Synthesis and Evaluation of Glyceride Prodrugs of Naproxen

doi:10.4236/ojmc.2013.33011

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Open Journal of Medicinal Chemistry, 2013, 3, 87-92

http://dx.doi.org/10.4236/ojmc.2013.33011 Published Online September 2013 (http://www.scirp.org/journal/ojmc)

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: *[email protected]

Received June 14, 2013; revised July 13, 2013; accepted July 28, 2013

Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

properly cited.

ABSTRACT

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.

V. K. REDASANI, S. B. BARI

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

Compounds

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, ﬁltered

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 ﬁltered 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) cm−1: 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) cm−1:

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

V. K. REDASANI, S. B. BARI

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.

V. K. REDASANI, S. B. BARI

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

equation,





obs

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 cm−1 and C-O stretching around 1232 cm−1

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-inﬂam-

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

evoked.

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).

V. K. REDASANI, S. B. BARI

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

Compounds

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 × 10−3664.30

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 × 10−4862.48

*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-

proxen.

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