Synthesis and Evaluation of Antibacterial Activity of 1, 4-Benzoxazine Analogues

 

Naveen Kadian*, Meenaxi Maste and A.R. Bhat

Department of Pharmaceutical Chemistry, KLEU’s College of Pharmacy, Belgaum, Karnataka, India.

*Corresponding Author E-mail: naveen20.a@gmail.com ,

 

Compound .{(3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)acetic acid.} and .{(6-chloro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)acetic acid.} was synthesized by reacting O-Amino Phenol and Maleic Anhydride. Futher eight Mannich Bases were synthesized by using substituted Aromatic Amines. The structures of Synthesized compounds were confirmed on basis of IR, NMR and Mass Spectra. All synthesized compounds were screened for their Antibacterial Activity against strains viz . E. coli, Staphylococcus aureus, Bacillus sps, Pseudomonas , K.Pneumonea and E. Faecalis.  Compounds [3-oxo-4-{[(4-(N-(2-pyridyl)sulfamoyl)phenyl) amino]methyl}-3,4-dihydro-2H-1,4-benaoxazin-2-yl] acetic acid,  [3-oxo-4-{[(4-(N-(5-methyl-1,2-oxazol-3-yl)sulfamoyl) phenyl) amino]methyl}-3,4-dihydro-2H-1,4-benaoxazin-2-yl] acetic acid, [6-chloro-3-oxo-4-{[4-(N-(2-pyridyl)sulfamoylmethyl)phenyl]amino} methyl}-3,4-dihydro-2H-1,4-benzoxazin 2-yl]acetic acid,[6-chloro-3-oxo-4-{[(4-(N-(5-methyl-1,2-oxazol-3-yl) sulfamoylmethyl)phenyl)amino]methyl}-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid showed good activity against the K .pneumonea and E. faecalis, compounds [3-oxo-4-{[(4-(N-(5-methyl-1,2-oxazol-3-yl)sulfamoyl) phenyl) amino]methyl}-3,4-dihydro-2H-1,4-benaoxazin-2-yl] acetic acid, [6-chloro-3-oxo-4-{[(4-(N-(5-methyl-1,2-oxazol-3-yl) sulfamoylmethyl)phenyl)amino]methyl}-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid showed good activity against the S.aureas, while other compound showed moderate activity against E. coli, klebeseila , Staphy. aureus, E. fecalis. While other derivative shown moderate antibacterial activity.

 

 

 

INTRODUCTION:

1,4 Benzoxazines have been extensively investigated for their pharmacological activity¹. Literature Survey revel that 1,4 Benzoxazine comprimises a important moiety and their pharmacological activity reported as antibacterial, Antiemetic, Antidepresant COX-2 inhibitor and Antinflammatory Effect.^2-7 The proposed work was aimed to synthesize  [3-oxo-4-({[4(sulfamoylmethyl)phenyl]amino}methyl)-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid using O-Aminophenol and Maleic Anhydride and  [6-chloro-3-oxo-4-{[4-(sulfamoylmethyl)phenyl]amino}methyl}-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid using 4-Chloro2-Aminophenol. Further Mannich Base condensation of these moieties along with Aromatic amines has been carried out.

 

Antibacterial Activity⁸:

All synthesized compounds were screened for their Antibacterial Activity against strains viz. E. coli, Staphylococcus aureus, Bacillus sps, Pseudomonas , K.Pneumonea and E. Faecalis. 

 

MATERIAL AND METHOD:

Melting points were determined in open capillary method and are uncorrected. IR spectra were recorded on Perkin-Elmer 1720 FT-IR using KBr pellets method. The ¹H-NMR spectra were recorded on Brucker AC 300 MHz using DMSO-d₆ as solvent and tetramethylsilane as internal standard

General method for preparation of 3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)acetic acid from o-Aminophenol(Step 1)⁹:

To a solution of maleic anhydride (0.05 mol) in diethyl ether (20 ml) a solution of o-aminophenol (0.05 mol) in diethyl ether (20 ml) was added. The reaction mixture was stirred at room temperature for 2 h. The precipitate was filtered and washed with ether and crystallized from ethanol to get needle shape crystals of pure.

 

 

Proposed Scheme: Step 1

a. Synthesis of (3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-yl)acetic acid

 

b. Synthesis of (6-chloro-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-yl)acetic acid.

 

 

Step 2

Synthesis of substituted 1,4-Benzoxazine  OR General Procedure for Mannich Base Reaction

 

 

General Procedure for Mannich base condensation (Step 2)^10,11:

A mixture of compound ethyl (3-oxo-3, 4-dihydro-2H-1, 4-benzothiazin-2-yl) acetate or 3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)acetic acid  or (3-oxo-3,4-dihydro-2H-1,4-benzothiazin-2-yl)acetate (0.01 mol) in ethanol, formaldehyde (0.02 mol) and substituted Sulphonamide was added. The reaction mixture was refluxed for 4-5 hrs. The solvent was poured in ice water, resulting solid were filtered off dried and recrystallized using appropriate solvent to get the final products (Table No.1).

 

Antibacterial screening⁸:

The antimicrobial activity of the synthesized compounds was determined MIC by agar plate dilution method.The antibacterial activity was determined against gram-positive organism, Enterococcus faecalis, Bacillus and gram-negative organism Escherichia coli, Klebsiella, Pseudomonas at 500µg/ml, 250µg/ml, 125µg/ml, 62.5µg/ml, 31.25µg/ml, 16.12µg/ml, 8.06µg/ml and 4.03µg/ml concentration of sample compounds. (Table No. 2-7)

 

Table No. 1 - IUPAC names of the synthesized compounds

Compound code	Structures	IUPAC Names
OX		(3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)acetic acid.
OA		[3-oxo-4-({[4-(sulfamoylmethyl)phenyl]amino}methyl)-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid
OB		[3-oxo-4-{[(4-(N-(2-pyridyl)sulfamoyl)phenyl) amino]methyl}-3,4-dihydro-2H-1,4-benaoxazin-2-yl] acetic acid
OC		[3-oxo-4-{[(4-(N-acetyl sulfamoyl)phenyl) amino]methyl}-3,4-dihydro-2H-1,4-benaoxazin-2-yl] acetic acid
OD		[3-oxo-4-{[(4-(N-(5-methyl-1,2-oxazol-3-yl)sulfamoyl) phenyl) amino]methyl}-3,4-dihydro-2H-1,4-benaoxazin-2-yl] acetic acid
OXC		(6-chloro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)acetic acid
OCA		[6-chloro-3-oxo-4-{[4-(sulfamoylmethyl)phenyl]amino}methyl}-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid
OCB		[6-chloro-3-oxo-4-{[4-(N-(2-pyridyl)sulfamoylmethyl)phenyl]amino}methyl}-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid
OCC		[6-chloro-3-oxo-4-{[(4-(N-acetyl sulfamoylmethyl)phenyl)amino]methyl}-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid
OCD		[6-chloro-3-oxo-4-{[(4-(N-(5-methyl-1,2-oxazol-3-yl) sulfamoylmethyl)phenyl)amino]methyl}-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid

 

Analytical Data:

OX IR (KBr) cm^-1: 3394.72 (N-H str), 2950.45 (COOH str), 1695.43 (C=O str), 1460.11 (C-C str ), 1355.96 (C-H str ), 1105.21, (C-N str)

OA IR (KBr) cm^-1: 3385.07( N-H str), 1728.22 (C=O str ), 1375.25( N-CH₂ str), 1320.34 (S=O str)

OB IR (KBr) cm^-1: 3410.05(N-H str), 1714.77(C=O str), 1633.76 (R-NH str), 1596.15(C-N str), 1391.69 (N-CH₂ str), 1323.21( S=O str), 1H NMR: 2.081(s, 2H -CH2), 3.395(s, 2H-CH2), 3.674(s, 1H, -NH), 5.56(s, 1H, - NH), 6.722- 8.081(m, 12H, Ar-H), 9.846 (s, 1H –CH), 11.227(s, 1H –COOH)

OC IR (KBr) cm^-1: 3316.71(N-H  str), 3232.80 (Aromatic ring  str), 1707.06( C=O str), 1597.11 (C-N str), 1395.54 (N-CH₂str ), 1317.43(S=O str)

OD IR (KBr) cm^-1: 3383.07 (N-H str), 1717.62 (C=O str ), 1418.88 (R-NH str), 1362.75 (C-N str), 1339.61( N-CH₂),1286.56 (S=O str)

OCX IR (KBr) cm^-1: 3396.64 (N-H str), 1691.57 (C=O str), 1608.63 (C-N str), 1246.02 (C-O str), 1409.96 (N-CH₂ str ), 742.80( C-Cl str)

OCA IR (KBr) cm^-1: 3373.50(N-H str), 1724.36 (C=O str), 1581.63 (C-Nstr), 1232.51 (C-Ostr), 1421.54( N-CH₂ str ), 780.02 (C-Cl str), 1302.03(S=O str)

OCB IR (KBr) cm^-1:3371.57 (N-H  str), 1722.43 (C=O str), 1597.06 (C-N str), 1232.51( C-O str), 1421.54 (N-CH₂ str ) ,808.96 (C-Cl str), 1328.95 (S=O str)

OCC IR (KBr) cm^-1: 3319.49 (N-H str), (1724.36 C=O str), 1606.70(C-N str), 1222.87 (C-O str), 1408.29 (N-CH₂ str ) 840.93 (C-Cl str) 1328.95 (S=O str)

OCC IR (KBr) cm^-1: 3381.21  ( N-H str) 1722.43 (C=O str), 1614.42 (C-N str) 1232.51 (C-O str) 1423.90 (N-CH₂ str ) 840.96 (C-Cl str) 1328.95 (S=O str)

 

Table No.2- MIC Result for Bacillus Species

Compounds	500µg/ml	250µg/ml	125µg/ml	62.5µg/ml	31.25µg/ml	16.12µg/ml	8.06µg/ml	4.03µg/ml
OA	S	S	S	R	R	R	R	R
OB	S	S	S	S	R	R	R	R
OC	S	S	S	S	R	R	R	R
OD	S	S	S	S	R	R	R	R
OCA	S	S	S	S	S	S	R	R
OCB	S	S	S	R	R	R	R	R
OCC	S	S	S	S	S	R	R	R
OCD	S	S	S	R	R	R	R	R

 

 

Table No. 3- MIC Result for Enterococcus faecalis

Compounds	500µg/ml	250µg/ml	125µg/ml	62.5µg/ml	31.25µg/ml	16.12µg/ml	8.06µg/ml	4.03µg/ml
OA	S	S	S	S	S	S	S	S
OB	S	S	S	S	S	S	S	S
OC	S	S	S	S	S	S	S	S
OD	S	S	S	S	S	S	S	S
OCA	S	S	S	S	S	S	S	S
OCB	S	S	S	S	S	R	R	R
OCC	S	S	S	S	S	R	R	R
OCD	S	S	S	S	S	S	S	S

 

Table No. 4- MIC Result for Staphylococcus aureus

Compounds	500µg/ml	250µg/ml	125µg/ml	62.5µg/ml	31.25µg/ml	16.12µg/ml	8.06µg/ml	4.03µg/ml
OA	S	S	S	R	R	R	R	R
OB	S	S	S	S	R	R	R	R
OC	S	S	S	S	R	R	R	R
OD	S	S	S	S	S	S	R	R
OCA	S	S	S	S	S	R	R	R
OCB	S	R	R	R	R	R	R	R
OCC	S	S	S	S	S	R	R	R
OCD	S	S	S	S	S	S	R	R

 

Table No. 5- MIC       Result for Escherichia coli

Compounds	500µg/ml	250µg/ml	125µg/ml	62.5µg/ml	31.25µg/ml	16.12µg/ml	8.06µg/ml	4.03µg/ml
OA	S	S	R	R	R	R	R	R
OB	S	R	R	R	R	R	R	R
OC	S	R	R	R	R	R	R	R
OD	S	R	R	R	R	R	R	R
OCA	S	R	R	R	R	R	R	R
OCB	S	R	R	R	R	R	R	R
OCC	S	R	R	R	R	R	R	R
OCD	S	R	R	R	R	R	R	R

 

Table No. 6- MIC Result for Klebsiella pneumoniae 

Compounds	500µg/ml	250µg/ml	125µg/ml	62.5µg/ml	31.25µg/ml	16.12µg/ml	8.06µg/ml	4.03µg/ml
OA	S	S	S	S	S	R	R	R
OB	S	S	S	S	R	R	R	R
OC	S	S	S	S	R	R	R	R
OD	S	S	S	S	S	S	R	R
OCA	S	S	S	S	S	S	R	R
OCB	S	S	S	S	R	R	R	R
OCC	S	S	S	S	S	S	R	R
OCD	S	S	S	S	S	S	R	R

 

Table No. 7- MIC Result for Pseudomonas

Compounds	500µg/ml	250µg/ml	125µg/ml	62.5µg/ml	31.25µg/ml	16.12µg/ml	8.06µg/ml	4.03µg/ml
OA	S	S	S	S	S	S	R	R
OB	S	S	S	S	S	R	R	R
OC	S	S	S	S	R	R	R	R
OD	S	S	S	S	S	S	R	R
OCA	S	S	S	S	S	R	R	R
OCB	S	S	S	S	S	R	R	R
OCC	S	S	S	S	R	R	R	R
OCD	S	S	S	S	S	R	R	R

MIC- Minimum inhibitory concentration is the lowest concentration of on antimicrobial agent that will significantly inhibit the visible growth of microorganism after a period of incubation.

 

 

RESULT AND DISCUSSION:

Compounds [3-oxo-4-{[(4-(N-(2-pyridyl)sulfamoyl)phenyl) amino]methyl}-3,4-dihydro-2H-1,4-benaoxazin-2-yl] acetic acid,  [3-oxo-4-{[(4-(N-(5-methyl-1,2-oxazol-3-yl)sulfamoyl) phenyl) amino]methyl}-3,4-dihydro-2H-1,4-benaoxazin-2-yl] acetic acid, [6-chloro-3-oxo-4-{[4-(N-(2-pyridyl) sulfamoylmethyl)phenyl]amino}methyl}-3,4-dihydro-2H-1,4-benzoxazin 2-yl]acetic acid,[6-chloro-3-oxo-4-{[(4-(N-(5-methyl-1,2-oxazol-3-yl)sulfamoylmethyl) phenyl) amino]methyl}-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid showed good activity against the K .pneumonea and E. faecalis, compounds [3-oxo-4-{[(4-(N-(5-methyl-1,2-oxazol-3-yl)sulfamoyl) phenyl) amino]methyl}-3,4-dihydro-2H-1,4-benaoxazin-2-yl] acetic acid, [6-chloro-3-oxo-4-{[(4-(N-(5-methyl-1,2-oxazol-3-yl)sulfamoylmethyl)phenyl)amino] methyl}-3,4-dihydro-2H-1,4-benzoxazin-2-yl]acetic acid showed good activity against the S.aureas, while other compound showed moderate activity against E. coli, klebeseila , Staphy. aureus, E. fecalis. While other derivative shown moderate antibacterial activity.

 

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Accepted on 22.02.2012         © AJRC All right reserved