INTRODUCTION

Some oxazolinone are reported as potent antibacterial, antifungal, antiviral and anti-inflammatory agent.^1-4 Here we have preparedsome novel 4-((5-oxo-2-phenyloxazol-4(5H)-ylidene)-susbstituted)-phenyl-2-substitutedbenzoate. 1,3-oxazole-5-one based compounds are known to exhibit excellent antimicrobial properties. C₂ and C₄ position of the oxazolone and also N-substituted show antimicrobial activity.

Hence it was thought of interest to synthesize oxazol-5-one derivatives and screen them for anti bacterial and antifungal activity.

MATERIALS AND METHODS:

All the solvents were of LR grade we are obtained from S.D fine, Finar Chem., Loba chemicals. The melting points were determined in open capillaries and were uncorrected. The purity of synthesized compound was confirmed by thin layer chromatography (TLC) silica gel G in developing solvent system of ethyl acetate: n-hexane and the spot visualized in UV light or iodine vapour. UV spectra were recorded on a UV visible spectrophotometer UV-1700 Shimadzu. The IR spectra of all compounds were recorded on FT-IR 8400S Shimadzu spectrophotometer using KBr. The ¹H-NMR was recorded on Bruker Advance II NMR-400MHz instruments using DMSO-d₆ as solvent and Tetra Methyl Silane (TMS) as internal standard, chemical shifts are express in values (parts per million). Splitting patterns are as follows: s (singlet), d (doublet), m (multiplet). Mass spectra was obtained using 2010EV LCMS Shimadzu instrument. Elemental analysis was obtained using Euro EA elemental analyser.

GENERAL PROCEDURE:

General procedure for substituted benzoylglycine [II (a-c)] (Schotten Baumann reaction):

Substituted benzoylchloride (9ml, 0.078mole) was gradually added to a solution of glycine (5g, 0.067mole) in sodium hydroxide solution (10%, 50ml) in a conical flask. After each addition the flask was stoppered and shaken vigorously. The mixture was cooled and acidified with concentrated hydrochloric acid. The crude product separated out and recrystallized with methanol.⁵

General procedure for 2-(4-substitutedphenyl)-4-(4-hydroxybenzylidene)-1,3-oxazol-5(4H)-one [III (a-c)](ErlenmeyerPlöchl Azlactone):

A mixture of p-hydroxybenzaldehyde (13ml, 0.125mol), substituted benzoylglycine (22.5g, 0.125mol), acetic anhydride (35.75ml, 0.75mol) and fresh powdered sodium acetate (10.25g, 0.125mol) was heated in a boiling water bath with constant shaking. After the mixture has become liquid, it was heated for two more hours. Ethylalcohol (50 ml) was slowly added and the mixture shaken and allowed to stand overnight. The separated product was filtered, washed with ice cold alcohol and recrystallized with DMF.⁶

General procedure for4-((5-oxo-2-phenyloxazol-4(5H)-ylidene)-susbstituted)-phenyl-2-substitutedbenzoate [IV(a-j)]

A mixture of 4-(4-hydroxybenzylidene)-2-phenyl-1, 3-oxazol-5(4H)-one (0.01mole) and substituted benzoylchloride (0.01mole) was mixed in pyridine refluxed for 9hrs. After cooling the contents, few drops of concentrated hydrochloric acid was gradually added to precipitate out the product and finally recrystallized with methanol. The reaction was monitored by TLC.

Linezolid Fluconazole

Synthesized Moiety

Structural Modulation of Standard Drugs with Synthesized Moiety:

SCHEME OF SYNTHESIS:

Where R= -H, -Cl, -OCH₃

R₁=-H, -Cl

R₂=-H,-Cl,-OCH_3, -Br

Table-1:Physical and spectral data of synthesized compounds (IIa-IIIc):

Compd. code	Mol. formula	Mol. Wt. (g/mol)	Melting point (^oC)	% yield (%w/w)	R_fvalue	UV (λ_max)	IR (ʋ,cm^-1)
IIa	C₉H₉O₃N	179.17	185-189 (187-188)	76.2	0.55	226 nm	2937.38 (COOH), 1610.45 (CONH)
IIb	C₉H₈ClNO₃	213.62	143-146 (144-146)	70	0.49	235 nm	2840.95(COOH),1633.59(CONH), 1164.28(Ar-pCl)
IIc	C₁₀H₁₁NO₄	209.2	157-159 (155-158)	72	0.47	248 nm	2825.52(COOH),1602.74(CONH),1261.36 (C-O)
IIIa	C₁₆H₁₁NO₃	265.26	200-202	62	0.50	325 nm	3319.26(Ar-O-H), 1743.5(Lactone), 1647.10(-C=C)
IIIb	C₁₆H₁₀ClNO₃	265.26	245-247	59	0.44	378 nm	3303.83(Ar-O-H), 1679.88(Lactone), 1647.10(-C=C), 1166.85(Ar-pCl)
IIIc	C₁₇H₁₃NO₄	295.28	221-223	60	0.45	248 nm	3506.35 (Ar-O-H), 1687.60(Lactone),1650.95(-C=C),1201.57(C-O)

Mobile phase: Ethyl acetate:n-Hexane(1:1)

Table-2: Physical characteristics of synthesized compounds (IVa-IVj):

Comp. code	R	R₁	R₂	Molecular formula	Mol. Wt. (g/mol)	Melting point (°C)	%yield (%w/w)	R_f
IVa	H	Cl	H	C₂₃H₁₄ClNO₄	403.81	279-281	64	0.56
IVb	H	H	Cl	C₂₃H₁₄ClNO₄	403.81	275-277	60	0.57
IVc	H	H	OCH₃	C₂₄H₁₇ClNO₅	399.39	262-264	58	0.53
IVd	H	H	Br	C₂₃H₁₄BrNO₄	448.26	270-272	56	0.51
IVe	Cl	Cl	H	C₂₃H₁₃Cl₂NO₄	438.25	287-289	56	0.44
IVf	Cl	H	Cl	C₂₃H₁₃Cl₂NO₄	438.25	290-294	58	0.46
IVg	Cl	H	OCH₃	C₂₄H₁₆ClNO₅	433.84	265-267	55	0.46
IVh	OCH₃	Cl	H	C₂₄H₁₆ClNO₅	433.84	262-264	55	0.52
IVi	OCH₃	H	Cl	C₂₄H₁₆ClNO₅	433.84	255-257	57	0.58
IVj	OCH₃	H	OCH₃	C₂₅H₁₉ClNO₆	429.42	243-245	55	0.52

Mobile phase: Ethyl acetate:n-Hexane(1:2)

Table-3: Spectral datas of synthesised compounds (IVa-IVj):

Comp. code	UV (λmax,nm)	IR ( υ,cm^-1)	Mass(m/z)	NMR(δ,ppm)
IVa	302	_{1691.46(lactone), 1666.38(-C=C), 1284.50(Ar-COOR-)}	402.9(M), 405(M+2)	δ: 8.910-8.895 (d, 1H, -CH), 7.763-7.755 (q, 2H, Ar-H), 7.656-7.637 (m, 1H, Ar-H), 7.596-7.574 (q, 2H, Ar-H), 7.462-7.457 (m, 6H, Ar-H), 7.033-7.005 (q, 2H,Ar-H)
IVb	305	_{1691.46(lactone), 1677.95(-C=C), 1265.22(Ar-COOR-),1168.78(Ar-pCl)}	403.2(M) 405(M+1)	-
IVc	290	_{1739.67(lactone), 1612.38(-C=C), 1371.29(C-O), 1267.14(Ar-COOR-)}	400.1 (M+1)	-
IVd	320	_{1693.38(lactone), 1668.31(-C=C), 1255.54(Ar-COOR-),1149.50(Ar-pBr)}	449.4 (M+1) 453(M+4)	-
IVe	299	_{1693.38(lactone), 1645.17(-C=C), 1263.29(Ar-COOR-),1176.50(Ar-pCl), 1041.49 (Ar-oCl)}	439.4 (M+1) 452(M+4)	-
IVf	310	_{1645.45(lactone), 1602.74 (-C=C), 1263.29(Ar-COOR-),1149.50(Ar-pCl)}	_{439.7 (M+1) 442 (M+4)}	-
IVg	295	_{1693.38(lactone), 1263.29(Ar-COOR-), 1305.72(C-O), 1149.50(Ar-pCl)}	_{434.5 (M) 437(M+2)}	δ:8.985-9.010 (d, 1H, -CH), 8.179-8.160 (m, 2H, Ar-H), 8.034-7.9570 (m, 2H, Ar-H), 7.954-7.950 (m, 2H, Ar-H), 7.725-7.68 (m, 2H, Ar-H), 7.640-7.610 (m, 2H,Ar-H), 3.771(s, 3H, -OCH₃)
IVh	290	_{1693.38(lactone), 1313.43(C-O), 1265.22(Ar-COOR-),1180.06 (Ar-oCl)}	_{434.5 (M) 437(M+2)}	-
IVi	297	_{1693.38(lactone), 1647.10(-C=C), 1323.08(C-O) 1263.29(Ar-COOR-), 1170.71(Ar-pCl)}	_{434.1 (M+1) 436(M+2)}	-
IVj	280	_{1658.67(lactone), 1602.40(-C=C), 1325.21(C-O) 1238.21(Ar-COOR-)}	_{(M) 429.3}	δ: 8.984-9.006 (d, 1H, -CH), 8.180-8.160 (m, 2H,Ar-H), 8.012-8.032 (d, 2H, Ar-H), 7.957-7.958 (d, 2H, Ar-H), 7.719-7.76 (m, 2H, Ar-H), 7.61-7.64 (m, 2H,Ar-H), 3.775 (s, 3H, -OCH₃)

Table-4: Elemental analysis data of synthesised compound (IVa):

_Compcode	R	R₁	R₂	_{Mol. Formula}	_{% Found(calculated)}
_Compcode	R	R₁	R₂	_{Mol. Formula}	_C	_H	_N	_O	_Cl
_IVa	H	Cl	H	C₂₃H₁₄ClNO₄	_{67.865(68.41)}	_3.671(3.49)	_{3.551 (3.47)}	_{15.667 (15.85)}	_9.011(8.78)

Antibacterial Activity:

The microbiological assay was based upon a comparison of inhibition of growth of microorganisms by measured concentrations of test compounds with that produced by known concentration of a standard antibiotic. Two methods generally employed were turbidometric (tube dilution) method and filter paper disc method. In the turbidometric method inhibition of growth of microbial culture in a uniform dilution of antibiotic in a fluid medium is measured. It was compared with the synthesized compounds. Here the presence or absence of growth was measured. The cylinder plate method depends upon diffusion of antibiotic from a vertical cylinder through a solidified agar layer in a petridish or plate to an extent such that growth of added micro-organisms is prevented entirely in a zone around the cylinder containing solution of the antibiotics. The cup-plate method is simple and measurement of inhibition of microorganisms was also easy. Here we have used this method for antibacterial screening of the test compounds.^7,8

Name of Microorganisms:

Gram +ve microorganisms

Staphylococcus aureus

Bacillus subtilis

Gram -ve microorganism

Escherichia coli

Preparation of medium:

Nutrient agar 2%

Peptone 1%

Beef extract 1%

Sodium chloride 0.5%

Distilled water up to 100ml

All the ingredients were weighed and added to water. This solution was heated on water bath for about one and half-hour till it became clear. This nutrient media was sterilized by autoclave at 121°C at 15psi.

Apparatus:

All the apparatus like petridishes, pipettes, glass rods, test-tubes etc. were properly wrapped with papers and sterilized in hot air oven.

Antibacterial screening method:

Disc Diffusion Method:

· All the Petri dishes were sterilized in oven at 160°C for 1 hour.

· Agar media, borer and test solutions were sterilized in autoclave at 121°C at 15psi.

· Molten sterile agar was poured in sterile petridishes aseptically.

· The agar was allowed to cool and the bacterial suspension was poured into the petridishes aseptically.

· Placing the sterile filter paper discs in the agar plate and solution of the compounds was added by using pipette (0.1ml) in appropriate four quadrants of petridishes aseptically.

· Petridishes were incubated at 37°C for antimicrobial and 24ºC for antifungal for 24 hrs and observed the zone of inhibition.

MIC:

Minimum inhibitory concentration is the lowest concentration of antimicrobial compound found to inhibit the growth of particular test organism. MIC of different antimicrobial compounds is determined by liquid dilution method. MIC of the synthesized compounds was determined by tube dilution techniques. Serial dilution of the substance under examination was placed into culture tubes containing suitable medium and inoculated with the test organism. After incubation, the minimum concentration of test compound that inhibited the growth of the organism was observed.

Antifungal Screening:

Culture:

The synthesized compounds were screened for their antifungal activity against fungi Candida albicans.

Apparatus:

All the apparatus like Petri dishes, pipettes, glass rods, test-tubes etc. were properly wrapped with papers and sterilized in hot air oven.

Preparation of Sabouraud Dextrose Broth:

Enzymatic digest of Casein 5g

Enzymatic digest of Animal Tissue 5g

Dextrose 20g

Final pH 5.6 ±0.2 at 25 °C

Purified water 1000ml

Preparation of standard solution:

The standard drug clotrimazole was dissolved in appropriate quantity of DMF to obtain the concentration range of 100, 250 and 500µg/ml and the zone of inhibition was checked.

Preparation of test solution:

Specified quantity (100mg) of the compound was accurately weighed and dissolved in 100ml of DMF to get the 1000µg/ml stock solution. Further dilution was made to obtain the concentration in the range 750µg/ml, 500µg/ml and 250µg/ml.

Procedure:

30g of the medium was suspended in 1000ml of purified water. The mixture was allowed to boil till it forms a homogeneous solution. The medium was autoclaved at 121°C for 15minutes at 15psi. Media was cooled to the temperature of approximately 40°C temperature and microorganisms were inoculated to the media. 150ml was transferred to petriplates aseptically. Two such plates were prepared for each organism. Plates were allowed to cool for 20 minutes. Here both high and low strength disks were applied for each compound to be tested. The Petridishes were then incubated at 24°C for 24 hours after which zone of inhibition was measured.^7,8

RESULTS AND DISCUSSION:

Screening of antibacterial activity:

Table-5: Zone of Inhibition ofIVa-IVj(Antibacterial):

Compound Code	Concentration (μg/ml)	Zone of inhibition (mm)
		Gram +ve			Gram-ve
		*B.subtilis*	*S.aureus*		*E.coli*
IVa	200	12		13		11
	400	15	16		12
	600	17	18		14
IVb	200	00		00		00
	400	11	13		12
	600	14	17		13
IVc	200	00		00		00
	400	12	10		08
	600	13	15		10
IVd	200	13		14		11
	400	15	16		13
	600	17	18		14
IVe	200	16		15		13
	400	18	16		14
	600	20	18		17
IVf	200	14		16		12
	400	17	18		13
	600	19	21		15
IVg	200	13		14		12
	400	16	15		13
	600	18	19		14
IVh	200	15		13		11
	400	17	16		13
	600	19	18		16
IVi	200	12		12		11
	400	14	15		12
	600	16	18		13
IVj	200	11		10		08
	400	12	13		10
	600	14	15		11
Linezolid	200	27		28		24
	400	29	31		25
	600	31	33		28

MIC of antibacterial screening:

Table-6: MIC of IVa-IVj (Antibacterial):

Minimum Inhibitory Concentrations (μg/ml)
Compound code	Gram +ve		Gram –ve
Compound code	*B.subtilis*	*S.aureus*	*E.coli*
IVa	200	200	200
IVb	250	250	300
IVc	300	250	350
IVd	250	250	300
IVe	100	100	200
IVf	100	100	150
IVg	150	100	150
IVh	200	200	250
IVi	300	300	300
IVj	350	350	300
Linezolid	50	50	50

Histogram of MIC (Antibacterial screening):

Figure-2: MIC of IVa-IVj (Antibacterial)

DISCUSSION:

· All synthesized compounds were screened for antibacterial activity against three microorganisms S.aureus, B.subtilis and E.coli by disc diffusion method at different concentration ranges from 200 to 600µg/ml.

· Among all the synthesized compounds IVe and IVf have shown highest activity against S.aureus and B.subtilis but less than Linezolid (standard drug).Compound IVc gives less activity against B.subtilis and S.aureus.

· Compound IVh shown higher activity while remaining all synthesized compounds is less active against E.coli.

Screening of antifungal activity:

Table-7: Zone of Inhibition of IVa-IVj (Antifungal)

Compound code	Concentration (μg/ml)	Zone of inhibition (mm)
Compound code	Concentration (μg/ml)	*C.albicans*
IVa	250	00
	500	09
	750	11
	1000	13
IVb	250	00
	500	08
	750	10
	1000	12
IVc	250	00
	500	00
	750	08
	1000	10
IVd	250	07
	500	08
	750	09
	1000	11
IVe	250	09
	500	10
	750	12
	1000	14
IVf	250	11
	500	13
	750	14
	1000	16
IVg	250	09
	500	10
	750	11
	1000	13
IVh	250	09
	500	10
	750	11
	1000	13
IVi	250	08
	500	09
	750	11
	1000	13
IVj	250	08
	500	09
	750	10
	1000	11
Fluconazole	250	16
	500	19
	750	20
	1000	22

_M_{IC
of Antifungal Screening}

Table-8: MIC of IVa-IVj (Antifungal)

Minimum Inhibitory Concentrations (μg/ml)
Compoundcode	*C.albicans*
IVa	400
IVb	450
IVc	750
IVd	250
IVe	200
IVf	200
IVg	250
IVh	250
IVi	250
IVj	500
Fluconazole	100

Histogram of MIC (Antifungal screening)

Figure-4: MIC of IVa-IVj (Antifungal)

DISCUSSION:

· All synthesized compounds were screened for antifungal activity against C.albicans by disc diffusion method at different concentration ranges from 250 to 1000µg/ml.

· Among all the compounds IVe and IVf have shown highest activity against C. albicans but less than Fluconazole (standard drug).

· Compound IVc has shown the lowest activity against C.albicans.

CONCLUSION:

· In antibacterial activity and antifungal activity, compounds IVe andIVf found to have better activity against S.aureus, B.subtilis and C.albicans as compared to other synthesized compounds.

· Compound IVc have shown less activity in both antibacterial and antifungal activity.

· Among all the synthesized compounds, compounds with p-chloro phenyl group at second position of oxazolinone ring are found to be more active compared to the p-methoxy phenyl and unsubstituted phenyl group at second position of oxazolinone.

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