Open Journal of Medicinal Chemistry, 2013, 3, 87-92 Published Online September 2013 (
Synthesis and Evaluation of Glyceride
Prodrugs of Naproxen
Vivekkumar K. Redasani1*, Sanjay B. Bari2
1R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, India
2H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur, India
Email: *
Received June 14, 2013; revised July 13, 2013; accepted July 28, 2013
Copyright © 2013 Vivekkumar K. Redasani, Sanjay B. Bari. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
The glyceride ester derivatives 6a and 6b were prepared by reacting 1,2,3-trihydroxy propane 1,3-dipalmitate/stearate
with (S)-naproxen as potential prodrugs. The synthesis was achieved successfully with the aid of N,N’-dicyclohexyl-
carbodiimide. These prodrugs were evaluated for anti inflammatory, analgesic and gastroprotective activity. It was
found that prodrugs 6a and 6b showed less irritation to gastric mucosa as indicated by ulcer index. The synthesized
glyceride esters were found to possess good pharmacological profile as shown by results of anti inflammatory and an-
algesic activity. The aqueous studies were performed in order to ensure the release of prodrugs. Both prodrugs were
found to stable at acidic pH while underwent hydrolysis at pH 7.4. These findings suggest that the glyceride prodrugs
6a and 6b might be used as potential biolabile derivatives.
Keywords: Naproxen; Glyceride Prodrugs; Anti-Inflammatory; Analgesic; Gastroprotective; Hydrolysis Kinetics
1. Introduction
The clinical utility of the conventional acidic non-ste-
roidal anti-inflammatory drugs (NSAIDs) continues to be
principally limited by their undesired side effects, par-
ticularly stomach ulceration, bleeding and perforation [1].
The gastric side effects related to the use of NSAIDs are
generally attributed to local and/or systemic mechanisms
[2]. This can be overcome to a considerable extent by
derivatization of the carboxylic function of the NSAIDs
to produce prodrug with adequate stability at the acidic
pH, thus preventing local irritation of the stomach mu-
cosa, and also capable of releasing the parent drug [3].
The utility of glyceride as a promoiety in the design of
prodrugs of carboxylic acids relates to the absorption of
natural triglycerides, thereby increasing stability in the
stomach and thus overall absorption of the drug [4].
Naproxen, ((S)-6-methoxy-α-methyl-2-naphthalene
acetic acid) is one of the most widely used NSAIDs for
relieving arthritic pain. Free carboxylic group of na-
proxen has severe gastrointestinal side effects on oral
administration that restricts its use [5]. To overcome this,
acidic group is temporarily masked by synthesizing
glyceride ester prodrugs, which can pass through the sto-
mach without releasing active drug in significant quantity
and also increase the absorption pertaining to the natu-
ral triglycerides.
Earlier, the glyceride prodrugs of some NSAIDs like
mefenamic acid [4], asprin [6], indomethacin [7], nif-
lumic acid [8], diclofenac [9], ibuprofen [10] and also
biphenyl acetic acid [11] were reported. In the present
study, we have reported the synthesis of glyceride prod-
rugs of (S)-naproxen, their evaluation for pharmacologi-
cal activity and hydrolysis kinetics studies.
2. Materials and Methods
2.1. General Experimental
Naproxen was obtained as gift sample form Cadila
Health Care Ltd., Ahmedabad (India). All other chemi-
cals and reagents were obtained from Loba Chemie Pvt.
Ltd., Mumbai (India). Dihydroxyacetone was procured
from Merck Specialities Pvt. Ltd., Mumbai (India). All
the solvents used were distilled and dried before use as
required. Infra red (FTIR) spectra were recorded using
KBr on FTIR-8400S Shimadzu. 1H NMR spectra record-
ed on Joel-FT-NMR-300MHZ in CDCl3 and mass spec-
tra were recorded on HELWETT PACKARD G180017
*Corresponding author.
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GCD system. The chromatographic system consisted of a
Shimadzu LC-20 AD solvent delivery system, PDA de-
tector with a Qualisil BDS C8 column (200 mm × 4.6
mm 5 µm) using acetonitrile: buffer (90:10 v/v) as mo-
bile phase for determination and flow rate of 1.0 mL/min
with UV detection at 232 nm.
2.2. Synthesis and Characterization of Title
1) Preparation of palmityl steryl chloride (1a and 1b)
Palmitic/steric acid (10 gm) was dissolved in chloro-
form and treated with thionyl chloride (10 mL). The re-
action mixture was refluxed for 4 hrs at 85˚C - 90˚C,
cooled and filtered to remove unreacted acid. Solvent
was evaporated. The oily product was dissolved in abso-
lute ether and evaporated to get corresponding acid chlo-
ride (1a and 1b).
2) Preparation of 1,3-dipalmitoyl-1,3-dihydroxy-pro-
pane-2-one (3a) and 1,3-distearyl-1,3-dihydroxy-propane-
2-one (3b)
Palmitoyl chloride/steryl chloride (20 mmol) was ad-
ded drop wise to reaction media, prepared under stirring
at 5˚C - 10˚C, containing 1,3-hydroxypropane-2-one
[DHA] (2) (90 mmol), pyridine (17 mL) and CHCl3. Re-
actions were stirred at room temperature for 48 h. Water
was added and organic layers were separated. Aqueous
layers were extracted with CHCl3 (3*50 mL). Organic
layers were joined, washed with water and treated with
0.1 N HCl (3*30 mL). The resulting organic layers were
separated, dried over anhydrous sodium sulfate, ltered
and the solvent was removed under reduced pressure.
3) Preparation of 1,3-dipalmitoyl-1,2,3-propanetriol
(4a) and 1,3-distearyl-1,2,3-propanetriol (4b)
Sodium borohydride (53 mmol) was added to reaction
media containing 3a and 3b respectively (20 mmol) in
tetrahydrofurane, benzene and distilled water. Reactions
were kept under stirring at 0˚C - 5˚C, and when presence
of reactants was no longer detected through TLC, ben-
zene (25 mL) and water (100 mL) were added. Organic
layers were separated and aqueous layers extracted with
CHCl3 (25 mL). The combined extracts were washed
with water and treated with 0.1 N HCl (15 mL). The re-
sulting organic layer was separated, dried over anhydrous
sodium sulfate and solvent evaporated under reduced
pressure to get 4a and 4b.
4) Preparation of glyceride prodrug of naproxen (6a)
Naproxen (5) (1 mmol) and 4a/4b (1.1 mmol) added to
reaction media containing 4-DMAP (0.1 mmol) in
CH2Cl2. After homogenization, DCC (0.0013 mol) in
CH2Cl2 (100 mL) was added drop wise, and the mixture
was kept under stirring at room temperature. After 24 h,
the precipitate DCU was ltered and the solvent removed
under reduced pressure to get solid mass (Scheme 1) (6a
and 6b). This was further recrystallized by petroleum
ether and characterized.
6a: [C49H80O7] mp 68˚C - 69˚C, IR (KBr) cm1: 2915,
2854 (C-H), 1740 (C=O ester), 1232 (C-O ester). 1H
NMR (300 MHz, CDCl3): δ (ppm): 0.96 (m, 6H, 2xCH3),
1.29 (m, 24xCH2), 1.58 (s, 3H, CH3), 1.68 (m, 4H,
2xCH2, β to CO), 2.25 (m, 4H, 2xCH2, α to CO), 3.73 (s,
3H, -OCH3), 3.78 (m, 1H, -CH), 4.73 (m, 1H, -CH), 4.32
(m, 4H, 2xCH2), 7.18 - 7.57 (6H, naphthalene). Mass:
(70 eV) m/z 780.
6b: [C53H88O7] mp 104˚C - 105˚C, IR (KBr) cm1:
2924, 2860 (C-H), 1744 (C = O ester), 1237 (C-O ester).
1H NMR (300 MHz, CDCl3): δ (ppm): δ 0.94 (m, 6H,
2xCH3), δ 1.31 (m, 28xCH2), δ 1.56 (s, 3H, CH3), δ 1.68
(m, 4H, 2xCH2, β to CO), δ 2.34 (m, 4H, 2xCH2, α to
CO), δ 3.73 (s, 3H, -OCH3), δ 3.88 (m, 1H, -CH), δ 4.32
(m, 4H, 2xCH2), δ 4.84 (m, 1H, -CH), δ 7.18 - 7.56 (6H,
naphthalene). Mass: (70 eV) m/z 836.
2.3. Pharmacological Screening
Pharmacological activity was done by using Wistar rats
of either sex weighing between 150 - 200 g and Swiss
albino mice weighing between 25 - 35 gm; procured
from animal house of the Institute (RCPIPER/IAEC/
2009-10/14). The paw edema volume was measured with
the help of Ugo Basile Plethysmometer (7140). All re-
sults were expressed as mean ± SEM. Statistical evalua-
tion was performed using analysis of variance followed
by Dunnett test for sub group comparison.
2.3.1. Anti-Inflammatory Acti vi t y
The anti-inflammatory activity was evaluated by carra-
geenan-induced rat paw oedema model [12]. Wistar rats
were divided into four groups of six animals each. Group
I serve as control and received only vehicle (0.5% w/v
CMC). Group II, III and IV received naproxen (10 mg/kg)
and glyceride prodrugs 6a and 6b respectively in dose
molecularly equivalent. All compounds were adminis-
tered through oral gavage. After 30 min of compound
administration, 0.1 mL of 1% carrageenan in normal sa-
line was injected into the sub planter region of left hind
paw and the edema volume was measured before injec-
tion (V0) and at the interval of every hour up to 6 h. The
percentages of swelling inhibition were calculated as:
% Inhibition = {[(Vt V0) control (Vt V0) treated]/
(Vt V0) control} × 100.
V0 and Vt are the average volume in the hind paw of
the rats before and after treatment respectively.
2.3.2. An al gesic Acti vity
Analgesic activity was carried out using acetic acid in-
duced writhing method [13]. Group I served as a control
and received vehicle 1% (v/v) Tween-80 in water at the
dose of 10 mL/kg of body weight while group II, III and
IV received naproxen (10 m/kg), glyceride prodrugs 6a g
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Scheme 1. Scheme of synthesis for title compounds 6a and 6b. DHA: dihydroxyacetone; DCC: N,N’-dicyclohexylcarbidiim-
ide; DMAP: 4-dimethy l amino py ridine; DCM: dichloromethane .
and 6b respectively in the dose molecularly equivalent.
Acetic acid (0.7%) at a dose of 0.1 mL/10g was adminis-
tered intraperitoneally 40 min after oral administration of
the test compounds. After an interval of 10 min, numbers
of writhing were counted for 10 min. Analgesic activity
was measured as percent decrease in writhing in com-
parison to control and calculated as:
Percent inhibition of writhing
1Wt Wc100 .
Where, Wc and Wt are average number of writhing pro-
duced by control and test groups.
2.3.3. Evaluation of Gastroprotective Effect
Gastro protective effect was determined by the reported
method [14]. The animals were given orally 40 mg/kg
body weight of naproxen or molecular equivalent of
glyceride prodrugs 6a and 6b as suspension in 0.5% aca-
cia. The animals were fasted 24 h prior to administration
of each of control, standard and test compounds. The
animals were sacrificed 6 h after administration of drug
and food and water were available ad libitum. The gastric
mucosa was opened, rinse with 5 mL saline and was ex-
amined by means of 4× binocular magnifier. The stom-
achs were carefully examined and ulcers were scored
according to severity. The ulcer index was calculated as
mean for all animals in the group.
2.4. In Vitro Hy d r o l y s i s St u d i e s
The in-vitro aqueous hydrolysis kinetics studies of prod-
rugs 6a and 6b were carried out at pH 7.4. The total
buffer concentration was 20 mmol and constant ionic
strength of 0.5 M for each sample was maintained by
adding KCl. Hydrolysis of prodrugs was initiated by
adding the samples to buffer solution. The mixtures were
equilibrated at 37˚C for 1 h and 100 mg of each sample
was added. The samples were withdrawn at appropriate
time interval (0.5, 1, 2, 3, 4, 5, 6, 7, 8 h), 0.1 mL of solu-
tion was removed and diluted with mobile phase up to 10
mL and 20 µl of this solution was injected for analysis by
HPLC [15]. Pseudo first-order rate constants (Kobs) for
the individual reactions were calculated with the help of
K2.303logtaa x
, Where, “a” is
initial concentration, “x” is the amount of drug hydro-
lyzed and “t” is time in minutes. The corresponding
half-life (t1/2) was then obtained from the equation:
1 2obs
0.693 Kt.
3. Results and Discussion
3.1. Chemistry
Triglycerides being the major constituents of dietary fat
and their absorption involve simple hydrolysis mainly by
pancreatic lipases to monoglycerides and free fatty acids.
These prodrugs, therefore, do not involve the risk of un-
wanted effects after they are hydrolyzed and release pro-
moiety [6].
The synthesis of targeted glyceride esters of naproxen
6a and 6b was achieved successfully using DCC as per
the method reported [16]. DCC proves to be an effective
catalyst for the conversion of carboxylic acid to esters
and amides. It functions by activating the free carboxylic
groups [17]. The purity and confirmation of structures of
the synthesized compounds were confirmed by TLC,
FTIR, 1HNMR and mass spectroscopy. Infrared spectra
showed the characteristics band of C=O stretching
around 1740 cm1 and C-O stretching around 1232 cm1
which confirms the formation of esters. The 1H NMR
spectra of synthesized compounds showed characteristic
chemical shifts, which anticipated their structures. Pres-
ence of parent peak in mass spectra further confirms the
molecular weight.
3.2. Pharmacological Screening
Carrageenan-induced paw oedema is a useful model to
assess the contribution of mediators involved in vascular
changes associated with acute inflammation. The inhibi-
tion of swelling in carrageenan-induced edema in rat paw
is brought by oral administration of drugs. The develop-
ment of edema in the rat hind paw following the injection
of carrageenan has been described as a biphasic event in
which various mediators operate in sequence to produce
this inflammatory response. Prodrugs 6a and 6b demon-
strate better anti-inflammatory activity with percentage
inhibition of 58% and 55% in comparison to 51% for
naproxen when studied up to 6 h. Increased anti-inam-
matory effect observed for glyceride derivatives might be
the result of either better absorption of esters from the
gastrointestinal tract or due to higher selectivity towards
the COX-2 enzyme than the parent drug [18].
The decrease in number of writhing expressed as per-
centage protection by test compounds with reference to
the control for analgesic activity. Glyceride derivatives
showed high value of percentage protection with 75%
and 74% respectively as compared to naproxen with 70%.
The abdominal constriction response induced by acetic
acid is a sensitive procedure to establish peripherally
acting analgesics. Results of analgesic activity of prod-
rugs shows better as compared to parent drug indicated
successful effects of derivatization [19].
The synthesized prodrugs showed less ulcer index
value of 0.91 and 1.33 as compared to 2.83 for naproxen
thus indicates minimized gastrointestinal side effects
obtained by modification to prodrug. This might be due
to inhibition of direct contact of carboxylic group of na-
proxen to gastric mucosa; mainly responsible for gastric
damage. This also supports to the successful masking of
carboxylic group of naproxen when coupled with that of
promoieties thus protect the gastric mucosa from injury
3.3. In Vitro Hy d r o l y s i s St u d i e s
The hydrolysis kinetics studies were carried out in aque-
ous buffer to determine the fate of prodrugs. Under the
experimental conditions the targeted compounds hydro-
lyzed to release the parent drug as evident by HPLC
analysis. At constant pH and temperature the reaction
displayed strict first order kinetics as the Kobs was fairly
constant and a straight plot was obtained on plotting log
concentration of residual prrug v/s time (Figure 1).
Copyright © 2013 SciRes. OJMC
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Figure 1. First order hydrolysis plot of naproxen prodrugs 6a and 6b in phosphate buffer pH 7.4.
Table 1. Biological activity and in vitro hydrolysis studies of glyceride prodrugs.
Biological activity*
Anti-inflammatory activity (% inhibition)a
In vitro
hydrolysis studyb
1 h 2 h 3 h 4 h 5 h 6 h
Ulcer indexaAnalgesic
activity a Kobs t
1/2 (min)
Naproxen 20.39 ± 0.77 32.69 ± 1.29 41.74 ± 0.80 49.60 ± 0.8251.82 ± 0.4551.74 ± 0.432.83 ± 0.1670.70 ± 4.93 - -
6a 26.74 ± 0.39 39.48 ± 0.75 46.88 ± 0.37 52.34 ± 0.4857.00 ± 0.5458.69 ± 0.370.91 ± 0.2075.87 ± 5.76 1.04 × 103664.30
6b 24.04 ± 0.40 30.11 ± 0.37 42.58 ± 0.30 48.75 ± 0.4653.05 ± 0.3855.04 ± 0.501.33 ± 0.2174.15 ± 4.42 8.03 × 104862.48
*Data represented as mean ± SEM, n = 6. aStatistical analysis was performed with ANOVA followed by Dunnett test P < 0.01 with respect to control; bat pH 7.4
and 37˚C.
5. Acknowledgements
The rate constant (Kobs) and the corresponding half-lives
(t1/2) for the respective prodrugs 6a and 6b were calcu-
lated and found to be 664 min and 862 min respectively
(Table 1). The release of drug at pH 7.4 indicates that the
prodrugs were resistant to acidic environment as desired
but would release the parent drug in the system. The ob-
tained values of t1/2 suggests a slow and sustained release
in the body and hence effective for longer duration [4].
The authors are thankful to Cadila Health Care Ltd.,
Ahmedabad (India) for providing gift sample of na-
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