Synthesis, hydrolysis studies and phamacodynamic profiles of amide prodrugs of dexibuprofen with amino acids

Article abstract of DOI:10.3109/14756366.2010.548327

The present investigation deals with the synthesis of novel prodrugs of dexibuprofen with amino acids with an aim to achieve potent anti-inflammatory activity and less gastrointestinal toxicity. Structures of synthesized compounds were confirmed by spectral and elemental analyses. In vitro hydrolytic studies in simulated intestinal fluid, 80% plasma and rat faecal matter showed satisfactory release of dexibuprofen due to enzymatic cleavage. The synthesized prodrugs were evaluated for anti-inflammatory activity, analgesia, ulcerogenicity and histopathology. The anti-inflammatory activity of dexibuprofen was 43.3% whereas an improved value of 73.4, 77.3, 72.8 and 64.5% was observed for the synthesized prodrugs. The percentage analgesia of the prodrugs increased, whereas a decrease in the mean ulcer index values than dexibuprofen was observed. The histopathological studies revealed less ulceration in the gastric region when treated with prodrugs. Thus, the prodrugs were proved to be better in action as compared with the parent drug.

Full text of DOI:10.3109/14756366.2010.548327

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(5): 688–695
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.548327
RESEARCH ARTICLE
Synthesis, hydrolysis studies and phamacodynamic profiles of
amide prodrugs of dexibuprofen with amino acids
Arun Rasheed1, C. K. Ashok Kumar1, and Ashutosh Mishra2
1Department of Pharmaceutical Chemistry, Sree Vidyanikethan College of Pharmacy, Sree Sainath Nagar, Tirupati,
Andhra Pradesh, India, and 2Department of Pharmaceutical Chemistry, Acharya Narendradev College of Pharmacy,
Babhnan, Gonda, Uttar Pradesh, India
Abstract
The present investigation deals with the synthesis of novel prodrugs of dexibuprofen with amino acids with an aim to
achievepotentanti-inflammatoryactivityandlessgastrointestinaltoxicity. Structuresofsynthesizedcompoundswere
confirmed by spectral and elemental analyses. In vitro hydrolytic studies in simulated intestinal fluid, 80% plasma and
rat faecal matter showed satisfactory release of dexibuprofen due to enzymatic cleavage. The synthesized prodrugs
were evaluated for anti-inflammatory activity, analgesia, ulcerogenicity and histopathology. The anti-inflammatory
activity of dexibuprofen was 43.3% whereas an improved value of 73.4, 77.3, 72.8 and 64.5% was observed for the
synthesized prodrugs. The percentage analgesia of the prodrugs increased, whereas a decrease in the mean ulcer
index values than dexibuprofen was observed. The histopathological studies revealed less ulceration in the gastric
region when treated with prodrugs. Thus, the prodrugs were proved to be better in action as compared with the
parent drug.
Keywords: NSAIDs, dexibuprofen, amide prodrugs, pharmacokinetics, ulcerogenicity
Introduction
into corresponding amides of amino acid, and it results
Dexibuprofen
(Dex),
a
novel
non-steroidal
in altering the physical properties of parent drug with
one or more of hydrolase enzymes serving as the in vivo
reconversion sites. e new prodrugs were expected
to have better lipophilicity, reduced gastric irritancy,
improved therapeutic index through the prevention of
GI irritation and bleeding, improved anti-inflammatory
activity, reduced GI erosive properties and synergistic
anti-inflammatory drug (NSAID), is a single and phar-
macologically effective enantiomer of racemic ibuprofen.
Racibuprofen and dexibuprofen differ in their physico-
chemical properties, pharmacological profiles and meta-
bolic activities1. e efficacy of dexibuprofen was found
to be same as that of other NSAIDs, such as diclofenac,
naproxen and celecoxib. Generally, the NSAIDs are asso-
ciated with various gastrointestinal (GI) side effects like
stomach ulceration, bleeding and perforation due to the
presence of its acidic group. e GI toxicity is produced
either by direct contact mechanism or by generalized sys-
temic action, which occur after absorption. To overcome
the GI side effects, prodrugs of NSAIDs can be developed
by hiding its acidic group through conjugation with
amino acids. Many amino acids possess site specificity,
marked anti-inflammatory activity and exhibit profound
healing effect on gastric toxicity2. During conjugation, the
NSAIDs having free carboxylic group can be derivitized
analgesic effect than the parent drug3,4
.
e synthesis of prodrugs of ketoprofen, diclofenac,
flubiprofen, naproxen, ibuprofen, etc., with various amino
acids via masking the carboxylic acid group to form ethyl
ester, methyl ester and glycolamide ester prodrug5-7 were
carried out successfully so far. e objectives of this study
are (i) to synthesize the amide prodrugs of Dex with methyl
esters of l-tryptophan, l-phenylalanine, glycine and
l-tyrosine to form Dex-1, Dex-2, Dex-3 and Dex-4, respec-
tively and (ii) to perform a detailed study on their various
physicochemical properties, hydrolysis kinetics and phar-
macological activities. e compounds synthesized will
Address for Correspondence: Arun Rasheed, Department of Pharmaceutical Chemistry, Sree Vidyanikethan College of Pharmacy, Sree
Sainath Nagar, Tirupati, Andhra Pradesh 517102, India. Tel.: 0919701425804. E-mail: arunrasheed@rediffmail.com
(Received 28 March 2010; revised 19 November 2010; accepted 09 December 2010)
688

Synthesis and evaluation of novel dexibuprofen amide prodrugs 689
act as prodrugs of dexibuprofen, which upon administra-
tion would release the parent drug as a result of hydrolysis
(enzymatic or non-enzymatic) in the body.
methanol by slow addition of 15–20 mL ether followed
by cooling at 0°C. e crystals were collected next day
and washed twice with ether: methanol mixture at 5:1
ratio followed by pure ether and dried under vacuum
to give pure tryptophan methyl ester hydrochloride
(2a). e same procedure was followed to synthesize
phenylalanine methyl ester hydrochloride, glycine
methyl ester hydrochloride and tyrosine methyl ester
hydrochloride.
Materials and methods
Materials
e amino acids l-tryptophan, l-phenylalanine, glycine
and l-tyrosine were obtained from M/s Hi-Media Ltd.,
India, and drug dexibuprofen was obtained as a gift
sample from Alkem Laboratories, India. Other reagents
and solvents were of analytical grade. e melting points
were recorded using melting point determination appa-
ratus by Sigma Instrument, India, and are uncorrected.
e elemental analysis was performed using Carlo-Erba
Model 1108 Analyzer (Italy). 1H NMR and 13C NMR spectra
were recorded in diemthylsulphoxide (DMSO) on NMR
spectrophotometer (Bruker DRX 300, USA). Chemical
shifts are expressed as δ (ppm) values. IR spectra were
recorded using IR spectrophotometer (Shimadzu FTIR-
8201PC (Kyoto, Japan)) in KBr phase and mass spectra
were recorded on mass spectrophotometer (Jeol SX-102
(FAB), Japan). e hydrolysis data and drug content
determination were performed by ELICO Double Beam
UV-VIS Spectrophotometers (Hyderabad, India).
Step 3: Synthesis of prodrugs of dexibuprofen with methyl
esters of l-tryptophan, l-phenylalanine, glycine and l-tyrosine
Ice cold, aqueous sodium hydroxide solution (5 %) was
taken in 250 mL beaker and 12.6 g (0.05 mol L−1) of methyl
ester of tryptophan hydrochloride was added to it. e
reaction mixture was mechanically stirred for 30min at
room temperature, after which the beaker was trans-
ferred to an ice bath kept on the mechanical stirrer, main-
taining the temperature at 10°C. To this, 22.3g (0.01 mol
L−1) of dex acid chloride was added in small portions with
continuous stirring for 7–8 h. e solid that separated
out was filtered off. e crude prodrug was recrystallized
from methanol. e same procedure was followed for
l-phenylalanine, glycine and l-tyrosine. e schematic
representation of synthesis of tryptophan-conjugated
dexibuprofen (Dex 1), phenylalanine-conjugated dexi-
buprofen (Dex 2), glycine-conjugated dexibuprofen
(Dex 3) and tyrosine-conjugated dexibuprofen (Dex 4)
are shown in Scheme 1.
Synthesis of amide prodrugs of dexibuprofen
Dexibuprofenis2-[4-(2-methylpropyl)phenyl]propanoic
acid and the synthesis of its amide prodrugs was carried
out by Schotten Baumann technique8 as explained below.
e purity of the compounds was checked by thin-layer
chromatography on pre-coated silica GF252 plates using
iodine vapour as a detecting agent. e structures of all
the synthesized prodrugs were confirmed by elemental
Spectral analysis
e spectral data obtained for Dex 1, Dex 2, Dex 3 and
Dex 4 are given as follows.
Dex 1: S(+) Methyl 3-(1H-indol-3-yl)-2-(2-(-
1
analysis and spectral analysis such as IR, H NMR, 13C
4-isobutylphenyl)propanamido)propanoate−
UV
NMR and mass spectroscopy.
Spectra (λmax) in simulated gastric fluid (SGF) 205 nm, in
simulated intestinal fluid (SIF) 225 nm, in 80% human
plasma 218 nm; IR (KBr, cm−1) 3290 (NH str. of amide),
3075 (aromatic CH str.), 1677 (C=O str. of ester), 1570
Step 1: Synthesis of dexibuprofen acid chloride
Dex (1) of 10.31 g (0.05 mol L−1) was dissolved in mini-
mum amount of chloroform and freshly distilled 6 mL
(0.05 mol L−1) thionyl chloride was added slowly to it. e
mixture was refluxed at 60–70°C with continuous stirring
on a magnetic stirrer. e viscous liquid was immediately
poured on petridish and was vacuum dried to give yellow
coloured crude dexibuprofen acid chloride (1a).
1
(C=N), 1292 (CO str.of ester); H NMR (δ ppm) (DMSO-
d6) 1.48 (d, J= 7.1 Hz, 3H, CH3), 1.52 (d, J= 6.8 Hz, 1H,
CH), 3.72 (d, J= 7.1 Hz, 2H, CH2), 7.18 (m, 5H, indol ring),
7.20 (m, 4H, aromatic ring), 9.77 (s, 1H, NH), 9.88 (s, 1H,
NH); 13C NMR (δ ppm) (DMSO-d6) 17.3, 18.3, 18.3, 36.2,
38.2, 43.5, 47.3, 48.2, 51.9, 125.2, 126.2, 126.2, 126.3, 126.8,
127.2, 127.2, 127.3, 128.2, 129.2, 129.2, 171.3, 178.2; Mass
(m/z) 406 (M+).
Step 2: Synthesis of methyl ester hydrochlorides of
l-tryptophan, l-phenylalanine, glycine and l-tyrosine
Dex 2: S(+) Methyl 2-(2-(4-isobutylphenyl)
Freshly distilled 6 mL (0.05 mol L−1) thionyl chloride
was slowly added to methanol (100 mL) with cooling
and 20.42 g (0.1 mol L−1) of l-tryptophan (2) was added
to it. e mixture was refluxed for 6–8 h at 60–70°C with
continuous stirring on magnetic stirrer. Excess thionyl
chloride and solvent was removed under reduced pres-
sure giving crude tryptophan methyl ester hydrochlo-
ride. It was treated with 20 mL portion of cold ether
at 0°C. e resulting solid product was collected and
dried under vacuum. It was recrystallized from hot
propanamido)-3-phenylpropanoate—UV
Spectra
(λmax) in SGF 210 nm, in SIF 217 nm, in 80% human
plasma 213 nm; IR (KBr, cm−1) 3326 (NH str. of amide),
2930 and 2906 (aromatic CH str.), 1726 (C=O str. of ester),
1630 (C=N), 1272 (CO str.of ester); 1H NMR (δ ppm)
(DMSO-d6) 1.42 (d, J= 7.1 Hz, 3H, CH3), 3.68 (q, J= 6.8 Hz,
1H, CH in ring), 3.72 (q, J= 7.2 Hz, 1H, CH), 7.28 (m, 4H,
Ar. H), 7.32 (m, 4H, Ar. ring), 9.77 (s,1H, NH); 13C NMR
(δ ppm) (DMSO-d6) 17.3, 17.3, 18.3, 35.2, 35.2, 42.3, 47.3,
47.8, 51.9, 119.2, 120.2, 125.2, 125.3, 125.3, 126.2, 126.6,
© 2011 Informa UK, Ltd.

690 A. Rasheed et al.
Step I: Synthesis of dexibuprofen acid chloride
H3C
H3C
O
O
SOCl2
+
OH
CH3
CH3
Cl
CH3
1a
2a
1
CH3
Step II: Synthesis of methyl ester of L-tryptophan
CH2 CH COOCH
SOCl2
CH2 CH COOCH3
+
NH2
NH3 Cl
CH3OH
N
H
N
2
H
Step III: Synthesis of prodrug: Conjugation of dexibuprofen acid chloride with methyl ester of L- tryptophan
H3C
O
CH2 CH COOCH3
+
CH3
+
NH3 Cl
Cl
N
H
NaOH
2–8°C
CH3
CH3
2a
1a
NH
CH2
CH
CH3
O
COOCH3
N
H3C
H
Dex-1
CH3
O
CH3
NH
NH
OH
CH
COOCH3
CH2
CH3
CH2 COOCH3
CH3
O
H3C
H3C
Dex-3
Dex-2
CH3
NH
CH
COOCH3
CH2
CH3
O
H3C
Dex-4
Scheme 1. Synthetic protocol of amide prodrugs of dexibuprofen.
127.2, 127.3, 127.5, 128.3, 128.4, 171.3, 172.9; Mass (m/z)
367 (M+).
120.6, 122, 122.2, 125, 125.6, 126, 126.2, 128.2, 128.4,
135.2, 171.2, 172.1; Mass (m/z) 383 (M+).
Dex 3: S(+) Methyl-2-(2-(4-isobutyl phenyl)pro-
panamido)acetate—UV Spectra (λmax) in SGF 212 nm, in
SIF 219 nm, in 80% human plasma 222 nm; IR (KBr, cm−1)
3280 (NH str. of amide), 3043 (aromatic CH str.), 2932
Physicochemical characterization of the synthesized
prodrugs
Solubility
1
(C=O str. of ester), 1630 (C=N), 1290 (CO str.of ester); H
Approximately 5 mg of prodrug was dissolved in 5 mL of
each solvent at 37 1°C in glass test tubes. e solvents
used were 0.1 N NaOH, 0.1 N HCl, ethanol, methanol,
ether, ethyl acetate, chloroform, acetone, DMF and
water. Test tubes were gently shaken and solubility was
observed. In case of any observed insoluble fractions, the
known amount of solvent was further added to ascertain
the solubility of the compound.
NMR (δ ppm) (DMSO-d6) 1.38 (d, J= 6.7 Hz, 6H, CH3),
2.42 (d, J= 7.3 Hz, 2H, CH2), 3.80 (q, J= 7.1 Hz, 1H, CH),
3.82 (q, J= 7.1 Hz, 1H, CH), 7.48 (d, J= 8 Hz, 4H, Ar.), 9.68
(s, 1H, NH); 13C NMR (δ ppm) (DMSO-d6) 17.3, 17.3, 17.8,
20.2, 35.2, 42.3, 47.8, 51.9, 63.2, 120, 120.8, 128.6, 128.9,
139.6, 172.9; Mass (m/z) 277 (M+).
Dex 4: S(+) Methyl 3-(4-hydroxyphenyl)-2-(2-(4-
isobutylphenyl)propanamido)
propanoate—UV
Spectra (λmax) in SGF 208 nm, in SIF 221 nm, in 80 %
human plasma 218 nm; IR (KBr, cm−1) 3362 (NH str. of
amide), 3046 (aromatic CH str.), 1745 (C=O str. of ester),
1532 (C=N), 1280 (CO str.of ester); 1H NMR (δ ppm)
(DMSO-d6) 1.28 (d, J= 6.7 Hz, 6H, CH3), 3.62 (q, J= 7.1 Hz,
1H, CH), 7.28 (d, J= 8 Hz, 4H, Ar. ring), 7.63 (d, J= 8 Hz,
4H, Ar. ring), 9.68 (s, 1H, NH); 13C NMR (δ ppm) (DMSO-
d6) 17.8, 18.2, 18.3, 36.2, 47.3, 48.2, 48.3, 51.9, 120, 120.2,
Protein binding studies
A solution of synthesized prodrug (10 mg mL−1) was
made in phosphate-buffered saline (PBS, pH 7.4). A
100 mL of this solution was taken in a beaker. e cello-
phane membrane (molecular weight cutoff in the range
of 10,000–12,000 Da obtained from Hi-Media, India) was
first washed with distilled water and then with buffer
solution (pH 7.4). It was tied at the opening end of dialysis
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and evaluation of novel dexibuprofen amide prodrugs 691
Hydrolysis rate determination in 80% human plasma10
A solution of 10 mg of prodrug was prepared in methanol
(2 mL) and was added to 88 mL of 80% human plasma
(pH 7.4 prepared by mixing 80 portion of plasma and
20 portion of phosphate buffer at pH 7.4). An aliquot of
15 mL of this solution was withdrawn and kept in test
tubes maintained at 37 0.5°C. At definite interval of time
(0–8 h), an aliquot of 22 mL was withdrawn and mixed
with 0.5 % ZnSO4 solution. Samples were centrifuged at
6000g for 10 min and a clear supernatant solution was
analyzed spectrophotometrically at 223 nm.
tube; the dialysis tube containing (6%) egg albumin was
dipped into the drug solution and covered. e whole
assembly was placed on a magnetic stirrer and switched
at low revolutions per minute. e temperature was
maintained at 37 0.5°C. After every 1 h, 1 mL of the PBS
containing drug solution was replaced with fresh 1 mL of
PBS. Withdrawn sample was diluted further with 1 mL
phosphate buffer and the concentration of prodrug was
estimated using spectrophotometer at 223 nm. Table 1
indicates the physicochemical properties of the synthe-
sized prodrugs.
Hydrolysis rate determination in SGF and SIF9
Hydrolysis rate determination in rat faecal matter11
e prodrug was dissolved in phosphate buffer so that
final concentration of the solution was 250 µg/mL. Fresh
faecalmaterialofratswereweighed(about1g)andplaced
in different sets of test tubes. To each test tube, 2 mL of
the prodrug solution was added and diluted to 5 mL with
phosphate buffer. e sets of test tubes were incubated at
37 0.5°C for different intervals of time (0–8 h). For analy-
sis, the free drug was extracted with 5 mL of methanol
and estimated on UV spectrophotometer at 210 nm.
In vitro hydrolysis studies of synthesized prodrugs were
carried out in SGF at pH 1.2 (USP 1970) and SIF at pH
7.4 (USP 1970). A solution of 10 mg of prodrug was pre-
pared in 90 mL of SIF (pH 7.4) or SGF (pH 1.2). An aliquot
of 15 mL of this solution was withdrawn repeatedly and
kept in test tubes maintained at 37 0.5°C. At a definite
interval of time (0.5 h, 1–8 h), an aliquot was withdrawn
from different test tubes and was transferred to micro-
centrifuge tubes followed by addition of methanol to
make up the volume. e tubes were placed in freezing
mixture in order to arrest further hydrolysis, followed
by vortexing at high speed for 5 min. After vortexing, the
tubes were centrifuged at high speed (3000g) for 5 min.
A 5 mL of clear supernatant obtained from each tube
was measured on UV spectrophotometer for the amount
of free Dex released after the hydrolysis of prodrugs in
SGF and SIF at 210 nm. e kinetics of hydrolysis was
monitored by increase in free drug concentration with
time, and order of reaction and half life (t1/2) were also
calculated. e rate of hydrolysis was calculated using
equation:
Pharmacological evaluations
Dex and the synthesized prodrugs were evaluated for
anti-inflammatory activity, analgesic activity, ulcero-
genicity and histopathology, and a comparative study
was performed. Test compounds and standard drugs
were administered in the form of a suspension by oral
route of administration for anti-inflammatory and
analgesic studies (1% carboxymethylcellulose as a
vehicle), as well as for ulcerogenicity studies (2% acacia
as vehicle). Wistar albino rats of six groups, including
a control and a standard group, each with six animals
were selected. e selected animals were housed in
acrylic cages at standard environmental conditions at
25 2°C, relative humidity of 45–55%, in a well-venti-
lated room maintained at 12:12 h light:dark cycle, fed
with standard rodent diet and water ad libitum. All the
r= (2.303/t) log [a/(a − x)]
where r represents hydrolysis constant, t is the time
in min, a is the initial concentration of prodrug, x is the
amount of prodrug hydrolyzed and (a − x) is the amount
of prodrug remaining.
Table 1. Physicochemical properties of prodrugs.
Elemental analysis
Protein
Prodrug Molecular
Melting*
code
formula
Mol. wt.
406
Colour
point (°C)
Yield (%) Rf # value binding (%) Calculated (%)
Found (%)
73.55
7.48
Dex-1
C25H30N2O3
Off white
68–69
58–59
62–64
56–58
74
85
78
64
0.56
0.64
0.58
0.67
74.08
68.45
56.78
58.78
C
73.86
7.44
6.89
75.17
7.95
3.81
69.29
8.36
5.05
72.04
7.62
3.65
H
N
C
6.91
Dex-2
Dex-3
Dex-4
C23H29NO3
C16H23NO3
C23H29NO4
367
277
383
Creamy white
Off white
75.23
7.96
H
N
C
3.73
69.32
8.24
H
N
C
5.13
Yellowish white
71.90
7.75
H
N
3.33
*Uncorrected #(n-hexane:water:ethyl acetate:glacial acetic acid is 30:10:10:2.5).
© 2011 Informa UK, Ltd.

692 A. Rasheed et al.
animals were acclimatized for a week before experi-
ment. All animal experiments were carried out accord-
ing to the guidelines of the Committee for the Purpose
of Control of Experiments on Animals and approval
of the Institutional Animal Ethics Committee, Sree
Vidyanikethan College of Pharmacy, Tirupati, India,
was obtained.
Ulcerogenicity
GI toxicity of the synthesized prodrugs was measured
and compared with the parent drug by measuring mean
ulcer index (UI)14. e control group was administered
orally in 2% acacia suspension. Test compounds and
standard were administered orally (at 10 times higher
dose) as a suspension in 2% acacia daily for 5 days. e
rats (130–150 g) were fasted after the administration of
last dose, thereafter they were killed by decapitation and
the stomach was removed, opened and washed with
distilled water. e lesions on the gastric mucosa were
counted by visual examination using a binocular mag-
nifier. Ulcers greater than 0.5 mm were recorded. e
mean UI was calculated by severity of gastric mucosal
lesions that are graded as (i) grade 1: >1 mm erosions
(ii) grade 2: 1–2 mm erosions and (iii) grade 3: <2 mm
erosions.
Anti-inflammatory activity
The anti-inflammatory activity was determined by
hind paw oedema method12 using carrageenan as
phlogistic agent. Wistar rats (100–200 g) were divided
into six groups, each comprising of six rats, including
a control and a standard group. The initial volume of
right hind paw of rat was measured by plethysmom-
eter without administration of the prodrug. A 1%
sodium carboxymethylcellulose suspension contain-
ing a dose equivalent to the 2.4 mg or 12 mg kg−1 body
weight of Dex was administered orally. An equivalent
quantity of prodrugs 4 mg of Dex-1, 4.36 mg of Dex-2,
3.52 mg of Dex-3 and 4.61 mg of Dex-4 were adminis-
tered to the test groups. After 30 min of administration
of the drugs, carrageenan (0.1 mL, 1% m/v) solution
in normal saline was injected into the planter sur-
face of right hind paw of each animal. The volume
of right hind paw of albino rat was measured after 2,
4 and 6 h. The mean difference in the volume of the
right hind paw of rats was compared with control and
standard. The percent inhibition of paw oedema was
calculated as
e UI was calculated as:
UI = [1 × (number of lesions of grade 1) + 2 × (number
of lesions of grade 2) + 3 × (number of lesions of grade
3)]/10.
Histopathological studies15
e histopathological studies of stomach of rats were
carried out using haemotoxylin and eosin stain at
Pathology Department, Sri Venkateswara Veterinary
University, Tirupati, India. e stomach tissues were
removed from the rats and fixed in 10% normal saline for
at least 48 h. ese were then processed routinely and
the tissues were embedded in paraffin wax. Histological
sections were cut at 5–6 μm and stained with routine
haematoxylin and eosin. ese were then examined
by a consultant histopathologist. e lesions observed
were assessed for the following mucosal atrophy, the
presence of inflammatory cells in the wall, oesinophils,
lymphocytes and plasma cells. Photomicrographs of
representative lesions at various magnifications were
taken on Zeiss optical microscope (Germany), Stemi
2000-C, with a resolution of 10 × 45X, attached with tri-
nocular camera.
Percent inhibition = (1 − Vt/Vc) × 100
where Vc is the mean relative change in paw edema
volume in control group and Vt is the mean relative
change in paw edema volume in test group.
Analgesic activity
e analgesic activity was determined by thermal stim-
ulus using tail flick method13. Analgesiometer was used
for the determination of pain threshold of albino mice.
Cold water was circulated through the water jackets
of the instrument to avoid heating of the area around
the hot wire. e rat (100–200 g) was placed in a holder
through which the tail of the rat was protruded out. e
reaction time was recorded at 1, 2, 3 and 4 h after the
treatment and cutoff time was 9 s. e normal reac-
tion time, i.e., the time taken to flick the tail was noted.
Animals showing delayed response were rejected. e
drug (dose equivalent to 2.4 mg or 12 mg kg−1 body
mass) was administered orally in 1% suspension of
sodium carboxymethylcellulose. An equivalent quantity
of prodrugs 4 mg of Dex-1, 4.36 mg of Dex-2, 3.52 mg of
Dex-3 and 4.61 mg of Dex-4 was administered to the test
groups. e percent analgesic activity was calculated by
the formula given as
Statistical analysis
Statistical analysis of the pharmacological activity of the
synthesized prodrugs on animals was evaluated using a
one-way analysis of variance (ANOVA). Student’s t-test
was applied for expressing the significance, and the
experimental data are expressed as mean SD.
Results and discussion
Synthesis and characterization
is study describes the synthesis of amide prodrugs
of Dex as Dex-1 {S(+) methyl-3-(1H-indol-3-yl)-2-(2-
(4-isobutylphenyl)propanamido)propanoate}, Dex-2
{S(+) methyl 2-(2-(4-isobutylphenyl)propanamido)-
3-phenylpropanoate}, Dex-3 {S(+) methyl-2-(2-(4-
isobutyl phenyl)propanamido)acetate} and Dex-4
{S(+) methyl 3-(4-hydroxy phenyl)-2-(2-(4-isobutyl
% Analgesic activity = [(T2 − T1)/(Tc − T1)] × 100
where T1 is the reaction time (s) before administration
of prodrug and T2 the reaction time (s) after administra-
tion of prodrug and Tc cutoff time (s).
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and evaluation of novel dexibuprofen amide prodrugs 693
phenyl)propanamido)propanoate}as portrayed in
Scheme 1. e prodrugs were subjected to solubility,
physicochemical characterization, protein binding and
hydrolytic studies. All the prodrugs were observed to be
highly soluble in organic solvents, sparingly soluble in
alkaline solution and insoluble in acidic solutions. e
high solubility of the prodrugs in organic solvents indi-
cates their lipophillic behaviour. e physicochemical
properties of synthesized prodrugs were determined,
respectively, in rat faecal matter. The hydrolysis study
revealed satisfactory hydrolysis of prodrugs in SIF and
very encouraging hydrolysis in 80% human plasma and
rat faecal matter. All the prodrugs followed first-order
kinetics. The percentage protein binding of Dex-1,
Dex-2, Dex-3 and Dex-4 was found as 74.08, 68.45,
56.78 and 58.78%, respectively. The prodrugs showed
low protein binding in comparison with Dex whose
value is found to be 85%. This increases the availability
of prodrugs for hydrolysis in plasma and the required
dose will be less.
1
as well as the IR, H NMR, 13C NMR and mass spectral
analyses were carried out. e results of elemental
analysis of synthesized prodrugs were in all case within
0.4% of theoretical value and were in confirmation of
desired structure.
Pharmacological studies
Anti-inflammatory activity
In vitro hydrolysis of Dex-1, Dex-2, Dex-3 and Dex-4
were carried out in SGF (pH 1.2), SIF (pH 7.4), 80%
human plasma (pH 7.4) and rat faecal matter (pH 7.4)
and their respective half life (t1/2) values in SIF were
observed as 4.5, 4.8, 4.5 and 4.6 h, respectively. The
comparative hydrolysis patterns of the synthesized
prodrugs are depicted in Figure 1. None of the prod-
rugs showed significant hydrolysis in SGF. The amount
of Dex regenerated by hydrolysis of Dex-1, Dex-2,
Dex-3 and Dex-4 in SIF was found as 71, 67, 76 and
73%, respectively, whereas 85, 82, 93 and 89%, respec-
tively, in 80% human plasma and 86, 82, 93 and 89%,
Table 2 shows that after 6 h of administration of Dex,
the percentage anti-inflammatory activity was found as
43.3%, whereas an improved value of 73.4, 77.3, 72.8 and
64.5% were observed for prodrugs Dex-1, Dex-2, Dex-3
andDex-4,respectively.emaximumanti-inflammatory
activity of prodrugs was observed at 6 h and remained
practically constant upto 8 h. e anti-inflammatory
activity of free Dex decreased with time, whereas that of
its prodrugs increased with time due to its higher bio-
availability compared with the parent drug. Statistical
significant testing using one-way ANOVA showed that
anti-inflammatory activity of Dex and its prodrugs were
In SGF
In SIF
b
90
80
70
60
50
40
30
20
10
0
a
35
30
25
20
15
10
5
Dex-1
Dex-2
Dex-3
Dex-4
Dex-1
Dex-2
Dex-3
Dex-4
0
240
0
30
60
90
120 150 180 210
Time (min.)
0
1
2
3
4
5
6
7
8
9
Time (h)
In 80% human plasma
In rat faecal matter
100
90
80
70
60
50
40
30
20
10
0
c
90
80
70
60
50
40
30
20
10
0
d
Dex-1
Dex-2
Dex-3
Dex-4
Dex-1
Dex-2
Dex-3
Dex-4
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Time (h)
Time (h)
Figure 1. Comparative hydrolysis pattern of prodrugs of dexibuprofen.
© 2011 Informa UK, Ltd.

694 A. Rasheed et al.
Table 2. Comparative anti-inflammatory and analgesic activity of prodrugs.
Anti-inflammatory activity (%)1
Analgesic activity (%)1
Group
I
Prodrug
Dose (mg)
1% CMC
2 h
0.0
4 h
0.0
6 h
0.0
1 h
0.0
2 h
0.0
3 h
0.0
4 h
0.0
Normal
control2
II
Dex
2.4
60.6 2.1
56.1 1.2
43.3 1.5
50.3 2.6
48.1 1.3
47.5 1.5
46.4 2.1
59.0 1.3*3 68.1 2.3*3 73.4 1.32*3 38.3 1.0*3 46.2 1.1*3 53.0 1.0*3 61.4 1.0*3
67.4 1.1*3 71.7 1.1*3 77.3 2.22*3 35.0 2.9*3 43.2 2.5*3 50.1 1.7*3 59.7 1.0*3
61.4 1.4*3 65.9 1.4*3 72.8 2.42*3 36.2 2.0*3 45.3 2.0*3 54.8 1.0*3 63.6 1.4*3
54.4 1.7*3 58.3 1.0*3 64.5 2.35*3 31.4 2.4*3 38.1 2.5*3 46.7 1.6*3 52.7 1.2*3
III
IV
V
Dex-1
Dex-2
Dex-3
Dex-4
4.0
4.36
3.52
4.61
VI
1Value were expressed as mean SD of 6 observations.
2Normal control animals were administered orally 1% carboxymethylcellulose.
3Comparison between group II vs group III, IV, V and VI.
*p<0.05, Statistical significant test comparison was done by one way annova followed by Dunnet ‘t’ test.
a
b
d
f
30
25
20
15
10
5
0
c
Prodrugs
Figure 2. Comparative ulcer index of dexibuprofen and its
prodrugs.
e
effective in comparison with control group. us, the
amino acid prodrugs proved to be a suitable promoiety
for Dex.
Analgesic activity
The percentage protection in mice brought about by
administration of drug is shown in Table 2. After 4 h
of the administration of Dex, the percentage analge-
sia was observed as 46.4%, whereas for Dex-1, Dex-2,
Dex-3 and Dex-4, corresponding values of 61.4, 59.7,
63.6 and 52.7% were observed. The study revealed
that analgesic activity of Dex decreased with time,
while the prodrugs showed an improved analgesic
activity.
Figure 3. Histopathological studies of dexibuprofen and its
prodrugs on rat stomach.
Histopathological studies
Ulcerogenicity
e histopathological studies were attempted and sum-
marized as follows. e gastric tissues were investigated
microscopically and the tissue samples of control group
rats showed normal histological findings (Figure 3).
Microscopic investigation of Dex group revealed a focal
erosive area in gastric mucosa and a zone (clear zone) in
the basal regions of the gastric glands stained pale. is
zone was parallel to the surface of the stomach lumen.
Dex has produced a mean UI of 25.3 while that of its
prodrugs resulted in producing very less values and is
shown in Figure 2. e study revealed that the minimized
side effect obtained in the prodrugs might be due to the
inhibition of direct contact of carboxylic acid group of the
drug to the gastric mucosa, which is mainly responsible
for the damage.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and evaluation of novel dexibuprofen amide prodrugs 695
In this zone, the structures of the gland were destroyed.
ey had disintegrated from the basal lamina and
fallen into the lumen. e nuclei of these cells became
smaller and dense, and their cytoplasms were stained as
dark eosinophilic bodies. Small hemorrhagic areas and
patches of inflammatory cell infiltrations were present in
the lumen of the glands and lamina propria. Normal his-
tological findings were displayed for Dex-1, Dex-2, Dex-3
and Dex-4 revealing that the prodrugs are not producing
any ulceration in the gastric region.
Declaration of interest
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.
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e authors thank M/s. Alkem Laboratories, Mumbai,
India, for providing gift sample of dexibuprofen. e
authors are grateful to Padmashree Dr. M. MohanBabu,
Chairman, Sree Vidyanikethan Educational Trust,
Tirupati, India, for providing the necessary facilities to
carry out this work.
© 2011 Informa UK, Ltd.