RETRACTED ARTICLE: Synthesis, kinetic studies and pharmacological evaluation of mutual azo prodrug of 5-aminosalicylic acid with d-phenylalanine for colon specific drug delivery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

Article abstract of DOI:10.1016/j.bmcl.2007.01.031

Mutual azo prodrug of 5-aminosalicylic acid with d-phenylalanine was synthesized by coupling d-phenylalanine with salicylic acid, for targeted drug delivery to the inflamed gut tissue in inflammatory bowel disease. The structure of synthesized prodrug was

Full text of DOI:10.1016/j.bmcl.2007.01.031

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1897–1902
Synthesis, kinetic studies and pharmacological evaluation of mutual
azo prodrug of 5-aminosalicylic acid with D-phenylalanine for colon
specific drug delivery in inflammatory bowel disease
Suneela S. Dhaneshwar,a,* Neha Gairola,a Mini Kandpal,a Lokesh Bhatt,b
Gaurav Vadnerkara and S. S. Kadama
aDepartment of Pharmaceutical Chemistry, Bharati Vidyapeeth University, Poona College of Pharmacy,
Pune 411038, Maharashtra, India
bDepartment of Pharmacology, Bharati Vidyapeeth University, Poona College of Pharmacy,
Pune 411038, Maharashtra, India
Received 31 July 2006; revised 27 December 2006; accepted 11 January 2007
Available online 19 January 2007
Abstract—Mutual azo prodrug of 5-aminosalicylic acid with D-phenylalanine was synthesized by coupling D-phenylalanine with sal-
icylic acid, for targeted drug delivery to the inflamed gut tissue in inflammatory bowel disease. The structure of synthesized prodrug
was confirmed by elemental analysis, IR and NMR spectroscopy. In vitro kinetic studies in HCl buffer (pH 1.2) showed negligible
release of 5-aminosalicylic acid, whereas in phosphate buffer (pH 7.4) only 15% release was observed over a period of 7 h. In rat fecal
matter the release of 5-aminosalicylic acid was almost complete (85%), with a half-life of 160.1 min, following first order kinetics.
The azo conjugate was evaluated for its ulcerogenic potential by Rainsford’s cold stress method. Therapeutic efficacy of the carrier
system and the mitigating effect of the azo conjugate were evaluated in trinitrobenzenesulfonic acid-induced experimental colitis
model. The synthesized prodrug was found to be equally effective in mitigating the colitis in rats as that of sulfasalazine without
the ulcerogenicity of 5-aminosalicylic acid.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Inflammatory bowel disease (IBD) is characterized by
chronic inflammation in the mucosal membrane of the
small and/or large intestine.1 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 inflamma-
tion in the colon that requires frequent intake of antiin-
flammatory 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 significant ad-
verse effects.2,3 Therefore, out of the need to overcome
this formidable barrier of GIT, colonic drug delivery
has evolved as an ideal drug delivery system for the top-
ical treatment of diseases of colon like Crohn’s disease,
ulcerative colitis, colorectal cancer and amaebiasis. To
achieve successful colonic delivery, a drug needs to be
protected from absorption and/or the environment of
upper GIT and then be abruptly released into proximal
colon, which is considered as the optimum site for
colon-targeted delivery of drug.4
Prodrug approach is one of the important approaches
for targeting drugs to colon. Colon-specific drug deliv-
ery 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 sulfa-
salazine (an azo prodrug 5-ASA and sulfapyridine) for
targeting drugs to colon.5 But majority of side effects
of sulfasalazine like hepatotoxicity, hypospermia and
severe blood disorders are due to sulfapyridine. Few
prodrugs of 5-ASA like basalazine, ipsalazine and olsal-
azine have been reported, but most of them suffer from
adverse effects due to the carriers used with them.6–8 The
Keywords: Mutual azo prodrug; 5-Aminosalicylic acid; Inflammatory
bowel disease; D-Phenylalanine.
*
Corresponding author. Tel.: +91 20 25437237/25436898; fax: +91 20
25439383; e-mail: suneeladhaneshwar@rediffmail.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.031

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need for a totally safe, colon specific prodrug of 5-ASA
with nontoxic carrier still remains. In the present work,
concept of mutual prodrug has been adopted for synthe-
sis of azo conjugate 5-ASA with D-phenylalanine (SP)
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 evaluate
the therapeutic efficacy of this drug-carrier system in
experimental colitis rat model. D-Phenylalanine was
chosen as a promoiety due to its marked antiinflamma-
tory activity.9 Being a natural component of our body, it
would be nontoxic and free from any side effects. Intro-
duction of azo linkage in the prodrug (similar to sulfa-
salazine) would ensure release of 5-ASA in colon by
the reductive action of azo reductases secreted by the
colonic microflora.
0
100
200
300
400
500
Time (min)
Figure 1. Release profile of 5-ASA from SP in rat fecal matter.
Table 1. Results of ulcerogenic activity
Compound
Dose (mg/kg)
Ulcer index SDa
The melting point of SP was found to be 230–235 ꢁC
(uncorrected). All the results of elemental analysis were
in an acceptable error range.
HC
—
2
1
59.6 4.7
5-ASA
Slz
SP
2290
3000
3050
9
1.33 1.1
2
The IR spectra of 5-ASA conjugate showed characteris-
tic peak at 1485 cmÀ1 of N@N-stretching (unsymmetric
p-substituted azobenzene) which confirms the formation
of azo bond. A broad peak of unbonded phenolic O–H
stretching at 3640–3526 cmÀ1 was also found. It also
showed carboxylate anion stretching at 1597 cmÀ1 and
HC, healthy control; 5-ASA, 5-amino salicylic acid; Slz, sulfasalazine;
SP, prodrug of 5-ASA with D-phenylalanine.
a Average of six readings.
in the ulcer index brought about by the conjugate was
comparable to that produced by sulfasalazine (9 2).
Statistical differences between the groups were calculat-
ed by Kruskal Wallis test followed by Dunn’s post hoc
test. All data are expressed as means SD. Differences
were considered at a P value of <0.01 in relation to
control.
1375 cmÀ1 and C–N stretching at 1030 cmÀ1
.
1H NMR spectra of SP showed chemical shifts for pro-
tons of aromatic OH at d 6.40 [d, 1H], CH-benzene d
6.34 [d, 1H], d 6.98 [d, 1H] and d 7.47 [s, 1H]. The signals
of CH-methine at d 2.8 [s, 1H], CH2-methylene at d 2.9
[d, 2H] were also found. The aqueous solubility was
found to be 0.25 g/ml and partition coefficient in n-oct-
anol/phosphate buffer (pH7.4) was found to be 0.30,
which was decreased as compared to 5-ASA (0.64).
The kinetics was monitored by the decrease in prodrug
concentration with time in HCl buffer (pH 1.2) at
236 nm and phosphate buffer (pH 7.4) at 294 nm. Kinet-
ic studies confirmed that the prodrug did not release the
parent drug in 0.05 M hydrochloric acid buffer (pH 1.2),
whereas in phosphate buffer (pH 7.4) only 15% 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 matter10 to confirm the colonic reduction of
azo prodrug. t 12 (average of four trials) of SP was found
to be 160.1 min, whereas rate constant (K) was found to
be 4.32 · 10À3 0.0001. Over a period of 7 h, SP gave
85% cumulated release of 5-ASA following first order
kinetics (Fig. 1). Thus in vitro kinetic studies confirmed
that the synthesized conjugate did not release 5-ASA at
all in HCl buffer (pH 1.2) but in phosphate buffer (pH
7.4), 15% release was observed. The release in rat fecal
matter was almost complete.
In order to study the feasibility of azo prodrug of 5-ASA
for targeted oral drug delivery to the inflamed tissue of
colon in IBD, TNBS-induced experimental colitis model
was selected.13–15 After inducing the experimental coli-
tis, the clinical activity score increased rapidly and
consistently for the next 3 days for all groups. All drug-
receiving groups showed a decrease of inflammation
severity after a lag time of 24–48 h. The difference
between the drug treated group and colitis control group
became significant on day 7. A significant lowering of
clinical activity was shown by SP (1.39 0.39), which
was comparable to sulfasalazine (0.83 0.42) but
distinctly more than 5-ASA (2.09 0.27). The positive
contribution of D-phenylalanine towards lowering effect
on clinical activity score (1.92 0.08) is obvious from
the gross difference in lowering effect of plain 5-ASA
and SP. To ensure the synergistic effect of D-phenylala-
nine further, two test groups of animals were subjected
to rectal administration of plain D-phenylalanine
and 5-ASA+ D-phenylalanine, respectively. The lower-
ing of clinical activity score by rectally administered
D-phenylalanine was (1.06 0.35) less than that of
sulfasalazine (0.83 0.42) but better than D-phenylala-
nine administered orally (1.92 0.08). Co-administra-
tion of 5-ASA+ D-phenylalanine showed comparable
lowering of clinical activity score as that of sulfasalazine
(0.83 0.42) but better than D-phenylalanine (1.92
0.08) or 5-ASA (2.09 0.27) administered orally. This
The synthesized compound was evaluated for ulcerogen-
ic activity by Rainsford’s method11 and the ulcer index
was determined12 (Table 1). The conjugate showed
remarkable reduction in the ulcer index (11.3 1.1) as
compared to its parent drug (59.6 4.7). This reduction

S. S. Dhaneshwar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1897–1902
1899
particular finding supports positive contribution of
D-phenylalanine and hence its synergistic effect. On
day 11 (24 h after the drug administration), the animals
were sacrificed and colon/body weight ratio was deter-
mined to quantify inflammation. The prodrug treated
group showed a distinct decrease in the colon/body
weight ratio compared to colitis control group (Fig. 3).
Decrease in colon/body weight ratio produced by
rectally administered D-phenylalanine as well as
5-ASA+ D-phenylalanine was comparable to sulfasala-
zine. During the evaluation of macroscopic damage of
colon segments in colitis control, the colons appeared
flaccid and filled with liquid stool. The cecum, colon
and rectum all had evidence of mucosal congestion,
erosion and haemorrhagic ulcerations and histopatholo-
gical features included transmural necrosis, oedema,
absence of epithelium, a massive mucosal/submucosal
infiltration of inflammatory cells. In vivo treatment with
SP resulted in the significant decrease in the extent and
severity of colonic damage. Its histopathological
features clearly indicated that the morphological distur-
bances associated with TNBS administration were cor-
rected by treatment with SP. These results were found
to be comparable with those obtained for sulfasalazine
treated group. Histopathological features of rectally
administered D-phenylalanine and 5-ASA + D-phenylal-
anine groups also indicated correction of disrupted
morphology of the colon. Statistical differences between
the groups were calculated by Kruskal Wallis test
followed by Dunn’s post hoc test. Differences were
considered at a P value of <0.01 in relation to control.
The feasibility of reduction of azo linkage by azo reduc-
tase secreted by intestinal microflora was tested with the
help of release study in rat fecal matter at 37 1 ꢁC. All
the kinetic studies were carried out in triplicate. The K
values from the plots were calculated separately and
average K and SD values were determined. The half lives
were calculated using software ‘PCP Disso’ developed
by Department of Pharmaceutics, Poona College of
Pharmacy, Pune. The process was validated as per
U.S.P. XXIV edition using different parameters like
accuracy, selectivity, sensitivity and reproducibility. SP
(10 mg) was introduced in 900 ml HCl buffer taken in
a basket and was kept in a constant temperature bath
at 37 1 ꢁC. The solution was occasionally stirred and
5 ml aliquot portions were withdrawn at various time
intervals. The aliquots were shaken with equal amount
of chloroform in order to remove the interference by
5-ASA which was supposed to be released by the synthe-
sized prodrug and the aliquots were estimated on UV
spectrophotometer at 294 nm for the amount of SP
remaining.
Same procedure as described earlier was followed;
except that the HCl buffer was replaced by phosphate
buffer. The kinetics was monitored by the decrease in
prodrug concentration with time.
To study the release of 5-ASA from SP in rat fecal mat-
ter,18 SP was dissolved in sufficient volume of phosphate
buffer (pH 7.4) so that final concentration of solution
was 250 lg/ml. Fresh fecal material of rats was weighed
(about 1 g) and placed in different sets of test tubes. To
each test tube containing weighed amount of rat fecal
matter, 1 ml of the prodrug solution was added and dilut-
ed to 5 ml with phosphate buffer (50 lg/ml). The test tubes
were incubated at 37 ꢁC for different intervals of time. For
analysis, the aliquots of SP were removed from the test
tubes at different time intervals and shaken with chloro-
form so as to extract free drug from the aliquots. The con-
centration of 5-ASA was directly estimated from the
chloroform layer on double-beam UV-spectrophotome-
ter (JASCO, V-530 model, Japan) at 322 nm.
The data generated as an outcome of this work demon-
strate that this new prodrug has a remarkable ameliorat-
ing effect on the disruption of colonic architecture and
suppresses the course of TNBS-induced colitis effectively.
The criterion for selection of D-phenylalanine as carrier
has also proven correct, as it has effectively delivered 5-
ASA to colon. Moreover, its synergistic ameliorating ef-
fect on disrupted colonic architecture strengthens the
hypothesis of mutual prodrug design.
1H NMR spectra of the synthesized compound were
recorded in DMSO using 1H NMR Varian Mercury
300 Hz with superconducting magnet using TMS as
internal standard. Chemical shift values are reported
in ppm downfield on dscale. The IR spectra of the syn-
thesized compound was recorded on JASCO, V-530
FTIR in potassium bromide (anh. IR grade). The absor-
bance maxima (kmax) of synthesized compound was
determined on JASCO V530, UV–visible double-beam
spectrophotometer in hydrochloric acid buffer (pH
1.2), phosphate buffer (pH 7.4) and distilled water. Par-
tition coefficient was determined in n-octanol/phosphate
buffer (pH 7.4), whereas the aqueous solubility was
determined in distilled water at room temperature
(25 1 ꢁC).
Pharmacological screening of the synthesized compound
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 approved by the Institutional Animal
Ethical Committee.
All chemicals used in the synthesis were of AR grade.
Sulfasalazine was obtained as gift sample from Wallace
Pharmaceutical Pvt. Ltd. Goa; salicylic acid and
D-phenylalanine were purchased from Loba Chemie,
Mumbai. The reactions were monitored on TLC, which
was performed on precoated silica gel plates-60 F264
(Merck) using solvent system of chloroform: methanol
(4:1.5) and iodine vapours/UV light as detecting agents.
In vitro stability studies were carried out in hydrochloric
acid buffer (pH 1.2), phosphate buffer (pH 7.4).16,17 The
total buffer concentration was 0.05 M and a constant
ionic strength (l) of 0.5 was maintained for each buffer
by adding a calculated amount of potassium chloride.
Synthesis of methyl ester hydrochloride of D-phenylala-
nine19 was carried out by adding thionyl chloride to
methanol followed by refluxing with D-phenylalanine
(1) at 60–70ꢁ C for 7 h. D-Phenylalanine methyl

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S. S. Dhaneshwar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1897–1902
Scheme 1.
4.5
4
HC
3.5
3
CC
5-ASA
2.5
2
Slz
PA oral
SP
1.5
1
5-ASA+ PA rectal
PA rectal
0.5
0
1
2
3
4
5
6
7
8
9
10 11
Figure 2. Clinical activity score rate. HC, healthy control; CC, colitis control; 5-ASA, 5-amino salicylic acid; Slz, sulfasalazine; PA oral,
D-phenylalanine oral; SP, prodrug of 5-ASA with D-phenylalanine; 5-ASA with PA rectal, co-administration of 5-ASA with PA by rectal route; PA
rectal, rectal administration of D-phenylalanine.
ester hydrochloride (2) was diazotised20 at 0–5 ꢁC in
cryostatic bath. The coupling20,21 of diazonium salt of
0.014
0.012
0.01
D-phenylalanine (3) with salicylic acid (4) was carried
out at 0–5 ꢁC in a cryostatic bath (Scheme 1). It was
recrystallized by methanol followed by cooling at 0 ꢁC.
Purified product (SP) was dried under vacuum.
0.008
0.006
0.004
0.002
0
The ulcerogenic activity was determined by Rainsford’s
cold stress method,11 which is an acute study model and
is used to determine ulcerogenic potency of a drug at ten
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 continuous 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 experi-
CC
5-ASA
Slz
SP
PA Oral PA Rectal
5-
ASA+PA
Rectal
Figure 3. Colon to body weight ratio. HC, healthy control; CC, colitis
control, 5-ASA, 5-amino salicylic acid; Slz, sulfasalazine; PA oral,
D-phenylalanine oral; SP, prodrug of 5-ASA with D-phenylalanine;
5-ASA with PA rectal, co-administration of 5-ASA with PA by rectal
route; PA rectal, rectal administration of D-phenylalanine.

S. S. Dhaneshwar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1897–1902
1901
mental groups of six animals each. Following oral
administration of 5 ml of the aqueous drug suspensions
(at 10 times the normal dose), the animals were stressed
by exposure to cold (À15 ꢁC for 1 h). The animals were
placed in separate polypropylene cages to ensure equal
cold exposure. After 2 h of drug administration, the ani-
mals 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 SD.
Figure 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, showing slight mucosal abscess and inflammatory
infiltrate on oral administration. (d) Sulfasalazine. (e) SP showing corrected morphology of colon with comparable results to that of sulfasalazine. (f)
D-Phenylalanine showing no mucosal injury with slight inflammatory infiltrate on oral administration. (g) D-Phenylalanine showing corrected
morphology of colon with comparable results to that of sulfasalazine on rectal administration. (h) Co-administration of 5-ASA and D-phenylalanine
by rectal route, showing comparable ameliorating effect to that of sulfasalazine.

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S. S. Dhaneshwar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1897–1902
In order to study the ameliorating effect of azo prodrug
of 5-ASA on the inflamed tissue of colon in IBD, trini-
trobenzenesulfonic acid (TNBS)-induced experimental
colitis model was selected which is simple and reproduc-
ible. Moreover, it is the most relevant model as it in-
volves the use of immunological haptens and develops
a chronic inflammation rather than an acute mucosal
injury.22 By this model in vivo characterization of the
azo carrier system under the influence of chronic inflam-
matory symptoms was possible. Sprague–Dawley rats
(average weight 200–230 g; 12–15 w; n = 6/group) were
used. They were distributed into six different groups,
i.e. healthy control, colitis control, two standard groups
and two 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 water ad libitum after the administration of TNBS.
To induce an inflammation, all the groups except
healthy control group were treated by a procedure dis-
cussed below. After light narcotizing with ether, the rats
were catheterized 8 cm intrarectal and 500 ll of TNBS
(Himedia Laboratories Pvt. Ltd., Mumbai) in ethanol
was injected into colon via rubber canula (dose was
150 mg/kg of body weight of TNBS in ethanol, 50%
solution). Animals were then maintained in a vertical
position for 30 sec 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
orally 5-ASA, sulfasalazine, D-phenylalanine and SP,
respectively, once daily for five continuous days at doses
equimolar to 5-ASA present in sulfasalazine. The
healthy control and colitis control groups received only
1% carboxymethylcellulose instead of free drug or pro-
drug. The animals of all groups were examined for
weight loss, stool consistency and rectal bleeding
throughout the 11 days study. Colitis activity was quan-
tified with a clinical activity score assessing these param-
eters (Fig. 2) 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).23 They were sacrificed 24 h after the
last drug administration by isoflurane anaesthesia and
a segment of colon, 8 cm long, was excised and colon/
body weight ratio was determined to quantify the inflam-
mation (Fig. 3). Tissue segments 1 cm in length were then
fixed in 10% buffered formalin for histopathological
studies. Histopathological studies (Fig. 4a–e) of the
colon were carried out using haematoxylin and eosin
stains, at Nucleus Pathology Laboratory, Pune. Col-
oured microscopical images of the colon sections were
taken on Zeiss optical microscope, Stemi 2000-C, with
resolution 5 · 20X, attached with trinocular camera at
Kolte Pathology Laboratory, Pune.
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