INTRODUCTION:

The rapid progress of organic fluorine chemistry since 1950 has been translated as a pathfinder to invent useful biodynamic agents in medicinal and biochemistry. The new generation antibiotics like Norfloxacin, Ciprofloxacin which were incorporated with fluorobenzene moiety proved their efficacy as potent bio active molecules. Now a day’s vast number of compounds with fluorobenzene moiety feature in diverse areas like antibacterial, antifungal, anti-inflammatory, psychoactive agents etc.

The extensive use of antimicrobial drugs resulted in drug resistance that threatens to reverse the miracles of the last half century. Drug-resistant pathogens are a growing menace to all people, regardless of age, gender, or socioeconomic background. They endanger people in affluent, industrial societies, as well as in less developed nations. If we do not act to address the problem of resistance, we may lose quick and reliable treatment of infections that have been a manageable problem in the present scenario.

Drug choice for the treatment of common infections will become increasingly expensive and in some cases, nonexistent. To counteract the resistance produced by microbes there is a need to invent new drugs which are more safe and effective.

Fluorine being the second smallest element next to hydrogen, closely mimics hydrogen in enzyme-receptor interactions. The substitution of fluorine for hydrogen increases lipid solubility which in turn increases the transport and absorption of drug in-vivo. The strong electron withdrawing inductive effect of fluorine influences stability and reactivity of functional groups which may in turn influence the reactivity for neighbouring reaction centres.

Benzamides are an important group of compounds with a wide range of biological activities such as anti tumour^1-3, anti-platelet activity⁴, polymerase inhibitor⁵, Arginine vasopressin V2 receptor agonists⁶, Angiotensin II ( AT2) receptor agonists⁷, Inhibition of PDGFR tyrosine kinase activity⁸, has been reported.

By considering the above facts, in this project, the syntheses of various substituted Fluoro-phenyl benzamide derivatives for better anti-bacterial activity has been carried out and also evaluated for anti-inflammatory activity using bovine serum albumin denaturation model and for anti-oxidant activity.

EXPERIMENTAL:

Melting points were determined by Thiel’s melting point tube (capillary tube method). The melting points were determined and are uncorrected. Infrared spectra (KBr disc) were performed on FTIR-8300 Shimadzu and the frequencies were expressed in cm^-1. 1H NMR spectra were recorded on Bruker-Avance 400 MHz instrument with TMS (0 ppm) as an internal standard. Completion of the reaction and the purity of the compounds were checked on Merck precoated silica gel 60 F-254. Yields were not optimized. Bovine serum albumin (Merck Limited) and other chemicals were of analytical grade. All the solvents and reagents were used without further purification.

Preparation of p-Nitro benzyl chloride (2):

Mix p-Nitro benzoic acid pure [10 g (0.06 moles)] and Phosphorous pentachloride pure [12.6 g (0.06 moles)] in a 500 ml round bottomed flask. Fit the flask with a condenser having calcium chloride guard tube, reflux for few minutes just to melt the mixture and stop the heating. After 15 minutes evaporate the Phosphorous pentachloride by heating at low temperature, and collect p-Nitro benzyl chloride as a yellow solid.

Percentage yield 72.5%, Melting point 74-76°C, TLC Solvent Pet ether: Ethyl acetate 2:1, Rf value 0.342

Preparation of N-(3-chloro-4-fluorophenyl)-4-nitro benzamide (4)⁹

Fluoro chloro aniline [2 g (0.01 moles)] was taken with 20 ml dry benzene and cooled in ice bath with stirring. p-Nitro benzyl chloride [1.4 g (0.01 moles)] mixed with 10ml toluene, added drop wise with stirring which was continued for half an hour after the completion of addition. The reaction mixture refluxed for 4 hour and toluene removed using rotary evaporator. The residue obtained was extracted with petroleum ether which was evaporated to get white solid.

Percentage yield 80%, Melting point 180°C, TLC Solvent Pet ether: Ethyl acetate 2:1 , Rf value 0.97.

Preparation of 4-Amino- N-(3-chloro-4-fluorophenyl)-4-benzamide (5):

N-(3-chloro-4-fluorophenyl)-4-nitro benzamide[1.075 g (0.005 M)] was dissolved in ethanol (50% w/w) in a two necked RB flask and iron [0.86 g (0.05 M)] was added. Stirred vigorously with heating. When started refluxing , dil.HCl [in 10 ml of 50% w/w ethanol] was added slowly drop by drop for 1 hour. After addition stirred for another hour, cooled and added 15% KOH in pure ethanol. Extracted the solution with ethyl acetate, dried on sodium sulphate and evaporate to get the title compound.

Preparation 4-Acetamido-N-(3-chloro-4-fluorophenyl) benzamide (6):

The Reduction compound [1 g (0.01M)] and Acetic anhydride [3ml (0.03 M)] is taken in a 50 ml RBF fitted with a air condenser and reflux for one hour, cool the reaction mixture to room temperature, then it is transferred to ice cooled water. Boil the solution and allowed to cool, filter the solution to get the acetylated compound.

Percentage yield 84 %, Melting point 140-145 °C, TLC Solvent Pet ether: Ethyl acetate 2:1, Rf value 0.563.

Preparation of 4-Acetamido-N-(3-substituted-4- fluorophenyl) benzamides (8a-8e):

4-Acetamido-N-(3-chloro-4-fluorophenyl) benzamide 1 g (1mole), different types of anilines (7a-7e) (1.5 moles), acetonitrile 20ml and 2-3 drops of triethylamine were taken in a RB flask. Refluxed the reaction mixture for 4-5 hours and monitored by TLC, then transferred to ice cooled water and filtered the resultant mixture to get the different derivatives.

4-Acetamido-N-(3-(3-chloro-4-fluorophenylamino)-4 fluorophenyl)benzamide:

IR spectral data (KBr) cm^-1: 3477.77, 3300.31, 3292.60 (NH str.), 1602.90, 1656.21, (C=O str), 1375.29, 1321.28, 1309.71 (C=C Ar str.). ¹HNMR spectral data (δ): 2H doublet at 9.126 δ CONH. 10H multiplet at 5.481 δ – 7.024 δ (Aromatic protons). 3H singlet at 1.109 δ CH₃.

4-Acetamido-N-(4-fluoro-3-(2nitrophenylamino) phenyl) benzamide:

IR spectral data (KBr) cm^-1: 3477.77, 3348.54, 3300.31 (NH str.), 1668.21, 1600.97, (C=O str), 1348.29, 1321.28, 1309.71 (C=C Ar str.).

4-Acetamido-N-(4-fluoro-3-(Pyridin-2-ylamino) phenyl) benzamide:

IR spectral data (KBr) cm^-1: 3446.91, 3329.25, 3211.59 (NH str.), 1681.98, 1654.98, (C=O str), 1396.51, 1369.50, (C=C Ar str.).

4-Acetamido-N-(4-fluoro-3- (-3nitrophenylamino) phenyl)benzamide:

IR spectral data (KBr) cm^-1: 3481.63, 3362.04, 3200.31 (NH str.), 1668.48, 1618.26, (C=O str), 1373.36, 1320.28, 1309.71 (C=C Ar str.), 1263.42 (NO2).

4-Acetamido-N-(4-fluoro-3-(4-nitrophenylamino)phenyl)benzamide:

IR spectral data (KBr) cm^-1: 3300.31, 3198.08, 3178.79 (NH str.), 1668.48, 1645.33, (C=O str), 1373.36, 1350.22, 1321.28 (C=C Ar str.), 1263.42 (NO2).

Preparation of 4-Acetamido-N-(4-fluoro-3-substituted-phenoxy phenyl) benzamide derivatives (10a-10e):

A mixture of 4-Acetamido-N-(3-chloro-4-fluorophenyl) benzamide 0.73g (0.0025moles) and freshly distilled phenols(9a-9e) (6 g) and powdered ammonium carbonate (2 g) was warmed on a steam bath to initiate the reaction. After the initial vigorous reaction was subsided, it was further heated on a water bath for 40-50 min the reaction product was treated with an excess of aqueous NaOH (10%) to remove any residual phenolic compound. The insoluble material was collected, washed thoroughly with water and recrystallised from suitable solvent.

4-Acetamido-N-(4-fluoro-3-phenoxy phenyl) benzamide.

IR spectral data (KBr) cm^-1:

3446.61, 3300.31, 3134.43 (NH str.), 1668.48, 1645.33 (C=O str), 1373.36, 1321.28, 1311.64 (C=C Ar str.),1265.35 (C-O-C str). ¹HNMR spectral data (δ): 2H doublet at 8.781 δ – 8.844 δ CONH. 12H multiplet at 5.875 δ – 6.804 δ (Aromatic protons). 3H singlet 0.903 δ CH3

4-Acetamido-N-(3-(4-chloro phenoxy)-4-fluorophenyl) benzamide:

IR spectral data (KBr) cm^-1: 3443.60, 3300.24, 3198.08 (NH str.), 1668.48, 1645.33 (C=O str), 1373.29, 1321.28, 1311.64 (C=C Ar str.), 1265.35 (C-O-C str).

4-Acetamido-N-(4-fluoro-3-(m-tolyoxy) phenyl) benzamide:

IR spectral data (KBr) cm^-1:

3446.91, 3421.83, 3300.31 (NH str.), 1668.48, 1645.33 (C=O str), 1375.29, 1321.28, 1311.64 (C=C Ar str.), 1263.42 (C-O-C str).

In vitro anti-microbial activity^{10, 11, 12}

Anti-microbial activity is determined based on the in-vitro activity against Staphylococcus aureus (Gram +ve) and Escherchia coli (Gram –ve). The media were cooled to room temperature and inoculated with test organism (20 ml of subculture medium/100 ml assay medium) 30 ml aliquots of inoculated media were distributed into each of petri plates and maintained at room temperature to solidify. The cups (8 mm) were bored using cork borer. The test solution and the standard drugs in two different concentrations viz., (50 mcg/ml and 10 mcg/ml) were placed in the so made cups. The volume of the test and standard solutions added was 0.1 ml using sterile pipettes. All the above operations were carried out in an aseptic area under laminar flow. The petri plates were kept in the refrigerator for 2 hours to allow uniform diffusion of the drug into the agar medium. Later they were taken out from the refrigerator and incubated for 24-36 hours at 37+/-1 °C. After the incubation period was over, the plates were observed for zone of inhibition and were measured using transparent scale or slide calipers, each reading was taken in triplet. The average mean zone of inhibition was listed in the table.

In vitro anti-inflammatory activity¹³

The test compounds were dissolved in minimum amount of Dimethyl formamide (DMF) and diluted with Phosphate buffer (0.2M, pH 7.4). Final concentration of DMF in all solution was 2% test solution (1ml) containing different concentrations of drug were mixed with 1 ml of 1% mM bovine serum albumin in phosphate buffer and incubated at 270 °C for 15 minutes. Denaturation was induced by keeping the reaction mixture at 60 °C in a water bath for 10 min. After cooling, the turbidity was measured at 660nm (Shimadzu UV visible spectrometer). Percentage of denaturation was calculated from the following formula:

% Inhibition = 100(1-Vt/Vc)

Where Vt = absorbance value in test solution.

Vc = absorbance value in control solution.

Antioxidant activity¹⁴

The reaction mixture containing o-phenanthroline (0.5m), ferric chloride (0.2mM) and different type fractions of test compound in a volume of 5 ml was incubated for 15-20 min at ambient temperature. The absorbance at 510nm was measured. In other set, sodium dithionite (0.3mM) was added instead of test samples and the absorbance was taken as equivalent to 100% reduction of all the ferric ions present.

Anti-oxidant activity can be calculated by the following formula:

% Activity = [At /As] × 100

Where,

At = Absorbance by Sodium dithionite (300 μg/ml) at 510 nm.

As = absorbance by standard drug solution at 510 nm.

RESULTS AND DISCUSSION:

By treating 4-Acetamido-N-(3-chloro-4-fluorophenyl) benzamide with various anilines (7(a-e)), various Preparation of 4-Acetamido-N-(3-substituted-4- fluorophenyl) benzamides 8(a-e) were obtained. The compounds were recrystallized with ethyl acetate. By treating with various phenols various -Acetamido-N-(4-fluoro-3-substituted-phenoxy phenyl) benzamide derivatives 10(a-e) were obtained. The compounds were recrystallized with ethyl acetate. All the compounds synthesized were identified and characterized by physical methods like Melting point (Table 1), Thin layer chromatography and spectral methods like IR and ¹HNMR spectra.

All the synthesized compounds were screened for anti-microbial activities against Staphylococcus aureus (gram +ve) and Escherchia coli (gram –ve). All the compounds were found to possess weak to moderate anti-bacterial activity against both micro-organisms when compared to Procaine penicillin (gram +ve) and Streptomycin (gram –ve) (Table 2). Compounds 8a, 8b and 10a have shown moderate activity against Staphylococcus aureus. The synthesized compounds were subjected to in vitro anti-inflammatory activity using bovine serum albumin denaturation model. 8a, 8c and 10a were found to possess good anti-inflammatory activity when compared to Diclofenec sodium (Table 3).

Among the synthesized four compounds 8a, 8b and 10a were subjected for the evaluation of anti- oxidant activity. All the three compounds were found to possess weak anti-oxidant activity (Table 4).

Table-1: Physicochemical properties of the synthesized compounds 8(a-e), 10(a-e)

Comp	R	m.p. (^°C)	% Yield	R_f value
8a	C₆H₄-4-F-3-Cl	170	63.5%	0.42
8b	C₆H₄-2- NO₂	205	73%	0.56
8c	2-yl-C₅H₄N	214	68 %	0.68
8d	C₆H₄-4-NO₂	208	58.3%	0.57
8e	C₆H₄-3- NO₂	194	65.7%	0.76
10a	C₆H₅	240	65.9 %	0.62
10b	C₆H₄-4-Cl	222	59.2%	0.71
10c	C₆H₄-4-CH₃	218	70.3%	0.83
10d	C₆H₄-2-CH₃	230	72.3%	0.67
10e	C₆H₄-3-CH₃	190	59.7%	0.56

Table-2: Antibacterial evaluation data of the synthesized compounds

Compound code	ZONE OF INHIBITION AFTER 24 Hrs. (in mm) &(ACTIVITY INDEX)
	*E.coli*		*S.aureus*
	50 mcg	100 mcg	50 mcg	100 mcg
8a	10 (0.58)	16 (0.64)	12 (0.63)	21 (0.75)
8b	12 (0.70)	19 (0.76)	11 (0.57)	19 (0.67)
8c	8 (0.47)	12 (0.48)	10 (0.52)	17 (0.60)
8d	11 (0.64)	15 (0.60)	14 (0.73)	23 (0.82)
8e	13 (0.76)	21 (0.84)	13 (0.68)	20 (0.71)
10a	14 (0.82)	23 (0.92)	15 (0.78)	24 (0.85)
10b	10 (0.58)	15 (0.60)	12 (0.63)	19 (0.67)
10c	10 (0.58)	17 (0.68)	11 (0.57)	17 (0.60)
10d	11 (0.64	18 (0.72)	13 (0.68)	19 (0.67)
10e	8 (0.47)	13 (0.52)	14 (0.73)	22 (0.78)
Procaine penicillin	17	25	-------	---------
Streptomycin	-------	-------	19	28

Table 3: Anti inflammatory activity

Sl.No	Compound Code	Absorbance value	Inhibition of Denaturation (%)
1	Control	0.040
2	8a	0.032	80.00
3	8b	0.026	65.00
4	8c	0.029	72.50
5	8d	0.021	52.50
6	8e	0.027	67.50
7	10a	0.031	77.50
8	10b	0.028	70.00
9	10c	0.016	40.00
10	10d	0.013	32.50
11	10e	0.020	50.00
14	Diclofenec Sodium	0.036	90.00

Table 4: Anti oxidant activity

Sr. No.	Sample Code with Concentration (μg/ml)	Absorbance At 510 nm (As)	Anti-Oxidant Activity (%)
01.	8a₁₀₀	0.038	5.82%
02.	8a₂₀₀	0.040	6.15%
03.	8a₃₀₀	0.036	5.53%
04.	8a₄₀₀	0.052	8.00 %
05.	8a₅₀₀	0.075	11.53%
06.	8b₁₀₀	0.019	2.92%
07.	8b₂₀₀	0.026	4.0%
08.	8b₃₀₀	0.031	4.76%
09.	8b₄₀₀	0.029	4.46%
10.	8b₅₀₀	0.041	6.30%
11.	10a₁₀₀	0.029	4.46%
12.	10a₂₀₀	0.035	5.38%
13.	10a₃₀₀	0.051	7.84%
14.	10a₄₀₀	0.068	10.46%
15.	10a₅₀₀	0.79	12.15%

CONCLUSION:

A series of targeted compounds were synthesized by the two schemes and all derivatives were identified and characterized. Compounds 8a, 8b and 10a have shown moderate activity against Staphylococcus aureus. Compounds 8a, 8c and 10a were found to possess good anti-inflammatory activity when compared to Diclofenec sodium. The study regarding anti oxidant activity shown that synthesised compounds are not anti-oxidants.

ACKNOWLEDGEMENT:

Authors want to express their thanks to Shushrutha Educational Trust’s, Nargund College of Pharmacy, Dattatreya Nagar, BSK III stage, Bangalore-560085, Karnataka, India.

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Received on 21.10.2013 Modified on 25.11.2013

Asian J. Research Chem. 7(2): February 2014; Page 132-136

INTRODUCTION:

2 Penning TD, et al. Discovery and SAR of 2-(1-propylpiperidin-4-yl)-1H-benzimidazole-4-arboxamide: A potent inhibitor of poly(ADP-ribose) polymerase (PARP) for the treatment of cancer. Bioorganic & Medicinal Chemistry. 16 (14); 2008: 6965–6975.

3 Andrianov V,et al. Novel amide derivatives as inhibitors of histone deacetylase: Design, synthesis and SAR. European Journal of Medicinal Chemistry. 44(3); 2009: 1067-1085.

6 Tsukamoto I, et al. Preparation of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetamide derivatives as novel arginine vasopressin V2 receptor agonists. Bioorganic & Medicinal Chemistry. 16(21); 2008: 9524–9535.

6 Tsukamoto I, et al. Preparation of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetamide derivatives as novel arginine vasopressin V₂ receptor agonists. Bioorganic & Medicinal Chemistry. 16(21); 2008: 9524–9535.