DrugRes/2014-11-0895/13.4.2015/MPS
Original Article
(s, 1H, -CH-CH3), δ 6.58–6.59 (m, 1H, Ar-H), δ 6.73–6.76 (m, 1H
Ar-H), δ 6.89–7.03 (m, 2H, Ar-H), δ 7.08–7.09 (m, 1H, Ar-H), δ
7.20–7.24 (m, 1H, Ar-H), δ 7.51–7.62 (m, 3H, Ar-H), δ 7.66–7.71
(m, 2H, Ar-H) δ 7.73–7.77 (m, 2H, Ar-H). Mass: (70eV) m/z 376.
each for the evaluation of anti-inflammatory activity and ulcero-
genic potential and Swiss albino mice were used for the evalua-
tion of analgesic activity. The animals were housed in standard
polypropylene cages in an air-conditioned room at 22±3°C with
55±5% humidity and provided with standard laboratory diet
and water ad libitum. The study protocol was approved by Insti-
tutional Animal Ethics Committee (448/01/c/CPCSEA/PRCOP/14-
15/12).
2-(3-Benzoylphenyl)propionic acid, 4-formyl-2-
methoxyphenylester (KTPVAN)
UV (λmax): (MeOH) 260nm, IR (KBr) cm− 1: 3052 (C-H str. aro-
matic), 2933 and 2850 (C-H str. aliphatic), 1760 (C=O str. ester),
1212 (C-O str. ester), 1653 (C=C str. aromatic), 1288 (C-H bend-
ing In plane), 819.96 (C-H bending out of plane). 1H NMR
(500MHz, DMSO): δ 1.39–1.40 (d, 3H, -C-CH3), δ 3.81–3.82 (q,
1H, Ar-CH-CH3), δ 3.84 (s, 3H, -OCH3), δ 6.39 (d, 1H, Ar-H), δ
7.41–7.43 (d, 1H, Ar-H), δ 7.51–7.54 (t, 1H, Ar-H), δ 756–7.62
(m, 4H, Ar-H), δ 7.68–7.71 (m, 2H, Ar-H), δ 7.73–7.76 (m, 2H,
Ar-H), δ 9.97 (s, 1H, -CHO). Mass: (70eV) m/z 387.
Anti-inflammatory activity
The anti-inflammatory activity of ketoprofen ester prodrugs was
determined by using carrageenan-induced rat paw edema
model [32,33]. Group I served as the control and received only
vehicle (0.5% w/v aq.CMC suspension). Group II received keto-
profen (25mg/kg) while, the groups III–VIII received prodrugs in
the doses molecularly equivalent to ketoprofen, p.o. After 30min
of compound administration, 0.1mL of 1% w/v carrageenan in
normal saline was injected into the sub planter region of left
hind paw and the edema volume was measured before injection
and at the several intervals up to 12h. The initial volume of right
hind paw was measured using a digital plethysmometer without
administration of drug/prodrug.
2-(3-Benzoylphenyl)propionic acid
1,3-dihydroisobenzofuran-5-yl ester (KTPSE)
UV (λmax): (MeOH) 262nm, IR (KBr) cm− 1: 3061 (C-H str. aro-
matic), 2931 and 2852 (C-H str. aliphatic), 1754 (C=O str. ester),
1248 (C-O str. ester), 1601 (C=C str. aromatic), 1283 (C-H bend-
ing In plane), 893 (C-H bending out of plane). 1H NMR (500MHz,
DMSO): δ 1.52–1.54 (d, 3H, -C-CH3), δ 4.19–4.23 (q, 1H, Ar-CH-
CH3), δ 6.05 (s, 2H, -CH2-, Sesamol), δ 6.46–6.48 (m, 1H, Ar-H), δ
6.67–6.69 (d, 1H Ar-H), δ 6.72–6.76 (m, 2H, Ar-H), δ 6.58–6.60
(d, 1H, Ar-H), δ 6.89–6.90 (d, 1H, Ar-H), δ 7.54–7.55 (m, 1H,
Ar-H), δ 7.55–7.59 (m, 3H, Ar-H), δ 7.60–7.62 (m, 2H, Ar-H), δ
7.68–7.70 (m, 2H, Ar-H). Mass: (70eV) m/z 378.
Analgesic activity
Analgesic activity was carried out by the acetic acid induced
writhing method [34] using the swiss albino mice model. A 1%
v/v aqueous solution of acetic acid was used to induce writhings.
The animals of either sex were used and were divided in 8
groups of 6 animals each. Group I served as a control group,
group II received standard drug, ketoprofen (25mg/kg) and all
other 6 groups received appropriate prodrugs in molecularly
equivalent doses, orally, in a 1% w/v aqueous suspension of
sodium carboxymethylcellulose. Acetic acid was administered
intraperitoneally at 1mL/100g body weight of the animal. Test
compounds were administered orally 3h prior to acetic acid
injection. The number of writhings in 10min within the control
group and the standard and test compounds groups, were
counted and compared. Analgesic activity was measured as per-
centage decrease in writhing as compared to the control.
Solubility and Partition coefficient determination
10mg of the synthesized prodrug was tested for solubility in
0.5ml of each of the solvents, viz., ethanol, methanol, chloroform
and dichloromethane in separate test tubes. After gentle shak-
ing, solubility was observed. Further, 0.5ml of solvent was added
if required to completely dissolve the compound. The partition
coefficients of synthesized prodrugs were determined in
n-octanol-phosphate buffer (1:1) (pH 7.4) as follows. The prod-
rug, 10mg, was added to 10ml of aqueous phase followed by
addition of 10ml of n-octanol. The contents were thoroughly
shaken for 2h at room temperature and left for 1h. The concen-
trations in the aqueous and organic phase was determined using
acetonitrile: phosphate buffer pH 3 (60:40v/v) as mobile phase
and flow rate 1.0ml/min with UV detection at 260nm by using
HPLC [30] and partition coefficient computed.
Ulcerogenic potential
Gastrointestinal toxicity of the synthesized prodrugs was com-
pared with that of the parent drug, ketoprofen by measuring the
ulcer index. For this albino Wistar rat of either sex, weighing
around 100–150g each, were divided in 8 groups of 6 animals
each and fasted for 24h prior to administration of drug/prodrug.
The ketoprofen (standard, 250mg/kg) and prodrugs (dose of
prodrugs molecularly equivalent to ketoprofen) were adminis-
tered orally as aqueous suspension in 0.5% w/v acacia. The con-
trol group was administered as 0.5% w/v acacia aqueous
suspension only. Animals were sacrificed 12h after the treat-
ment. The stomach was removed, opened along the curvature,
rinsed with 5ml saline and examined by means of a magnifier.
The ulcer index was calculated as mean for all animals in the
group [35].
In vitro hydrolysis
The hydrolysis kinetics of the prodrugs was studied in aqueous
buffer solutions at pH 1.2 and pH 7.4 at 37°C using hydrochloric
acid-buffer and phosphate buffer, respectively. Solutions of
10mg of the prodrug prepared in 90mL of hydrochloric acid-
buffer (pH 1.2) or phosphate buffer (pH 7.4) were kept in screw
capped tubes maintained at 37±0.5°C. At definite time intervals
(15, 30, 60, 120, 240min), aliquots were withdrawn from tubes
and analyzed by HPLC for the amount of drug released after the
hydrolysis of the prodrug. Pseudo first order rate constants (Kobs
and half-life (t1/2) were calculated [31].
)
Statistical analysis
Statistical analysis was carried out for pharmacological evalua-
tion data using analysis of variance (ANOVA) test, followed by
Dunnet’s Test for determining level of significance. P-values<0.05
were considered statistically significant.
Pharmacological evaluations
The title prodrugs were evaluated for analgesic, anti-inflamma-
tory and ulcerogenic potential. Wistar rats (albino rats) of either
sex weighing 100–200g were divided into 8 groups of 6 animals
Dhokchawle BV et al. Ketoprofen Ester Prodrugs… Drug Res