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S.S. Dhaneshwar et al. / European Journal of Medicinal Chemistry 44 (2009) 3922–3929
Table 1
Physico-chemical and structural characterization of the synthesized prodrugs.
Code M.p.a Rf
%
Aqueous
log P
Elemental analysis
IR (KBr) cmꢃ1
1H NMR [300 MHz, DMSO-d6,
d (ppm), J
(Hz)]
(ꢀC)
Yield solubility
(g/mL)
ST
SP
TS
240– 0.60
245 CHCl3
74
72
78
0.28 ꢁ 0.02 0.26 ꢁ 0.04 Calculated: C, 54.42; H, 3.30; 3590; indole N–H stretching, 3466–3422; 2.5 [d, J ¼ 7.3, 2H] CH2-methylene, 2.6 [t,
N, 10.58; Na, 11.57; O, 20.14. unbonded phenolic OH stretching, 2320; J ¼ 7.42,1H] CH-methine, 6.64 [d, J ¼ 7.15,
Found: C, 54.50; H, 3.35; N, aromatic C–H stretching, 1597, 1383 cmꢃ1 1H], 7.2 [d, J ¼ 7.3, 1H], 7.4 [d, J ¼ 7.4, 1H]
(d)
ꢃMeOH
(4:1.5)
10.63; Na, 11.53; O, 20.20
carboxylate anion stretching, 1487; N]N CH-aromatic, 6.59 [d, J ¼ 7.3, 1H] aromatic
stretching (unsymmetric p-substituted
azobenzene), 1030; C–N stretching
OH, 6.9 [d, J ¼ 7.4, 1H], 7.10–7.16 [m, 4H]
CH-indole, 10.65 [d, J ¼ 7.3, 1H] NH-indole
2.8 [d, J ¼ 7.3, 2H] CH2-methylene, 2.9 [s,
230– 0.53,
235 CHCl3
0.25 ꢁ 0.04 0.30 ꢁ 0.05 Calculated: C, 53.64; H, 3.38; 3640–3526; unbonded phenolic OH
N, 7.82; Na, 12.83; O, 22.33. stretching, 1597, 1375; carboxylate anion 1H] CH-methine, 6.34 [d, J ¼ 7.3, 2H], 6.93
(d)
ꢃMeOH
Found: C, 53.67; H, 3.40; N, stretching, 1485; N]N stretching
7.68; Na, 12.89; O, 22.36
[d, J ¼ 7.3, 2H], 7.18 [s, 1H] CH-aromatic,
(4:1.5)
(unsymmetric p-substituted azobenzene), 6.40 [d, J ¼ 6.1, 1H] aromatic OH, 7.0 [d,
1030; C–N stretching
J ¼ 7.15, 1H], 7.47 [s, 1H], 7.5 [s, 1H] CH-
aromatic
283– 0.62,
285 CHCl3
0.26 ꢁ 0.06 0.35 ꢁ 0.02 Calculated: C, 51.35; H, 3.23; 3558–3219; unbonded phenolic OH
2.66 [d, J ¼ 7.32, 1H] CH2-methylene, 2.93
N, 7.49; Na, 12.29; O, 25.65. stretching, 3018; CH stretching (aromatic), [t, J ¼ 7.7, 1H] CH-methine, 5.80 [s, 1H]
(d)
ꢃMeOH
Found: C, 51.29; H, 3.28; N,
7.50; Na, 12.34; O, 25.70
1593, 1390; carboxylate anion stretching, aromatic OH, 5.83 [s, 1H] aromatic OH,
(4:1.5)
1494; N]N stretching (unsymmetrical
p-substituted azobenzene), 1276; out of
plane, CH bending aromatic ring, 1095;
C–N stretching
6.48 [d, J ¼ 7.3, 2H], 7.01 [d, J ¼ 7.3, 2H],
CH-aromatic, 7.74 [s, 1H], 7.55 [d, J ¼ 6.10,
1H], 7.53 [d, J ¼ 7.3, 1H], CH-aromatic
a
Uncorrected.
which were found to be diminished in D2O exchange thus con-
firming that the aromatic OH was unbonded. The other chemical
shifts were in accordance with the anticipated structures. The
enhanced aqueous solubility of all the prodrugs (0.25 ꢁ 0.02–
0.28 ꢁ 0.06 g/mL) and decreased partition coefficients (log P) in
n-octanol/phosphate buffer (pH 7.4) (0.26 ꢁ 0.05–0.35 ꢁ 0.02) as
compared to 5-ASA (aqueous solubility ¼ 1.85 ꢁ 0.2 mg/mL,
log P ¼ 0.64 ꢁ 0.04) suggest that their absorption from the upper
GIT would be minimum thus facilitating the passage of prodrugs
directly to colon (Table 1).
In vitro stability studies confirmed that the prodrugs were stable
in 0.05 M hydrochloric acid buffer (pH 1.2), whereas in phosphate
buffer (pH 7.4) 15–18% release was observed after 7 h. Cleavage of
azo bond by the colonic reduction in the presence of azo reductases
secreted by colonic microflora was confirmed by 85–88% cumu-
lated release of 5-ASA over a period of 6–7 h. The half-lives (average
of four trials) were in the range of 140–160 min with TS showing
lowest t1/2 and SP showing the highest (Table 2).
The conjugates showed remarkable reduction in the ulcer index
(10.76 ꢁ 0.55–13 ꢁ 1.1) as compared to very high ulcer index of
5-ASA (60.03 ꢁ1.15). These results were comparable to sulfasalazine
(9 ꢁ 2). Statistical differences between the groups were calculated
by Kruskal Wallis test followed by Dunnet’s post hoc test. All data are
expressed as mean ꢁ S.D. The results were found to be statistically
significant in relation to control (Table 3). The lower ulcer indices are
in accordance with stability of prodrugs in acidic pH.
Three important parameters i.e. clinical activity score, colon/
body weight ratio and MPO activity were studied in TNBS-induced
experimental colitis rat model to quantify the severity of colonic
inflammation and compare mitigating effects of synthesized pro-
drugs with sulfasalazine and 5-ASA on disrupted colonic architec-
ture. There is an inverse proportion between clinical activity score
and extent of mitigating effect on inflamed colon. A significant
lowering of clinical activity was shown by all the prodrugs amongst
which; results of TS (0.9 ꢁ 0.13) were similar while those of SP
(1.06 ꢁ 0.51) and ST (1.39 ꢁ 0.39) were comparable to sulfasalazine
(0.83 ꢁ 0.42) but distinctly more than 5-ASA (2.09 ꢁ 0.27). The
prominent differences in lowering effect of plain 5-ASA and the
prodrugs indicate the positive contribution of carriers. To check the
correctness of mutual prodrug hypothesis, animals of six separate
test groups were subjected to rectal administration of plain amino
acids and 5-ASA þ one amino acid each respectively. The lowering
of clinical activity score by rectally administered amino acids was
less than that of sulfasalazine (0.83 ꢁ 0.42) but better than amino
acids administered orally. Rectal co-administration of 5-ASA -
þ amino acid showed comparable lowering of clinical activity score
as that of sulfasalazine but better than prodrugs, amino acids or
5-ASA administered orally. These results are in accordance with the
obvious advantage of availability of 5-ASA and amino acids in their
effective concentrations in colon by rectal administration and
indicate positive contribution of amino acids that seems to be
responsible for the observed synergistic effect and also justify the
soundness of concept-based mutual prodrug design that has been
adopted for this work. Colon/body weight is inversely proportional
to ameliorating effect on the inflamed colon. The prodrug treated
groups showed a distinct decrease in the colon/body weight ratio
(TS > SLZ ¼ ST > SP) compared to colitis control group. MPO activity
is an important quantitative index for colonic inflammation and was
determined in terms of mU/100 mg tissue. Prodrugs showed
comparable MPO activity to sulfasalazine (SLZ < TS ¼ ST < SP)
Table 3
Ulcerogenic activity by Rainsford’s cold stress method.
Code
Dose (mg/kg)a
Ulcer index ꢁ S.D.b
Table 2
Healthy control
Salicylic acid
Sulfasalazine
5-ASA
4-ASA
ST
SP
TS
–
2290
3000
1154.30
1154.30
2829
2 ꢁ 1
59.6 ꢁ 4.7
9 ꢁ 2
In vitro release studies in rat fecal matter.
Code Order of reaction Half-life Rate constanta
% Cumulated release
of 5-ASA over a
period of 7 h
60.03 ꢁ 1.15
69.61 ꢁ 1.46
13 ꢁ 1.1
(min)
(k) minꢃ1
ST
SP
TS
First order
First order
First order
144
160
140
4.82 ꢂ 10ꢃ3 ꢁ 0.0001 88
4.32 ꢂ 10ꢃ3 ꢁ 0.0001 85
4.94 ꢂ 10ꢃ3 ꢁ 0.0001 87
3050
2647.70
11.3 ꢁ 1.1
10.76 ꢁ 0.55
a
At ten times normal dose.
Average of six readings; P < 0.01.
a
b
Average of three trials.