 37 ± 0.1 °C. The buffers used were hydrochlo-
ric acid, citrate, phosphate and borate. The total buffer
concentration was 0.02 M and a constant ionic strength of
0.5 µ for each buffer was maintained by adding a calcu-
lated amount of potassium chloride. Degradation of the
studied compounds was followed by an HPLC procedure
capable of determining intact ester and all products of
hydrolysis. The reactions were initiated by adding 0.5 mL
of stock solutions to 5 mL of the preheated buffer
solutions in screw capped tubes, the final concentrations
of the compounds being about 1 × 10–4 M. At appropriate
intervals 20 µL samples of the reaction solutions were
injected into the chromatographic system and analysed
for the remaining ester prodrug. Pseudo-first-order rate
constants for the hydrolysis were calculated from the
slopes of the linear plots of log (% residual prodrugs) vs.
time.
5.5. In vivo ulcerogenicity study
A Joel, JSM-54000LV scanning electron microscope
(electron microscope unit Assiut University, Assiut,
Egypt) was used for the scanning micrographs. Wistar
rats (200–250 g) from the animal house, Faculty of
Medicine, Assiut University. Seven groups, each of 3 rats
were used.
The tested drugs 1a and 1d, as well as their respective
ester prodrugs 5a, 6a, 5d and 6d were suspended in 0.5%
carboxymethylcellulose solution. The dose (1 mL suspen-
sion) was equivalent to: 100 mg/kg of 1a; 6.75 mg/kg of
1d or the equivalent amounts of the respective prodrugs
5a, 6a, 5d and 6d.
5.3.2. Enzymatic hydrolysis
In 80% human plasma: the rate of enzymatic hydroly-
sis of the ester prodrugs 5a–d and 6a–d was studied in
80% human plasma containing isotonic phosphate buffer
of pH 7.4 at 37 ± 0.1 °C. The reaction was initiated by
adding 100 µL of stock methanolic solution (1 × 10–4 M)
of the respective derivative to 1 mL of plasma solution.
At appropriate time intervals, samples of 50 µL were
withdrawn, mixed with 50 µL of acetonitrile for depro-
teinization and centrifuged at 1 × 104 rpm for 10 min.
20 µL of the clear supernatant was analysed by HPLC for
the remaining ester prodrug as described above.
The animals were fasted for 12 h and then the first 6
groups were administered a daily oral dose of 1 mL of the
drug suspensions for 4 successive days. The seventh
group received an equal amount of the dispersion me-
dium and was considered as control. All groups were
denied access to food throughout this period and 24 h
after the last dosing. The rats were sacrificed by spinal
decapitation, the stomach and duodenum were removed,
opened along the greater curvature and cleaned gently by
dipping in saline. Randomly selected specimens were
then taken and prepared for scanning in an electron
microscope. The preparation method involves, firstly,
fixation by soaking in glutaraldehyde solution (5% in
cacodylate buffer, pH 7.2) for 24 h followed by three
washings each for 20 min with cacodylate buffer. The
specimens were then treated with osmium tetraoxide (1%
solution) for 2 h and washed in the same sequence with
cacodylate buffer solution. Secondly, dehydration of the
In 10% rat liver homogenate: male Wistar rat livers
were homogenized with ice-cooled saline to give a
concentration of 40% w/v, and were then centrifuged at
1 × 104 rpm for 15 min. The supernatant was collected
and stored at –40 °C until use. Homogenate was thawed
10 min before the experiments and diluted with saline to
give a preparation of 10% w/v concentration. The hy-
drolysis studies in rat liver homogenate were performed
as described for 80% human plasma solution. The kinetic
data are the average of three experiments and are given in
table III.

562
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