INTRODUCTION:

N-aryl anthranilic acid and its derivatives show a wide spectrum of pharmacological activities such as skeletal muscle relaxant¹, anti-inflammatory activity^2-4, anti diabetic activity, antipyretic activity^5,6,antiangiogenic and anti tumor activity⁷, antiproliferative activity⁸, analgesic activity, trichomonoas vaginalis, antibacterial and antifungal activities

N-aryl anthranilic acid nucleus is an effective NSAID, used for skeletal muscle relax tent also pain relief agents. Mefenamic acid, Flufenamic Acid and Glafenine is a non-steroidal anti-inflammatory Agent with analgesic, anti-inflammatory and antipyretic properties. Anthranilic acid is the biochemical precursor to the amino acid tryptophan.Anthranilic acid is vitamin-L is thought to be necessary for lactation in human females that is reason for the “L” Ullmann⁹–type arylamination has been the reaction of choice for the preparation of N-aryl anthranilic acid derivatives since the discovery in 1903¹⁰

Synthesis of N-aryl anthranilic acid derivatives

Para -substituted N-aryl anthranilic acid derivatives were synthesized by refluxing a mixture of O-Chlorobenzoic acid 3.12gm (0.02mol), various P-substituted anilines X gm (0.022mol) cupric oxide (20-25 mg) and anhydrous Potassium carbonate (80-100 mg) was heated under reflux for 7-8 hrs.

Add water the undissolved materials were removed by filtration. Finally the compounds were precipitated with the help of dilute hydrochloric acid, filtered, dried and recrystallized from ethanol (95%) .

ANTI INFLAMMATORY ACTIVITY:^11-14

Male albino rats weighting approximately, 150-200 gm were divided in to 18 groups and each of 6 animals. A mark was made on the hid paw just behind tibiotarsal junction so that every the Paw was dipped in the mercury column up to the fixed mark to ensure constant Paw volume. The paw volume of each animal was measured before the administration of the drug.

The dosage of the drug administered to different groups were as follows

Group –I: A control group received orally 0.2 ml of DMSO

Group – II: The standard group received orally 10mg /kg of body weight of diclofenac sodium.

Table 1: Anti inflammatory activity of various synthesized compounds using Carregenan induced paw odema method

Group	Compound	Dose (mg/kg)	Basal paw volume	Paw volume after the administration (Mean± SEM) (ml)
Group	Compound	Dose (mg/kg)	Basal paw volume	0min	30min	60min	90min	120min
I	Control	0.2ml	0.344±0.042	0.718±0.019	0.732±0.017	0.746±0.009	0.762±0.012	0.780±0.021
II	Standard	10ml	0.312±0.021	0.712±0.014	0.702±0.024	0.550±0.021	0.396±0.011	0.332±0.014
III	Ib	10ml	0.330±0.019	0.730±0.015	0.726±0.050	0.632±0.038	0.516±0.042	0.472±0.040
IV	Id	10ml	0.341±0.022	0.734±0.022	0.721±0.022	0.692±0.046	0.606±0.042	0.522±0.018
V	Ie	10ml	0.314±0.016	0.727±0.020	0.712±0.022	0.662±0.046	0.586±0.042	0.502±0.018
VI	Ig	10ml	0.320±0.022	0.723±0.017	0.710±0.050	0.628±0.038	0.512±0.042	0.452±0.032
VII	Ih	10ml	0.316±0.032	0.731±0.021	0.720±0.042	0.668±0.028	0.602±0.011	0.532±0.010

Table 2: Anti inflammatory activity of various synthesized compounds using Carregenan induced paw odema method

Group	Compound	Dose(mg/kg)	Paw volume after the administration ( %Mean)
Group	Compound	Dose(mg/kg)	0min	30min	60min	90min	120min
I	Control(DMSO)	0.2ml	--	--	--	--	--
II	Standard(Diclofenac sodium)	10ml	4.10%	26.27%	48.03%	57.44%	***
VIII	Ib	10ml	0.82%	15.28%	32.28%	39.49%	**
IX	Id	10ml	1.50%	7.23%	20.47%	33.08%	*
X	Ie	10ml	1.53%	11.26%	23.09%	35.64%	*
XI	Ig	10ml	3..01%	15.82%	32.80%	42.05%	***
XII	Ih	10ml	1.64%	10.46%	20.99%	31.79%	*

* - p<0.05 significant ** - p<0.01 moderate significant *** - p<0.001 highly significant from control

Group –III to VII: The III to VII groups received compound code Ib, Ic, Id, Ie, Ig and Ih drugs respectively. All the above test compounds were dissolved in 0.5ml solution of DMSO and given 30minutes before the commencement of the study. After that 0.1ml of 1% w/v Carrageenan solution in normal saline was injection into the sub plantar tissue of the left final paw of the rat. The volume of the mercury displaced in the plethmograph was measured at 0 min, 30 min, 60min, 120min and 240min. (Table 1 and 2)

ANALGESIC ACTIVITY:^{15, 16}

Albino mice of same sex were divided into twelve groups. Each group consists of three animals. The paw licking of jump response of the animal, when placed on the hot plate maintained at constant temperature of 55oC was considered as basal reaction. The jump response or paw licking was noted. The dosage of drug administered to different groups was as follows.

Group I: A control group received orally 0.5ml of carboxy methyl cellulose (1%w/v)

Group II: The standard group received orally 2.5ml of Diclofenac sodium

Group III to VII: The III to VII groups received compound code Ib, Ic, Id, Ie, Ig and Ih, drugs respectively. The reaction time of animals on the hot plate at 30, 60, 90 and 120mts after drug administration was noted. (Table 3)

Statistical analysis:¹⁷

All the data were presented as mean ± SEM and analyzed by Dunnett’s test and unpaired Students t-test for the possible significant inter relation between the various groups. The value of p<0.01 was considered statistically significant.

ANTI BACTERIAL ACTIVITY:^18-23

Preparation of Test solutions:

For gram positive bacteria: Each compound of 10mg was weighed in sterile boiling tubes, dissolved in DMSO and made up to 10ml. 1ml of the test solution contains 1mg of test compound.

For gram negative bacteria: Each compound of 10mg was weighed in sterile boiling tubes, dissolved in DMSO and made up to 20ml. 1ml of the test solution contains 0.5mg of test compound.

Protocol:

Assay was carried out by disc diffusion method. The method followed was spread plate technique. Prepare Muller Hinton agar medium and sterilized in the autoclave and dispensed 15ml into each Petri-plate. Label with appropriate name of the organisms by sterile technique inoculate each label plate with the respective organism by spread method. Using sterile forceps, place the standard disc of ofloxacin over the agar surface in the Petri-plates. Then the filter paper discs (sterile) of 5mm were soaked in 1ml (1mg/ml) of the test solution and control DMSO solvent. After evaporating the solvent in a sterile atmosphere the drug impregnated discs were placed in Petri-plates. Gently press each disc down with a wooden end of a cotton swap (or) sterile forceps to ensure that the discs adhere to the surface of the agar. The plates were refrigerated for 1hrs to arrest the growth and for easier diffusion of test compounds. Then the plates were removing from refrigerator and incubate all plates in an incubator and inverted position for 24hrs at 37oC. The zones of inhibition were showed on the (Figure-II). The clear zones of inhibition were measured using Hi media zone reader scale. The values are shown in the (Table-4). The zones of test solutions were compared with standard Ofloxacin. (Table 4)

Table 3: Analgesic activity of various synthesized compounds using Hot plate method

Group	Compound	Dose(mg/kg)	Basal reaction Time (sec)	Rectal temperature after the drug administration(mean± SEM)
Group	Compound	Dose(mg/kg)	Basal reaction Time (sec)	0min	30min	60min	90min	120min
I	Control	0.2ml	3.3±0.33	3.3±0.30	3.0±0.28	3.2±0.34	3.3±0.32	3.3±0.30
II	Standard	10ml	3.1±0.30	3.3±0.32	5.7±0.33	7.3±0.30	10.6±0.29	9.6±0.32
III	Ib	10ml	3.0±0.32	3.0±0.31	3.9±0.28	5.4±0.31	7.1±0.33	8.9±0.30
IV	Id	10ml	3.3±0.31	3.3±0.28	3.7±0.30	4.8±0.33	6.2±0.29	6.9±0.32
V	Ie	10ml	3.1±0.30	3.1±0.32	3.5±0.28	4.9±0.31	6.5±0.32	6.7±0.32
VI	Ig	10ml	2.9±0.29	2.9±0.33	4.1±0.32	5.6±0.30	7.8±0.29	9.7±0.31
VII	Ih	10ml	3.2±0.33	3.2±0.30	4.0±0.30	5.1±0.31	6.9±0.32	7.2±0.29

** - p<0.01 significant *** - p<0.001 highly significant from control

Table 4: Anti bacterial activity of N-aryl anthranilic acid derivatives

Compound Code	Organism used			Inference
	Gram positive	Gram Negative
	*Staphylococcus aureus* (1mg/ml)	*Klebsiella aerogenes* (0.5mg/ml)	*E. Coli* (0.5mg/ml)
Control	O mm	Omm	Resistant	A
Standard	16mm	20mm	Resistant	A* and A*
Ia	17mm	16mm	18mm	A*
Ib	20mm	14mm	12mm	A*
Ic	18mm	8mm	12mm	A*
Id	17mm	15mm	9mm	A*
Ie	13mm	14mm	4mm	A*
If	16mm	11mm	10mm	A*
Ig	18mm	6mm	3mm	A*
Ih	14mm	12mm	12mm	A*

Control- DMSO , A – Inactive, Standard- Ofloxacin 10mg/disc, A* -Active

RESULT AND DISCUSSION:

Anti –Inflammatory Activity: The compounds such IIg possess high significant anti-inflammatory activity, because these above mentioned compounds markedly reduce the paw volume than compared to control. The compounds Ib and Ih possess moderate significant reduction of inflammation when compare to the compounds Ig possess least reduction of inflammation but when compared to control it possess significant anti-inflammatory activity. The compounds Id, Ie and Ih possess anti-inflammatory activity significantly.

Analgesic Activity: The compounds such Ib, and Ig possess highly significant analgesic activity Id, Ie and Ih possess significantly activity.

Fig-1

Antibacterial activity: The antibacterial activity of the compounds was evaluated against gram positive organism Staphylococcus aureus and negative organism Klebsiella aerogenes and E. Coli the zone of inhibition was measured as parameter of activity. Ofloxacin 10µg/disc was used as standard compound.

For Staphylococcus aureus: The compounds Ia, Ib, Ic, Id, If and Ig shown higher antibacterial activity than standard ofloxacin, while compounds Ie and Ih shown least activity.

For Klebsiella aerogenes: The compounds, Ia, Ib, Ic, Id, Ie, If, Ig and Ih exhibited activity. For E. coli: The compounds Ia, Ib, Ic and Ih possessed potent activity than the standard ofloxacin, while compounds Id, Ie, If and Ig exhibited good activity.

CONCLUSION:

Test compounds Ig were found to exhibit high anti-inflammatory activity. Test compounds Ib and Ih were found to exhibit moderate anti inflammatory activity. The compounds such Ib, and Ig possess highly significant activity, Id, Ie and Ih possess significantly analgesic activity. Test compounds Ia, Ib, Ic, Id, If and Ig shown high antibacterial activity, while compounds Ie and Ih shown least activity against Staphylococcus aureus. Test compounds Ia, Ib, Ic, Id, Ie, If, Ig and Ih exhibited least activity against Klebsiella aerogenes. Test compounds Ia, Ib, Ic and Ih possessed potent activity, while compounds Id, Ie, If and Ig exhibited good activity against E. coli. As the N-aryl anthranilic acid derivatives possess wide spectrum of antibacterial activities and anti-inflammatory activities, the synthesized compounds lead to a promising tool for extrapolating the biological activities.

Anti–Inflammatory Activity: The compounds such Ig possess high significant anti-inflammatory activity, because these above mentioned compounds markedly reduce the paw volume than compared to control. ***p<0.001 the standard used is Diclofenac sodium. The compounds Id, Ie and Ih possess anti-inflammatory activity significantly.*p<0.05

Analgesic Activity: The compounds such Ib, and Ig possess highly significant analgesic activity Id, Ie and Ih possess significantly activity.*p<0.01 the standard used is Diclofenac sodium.

Antibacterial activity: The antibacterial activity of the compounds was evaluated against gram positive organism Staphylococcus aureus and negative organism Klebsiella aerogenes and E. Coli The zone of inhibition was measured as parameter of activity. Ofloxacin 10µg/disc was used as standard compound.

Staphylococcus aureus: The compounds Ia, Ib, Ic, Id, If and Ig shown higher antibacterial activity than standard Ofloxacin, while compounds Ie and Ih shown least activity.

Klebsiella aerogenes: The compounds, Ia, Ib, Ic, Id, Ie, If, Ig and Ih exhibited activity. E.coli: The compounds Ia, Ib, Ic and Ih possessed potent activity than the standard Ofloxacin, while compounds Id, Ie, If and Ig exhibited good activity.

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Received on 03.11.2009 Modified on 23.01.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 740-743