Synthesis, characterization and in vitro hydrolysis studies of ester and amide prodrugs of dexibuprofen

Article abstract of DOI:10.1007/s00044-011-9866-z

Ten prodrugs of dexibuprofen having ester and amide moieties instead of free carboxylic acid which involves in gastrointestinal side effects have been synthesized. Dexibuprofen acid chloride was condensed with different amino acid methyl ester hydrochlorides and five alcohols to afford the amide and ester prodrugs. All of the synthesized prodrugs were characterized by their mp, Rf, elemental analysis, FTIR, ¹H NMR, and ¹³C NMR spectroscopy. The in vitro hydrolysis studies in plasma reflect prodrugs have been varied in terms of reactivity toward hydrolysis, owing to the different chemical structures. In alkyl substitution the branched chain alkyl substituents or aromatic substituents resulted in enhanced lipophilicity but diminished dissolution and hydrolysis rate. The amide prodrugs with branched and aromatic substitution can also be considered for sustained release. Prodrugs are less irritating to gastric mucosa than dexibuprofen. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9866-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3361–3368
DOI 10.1007/s00044-011-9866-z
ORIGINAL RESEARCH
Synthesis, characterization and in vitro hydrolysis studies of ester
and amide prodrugs of dexibuprofen
•
Zaman Ashraf Muhammad Imran
•
Shahid Amin
Received: 27 May 2011 / Accepted: 31 October 2011 / Published online: 17 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Ten prodrugs of dexibuprofen having ester and
amide moieties instead of free carboxylic acid which
involves in gastrointestinal side effects have been synthe-
sized. Dexibuprofen acid chloride was condensed with
different amino acid methyl ester hydrochlorides and five
alcohols to afford the amide and ester prodrugs. All of the
synthesized prodrugs were characterized by their mp, Rf,
with analgesic & antipyretic effects and which have, in
higher doses, anti-inflammatory effects (Vane, 1996).
Since the introduction of NSAIDs in the market, enormous
literature has been published regarding their side effects
(Benu and Sharma, 2009). The most common, widely
studied, reported and reviewed side effects are related
to gastrointestinal tract (GIT) (Clive and Stoff, 1984;
Johnson, 1997; Pope et al., 1993; Polonia, 1997).
1
elemental analysis, FTIR, H NMR, and 13C NMR spec-
troscopy. The in vitro hydrolysis studies in plasma reflect
prodrugs have been varied in terms of reactivity toward
hydrolysis, owing to the different chemical structures. In
alkyl substitution the branched chain alkyl substituents or
aromatic substituents resulted in enhanced lipophilicity but
diminished dissolution and hydrolysis rate. The amide
prodrugs with branched and aromatic substitution can also
be considered for sustained release. Prodrugs are less irri-
tating to gastric mucosa than dexibuprofen.
Ibuprofen (R, S-2-(4-isobutylphenyl)-propionic acid), is
among the widely used NSAIDs, contains a chiral carbon
and exists in two enantiomeric form, which are present in
the racemate (Gabard et al., 1995). S (?)-Ibuprofen or
dexibuprofen, is the pharmacologically effective enantio-
mer of racemic ibuprofen. Racemic ibuprofen and dex-
ibuprofen differ in their physico-chemical properties. In the
last 5 years 4,836 patients have been exposed to dexibu-
profen in clinical trials and post marketing surveillance
(PMS) trials. Only in 3.7% of patients adverse drug reac-
tions have been reported and three serious adverse drug
reactions (0.06%) were observed. In the dose ratio of 1:0.5
(Racemic ibuprofen vs. dexibuprofen) at least equivalent
efficacy was proven in acute mild to severe somatic and
visceral pain models. Dexibuprofen has proven at least
comparable efficacy to diclofenac, naproxen, and celecoxib
and has shown a favorable tolerability (Kaehler et al.,
2003).
Keywords Prodrugs ꢀ Dexibuprofen ꢀ NSAIDs
Introduction
The non-steroidal anti-inflammatory drugs (NSAIDs) are
among the most widely used drugs (Bonabello et al., 2003),
Dexibuprofen has a tendency of GIT disturbance, peptic
ulceration, and GIT bleeding properties (Rasheed et al.,
2009). These side effects are attributed to the local irrita-
tion produced by the free carboxylic group (–COOH) in the
molecular structure (Bernard, 1999). A number of attempts
have been made to mask this free carboxylic group (Peng
et al., 2006; Zgoda et al., 2006; Zhao et al., 2006).
Prodrugs are designed to overcome pharmaceutical,
pharmacokinetic, or pharmacodynamic barriers such as
Z. Ashraf (&)
Chemistry Department, Allama Iqbal Open University,
H-8, Islamabad, Pakistan
e-mail: mzchem@yahoo.com
M. Imran
Riphah Institute of Pharmaceutical Sciences,
Riphah International University, Islamabad, Pakistan
S. Amin
Kohat University of Science and Technology, Kohat, Pakistan
123

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Med Chem Res (2012) 21:3361–3368
Synthesis of methyl ester hydrochlorides of glycine,
alanine, valine, leucine, and phenyl alanine
insufficient chemical stability, poor solubility, unaccept-
able taste or odor, irritation or pain, insufficient oral
absorption, inadequate blood–brain barrier permeability,
marked pre-systemic metabolism, and toxicity (Helieh and
Jeffrey, 2008). Furthermore, by attachment of a pro-moiety
to the active moiety, a prodrug is formed which is designed
to overcome the barriers that hamper the optimal use of the
active principle (Marinella et al., 2008). Hence, it is a
promising way of overcoming gastrotoxicity associated
with long term oral use of NSAIDs like ibuprofen (Helieh
and Jeffrey, 2008; Bansal et al., 2001).
Freshly distilled thionyl chloride (0.05 M) was slowly
added to methanol (100 ml) with cooling. The amino acids
(1a–e) (0.1 M) were also added to this mixture and the
reaction mixture was refluxed for 6–8 h with continuous
stirring on magnetic stirrer. After the completion of reac-
tion the excess thionyl chloride and solvent was evaporated
under reduced pressure to afford crude different amino
acids methyl ester hydrochlorides. Then, crude products
were washed with 20 ml portion of the cold diethyl ether to
remove dimethyl sulfate. The resulting solid products were
collected and dried under vacuum. The recrystallization of
the products was carried out in methanol by slow addition
of 15–20 ml of ether followed by cooling at 0°C.
The crystals of amino acid methyl ester hydrochlorides
(2a–e) were collected on next day, washed with cold ether,
and dried under vacuum.
A number of functional groups have been employed for
the preparation of prodrugs. These functionalities must be
labile and cleaved in body releasing active drug. Esters and
amides are the excellent examples of such moieties
(Shanbhag et al., 2006; Mahfouz et al., 1999; Arun and
Ashok, 2009).
Keeping in mind the applications and side effects
associated with dexibuprofen, we planned to synthesize
both ester and amide prodrugs of dexibuprofen. Dexibu-
profen was condensed with different amino acids like
glycine, alanine, valine, leucine, and phenylalanine to
afford amide prodrugs. There are certain advantages of
conjugation of amino acids with NSAIDs, i.e., amino acids
are dietary constituents, many amino acids possess marked
anti-inflammatory activity against gelatin induced hind
paw edema in rats and by using different amino acids like
non-polar, polar, acidic and basic, the drug molecule can be
made more or less polar, or more or less soluble in given
solvents (Rooseboom et al., 2004). The ester prodrugs have
been synthesized by the esterification of dexibuprofen
using different alcohols like, isopropyl, n-butyl, isobutyl,
benzyl alcohol, and glycerol. All of the synthesized pro-
Synthesis of dexibuprofen acid chloride (3)
Dexibuprofen (0.05 M) was dissolved in minimum amount
of anhydrous benzene and freshly distilled thionyl chloride
(0.1 M) was then slowly added to it. The reaction mixture
was refluxed for 10–12 h with continuous stirring on
magnetic stirrer. The unreacted thionyl chloride and sol-
vent was rotary evaporated to afford the dexibuprofen acid
chloride (3).
Synthesis of amide prodrugs (4a–e)
General procedure
1
drugs were characterized by FTIR, H NMR, 13C NMR
The amino acids methyl ester hydrochlorides (2a–e)
(0.05 M) were added to an ice cold 5% aqueous sodium
hydroxide solution (50 ml) and stirred the mixture for
30 min at room temperature. The mixture was then trans-
ferred on ice bath stirred at 10°C. Dexibuprofen acid
chloride (3) (0.01 M) was slowly added to reaction flask
with continuous stirring for 7–8 h. The amide prodrugs
(4a–e) were then extracted with (20 ml 9 3) ethyl acetate,
solvent was rotary evaporated to afford the amide prodrugs.
The schematic representation of the amide prodrugs is
shown in Scheme 1.
spectroscopy, and elemental analysis. The comparative
stabilities of ester and amide prodrugs were also evaluated
by their in vitro hydrolysis studies. We have examined that
ester prodrugs are labile or easily hydrolyze as compared to
amide prodrugs; this depicts the high stability of amide
functionality over ester functionality.
Experimental
Melting points were recorded using a digital Gallenkamp
(SANYO) model MPD BM 3.5 apparatus and are uncor-
rected. FT IR spectra were recorded using an FTS 3000
Synthesis of methyl ({2-[4-(2-methylpropyl)phenyl]propa-
noyl}amino)acetate (4a) Yield 83%, oil, Rf 0.6 (pet.
ether:ethyl acetate 4:1), IR mmax cm-1 (KBr): 3,324 (N–H),
2,955 (C–H), 1,739 (C=O ester) 1,654 (C=O amide), 1,511
1
MX spectrophotometer, H NMR and 13C NMR spectra
were determined as CDCl3 solutions at 300 MHz on a
Bruker AM-300 spectrophotometer and elemental analysis
with a LECO-183 CHNS analyzer. All the compounds
were purified by thin layer chromatography using silica gel
HF-254 from Merck.
1
(C=C); HNMR (CDCl3, d ppm) 7.24 (d, J = 6.0 Hz, 2H,
H-20 aromatic ring), 7.15 (d, J = 8.4 Hz, 2H, H-30 aromatic
ring), 6.04 (s, 1H, N–H), 3.98 (q, J = 5.1 Hz, 1H, H-1), 3.72
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Med Chem Res (2012) 21:3361–3368
3363
H
H
R
Scheme 1 Synthesis of amide
prodrugs (4a–e)
O
O
CH3OH
Cl-
NH
+
SOCl2
H N
+
2
3
OH
OCH
3
R
(1a-e)
(2a-e)
R= same as 1a-e
R=
1a = H
1b = CH3
1c = CH-(CH3)2
1d = CH2CH-(CH3)2
1e = CH2C6H5
CH3
CH3
O
O
dry benzene
CH3
CH3
SOCl2
+
OH
Cl
H3C
H3C
(3)
CH3
CH3
O
H
R
O
CH3
O
H
CH3
Cl-
+
HN
NH
+
3
H3C
O
Cl
H3C
OCH
R
3
OCH3
(2a-e)
(4a-e)
(3)
(s, 3H, ester –OCH3), 2.41 (d, J = 7.8 Hz, 2H, H-40), 2.05 (s,
2H, H-3), 1.89 (m, 1H, H-5), 1.52 (d, J = 7.2 Hz, 3H, H-2),
0.91 (d, J = 3.3 Hz, 6H, H-6); 13CNMR (CDCl3, d ppm)
171.5 (ester C=O), 169.6 (amide C=O), 139.3 (C-40 aro-
matic), 133.2 (C-10 aromatic), 129.1 (C-30 aromatic), 128.3
(C-20 aromatic), 51.6 (C–OCH3), 45.7 (C-4), 42.6 (C-1), 40.8
(C-3), 29.1 (C-5), 22.8 (C-6), 17.1 (C-2); Elemental analysis:
C16H23O3N; Calculated; C 69.31%, N 5.10%, H 8.30%,
Found; C 69.24%, N 5.02%, H 8.21%.
oil, Rf 0.74 (pet. ether:ethyl acetate 4:1), IR mmax cm-1
(KBr): 3,313 (N–H), 2,955 (C–H), 1,737 (C=O ester) 1,621
1
(C=O amide), 1,512 (C=C); HNMR (CDCl3, d ppm) 7.13
(d, J = 3.8 Hz, 2H, H-20 aromatic ring), 7.07 (d,
J = 8.1 Hz, 2H, H-30 aromatic ring), 6.77 (s, 1H, N–H),
4.41 (d, J = 6.5 Hz, 1H, H-3), 3.91 (s, 3H, ester –OCH3),
3.81 (q, J = 5.6 Hz, 1H, H-1), 3.09 (m, 1H, H-4), 2.51 (d,
J = 4.7 Hz, 2H, H-6), 2.22 (m, 1H, H-7), 1.52 (d,
J = 7.5 Hz, 2H, H-2), 1.16 (d, J = 7.2 Hz, 6H, H-5), 0.90
(d, J = 7.1 Hz, 6H, H-8); 13CNMR (CDCl3, d ppm) 172.1
(ester C=O), 169.8 (amide C=O), 139.5 (C-40 aromatic),
132.3 (C-10 aromatic), 127.9 (C-30 aromatic), 125.4 (C-20
aromatic), 52.5 (C–OCH3), 49.8 (C-3), 44.8 (C-6), 41.7
(C-1), 30.5 (C-4), 29.3 (C-7), 22.8 (C-8), 17.5 (C-5), 16.8
(C-2); Elemental analysis: C19H29O3N; calculated; C
71.47%, N 4.39%, H 9.10%, Found; C 71.39%, N 4.31%,
H 9.01%.
Synthesis of methyl 2-({2-[4-(2-methylpropyl)phenyl]pro-
panoyl}amino)propanoate (4b) Yield 78%, oil, Rf 0.7
(pet. ether:ethyl acetate 4:1), IR mmax cm-1 (KBr): 3,345
(N–H), 2,926 (C–H), 1,737 (C=O ester), 1,706 (C=O
1
amide), 1,511 (C=C); HNMR (CDCl3, d ppm) 7.23 (d,
J = 4.2 Hz, 2H, H-20 aromatic ring), 7.15 (d, J = 8.1 Hz,
2H, H-30 aromatic ring), 5.98 (s, 1H, N–H), 4.55 (q,
J = 7.2 Hz, 1H, H-3), 3.69 (s, 3H, ester –OCH3), 3.56 (q,
J = 6.9 Hz, 1H, H-1), 2.47 (d, J = 3.9 Hz, 2H, H-5), 1.84
(m, 1H, H-6), 1.54 (d, J = 7.2 Hz, 2H, H-2), 1.36 (d,
J = 7.2 Hz, 2H, H-4), 0.90 (d, J = 6.6 Hz, 6H, H-7);
13CNMR (CDCl3, d ppm) 171.6 (ester C=O) 170.2 (amide
C=O), 138.5 (C-40 aromatic), 134.3 (C-10 aromatic), 128.6
(C-30 aromatic), 126.5 (C-20 aromatic), 51.9 (C–OCH3),
48.5 (C-3), 45.5 (C-5), 42.9 (C-1), 28.5 (C-6), 24.3 (C-7),
18.6 (C-2), 17.2 (C-4); Elemental analysis: C17H25O3N;
Calculated; C 70.10%, N 4.81%, H 8.60%, Found; C
70.03%, N 4.73%, H 8.52%.
Synthesis of methyl 4-methyl-2-({2-[4-(2-methylpropyl)
phenyl]propanoyl}amino)pentanoate (4d) Yield 76%,
oil, Rf 0.7 (pet. ether:ethyl acetate 4:1); IR mmax cm-1
(KBr): 3,324 (N–H), 2,955 (C–H), 1,736 (C=O ester) 1,643
1
(C=O amide), 1,511 (C=C); HNMR (CDCl3, d ppm) 7.25
(d, J = 4.5 Hz, 2H, H-20 aromatic ring), 7.13 (d, J =
7.5 Hz, 2H, H-30 aromatic ring), 6.50 (s, 1H, N–H), 4.38 (t,
J = 6.3 Hz, 1H, H-3), 3.89 (s, 3H, ester –OCH3), 3.67 (q,
J = 6.5 Hz, 1H, H-1), 2.51 (d, J = 3.8 Hz 1H, H-7), 2.22
(m, 1H, H-8), 1.86 (dd, J = 5.4, 4.8 Hz, 2H, H-4), 1.72 (m,
1H, H-5), 1.52 (d, J = 6.8 Hz, 3H, H-2), 1.01 (d,
J = 7.5 Hz, 6H, H-6), 0.90 (d, J = 6.5 Hz, 6H, H-9);
13CNMR (CDCl3, d ppm) 171.6 (ester C=O), 170.5 (amide
Synthesis of methyl 3-methyl-2-({2-[4-(2-methylpro-
pyl)phenyl]propanoyl}amino)butanoate (4c) Yield 74%,
123

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Med Chem Res (2012) 21:3361–3368
C=O), 138.5 (C-40 aromatic), 131.7 (C-10 aromatic), 128.9
(C-30 aromatic), 127.4 (C-20 aromatic), 51.9 (C-OCH3),
50.1 (C-3), 45.7 (C-7), 42.9 (C-1), 40.6 (C-4), 29.1 (C-8),
22.8 (C-9), 22.5 (C-5), 19.5 (C-6), 17.1 (C-2); Elemental
analysis: C20H31O3N; calculated; C 72.07%, N 4.20%, H
9.30%, Found; C 71.98%, N 4.13%, H 9.22%.
NaOH solution, and again with water. The organic layer
was dried over anhydrous magnesium sulfate and evapo-
rated under reduced pressure. The crude products were
further purified by preparative thin layer chromatography
using silica gel as stationary phase and chloroform as
mobile phase. The schematic representation of the ester
prodrugs is shown in Scheme 2.
Synthesis of methyl 3-phenyl-2-({2-[4-(2-methylpropyl)
phenyl]propanoyl}amino)propanoate (4e) Yield 85%,
oil, Rf 0.65 (pet. ether:ethyl acetate 4:1), IR mmax cm-1
(KBr): 3,331 (N–H), 2,955 (C–H), 1,739 (C=O ester) 1,654
(C=O amide), 1,511 (C=C); 1HNMR (CDCl3, d ppm)
7.2–7.3 (m, 5H, aromatic ring), 7.15 (d, J = 7.5 Hz, 2H,
H-30 aromatic ring), 7.03 (d, J = 6.2 Hz, 2H, H-30 aro-
matic ring), 6.71 (s, 1H, N–H), 4.81 (t, J = 5.4 Hz, 1H,
H-3), 3.90 (s, 3H, ester –OCH3), 3.78 (q, J = 7.1 Hz, 1H,
H-1), 3.29 (d, J = 4.6 Hz 2H, H-4), 2.51 (d, J = 5.1 Hz
2H, H-5), 2.18 (m, 1H, H-6), 1.52 (d, J = 6.7 Hz, 3H,
H-2), 1.01 (d, J = 7.1 Hz, 6H, H-7); 13CNMR (CDCl3, d
ppm) 172.5 (ester C=O), 171.1 (amide C=O), 139.5 (C-40
aromatic), 136.2 (C-100 aromatic), 132.4 (C-10 aromatic),
129.9 (C-30 aromatic), 128.4 (C-20 aromatic), 126.4 (C-200
aromatic), 125.8 (C-300 aromatic), 123.3 (C-400 aromatic),
53.4 (C-3), 51.7 (C–OCH3), 45.4 (C-5), 42.3 (C-1), 37.1
(C-4), 29.5 (C-6), 22.4 (C-7), 17.4 (C-2); Elemental anal-
ysis: C23H29O3N; calculated; C 75.20%, N 3.87%, H
7.90%, Found; C 75.12%, N 3.79%, H 7.81%.
Synthesis of propan-2-yl 2-[4-(2-methylpropyl)phenyl]
propanoate (6a) Yield 87%, oil, Rf 0.8 (pet. ether:ethyl
acetate 4:1), IR mmax cm-1 (KBr): 2,955 (C–H), 1,729
1
(C=O ester), 1,464 (C=C); HNMR (CDCl3, d ppm) 7.21
(d, J = 5.8 Hz, 2H, H-20 aromatic ring), 7.15 (d,
J = 6.7 Hz, 2H, H-30 aromatic ring), 4.01 (m, 1H, H-3),
3.78 (q, J = 5.1 Hz, 1H, H-1), 3.23 (d, J = 5.2 Hz 6H,
H-4), 2.31 (d, J = 4.3 Hz, 2H, H-5), 1.63 (d, J = 5.7 Hz,
3H, H-2), 1.23 (m, 1H, H-6), 1.01 (d, J = 7.1 Hz, 6H,
H-7); 13CNMR (CDCl3, d ppm) 173.7 (ester C=O), 139.2
(C-40 aromatic), 132.5 (C-10 aromatic), 129.4 (C-30 aro-
matic), 128.3 (C-20 aromatic), 68.4 (C-3), 45.7 (C-5), 40.3
(C-1), 30.3 (C-6), 24.5 (C-4), 22.4 (C-7), 17.1 (C-2); Ele-
mental analysis: C16H24O2; calculated; C 77.41%, H
9.68%, Found; C 77.33%, H 9.60%.
Synthesis of butyl 2-[4-(2-methylpropyl)phenyl]propanoate
(6b) Yield 84%, oil, Rf 0.85 (pet. ether:ethyl acetate 4:1),
IR mmax cm-1 (KBr): 2,945 (C–H), 1,732 (C=O ester),
1,458 (C=C); 1HNMR (CDCl3,
d ppm) 7.25 (d,
Synthesis of ester prodrugs (6a–e)
J = 7.1 Hz, 2H, H-20 aromatic ring), 7.17 (d, J = 5.2 Hz,
2H, H-30 aromatic ring), 4.21 (t, J = 5.6 Hz, 2H, H-3),
3.85 (q, J = 6.5 Hz, 1H, H-1), 3.01 (p, J = 4.8 Hz 2H,
H-4), 2.56 (d, J = 5.3 Hz, 2H, H-7), 1.58 (d, J = 5.7 Hz,
3H, H-2), 1.25 (m, 1H, H-8), 1.17 (m, 2H, H-5), 1.01 (d,
J = 6.5 Hz, 6H, H-9), 0.91 (t, J = 4.7 Hz, H-6); 13CNMR
(CDCl3, d ppm) 173.3 (ester C=O), 138.1 (C-40 aromatic),
132.3 (C-10 aromatic), 129.0 (C-30 aromatic), 127.5 (C-20
aromatic), 65.4 (C-3), 44.5 (C-7), 40.3 (C-1), 31.2 (C-4),
28.2 (C-8), 22.8 (C-9), 19.4 (C-5), 16.5 (C-2), 13.8 (C-6);
Elemental analysis: C17H26O2; calculated; C 77.86%, H
9.92%, Found; C 77.79%, H 9.85%.
General procedure
The ester prodrugs of dexibuprofen have been synthesized
by coupling it with alcohols in the presence of dicyclo-
hexylcarbodiimide (DCC). A solution of dexibuprofen
(6.7 mmol), N,N-dicyclohexylcarbodiimide (7.4 mmol),
respective alcohol (7.4 mmol) and dimethylaminopyridine
(DMAP, 0.7 lmol) in anhydrous dichloromethane (30 ml)
was taken in round bottom flask. The reaction mixture was
stirred at room temperature and completion of the reaction
was monitored by thin layer chromatography. After the
completion of reaction, N,N-dicyclohexylurea was filtered
off and filtrate was washed with water, acetic acid, 1%
Synthesis of butan-2-yl 2-[4-(2-methylpropyl)phenyl]pro-
panoate (6c) Yield 81%, oil, Rf 0.75 (pet. ether:ethyl
CH3
Scheme 2 Synthesis of ester
prodrugs (6a–e)
CH3
O
O
CH3
DCC
CH3
R-OH
+
OR
DMAP
OH
H3C
H3C
(5a-e)
(6a-e)
R= same as 5a-e
R=
1a = CH-(CH3)2
1b = C4H7
1c = CH-C2H5(CH3)
1d = CH2C6H5
1e = CH2CH(OH)CH2OH
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Med Chem Res (2012) 21:3361–3368
3365
acetate 4:1), IR mmax cm-1 (KBr): 2,956 (C–H), 1,734
(C=O ester), 1,465 (C=C); HNMR (CDCl3, d ppm) 7.24
solvent selected were 0.1 N NaOH, 0.1 N HCl, ethanol,
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 sol-
ubility of the prodrug.
1
(d, J = 8.1 Hz, 2H, H-20 aromatic ring), 7.12 (d,
J = 8.4 Hz, 2H, H-30 aromatic ring), 3.86 (m, 1H, H-3),
3.73 (q, J = 7.2 Hz, 1H, H-1), 2.45 (d, J = 7.2 Hz 3H,
H-7), 1.93 (m, 2H, H-4), 1.51 (d, J = 7.2 Hz, 3H, H-2),
1.28 (m, 1H, H-8), 1.06 (d, J = 6.6 Hz, 2H, H-6), 0.92 (d,
J = 6.6 Hz, 6H, H-9), 0.85 (t, J = 6.9 Hz, 3H, H-5);
13CNMR (CDCl3, d ppm) 171.7 (ester C=O), 140.5 (C-40
aromatic), 131.7 (C-10 aromatic), 127.1 (C-30 aromatic),
126.5 (C-20 aromatic), 74.5 (C-3), 47.2 (C–C-7), 41.8 (C-
1), 30.2 (C-4), 28.2 (C-8), 22.8 (C-9), 21.4 (C-6), 16.5 (C-
2), 10.5 (C-5); Elemental analysis: C17H26O2; calculated; C
77.86%, H 9.92%, Found; C 77.79%, H 9.85%.
Protein binding studies (Martin et al., 1993)
A solution of synthesized prodrug (20 lg/ml) was made in
phosphate buffered saline (PBS pH 7.4). A 100 ml of this
solution was taken in a beaker. The prepared membrane
was first washed with distilled water and then with buffer
solution (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 rpm. The temperature was maintained at 37 0.5°C.
After every 2 h, 1 ml of the PBS containing drug solution
was replaced of the beaker with fresh 1 ml PBS. With-
drawn sample was diluted further with 1 ml phosphate
buffer and the concentration of the drug was estimated
spectrophotometrically using spectrophotometer (Shima-
dzu-1601).
Synthesis of benzyl 2-[4-(2-methylpropyl)phenyl]propano-
ate (6d) Yield 86%, oil, Rf 0.8 (pet. ether:ethyl acetate
4:1), IR mmax cm-1 (KBr): 2,953 (C–H), 1,734 (C=O ester),
1
1,453 (C=C); HNMR (CDCl3, d ppm) 7.28–7.39 (m, 5H,
H-200–H-600), 7.22 (d, J = 3.9 Hz, 2H, H-20 aromatic ring),
7.10 (d, J = 8.1 Hz, 2H, H-30 aromatic ring), 4.01 (s, 2H,
H-3), 3.79 (q, J = 7.2 Hz, 1H, H-1), 2.46 (d, J = 2.2 Hz
2H, H-4), 1.52 (d, J = 6.9 Hz, 3H, H-2), 1.28 (m, 1H,
H-5), 0.91 (d, J = 6.9 Hz, 6H, H-6); 13CNMR (CDCl3, d
ppm) 169.5 (ester C=O), 141.2 (C-100), 139.5 (C-40 aro-
matic), 132.3 (C-10 aromatic), 129.5 (C-30 aromatic), 128.5
(C-20 aromatic), 127.5 (C-300, 500), 126.2 (C-200, 600), 122.4
(C-400), 68.8 (C-3), 44.6 (C-4), 40.8 (C-1), 39.2 (C-5), 22.5
(C-6), 17.2 (C-2); Elemental analysis: C20H24O2; calcu-
lated; C 81.08%, H 8.10%, Found; C 81.01%, H 8.03%.
In vitro hydrolysis studies of synthesized prodrugs
Hydrolytic behavior of the synthesized prodrugs was
studied in simulated gastric fluid (SGF pH 1.2), simulated
intestinal fluid (SIF pH 7.4), and 80% human plasma. The
following method was adopted for invitro hydrolysis
studies.
Synthesis of 2,3-dihydroxypropyl 2-[4-(2-methylpro-
pyl)phenyl]propanoate (6e) Yield 71%, oil, Rf 0.4 (pet.
ether:ethyl acetate 4:1), IR mmax cm-1 (KBr): 3,396 (O–H),
1
Method for the determination of hydrolysis rate in SGF and
SIf A solution of 10 mg of prodrug was prepared in
acetonitrile (2 ml) and was added to 88 ml of SGF (pH 1.2)
or SIF (pH 7.4). An aliquot of 15 ml of this solution was
withdrawn at definite interval of 15 min and was kept in
test tubes maintained at 37 0.5°C. An aliquot of
0.225 ml was withdrawn from different test tubes and was
transferred to microcentrifuge tubes followed by the addi-
tion of methanol to make up the volume. The tubes were
placed in freeing mixture to arrest further hydrolysis, fol-
lowed by vortexing at high speed (3,000 rpm) for 5 min.
Clear supernatant (0.020 ml) obtained from each test tube
was subjected for HPLC analysis using C18 column and
mobile phase acetonitrile : water 70 : 30 (pH 2.3 adjusted
with acetic acid). Flow rate of mobile phase was kept at
1 ml/min at pressure 120–135 psi and UV detector was
used and retention time and peak area were noted at
247 nm. Figure 1 depicts the hydrolysis pattern of ester
and amide prodrugs in SIF (pH 7.4).
2,955 (C–H), 1,714 (C=O ester), 1,478 (C=C); HNMR
(CDCl3, d ppm) 7.19 (d, J = 6.4 Hz, 2H, H-20 aromatic
ring), 7.05 (d, J = 7.5 Hz, 2H, H-30 aromatic ring), 4.34 (d,
J = 4.5 Hz, 2H, H-3), 3.90 (p, J = 6.2 Hz, 1H, H-4), 3.78
(q, J = 6.2 Hz 2H, H-1), 3.56 (d, J = 4.9 Hz, 2H, H-5),
2.51 (d, J = 7.3 Hz 2H, H-6), 2.22 (m, 1H, H-7), 2.01 (s,
2H, O–H), 1.51 (d, J = 3.6 Hz, 3H, H-2), 1.01 (d,
J = 6.9 Hz, 6H, H-8); 13CNMR (CDCl3, d ppm) 168.6
(ester C=O), 139.2 (C-40 aromatic), 132.6 (C-10 aromatic),
129.5 (C-30 aromatic), 128.9 (C-20 aromatic), 71.2 (C-4),
67.5 (C-3), 64.6 (C-5), 46.2 (C-6), 40.5 (C-1), 29.1 (C-7),
22.8 (C-8), 16.5 (C-2); Elemental analysis: C16H24O4;
calculated; C 68.57%, H 8.57%, Found; C 68.49%, H
8.49%.
Dissolution studies
Approximately 5 mg of each prodrug was dissolved in
5 ml of each solvent at 37 1°C in glass test tubes. The
123

3366
Med Chem Res (2012) 21:3361–3368
110
100
90
Table 1 Physical data table
Prodrug
Molecular
formula
MW
calculated
Physical
state
%
yield
Rf
valuea
80
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
a
C16H23O3N
277
291
319
333
367
248
262
262
296
280
Oil
Oil
Oil
Oil
Oil
Oil
Oil
Oil
Oil
Oil
83
78
74
76
85
87
84
81
86
71
0.6
70
C
C
C
C
C
C
C
C
C
17H25O3N
19H29O3N
20H31O3N
23H29O3N
16H24O2
17H26O2
17H26O2
20H24O2
16H24O4
0.7
60
0.74
0.7
50
0
10 20 30 40 50 60 70 80 90 100
0.65
0.8
time/min
4a
6b
4b
6c
4c
6d
4d
6e
4e
6a
0.85
0.75
0.8
Fig. 1 Invitro hydrolytic pattern of the ester and amide prodrugs in
SIF (pH 7.4)
0.4
TLC (Adsorbent Silica Gel HF-254, Solvent System Petroleum
Ether:Ethyl Acetate; 4:1)
Method for the determination of hydrolysis rate in 80%
human plasma (pH 7.4) A solution of 10 mg of prodrug
was prepared in acetonitrile (2 ml) and was added to the
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 with-
drawn and kept in test tubes maintained at 37 0.5°C. An
aliquot of 0.225 ml was withdrawn from different tubes at
interval 15 min and was transferred to microcentrifuge
tubes. Then the solution was treated as above mentioned
procedure. The comparative study of rate of hydrolysis of
ester and amide prodrugs in 80% human plasma (pH 7.4) is
shown in Fig. 2.
preparative thin layer chromatography using silica gel HF-
254 and physical data is presented in Table 1. Two series
of prodrugs, five amide and five ester prodrugs of dex-
ibuprofen were synthesized. The amino acids glycine,
alanine, valine, leucine, and phenyl alanine were selected
to synthesized amide prodrugs. The amino acids were
converted into their corresponding methyl ester hydro-
chlorides by reacting with methanol in the presence of
thionyl chloride. These hydrochlorides were then con-
densed with dexibuprofen acid chloride to afford amide
prodrugs (4a–e). The ester prodrugs (6a–d) were synthe-
sized by reacting dexibuprofen with different alcohols
(isopropyl, n-butyl, isobutyl, benzyl, and glycerol) in the
presence of DCC and DMAP. The structures of synthesized
prodrugs were confirmed by FTIR, 1H NMR, and 13C NMR
and elemental analysis.
Results and discussion
In the present study, the free carboxylic acid group of the
dexibuprofen was temporarily masked by a promoiety so as
not to expose stomach’s mucosa to this free acidic group.
Purity of synthesized prodrugs was ascertained by
All of the synthesized amide and ester prodrugs of
dexibuprofen were subjected to solubility studies, protein
binding, and hydrolytic studies. Solubility studies showed
that only dexibuprofen was found highly soluble in 0.1 N
NaOH. On contrary every prodrug was found slightly
soluble in 0.1 N NaOH. The parent drug and the synthe-
sized prodrugs were found insoluble in water and 0.1 N
HCl. But they showed moderate to high solubility in var-
ious solvents such as ethanol, ethyl acetate, DMF, and
chloroform. The greater solubility of dexibuprofen in
NaOH is due to the presence of free carboxylic acid group,
which forms sodium salt and make the compound soluble.
But all prodrugs and the standard drug showed moderate to
high solubility in various organic solvents, which indicate
lipophilic behavior of the compounds.
110
100
90
80
70
60
50
40
30
20
0
10 20 30 40 50 60 70 80 90 100
time/min
The ester prodrugs showed smaller protein binding as
compared to amide prodrugs. The prodrugs 4a, 4b, 6b, 6e
showed 20–40% protein binding and 4c, 4d, 4e, 6d showed
40–60% protein binding. The prodrugs 6a, 6c showed 65
and 67% protein binding. Prodrugs showed comparatively
4a
6b
4b
6c
4c
6d
4d
6e
4e
6a
Fig. 2 Invitro hydrolytic pattern of the ester and amide prodrugs in
80% human plasma (pH 7.4)
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Med Chem Res (2012) 21:3361–3368
3367
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. The per-
centage protein binding of the prodrugs is presented in
Table 2.
The HPLC method was adopted for the estimation of
remaining prodrugs and regenerated drug. The spectro-
photometric method was not suitable because both the drug
and prodrug have very close kmax values, this method was
used only to find whether the hydrolysis was taking place
or not in acidic buffer (pH 1.2) and in phosphate buffer (pH
7.4).
Hydrolytic studies in PBS (pH 7.4) of the prodrugs
showed the t1/2 values (time required for 50% hydrolysis)
of dexibuprofen prodrugs 4c, 4d, and 4e were found
198.21, 189.58, and 178.43 min. respectively; t1/2 values of
4a and 4b were found from 163 to 155 min and the t1/2
values of ester prodrugs 6a–6e were found from
126–143 min. Hydrolytic studies of prodrugs in 80%
human plasma (pH 7.4) showed t1/2 value of amide pro-
drugs 4a–4e were found 61–53 min; t1/2 values of ester
prodrugs 6a, 6c, and 6d were found 46–39 min and ester
prodrugs 6b and 6e showed t1/2 values 33–29 min.
The amount of dexibuprofen regenerated on hydrolysis
after 1 h (in SIF, pH 7.4) of prodrugs 4c, 4d, and 4e was
found from 6.34 to 8.02%; in prodrugs 4a and 4b drug
regenerated was found 13.5–17.56%; prodrug 6a and 6c
showed regeneration from 19.03 to 21.16% and amount
regenerated in prodrug 6b, 6d, and 6e was found
23.51–25.67%, respectively.
Conclusion
The ester and amide prodrugs of dexibuprofen have been
successful synthesized and structures were confirmed by
physical data and spectral analysis. The ester prodrugs are
more labile than amides but all the synthesized prodrugs
showed encouraging hydrolysis rate in SIF ? 80% plasma.
The prodrug synthesis also prevent the accumulation of the
drug in gastric mucosa results in decrease of gastrointes-
tinal irritent without loss of pharmacological response. The
amide prodrugs showed the controlled hydrolysis rate and
it depicts these prodrugs can be considered as sustained
release, it calls for further extensive studies.
The amount of dexibuprofen regenrated on hydrolysis
after 1 h (in 80% human plasma, pH 7.4) of prodrugs 6b,
6d, and 6e was found from 69.4 to 64.7%; in prodrugs 6a
and 6c the amount of regenerated drug was obtained from
61.5 to 58.8%; the amount of regenerated drug was found
from 51.6 to 46.5% in prodrugs 4a, 4b, and 4e; and the
prodrugs 4c and 4d showed 36.41 and 38.56% regeneration
of drug, respectively. None of prodrugs showed hydrolysis
in SGF (pH 1.2). Satisfactory hydrolysis was observed in
SIF (pH 7.4) and the regeneration of drug was found from
13 to 58%. All prodrugs showed very encouraging hydro-
lysis rate in 80% human plasma (pH 7.4) and the regen-
eration of active drug was found from 61 to 92%.
References
Arun R, Ashok KCK (2009) Synthesis, hydrolysis and pharmacody-
namic profiles of novel prodrugs of mefenamic acid. Int J Cur
Pharm Res 1(1):47–55
Bansal AK, Khar RK, Dubey R, Sharma AK (2001) Benzyl ester
prodrug of Ibuprofen: pharmacological and toxicological profile.
Boll Chim Farm 140(2):79–82
Benu M, Sharma PD (2009) Design, synthesis and evaluation of
diclofenac-antioxidant mutual prodrugs as safer NSAIDs. Indian
J Chem 48B:1279–1287
Bernard S (1999) Profile and mechanism of gastrointestinal and other
side effects of nonsteroidal antiinflammatory drugs (NSAIDs).
Am J Med 17:27–35
Bonabello A, Galmozzi MR, Canaparo R, Isaia GC, Serpe L, Muntoni
E, Zara GP (2003) Desibuprofen (S(?)-isomer ibuprofen)
reduces gastric damage and improves analgesic and anti-
inflammatory effects in rodents. Anesth Analg 97:402–408
Clive DM, Stoff JS (1984) Renal syndromes associated with non-
steroidal anti-inflammatory drugs. N Engl J Med 310:563–572
Gabard B, Nirnberger G, Schiel H, Mascher H, Kikuta Ch, Mayer JM
(1995) Comparison of the bioavailability of dexibuprofen
administered alone or as part of racemic ibuprofen. Eur J Clin
Pharmacol 48:505–511
Helieh SO, Jeffrey LE (2008) Application of prodrugs to inflamma-
tory diseases of the gut. Mole 13:452–474
Johnson AG (1997) NSAIDS and increased blood pressure: what is
the clinical significance? Drug Saf 17:277–289
Kaehler ST, Phleps W, Hesse E (2003) Dexibuprofen: pharmacology,
therapeutic uses and safety. Inflam pharm 11:371–383
Mahfouz NM, Omar FA, Aboul-Fadl T (1999) Cyclic amide
derivatives as potential prodrugs II: N-hydroxymethylsuccini-
mide and N-hydroxymethylisatin esters of some NSAIDs as
prodrugs with an improved therapeutic index. Eur J Med Chem
34(7–8):551–562
Table 2 Percent protein binding
S. Prodrug Initial
Amount of
prodrug
prodrug/mg bound/mg
Amount of
prodrug
unbound/mg
%
protein
binding
no.
amount of
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
6a
6b
6c
6d
20
20
20
20
20
20
20
20
20
20
4.7
7.2
15.3
12.8
11.0
9.8
24
36
45
51
47
65
35
67
37
23
9.0
10.2
9.4
9.6
13
7
7.0
13.0
6.6
13.4
7.4
12.6
15.4
10 6e
4.6
123

3368
Med Chem Res (2012) 21:3361–3368
Marinella GS, Chady BS, Maryam T (2008) Prodrug in cardiovas-
cular therapy. Mole 13:1156–1178
Martin AN, Bustamante P, Chine AHC (1993) Colloids. In: Physical
pharmacy. BI Waverly Pvt Ltd, New Delhi
Peng YS, Lin SC, Huang SJ, Wang YM, Lin YJ, Wang LF, Chen JS
(2006) Chondroitin sulfate-based anti-inflammatory macromo-
lecular Prodrugs. Eur J pharm Sci 29(1):60–69
Rooseboom M, Commandeur JN, Vermeulen NP (2004) Enzyme-
catalyzed activation of anticancer prodrugs. Pharmacol Rev
56(1):53–102
Shanbhag VR, Crider AM, Gokhale R, Harpalani A, Dick RM (2006)
Ester and amide prodrugs of ibuprofen and naproxen: synthesis,
anti-inflammatory activity, and gastrointestinal toxicity. J Pharm
Sci 81:149–154
Polonia J (1997) Interaction of antihypertensive drugs with anti-
inflammatory drugs. J Cardiol 88:47–51
Vane JR (1996) Introduction: mechanism of action of NSAIDs. Br J
Rheum 35:1–3
Pope JE, Anderson JJ, Felson D (1993) A meta-analysis of the effects
of nonsteroidal anti-inflammatory drugs on blood pressure. Arch
Intern Med 153:477–484
Rasheed A, Aishwarya K, Basha N, Reddy, Sravya B, Swetha A
(2009) Dexibuprofen-dextran macromolecular prodrugs: synthe-
sis, characterization and pharmacological evaluation. Der Pharm
Chem 1(2):124–132
Zgoda MM, Lukosek M, Nachajski MJ (2006) Micellar solubilization
of selected non-steroidal therapeutic agents by new surface-
active agents of the class of the products of oxyethylation of
ursodeoxycholic acid. Polim Med 36(4):13–30
Zhao X, Tao X, Wei D, Song Q (2006) Pharmacological activity and
hydrolysis behavior of novel ibuprofen glucopyranoside conju-
gates. Eur J Med Chem 41(11):1352–1358
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