R. Sharma et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5332–5338
5335
O
C
OH
OH
O
C
OH
OH
(a)
(13)
C
O
HO
HO
N
N
N
C
O
(12)
H2N
(b)
O
C
O
Ba
C
O
O
N
(14)
(15)
(c)
O
C
H2
O
N
Pt
C
O
O
N
HO
N N
H2
Scheme 3. Synthesis of azo prodrug of RTB-4 (15)30. Reagents and conditions: (a) HCl/NaNO2, (0–5 °C), C6H5OH; (b) Ba(OH)2Á8H2O; (c) (DACH)Pt(SO4), yield 61%.
N@N stretching (1589–1640 cmÀ1) and the platinum complex was
examined with confirmation of frequency of NH2, which were
shifted to lower frequencies in comparison to free amino group
due to the coordination of platinum (II), that is, near 1663–
1610 cmÀ1. The characteristics chemical shifts of aromatic OH
were observed at d (4.16–5.32) in 1H NMR. In mass spectrometry,
all the compounds showed their molecular ion peak (M+1)+ which
are correspond to their molecular weight. The elemental data of all
these compounds were found to be in good agreement with the
calculated values of proposed structure.
The stability of the synthesized prodrugs of (10, 11 and 15) was
checked in 0.1 N HCl solution and 7.4 phosphate buffer in simu-
lated gastric and intestinal fluid, respectively. Weighed amount
of prodrugs of methotrexate (10), gemcitabine (11) and RTB-4
(15) was taken in separate baskets having 900 mL solution of
0.1 N HCl which was kept at constant temperature,that is,
37 1 °C with occasional stirring. At various time intervals, 5 mL
aliquots were withdrawn and estimated on UV spectrophotometer
(Shimadzu, UV-1700) at 306 nm, 256 nm (0.1 N HCl, 7.4 pH) for
(6); 274 nm, 284 nm (0.1 N HCl, 7.4 pH) for (7); 280 nm, 260 nm
(0.1 N HCl, 7.4 pH) for (8) for the estimation of decrease in concen-
tration of prodrugs in GIT medium.
dye was reduced with colonic microflora due to the presence
of azoreductase enzyme which was absent in the flora free
medium. Therefore, similar experimental protocol was fol-
lowed with newly synthesized azo based prodrugs (10, 11
and 15) instead of amaranth and analyzed by UV Spectro-
photometer at 256 nm (6), 284 nm (7) and 260 nm (8). This
experiment clearly defined the mechanism of azo based
drugs that the azo bond was only reduced in the presence
of specific azoreductase enzyme which is present the rat’s
fecal material (intestinal microflora) and releases the drug
molecules to the colon site.
(2) The cecal content, SI content were removed from wistar rats
and suspended in cold PBS, pH 7.4 and 10% (w/v) slurry was
maintained. The cecal content was bubbled with nitrogen
and stored at À20 °C. The suspension of SI content, LI
mucosa and SI mucosa was stored at À20 °C after homogeni-
zation and centrifugation with final concentration of (2% w/
w). The % release of drugs from their prodrugs were evalu-
ated and represented graphically.
The release behavior of methotrexate (6), gemcitabine (7) and
RTB-4 (8) from their prodrugs, fresh fecal content of rats (about
1 g) was taken in different sets of test tubes. The final concentra-
tion of prodrugs solution was prepared up to 250 lg/mL after dis-
solving them into phosphate buffer (pH 7.4). The prodrugs
solutions (1 m) were added to each test tube and diluted it further
with 5 ml of phosphate buffer (50 lg/ml). The test tubes were al-
lowed to incubate at 37 °C. Aliquots were removed from the test
tubes at different time intervals and were estimated on double
beam UV spectrophotometer at above mentioned wavelengths.26
The cytotoxicity of synthesized prodrugs were determined by
means of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tet-
razolium bromide) colorimetric microculture assay as per standard
procedure.27 The cell lines COLO 205, COLO 320 DM and HT-29
were chosen for the cytotoxicity study. Cells were plated in
(5 Â 105 well) in 96 well plates. After 48 h, cells were incubated
in 0.1% DMSO for 37 °C. After the removal of sample solution, it
was washed properly with phosphate saline buffer (pH 7.4) and
then cells were treated with MTT solution. The isopropranol solu-
tion (0.04 M) was added after the 4 h of incubation. The viability of
cells was determined by the absorbance at 595 nm with Elisa
microplate reader (Bio Rad). All the measurements were performed
in triplicates and IC50’s were expressed in mean SD and p <0.05
were considered as significant value.
The azoreductase assay was performed by two different ways
which confirmed the reduction of azo bond by azoreductase en-
zyme as well as the release of drugs (10, 11 and 15) from the azo
bond to the target site. The azoreductase assay was carried out
(1) in the presence of rat fecal matter with Gram anaerobic
medium23,24 (2) in the freeze dried cecum content and in other dif-
ferent parts of intestinal contents, that is; SI content, SI mucosa,
and LI mucosa.25
(1) The degradation pattern of all three azo based prodrugs was
evaluated in the presence of azoreductase which was cul-
tured after the preparation of Gram anaerobic medium
(GAM).23,24 More than 70% of human feces15 and rat feces14
contain colonic microflora. Therefore, rat fecal matter was
used as a source of azoreductase enzyme in colonic micro-
flora. Rat feces (1 g) were poured into 9 mL of GAM which
was further diluted with GAM to get the standard solution
of microflora. A few mL of this solution was transferred to
the cultured tube which was diluted 200 times with the
gram anaerobic medium and kept aside under anaerobic
conditions at 37 °C for a day. The pH (7.2–7.4) was main-
tained throughout the experiment. The reductive behavior
of azoreductase was confirmed by incubating the amaranth
(an azo dye) and analyzed by UV at kmax 520 nm. The azo
Colon targeting azo prodrugs have the challenge to survive
themselves while passage from stomach and small intestine as
Sharma, Rajiv
