Colon-specific mutual amide prodrugs of 4-aminosalicylic acid for their mitigating effect on experimental colitis in rats

Article abstract of DOI:10.1016/j.ejmech.2008.03.035

Mutual amide prodrugs of 4-aminosalicylic acid with d-phenylalanine and l-tryptophan were synthesized for targeted drug delivery to the inflamed gut tissue in inflammatory bowel disease. Stability studies in aqueous buffers (pH 1.2 and 7.4) showed that the synthesized prodrugs were stable in both the buffers over a period of 10 h. In rat fecal matter the release of 4-aminosalicylic acid from the prodrugs was in the range of 86-91% over a period of 20 h, with half-lives ranging between 343 and 412 min following first order kinetics. Targeting potential of the carrier system and the ameliorating effect of the amide conjugates were evaluated in trinitrobenzenesulfonic acid-induced experimental colitis model in rats. The prodrugs were assessed for their probable damaging effects on pancreas and liver with the help of histopathological analysis and for their ulcerogenic potential by Rainsford's cold stress method. They were found to have improved safety profile than sulfasalazine, oral 4- and 5-aminosalicylic acid with similar pharmacological spectrum and advantages of sulfasalazine.

Full text of DOI:10.1016/j.ejmech.2008.03.035

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 131e142
http://www.elsevier.com/locate/ejmech
Original article
Colon-specific mutual amide prodrugs of 4-aminosalicylic acid for
their mitigating effect on experimental colitis in rats
a
a
b
a
Suneela S. Dhaneshwar a, , Mukta Chail , Mahavir Patil , Salma Naqvi , Gaurav Vadnerkar
*
a Department of Pharmaceutical Chemistry, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune 411038, Maharashtra, India
b Department of Pharmacology, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune 411038, Maharashtra, India
Received 1 January 2008; received in revised form 19 March 2008; accepted 20 March 2008
Available online 8 April 2008
Abstract
Mutual amide prodrugs of 4-aminosalicylic acid with D-phenylalanine and L-tryptophan were synthesized for targeted drug delivery to the
inflamed gut tissue in inflammatory bowel disease. Stability studies in aqueous buffers (pH 1.2 and 7.4) showed that the synthesized prodrugs
were stable in both the buffers over a period of 10 h. In rat fecal matter the release of 4-aminosalicylic acid from the prodrugs was in the range of
86e91% over a period of 20 h, with half-lives ranging between 343 and 412 min following first order kinetics. Targeting potential of the carrier
system and the ameliorating effect of the amide conjugates were evaluated in trinitrobenzenesulfonic acid-induced experimental colitis model in
rats. The prodrugs were assessed for their probable damaging effects on pancreas and liver with the help of histopathological analysis and for
their ulcerogenic potential by Rainsford’s cold stress method. They were found to have improved safety profile than sulfasalazine, oral 4- and 5-
aminosalicylic acid with similar pharmacological spectrum and advantages of sulfasalazine.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Mutual amide prodrug; 4-Aminosalicylic acid; Inflammatory bowel disease; D-Phenylalanine; L-Tryptophan; TNBS-induced colitis
1. Introduction
only in reducing the inflammation and accompanying symp-
toms in up to 80% of patients. The primary goal of drug ther-
apy is to reduce inflammation in the colon that requires
frequent intake of anti-inflammatory drugs at higher doses.
5-Aminosalicylic acid (5-ASA) is an active ingredient of
agents used for the long term maintenance therapy to prevent
relapses of IBD. But its oral administration results in extensive
and rapid absorption from the upper gastrointestinal tract
(GIT) before it reaches the colon leading to low drug bioavail-
ability and efficiency with significant systemic side effects
[3,4]. This formidable barrier of GIT can be circumvented
by delivering the drug site-specifically to colon utilizing pro-
drug approach.
The earliest reported colon-specific prodrug of 5-ASA is
sulfasalazine (Slz) (Fig. 1), an azo conjugate of 5-ASA with
sulfapyridine (SP) [5]. But its sulfapyridine part used as a car-
rier is completely absorbed through colon and produces ad-
verse effects like hepatitis and immunoallergic reactions [6].
A total of 2400 suspected adverse drug reactions were reported
such as interstitial nephritis, skin reaction, pancreatitis and
Ulcerative colitis and Crohn’s disease collectively referred
to as inflammatory bowel disease (IBD) are chronic relapsing
conditions characterized by up-regulated proinflammatory me-
diators and dysregulated immune responses resulting in tissue
damage [1] with a high morbidity and remain largely incur-
able. Routine treatment has not changed significantly over
the last 40 years and still relies heavily on steroids and amino-
salicylates [2] which do not treat the cause but are effective
Abbreviations: IBD, inflammatory bowel disease; 5-ASA, 5-aminosalicylic
acid; GIT, gastrointestinal tract; Slz, sulfasalazine; 4-ASA, 4-
aminosalicylic acid; BOC, di-tert-butyl dicarbonate; TNBS, 2,4,6-trinitroben-
zenesulfonic acid; 4AP, amide conjugate of 4-ASA with D-phenylalanine; 4AT,
amide conjugate of 4-ASA with L-tryptophan; MPO, myeloperoxidase;
DMSO, dimethyl sulfoxide.
* Corresponding author. Tel.: þ91 20 25437237/6898; fax: þ91 20
25439383.
E-mail address: suneeladhaneshwar@rediffmail.com (S.S. Dhaneshwar).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.035

132
S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 131e142
HOOC
HO
COONa
OH
NaOOC
N
N
N
SO2 NH
HO
N
N
Fig. 1. Sulfasalazine.
Fig. 3. Olsalazine.
blood dyscrasias for sulfasalazine to the Committee on Safety
of Medicines (CSM) of the UK between 1st Jan 1991 and 31st
Dec 1998 compared with 1100 for mesalazine [7]. Due to
adverse effects caused by SP, the efforts were focused on re-
placing it with 4-aminobenzoyl-b-alanine in balsalazide
(Fig. 2) [8] and by another 5-ASA molecule in olsalazine
(Fig. 3) [9]. We have reported azo conjugates of 5-ASA with
D-phenylalanine [10] and L-tryptophan [11] where mutual pro-
drug concept has been utilized and these prodrugs have been
found to possess comparable effect to that of sulfasalazine
on TNBS-induced colitis in rats. Polymeric prodrugs of 5-
ASA linked with dextran [12] and polyanhydride [13] have
been developed for site specific delivery to colon. Colon-
specific amide conjugates of 5-ASA with glutamic acid [14],
glycine [15] and taurine [16] are also reported in literature.
4-Aminosalicylic acid (4-ASA) differs from its 5-ASA
counterpart by the position of the NH2 group and is considered
as a second line anti-tuberculosis agent in the treatment of
drug-resistant tuberculosis caused by Mycobacterium tubercu-
losis [17]. 4-ASA has been used in the treatment of mild to
moderate ulcerative colitis in patients who are intolerant of
sulfasalazine and in the treatment of Crohn’s disease; the
drug is designated as an orphan drug by the FDA for use in
mild to moderate ulcerative colitis [18,19]. 4-ASA has been
claimed to be beneficial in the topical treatment of ulcerative
colitis and in contrast to 5-ASA, has no effect on arachidonic
acid metabolism in human neutrophils or on the free radical
1,1-diphenyl-2-picrylhydrazyl [20]. 4-ASA has been sug-
gested as an effective treatment for both active and quiescent
ulcerative colitis with lesser side effects [21]. 5-ASA-induced
pancreatitis is well described on the World Health Organiza-
tion’s adverse reaction database [7,22], which might be of im-
munoallergic origin [19]. On the other hand, IBD patients on
subsequent treatment with 4-ASA enemas show no recurrence
of pancreatitis. 4-ASA enema is a safer and well-tolerated
therapeutic alternative whenever 5-ASA-induced pancreatitis
occurs [19]. Very recently the focus of IBD research seems
to have shifted from 5-ASA to 4-ASA with very few of its pro-
drugs reported in the literature like azo derivatives of 4-ASA
with salicylic acid, hydroxybenzene, N-salicyloyl glycine
acid [23], amide conjugates of 4-ASA with glycine [24],
ursodeoxycholic acid [25] and arginine salt of 4-ASA [26].
Research studies suggest that 4-ASA may provide a stable,
inexpensive alternative to 5-ASA for the topical treatment of
ulcerative colitis or for linking to carrier molecules for release
in the colon [27]. Though 4-ASA has so many advantages over
5-ASA, its potential has not yet been fully explored.
The present work describes concept-based mutual prodrug
design and synthesis of amide conjugates of 4-ASA with
amino acids for its colon-targeted delivery, with improved
safety profile than sulfasalazine, 5-ASA and 4-ASA. The
aim of this project was to test, in vivo the targeting potential
of amide conjugates to inflamed tissue of colon and compara-
tively evaluate the therapeutic efficacy of this drug-carrier sys-
tem over azo prodrugs of 5-ASA (with same carriers as used in
the present study) reported by us, in experimental rat colitis
model. The amino acid carriers have been found to increase
the bioavailability and efficacy of the drugs and at the same
time reduces their toxicity. D-Phenylalanine and L-tryptophan
were chosen as promoieties due to their marked anti-inflam-
matory activity [10,11,28]. When linked with 4-ASA, they
would improve its efficiency in mitigating the colonic inflam-
mation. Being the natural components of our body, they would
be nontoxic and free from any side effects. N-Aromatic acyl-
amino acid conjugates are reported in the literature, they are
stable in upper intestine and hydrolyzed when incubated
with mammalian cecal content [29]. Introduction of amide
linkage in the prodrug would increase aqueous solubility
thus transcellular absorption by lipid membrane permeation
might be limited in upper intestine. This would facilitate deliv-
ery of intact prodrug to colon. Microbial degradation of amide
prodrugs by hydrolytic action of amidases secreted by the co-
lonic microflora would further ensure the release of 4-ASA
only in colon.
2. Materials and methods
2.1. Synthesis
2.1.1. Materials and general methods
1H NMR spectra of the synthesized compounds were re-
corded in DMSO using 1H NMR Varian Mercury 300 Hz
with super conducting magnet using TMS as internal standard.
Chemical shift values are reported in ppm downfield on d scale.
The IR spectra of the synthesized compounds were recorded
on JASCO, V-530 FTIR in potassium bromide (anhy. IR
grade). The reactions were monitored on TLC, which was per-
formed on precoated silica gel plates-60 F264 (Merck) using
solvent system of chloroform:methanol (4:1.5) and iodine va-
pors as detecting agent.
HOOC
All chemicals used in the synthesis were of AR grade. 4-
ASA and D-phenylalanine were purchased from Himedia Lab-
oratories Pvt. Ltd., Mumbai while L-tryptophan was purchased
from Loba Chemie, Mumbai.
HO
N
N
CONHCH2CH2COONa
Fig. 2. Balsalazide.

S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 131e142
133
2.1.2. Synthesis of methyl ester hydrochloride of
1590 cmꢁ1 C]O stretching (amide II), 1470 cmꢁ1 CeH bend-
ing CH2, 1430 cmꢁ1 and 1370 cmꢁ1 CeH bending CH3,
phenylalanine (PME$HCl) and tryptophan (TME$HCl)
Freshly distilled thionyl chloride (0.05 mol þ 30% extra)
was slowly added to methanol (100 ml) with cooling and amino
acid (0.1 mol) was added to it. The mixture was refluxed for 7 h
at 60e70 ꢀC with continuous stirring on a magnetic stirrer.
Excess of thionyl chloride and solvent was removed under
reduced pressure giving crude methyl ester hydrochlorides of
tryptophan and phenylalanine, respectively. The crude products
were triturated with 20 ml portions of cold ether at 0 ꢀC, until
excess dimethyl sulphite was removed. The resulting solid
products were collected and dried under high vacuum. The
products were then recrystallized from hot methanol by slow
addition of 15e20 ml of ether, followed by cooling at 0 ꢀC.
Crystals were collected on the next day and washed twice
with ether:methanol mixture (5:1) followed by pure ether and
dried under vacuum to give pure PME$HCl and TME$HCl.
PME$HCl: m.p.: 150e156 ꢀC (uncorrected), Rf ¼ 0.81 in
chloroform:methanol (2:1), % yield: 70, IR (KBr): 3390 cmꢁ1
and 3270 cmꢁ1 NeH stretching primary aromatic amine,
3070 cmꢁ1 aromatic CeH stretching, 1740 cmꢁ1 C]O satu-
rated ester stretching, 1470 cmꢁ1 CeH bending CH2,
1250 cmꢁ1 CeO saturated ester stretching, 1H NMR (DMSO-
d6): d 7.26 [m, 5H] aromaticeCH, d 3.84 [m, 1H] methine,
d 3.67 [s, 3H] methyl, d 3.16 [d, 2H] methylene, d 2.0 [s, 2H]
amine.
1
1360 cmꢁ1 tert-butyl stretching, H NMR (DMSO-d6): d 11.0
[s, 1H] OH-carboxylic acid, d 8.0 [s, 1H] NH-sec. amide,
d 7.94 [m, 4H] CH-methine, d 5.0 [s, 1H] CeOH aromatic,
d 1.40 [t, 3H] CH3-methyl.
2.1.4. Protection of hydroxyl group of BOC-4-ASA
by acetylation
Well dried BOC protected 4-ASA (0.040 mol) was placed
carefully in freshly prepared admixture of 10 ml each of acetic
anhydride and glacial acetic acid in a clean and dry 100 ml
round bottom flask and stirring was continued at room temper-
ature for the duration of 12 h. The resulting mixture was di-
rectly poured into 100 ml cold water, contained in a 500 ml
beaker in one lot; the contents were stirred vigorously with
clean glass rod till the shining tiny crystals separated out.
The product was filtered off on a Buchner funnel fitted with
an air suction device and the residue was washed with suffi-
cient cold water, was drained well and finally the excess of
water removed by pressing it between the folds of filter paper
and spreading it in the air to allow it to dry completely. It was
recrystallized by dissolving in methanol and cooling at 0 ꢀC.
M.p.: 105 ꢀC, Rf ¼ 0.45 in benzene:chloroform:ethyl ace-
tate (2:1:1 v/v/v), % yield: 85, IR (KBr): 3060 cmꢁ1 aromatic
CeH stretching, 1688 cmꢁ1 C]O stretching (amide I),
1595 cmꢁ1 C]O stretching (amide II), 1472 cmꢁ1 CeH
bending CH2, 1434 cmꢁ1 and 1370 cmꢁ1 CeH bending
TME$HCl: m.p.: 220e223 ꢀC (uncorrected), Rf ¼ 0.78 in
chloroform:methanol (2:1), % yield: 73, IR (KBr): 3590 cmꢁ1
indole NeH stretching, 1735 cmꢁ1 C]O saturated ester
stretching, 1470 cmꢁ1 CeH bending CH2, 1430 cmꢁ1 and
1370 cmꢁ1 CeH bending CH3, 1240 cmꢁ1 CeO saturated ester
1
CH3, 1368 cmꢁ1 tert-butyl stretching, H NMR (DMSO-d6):
d 8.0 [s, 1H] NH-sec. amide, d 7.94 [m, 4H] CH-methine,
d 1.40 [t, 3H] CH3-methyl.
1
stretching, H NMR (DMSO-d6): d 10.65 [d, 1H] NH-indole,
d 7.10e7.16 [m, 4H] and d 6.9 [d, 1H] CH-indole, d 4.15
[s, 3H] CH3-methyl, d 2.5 [t, 1H] CH-methine, d 2.6 [d, 2H]
CH2-methylene.
2.1.5. Synthesis of acid chloride of protected 4-ASA
Redistilled thionyl chloride (3.3 mmol) in methylene chlo-
ride (2 ml) was added dropwise to a suspension (3.32 mmol)
of protected 4-ASA in 15 ml of anhydrous methylene chloride
cooled at 0 ꢀC containing pyridine (0.4 mmol). The mixture
was allowed to attain room temperature and stirring was con-
tinued for 24 h. The product was dried under vacuum and im-
mediately used in the next step.
2.1.3. Protection of amino group of 4-ASA by BOC
A solution of 4-ASA (10 mmol) in a mixture of dioxane
(20 ml), water (10 ml) and 1 M sodium hydroxide (10 ml)
was stirred and cooled in an ice-water bath. Di-tert-butyl
dicarbonate (11 mmol) was added dropwise over a period of
3 h at room temperature and further stirring was continued
at room temperature for 30 min. The solution was concen-
trated under high vacuum to about 10e15 ml, cooled in ice-
water bath, covered with a layer of ethyl acetate (30 ml) and
acidified with dilute aqueous potassium hydrogen sulphate
solution to pH 2e3 (Congo red). The aqueous phase was
extracted with ethyl acetate (2 ꢂ 15 ml). The ethyl acetate ex-
tracts were pooled, washed with water (3 ꢂ 20 ml), dried over
anhydrous sodium sulphate and dried under vacuum to give
BOC protected 4-ASA (N-tert-butylcarbonyl-4-ASA). It was
recrystallized by dissolving in mixture of ethyl acetate and
hexane (4:1 v/v) and cooling at 0 ꢀC.
M.p.: 200 ꢀC (melts with decomposition), Rf ¼ 0.62, chloro-
form:methanol:benzene (4:1:1 v/v/v), % yield: 82%, IR (KBr):
3060 cmꢁ1 aromatic CeH stretching, 1810 cmꢁ1 C]O stretch-
ing (acid halide), 1688 cmꢁ1 C]O stretching (amide I),
1595 cmꢁ1 C]O stretching (amide II), 1472 cmꢁ1 CeH bend-
ing CH2, 1434 cmꢁ1 and 1370 cmꢁ1 CeH bending CH3,
1
1368 cmꢁ1 tert-butyl stretching, H NMR (DMSO-d6): d 8.0
[s, 1H] NH-sec. amide, d 8.56 [m, 4H] CH-methine, d 1.40
[t, 3H] CH3-methyl.
2.1.6. Coupling of amino acid methyl ester hydrochloride
with protected 4-ASA acid chloride
Methyl ester hydrochloride of amino acid (0.11 mol) was
accurately weighed and dissolved in 4 ml water containing so-
dium hydroxide (0.11 mol) and the resultant solution was
cooled in ice bath. Acid chloride of protected 4-ASA
(0.12 mol) and solution of sodium hydroxide (0.11 mol) in
4-ASA-BOC: m.p.: 125 ꢀC, Rf ¼ 0.66 in benzene:chlorofor-
m:ethyl acetate (2:1:1 v/v/v), % yield: 76, IR (KBr):
3620 cmꢁ1 phenolic OeH stretching, 3070 cmꢁ1 aromatic
CeH stretching, 1685 cmꢁ1 C]O stretching (amide I),

134
S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 131e142
2 ml of water were added in small lots over a period of 2 h,
into the previously chilled amino acid ester solution with con-
stant stirring. The stirring was continued for a further duration
of 36 h at 5e10 ꢀC so as to complete the reaction. The resul-
tant reaction mixture was acidified to Congo red by careful
dropwise addition of diluted HCl in chilled condition. The re-
sultant solid was filtered off on Buchner funnel fitted with an
air suction device and the residue was washed with sufficient
cold water, drained and finally the excess of water removed by
pressing it between the folds of filter paper and was dried un-
der vacuum. The crude product was purified by preparative
chromatography using the solvent system dichloromethane:-
methanol:benzene (4:3:1 v/v/v) and amino acid conjugate of
protected 4-ASA was obtained.
4AT: m.p.: 165 ꢀC (melts with decomposition), Rf ¼ 0.66,
chloroform:methanol:benzene (5:1:1 v/v/v), % yield: 73, IR
(KBr): 3616 cmꢁ1 unbonded phenolic OH stretching,
3414 cmꢁ1 NH stretching(sec. amide), 3018 cmꢁ1 aromatic Ce
H stretching, 1631 cmꢁ1 amide C]O stretching, 1521 cmꢁ1
carboxylate anion stretching, 1473 cmꢁ1 CeH bending CH2,
1432 cmꢁ1 and 1364 cmꢁ1 CeH bending CH3, 1H NMR
(DMSO-d6): d 5.31 [s, 1H] aromatic OH, d 6.29 [s, 1H],
d 6.28 [s, 1H], d 6.99 [t, 3H], d 7.12 [t, 3H], d 7.31 [d, 3H],
d 7.34 [d, 3H] and d 7.63 [d, 3H] CH-benzene, d 3.30 [t, 2H]
CH-methine, d 3.02 [d, 1H] CH2-methylene, d 3.91 [s, 1H]
NH2-primary aromatic amine stretching, d 9.98 [s, 1H] NH-
stretching in benzamide.
Protected 4AP: m.p.: 150 ꢀC (melts with decomposition),
Rf ¼ 0.68, dichloromethane:methanol:benzene (4:3:1 v/v/v),
% yield: 72, IR (KBr): 3065 cmꢁ1 aromatic CeH stretching,
1682 cmꢁ1 C]O stretching (amide I), 1590 cmꢁ1 C]O
stretching (amide II), 1474 cmꢁ1 CeH bending CH2,
1430 cmꢁ1 and 1368 cmꢁ1 CeH bending CH3, 1360 cmꢁ1
2.2. Partition coefficient and aqueous solubility
Partition coefficient was determined in n-octanol/phosphate
buffer (pH 7.4) whereas the aqueous solubility was determined
in distilled water at room temperature (25 ꢃ 1 ꢀC).
1
tert-butyl stretching, H NMR (DMSO-d6): d 8.0 [s, 1H] NH-
2.3. In vitro stability studies
sec. amide, d 8.56 [m, 4H] CH-methine, d 3.40 [d, 2H] CH2-
methylene, d 2.0 [s, 1H] NH-amine, d 1.40 [t, 3H] CH3-methyl.
Protected 4AT: m.p.: 158 ꢀC Rf ¼ 0.67, chloroform:metha-
nol:benzene (4:1:1 v/v/v), % yield: 78, IR (KBr): 3592 cmꢁ1 in-
dole NeH stretching, 3069 cmꢁ1 aromatic CeH stretching,
1680 cmꢁ1 C]O stretching (amide I), 1595 cmꢁ1 C]O
stretching (amide II), 1478 cmꢁ1 CeH bending CH2,
1432 cmꢁ1 and 1367 cmꢁ1 CeH bending CH3, 1365 cmꢁ1
tert-butyl stretching, 1H NMR (DMSO-d6): d 10.1 [s, 1H]
NH-indole, d 8.56 [m, 4H] CH-methine, d 8.0 [s, 1H] NH-sec.
amide, d 7.55e6.45 [s, 1H] CH-indole, d 3.40 [d, 2H] CH2-
methylene, d 2.0 [s, 1H] NH-amine, d 1.40 [t, 3H] CH3-methyl.
The absorbance maxima (lmax) of synthesized compounds
were determined on JASCO V530, UVevisible double-beam
spectrophotometer in hydrochloric acid buffer (pH 1.2), phos-
phate buffer (pH 7.4) and distilled water. All chemicals used in
the preparation of buffer were of AR grade.
In vitro stability studies were carried out in hydrochloric
acid buffer (pH 1.2) and phosphate buffer (pH 7.4) [30,31].
The total buffer concentration was 0.05 M and a constant ionic
strength (m) of 0.5 was maintained for each buffer by adding
a calculated amount of potassium chloride. The feasibility of
hydrolysis of amide linkage by amidases secreted by intestinal
microflora was tested with the help of release study in rat fecal
matter at 37 ꢃ 1 ꢀC. All the kinetic studies were carried out in
triplicate. The K values from the plots were calculated sepa-
rately and average K and SD values were determined. The
half-lives were calculated using software ‘PCP Disso’ devel-
oped by Department of Pharmaceutics, Poona College of Phar-
macy, Pune. The process was validated as per U.S.P. XXIV
edition using different parameters like accuracy, selectivity,
sensitivity and reproducibility. 4AP and 4AT (10 mg each)
were introduced in 900 ml of HCl buffer taken in two different
baskets and kept in a constant temperature bath at 37 ꢃ 1 ꢀC.
The solution was occasionally stirred and 5 ml aliquot portions
were withdrawn at various time intervals. The aliquots were
directly estimated on UV spectrophotometer at 251 nm and
285 nm, respectively, for the amount of 4AP and 4AT remain-
ing. 4-ASA, which was supposed to be released by the synthe-
sized prodrugs, did not interfere with absorption of 4AP and
4AT because its lmax was found to be 274 nm, which was sub-
stantially different from 4AP and 4AT. Therefore simultaneous
estimation of prodrugs in presence of released 4-ASA was car-
ried out over a period of 10 h. Same procedure as described
earlier was followed; except that the HCl buffer was replaced
by phosphate buffer. The kinetics was monitored by the de-
crease in prodrugs concentration with time over a period of
2.1.7. Deprotection of acetyl and BOC groups to form
final prodrug
Protected 4-ASA amino acid conjugate (0.0031 mol) was
treated with 30 ml of 1 M solution of hydrochloric acid in ace-
tic acid and was stirred for 12 h at room temperature. Then it
was neutralized with 25% sodium hydroxide solution to give
the crude product which was purified by preparative chroma-
tography using the solvent system chloroform:methanol (4:1)
to give pure final product, which was stored in well-closed
amber coloured bottle in refrigerator.
4AP: m.p.: 160 ꢀC (melts with decomposition), Rf ¼ 0.62,
chloroform:methanol:benzene (5:1:1 v/v/v), % yield: 78, IR
(KBr): 3616 cmꢁ1 unbonded phenolic OH stretching,
3425 cmꢁ1 amide NH stretching, 3018 cmꢁ1 aromatic CeH
stretching, 1633 cmꢁ1 amide C]O stretching, 1574 cmꢁ1 car-
boxylate anion stretching, 1474 cmꢁ1 CeH bending CH2,
1430 cmꢁ1 and 1368 cmꢁ1 CeH bending CH3, 1H NMR
(DMSO-d6): d 5.21 [s, 1H] aromatic OH, d 6.29 [s, 1H],
d 6.38 [s, 1H], d 6.98 [t, 3H], d 7.11 [t, 3H], d 7.22 [d, 3H],
d 7.34 [d, 3H], and d 7.66 [d, 3H] CH-benzene, d 3.26
[t, 2H] CH-methine, d 3.02 [d, 1H] CH2-methylene, d 4.31
[s, 1H] NH2-primary aromatic amine, d 9.88 [s, 1H] NH
stretching in benzamide.

S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 131e142
135
10 h. The aliquots were directly estimated on UV spectropho-
tometer at 255 nm and 290 nm, respectively, for the amount of
4AP and 4AT remaining. To study the release of 4-ASA from
4AP and 4AT in rat fecal matter [32], prodrugs were dissolved
in sufficient volume of phosphate buffer (pH 7.4) so that final
concentration of solution was 250 mg/ml. Fresh fecal material
of rats was weighed (1 g) and placed in different sets of test
tubes. To each test tube containing weighed amount of rat fe-
cal matter, 1 ml of the prodrug solution was added and diluted
to 5 ml with phosphate buffer (50 mg/ml). The test tubes were
incubated at 37 ꢀC under anaerobic conditions in CO2 incuba-
tor for different intervals of time. For analysis, the aliquots of
4AP and 4AT were removed from the test tubes at different
time intervals and estimated directly on double-beam UV
spectrophotometer (JASCO, V-530 model, Japan) at 255 nm
and 290 nm, respectively. The concentration of prodrugs re-
maining was determined from the calibration curve of 4AP
and 4AT in phosphate buffer.
sulfasalazine. The healthy control and colitis control groups
received only 1% carboxymethylcellulose instead of free
drug or prodrugs. The animals of all groups were examined
for weight loss, stool consistency and rectal bleeding through-
out the 11 days study. Colitis activity was quantified with
a clinical activity score assessing these parameters (Fig. 4)
by clinical activity scoring rate. The clinical activity score
was determined by calculating the average of the above three
parameters for each day, for each group and was ranging from
0 (healthy) to 4 (maximal activity of colitis) [34]. They were
sacrificed 24 h after the last drug administration by isoflurane
anesthesia and a segment of colon, 8 cm long was excised and
colon/body weight ratio was determined to quantify the in-
flammation (Fig. 5). Tissue segments, 1 cm in length were
then fixed in 10% buffered formalin for histopathological
studies. Histopathological studies of the colon (Fig. 6AeN),
pancreas (Fig. 7AeC), and liver (Fig. 8AeC) were carried
out using haematoxylin and eosin stains, at Kolte Pathology
Laboratory, Pune. Colored microscopical images of the colon
sections were taken on Nikon Electronic microscope
ECLIPSE E200 with resolution (10ꢂ), attached with Nikon
COOL-PIX 5400 (Type: E5400 Digital Camera) of magnifica-
tion, 0.26e0.92ꢂ.
2.4. Biological investigations
2.4.1. General methods
Pharmacological screening of the synthesized compounds
was carried out in the Department of Pharmacology, Poona
College of Pharmacy and its animal facility is approved by
CPCSEA. The experimental protocols for the same were ap-
proved by the Institutional Animal Ethical Committee.
2.4.3. Myeloperoxidase (MPO) activity
Myeloperoxidase (MPO) activity was determined by the
method reported by Krawisz et al. [35], the results of which
are depicted in Fig. 9.
2.4.2. Trinitrobenzenesulfonic acid (TNBS)-induced
experimental colitis
2.4.4. Ulcerogenic activity
Trinitrobenzenesulfonic acid (TNBS)-induced experimental
colitis model was selected to study the ameliorating effect of
amide prodrugs of 4-ASA and in vivo characterization of the
amide carrier system on the inflamed tissue of colon in IBD.
It is the most relevant model for chronic inflammation of colon
[33]. Wistar rats (average weight 200e250 g; 12e15 weeks;
n ¼ 6/group) were used. They were distributed into 14 differ-
ent groups, i.e. healthy control, colitis control, three standard
groups and nine test groups. They were housed in a room
with controlled temperature (22 ꢀC). The animals were food
fasted 48 h before experimentation and allowed food and wa-
ter ad libitum after the administration of TNBS. To induce an
inflammation, all the groups except healthy control group were
treated by a procedure discussed below. After light narcotizing
with ether, the rats were catheterized 8 cm intrarectal and
500 ml of TNBS (SigmaeAldrich Chemicals Pvt. Ltd.,
Schweiz) in ethanol was injected into colon via rubber canula
(dose was 100 mg/kg of body weight of TNBS in ethanol, 50%
solution). Animals were then maintained in a vertical position
for 30 s and returned to their cages. For 3 days the rats were
housed without treatment to maintain the development of
a full inflammatory bowel disease model. The animals of
standard and test groups received 4-ASA, 5-ASA, sulfasala-
zine, D-phenylalanine, L-tryptophan, 4AP and 4AT orally while
4-ASA, D-phenylalanine, L-tryptophan, 4-ASA þ D-phenylala-
nine and 4-ASA þ L-tryptophan rectally, once daily for five
continuous days at doses equimolar to 5-ASA present in
The ulcerogenic activity was determined by Rainsford’s
cold stress method [36] at 10 times higher dose as described
in our earlier work [37] with 4-ASA, 5-ASA and sulfasalazine
as standards. Wistar rats (average weight 120e150 g; n ¼ 6/
group) were used for the same.
3. Results and discussion
3.1. Chemistry
Synthesis of methyl ester hydrochlorides of amino acids
[38] was carried out by adding thionyl chloride to methanol
followed by refluxing with amino acid (1) at 60e70 ꢀC for
7 h. Amino group of 4-ASA was protected by using di-tert-
butyl dicarbonate (BOC). The reaction was carried out at
ambient temperature for 30 h [39]. The hydroxyl group of
BOC-4-ASA (2) was protected by using acetic anhydride in
presence of glacial acetic acid [40]. The reaction was carried
out at ambient temperature for 12 h. The carboxylic group
of protected 4-ASA (3) was activated by treating it with thi-
onyl chloride [41]. The reaction was carried out at room tem-
perature for 24 h. The coupling between acid chloride of
protected 4-ASA (4) and methyl ester hydrochloride of amino
acid was carried out by SchotteneBauman procedure using
10% sodium hydroxide [40]. Reaction was carried out at 5e
10 ꢀC for 36 h. Finally, the amino and hydroxyl groups were

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S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 131e142
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
1
2
3
4
5
6
Days
7
8
9
10
11
CC
HC
4AP
PA(r)
4ASA(o)
PA(o)
4ASA+PA(r)
5ASA(o)
TP(o)
4ASA+TP(r)
Slz
4ASA(r)
4AT
TP(r)
Fig. 4. Clinical activity score rate. Average of six readings; P < 0.05. HC: healthy control; CC: colitis control; 4-ASA (o): administration of 4-aminosalicylic acid
by oral route; 5-ASA (o): administration of 5-aminosalicylic acid by oral route; Slz (o): administration of sulfasalazine by oral route; 4AP (o): administration of
amide conjugate of 4-ASA with D-phenylalanine by oral route; 4AT (o): administration of amide conjugate of 4-ASA with L-tryptophan by oral route; PA (o):
administration of D-phenylalanine by oral route; TP (o): administration of L-tryptophan by oral route; PA (r): administration of D-phenylalanine by rectal route;
TP (r): administration of L-tryptophan by rectal route; 4-ASA (r): administration of 4-aminosalicylic acid by rectal route; 4-ASA þ PA (r): co-administration of 4-
aminosalicylic acid þ D-phenylalanine by rectal route; 4-ASA þTP (r): co-administration of 4-aminosalicylic acid þ L-tryptophan by rectal route.
0.016
0.014
0.012
0.01
0.008
0.006
0.004
0.002
0
Groups
HC
5-ASA(oral)
4AT (oral)
PA (rectal)
4-ASA(oral)
PA (oral)
CC
Slz (oral)
TP (oral)
4AP (oral)
4-ASA(rectal)
4-ASA+TP(rectal)
TP (rectal)
4-ASA+PA (rectal)
Fig. 5. Colon to body weight ratio. Average of six readings; P < 0.05. HC: healthy control; CC: colitis control; 4-ASA (o): administration of 4-aminosalicylic acid
by oral route; 5-ASA (o): administration of 5-aminosalicylic acid by oral route; Slz (o): administration of sulfasalazine by oral route; 4AP (o): administration of
amide conjugate of 4-ASA with D-phenylalanine by oral route; 4AT (o): administration of amide conjugate of 4-ASA with L-tryptophan by oral route; PA (o):
administration of D-phenylalanine by oral route; TP (o): administration of L-tryptophan by oral route; PA (r): administration of D-phenylalanine by rectal route;
TP (r): administration of L-tryptophan by rectal route; 4-ASA (r): administration of 4-aminosalicylic acid by rectal route; 4-ASA þ PA (r): co-administration of 4-
aminosalicylic acid þ D-phenylalanine by rectal route; 4-ASA þTP (r): co-administration of 4-aminosalicylic acid þ L-tryptophan by rectal route.

S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 131e142
137
Fig. 6. Histology of colon of rats subjected to TNBS. (A) Healthy control and (B) colitis control showing mucosal injury characterized by absence of epithelium
and a massive mucosal/submucosal infiltration of inflammatory cells. (C) 5-ASA, (D) 4-ASA, (E) D-phenylalanine and (F) L-tryptophan, all showing slight mucosal
abscess and inflammatory infiltrate on oral administration. (G) Sulfasalazine, (H) 4AP and (I) 4AT showing corrected morphology of colon with comparable results
to that of sulfasalazine. (J) d-Phenylalanine, (K) 4-ASA, (L) L-tryptophan, (M) 4-ASA þ D-phenylalanine and (N) 4-ASA þ L-tryptophan show comparable results
as that of colon-specific prodrugs thus indicating a positive contribution of carriers towards anti-inflammatory activity on rectal administration.
deprotected using acetic acid [42]. Reaction was carried out at
room temperature for 12 h (Scheme 1).
amide C]O stretching, 3425 cmꢁ1 and 3414 cmꢁ1 for amide
NH-stretching, 3616 cmꢁ1 and 3501 cmꢁ1 for unbonded phe-
nolic OeH stretching, 1574 cmꢁ1 and 1521 cmꢁ1 for carbox-
ylate anion stretching, 3468 cmꢁ1 and 3305 cmꢁ1 for NH
stretching in primary aromatic amine, respectively.
1H NMR spectra of 4AP showed chemical shifts for protons
of aromatic OH at d 5.21 [s, 1H], CH-benzene d 6.29 [s, 1H],
The melting points of 4AP and 4AT were found to be
160 ꢀC and 180 ꢀC, respectively, which melted with decompo-
sition (uncorrected). All the results of elemental analysis were
in an acceptable error range. The IR spectra of 4AP and 4AT
showed characteristic peaks at 1633 cmꢁ1 and 1631 cmꢁ1 for

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S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 131e142
Fig. 7. Histology of pancreas of rats after treatment with prodrugs. (A) Healthy
control, (B) 4AP and (C) 4AT showing no evidence of pancreatitis.
Fig. 8. Histology of rat livers after treatment with prodrugs. (A) Healthy con-
trol, (B) 4AP and (C) 4AT showing no adverse effects on liver.

S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 131e142
139
found to be 2.015 ꢂ 10ꢁ3 ꢃ 0.0001 sꢁ1 and 1.673 ꢂ 10ꢁ3
ꢃ
180
160
140
120
100
80
150
0.0001 sꢁ1, respectively. The cumulative percent release of
4-ASA from the prodrugs followed first order kinetics
(Fig. 10) with 86% release from 4AP and 91% from 4AT. In
vitro kinetic studies confirmed that the release of 4-ASA
from synthesized conjugates in rat fecal matter was almost
complete over a period of 20 h.
72.25
70.51
61.43
60.85
49.24
46.63
46.57
60
3.3. Biological results
19.92
40
20
In order to evaluate the feasibility of orally administered
amide prodrugs of 4-ASA for targeted drug delivery to the in-
flamed colon in IBD, 2,4,6-trinitrobenzene sulphonic acid
(TNBS)-induced experimental colitis model was chosen
[30,44,45]. Severity of inflammation was quantified by deter-
mining clinical activity score, colon to body weight ratio
and myeloperoxidase activity. There is direct correlation be-
tween the severity of inflammation and these parameters, i.e.
more severe the inflammation, higher are the values for these
parameters and lower the mitigating effect of the prodrugs.
After induction of experimental colitis, the animals were
housed without treatment for the next 3 days to maintain the
development of a full IBD model. During these 3 days, the
clinical activity score for all groups increased consistently. Af-
ter a lag time of 24e48 h, all drug-receiving groups showed
a decrease in severity of inflammation. On 7th day, a significant
difference between the drugs treated groups and a colitis con-
trol group was observed. 4AP produced a comparable lower-
ing of clinical activity (0.8 ꢃ 0.09) to sulfasalazine
(0.9 ꢃ 0.7) while effect of 4AT on lowering of clinical activity
was moderate (1.3 ꢃ 0.12), but for both, it was distinctly more
than 4-ASA (2.0 ꢃ 0.08). The positive contribution of carriers
towards lowering effect on clinical activity score (2.4 ꢃ 0.13
and 2.2 ꢃ 0.08 for D-phenylalanine and L-tryptophan, respec-
tively) is obvious from the gross difference in lowering effect
of plain 4-ASA, 4AP and 4AT. To ensure the synergistic effect
of these carriers further, four test groups of animals were
subjected to rectal administration of plain D-phenylalanine
and L-tryptophan, 4-ASA þ D-phenylalanine and 4-ASA þ L-
tryptophan, respectively. The lowering of clinical activity
score by rectally administered D-phenylalanine and L-trypto-
phan was 2.1 ꢃ 0.03 and 1.8 ꢃ 0.15, respectively, which was
less than that of sulfasalazine (0.9 ꢃ 0.07) but better than D-
phenylalanine (2.4 ꢃ 0.08) and L-tryptophan (2.2 ꢃ 0.08)
administered orally. Co-administration of 4-ASA þ D-phenyl-
alanine and 4-ASA þ L-tryptophan showed comparable lower-
ing of clinical activity score (1.2 ꢃ 0.07 and 1.3 ꢃ 0.04,
respectively) as that of sulfasalazine (0.9 ꢃ 0.07) but better
than 4-ASA or carriers administered orally. This particular
finding supports the positive contribution of carriers, their syn-
ergistic effect and hence the mutual prodrug hypothesis. On
day 11 (24 h after the drug administration), the animals were
sacrificed and colon/body weight ratio was determined to
quantify inflammation. 4AP (0.0073 ꢃ 0.00049) and 4AT
(0.0071 ꢃ 0.00060) treated groups showed a distinct decrease
in the colon/body weight ratio compared to colitis control
group (Fig. 5). Significant decrease in colon/body weight ratio
0
Groups
Fig. 9. Myeloperoxidase activity. HC: healthy control; CC: colitis control; 4-
ASA (o): administration of 4-aminosalicylic acid by oral route; 5-ASA (o): ad-
ministration of 5-aminosalicylic acid by oral route; Slz (o): administration of
sulfasalazine by oral route; 4AP (o): administration of amide conjugate of 4-
ASA with D-phenylalanine by oral route; 4AT (o): administration of amide
conjugate of 4-ASA with L-tryptophan by oral route; PA (o): administration
of D-phenylalanine by oral route; TP (o): administration of L-tryptophan by
oral route.
d 6.38 [s, 1H], d 6.98 [t, 3H], d 7.11 [t, 3H], d 7.22 [d, 3H],
d 7.34 [d, 3H], and d 7.66 [d, 3H]. The signals of CH-methine
at d 3.26 [t, 2H], CH2-methylene at d 3.02 [d, 1H], NH2-pri-
mary aromatic amine stretching at d 4.31 [s, 1H], NH-stretch-
1
ing in benzamide at d 9.88 [s, 1H] were also found. H NMR
spectra of 4AT showed chemical shifts for protons of aromatic
OH at d 5.31 [s, 1H], CH-benzene d 6.29 [s, 1H], d 6.28[s,
1H], d 6.99 [t, 3H], d 7.12 [t, 3H], d 7.31 [d, 3H], d 7.34 [d,
3H] and d 7.63 [d, 3H]. The signals of CH-methine at
d 3.30 [t, 2H], CH2-methylene at d 3.02 [d, 1H] NH2-primary
aromatic amine stretching at d 3.91 [s, 1H], NH-stretching in
benzamide at d 9.98 [s, 1H] were also found.
The aqueous solubilities of 4AP and 4AT were found to be
0.33 g/ml and 0.26 g/ml, respectively. Partition coefficients
(log P) of 4AP and 4AT in n-octanol/phosphate buffer (pH
7.4) were found to be ꢁ0.21 and ꢁ0.19, which were much
lower as compared to 4-ASA (1.012).
3.2. Kinetic study results
In order to check the stability of these prodrugs at acidic
and alkaline pH, their release kinetics was studied by monitor-
ing the decrease in concentration of 4AP and 4AT with time, in
HCl buffer (pH 1.2) at 251 nm and 285 nm and in phosphate
buffer (pH 7.4) at 255 nm and 290 nm, respectively.
Kinetic studies confirmed that these prodrugs were stable
and did not release 4-ASA in aqueous buffers of pH 1.2 and
7.4. Thus, the objective of bypassing the upper GIT without
any free drug release was achieved. The hydrolysis kinetics
was further studied in rat fecal matter [43] to confirm the
colonic hydrolysis of amide prodrugs, over a period of 20 h.
Half-lives (average of four trails) of 4AP and 4AT were found
to be 412 min and 343 min whereas rate constants (K ) were

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S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 131e142
OCH3
OH
60 - 70 °C
8h
Ar/R. HCl
CH3OH + SOCl2
Ar/R. HCl+
O
NH2
Amino acid
NH2
(1)
HOOC
t-Bu-COO-O-OOC-Bu-t
HOOC
HO
in 1M NaOH + Dioxane + H2O
(Ambient temp.), 30h
HO
NHCO2-t-Bu
NH2
(2)
4-ASA
(Ambient temp.), 12h
CH3-CO-O-OC-CH3
ClOC
HOOC
SOCl2, Methylene Chloride
(Ambient temp.), 24h
NHCO2-t-Bu
H3COCO
H3COCO
NHCO2-t-Bu
(4)
(3)
OCH3
ONa
10% NaOH, 36h
Ar/R. HCl
Ar/R
O
O
NH2
NH
O
H3COCO
NHCO2-t-Bu
(5)
(Room Temp.), 12h
ONa
1. 1M HCl in acetic acid
2. 10% NaOH
Ar/R
O
NH
Ar =
N
H
O
4AT
4AP
HO
NH2
Scheme 1.
produced by rectally administered carriers as well as co-ad-
ministration of 4-ASA with carriers was comparable to sulfa-
salazine and indicated an intestinal anti-inflammatory activity.
Myeloperoxidase (MPO) activity was measured according to
the technique described by Krawisz et al.; the results were ex-
pressed as MPO units per gram of wet tissue and one unit of
MPO activity was defined as that degrading 1 mmol minꢁ1
of hydrogen peroxide at 25 ꢀC [35]. Colonic MPO activity
for 4AP and 4AT in mU/100 mg tissue was found to be
46.57 and 49.24, respectively, which was comparable to sulfa-
salazine (46.63), but much less than plain 4-ASA (70.5) sug-
gesting a lower neutrophil infiltrate in the inflamed colon.
Macroscopic damage of colon segments in colitis control
was characterized by severe mucosal necrosis and ulceration
with thickening of bowel wall accompanied by hyperemia,
edema of submucosa, epithelial disruption, mucosal erosions
with goblet cell depletion and a mixed inflammatory infiltrate
containing polymorphonuclear leucocytes and lymphocytes. In
100
90
80
70
60
50
40
30
20
10
0
4AP
4AT
Time (min)
Fig. 10. Release profile of 4-ASA from 4AP and 4AT in rat fecal matter. 4AP:
amide conjugate of 4-ASA with D-phenylalanine; 4AT: amide conjugate of 4-
ASA with L-tryptophan.

S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 131e142
141
vivo treatment with 4AP and 4AT resulted in the significant de-
crease in the extent and severity of colonic damage. Its histo-
pathological features clearly indicated that the morphological
disturbances associated with TNBS administration were cor-
rected by treatment with 4AP and 4AT. These results were
found to be comparable with those obtained for sulfasalazine
treated group. Histopathological features of rectally adminis-
tered D-phenylalanine, L-tryptophan, 4-ASA þ D-phenylala-
nine and 4-ASA þ L-tryptophan groups also indicated
correction of disrupted morphology of the colon. To evaluate
and compare the safety profile of synthesized prodrugs with
respect to 5-ASA-induced pancreatitis and sulfapyridine-in-
duced hepatitis, the prodrugs were assessed for their probable
damaging effects on pancreas and liver with the help of histo-
pathological analysis. The histopathological sections of pro-
drug-treated rat livers and pancreas showed no adverse
effects on liver or any signs of pancreatitis. From these partic-
ular findings, it can be concluded that the synthesized prodrugs
have improved safety profile than 5-ASA or sulfasalazine. Sta-
tistical differences between the groups were calculated by
One-way ANOVA followed by Dunnett’s post hoc test. Differ-
ences were considered at a P value of <0.05 in relation to
control.
The synthesized compounds were evaluated for ulcerogenic
activity by Rainsford’s cold stress method [38] and the ulcer
index was determined [46,47] according to the method re-
ported by Cioli et al. (Table 1). 4AP and 4AT showed remark-
able reduction in the ulcer index 6.0 ꢃ 2.74 and 8.4 ꢃ 2.608,
respectively, as compared to 4-ASA (69.61 ꢃ 14.6). Their ul-
cer indices were comparable to sulfasalazine (5.83 ꢃ 0.47).
One-way ANOVA followed by Dunnett’s post hoc test was
used for calculation of statistical differences between the
groups. All data were expressed as mean ꢃ SD. Differences
were considered at a P value of <0.01 in relation to control.
A comparative study was carried out between amide pro-
drugs of 4-ASA (4AP and 4AT) and azo prodrugs of 5-ASA
with D-phenylalanine (SP) and L-tryptophan (ST) which were
reported earlier by us [10,11] with respect to their therapeutic
efficacy in TNBS-induced colitis. The comparative data re-
vealed that amide prodrugs of 4-ASA with same carriers re-
quired more time for releasing 4-ASA by colonic hydrolysis
(t1/2 6e7 h) than 5-ASA azo prodrugs to release 5-ASA by
colonic reduction (t1/2 2 h) which could be accounted by the
fact that amides are reported to be resistant to hydrolysis.
Amongst the two amino acids, D-phenylalanine proved to be
the better carrier, may it be an amide or an azo prodrug, as
it showed better results than L-tryptophan with respect to syn-
ergistic mitigating effect on TNBS-induced colitis. Out of the
four prodrugs, 4AP and SP (both having the carrier D-phenyl-
alanine) showed comparable results to each other and sulfasa-
lazine. ST and 4AT (both having the carrier L-tryptophan)
showed slightly lower activity spectrum.
4. Conclusion
The amide prodrugs of 4-ASA demonstrated a significant
ameliorating effect on TNBS-induced colitis in rats. The syn-
ergistic ameliorating effect of D-phenylalanine and L-trypto-
phan on disrupted colonic architecture strengthens the
hypothesis of concept-based mutual prodrug design. The syn-
thesized prodrugs have noticeably improved safety profiles
than sulfasalazine, oral 4- and 5-aminosalicylic acid with sim-
ilar pharmacological spectrum and advantages of
sulfasalazine.
Future prospects. Additional work is in progress with re-
spect to in vivo kinetic studies and synthesis of a series of
novel mutual prodrugs of 4-ASA for their potential use in
the management of IBD.
Acknowledgement
Authors thank the All India Council for Technical Educa-
tion (AICTE) for providing financial assistance and to Wallace
Pharmaceutical Pvt. Ltd., Goa, for providing the gift sample of
sulfasalazine.
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