Synthesis and antimicrobial activity of 5-imidazolinone derivatives.

Several 4-arylidene-2-phenyl-1-(2,4,5-trichlorophenyl)-1H-imidazol-5(4H)-ones (4a-q), N-(4-benzylidene-5-oxo-2-phenyl-4,5-dihydroimidazol-1-yl)-4-chlorobenzamides (5a-o) and N-(4-benzylidene-5-oxo-2-phenyl-4,5-dihydroimidazol-1-yl)-2,4-dichlorobenzamides (6a-m) were prepared. All newly synthesized compounds have been tested for their antibacterial activity against gram (+)ve and gram (-)ve bacteria and also on different strains of fungi. Introduction of OH, OCH(3), NO(2), Cl and Br groups to the heterocyclic frame work enhanced antibacterial and antifungal activities.

Imidazolinone ring system is of biological and chemical interest since long. The imidazolinones[1] are associated with a wide range of therapeutic activities[2-7] such as anticonvulsant, sedative and hypnotic, potent CNS depressant, antihistamine, antifilarial, bactericidal, fungicidal, antiinflammatory, MAO inhibitory, antiparkinsonian, antihypertensive and anthelmintic. Recently some new imidazolinone derivatives have been reported as antiinflammatory, herbicidal and hypertensive activities. Some workers have recognized 5-imidazolone as having anticancer activity[8]. The therapeutic importance of the compounds inspired us to synthesize some potential imidazolinones[9-13].

Desai et al.[14] have synthesized 4-benzylidene-2-phenyloxazole-5-one based on the methods descried in the literature which is a special type of Perkin condensation in which reaction between aldehyde and benzoylglycine proceeds first followed by ring closer. It is observed that aldehyde condenses under the influence of a base with the reactive methylene group in the azalactone which is formed by the dehydration of benzoylglycine, when the latter reacts with Ac2 O in presence of sodium acetate. In view of these observations, we have synthesized imidazol-5-ones (Scheme I, Table 1).

Scheme 1

Synthetic pathway for synthesis of 5-imidazolone derivatives

TABLE 1

PHYSICAL CONSTANTS AND ELEMENTAL ANALYSIS OF 5-IMIDAZOLNES4a-q, 5a-o AND 6a-m

Sr No	Ar-	Molecular Formula	M.P.	Yield (%)	Elemental Analysis
					% Carbon		% Nitrogen
					Cal.	Found	Cal.	Found
4a	C6H5	C22H13Cl3N2O	173	65	61.78	61.69	6.55	6.41
4b	2-OH-C6H4	C22H13Cl3N2O2	170	60	59.55	59.47	6.31	6.25
4c	4-OCH3-C6H4	C23H15Cl3N2O2	160	55	60.35	60.28	6.12	6.03
4d	3-Cl-C6H4	C22H12Cl4N2O	185	54	57.17	57.06	6.06	6.01
4e	3-OCH3-C6H4	C23H15Cl3N2O2	190	50	60.35	60.21	6.12	6.03
4f	2-Cl-C6H4	C22H12Cl4N2O	195	53	57.17	57.06	6.06	5.98
4g	4-Cl-C6H4	C22H12Cl4N2O	210	54	57.17	57.05	6.06	6.01
4h	2-NO2-C6H4	C22H12Cl3N3O3	180	57	55.90	55.79	8.89	8.80
4i	3-NO2-C6H4	C22H12Cl3N3O3	230	55	55.90	55.74	8.89	8.78
4j	3-OCH3-4-OH-C6H3	C23H15Cl3N2O3	170	65	58.31	58.21	5.91	5.70
4k	5-Br-3OCH3-4-OH-C6H2	C23H14BrCl3N2O3	235	68	49.99	49.85	5.07	4.98
4l	4-OH-C6H4	C22H13Cl3N2O2	145	57	59.55	59.36	6.31	6.21
4m	5-Br-2OH-C6H3	C22H12BrCl3N2O2	175	50	50.56	50.45	5.36	5.22
4n	3-OC6H5-C6H4	C28H17Cl3N2O2	160	48	64.70	64.64	5.39	5.34
4o	2,4,5 (OCH3)3-C6H2	C25H19Cl3N2O4	198	45	57.99	57.90	5.41	5.33
4p	3,4,5 (OCH3)3-C6H2	C25H19Cl3N2O4	185	45	57.99	57.89	5.41	5.35
4q	3-OH-C6H4	C22H13Cl3N2O2	165	58	59.55	57.47	6.31	6.26
5a	C6H5	C23H16ClN3O2	233	60	68.74	68.62	10.46	10.35
5b	2-OH-C6H4	C23H16ClN3O3	235	65	66.11	66.05	10.06	9.97
5c	3-Cl-C6H4	C23H15Cl2N3O2	237	66	63.32	63.20	9.63	9.51
5d	3-OCH3-C6H4	C24H18ClN3O3	246	55	66.75	66.66	9.37	9.29
5e	2-Cl-C6H4	C23H15Cl2N3O2	212	62	63.32	63.19	9.63	9.51
5f	4-Cl-C6H4	C23H15Cl2N3O2	214	64	63.32	63.23	9.63	9.54
5g	2-NO2-C6H4	C23H15ClN4O4	248	50	61.82	61.73	12.54	12.45
5h	3-NO2-C6H4	C23H15ClN4O4	223	56	61.82	61.69	12.54	12.47
5i	3-OCH3-4-OH-C6H3	C24H18ClN3O4	226	45	64.36	64.25	9.38	9.25
5j	4-OH-C6H4	C23H16ClN3O3	231	47	66.11	66.01	10.06	9.98
5k	3-OC6H5-C6H4	C29H20ClN3O3	186	48	70.52	70.40	8.51	8.39
5l	2,4,5 (OCH3)3-C6H2	C26H22ClN3O5	245	46	63.48	63.40	8.54	8.47
5m	3,4,5 (OCH3)3-C6H2	C26H22ClN3O5	210	50	63.48	63.41	8.54	8.45
5n	3-OH-C6H4	C23H16ClN3O3	182	57	66.11	66.02	10.06	9.98
5o	4-N(C2H5)2-2-OH-C6H3	C27H25ClN4O3	176	43	66.21	66.21	11.46	11.40
6a	2-OH-C6H4	C23H15Cl2N3O3	175	60	61.08	61.01	9.29	9.93
6b	3-Cl-C6H4	C23H14Cl3N3O2	208	58	58.68	58.61	8.39	8.88
6c	3-OCH3-C6H4	C24H17Cl2N3O3	202	55	61.82	61.70	9.01	5.90
6d	2-Cl-C6H4	C23H14Cl3N3O2	205	56	58.68	58.58	8.93	8.85
6e	4-Cl-C6H4	C23H14Cl3N3O2	244	55	58.68	58.59	8.93	8.87
6f	2-NO2-C6H4	C23H14Cl2N4O4	216	58	57.40	57.31	11.64	11.55
6g	3-NO2-C6H4	C23H14Cl2N4O4	233	60	57.40	57.32	11.64	11.56
6h	3-OCH3-4-OH-C6H3	C24H17Cl2N3O4	237	48	59.77	59.65	8.74	8.65
6i	4-OH-C6H4	C23H15Cl2N3O3	236	45	61.08	61.01	9.29	9.22
6j	3-OC6H5-C6H4	C29H19Cl2N3O3	224	48	65.92	65.80	7.95	7.81
6k	2,4,5 (OCH3)3-C6H2	C26H21Cl2N3O5	238	43	59.33	59.27	7.98	7.90
6l	3,4,5 (OCH3)3-C6H2	C26H21Cl2N3O5	212	45	59.33	59.26	7.98	7.88
6m	3-OH-C6H4	C23H14Cl2N3O3	190	48	61.08	61.01	9.29	9.20

Various 4-arylidene-2-phenyl-1-(2,4,5-trichlorophenyl)-1H-imidazol-5(4H)-ones (4a-q) were prepared by the reaction of 2,4,5-trichlorobenzenamine with 4-arylidene-2-phenyloxazol-5(4H)-ones (3a-q). N-(4-benzylidene-5-oxo-2-phenyl-4,5-dihydroimidazol-1-yl)-4-chlorobenzamide (5a-o) were synthesized by the reaction of 4-chlorobenzohydrazide and 4-arylidene-2-phenyloxazol-5(4H)-ones (3a-q). N-(4-benzylidene-5-oxo-2-phenyl-4,5-dihydroimidazol-1-yl)-2,4-dichlorobenzamides (6a-m) were obtained by the reaction of 2,4-dichlorobenzohydrazide with 4-arylidene-2-phenyloxazol-5(4H)-ones (3a-q).

Melting points were taken in open capillaries using paraffin bath and are uncorrected. IR spectra were recorded on FTIR-Perkin-Elmer spectrometer (Vmax cm−1); 1H NMR spectra were recorded on Bruker Avance 300 FT-NMR spectrometer using CDCl3 as a solvent and mass spectra carried out on JEOL SX 102/DA-600 mass spectrometer, respectively. All the compounds were analyzed for carbon, hydrogen and nitrogen and the results were within ±0.4% of theoretical values. Purity was checked by TLC using TLC aluminum sheets silica gel 60, supplied by E. Merck, Mumbai, India. The spots were located by keeping the plates in iodine vapor and 2,4,5-trichlorobenzenamine was supplied by S. D. Fine Chem Limited, Mumbai, India. 4-Chlorobenzohydrazide, 2,4-dichlorobenzo hydrazide and 4-arylidene-2-phenyloxazol-5(4H)-one (3a-q), were prepared as given in literature method[15-20].

4-Arylidene-2-phenyl-1-(2,4,5-trichlorophenyl)-1H-imidazol-5(4H)-one (4) were synthesized as follows; A mixture of 2,4,5-trichloroaniline (0.01 mol) and 4-(arylidene)-2-phenyloxazol-5(4H)-ones (0.01 mol) was placed in a round bottom flask and 10 ml of pyridine were added to it. The reaction mixture was refluxed on a sand bath for 6 h. (scheme I) and the mixture was poured into ice-cold water and then a required amount of conc. hydrochloric acid was added to neutralize the reaction mixture. The progress of the reaction and the purity of compounds were routinely checked on TLC. The solid obtained was left overnight, filtered and washed with water. The product was dried and recrystallized from ethanol (99%). m.p.195° Yield 53% anal. found: C, 57.06; N, 5.98; calc for C22 H12 Cl4 N2 O: C, 57.17; N, 6.06%.

Compound 4f: IR (KBr): 3062 cm−1 (-C-H str., aromatic), 1643 cm−1 (>C=O str., cyclic ring), 1359 cm−1 (>C=N str., imidazol ring), 1284 cm−1 (-C-N tertiary amine), 1074 cm−1 (-C-Cl str., aromatic), 744 cm−1 (>C=CH medium), 704, 688, 613 cm−1 (trisubstituted aromatic). 1H NMR (CDCl3): δ7.2 (s, 1H, -CH), 7.26-8.54 (m, 11H, Ar-H, C=C-Ar) ppm. MS: m/z 461 with 62% relative intensity (base peak) & 462 with 47% relative intensity (M+). Other compounds of the series were prepared by using a similar method and their physical data are recorded in Table 1.

N-(4-benzylidene-5-oxo-2-phenyl-4,5-dihydroimidazol-1-yl)-4-chlorobenzamides (5)/ N-(4-benzylidene-5-oxo-2-phenyl-4,5-dihydroimidazol-1-yl)-2,4-dichlorobenzamides (6) were prepared using the following procedure; A mixture of 4-chlorobenzohydrazide/ 2,4-dichlorobenzohydrazide (0.01 mol) and 4-(arylidene)-2-phenyloxazol-5(4H)-ones (0.01 mol) was placed in a round bottom flask and 10 ml of pyridine was added to this mixture. The reaction mixture was refluxed on a sand bath for 6 h (Scheme I). The mixture was poured into ice-cold water and then required amount of con. hydrochloric acid was added to neutralize the reaction mixture. The solid obtained was left overnight, filtered and washed with water. The product was dried and recrystallized from ethanol (99%).

Compound 5f: IR (KBr): 3249 cm−1 (medium –CONH-), 3033 cm−1 (-C-H str., aromatic), 1656 cm−1 (>C=O str., cyclic ring), 1625 cm−1 (>C=N str., imidazol ring), 1490 cm−1 (>NH weak), 1299 cm−1 (-C-N tertiary amine), 1095 cm−1 (-C-Cl str., aromatic), 754 cm−1 (>C=CH medium), 707 cm−1 (monosubstituted aromatic). 1H NMR (CDCl3): δ7.28 (s, 1H, -CH), 7.26-8.54 (m, 13H, Ar-H, -C=C-Ar), 10.02 (s, 1H, -NH-CO-) ppm. MS: m/z 436 with 45% relative intensity (base peak) & 437 with 32% relative intensity (M+).

Compound 6e: IR (KBr): 3213 cm−1 (medium, –CONH-), 2993 cm−1 (-C-H str., aromatic), 1662 cm−1 (>C=O str., cyclic ring), 1635 cm−1 (>C=N str., imidazol ring), 1473 cm−1 (>NH weak), 1305 cm−1 (-C-N tertiary amine), 1109 cm−1 (-C-Cl str., aromatic), 925 cm−1 (>C=CH medium), 825, 713 cm−1 (disubstituted aromatic), 707 cm−1 (monosubstituted aromatic). 1H NMR (CDCl3): δ7.2 (s, 1H,-CH), 7.32-8.05 (m, 12H, Ar-H, C=C-Ar), 10.02 (s,1H, -NH-CO-) ppm. MS: m/z 471 with 79% relative intensity (base peak) and 472 with 51% relative intensity (M+). Other compounds of the series were prepared by using a similar method and their physical data are recorded in Table 1.

Antibacterial activity was carried out by broth dilution method[21]. The strains used for the activity were procured from Institute of Microbial Technology, Chandigarh. The compounds 4a-q, 5a-o and 6a-m were screened for their antibacterial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Staphylococcous pyogenes at concentrations of 1000, 500, 200, 100, 50, 25, 12.5 µg/ml respectively (Table 2). Same compounds were tested for antifungal activity against C. albicans, A. niger and A. clavatus at concentrations of 1000, 500, 200, and 100 µg/ml respectively (Table 2). The results are recorded in the form of primary and secondary screening.

TABLE 2

ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF THE SYNTHESIZED COMPOUNDS*

Sr. No.	Minimal bactericidal concentration (MBC) in μg/ml				Minimal fungicidal concentration (MFC) in μg/ml
	E. coli MTCC-443	P. aeruginosa MTCC-1688	S. aureus MTCC-96	S. pyogenus MTCC-442	C. albicans MTCC-227	A. niger MTCC-282	A. clavatus MTCC-1323
4a	25	-	-	-	-	-	-
4b	25	50	-	-	-	-	-
4f	100	-	-	-	100	100	100
4i	25	-	-	-	-	-	-
4j	25	-	-	-	-	-	-
4k	50	100	50	-	-	-	-
4q	50	100	-	-	-	-	-
5b	-	-	-	-	100	100	100
5e	50	50	-	-	-	-	-
6f	100	-	-	-	100	100	100
6j	-	-	-	-	-	-	100
6l	-	-	-	-	100	-	100
6m	-	-	-	-	100	-	-

Gentamycin is used as standard for antibacterial activity which showed (0.05, 0.25, 0.5 and 1 μg/ml) MBC against E. coli, S. aureus, S. pyogenus and P. aeruginosa respectively. K nystatin was used as the standard for antifungal activity which showed 100 μg/ml MFC against fungi, used for the antifungal activity.

All the compounds were tested for the antibacterial and antifungal activities but data of active compounds have been reported as present protocol

The synthesized compounds found to be active in the primary screening were further tested in a second set of dilution against all microorganisms. The compounds found active in primary screening were similarly diluted to obtain 100, 50, 25 μg/ml concentrations. Ten microlitres suspensions from each well were further inoculated on appropriate media and growth was noted after 24 and 48 h. The lowest concentration, which showed no growth after spot subculture was considered as MBC/MFC for each drug. The highest dilution showing at least 99% inhibition was taken as MBC/MFC. The result of this test is affected by the size of the inoculums. The test mixture should contain 108 organisms/ml. For antibacterial activity, in present protocol 50 µg/ml is considered as active as compared to the standard drug gentamycin. For antifungal activity, 100 µg/ml is considered as active as compared to standard nystatin. Compounds 4a, 4b, 4i, 4j, 4k, 4q and 5e are active on E. coli where as 4b and 5e are active on P. aeruginosa. Compound 4k is active on S. aureus and 6m is also active on S. pyogens. Compounds 4f, 5b, 6f, 6l, and 6m are active on fungi strains. On the basis of biological activity results, it may be concluded that the introduction of OH, OCH3, NO2, Cl and Br groups to the heterocyclic frame work enhanced antibacterial and antifungal activities.