RETRACTED ARTICLE: Synthesis, kinetic studies and pharmacological evaluation of mutual azo prodrug of 5-aminosalicylic acid for colon-specific drug delivery in inflammatory bowel disease

Article abstract of DOI:10.1016/j.ejmech.2006.11.017

Mutual azo prodrug of 5-aminosalicylic acid with l-tyrosine was synthesized by coupling l-tyrosine with salicylic acid, for targeted drug delivery to the inflamed gut tissue in inflammatory bowel disease. The structure was confirmed by elemental analysis, IR and NMR spectroscopy. In vitro kinetic studies in rat fecal matter showed 87.18% release of 5-aminosalicylic acid with a half-life of 140.28 min, following first order kinetics. Therapeutic efficacy of the carrier system and the mitigating effect of the azo conjugate were evaluated in trinitrobenzenesulfonic acid-induced experimental colitis model. Myeloperoxidase activity was determined by the method of Krawisz et?al. The synthesized prodrug was found to produce comparable mitigating effect as that of sulfasalazine on colitis in rats.

Full text of DOI:10.1016/j.ejmech.2006.11.017

European Journal of Medicinal Chemistry 42 (2007) 885e890
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, kinetic studies and pharmacological evaluation of mutual
azo prodrug of 5-aminosalicylic acid for colon-specific drug
delivery in inflammatory bowel disease
a
a
Suneela S. Dhaneshwar a, , Mini Kandpal , Gaurav Vadnerkar ,
*
Badal Rathi b, S.S. Kadam a
a Department of Pharmaceutical Chemistry, Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Pune-411038, Maharashtra, India
b Department of Pharmacology, Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Pune-411038, Maharashtra, India
Received 13 September 2006; accepted 30 November 2006
Available online 15 December 2006
Abstract
Mutual azo prodrug of 5-aminosalicylic acid with L-tyrosine was synthesized by coupling L-tyrosine with salicylic acid, for targeted drug
delivery to the inflamed gut tissue in inflammatory bowel disease. The structure was confirmed by elemental analysis, IR and NMR spectroscopy.
In vitro kinetic studies in rat fecal matter showed 87.18% release of 5-aminosalicylic acid with a half-life of 140.28 min, following first order
kinetics. Therapeutic efficacy of the carrier system and the mitigating effect of the azo conjugate were evaluated in trinitrobenzenesulfonic acid-
induced experimental colitis model. Myeloperoxidase activity was determined by the method of Krawisz et al. The synthesized prodrug was
found to produce comparable mitigating effect as that of sulfasalazine on colitis in rats.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Mutual azo prodrug; 5-Aminosalicylic acid; Inflammatory bowel disease; L-Tyrosine
1. Introduction
significant adverse effects. Therefore, out of the need to over-
come this formidable barrier of GIT, colonic drug delivery has
evolved as an ideal drug delivery system for the topical treat-
ment of diseases of colon like Crohn’s disease, ulcerative
colitis, colorectal cancer and amaebiasis. To achieve success-
ful colonic delivery, a drug needs to be protected from absorp-
tion and/or the environment of upper GIT and then abruptly
released into proximal colon, which is considered as the opti-
mum site for colon-targeted delivery of the drug [1].
Prodrug approach is one of the important approaches for
targeting drugs to colon. Colon-specific drug delivery through
colon-specific prodrug activation may be accomplished by the
utilization of high activity of certain enzymes at the target site
relative to non-target tissues for prodrug to drug conversion.
Prodrug approach has been successfully utilized in sulfasala-
zine (an azo prodrug 5-ASA and sulfapyridine) for targeting
drugs to colon [2]. But majority of side effects of sulfasalazine
like hepatotoxicity, hypospermia and severe blood disorders
Inflammatory bowel disease (IBD) is characterized by
chronic inflammation in the mucosal membrane of the small
and/or large intestine. Although many treatments have been
recommended for IBD, they do not treat the cause but are
effective only in reducing the inflammation and accompanying
symptoms in up to 80% of patients. The primary goal of drug
therapy is to reduce inflammation in the colon that requires
frequent intake of anti-inflammatory drugs at higher doses.
5-Aminosalicylic acid (5-ASA) is very effective in IBD but
it is absorbed so quickly in the upper gastrointestinal tract
(GIT) that it usually fails to reach the colon leading to
* Corresponding author. Tel.: þ91 20 25437237/25436898; fax: þ91 20
25439383.
E-mail address: suneeladhaneshwar@rediffmail.com (S.S. Dhaneshwar).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.11.017

886
S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 42 (2007) 885e890
are due to sulfapyridine. Even though few prodrugs of 5-ASA
like balsalazide, ipsalazine and olsalazine [3e5] have been
reported, none of them have reached beyond the stage of clin-
ical trials. The need for a totally safe, colon-specific prodrug
of 5-ASA with nontoxic carrier still remains.
3. Biological investigations
The ulcerogenic activity was determined by Rainsford’s
cold stress method [8], which is an acute study model and is
used to determine ulcerogenic potency of a drug at 10 times
higher dose. 5-ASA and sulfasalazine were taken as standards.
The test compounds and standards were administered orally,
as fine particles suspended in carboxymethylcellulose by con-
tinuous stirring. The volume of vehicle or suspensions was
kept constant. Wistar rats of either sex weighing between
120 and 150 g were randomly distributed in control and exper-
imental groups of six animals each. Following oral administra-
tion of 5 ml of the aqueous drug suspensions (10 times the
normal dose), the animals were stressed by exposure to cold
(ꢁ15 ꢀC for 1 h). The animals were placed in separate poly-
propylene cages to ensure equal cold exposure. After 2 h of
drug administration, the animals were sacrificed. The stomach
and duodenal part were opened along the greater curvature and
the number of lesions was examined by means of a magnifying
lens. All ulcers larger than 0.5 mm were counted. Average of
six readings was calculated and was expressed as mean ꢂ S.D.
In order to study the ameliorating effect of azo prodrug of
5-ASA on the inflamed tissue of colon in IBD, trinitrobenze-
nesulfonic acid (TNBS)-induced experimental colitis model
was selected which is simple and reproducible. Moreover it
is the most relevant model as it involves the use of immuno-
logical haptens and develops a chronic inflammation rather
than an acute mucosal injury [16]. By this model in vivo char-
acterization of the azo carrier system under the influence of
chronic inflammatory symptoms was possible. Wistar rats
(average weight 200e230 g; 12e15 weeks; n ¼ 6 per group)
were used. They were distributed into six different groups
i.e. healthy control, colitis control, two standard groups and
In the present work, concept of mutual prodrug has been
adopted for the synthesis of azo conjugate of 5-ASA with L-ty-
rosine (TS) for its colon-targeted delivery, which would be
safer with comparable activity to sulfasalazine. The aim of
this project was to test in vivo the targeting potential of azo
conjugate to inflamed tissue of colon and to evaluate the ther-
apeutic efficacy of this drug-carrier system in experimental co-
litis rat model. L-Tyrosine was chosen as a promoiety due to its
marked anti-inflammatory activity [6]. Being a natural compo-
nent of our body, it would be nontoxic and free from any side
effects. Introduction of azo linkage in the prodrug (similar to
sulfasalazine) would ensure release of 5-ASA in colon by
the reductive action of azo reductases secreted by the colonic
microflora.
2. Chemistry
The synthesis of derivative is outlined in Scheme 1. Synthe-
sis of methyl ester hydrochloride of L-tyrosine [13] was carried
out by adding thionyl chloride to methanol followed by reflux-
ing with L-tyrosine 1 at 60e70 ꢀC for 7 h. L-Tyrosine methyl
ester hydrochloride 2 was diazotised [14] at 0e5 ꢀC in cryo-
static bath. The coupling [14,15] of diazonium salt of L-tyro-
sine 3 with salicylic acid 4 was carried out at 0e5 ꢀC in
a cryostatic bath (Scheme 1). It was recrystallized by methanol
followed by cooling at 0 ꢀC. Purified product (TS) was dried
under vacuum.
OH
O
OCH3
60-70°C
+
CH3OH+ SOCl2
O
. HCl
NH2
8h
NH2
OH
OH
(1)
(2)
0-5° C
NaNO2, 2HCl
OH
(4)
COOH
A
N
N
HO
B
OH
O
ONa
OH
NaOOC
2M NaOH
O
(TS)
N+
OH
NCl-
(3)
Scheme 1.

S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 42 (2007) 885e890
887
two test groups. They were housed in a room with controlled
temperature (22 ꢀC). The animals were fasted 48 h before ex-
perimentation and allowed food and water ad libitum after the
administration of TNBS. To induce an inflammation, all the
groups except healthy control group were treated by a proce-
dure discussed below. After light narcotizing with ether, the
rats were catheterized 8 cm intrarectal and 0.25 ml of TNBS
(Himedia Laboratories Pvt. Ltd., Mumbai) in ethanol was in-
jected into colon via rubber cannula (dose was 100 mg/kg of
body weight of TNBS in 50% v/v ethanol). Animals were
then maintained in a vertical position for 30 s and returned
to their cages. For three 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 orally 5-ASA, sulfasalazine, L-tyrosine and TS,
respectively, once daily for five continuous days at doses equi-
molar to 5-ASA present in sulfasalazine. The healthy control
and colitis control groups received only 1% carboxymethylcel-
lulose instead of free drug or prodrug. The animals of all
groups were examined for weight loss, stool consistency and
rectal bleeding throughout the 11 days study. Colitis activity
was quantified with a clinical activity score assessing these pa-
rameters (Fig. 1) by clinical activity scoring rate. The clinical
activity score [17] 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).
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 quan-
tify the inflammation (Fig. 3). Tissue segments 1 cm in length
were then fixed in 10% buffered formalin for histopathological
studies. Histopathological studies (Fig. 4aee) of the colon
were carried out using haematoxylin and eosin stains, at Kolte
Pathology Laboratory, Pune. Colored microscopical images of
the colon sections were taken on Zeiss optical microscope,
Stemi 2000-C, with resolution 10ꢃ45 X, attached with trinoc-
ular camera.
proportional to the ameliorating effect on disrupted colonic
architecture. The intestinal tissue samples (approximately
50e100 mg) were homogenised on ice using a polytron
(13,500 rpm, 1 min) in a solution of 0.5% hexadecyltrimethyl
ammonium bromide (HTAB, Loba Chemie, Mumbai) in
50 mM potassium phosphate buffer (pH 6.0, 1 ml per 50 mg tis-
sue). The resulting homogenate was subjected to three rapid
freezing (70 ꢀC) and thawing (immersion in warm water,
37 ꢀC) cycles. The samples were then centrifuged (4000 rpm,
15 min, 4 ꢀC) to remove insoluble material. The MPO contain-
ing supernatant (0.1 ml) was assayed spectrophotometrically
after addition of 2.88 ml phosphate buffer (50 mM, pH 6.0)
containing 0.167 mg/ml o-dianisidine hydrochloride (Himedia
Laboratories Pvt. Ltd., Mumbai) and 0.0005% hydrogen perox-
ide. The kinetics of absorbance changes at 470 nm was mea-
sured. Sample enzyme activity was calculated with a standard
curve of known MPO unit activity. One unit of MPO activity,
defined as the quantity of enzyme able to convert 1 mmol of
hydrogen peroxide to water in 1 min at room temperature, was
expressed in mU/100 mg of tissue.
For determination of antiarthritic activity, Freund’s adju-
vant-induced arthritis model was used [19]. Wistar rats of
either sex weighing 150e200 g were divided into three groups
viz. arthritic control, standard and test containing six animals
each. On day one, 0.1 ml of complete Freund’s adjuvant (F-
5881, SigmaeAldrich Corporation, USA) was injected into
the subplanar region of hind paw of rats. The animals were
housed in cages to allow the development of full arthritis up
to 13 days. The paw volumes were measured on 5th and
13th day using UGO BASILE Plethysmometer 7140, Italy.
On 13th day the drug administration was started and continued
up to 21st day. Animals of the standard groups received sulfa-
salazine and 5-ASA, respectively, while test group received
TS. All the doses were calculated on equimolar basis of
5-ASA present in sulfasalazine. The arthritic control group re-
ceived 1% carboxymethylcellulose only. Finally, paw volume
was again measured on 21st day.
Further myeloperoxidase (MPO) activity was determined by
the method of Krawisz et al. [18]. MPO activity is inversely
4. Results and discussion
The melting point of TS was found to be 283e285 ꢀC (un-
corrected). All the results of elemental analysis were in an ac-
ceptable error range.
4.5
HC
4
3.5
3
CC
5-ASA
C1
The IR spectra of TS showed characteristic peak at
1494 cmꢁ1 of N]N stretching (unsymmetric p-substituted
azobenzene) which confirms the formation of azo bond. A
broad peak of unbonded phenolic OH stretching at 3558e
3219 cmꢁ1 was also found. It also showed carboxylate anion
stretching at 1593 and 1390 cmꢁ1 and CeN stretching at
Slz
TS
2.5
2
1.5
1
1095 cmꢁ1
.
1H NMR spectra of TS showed chemical shifts for protons
of Ring A, aromatic OH at d 5.80 [s, 1H] and Ring B, aromatic
OH at 5.83 [s, 1H]; Ring A, CH-benzene at d 7.74 [s, 1H],
d 7.55 [d, 1H] and d 7.53 [d, 1H] and Ring B, CH-benzene
at d 6.48 [d, 1H] and d 7.01 [d, 1H]. The signals of CH (me-
thine) at d 2.66 [t, 1H] and CH2 (methylene) at d 2.93 [d, 2H]
were also found.
0.5
0
1
2
3
4
5
6
7
8
9
10
11
Day
Fig. 1. Clinical activity score rate. HC: healthy control, CC: colitis control, 5-
ASA: 5-aminosalicylic acid, C1: carrier (L-tyrosine), Slz: sulfasalazine, TS:
prodrug of 5-ASA with L-tyrosine.

888
S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 42 (2007) 885e890
Table 1
Results of ulcerogenic activity
The aqueous solubility was found to be 0.26 g/ml and
partition coefficient in n-octanol/phosphate buffer (pH 7.4)
was found to be 0.35, which was decreased as compared to
5-ASA (0.64).
Compound
Dose (mg/kg)a
Ulcer index ꢂ S.D.b
HC
e
1.78 ꢂ 0.60
60.03 ꢂ 1.15
5.83 ꢂ 0.47
10.76 ꢂ 0.55
5-ASA
Slz
TS
2290
3000
3050
The kinetics was monitored by the decrease in prodrug con-
centration with time in HCl buffer (pH 1.2) at 264 nm and
phosphate buffer (pH 7.4) at 272 nm. Kinetic studies con-
firmed that the prodrug did not release the parent drug in
0.05 M hydrochloric acid buffer (pH 1.2), whereas in phos-
phate buffer (pH 7.4) only 15.17% release was observed after
7 h. Thus, the objective of bypassing the upper GIT without
any free drug release was achieved. The release kinetics was
further studied in rat fecal matter [7] to confirm the colonic re-
HC: healthy control, 5-ASA: 5-aminosalicylic acid, Slz: sulfasalazine, TS:
prodrug of 5-ASA with L-tyrosine.
a
Ten times the normal dose.
Average of six readings.
b
became significant on day seven. A significant lowering of clin-
ical activity was shown by TS (0.9 ꢂ 0.13), which was compa-
rable to sulfasalazine (0.7 ꢂ 0.22) but distinctly more than
5-ASA (1.8 ꢂ 0.07). The positive contribution of L-tyrosine to-
wards lowering effect on clinical activity score (1.8 ꢂ 0.06) is
obvious from the gross difference in lowering effect of plain
5-ASA and TS. On day 11 (24 h after the drug administration),
the animals were sacrificed and colon/body weight ratio was de-
termined to quantify inflammation. The prodrug treated group
showed a distinct decrease in the colon/body weight ratio com-
pared to colitis control group (Fig. 3).
duction of azo prodrug. t (average of four trails) of TS was
½
found to be 140.28 min whereas rate constant (K ) was found
to be 4.94 ꢃ 10ꢁ3 ꢂ 0.0001. Over a period of 7 h, TS gave
87.18% cumulated release of 5-ASA following first order
kinetics (Fig. 2). Thus in vitro kinetic studies confirmed that
the synthesized conjugate did not release 5-ASA at all in
HCl buffer (pH 1.2) but released it in a very negligible extent
in phosphate buffer (pH 7.4), whereas the release in rat fecal
matter was almost complete.
The synthesized compound was evaluated for ulcerogenic
activity by Rainsford’s method [8] and the ulcer index was
determined that has been shown in Table 1 [9]. The conjugate
showed remarkable reduction in the ulcer index (10.76 ꢂ
0.55) as compared to its parent drug (60.03 ꢂ 1.15). This reduc-
tion in the ulcer index brought about by the conjugate was com-
parable to that produced by sulfasalazine (5.83 ꢂ 0.47).
Statistical differences between the groups were calculated by
One-Way ANOVA followed by Dunnett’s post hoc test. All
data are expressed as mean ꢂ SD. Differences were considered
at a P value of <0.01 in relation to control.
In order to study the feasibility of azo prodrug of 5-ASA for
targeted oral drug deliveryto the inflamed tissue of colon in IBD,
TNBS-induced experimental colitis model was selected [10e
12]. After inducing the experimental colitis, the clinical activity
score increased rapidly and consistently for the next three days
for all groups. All drug-receiving groups showed a decrease of
inflammation severity after a lag time of 24e48 h. The differ-
ence between the drug treated group and colitis control group
During the evaluation of macroscopic damage of colon seg-
ments in colitis control, the colons appeared flaccid and filled
with liquid stool. The cecum, colon and rectum all had evi-
dence of mucosal congestion, erosion and hemorrhagic ulcer-
ations and histopathological features included transmural
necrosis, edema, absence of epithelium, a massive mucosal/
submucosal infiltration of inflammatory cells. In vivo treat-
ment with TS resulted in the significant decrease in the extent
and severity of colonic damage. Its histopathological features
clearly indicated that the morphological disturbances associ-
ated with TNBS administration were corrected by treatment
with TS (Fig. 4f). These results were found to be comparable
with those obtained for sulfasalazine treated group. Statistical
differences between the groups were calculated by One-Way
ANOVA followed by Dunnett’s post hoc test. Differences
were considered at a P value of <0.05 in relation to control.
Myeloperoxidase (MPO) activity, which is an important
quantitative index for colonic inflammation was determined
0.016
0.014
0.012
0.01
100
90
80
70
60
50
40
30
20
10
0
0.008
0.006
0.004
0.002
0
HC
CC
5-ASA
C1
Slz
TS
Groups
0
50
100
150
200
250
30
0
3
50
400
450
Fig. 3. Colon to body weight ratio. HC: healthy control, CC: colitis control, 5-
ASA: 5-aminosalicylic acid, C1: carrier (L-tyrosine), Slz: sulfasalazine, TS:
prodrug of 5-ASA with L-tyrosine.
Time (min)
Fig. 2. Release profile of 5-ASA from TS in rat fecal matter.

S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 42 (2007) 885e890
889
Fig. 4. Histology of colon of rats subjected to TNBS: (a) healthy control; (b) colitis control showing mucosal injury characterized by absence of epithelium and
a massive mucosal/submucosal infiltration of inflammatory cells; (c) 5-ASA; (d) L-tyrosine, both showing slight mucosal abscess and inflammatory infiltrate. (e)
Sulfasalazine; (f) TS showing corrected morphology of colon with comparable results to that of sulfasalazine.
in terms of mU/100 mg tissue. MPO activity for TS was found
to be 53.51 mU/100 mg tissue, which is comparable to sulfa-
salazine (46.63 mU/100 mg tissue), but much less than plain
160
140
5-ASA (60.85 mU/100 mg tissue). The results are depicted
120
100
80
60
40
20
0
in Fig. 5. Finally, antiarthritic activity was determined in
Freund’s adjuvant-induced arthritis, where sulfasalazine was
taken as the standard disease modifying anti-rheumatoid
drug. Percent inhibition of arthritis using TS was found to
be 62%, which is slightly less than sulfasalazine (73%), while
plain 5-ASA showed only 47% inhibition of arthritis (Table 2).
HC
CC
5-ASA
C1
Slz
TS
5. Conclusion
Groups
The data generated as an outcome of this work demon-
strates that this new prodrug has a remarkable ameliorating
effect on the disruption of colonic architecture and suppresses
Fig. 5. Myeloperoxidase activity. HC: healthy control, CC: colitis control, 5-
ASA: 5-aminosalicylic acid, C1: carrier (L-tyrosine), Slz: sulfasalazine, TS:
prodrug of 5-ASA with L-tyrosine.

890
S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 42 (2007) 885e890
Table 2
Antiarthritic activity
Compounds
Paw volume
1 day
Difference in paw volumea
% Inhibition
13 day
5 day
13 day
21 day
13 day
21 day
21 day
Arthritic control
2.27 ꢂ 0.03
2.27 ꢂ 0.02
2.17 ꢂ 0.03
2.23 ꢂ 0.02
2.53 ꢂ 0.11
2.94 ꢂ 0.05
2.64 ꢂ 0.03
2.79 ꢂ 0.04
3.12 ꢂ 0.05
3.02 ꢂ 0.04
2.87 ꢂ 0.06
2.97 ꢂ 0.03
3.58 ꢂ 0.06
2.96 ꢂ 0.03
2.53 ꢂ 0.02
2.72 ꢂ 0.04
0.85 ꢂ 0.03
0.75 ꢂ 0.04
0.70 ꢂ 0.04
0.74 ꢂ 0.02
1.31 ꢂ 0.02
0.69 ꢂ 0.02
0.36 ꢂ 0.05
0.49 ꢂ 0.06
e
e
5-ASA
Slz
TS
12
18
13
47
73
62
a
Average of six readings.
the course of TNBS-induced colitis effectively. The criteria for
the selection of L-tyrosine as carrier has also proven correct, as
it has effectively delivered 5-ASA to colon.
Acknowledgement
Authors thank the AICTE for providing financial assistance
and to Wallace Pharmaceutical Pvt. Ltd., Goa, for providing
the gift sample of sulfasalazine.
6. Experimental protocols
1H NMR spectra of the synthesized compound were
1
recorded in DMSO using H NMR Varian Mercury 300 Hz
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