INTRODUCTION:

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most common pharmacological group that has three primary therapeutic effects including anti-inflammatory, anti-pyrexia, and analgesia. Currently, available NSAIDs like, ibuprofen, flurbiprofen, fenbufen and naproxen exhibit gastric toxicity. Long-term use of these drugs has been associated with gastro-intestinal (GI) ulceration, bleeding and nephrotoxicity¹. The GI damage from NSAIDs is generally attributed to two factors, i.e. local irritation by the carboxylic acid moiety, common to most NSAIDs (topical effect) and decreased tissue prostaglandin production, which undermines the physiological role of cytoprotective prostaglandins in maintaining GI health and homeostasis^2,3. The pharmacological activity of NSAIDs is related to the suppression of prostaglandin biosynthesis from arachidonic acid by inhibiting cyclooxygenases (COXs)⁴. The chronic use of NSAIDs including naproxen may elicit appreciable GI toxicity⁵. Naproxen is one of the most important NSAID which is an orally administered which effect on a variety of inflammatory mediators. It is an effective analgesic and anti-inflammatory agent with a good tolerability profile.

It is absorbed from the gastrointestinal tract, but like other NSAIDs it has some side effects like it affect GI system⁶, dyspepsia and low bioavailability⁷. These side effects can be minimized by “Prodrug approach”. The results from the literature survey indicated that prodrugs have good chemical stability towards hydrolysis and more bioavailability⁸. Considerable research has been directed at designing prodrugs of NSAIDs with the aim of reducing their gastrointestinal toxicity. Therefore synthetic approaches based upon NSAIDs chemical modification has been taken with the aim of improving NSAID safety profile and pharmacological activity of the drug.

MATERIAL AND METHODS:

All the chemicals were of analytical grade: naproxen, substituted aniline, dry redistilled pyridine, silicon tetrachloride. Melting points were determined by open capillary method and are uncorrected. The purity of the compounds was monitored by thin layer chromatography (TLC) using silica gel G plates. The spots were visualized under UV light and by the exposure to iodine vapors. The homogeneity of the compounds were checked on silica gel-G coated plate by using Chloroform: Methanol (7:3) as solvent. All IR spectra were recorded in Alpha Bruker using ATR method. ¹H NMR spectra were recorded on Bruker spectrophotometer (400 MHz) in DMSO-d₆ solvent using tetra methyl silane (TMS) as an internal standard.. Mass spectra was recorded by LCMS method.

General Procedure:

Synthesis of Substituted 2-(6-methoxynaphthalen-2-yl)-N-phenylpropanamide⁹ (NAJ1-NAJ8)

A mixture of naproxen (0.01 mol) and different substituted amines (0.01 mol) was refluxed for 24 hours with stirring in 10 ml of dry redistilled pyridine and 5-6 drops of silicon tetrachloride. It was then cooled and poured into ice cold water with stirring. The product obtained was filtered, washed with water and recrystallised from ethanol.

Figure 1: Reaction scheme for carboxamide derivatives of naproxen

Spectral data

2-(6-methoxynaphthalen-2-yl)-N-phenylpropanamide (NAJ1)

IR KBr (cm^-1): 1506(Ar C=C str), 829 (Ar C-H bend), 3036 (Ar C-H str), 3163 (NH str 2˚amide), 1223 (C-O str).

¹H NMR (400 MHz, DMSO-d₆): δ 7.18-7.90 (m, 11H, Ar-H), 7.23 (s, 1H, -NH), 3.81 (s, 3H, OCH₃), 1.34 (s, 3H, CH₃).

MS (M⁺): m/z 305.

N-(3-Nitrophenyl)-2-(6-methoxynaphthalen-2-yl)propanamide (NAJ4)

IR KBr (cm^-1): 1506(Ar C=C str), 829 (Ar C-H bend), 3036 (Ar C-H str), 3163 (NH str 2˚amide), 1223 (C-O str), 1360 (Ar-NO₂ C-H str).

¹H NMR (400 MHz, DMSO-d₆): δ 7.18-8.72 (m, 10H, Ar-H), 7.23 (s, 1H, -NH), 3.81 (s, 3H, OCH₃), 1.34 (s, 3H, CH₃).

MS (M⁺): m/z 350.

N-(3-Methylphenyl)-2-(6-methoxynaphthalen-2-yl)propanamide (NAJ7)

IR KBr (cm^-1): 1506(Ar C=C str), 829 (Ar C-H bend), 3036 (Ar C-H str), 3163 (NH str 2˚amide), 1223 (C-O str), 1377 (Ar-CH₃str).

¹H NMR (400 MHz, DMSO-d₆): δ 7.12-7.92 (m, 10H, Ar-H), 7.23 (s, 1H, -NH), 3.81 (s, 3H, OCH₃), 1.34 (s, 3H, CH₃), 2.35 (s, 3H, Ar-CH₃),.

MS (M⁺): m/z 319.

Pharmacological screening

Acute toxicity studies

The preliminary pharmacological studies were conducted to assess the acute pharmacological effects and LD₅₀ of the drug. To assess the safety of the synthesized compounds under study acute toxicity studies were carried out as per OECD guidelines 425¹⁰ in healthy adult Wistar rats by "up and down" method. The synthesized naproxen derivatives were given to rats in the form of suspension at a dose of 200 mg/kg body weight by oral route. The standard drug Naproxen was given to rats at a dose of 200 mg/kg body weight by oral route.

Anti-inflammatory activity

The anti-inflammatory activity of the test compounds was carried out using carrageenan-induced rat paw edema¹¹ model according to Winter et al. by employing 1% Carrageenan solution as phlogistic agent. Edema was induced in the left hind paw of Wistar rats (150-200 g) of either sex by the sub-plantar injection of 0.1 ml of 1% Carrageenan in distilled water. Each group composed of six animals. The animals which were bred in our laboratory were housed under standard conditions and received a diet of commercial food pellets and water ad libitum during the maintenance but they were entirely fasted during the experiment period. Our studies were conducted in accordance with recognized guidelines on animal experimentation.

The test compounds were given intraperitoneally 30 min after Carrageenan injection. Naproxen was taken as the standard at a dose of 13.5 mg/kg body weight (p.o). The rat paw volume was measured after 1hr, 2hr, 3hr and 4hrs respectively after Carrageenan injection by using Plethysmometer. The difference between the paw volume at 4 hr and 0 hr measurement was calculated and taken as edema volume. Percentage inhibition in the paw edema was calculated by using the formula,

% Edema inhibition= 100(1-V_t/V_c), where V_trepresents mean increase in paw volume of test and V_c represents mean increase in paw volume of control.

Statistical analysis

All experimental groups were composed of six animals. Data obtained from animal experiments were expressed as mean ± SEM. The statistical significance of difference between groups were assessed by means of analysis of variance (ANOVA) followed by Dunnet's test.

Analgesic Activity

The analgesic activity of the test compounds was carried out using acetic acid by writhing method¹². Pain is produced by injection of acetic acid into peritoneal cavity of mice. The animals react with characteristic stretching behavior, which is called writhing. In this method male albino mice weighing between 20-25 g body weights were selected for the study. The animals were divided into 9 groups of six animals each. All animals received 0.1 ml acetic acid 0.6% v/v i.p and first group served as control. Second group served as standard and received naproxen. The remaining groups of animals received 300 mg/kg body weight of naproxen derivatives,30 min prior to the administration of acetic acid injection. The writhing effect was indicated by the stretching of abdomen with simultaneous stretching of at least one hind limb. This was observed for 30 min and change in number of writhings in test group compared with standard and control treated groups. The percentage inhibition was calculated by using the formula.

% protection = 1 - [1 - R_t ÷ Rc ] X 100

Rt = Mean number of writhings in treated group

Rc = Mean number of writhings in control group

Gastric Irritancy Test¹³

Rats were kept for fasting for 48 hours before the experiment. On the day of experiment rats received orally either vehicle or naproxen and derivatives (clinically recommended dose computed for rats). Four hours after administration of the drug, animals were sacrificed by cervical dislocation and their abdomen opened. The stomachs were isolated and opened along the lesser curvature. The contents were washed under thin running water. The stomach were stretched over a frog board. With the help of a magnifying lens and an mm scale, length of each ulcer or lesion was measured. By totaling all lesions in a stomach the gastric irritancy size was estimated. Total numbers of ulcer per stomach were counted. Then calculated production of ulcer size and ulcer number and called it as gastric irritancy index (G.I.I). Then ulcerogenic potential was calculated using the formula.

Ulcerogenic potential = MGII of drug – MGII of control

MGII of control

Statistical Analysis:

The results were expressed as mean ± SEM. The total variation present in the data was analyzed by one way analysis of variance (ANOVA) followed by Dunnett’s test.

RESULTS:

Table 1: Physicochemical data of the compounds NAJ1-NAJ8

Comp. code	R	Mol. formula	Mol. wt	M.P ^oC	R_f Value	% Yield
NAJ-1	H	C₂₀H₁₉NO₂	305	195-196	0.68	82
NAJ-2	3-Cl	C₂₀H₁₈ClNO₂	339	198-199	0.58	76
NAJ-3	4-Cl	C₂₀H₁₈ClNO₂	339	205-206	0.60	72
NAJ-4	3-NO₂	C₂₀H₁₈N₂O₄	350	170-172	0.52	78
NAJ-5	4-NO₂	C₂₀H₁₈N₂O₄	350	187-188	0.56	80
NAJ-6	2-CH₃	C₂₁H₂₁NO₂	319	210-212	0.78	75
NAJ-7	3-CH₃	C₂₁H₂₁NO₂	319	218-220	0.70	62
NAJ-8	4-OCH₃	C₂₁H₂₁NO₃	335	202-204	0.62	65

Table 2: Anti-inflammatory effect of Carboxamide derivatives of Naproxen (NAJ1-NAJ8) using Carrageenin induced paw edema in rats.

Treatment	Dose mg/kg	Change in paw volume in ml after 4 hr	% inhibition
Control	-	0.330 ± 0.004
Naproxen	13.5	0.240 ± 0.004	27.27
NAJ1	200	0.230 ± 0.004*	30.30
NAJ2	200	0.260 ± 0.004	21.21
NAJ3	200	0.220 ± 0.004**	33.33
NAJ4	200	0.210 ± 0.003**	36.63
NAJ5	200	0.267 ± 0.004	19.09
NAJ6	200	0.250 ± 0.004	24.24
NAJ7	200	0.258 ± 0.004	21.81
NAJ8	200	0.235 ± 0.004*	28.78

All values are expressed as mean ± SEM (n = 6). *P < 0.05 significant compared to control. **P < 0.01 significant compared to control.

Table 3: Analgesic activity of Carboxamide derivatives of Naproxen (NAJ1-NAJ8) using writhing method in mice.

Treatment	Dose mg/kg	Mean wriths in 20 min	% Protection
Control	-	36.00 ± 0.57
Naproxen	150	11.00 ± 0.57	30.55
NAJ1	150	10.50 ± 0.57	29.16
NAJ2	150	11.30 ± 0.56	31.3
NAJ3	150	10.00 ± 0.57	27.77
NAJ4	150	11.00 ± 0.57	30.55
NAJ5	150	11.50 ± 0.57	31.9
NAJ6	150	10.30 ± 0.57	28.61
NAJ7	150	9.70 ± 0.57	26.94
NAJ8	150	11.80 ± 0.57	32.77

Table 4: Gastric irritancy test of Carboxamide derivatives of Naproxen (NAJ1-NAJ8).

Treatment	Dose mg/kg	Mean ulcer number	Mean gastric irritation size (mm) (MGIS)	Mean gastric irritation Index (MGII)	Ulcerogenic Potential
Control	-	3	0.2	0.6
Naproxen	150	6	0.4	2.4	3
NAJ1	150	5	0.35	1.9	2.16
NAJ2	150	4.5	0.3	1.35	1.25
NAJ3	150	5.5	0.38	2.1	2.5
NAJ4	150	3.5	0.26	0.91	0.52
NAJ5	150	4.0	0.28	1.12	0.86
NAJ6	150	4.8	0.31	1.7	1.83
NAJ7	150	3.7	0.27	0.98	0.63
NAJ8	150	5.2	0.37	2.0	2.33

DISCUSSION:

Carboxamide derivatives of naproxen was synthesized by treating naproxen with different substituted anilines in the presence of dry redistilled pyridine and silicon tetrachloride as coupling reagent. The use of silicon tetrachloride as dehydrating agent for amide formation appears to be quite feasible from theoretical consideration. Halosilanes are known to react exothermically with carboxylic acid and amine to form acyloxy and aminosilanes respectively together with the evolution of hydrogen chloride gas. The percentage yield of the synthesized carboxamide derivatives were obtained in the range of 62-82%. The physicochemical data is presented in Table 1.

Anti-inflammatory activity

All the synthesized compounds were tested for their anti-inflammatory activity using Carrageenan induced rat hind paw edema method. Data of anti-inflammatory activity was expressed as mean ± SEM, and the student’s t-test was applied to determine the significance of the difference between the control group and rats treated with the test compounds. The anti-inflammatory activity of the newly synthesised compounds NAJ1-NAJ8 was compared with the standard naproxen 200 mg/kg body weight, showing 27.27% inhibition of rat paw edema whereas tested compounds showed inhibition ranging from 19.09 to 36.63% after 120 min. The amide prodrugs of naproxen NAJ1, NAJ3, NAJ4 and NAJ8 showed significant anti-inflammatory activity compared to the standard drug naproxen. There is however little but a marked change observed in anti-inflammatory activity of carboxamide derivatives than that of parent drug naproxen. The results of the anti-inflammatory activity are summarized in Table 3.

Analgesic activity

The studies of analgesic activity showed that compounds, NAJ2, NAJ5 and NAJ8 has slightly higher analgesic activity than the parent drug naproxen and compound NAJ4 is found to be equipotent to that of naproxen whereas the compounds NAJ1, NAJ3, NAJ5 and NAJ7 are having slightly less analgesic activity than that of naproxen. There is however, no remarkable change in analgesic activities of the derivatives than the parent drug. The results of the analgesic activity are summarized in Table 3.

Gastric irritation test

Studies on gastric irritation indicated that naproxen also show ulcerogenic activity, among all the compounds tested, Naproxen has shown maximum number of ulcers in each animal and its ulcerogenic potential is 3. The carboxamide derivatives of naproxen however caused less number of ulcers in animals and their ulcerogenic potentials are less than that of naproxen. Hence the derivatives of naproxen showed better ulcerogenic potential than the standard naproxen. The results of the gastric irritation test are summarized in Table 4.

CONCLUSION:

The above results proved that novel carboxamide derivatives synthesized from naproxen are found to be interesting lead molecules as analgesic and anti-inflammatory agents and showed better ulcerogenic potential. The study reports the successful synthesis of carboxamide derivatives of naproxen with moderate yields. It can be assumed that derivatives of naproxen are absorbed intact. Hence, it appears that gastrointestinal irritation produced by these compounds probably arises from systemic inhibition of prostaglandin synthesis, due to the conversion of derivatives to naproxen following the absorption from gastro intestinal tract. Thus, the results of this study strongly support the existence of a direct contact mechanism and a systemic action of gastro intestinal irritation. Thus the formation of amide prodrugs of naproxen represent a potentially useful method to decrease gastro intestinal side effects without altering the pharmacological profile of the drug.

ACKNOWLEDGEMENTS:

The authors are thankful to Divis laboratory, Hyderabad for the gift sample (Naproxen) and Nitte University for providing the necessary facilities to carry out this research. The authors are grateful to Sequent Research Ltd, Mangalore and Central Instrumentation Facility, MIT Manipal for providing spectroscopic data.

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Received on 17.03.2014 Modified on 01.05.2014

Research J. Pharm. and Tech. 7(6): June, 2014; Page 631-634