Synthesis, characterization and pharmacological evaluation of amide prodrugs of ketorolac

Article abstract of DOI:10.1016/j.ejmech.2007.09.011

Ketorolac (KC) suffers from the general side effects of NSAIDs, owing to presence of free carboxylic acid group. The study aimed to retard the adverse effects of gastrointestinal origin. Ten prodrugs of KC were synthesized by amidation with ethyl esters of amino acids, namely, glycine, l-phenylalanine, l-tryptophan, l-valine, l-isoleucine, l-alanine, l-leucine, l-glutamic acid, l-aspartic acid and β-alanine. Purified synthesized prodrugs were characterized by m.p., TLC, solubility, partition coefficients, elemental analyses, UV, FTIR, NMR and MS. Synthesized prodrugs were subjected for biopharmaceutical studies, analgesic, anti-inflammatory activities and ulcerogenic index. Marked reduction of ulcerogenic index and comparable analgesic, anti-inflammatory activities were obtained in all cases as compared to KC. Among synthesized prodrugs, viz. AR-11, AR-19 and AR-20 showed excellent pharmacological response and encouraging hydrolysis rate both in SIF and in 80% human plasma. Prodrugs with increased aliphatic side chain length or introduction of aromatic substituent showed enhanced partition coefficient but diminished dissolution and hydrolysis rates. Such prodrugs can be considered for sustained release purpose.

Full text of DOI:10.1016/j.ejmech.2007.09.011

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2464e2472
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization and pharmacological
evaluation of amide prodrugs of ketorolac
Ashutosh Mishra, Ravichandran Veerasamy, Prateek Kumar Jain,
*
Vinod Kumar Dixit, Ram Kishor Agrawal
Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutical Sciences, Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.) 470 003, India
Received 10 May 2007; received in revised form 7 September 2007; accepted 13 September 2007
Available online 26 September 2007
Abstract
Ketorolac (KC) suffers from the general side effects of NSAIDs, owing to presence of free carboxylic acid group. The study aimed to retard
the adverse effects of gastrointestinal origin. Ten prodrugs of KC were synthesized by amidation with ethyl esters of amino acids, namely, gly-
cine, L-phenylalanine, L-tryptophan, L-valine, L-isoleucine, L-alanine, L-leucine, L-glutamic acid, L-aspartic acid and b-alanine. Purified synthe-
sized prodrugs were characterized by m.p., TLC, solubility, partition coefficients, elemental analyses, UV, FTIR, NMR and MS. Synthesized
prodrugs were subjected for biopharmaceutical studies, analgesic, anti-inflammatory activities and ulcerogenic index. Marked reduction of
ulcerogenic index and comparable analgesic, anti-inflammatory activities were obtained in all cases as compared to KC. Among synthesized
prodrugs, viz. AR-11, AR-19 and AR-20 showed excellent pharmacological response and encouraging hydrolysis rate both in SIF and in
80% human plasma. Prodrugs with increased aliphatic side chain length or introduction of aromatic substituent showed enhanced partition
coefficient but diminished dissolution and hydrolysis rates. Such prodrugs can be considered for sustained release purpose.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Ketorolac; Prodrugs; Amino acid conjugates; Pharmacokinetics; Pharmacodyanamics
1. Introduction
first mechanism involves a local action composed of a direct
contact while the other has indirect effect on the GI mucosa.
The direct contact effect can be attributed to a combination of
a local irritation produced by acidic group of NSAIDs and local
inhibition of prostaglandin synthesis in the GI tract. The indi-
rect effect can be attributed to combination of an ion trapping
mechanism of NSAIDs from the lumen into the mucosa. The
second mechanism is based on a generalized systemic action
occurring after absorption, which can be demonstrated follow-
ing intravenous dosing. Recently, considerable attention has
been focused in the development of bio-reversible derivatives,
by temporarily masking the acidic group of NSAIDs, as
a promising mean of reducing or abolishing the GI toxicity.
In the present study well-recognized NSAID, viz. KC was
selected, which suffers with the gastrointestinal side effects.
Literature reveals that many efforts had been made to synthe-
size prodrug via masking carboxylic acid group by forming
ethyl esters and [(N,N-dimethylamino)carbonyl]methyl ester
Ketorolac (KC), one of non-steroidal anti-inflammatory
drug, could not be used as up to its potential, because of its ad-
verse reactions offered due to presence of free carboxylic acid
group. The non-steroidal anti-inflammatory drugs (NSAIDs)
are widely used for indications extending from inflammation
and pain to cardiovascular and genitourinary diseases. In the re-
cent years a number of NSAIDs have been introduced into clin-
ical practice. Hunt is on to relieve pain and inflammation with
freedom of undesirable effects. Gastrointestinal side effects
constitute the most frequent of all the adverse reactions of
NSAIDs and often these reactions lead to GIT ulceration and
hemorrhage. GI mucossal injury produced by NSAIDs is gener-
ally believed to be caused by two different mechanisms [1]. The
* Corresponding author. Tel./fax: þ91 7582233934.
E-mail address: dragrawal2001@yahoo.co.in (R.K. Agrawal).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.09.011

A. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 2464e2472
2465
[2,3] and amide prodrug [4] using various amines. Some alkyl
esters had been synthesized for topical delivery of KC [5].
However, no attempts were made to develop amide prodrugs
using amino acids, which have been utilized as a major tool
with other NSAIDs [6e8]. Apart from the amino acid conju-
gates of NSAIDs this approach is widely adopted for synthesis
of prodrugs of anticancer drugs [9]. The salient features of the
usefulness of conjugation of amino acids with drugs are as
follows [10]:
esters’ HCl salts of amino acids were condensed with anhy-
dride or acyl chloride of KC along with prior or simultaneous
neutralization of HCl salts. Schematic representation of the
reaction is given in Scheme 1.
Amino acid contains both acidic and basic groups in the
same molecular and exists in the zwitterionic form. The
non-availability of the free amino group in the zwitterionic
form of the amino acid restricts its use in the formation of am-
ide. Esterification of amino acid in presence of HCl produces
amino acid ester hydrochloride in which neutralization of HCl,
using aqueous alkali, pyridine or triethylamine, generates free
amino group to react as nucleophile in the synthesis of amide
[11,12].
(i) amino acids are normal dietary constituent and they are
non-toxic in moderate doses as compared to other
promoities;
(ii) amino acids have healing effect on gastric toxicity;
(iii) a drug with free carboxyl group can be derivatized into
corresponding esters or amide of amino acids, so as to
alter the physical properties of a parent drug with one or
more of the hydrolase enzymes serving as the in vivo
reconversion site(s);
(iv) being a nutritional substance, the use of amino acids as
a derivatizing group might also permit more specific
targeting site for enzymes involved in the terminal
phase of digestion;
2.3. General procedures used in acylation of amino acid
ethyl ester hydrochloride
Acylation of amino acid ethyl ester hydrochloride was done
as described in literature by six methods using acid anhydride
of KC as acid chloride of the KC could not be formed satisfac-
torily and triethylamine as base, acid chloride of KC and po-
tassium carbonate as base, acid chloride of KC and pyridine
as base, DCC, drug (1:1) and triethylamine as base and acid
anhydride of KC and triethylamine as base.
(v) many amino acids possess marked anti-inflammatory
activity against carrageenan induced hind paw edema
in rats; and
2.3.1. Method 1
(vi) by using different types of amino acids, viz. non-polar,
polar, acidic and basic, the drug molecule can be made
more or less polar, or more or less soluble in given
solvent.
Amino acid ester hydrochloride (0.005 M) and triethyl-
amine (2 mL) were dissolved in 10 mL of dichloromethane
and stirred mechanically for 10 min to neutralize the ester hy-
drochloride. This solution was added slowly to the freshly syn-
thesized acid anhydride of KC in dichloromethane at 0 ꢀC for
half an hour, followed by stirring at room temperature for 24 h.
Precipitated dicyclohexyl urea (DCU) was filtered off and the
solvent was removed at reduced pressure, 10 mL ethyl acetate
was further added to the dried product and filtered to remove
the triethylamine hydrochloride and remaining DCU. The fil-
trate was washed three times with 10% sodium bicarbonate
and three times with water and dried over anhydrous magne-
sium sulphate and the solvent was removed under reduced
pressure. White amorphous mass was obtained, which was
dried under high vacuum. Crude material was dissolved in
minimum amount of alcohol, followed by the slow addition
of distilled water until further precipitation stopped. After fil-
tration it was dried in air to get a solid product. This method
was used to synthesize AR-11.
Thus present work aims to synthesize amide prodrugs of
KC using amino acid ester with the expectation to get non-
toxic prodrugs with minimized GIT disturbances while main-
taining the useful anti-inflammatory and analgesic activities.
Various proteolytic enzymes will help in release of KC by hy-
drolysis of peptide linkage, without producing any xenobiotic
substance within serum or GIT. The list of synthesized
prodrugs along with product codes, chemical names, possible
trivial names and molecular structures is given in Table 1.
2. Materials and methods
2.1. Materials
All the amino acids, namely, glycine, L-phenylalanine, L-tryp-
tophan, L-valine, L-isoleucine, L-alanine, L-leucine, L-glutamic
acid, L-aspartic acid and b-alanine were procured from M/S Hi-
Media Ltd., Mumbai. Drug KC was obtained as gift sample
from M/S Knoll, Mumbai. Other reagents and solvents used
wereofanalytical/spectroscopic/HPLC gradeasthe casedesired.
2.3.2. Method 2
KC (0.01 M) was dissolved in 40 mL dichloromethane
followed by addition of DCC (0.01 M). Reaction mixture
was stirred for half an hour. To this solution was added a mix-
ture of amino acid hydrochloride (0.01 M) and triethylamine
(2 mL) in 20 mL of dichloromethane in drop wise manner.
Reaction mixture was stirred at 0 ꢀC initially for 2 h followed
by stirring at room temperature for whole night. Precipitated
dicyclohexyl urea was filtered off. Solvent was removed at
reduced pressure. Ethyl acetate (10 mL) was added to the
dried product to remove the remaining DCU. Ethyl acetate
2.2. Synthesis of ethyl esters of amino acid hydrochloride
salts
The prodrugs were synthesized first by converting amino
acids into their ethyl esters’ HCl salts. Thereafter, these ethyl

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A. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 2464e2472
Table 1
List of synthesized prodrugs of ketorolac
O
C
O
C
R2
N
H
N
Code
Chemical name
R2
AR-11
(ꢁ)-5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-[N-(ethylacetate)] carboxamide
(ꢁ)-5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-[N-(2-ethylpropionate)] carboxamide
(ꢁ)5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-[N-(3-ethylpropionate)] carboxamide
eCH2COOC2H5
CH3
AR-12
AR-13
C
COOC2H5
H
eCH2CH2COOC2H5
CH2
NH
AR-14
AR-15
(ꢁ)5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-[N-(2-(3-(3-benzimidazole)) ethylpropionate)] carboxamide
CHCH2
(ꢁ)5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-[N-(2(3-phenyl)ethylpropionate)]carboxamide
COOC2H5
CH3
CHCHCH3
COOC2H5
AR-16
AR-17
(ꢁ)5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-[N-(2-(3-methyl)ethyl butyrate)]carboxamide
CH3
CHCH2CHCH3
COOC2H5
(ꢁ)5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-[N-(2-(4-methyl)ethyl pentoate)]carboxamide
CH3
CHCHCH2CH3
COOC2H5
AR-18
(ꢁ)5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-[N-(2-(3-methyl)ethylpentoate)]carboxamide
CHCH2COOC2H5
COOC2H5
AR-19
AR-20
(ꢁ)5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-[N-(2-diethyl succinate)]carboxamide
(ꢁ)5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-[N-(20-diethylglutamate)]carboxamide
CHCH2CH2COOC2H5
COOC2H5
layer was washed with 10% NaHCO3 and distilled water to
remove the triethylamine hydrochloride and traces of alkali.
Ethyl acetate layer was dried over anhydrous magnesium sul-
phate and filtered off. Solvent was removed at reduced pres-
sure to get the crude product. Product was recrystallized by
selective precipitation using alcoholewater mixture, dried
and stored in cold condition in tightly closed container.
This method was used to synthesize AR-14, AR-15 and
AR-20.
2.3.3. Method 3
Amino acid hydrochloride (0.01 M), triethylamine (2 mL)
and KC (0.02 M) were dissolved in 40 mL of dichlorome-
thane. The reaction mixture was stirred at 0 ꢀC for half an

A. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 2464e2472
2467
OH
H2N
Ethyl ester of Amino Acid
R
+
Ar
O
---CH2 CH2 COOC2 H5 (AR-11)
R = -
---CH2 CH2 COOC2 H5 (AR-12)
CH3
C
COOC2 H5 (AR-13)
H
CH2
NH
Ar=
CH CH2
(AR-15)
(AR-14)
COOC2 H5
CH3
H
N
CH3
(AR-17)
N
R
CHCH2CHCH3
COOC2 H5
Ar
CHCHCH3
COOC2 H5
C
O
O
(AR-16)
CH3
CHCH2COOC2 H5
(AR-19)
(AR-18)
CHCHCH2 CH3
COOC2 H5
COOC2H5
CHCH2 CH2COOC2 H5
COOC2 H5
Scheme 1. Chemical reaction adopted for syntheses of amide prodrugs of ketorolac.
(AR-20)
hour. To this solution was added, DCC (0.01 M) in 10 mL of
dichloromethane slowly in a drop wise manner. Reaction mix-
ture was stirred for 24 h. Precipitated dicyclohexyl urea was
filtered off and the solvent was distilled off at reduced pres-
sure. Product thus obtained was again dissolved in 10 mL of
ethyl acetate. White precipitate was again obtained, which
was found to be a mixture of DCU and triethylamine hydro-
chloride. Filtrate was washed with 10% NaHCO3 and distilled
water in order to remove KC, triethylamine hydrochloride and
traces of alkali present. Ethyl acetate layer was dried over an-
hydrous magnesium sulphate and filtered to get a clear solu-
tion of product in ethyl acetate. Solvent was evaporated
under reduced pressure and the crude product, thus obtained,
was recrystallized by dissolving it in ethyl alcohol followed
by the addition of water until further precipitation stopped.
Product was filtered, dried and stored in tightly closed con-
tainer in cold condition. This method was used to synthesize
AR-12, AR-13, AR-16, AR-17, AR-18 and AR-19.
case of any observed insoluble fraction, the known amount
of solvent was further added to ascertain the solubility of
the compound.
2.4.2. Partition coefficient
A 20 mg of prodrug was weighed and dissolved in 20 mL
chloroform. This solution was divided in two parts and in
each part was added 10 mL acidic buffer (pH 1.2) and phos-
phate buffer (pH 7.4) separately. The contents were thoroughly
shaken for 24 h at room temperature followed by transferring
in separating funnel. The chloroform layer was dried under
high vacuum and the residue obtained was again dissolved
in methanol (10 mL). A 20 mL of this solution was further di-
luted to 1000 mL with methanol. From this solution an aliquot
of 250 mL was withdrawn and was mixed with 45 mL solution
of acidic buffer (pH 1.2) or phosphate buffer (pH 7.4) and ace-
tonitrile in 44:1. Volume was finally made to 1000 mL by ad-
dition of methanol. A 20 mL of this solution was filtered and
injected into HPLC column (C18 ODS reversed phase). The
mobile phase acetonitrile:water 50:50 was used for KC pro-
drugs. The peak area for drug as well as prodrug was observed
at 311 nm for KC prodrugs using UV detector (DAD, SPD-
M10A with D2 lamp).
2.4. Characterization of the synthesized prodrugs
2.4.1. Solubility
Approximately 10 mg of compound was dissolved in
0.1 mL of each solvent at 37 ꢁ 1 ꢀC in glass test tubes. Test
tubes were gently shaken and solubility was observed. In
The synthesized compounds were subjected to thin layer
chromatography in order to check their purity. The prepared

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A. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 2464e2472
plates of silica gel G adsorbents were dried and activated. The
solvent system chloroform:methanol:ammonia 75:3:0.5 was
used for KC. Iodine vapor was used as detecting agent. All
compounds were shown as single spot. The melting points
of the synthesized prodrugs were determined by open capillary
tube using Toshniwal melting point apparatus and are uncor-
rected. The data are shown in Table 2.
The IR spectra of the compounds were obtained on IR spec-
trophotometer (Shimadzu 820 IPC) in KBr phase and data are
shown in Table 3. The PMR spectral analyses of the synthe-
sized prodrugs were done on NMR spectrophotometer (Bruker
DRX300) using CDCl3 as solvent and data are shown in Table 3.
The mass was determined on Jeol SX102-FAB mass spec-
trometer and the results are shown in Table 3.
test apparatus. Five milliliter samples were withdrawn after
every 10 min and the volume was replaced by fresh simulated
gastric fluid every time. Samples withdrawn from dissolution
test apparatus were subjected to HPLC. The measurements
of peak area of the prodrugs were noted at 311 nm using
UV detector.
The samples of synthesized prodrugs as well as drug from
the dissolution study in SGF did not show their presence up to
2 h, which is considered as maximum time for any drug that
remains in stomach, so after 2 h. SGF was replaced by simu-
lated intestinal fluid (SIF, 900 mL) and the dissolution test was
run again, under similar conditions of temperature and speed.
The same procedure was followed for all.
2.5.3. Studies on hydrolytic behaviour of synthesized
prodrugs
2.5. Biopharmaceutical evaluations
Hydrolytic behaviour of synthesized prodrugs was studied
in SGF (pH 1.2; USP 1970), SIF (pH 7.4; USP 1970) and
80% human plasma. The method adopted for the studies is
as follows.
2.5.1. Plasmaeprotein binding studies
A solution of synthesized prodrug (20 mg/mL) was made in
phosphate buffered saline (PBS, pH 7.4) [13]. A 100 mL of
this solution was taken in a beaker. The prepared membrane
was first washed with distilled water and then with buffer so-
lution (pH 7.4). It was tied at the opening end of dialysis tube;
the dialysis tube containing (6%) egg albumin was dipped into
the drug solution and covered. The whole assembly was placed
on a magnetic stirrer and switched at low revolutions per min-
ute. The temperature was maintained at 37 ꢁ 0.5 ꢀC. After ev-
ery 2 h, 1 mL of the PBS containing drug solution was
replaced with fresh 1 mL of PBS. Withdrawn sample was di-
luted further with 1 mL phosphate buffer and the concentra-
tion of the prodrug was estimated spectrophotometrically
using spectrophotometer (Shimadzu-1601).
2.5.3.1. Method for the hydrolysis rate determination. A solu-
tion of 10 mg of prodrug was prepared in acetonitrile (2 mL)
and was added to 88 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 (15, 30, 45, 60, 75 and 90 min) an aliquot of
225 mL was withdrawn from different test tubes and was
transferred to microcentrifuge tubes (Eppendrof’s tube) fol-
lowed by the addition of methanol to make up the volume.
The 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 (3000 rpm) for 5 min. Clear supernatant (20 mL) ob-
tained from each tube was then injected in to run through
HPLC column (C18 ODS reversed phase) of HPLC instrument
(Shimadzu liquid chromatograph LC-10AT). Mobile phase
acetonitrile:water 50:50 was used for KC and prodrugs.
Flow rate of mobile phase was kept at 1 mL/min at pressure
120e135. UV detector (DAD, SPD-M10A with D2 lamp)
2.5.2. Dissolution rate studies
Dissolution test apparatus (USPXXI) was adjusted to
100 rpm speed and temperature 37 ꢀC. Prodrug (100 mg)
was suspended in 2 mL simulated gastric fluid (SGF, pH
1.2) in a dialysis bag (cut off size 5000) kept inside the basket.
The basket was dipped in simulated gastric fluid (SGF,
900 mL), which was kept in the internal jar of the dissolution
Table 2
Physical constants of synthesized prodrugs of ketorolac
Prodrug code
Molecular formula
MW calculated
Melting range(ꢀC)a
% Yield
Colour
Rfb value
% Nitrogen
Found
Calculated
AR-11
AR-12
AR-13
AR-14
AR-15
AR-16
AR-17
AR-18
AR-19
AR-20
a
C19H20N2O4
C20H22N2O4
C20H22N2O4
C28H27N3O4
C26H26N2O4
C22H26N2O4
C23H28N2O4
C23H28N2O4
C23H26N2O4
C24H28N2O4
340
354
354
469
430
382
396
396
426
440
144e146
84e86
84
83
54
70
88
62
71
51
63
59
White
0.67
0.73
0.69
0.8
8.18
7.82
7.87
6.19
6.49
7.29
7.11
7.01
6.47
6.31
8.23
7.90
7.90
6.15
6.51
7.32
7.07
7.07
6.57
6.36
Light yellow
Creamy white
Light yellow
Creamy white
Light yellow
Dirty white
Dirty white
Creamy white
Yellowish white
116e118
108e110
84e86
0.83
0.79
0.84
ꢂ0.83
0.72
0.74
96e98
92e94
98 with dec.
98e100
82e84
Melting points are uncorrected.
b
Solvent system e chloroform:methanol:ammonia 75:3:5.

A. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 2464e2472
2469
Table 3
IR, 1H NMR and mass spectral characterization of synthesized prodrugs of ketorolac
Prodrug code
AR-11
Characteristic peaks of IR spectra
Characteristic peaks of 1H NMR spectra and mass spectra
3296 (NH str. of amide), 3062 (aromatic CH str.), 2915,
2830 (aliphatic CH str.), 1735 (C]O str. of ester), 1646
(amide I), 1578 (amide II), 1424, 1378 (CH bend,
aliphatic), 1292 (CeO str. of ester)
7.29e7.86 (m, 5H, aromatic ring), 6.68 (d, 1H, CH in ring), 5.66 (d, 1H, CH in
ring), 3.81 (t, 2H, CH2 in ring), 2.56e2.86 (m, 2H, CH2 in ring), 7.89 (br, NH ),
4.17 (q, 2H, OCH2CH3), 1.28 (t, 3H, OCH2CH3) and m/z 340
AR-12
AR-13
AR-14
AR-15
AR-16
3312 (NH str. of amide), 3053 (aromatic CH str.), 2956,
2923, 2861 (aliphatic CH str.), 1723 (C]O str. of ester),
1654 (amide I), 1569.29 (amide II), 1424, 1378 (CH
bend, aliphatic), 1292 (CeO str. of ester)
7.25e7.87 (m, 5H, aromatic ring), 6.59 (d, 1H, CH in ring), 5.69 (d, 1H, CH in
ring), 3.79 (t, 2H, CH2 in ring), 2.99e3.24 (m, 2H, CH2 in ring), 7.83 (br, NH ),
4.17 (q, 2H, OCH2CH3) 1.28 (t, 3H, OCH2CH3), 4.59e4.65 (m, 1H, CHeCH3),
1.38 (d, 3H, CHeCH3) and m/z 354
3300 (NH str. of amide), 3020 (aromatic CH str.), 2985,
2892 (aliphatic CH str.), 1727 (C]O str. of ester), 1655
(amide I), 1605(amide II), 1450, 1380 (CH bend,
aliphatic), 1260 (CeO str. of ester)
7.28e7.81 (m, 5H, aromatic ring), 6.55 (d, 1H, CH in ring), 5.61 (d, 1H, CH in
ring), 3.73 (t, 2H, CH2 in ring), 2.94e2.98 (m, 2H, in CH2 in ring), 7.76 (br, NH ),
4.13 (q, 2H, OCH2CH3), 1.27 (t, 3H, OCH2CH3) and m/z 356
3359 (NH str. of amide), 3276 (aromatic CH str.), 2946,
2909 (aliphatic CH str.), 1738 (C]O str. of ester), 1661
(amide I), 1607.5(amide II), 1438, 1376 (CH bend,
aliphatic), 1269 (CeO str. of ester)
7.30e7.88 (m, 5H, aromatic ring), 6.60 (d, 1H, CH in ring), 5.63 (d, 1H, CH in
ring), 3.82 (t, 2H, CH2 in ring), 2.83e2.96 (m, 2H, CH2 in ring), 7.83 (br, NH ),
7.20 (q, 2H, OCH2CH3), 1.21 (t, 3H, OCH2CH3), 10.10 (br, NH ring), 7.18e7.24
(m, 4H, aromatic ring of indole nucleus), 4.79e4.82 (m, 1H, CHCH2) and m/z 470
7.33e7.77 (m, 5H, aromatic ring), 6.53 (d, 1H, CH in ring), 5.52 (d, 1H, CH in
ring), 3.66 (t, 2H, CH2 in ring), 2.97e3.12 (m, 2H, CH2 in ring), 8.21 (br, NH ),
4.20 (q, 2H, OCH2CH3), 1.32 (t, 3H, OCH2CH3), 7.12e7.27 (m, 5H, aromatic
ring), 4.91 (t, 2H, CH2Ph) and m/z 430
3306 (NH str. of amide), 3057 (aromatic CH str.), 2996,
2930 (aliphatic CH str.), 1750 (C]O str. of ester), 1653
(amide I), 1547 (amide II), 1489, 1375 (CH bend,
aliphatic), 1283 (CeO str. of ester)
3299 (NH str. of amide), 3020 (aromatic CH str.), 2964,
2852 (aliphatic CH str.), 1738 (C]O str. of ester), 1655
(amide I), 1574 (amide II), 1432, 1396 (CH bend,
aliphatic), 1271 (CeO str. of ester)
7.28e7.83 (m, 5H, aromatic ring), 6.60 (d, 1H, CH in ring), 5.64 (d, 1H, CH in
ring), 3.83 (t, 2H, CH2 in ring), 2.88e2.94 (m, 2H, CH2 in ring), 8.12 (br, NH ),
4.19 (q, 2H, OCH2CH3), 1.28 (t, 3H, OCH2CH3), 4.39e4.59 (m, 1H,
CHCH(CH3)2), 3.08e3.42 (m, 1H, CHCH(CH3)2), 1.01 (d, 6H, CHCH(CH3)2)
and m/z 382
AR-17
AR-18
3315 (NH str. of amide), 3052 (aromatic CH str.), 2923,
2861 (aliphatic CH str.), 1723 (C]O str. of ester), 1653
(amide I), 1559 (amide II), 1437, 1376 (CH bend,
aliphatic), 1273 (CeO str. of ester)
7.26e7.86 (m, 5H, aromatic ring), 6.23 (d, 1H, CH in ring), 6.62 (d, 1H, CH in
ring), 3.90 (t, 2H, CH2 in ring), 2.80e2.92 (m, 2H, CH2 in ring), 8.02 (br, NH ),
4.18 (q, 2H, OCH2CH3), 1.28 (t, 3H, OCH2CH3), 4.45e4.64 (m, 1H,
CHCH2CH(CH3)2), 1.77e1.94 (m, 2H, CH2(CH3)2), 1.03 (d, 6H, CH(CH3)2) and
m/z 395
3326 (NH str. of amide), 3027 (aromatic CH str.), 2930,
2853 (aliphatic CH str.), 1736 (C]O str. of ester), 1648
(amide I), 1574 (amide II), 1430, 1400 (CH bend,
aliphatic), 1270 (CeO str. of ester)
7.25e7.90 (m, 5H, aromatic ring), 6.61 (d, 1H, CH in ring), 5.56 (1H, CH in ring),
3.80, 2H, CH2 in ring), 2.83e2.95 (m, 2H, in CH2 in ring), 8.04 (br, NH ), 4.20 (q,
2H, OCH2CH3), 1.28 (t, 3H, OCH2CH3), 4.39e4.56 (m, 1H, CH(CH3)CH2CH3),
1.13e1.22 (m, 2H, CHCH2CH3), 0.89 (t, 3H, CH(CH3)CH2CH3), 2.89e2.93 (m,
1H, CH(CH3)CH2CH3), 1.08 (q, 3H, CH(CH3)CH2CH3) and m/z 396
7.26e7.85 (m, 5H, aromatic ring), 6.57 (d, 1H, CH in ring), 5.56 (d, 1H, CH in
ring), 3.87 (t, 2H, CH2 in ring), 2.81e2.98 (m, 2H, CH2 in ring), 8.02 (br, NH ),
4.20 (q, 2H, OCH2CH3) 1.30 (t, 3H, OCH2CH3), 2.70 (d, 2H, CHCH2), 4.78e4.99
(m, 1H, CHCH2) and m/z 425
AR-19
AR-20
3328 (NH str. of amide), 3031 (aromatic CH str.), 2960,
2906 (aliphatic CH str.), 1723 (C]O str. of ester), 1646
(amide I), 1554 (amide II), 1438, 1390 (CH bend,
aliphatic), 1296 (CeO str. of ester)
3296 (NH str. of amide), 3015 (aromatic CH str.), 2960,
2914 (aliphatic CH str.), 1738 (C]O str. of ester), 1661
(amide I), 1562 (amide II), 1438, 1376 (CH bend,
aliphatic), 1269 (CeO str. of ester)
7.25e7.85 (m, 5H, aromatic ring), 6.57 (d, 1H, CH in ring), 5.59 (d, 1H, CH in
ring), 3.82 (t, 2H, CH2 in ring), 2.87e3.12 (m, 2H, CH2 in ring), 7.85 (br, NH ),
4.18 (q, 2H, OCH2CH3), 1.28 (t, 3H, OCH2CH3), 2.92 (t, 2H, CHCH2CH2), 2.21e
2.64 (m, 2H, CHCH2CH2), 4.44e4.97 (m, 1H, CHCH2CH2) and m/z 430
was used and retention time and peak area were noted at
247 nm for KC prodrugs.
After vortexing, the tubes were centrifuged at high speed
(6000 rpm) for 5 min. Clear supernatant (20 mL) obtained
from each tube was then injected in to run through HPLC col-
umn (C18 ODS reversed phase). Mobile phase acetonitrile:-
water 50:50 was used for KC and prodrugs. UV detector
was used and retention time and peak area were noted at
247 nm for the prodrugs.
2.5.3.2. Method for the hydrolysis rate determination of pro-
drugs in 80% human plasma (pH 7.4). A solution of 10 mg
of prodrug was prepared in acetonitrile (2 mL) and was added
to 88 mL of 80% human plasma (pH 7.4, prepared by mixing
80 portion of plasma with 20 portion of phosphate buffer 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 a definite
interval of time (10, 20, 30, 40, 50 and 60 min) an aliquot of
225 mL was withdrawn from different boiling tubes and was
transferred to microcentrifuge tubes (Eppendrof’s tube). The
tubes were placed in freezing mixture in order to arrest further
hydrolysis, followed by vortexing at high speed for 5 min.
2.6. Pharmacological evaluations
All the synthesized prodrugs along with KC were evaluated
for analgesic, anti-inflammatory activities and ulcerogenic in-
dex. The prodrugs were compared with KC for these activities.
The methods employed for this purpose were as follows.

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A. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 2464e2472
2.6.1. Anti-inflammatory activity
attempts a white sticky mass was observed during the reaction,
possibly due to highly thermo-sensitive nature of the drug.
Acylation of amino acid ester was performed using various
conditions, viz. in the presence of 5% K2CO3 at 0e2 ꢀC,
non-aqueous acylation reaction in presence of pyridine, trie-
thylamine and acylation reaction in presence of excess
pyridine. All these methods suffer with the drawback of hydro-
lysis of acid chloride, slow rate of reaction and difficulty in the
collection of final product due to lump formation. The acyla-
tion of amino acid ester using anhydride (formed by reaction
of drug with DCC in 2:1 ratio) suffers with the problem of
poor yield. When equimolar drug and DCC were used, it
gave satisfactory results. Recrystallisation of the compounds
was performed by selective precipitation with waterealcohol
mixture. In some cases the oily products were obtained which
were crystallized by induced crystallization using n-hexane
and/or petroleum ether in the ethanolic solution of the prodrug.
Purity of synthesized prodrugs was ascertained by TLC using
silica gel G and physical properties are shown in Table 2. Es-
timation of percentage nitrogen, IR, NMR and mass spectral
analyses were in confirmation of desired structure.
The anti-inflammatory activity of synthesized prodrugs was
determined by hind paw edema method utilizing carrageenan
as phlogistic agent (0.1 mL, 1%). The animals used were Wis-
tar rats (albino rats). Rats (100e200 g) were divided into
seven groups, each comprising of three rats, including a control
and standard group. The initial volume of right hind paw of
albino rats was measured by plethysmometer, without admini-
stration of the drug/prodrug.
2.6.2. Analgesic activity
The analgesic activity of the synthesized prodrugs was de-
termined by tail flick method using thermal stimulus. A hot
water analgesiometer was used for the determination of pain
threshold of Wistar rats (albino rats). Cold water was circu-
lated through the water jackets of the instrument to avoid
the heating of the area around the hot wire. Rats (100e
200 g) were divided into seven groups, each comprising of
three rats. The rat was placed in a holder through which the
tail of the rat was protruded out. The normal reaction time,
i.e. the time taken to flick the tail was noted. The current
was adjusted so that more than 90% rats flick the tail within
range of 5e9 s. Animals showing delayed response were re-
jected. The drug/prodrug (dose of each prodrug was calculated
to equivalent to 1.2 mg/kg body weight) was administered
orally in 1% suspension of sodium carboxymethylcellulose.
Percent analgesia was calculated with the formula:
The synthesized prodrugs of KC were subjected to solubil-
ity, partition coefficient, dissolution and hydrolytic studies.
Solubility studies showed that only KC was found highly sol-
uble in 0.1 N NaOH. On contrary every synthesized prodrug
was found slightly soluble in 0.1 N NaOH. Both drugs and
all prodrugs were found insoluble in water and in 0.1 N
HCl. But they showed moderate to high solubility in various
solvents such as methanol, ethanol, chloroform, dichlorome-
thane and benzene. The greater solubility of the standard
drug KC is mainly due to presence of free carboxyl group,
which forms sodium salt and makes the compound ionic.
But all prodrugs and standard drug showed moderate to high
solubility in various organic solvents, which indicate lipophilic
%Analgesia ¼ ½1 ꢂ ðT2=T1Þꢃ100
where T1 is the reaction time in second before administration
of drug/prodrug, and T2 is the reaction time in second after
administration of drug/prodrug.
1
2.6.3. Ulcerogenic index
behaviour of the compounds. FTIR, H NMR and mass spec-
Gastrointestinal toxicity of the synthesized prodrugs was
measured and compared with the drug by measuring ulcero-
genic index. For the purpose male albino rats (Wistar rats)
were selected, weighing between 130 and 150 g, the rats
were divided into 23 groups each comprising of three rats, in-
cluding a control and standard group. The prodrug/drug
(100 mg) was suspended in 100 mL of 2% w/v suspension
of acacia. Measured volume of the suspension containing
dose equivalent to 1.2 mg/kg of body weight of KC was ad-
ministered orally to the test group daily for 5 days. The rats
were fasted after the administration of last dose, thereafter
they were sacrificed by decapitation and the stomach was re-
moved, opened and washed with distilled water. The lesions
on the gastric mucosa were counted by visual examination us-
ing a 2 ꢄ 2 binocular magnifier. Ulcers greater than 0.5 mm
were recorded.
troscopic data of the synthesized compounds are listed in
Table 3. These data are in conformity with the structure.
The partition coefficient of the drug in CHCl3ePBS (pH
7.4) was found as 6.022. The partition coefficient (K ) value
of prodrugs of KC, viz. AR-11, AR-12, AR-13, AR-19 and
AR-20 was found as 13.81, 15.61, 15.33, 18.26 and 18.08;
K value of AR-14, AR-15, AR-16, AR-17 and AR-18 was
found as 21.42, 23.18, 20.0, 23.09 and 23.87. The partition co-
efficient in CHCl3eacidic buffer (pH 1.4) of KC was 75.62.
The partition coefficients of prodrugs of KC, viz. AR-11,
AR-12, AR-13, AR-19 and AR-20 were found as 215.9,
256.73, 250.25, 330.12 and 319.5. The partition coefficients
of AR-14, AR-15, AR-16, AR-17 and AR-18 were found as
429.90, 504.05, 380.6, 640.0 and 656.8. Partition coefficient
study of prodrugs showed that the major fraction of the pro-
drugs was partitioned towards the organic phase. It indicates
enhancement in the lipophilic property, which might be favor-
able to biological absorption.
3. Results and discussion
The dissolution studies revealed that prodrugs did not show
any dissolution in SGF (pH 1.2), which might be useful in
minimizing the GIT disturbances, however, this may retard ab-
sorption of the prodrugs reverse of postulation of higher
Schematic representation of the reaction used for the syn-
thesis is given in Scheme 1. Acid chloride of the KC could
not be prepared using any of the reported methods. In all

A. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 2464e2472
2471
absorption due to very high partition coefficients. Prodrugs
showed reduced dissolution rate as compared to KC due to en-
hancement in lipophilicity in SIF (pH 7.4), however, enough to
get absorbed easily. The result of dissolution study showed
that prodrugs AR-11, AR-12, AR-13, AR-17, AR-19, and
AR-20 dissolved more than 25% in 2 h.
100
95
90
85
80
75
70
65
60
55
The protein binding of ketorolac prodrugs, viz. AR-12, AR-
13 was found as 18.9 and 37.9%, respectively; and in prodrugs
AR-20, AR-19, AR-17, AR-11, AR-15, AR-18, AR-16 and
AR-14 protein binding was found from 45 to 79.8%. Prodrugs
showed comparatively very low protein binding in comparison
to standard drug. This will increase availability of the prodrug
for hydrolysis in plasma and the required dose will be less.
Hydrolytic studies of the prodrugs were carried out in sim-
ulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid
(SIF, pH 7.4) and in 80% human plasma (pH 7.4). The t1/2
value of ketorolac prodrugs, viz. AR-14 was found as
178.85; the t1/2 value of AR-17, AR-16, AR-13 and AR-12
was found between 170 and 160 min and it was found in be-
tween 154 and 128 in case of AR-18, AR-15, AR-19, AR-20
and AR-11. Hydrolytic pattern of these prodrugs is depicted
in Fig. 1. Hydrolytic studies of prodrugs in 80% human plasma
(pH 7.4) were carried out. The t1/2 values of AR-17 and AR-12
were found as 61.11 and 59.20 min, respectively; t1/2 values of
AR-13 and AR-14 were found as 52.90 and 49.50 min, respec-
tively; prodrugs AR-16, AR-11 and AR-18 showed t1/2 values
from 41 to 39 min and the t1/2 values of AR-15, AR-20 and
AR-19 were found from 35 to 27 min. Comparative pattern
of hydrolysis in 80% human plasma is shown in Fig. 2.
The amount of KC regenerated on hydrolysis (in SIF, pH
7.4) of KC prodrugs, viz. AR-11 and AR-20 was found as
22.14 and 20.13%, respectively; in prodrugs AR-19, AR-15,
AR-12 and AR-13 the amount of regenerated drug was found
from 17.7 to 13.8%; and in prodrug AR-17, AR-14, AR-18
and AR-16 the amount of regenerated drug was found from
10.25 to 5.92%.
0
20
40
60
80
100
Time (min)
Fig. 2. Comparative pattern of hydrolysis of ketorolac amide prodrugs in 80%
human plasma.
AR-11, AR-12 and AR-17 the amount of regenerated drug
was found from 39.03 to 31.66%.
None of prodrugs showed hydrolysis in SGF (pH 1.2). Sat-
isfactory hydrolysis was observed in SIF (pH 7.4) and the re-
generation of drug was found from 14 to 63%. All prodrugs
showed very encouraging hydrolysis rate in 80% human
plasma (pH 7.4) and the regeneration of the active drug was
found from 63 to 93%. All prodrugs followed first order kinet-
ics. Dissolution profiles of synthesized prodrugs of ketorolac
are shown in Fig. 3.
Pharmacological investigations of the synthesized prodrugs
were done for anti-inflammatory, analgesic and ulcerogenic
activities. In each study albino rats of either sex weighing be-
tween 100 and 200 g were used. The dose of drug/prodrug in
1% CMC suspension was given through oral route. The dose
administered of prodrugs of KC was equivalent to 2.5 mg/kg
body weight of KC. Anti-inflammatory, analgesic and ulcero-
genic activities of the synthesized prodrugs were determined.
The anti-inflammatory activities obtained after 2 and 6 h of
the administration of standard drug KC were found as 64.24,
and 54.21%. The percentage anti-inflammatory activity after
2 h administration of ketorolac prodrugs, viz. AR-20, AR-19
and AR-15 was found between 48.7 and 44%; prodrugs AR-
18, AR-11, AR-16 and AR-13 showed anti-inflammatory
The amount of KC regenerated on hydrolysis (in 80% hu-
man plasma, pH 7.4) of KC prodrugs, viz. AR-19 and AR-
18 was found as 64.32 and 64.17%, respectively; in prodrugs
AR-15, AR-18 and AR-14 the amount of regenerated drug was
found from 59.32 to 52.75% and in prodrugs AR-13, AR-14,
40
35
30
25
20
15
10
5
100
95
90
85
80
75
70
65
60
55
0
20
40
60
80
100
0
0
20
40
60 80
Time (min)
100
120
140
Time (min)
Fig. 1. Comparative hydrolytic pattern of amide prodrugs in simulated intestinal
fluid of pH 7.4.
Fig. 3. Dissolution profiles of synthesized prodrugs of ketorolac.

2472
A. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 2464e2472
30
25
20
15
10
5
activities from 41 to 33% and inhibition in AR-14, AR-17 and
AR-12 was found from 30 and 27%. The anti-inflammatory
activity of ketorolac prodrugs after 6 h administration, viz.
AR-19 was found as 57.72%; in prodrugs AR-15, AR-20,
AR-15, AR-11, AR-16, AR-18 and AR-13 inhibition was
found from 54 to 49% and inhibition was found from 45
and 43% in AR-14, AR-17 and AR-12. The analgesic activity
of synthesized prodrugs, viz. AR-20, AR-19 and AR-15 was
found from 59 to 54%; prodrug AR-18, AR-16 and AR-11
showed analgesia from 47 to 36% and analgesic effect in pro-
drugs AR-14, AR-17 and AR-12 was found from 30 to 26% of
KC. The analgesic activity of synthesized prodrugs of KC, viz.
AR-20, AR-19 and AR-15 was found from 59 to 54%; pro-
drugs AR-18, AR-16 and AR-11 showed analgesia from 47
to 36% and analgesic effect in prodrugs AR-14, AR-17 and
AR-12 was found from 30 to 26%. Ulcerogenic index of the
synthesized prodrugs was recorded to observe the extent of
gastrointestinal side effects. The ulcerogenic index of standard
drug KC was found as 23.2. Comparative ulcerogenic index of
the synthesized prodrug is shown as bar diagram in Fig. 4.
Characterization of the prodrugs establishes that the pro-
drugs had been formed in pure form and conforms to expected
properties. Spectroscopic data are suggestive of the formation
of desired prodrugs. Biopharmaceutical studies revealed that
the prodrugs have lesser protein binding and better absorption
but slow onset of action. After prodrugs administration all the
prodrugs shown lesser anti-inflammatory activity in the initial
phase, but after some time the activity was increased gradu-
ally. After 6 h of administration, prodrugs showed nearly equal
effect in comparison to standard drug. This might be due to
various factors including hydrolysis rate, dissolution rate,
etc. Similar pattern of results was obtained in analgesic activ-
ity, which might be due to same factor involved in the anti-in-
flammatory activity. Ulcerogenic index of the prodrug was
found much lesser in comparison to standard drug. The mini-
mized side effects obtained in the prodrug might be due to in-
hibition of direct contact of carboxyl group of the drug to the
gastric mucosa, which is mainly responsible for the damage. It
is also due to negligible dissolution as well as hydrolysis in
acidic buffer (pH 1.2).
0
Control
AR-11
AR-13
AR-15
Prodrug code
AR-17
AR-19
Fig. 4. Ulcerogenic indices of ketorolac and its amide prodrugs.
and encouraging hydrolysis rate both in SIF and in 80% hu-
man plasma. On contrary prodrugs with increased aliphatic
side chain length showed enhanced partition coefficient but di-
minished dissolution and hydrolysis rates. Such prodrugs can
be considered for sustained release purpose.
Acknowledgements
One of the author’s Mr. Ashutosh Mishra thankful to Uni-
versity Grants Commission, New Delhi, India for providing
financial support to carry out this work.
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Out of all the synthesized compounds, prodrug, viz. AR-11,
AR-19 and AR-20 showed excellent pharmacological response