Synthesis and evaluation of novel prodrugs of naproxen

Article abstract of DOI:10.1007/s00044-010-9364-8

A series of novel prodrugs of naproxen has been synthesized. Naproxen (1) was reacted with thionyl chloride to yield acid chloride (2) which was further reacted with glucose to form the glucosyl naproxen (3). Tetra-acetate of glucosyl naproxen was prepared and finally reacted with different amino acids to yield the title compounds. These compounds were evaluated for analgesic, anti-inflammatory activities, and for possible GI toxicity. Compound 5b depicted most potent analgesic activity with percentage inhibition of 98.15%. Compound 5a was found to be most potent anti-inflammatory agent with 76% inhibition. Compound 5n was second most active analgesic (92.26%) and anti-inflammatory (73%) agent. In vitro hydrolysis pattern of synthesized prodrugs was studied in phosphate buffer of pH 7.4 and acetate buffers of pH 3.0, 4.0, and 5.0, respectively. Selected compounds were evaluated for their ulcerogenic potential and all the tested derivatives were significantly less irritating to gastric mucosa than the parent drug. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9364-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:648–655
DOI 10.1007/s00044-010-9364-8
ORIGINAL RESEARCH
Synthesis and evaluation of novel prodrugs of naproxen
•
Prabodh Chander Sharma Sonia Yadav
•
•
Rakesh Pahwa Dhirender Kaushik
•
Sandeep Jain
Received: 16 November 2009 / Accepted: 3 May 2010 / Published online: 30 May 2010
Ó Springer Science+Business Media, LLC 2010
Abstract A series of novel prodrugs of naproxen has
been synthesized. Naproxen (1) was reacted with thionyl
chloride to yield acid chloride (2) which was further
reacted with glucose to form the glucosyl naproxen (3).
Tetra-acetate of glucosyl naproxen was prepared and
finally reacted with different amino acids to yield the title
compounds. These compounds were evaluated for analge-
sic, anti-inflammatory activities, and for possible GI tox-
icity. Compound 5b depicted most potent analgesic activity
with percentage inhibition of 98.15%. Compound 5a was
found to be most potent anti-inflammatory agent with 76%
inhibition. Compound 5n was second most active analgesic
(92.26%) and anti-inflammatory (73%) agent. In vitro
hydrolysis pattern of synthesized prodrugs was studied in
phosphate buffer of pH 7.4 and acetate buffers of pH 3.0,
4.0, and 5.0, respectively. Selected compounds were
evaluated for their ulcerogenic potential and all the tested
derivatives were significantly less irritating to gastric
mucosa than the parent drug.
Introduction
Naproxen [(?)-6-methoxy-a-methyl-2-naphthaleneacetic
acid] is a nonsteroidal anti-inflammatory drug (NSAID)
with analgesic, antipyretic properties, and is frequently
used for treatment of rheumatoid arthritis and osteoar-
thritis (Rautio et al., 2000; Todd and Clissold, 1990).
Oral administration of this drug leads to formation of
crystals that coat the digestive mucus. Due to acidity and
low solubility, crystals dissolve slowly causing irritation
and damage to the stomach walls which on prolonged use
can lead to the formation of ulcerations in the mucus
(Otero Espinar et al., 1991). Generally, undesired GI
irritation limits clinical utility of most of the conventional
NSAIDs. Moreover, the main causes of NSAID-induced
gastropathy are reduced mucosal cytoprotective prosta-
glandin (PG) levels, increased gastric acidity and motil-
ity. This enhanced gastric motility leads to reduction in
mucosal blood flow, hypoxia, and destruction of mucous
bicarbonate barrier, which prevents back diffusion of
pepsin and hydrogen ions from lumen into the mucosal
layer (Takeuchi, 1989). The GI side effects produced by
NSAIDs are principally caused by two different mecha-
nisms: a direct contact mechanism on the GI mucosa
through oral dose and a generalized systemic action
appearing after intravenous dosing (Bundgaard and
Nielsen, 1988).
Keywords Naproxen ꢀ Prodrugs ꢀ Analgesic activity ꢀ
Anti-inflammatory activity ꢀ In vitro hydrolytic studies
To overcome these undesired characteristics of available
NSAIDs, several prodrugs have been developed and eval-
uated worldwide. Prodrug concept involves chemical
modification of a drug into some bioreversible form in
order to change its pharmaceutical and pharmacokinetic
properties, thus enhancing its delivery (Bonina et al.,
2001). In recent years, much attention has been focused on
the development of prodrugs of NSAIDs so as to reduce
P. C. Sharma (&) ꢀ S. Yadav ꢀ R. Pahwa ꢀ D. Kaushik
Institute of Pharmaceutical Sciences, Kurukshetra University,
Kurukshetra 136119, India
e-mail: sharma_prabodh@rediffmail.com
S. Jain
Department of Pharmaceutical Sciences, Guru Jambheshwar
University of Science and Technology, Hisar 125001, India
123

Med Chem Res (2011) 20:648–655
649
Synthesis of glucosyl naproxen
their GI adversities. The esterification of NSAIDs, how-
ever, results in reduced GI toxicities. The esters being
pharmacologically inactive get readily hydrolyzed fol-
lowed by their absorption to release parent drug in the
blood. These esters should also possess desirable physi-
cochemical properties such as aqueous solubility and
lipophilicity (Bundgaard and Nielsen, 1988).
Naproxen (2 g) 1 was dissolved in benzene (20 ml) in a
100 ml round-bottomed flask. Redistilled thionyl chloride
(2.5 ml) was added drop wise. The reaction was carried out
by heating under reflux for 3 h maintaining anhydrous
conditions to get 2-(6-methoxy-2-naphthyl)-propionyl
chloride 2. Then, anhydrous glucose and pyridine were
added to compound 2 and stirred over night to get glucosyl
naproxen (3).
Various prodrugs of naproxen synthesized in the form of
esters, amides, and glycolamides are reported. (Bundgaard
and Nielsen, 1988; Giammona et al., 1989; Otero Espinar
et al., 1991; Chang and Tsai, 1997; Chang et al., 1998;
Rautio et al., 2000; Bonina et al., 2001; Sheha et al., 2002;
Khan and Khan, 2002; Chang et al., 2004; Swart et al.,
2005; Ranatunge et al., 2006; Bhushan and Tanwar, 2008;
Stefanko et al., 2008). Moreover, conjugation with amino
acid such as L-aspartic acid has also been studied (El-Ka-
mel et al., 2008). Although considerable research has been
carried out on designing prodrugs of naproxen with
reduced GI toxicity, yet no one seems to be an ideal and
hence, there is a need for the development of newer pro-
drugs with better pharmaceutical and pharmacological
profile. Previous studies of b-D-glucopyranosyl derivative
of ibuprofen have demonstrated that the prodrugs possess
increased anti-inflammatory and analgesic activity with
reduction in GI toxicity.
Synthesis of tetracetate
Further, anhydrous zinc chloride (0.5 g) and acetic anhy-
dride (13.5 g; 12.5 ml; 0.13 mol) were placed in a 200 ml
round-bottomed flask. A double surface condenser was
attached and mixture was heated on a water bath until a
clear solution was obtained and to this, compound 3 was
added, shaking the mixture from time to time. Heating was
further continued for around 2 h till a clear solution was
obtained and then the reaction mixture was poured onto
250 ml of crushed ice. It was allowed to stand for 1 h,
stirring occasionally to break up the solid lumps of tetra-
acetate of glucose 4 which were separated, filtered off,
washed well with cold water, and recrystallized from
methanol.
Hence in the light of these findings, the present study
was undertaken in order to minimize the ulcerogenic
potential of naproxen and to render it water soluble prop-
erties through glucosidation and Schiff’s base formation for
better absorption and sustained release characteristics.
Specially designed prodrug was with expected advantages
of affording gastric protection by (1) temporarily masking
the acidic carboxylic group until absorption and (2) inhi-
bition of gastric secretion.
Synthesis of Schiff base of naproxen
For this purpose different amino acids namely glycine,
valine, alanine, cysteine, leucine, threonine, phenylalanine,
tyrosine, histidine, tryptophan, aspartic acid, glutamic acid,
ornithine, and 3,4-dihydroxyphenylalanine (DOPA) were
employed. Appropriate quantity of amino acid (0.005 mol)
was added to the compound 4 after dissolving it in a
solution of water and ethanol and the solution was refluxed
for 2 h. The product was allowed to evaporate till title
compounds 5a–n were obtained.
Experimental
Chemicals and all solvents used in this study were pro-
cured from Merck AG, SD Fines, Rankem labs, and
Qualigens. Distilled water was used in preparation of
buffer solutions. Melting points were determined on a
Labindia MR-VIS visual melting range apparatus and are
uncorrected. The absorbance against wavelength was
taken on a Systronic double beam UV spectrophotometer.
IR spectra (KBr disc) were obtained with a Perkin Elmer
IR spectrophotometer. NMR spectra were recorded using
Bruker 400 spectrometer and chemical shifts are expres-
sed as d (ppm) with tetramethylsilane as internal standard
for solutions in CDCl3. Drug sample was procured as a
gift sample from M/s M. J. Biopharma Private Limited,
Navi Mumbai, India. Mass spectra were recorded on
Waters Q-TOF micro mass spectrometer using electron
spray ionization method.
N-{6-[100-methyl-100-(6-methoxynaphth-1-yl)]ethoxy}-20,
30,40,50-tetraacetylhex-10-yl-iminoethanoic acid 5a. Yield
64%; m.p. 83–86°C. IR (KBr, cm-1): 1749.2 (Carboxylic
C=O str), 1631.5 (C=N str), 1165 (C–O), 1038 (C–O–C
1
str). H-NMR: d (ppm) (CDCl3): 1.51–1.82 (3H, d, CH–
CH3), 2.08–2.18 (12H, s, OCOCH3), 3.65 (3H, s, OCH3),
3.84–4.10 [5H, m, (1H, CH–CH3). (2H, COOCH2) and
(2H, N–CH2)], 4.24 (1H, s, CH–OCOCH3 {C4}), 5.09–
5.20 (3H, m, CH–OCOCH3 {C1–C3}, 6.91–7.46 [7H, m,
(6H, ArH) and (1 H, N=CH)]. MS, m/z: 617.
2-N-{6-[100-methyl-100-(6-methoxynaphth-1-yl)]ethoxy}-
20,30,40,50-tetraacetylhex-10-yl-2-iminopropanoic acid 5b.
Yield 59%; m.p. 76–79°C. IR (KBr, cm-1): 1749.6 (Car-
boxylic C=O str), 1632.8 (C=N str), 1159.9 (C–O), 1039.3
1
(C–O–C str). H-NMR: d (ppm) (CDCl3): 1.55–1.58 (6H,
123

650
Med Chem Res (2011) 20:648–655
m, CH–CH3), 2.01–2.21 (12H, s, OCOCH3), 2.62 (1H, q,
=N–CH), 3.70 (3H, s, OCH3), 3.81–3.91 (1H, q, CH–CH3),
4.12–4.26 [3H, m, (2H, COOCH2) and (1H, CH–OCOCH3
{C4})], 5.13–5.25 [3H, m, (1H, CH–OCOCH3 {C3}) and
(2H, CH–OCOCH3 {C1–C2}], 7.12–7.70 [7H, m, (6H,
ArH) and (1H, N=CH)]. MS, m/z: 631.
{C1–C2})], 6.80–7.51 [7H, m, (6H, ArH) and (1H, d,
N=CH)]. MS, m/z: 661.
3-phenyl-2-N-{6-[100-methyl-100-(6-methoxynaphth-1-yl)]
e t h o x y } -20,30,40,50-tetraacetylhex-10-yl-2-iminopropanoic
acid 5g. Yield 61%; m.p. 76–78°C. IR (KBr, cm-1): 1765.2
(Carboxylic C=O str), 1632.1 (C=N str), 1151.4 (C–O),
1037.5 (C–O–C str). 1H-NMR: d (ppm) (CDCl3): 1.51–1.83
(3H, d, CH–CH3), 2.09–2.31 [13H, m, (12H, –OCOCH3)
and (1H, =N–CH)], 2.63 (2H, m, –CH2–C6H5), 3.98 (3H, s,
OCH3), 4.04–4.27 [4H, m, (1H, CH–CH3), (2H, COOCH2)
and (1H, CH–OCOCH3 {C4})], 5.08–5.18(1H, s, CH–OC-
OCH3 {C3}), 5.12–5.18 (2H, m, CH–OCOCH3 {C1–C2}),
7.21–7.80 [12H, m, (11H, ArH) and (1H, N=CH). MS, m/z:
707.
3-(40-hydroxyphenyl)-2-N-{6-[100-methyl-100-(6-meth-
oxynaphth-1-yl)]ethoxy}-20,30,40,50-tetraacetylhex-10-yl-
2-iminopropanoic acid 5h. Yield 68%; m.p. 75–76°C. IR
(KBr, cm-1): 1755.3 (Carboxylic C=O str), 1632 (C=N
str), 1152.5 (C–O), 1041.7 (C–O–C str). 1H-NMR: d (ppm)
(CDCl3): 1.52–1.80 (3H, d, CH–CH3), 2.11–2.20 (12H, s,
OCOCH3), 2.63 (2H, m, –CH2–C6H4), 4.07–4.13 [7H, m,
(3H, –OCH3), (1H, CH–CH3), (2H, COOCH2) and (1H,
=N–CH)], 4.24–4.30 (1H, s, CH–OCOCH3 {C4}), 5.08–
5.16 [3H, m, (1H, CH–OCOCH3 {C3}) and (2H, CH–
OCOCH3 {C1–C2})], 7.04–7.81 [11H, m, (10H, ArH) and
(1H, N=CH)]. MS, m/z: 723.
3-methyl-2-N-{6-[100-methyl-100-(6-methoxynaphth-1-yl)]
ethoxy}-20,30,40,50-tetraacetylhex-10-yl-iminobutanoic acid
5c.Yield 66%; m.p. 77–78°C. IR (KBr, cm-1): 1755.9
(Carboxylic C=O str), 1632.7 (C=N str), 1163.1 (C–O),
1
1065.4 (C–O–C str). H-NMR: d (ppm) (CDCl3): 1.25–
1.58, 9H, m, [(6H, –CH(CH3)) and (3H, CH–CH3)], 2.01–
2.03 (1H, q, –CH(CH3)), 2.07–2.20 (12H, s, OCOCH3),
3.66 (1H, d, =N–CH), 3.79 (3H, s, OCH3), 3.82–4.22 [3H,
m, (1H, CH–CH3) and (2H, COOCH2)], 4.26–4.29 (1H, s,
CH–OCOCH3 {C4}), 5.07–5.17 [3H, m, (1H, CH–OC-
OCH3 {C3}) and (2H, CH–OCOCH3 {C1–C2})], 7.01–7.72
[(7H, m, (6H, ArH) and (1H, N=CH)]. MS, m/z: 659.
3-sulphanyl-2-N-{6-[100-methyl-100-(6-methoxynaphth-1-
yl)]ethoxy}-20,30,40,50-tetraacetylhex-10-yl-2-iminopropanoic
acid 5d. Yield 61%; m.p. 70–71°C. IR (KBr, cm-1):
1749.2 (Carboxylic C=O str), 1632.7 (C=N str), 1152.5
1
(C–O), 1038 (C–O–C str). H-NMR: d (ppm) (CDCl3):
0.88–0.91 (1H, s, SH), 1.51–1.82 (3H, d, CH–CH3), 2.02–
2.18 (12H, s, OCOCH3), 2.49–2.63 (2H, d, CH–CH2), 3.84
(3H, s, OCH3), 4.11–4.14 (1H, q, CH–CH3), 3.92 (1H, t,
=N–CH), 4.14–4.30 [3H, m, (2H, COOCH2) and (1H, CH–
OCOCH3 {C4})], 5.09–5.17 [3H, m, (1H, CH–OCOCH3
{C3}) and (2H, CH–OCOCH3 {C1–C2})], 6.8–7.6 [7H, m,
(6H, ArH) and (1H, N=CH). MS, m/z: 663.
3-(1H-imidazol-40-yl)-2-N-{6-[100-methyl-100-(6-methox
ynaphth-1-yl)]ethoxy}-20,30,40,50-tetraacetylhex-10-yl-2-imi
nopropanoic acid 5i. Yield 67%; m.p. 81–83°C. IR (KBr,
cm-1): 1749 (Carboxylic C=O str), 1632.1 (C=N str),
1
4-methyl-2-N-{6-[100-methyl-100-(6-methoxynaphth-1-yl)]
ethoxy}-20,30,40,50-tetraacetylhex-10-yl-iminopentanoic acid
5e. Yield 64%; m.p. 75–76°C. IR (KBr, cm-1): 1749.1
(Carboxylic C=O str), 1632 (C=N str), 1160.8 (C–O), 1040
1160.6 (C–O), 1038.1 (C–O–C str). H-NMR: d (ppm)
(CDCl3): 1.44–1.54 (3H, d, CH–CH3), 2.0–2.19 (12H, s,
OCOCH3), 2.58–2.62 (2H, m, –CH2), 3.65 (1H, t, =N–
CH), 3.77 (3H, s, OCH3), 3.81–4.28 [4H, m, (1H, CH–
CH3), (2H, COOCH2) and (1H, CH–OCOCH3 {C4})],
5.05–5.13 [3H, m (1H, CH–OCOCH3 {C3}) and (2H,
CH–OCOCH3 {C1–C2})], 7.14–7.75 [7H, m, (6H, ArH)
and (1H, N=CH)]. MS, m/z: 697.
1
(C–O–C str). H-NMR: d (ppm) (CDCl3): 1.55–1.88 [9H,
m, (6H –CH(CH3)2) and (3H CH–CH3)], 1.95–2.02 [3H, m,
(2H, –CH2–CH) and (1H, –CH(CH3))], 2.08–2.22 (12H, s,
OCOCH3), 3.70 (3H, s, OCH3), 3.76–4.14 [4H, m, (1H,
=N–CH), (1H, CH–CH3) and (2H, COOCH2)], 4.24–4.36
(1H, s, CH–OCOCH3 {C4}), 5.06–5.15 [3H, m, (1H, CH–
OCOCH3 {C3}) and (2H, m, CH–OCOCH3 {C1–C2})],
7.12–7.72 [7H, m, (6H, ArH) and (1H, N=CH)]. MS, m/z:
673.
3-(1H-indol-3-yl)-2-N-{6-[100-methyl-100-(6-methoxyna-
phth-1-yl)]ethoxy}-20,30,40,50-tetraacetylhex-10-yl-2-imino-
propanoic acid 5j. Yield 61%; m.p. 79–80°C. IR (KBr,
cm-1): 1749.1 (Carboxylic C=O str), 1631.8 (C=N str),
1
1160.8 (C–O), 1037.9 (C–O–C str). H-NMR: d (ppm)
3-hydroxy-2-N-{60-[100-methyl-100-(6-methoxynaphth-1-yl)]
ethoxy}-20,30,40,50-tetraacetylhex-10-yl-iminobutanoic acid 5f.
Yield 65%; m.p. 71–72°C. IR (KBr, cm-1): 1756.2 (Car-
boxylic C=O str), 1632.2 (C=N str), 1150.9 (C–O), 1041.5
(CDCl3): 1.58–1.60 (3H, d, CH–CH3), 2.01–2.10 (12H, s,
OCOCH3), 2.63 (2H, d, –CH2), 3.68 (1H, t, =N–CH), 3.76
(3H, s, OCH3), 4.03–4.26 [4H, m, (1H, CH–CH3), (2H,
COOCH2) and (1H, CH–OCOCH3 {C4})], 5.11–5.14 [3H,
m, (1H, CH–OCOCH3 {C3}) and (2H, CH–OCOCH3 {C1–
C2})], 6.92–7.71 [12H, m, (11H, ArH) and 7.26 (1H,
N=CH)]. MS, m/z: 746.
2-N-{6-[100-methyl-100-(6-methoxynaphth-1-yl)]ethoxy}-
20,30,40,50-tetraacetylhex-10-yl-2-iminobutanedioic acid 5k.
Yield 65%; m.p. 75–76°C. IR (KBr, cm-1): 1755.6
1
(C–O–C str). H-NMR: d (ppm) (CDCl3): 1.18–1.25 (1H,
d, –CH–CH3), 1.61 (3H, d, CH–CH3), 2.05–2.19 (12H, s,
OCOCH3), 2.30–2.49 (1H, d, =N–CH), 2.72 (1H, m, CH–
CH3), 3.77 (3H, s, OCH3), 4.07–4.28 [4H, m, (1H, CH–CH3),
(2H, COOCH2) and (1H, CH–OCOCH3 {C4})], 5.09–5.19
[3H, m, (1H, CH–OCOCH3 {C3}) and (2H, CH–OCOCH3
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Med Chem Res (2011) 20:648–655
651
(Carboxylic C=O str), 1632.4 (C=N str), 1151.7 (C–O),
1
4.0 ml of methanol and absorbance was measured at
231 nm (kmax) against a blank obtained by similar treat-
ment (Rautio et al., 1998).
1040.6 (C–O–C str). H-NMR: d (ppm) (CDCl3): 1.51–
1.80 (3H, d, CH–CH3), 2.05–2.20 (12H, s, OCOCH3),
2.34–2.71 [3H, m, (2H, –CH2–C6H6) and (1H, =N–CH)],
3.86 (3H, s, OCH3), 3.96–4.13 [3H, m, (1H, CH–CH3) and
(2H, COOCH2)], 4.19–4.28 (1H, s, CH–OCOCH3 {C4}),
5.08–5.17 [3H, m, (1H, CH–OCOCH3 {C3}) and (2H, CH–
OCOCH3 {C1–C2})], 7.05–7.76 [7H, m, (6H, ArH) and
(1H, N=CH)]. MS, m/z: 675.
Biological evaluation
Naproxen and its prodrugs were administered orally as a
suspension in Tween 80 (0.5% w/v) solutions. Protocol of
the animal experimentation was approved by the IAEC
(Institutional Animal Ethics Committee) as registered
under CPCSEA (Committee for the Purpose of Control and
Supervision of Experiments on Animals), Government of
India.
5-amino-5-oxo-2-N-{6-[100-methyl-100-(6-methoxynaphth-
1-yl)]ethoxy}-20,30,40,50-tetraacetylhex-10-yl-2-iminopen-
tanoic acid 5l. Yield 60%; m.p. 76–77°C. IR (KBr, cm-1):
1752.9 (Carboxylic C=O str), 1632.4 (C=N str), 1151.5
1
(C–O), 1041 (C–O–C str). H-NMR: d (ppm) (CDCl3):
1.03–1.18 (2H, m, –CH2), 1.54–1.72 (3H, d, CH–CH3),
2.03–2.17 [14, m, (2H, –CH2) and (12H, OCOCH3)], 2.31
(1H, t, =N–CH), 3.70 (3H, s, OCH3), 3.81 (1H, q, CH–
CH3), 4.11 (2H, d, COOCH2), 4.25–4.28 (1H, s, CH–OC-
OCH3 {C4}), 5.09–5.19 [3H, m, (1H, CH–OCOCH3 {C3})
and (2H, CH–OCOCH3 {C1–C2})], 6.34 (2H, s, –CONH2),
6.80–7.57 [7H, m, (6H, ArH) and (1H, N=CH)]. MS, m/z:
688.
Analgesic activity
Screening of the synthesized compounds for analgesic
activity was carried out by acetic acid induced writhing
method (Seigmund et al., 1957). The animals were divided
in groups (control, standard and test) consisting of six mice
each having weight 18–22 g. Control group received 0.5%
w/v Tween 80 (10 ml/kg), standard group received
naproxen (50 mg/kg) and test groups received dose
equivalent to molecular weight of the prodrugs 5a–n by
oral route. After 1 h of drug administration, the writhing
response was elicited by the intraperitonieal injection of
(10 ml/kg body weight of 0.6% v/v in normal saline) acetic
acid. Number of writhes for each mice was counted for a
period of 10 min, and the average number of writhes was
determined for each group and the degree of analgesia was
expressed as percentage inhibition calculated according to
the formula:
5-amino-2-N-{6-[100-methyl-100-(6-methoxynaphth-1-yl)]
ethoxy}-20,30,40,50-tetraacetylhex-10-yl-2-iminopentanoic
acid 5m. Yield 67%; m.p. 75–78°C. IR (KBr, cm-1):
1741.3 (Carboxylic C=O str), 1632.4 (C=N str), 1151.9
1
(C–O), 1042.1 (C–O–C str). H-NMR: d (ppm) (CDCl3):
1.58–1.61 (3H, d, CH–CH3), 2.02 (2H, t, –NH2), 2.09–2.18
(12H, s, OCOCH3), 2.34–2.92 (1H, t, =N–CH), 3.86 (3H, s,
OCH3), 3.90–4.30 [4H, m (1H, CH–CH3), (2H, COOCH2)
and (1H, CH–OCOCH3 {C4})], 5.06–5.17 [3H, m, (1H,
CH–OCOCH3 {C3}) and (2H, CH–OCOCH3 {C1–C2})],
7.15–7.84 [7H, m. (6H, ArH) and (1H, N=CH)]. MS, m/z:
646.
3-(3,4-dihydroxyphenyl)-2-N-{6-[100-methyl-100-(6-meth-
oxynaphth-1-yl)]ethoxy}-20,30,40,50-tetraacetylhex-10-yl-2-
iminopropanoic acid 5n. Yield 68%; m.p. 75–76°C. IR
(KBr, cm-1): 1756.4 (Carboxylic C=O str), 1632.4 (C=N
str), 1151.7 (C–O), 1042.9 (C–O–C str). 1H-NMR: d (ppm)
(CDCl3): 1.58–1.88 (3H, d, CH–CH3), 2.02–2.18 (12H, s,
OCOCH3), 2.37–2.41 [3H, m (1H, =N–CH) and (2H,
–CH2–C6H3)], 4.08–4.12 [6H, m, (3H, OCH3), (1H, CH–
CH3) and (2H, COOCH2)] 4.24–4.30 (1H, s, CH–OCOCH3
{C4}), 5.08–5.21 [3H, m, (1H, CH–OCOCH3 {C3}) and
(2H, CH–OCOCH3 {C1–C2})] 7.14–7.78 [7H, m, (6H,
ArH) and (1H, N=CH)]. MS, m/z: 739.
Percentage inhibition of writhing ¼ ð1 ꢁ T=SÞ ꢂ 100
where S and T are the number of writhes in the control and
drug treated group, respectively.
Anti-inflammatory activity
Carrageenan induced rat paw edema method was used to
determine anti-inflammatory activity of the compounds
(Winter et al., 1962). The experiment was carried out on
healthy Wistar rats of either sex weighing between 150 and
200 g. The rats were divided by randomization into dif-
ferent groups consisting of 6 animals in each. The animals
were fasted for 24 h with water ad libitum. Each animal
was injected with carrageenan suspension (0.1 ml, 1% w/v;
s.c.) into the subplantar region of the left hind paw after
30 min of drug administration. The paw volume was
measured after a time interval of 15, 30, 60, 90, and
120 min using a plethysmometer (Ugo Basile, Italy).
Naproxen 1 was given at a dose of 50 mg/kg body weight
and its prodrugs at dose equivalent to 50 mg/kg of
Hydrolysis studies
The rates of chemical hydrolysis of selected prodrugs were
determined in phosphate buffer of pH 7.4 and acetate
buffers of pH 3.0, 4.0, 5.0 at 37°C. Samples (1.0 ml) were
withdrawn at appropriate time intervals and diluted with
123

652
Med Chem Res (2011) 20:648–655
naproxen. Results have been expressed as percentage
inhibition of edema formation, calculated via the formula:
protons (COOCH2) was observed as a doublet at d 3.9
whereas signal for two protons (=N–CH2) was obtained
as a singlet at d 3.9–4.0. Further, the signal of one proton
(N=CH) was found as a doublet at d 7.3. The IR spec-
trum of compound 5b showed characteristic absorption
peak at 3020.7 (Ar CH), 2973.6–2843.3 (Aliphatic CH),
1749.6 (Carboxylic C=O), 1632.5 (C=N), 1159.9 (C–O),
Percentage inhibition of paw edema
¼ 1 ꢁ EDdrug=EDcontrol ꢂ 100
À
Á
where EDdrug and EDcontrol are the edema volume in drug
treated and control groups, respectively.
1
1039.3 (C–O–C). Its H-NMR spectrum, showed doublet
Ulcerogenicity
at d 1.58 for CH–CH3 proton. The two protons (CO-
OCH2) were observed as doublet at d 4.12. The signal for
one proton (N=CH) was seen as doublet at d 7.43–7.45.
The IR spectrum of compound 5c depicted characteristic
absorption peak at 3020.7 (Ar CH), 1755.9 (Carboxylic
C=O), 1632.7 (C=N), 1163.1 (C–O), 1065.4 (C–O–C).
The structure was further confirmed by its 1H-NMR
spectrum, which showed doublet at d 1.25–1.56 CH–CH3
proton. The two protons (COOCH2) were obtained as
doublet at d 4.11–4.24. The signal for one proton
(N=CH) was obtained as doublet at d 7.3. Mass spectral
data of synthesized compounds was also in agreement
with the proposed structures and hence from spectral
analysis, the assigned structures of synthesized com-
pounds were confirmed.
The ulcerogenic potential of naproxen and synthesized
prodrugs was evaluated by the method of Kunchandy et al.
1984. Healthy Wistar rats of either sex weighing between
150 and 200 g were used and divided by randomization
into different groups with six animals in each. All the test
compounds and standard drug in vehicle were administered
orally to rats over a period of seven successive days. All
the rats were fasted for 24 h on 8th day. The animals were
killed with excessive anesthesia. The stomach was
removed, opened along the greater curvature and washed
gently in running tap water. The gastric mucosa of rats was
examined by means of magnifying lens. The ulcer score
was measured. The score of ulcers were as followed:
0.0 = normal colored stomach, 0.5 = pink to red colora-
tion of stomach, 1.0 = spot ulcer, 1.5 = haemorrhagic
streak, 2.0 = number of ulcers\5, 3.0 = number of ulcers
[5, 4.0 = ulcers with bleeding.
In vitro hydrolysis studies
These studies were carried out on six compounds 5a to 5d,
5h, 5j in aqueous buffers to investigate the extent of
hydrolysis of novel prodrugs. In vitro hydrolysis kinetics of
the synthesized prodrugs were evaluated in different
aqueous buffer solution of pH 3.0, 4.0, 5.0, and 7.4. At
constant pH and temperature, the reaction displayed strict
first order kinetics with fair rate constant (Kobs) and a
straight plot was obtained on plotting log concentration of
residual prodrug versus time. The Kobs and corresponding
half-lives (t1/2) for the linear regression equation correlat-
ing the log concentration of residual prodrug versus time
was calculated.
Results and discussion
Synthesis
In the present study, a series of 14 novel compounds has
been synthesized. Scheme 1 illustrates the synthetic route
for the preparation of target compounds. Reaction of
naproxen with glucose to form ester was followed by
acetylation in presence of ZnCl2 as a catalyst. The tetra-
acetate thus formed was reacted with different amino
acids to yield the title compounds. All the synthesized
compounds were characterized by suitable spectral
methods such as IR, NMR, and MS. The IR spectrum of
compounds 5a–n showed absorption peaks at 1,631–
1,632 cm-1 and 1,749–1,765 cm-1 due to C=N and C=O
stretching vibrations. The structure was further supported
To examine the degradation of prodrugs of naproxen in
stomach, pH 3, 4, 5, and 7.4 were selected. The corre-
sponding half-lives in buffer solution are mentioned in
Table 1. In vitro hydrolytic studies revealed that the
compounds depict a remarkable stability under acidic
conditions and this indicates that compounds fulfill the
requirements for oral delivery system of NSAIDs, whereby
the masking group should be acid stable to prevent the
direct contact effects on gastric mucosa. This would pre-
vent the intracellular entrapment of these compounds and
hence leading to local inhibition of the cytoprotective PG
synthesis. Results indicate the possibility of these esters to
survive in GI conditions and enter the circulation as intact
compounds, thereby successfully overcoming the local GI
irritation.
1
by H-NMR spectrum, which showed doublet at d 3.7–
4.24 and d 7.26–7.45 for COOCH2, –CH=N–, respec-
tively. The IR spectrum of compound 5a showed char-
acteristic absorption peak at 3020.7 (Ar CH), 2974
(Aliphatic CH), 1749.2 (Carboxylic C=O), 1631.5 (C=N),
1165 (C–O), 1038 (C–O–C). The structure was further
confirmed by its 1H-NMR spectrum, which showed a
doublet at d 1.5–1.8 CH–CH3 proton. The signal of two
123

Med Chem Res (2011) 20:648–655
653
Scheme 1 Synthetic route of
Naproxen Prodrugs, reagents
and reaction conditions: (a)
SOCl2; (b) Benzene; (c)
CH3
COOH
CH3
COCl
a, b
Glucose; (d) Pyridine; (e)
(CH3CO)2O; (f) ZnCl2; (g)
Amino acid; (h) Ethanol
H3CO
H3CO
2
1
OHC
H
HO
H
OH
H
OHC
H
c, d
OCOCH3
H
OH
CH3
CH3COO
H
OH
H
H
OCOCH3
CH3
COO
OCOCH3
e, f
COO
H3CO
H3CO
3
4
N
R
g, h
CH
H
CH3COO
OCOCH3
H
H
H
OCOCH3
CH3
OCOCH3
COO
H3CO
5 a-n
=N-R
Comp
5a
Comp
=N-R
=N-R
Amino
acid
R
R
Comp
5k
R
Amino acid
Amino acid
H2C
CH
OH
CH2 COOH
Aspartic
acid
COOH
COOH
CH2
Glycine
5f
Threonine
COOH
HC
COOH
CH2 CONH2
COOH
CH2
HC
CH3
CH
Phenylalanine
Glutamic
acid
5b
5c
Valine
5g
5l
CH2
HC
HO
COOH
H3C
CH3
CH2
NH2
CH2
CH2
HC
CH
CH
Alanine
5h
Tyrosine
5m
Ornithine
DOPA
COOH
COOH
CH2
HC
COOH
OH
H
N
OH
SH
COOH
N
H2C
CH
5d
5e
Cysteine
Leucine
5i
5j
Histidine
5n
CH2
HC
H2
C
COOH
CH COOH
H
N
H3C
CH3
CH
H2C
CH
Tryptophan
COOH
HC
COOH
Hydrolytic pattern of the tested compounds indicated that
at pH 3.0, compound 5b demonstrated a swift hydrolysis
with t1/2 of 15 h followed by compound 5h (t1/2 22 h).
Compound 5a depicted least tendency of hydrolysis with a
long t1/2 of 115 h. At pH 4.0 compound 5b and 5d were most
readily hydrolyzed (t1/2 15 h) and at the same pH compound
5c and 5j depicted slowest rate of hydrolysis with t1/2 115 h.
The t1/2 of 50 and 88 h were observed for all the tested
compounds at pH 5.0. Under alkaline conditions (pH 7.4) all
the compounds depicted facile hydrolytic conversion with
t1/2 ranging from 18 to 30 h. In general, time required for
hydrolysis of compounds under acidic condition was longer
as compared to alkaline condition indicating that the
synthesized prodrugs are capable of tolerating acidic
123

654
Med Chem Res (2011) 20:648–655
Table 1 Chemical hydrolysis of prodrugs
Table 3 Ulcerogenic potential of naproxen and its prodrugs (5a–e)
Compound t1/2(h) pH
3.0
t1/2(h) pH
4.0
t1/2(h) pH
5.0
t1/2(h) pH
7.4
Treatment Dose (mg/kg) Average number of ulcer score SEM
Control
–
0.00 0.00
2.7 .0.26*
0.4 0.19D
0.5 0.16D
0.2 0.12D
0.3 0.20D
0.4 0.19D
5a
5b
5c
5d
5h
5j
115
15
44
44
22
88
44
88
77
88
77
50
57
25
22
19
30
18
18
Naproxen 50
15
115
15
5a
5b
5c
5d
5e
62.8
63.2
66.0
66.4
69.4
67
115
Ulcerogenic potential is expressed as average number of ulcer
score SEM
D
* P \ 0.01 as compared to control; P \ 0.01 as compared to
naproxen
environment of stomach. However, swift conversion with
alkaline pH indicates their suitability to act as prodrugs.
All the compounds were evaluated for their anti-
inflammatory activity by carrageenan induced paw edema
method in rats. The derivatives showed highly potent anti-
inflammatory activity. At 120 min, all compounds (except
5f, 5i, and 5k) were more potent than parent drug. Com-
pound 5a was most active compound of the series with
percentage inhibition of 76% as compared to naproxen
(58% inhibition). It was followed by 5n (73%) and 5j
(70%). Compound 5k (45%) was observed to be least
active whereas compound 5f and 5i showed comparable
percentage inhibition of edema of 58% (Table 2). Research
findings also reveal that naproxen and its prodrugs showed
their maximum inhibitory effects at 120 min. In addition,
the synthesized prodrugs protected edema formation in a
Biological evaluation
All the newly synthesized novel compounds 5a–n were
evaluated for analgesic and anti-inflammatory activity. In
acetic acid induced writhing method for evaluation of
peripheral analgesic activity, compound 5b was found to be
most active with percentage protection of 98.15 followed
by 5n (92.26%), 5g (91.24%), 5h (82.03%), and 5m
(82.03%), respectively. Compound 5c and 5f showed
comparable activity with percentage protection of 63.59
and 60.38% as compared to naproxen (61.36%) whereas 5d
(30.41%) was least active compound of the series. The
observations are presented in Table 2.
Table 2 Analgesic and anti-inflammatory activities of synthesized prodrugs (5a–n)
Compound Dose No. of writhing (% inhibition) Edema volume (% inhibition)
30 min
60 min
90 min
120 min
Control
–
36.17 0.6
0.50 0.05
0.62 0.06
0.73 0.05
0.84 0.06
Naproxen
5a
50.0
62.8
63.2
66.0
66.4
67.4
66.2
67.8
69.4
67.0
71.9
67.6
68.9
64.7
69.6
13.67 1.97**(61.36)
13.17 3.5**(63.59)
0.67 0.7**(98.15)
13.17 2.4**(63.59)
25.2 2.6**(30.41)
10.67 3.6**(70.5)
14.33 5.2**(60.38)
3.2 1.7**(91.24)
6.5 2.8**(82.03)
12.33 1.2**(65.91)
9.5 4.8**(73.74)
9.0 5.2**(75.12)
13.0 1.5**(64.06)
6.5 2.9**(82.03)
2.8 1.2**(92.26)
D
0.45 0.04**(10) 0.42 0.05*(32)
0.42 0.04**(42) 0.35 0.05**(58)
0.21 0.02*(58)
0.19 0.04D(62)
0.39 0.03**(37) 0.30 0.04**(58) 0.20 0.04**(76)
0.44 0.06D(29)
5b
0.37 0.07**(49) 0.27 0.06**(67)
0.41 0.03**(43) 0.30 0.03**(64)
0.31 0.03**(57) 0.27 0.02**(67)
0.36 0.03**(32) 0.30 0.03**(64)
0.44 0.02**(39) 0.35 0.03**(58)
0.38 0.04**(47) 0.26 0.03**(69)
0.38 0.08**(47) 0.27 0.08**(67)
0.44 0.05**(39) 0.35 0.05**(58)
0.46 0.04**(36) 0.25 0.06**(70)
5c
0.35 0.02**(30) 0.50 0.02D(19)
0.29 0.05**(42) 0.43 0.03D(30)
5d
5e
0.27 0.02D(46)
0.41 0.03D(18)
0.43 0.02*(30)
0.47 0.03D(24)
5f
5g
0.39 0.03**(22) 0.49 0.03D(13)
5h
0.20 0.05D(60)
0.37 0.05D(26)
0.41 0.04D(18)
0.40 0.05D(20)
0.34 0.04D(32)
0.38 0.03D(24)
0.46 0.08D(25)
0.49 0.05D(13)
0.52 0.04D(16)
0.61 0.05D(1)
0.42 0.03*(32)
0.42 0.03*(32)
5i
5j
5k
0.52 0.04*(28)
0.46 0.05**(45)
5l
0.34 0.03**(53) 0.28 0.04**(66)
0.36 0.04**(50) 0.27 0.04**(67)
5m
5n
0.28 0.04**(44) 0.36 0.03**(41) 0.29 0.04**(50) 0.22 0.04**(73)
** P \ 0.01, * P \ 0.05, P [ 0.05 as compared to control
Results are expressed as mean SEM
123

Med Chem Res (2011) 20:648–655
655
El-kamel AH, Abdel-Aziz AAM, Fatani AJ, El-Subbagh HI (2008)
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dose dependent manner. All tested doses exhibited highly
significant activities as compared to control. Remarkable
reduction was observed in the ulcerogenic potential of
compounds 5a–e when compared to their parent drug
(Table 3). Results indicated that GI toxicity due to direct
contact of the carboxylic group has been protected.
In conclusion, the present study undertakes the synthesis
of 14 novel prodrugs of naproxen. All the newly synthesized
compounds have been screened for their analgesic and anti-
inflammatory activities. Selected compound were also tested
for their ulcerogenic potential and in vitro hydrolysis pat-
tern. From the observations of present investigation, it can
be inferred that these novel prodrugs represent potentially
useful naproxen derivatives for oral administration as they
(1) are stable in aqueous solutions, (2) retain the analgesic
and anti-inflammatory activity of parent acid, and (3) nota-
bly inhibit GI irritation induced by naproxen.
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Acknowledgments M/s M. J. Biopharma Private Limited, Navi
Mumbai, India is duly acknowledged for providing gift sample of
naproxen. The authors also thank Professor Om Prakash, Director,
Institute of Pharmaceutical Sciences, Kurukshetra University,
Kurukshetra-136119, India for providing necessary facilities.
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