Benzothiazole incorporated thiazolidin-4-ones and azetidin-2-ones derivatives: Synthesis and in vitro antimicrobial evaluation

2.1 General

All the solvents were of AR grade and were obtained from Merck, CDH and S.D. Fine chemicals. Melting points were determined in open capillary tubes and are uncorrected. All the compounds were subjected to elemental analysis (CHN) and the measured values agreed within ±0.4% with the calculated ones. Thin layer chromatography was performed on silica gel G (Merck). The spots were developed in an iodine chamber and visualized with an ultraviolet lamp. The solvent systems used were benzene–acetone (8:2, v/v) and toluene–ethyl acetate–formic acid (5:4:1, v/v). Ashless Whatman No. 1 filter paper was used for vacuum filtration. The spots were developed in an iodine chamber and visualized under an ultraviolet (UV) lamp. The IR spectra were recorded in KBr pellets on a (BIO-RAD FTS 135) WIN-IR spectrophotometer. The FAB mass spectra of all the compounds were recorded on a JEOL SX102/ /DA-600 mass spectrometer using argon/xenon (6 kV, 10 mA) as the FAB gas. ¹H NMR spectra (d, ppm) were recorded in DMSO-d₆ solutions on a Varian-Mercury 300 MHz spectrometer using tetramethylsilane as the internal reference. ¹³C NMR spectra were recorded in DMSO-d₆ solutions on a Bruker Avance II 400 spectrometer at 400 MHz using tetramethylsilane as the internal reference. 2-Amino-6-chloro-benzothiazole 1 was synthesized by the literature procedure (Gilani et al., 2011b).

2.2 Synthesis of 1-(6-chlorobenzo[d]thiazol-2-yl)urea (2)

To the solution of sodium cyanate in minimum quantity of water, glacial acetic acid (5 mL) was added. This solution was heated with 2-amino-6-chloro-benzothiazole 1 (0.01 mol) in alcohol till the contents of the mixture became turbid and the volume remained half of the original volume. The contents were added to ice cool water. The solid obtained was filtered off, dried and recrystallised from a suitable solvent.

IR (KBr) λ_max (cm⁻¹): 3310 (NH), 1628 (C⚌O), 1560 (C⚌N), 830 (C–Cl), 645 (C–S–C benzothiazole); ¹H NMR (300 MHz, DMSO-d₆) δ ppm: 9.14 (s, 1H, NHC⚌O), 6.68–6.70 (m, 3H, Ar-H, J = 6 Hz), 6.34 (s, 2H, NH₂).

2.3 Synthesis of N-(6-chlorobenzo[d]thiazol-2-yl)hydrazine carboxamide (3)

To the warm hydrazine hydrate solution of compound 2 in alcohol, conc. NaOH was added and refluxed for 6 h. The reaction mixture was poured into crushed ice and solid obtained was filtered off and dried. The solid collected out was recrystallized from a suitable solvent to get the compound N-(6-chlorobenzo[d]thiazol-2-yl)hydrazine carboxamide.

IR (KBr) λ_max (cm⁻¹): 3300 (NH), 1660 (C⚌O), 1588 (C⚌N), 817 (C–Cl), 657 (C–S–C benzothiazole); ¹H NMR (300 MHz, DMSO-d₆) δ ppm: 9.12 (s, 1H, NHC⚌O), 7.28 (s, 1H, NHNH₂), 7.70–7.74 (m, 3H, Ar-H, J = 12 Hz).

2.4 Synthesis of 2-Substituted benzylidene-N-(6-chlorobenzo[d]thiazol-2-yl)hydrazinecarboxamide (4a–g)

To an equimolar methanolic solution of N-(6-chlorobenzo[d]thiazol-2-yl)hydrazine carboxamide (3) (0.1 mol) and substituted benzaldehyde (0.1 mol), a few drops of glacial acetic acid were added. The mixture was then refluxed on a water bath for 5–6 h. It was then allowed to cool, poured onto crushed ice and recrystallised from methanol.

2.5 Synthesis of 1-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-oxo-2-substituted phenylthiazolidin-3-yl)urea (5a–g)

A mixture of 4 (0.01 mol) and thioglycollic acid (0.01 mol) was heated on an oil-bath at 120–125 °C for 12 h. The reaction mixture was cooled and treated with 10% sodium bicarbonate solution. The product was isolated and recrystallised from methanol–dioxane (4:1).

2.6 1-(3-Chloro-2-oxo-4-substituted phenylazetidin-1-yl)-3-(6-chlorobenzo[d]thiazol-2yl)urea (6a–g)

A solution of 4 (0.01 mol) in dioxane (20 mL) was added to a well stirred mixture of chloroacetylchloride (0.012 mol) and triethylamine (Et₃N) (0.012 mol) in dioxane (10 mL) at 0–5 °C. The reaction mixture was then stirred for 8 h, kept for 2 days at room temperature and then treated with cold water. The solid thus obtained was filtered, washed with water and recrystallised from methanol.

2.7 Antimicrobial activity

All the synthesized compounds were tested for their in vitro antimicrobial activity against the Gram positive bacteria Staphylococcus aureus (ATCC-25923), the Gram-negative bacteria Escherichia coli (ATCC-25922), Pseudomonas aeruginosa (ATCC-27853) and Klebsiella pneumoniae (ATCC-700603) in the nutrient agar media, and fungi Candida albicans (ATCC-2091), Aspergillus niger (MTCC-281), Aspergillus flavus (MTCC-277), Monascus purpureus (MTCC 369) and Penicillium citrinum (NCIM-768) in Sabouraud dextrose medium at 200, 100, 50, 25 and 12.5 μg/mL concentrations by using serial plate dilution method. The minimum inhibitory concentrations (MIC’s) values were determined by comparison to ofloxacin and ketoconazole as the reference drugs for bacterial and fungal activity, respectively, as shown in Tables 2 and 3. Standard antibiotics ofloxacin and ketoconzole were used as reference drugs at 50, 25 and 12.5 μg/mL concentrations. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the compounds that inhibited the visible growth of microorganisms on the plate.

3.1 Chemistry

1-(6-Chlorobenzo[d]thiazol-2-yl)-3-(4-oxo-2-substituted phenylthiazolidin-3-yl)urea (5a–g) and 1-(3-chloro-2-oxo-4-substituted phenylazetidin-1-yl)-3-(6-chlorobenzo[d]thiazol-2yl)urea (6a–g) were prepared according to the procedure outlined in Scheme 1. The required 2-substituted benzylidene-N-(6-chlorobenzo[d]thiazol-2-yl)hydrazinecarboxamide (4a–g) was synthesized by reacting N-(6-chlorobenzo[d]thiazol-2-yl)hydrazine carboxamide (3) with substituted aromatic aldehyde in ethanol. This salt underwent ring closure by condensation with thioglycollic acid to give the required title compounds (5a–g). The synthesis of compounds (6a–g) was also accomplished in a single step by reacting the 2-substituted benzylidene-N-(6-chlorobenzo[d]thiazol-2-yl)hydrazinecarboxamide (4a–g) with chloroacetyl chloride and triethylamine in the presence of dioxane, respectively. The structure of the synthesized compounds was confirmed by elemental analysis and spectral data (IR, ¹H NMR, ¹³C NMR & Mass). Physicochemical data for the 2-substituted benzylidene-N-(6-chlorobenzo[d]thiazol-2-yl)hydrazinecarboxamide derivatives (4a–g, 5a–g, 6a–g) are given in Table 1.

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Scheme 1

Synthetic route for the synthesis of title compounds.

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Table 1 Physical and analytical data of benzothiazole derivatives 4, 5 & 6a–g.

Compound	R	Mol. formula	Yield (%)	M.P. (°C)	Mol. weight	Rf	Analysis % found (calculated)
							C	H	N
4a		C15H11ClN4OS	84	153	330.79	0.8	54.46	3.35	16.94
							(54.50)	(3.37)	(16.63)
4b		C15H10Cl2N4OS	88	198	365.24	0.6	49.33	2.76	15.34
							(49.38)	(2.71)	(15.39)
4c		C15H9Cl3N4OS	80	136	399.68	0.7	45.08	2.27	14.02
							(45.11)	(2.29)	(14.08)
4d		C16H13ClN4OS	76	149	344.82	0.9	55.73	3.80	16.25
							(55.67)	(3.76)	(16.28)
4e		C17H13ClN4O3S	83	164	388.83	0.6	52.51	3.37	14.41
							(52.54)	(3.39)	(14.44)
4f		C16H13ClN4O2S	79	157	360.82	05	53.26	3.63	15.53
							(53.29)	(3.57)	(15.50)
4g		C15H10ClN5O3S	81	181	375.79	0.8	47.94	2.68	18.64
							(47.78)	(2.62)	(18.67)
5a		C17H13Cl N4O2S2	77	222	404.89	0.7	50.43	3.24	13.84
							(50.46)	(3.28)	(13.66)
5b		C17H12Cl2N4O2S2	79	238	439.34	0.6	46.47	2.75	12.75
							(46.49)	(2.71)	(12.69)
5c		C17H11Cl3N4O2S2	70	208	473.78	0.8	43.10	2.34	11.83
							(43.14)	(2.37)	(11.76)
5d		C18H15ClN4O2S2	66	258	418.92	0.7	51.61	3.61	13.37
							(51.58)	(3.64)	(13.39)
5e		C19H15ClN4O4S2	71	214	462.93	0.9	49.30	3.27	12.10
							(49.34)	(3.31)	(12.14)
5f		C18H15ClN4O3S2	74	280	434.92	0.6	49.71	3.48	12.88
							(49.67)	(3.50)	(12.74)
5g		C17H12ClN5O4S2	80	202	449.89	0.8	46.40	3.46	15.03
							(46.43)	(3.51)	(15.09)
6a		C17H12Cl2N4O2S	71	226	407.27	0.7	50.13	2.97	13.76
							(50.17)	(2.88)	(13.64)
6b		C17H11Cl3N4O2S	78	232	441.72	0.9	46.22	2.51	12.68
							(46.28)	(2.54)	(12.65)
6c		C17H10Cl4N4O2S	73	262	476.16	0.5	42.88	2.12	11.77
							(42.79)	(2.16)	(11.72)
6d		C18H14Cl2N4O2S	76	217	421.30	0.7	51.32	3.35	13.30
							(51.37)	(3.39)	(13.34)
6e		C19H14Cl2N4O4S	77	238	465.31	0.9	49.04	3.03	12.04
							(49.10)	(3.07)	(12.08)
6f		C18H14Cl2N4O3S	72	219	437.30	0.8	49.44	3.23	12.81
							(49.47)	(3.27)	(12.76)
6g		C17H11Cl2N5O4S	75	210	452.27	0.6	45.15	2.45	15.48
							(45.19)	(2.48)	(15.51)

The IR spectra of compounds 4a–g showed absorption peaks at 3318 and 1668 cm^–1 due to N–H, C⚌O and –N⚌CH stretching vibrations. The appearance of the stretching of the C⚌O of thiazolidinone and β-lactam ring at 1726 and 1745 cm^–1, respectively, in the spectra of the derivatives, together with the C⚌O stretching at 1664 and 1670 cm^–1 confirmed the formation of the compounds 5a–g and 6a–g.

The ¹H-NMR spectra of compounds 4a–g revealed a multiplet at δ 7.77–7.81 ppm for the aromatic ring and singlets at δ 6.10 and 7.90 ppm for –NH and –N⚌CH, respectively. The disappearance of the singlet peak of –N⚌CH and the presence of a singlet peak at δ 2.48 and 7.70 of –CH₂ of thiazolidinone and –N–CH of β-lactam ring proved that these compounds participated in the cyclisation reaction and formed the desired compounds.

This was further confirmed by ¹³C–NMR spectrum of the title compounds 5a–g and 6a–g which showed peaks at 35.2 and 163.5 ppm suggesting the presence of –CH₂ and –C⚌O of thiazolidine and β-lactam ring, respectively.

The elemental analysis and molecular ion peaks of compounds 5a–g and 6a–g were consistent with the assigned structure.

3.2 Antimicrobial activity

The investigation of antibacterial and antifungal screening data revealed that all the tested compounds 5a–g and 6a–g showed good to moderate inhibition at 12.5–200 μg/mL in DMSO. The compounds 5c, 5d, 5g, 6c and 6f showed comparatively good activity against all the bacterial strains. The good activity is attributed to the presence of pharmacologically active 2,4-dichloro (5c), methyl (5d), 4-nitro (5g) groups attached to the phenyl group at position 2 of the thiazolidin-4-one ring, whereas 2,4-dichlorophenyl (6c) and phenoxy group (6f) attached at the fourth position of the β-lactam moiety. When these groups were replaced by 2-chlorophenyl (5b, 6b) and acetyl phenyl (5e, 6e), a sharp decrease in activity against most of the strains was observed. Compounds 5a, 5f, 6a, 6d and 6g exhibited moderate activity compared to that of standard ofloxacin against all the bacterial strains. Further, the result showed that Gram-negative exhibited better activity than Gram positive organisms.

Compounds 5c, 5d, 5f, 6e and 6g showed comparatively good activity against all the fungal strains. The structure of these compounds contains biologically active 2,4-dichlorophenyl (5c), methylphenyl (5d) and phenoxy (5f) groups attached at position 2 of the thiazolidin-4-one ring and acetyl (6e) and 4-nitrophenyl (6g) groups attached at the fourth position of β-lactam ring, respectively. Compounds 5a, 5b, 5e, 5g, 6a, 6b and 6c showed moderate activity compared to that of the standard against all the fungal strains. All the compounds showed good to moderate activity against all pathogenic fungal strains except compounds 6d and 6f having methyl and phenoxy groups attached at the fourth position of the β-lactam nucleus.