Synthesis and antimicrobial activity of Schiff bases and 2-azetidinones derived from quinazolin-4(3H)-one

2.1 General

Melting points were determined in one-end-open capillary tubes on a Mel-Temp apparatus and are uncorrected. Infrared (IR) absorption spectra were recorded on Perkin–Elmer RX-1 FTIR spectrometer using potassium bromide (KBr) pellet and the wave numbers were given in cm⁻¹. The ¹H NMR spectra were recorded in deutero chloroform (CDCl₃) on a Bruker Avance II 400 NMR spectrometer (400 MHz). The ¹³C NMR spectra were recorded in deutero chloroform (CDCl₃) on a Bruker Avance II 400 NMR spectrometer operating at 100 MHz. The chemical shifts are reported in parts per million (δ ppm) using tetramethylsilane (TMS) as an internal standard. The microanalyses were performed on a Carlo Erba 1108 elemental analyzer. The purities of the compounds were checked by thin layer chromatography (TLC) using ready-made silica gel plates (Merck) and benzene:methanol (8:2) as a solvent system. The spots were developed in an iodine chamber and visualized under ultraviolet (UV) lamp. 2-[(2,6-Dichlorophenyl)amino]phenyl acetyl chloride 1 was synthesized by the literature procedure (Furniss et al., 1989).

2.2 Synthesis of 6-bromo-2-[2-(2,6-dichlorophenyl)amino]benzyl-4H-3,1-benzoxazin-4-one (2)

A mixture of 2-[(2,6-dichlorophenyl)amino]phenyl acetyl chloride 1 (0.02 mol) and 5-bromo anthranilic acid (0.02 mol) in pyridine (40 ml) was stirred at 0–5 °C for 1 h, and further stirred for 1 h at room temperature. A pasty mass was obtained which was washed thoroughly with sodium bicarbonate (5%) to remove the unreacted acid. A solid was separated and was filtered, dried, and recrystallized from methanol.

Yield 55%, m.p. 194–198 °C. IR (KBr): t = 3446 (NH), 2926, 2850 (CH₂), 1740 (C⚌O), 1618 (C⚌N), 1153 (C–O), 743 (C–Cl), 565 (C–Br) cm⁻¹. ¹H NMR (400 MHz, CDCl₃, 25 °C, TMS): δ = 3.53 (s, 2H, CH₂), 6.38–8.16 (m, 10H, Ar–H), 9.10 (bs, 1H, NH) ppm; ¹³C NMR (100 MHz, CDCl₃, 25 °C, TMS) δ = 32.43 (CH₂), 116.31, 118.64, 120.62, 121.67, 124.31, 124.57, 126.54, 127.17, 127.43, 129.41, 131.12, 135.22, 137.29, 138.23, 141.78, 148.73 (Ar–C), 159.23 (C⚌O), 164.33 (C⚌N) ppm. Anal. For C₂₁H₁₃BrCl₂N₂O₂ (476.15) calcd. C52.97, H2.75, N5.88. Found C52.94, H2.71, N5.92.

2.3 Synthesis of 3-amino-6-bromo-2-[2-(2,6-dichlorophenyl)amino]benzyl-quinazolin-4(3H)-one (3)

A mixture of 2 (0.01 mol) and hydrazine hydrate (0.02 mol) in absolute methanol (20 ml) was refluxed on water bath for 4–6 h, excess of solvent was distilled off and the residue was cooled whereupon a crystalline solid was separated out, which was recrystallized from methanol.

Yield 58%, m.p. 178–182 °C. IR (KBr): t = 3495–3405 (NH and NH₂), 2922, 2848 (CH₂), 1684 (C⚌O), 1610 (C⚌N), 755 (C–Cl), 570 (C–Br). ¹H NMR (400 MHz, CDCl₃, 25 °C, TMS): δ = 3.56 (s, 2H, CH₂), 5.75 (bs, 2H, NH₂), 6.39–8.15 (m, 10H, Ar–H), 9.15 (bs, 1H, NH) ppm; ¹³C NMR (100 MHz, CDCl₃, 25 °C, TMS) δ = 30.57 (CH₂), 116.14, 120.45, 121.52, 123.14, 124.23, 124.55, 126.74, 127.28, 127.49, 129.36, 131.13, 132.22, 136.45, 137.26, 141.78, 146.22 (Ar–C), 161.28 (C⚌O), 164.53 (C⚌N) ppm. Anal. For C₂₁H₁₅BrCl₂N₄O (490.18) calcd. C51.46, H3.08, N11.43. Found C51.38, H3.02, N11.39.

2.4 General procedure for the synthesis of Schiff bases (4a–k)

To a mixture of 3 (0.005 mol) and substituted aromatic aldehydes (0.005 mol) in absolute ethanol (40 ml) was added a few drops of glacial acetic acid. Then the mixture was refluxed on water bath for 5–6 h. The excess of solvent was distilled off and poured onto ice cold water. The separated solid was filtered, washed, and recrystallized from ethanol.

2.5 General procedure for the synthesis of 2-azetidinones (5a–k)

A solution of 4a–k (0.0025 mol) in dry dioxane (20 ml) was added to a well-stirred mixture of chloro acetyl chloride (0.0025 mol) and triethylamine (0.0025 mol) in dry dioxane (20 ml) at 0–5 °C. The reaction mixture was stirred for 10–12 h and kept for two days at room temperature. The product was isolated and recrystallized from ethanol.

2.6 Antimicrobial activity

The in vitro antimicrobial activity of compounds 4a–k and 5a–k was carried out by cup-plate method (Barry, 1976). Antibacterial activity was screened against two gram-positive bacteria (Staphylococcus aureus ATCC 9144 and Bacillus subtilis ATCC 6633) and two gram-negative bacteria (Pseudomonas aeruginosa ATCC 9027 and E. coli ATCC 25922), while antifungal activity was screened against the fungi Candida albicans ATCC 10231 by measuring the zone of inhibition on agar plates at two different concentrations: 100 and 50 μg/ml. Penicillin-G was used as a standard antibacterial agent whereas amphotericine-B was used as a standard antifungal agent. Microbial cultures were obtained from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune. The potency of the compounds was calculated by using the following formula as per Edwin and Marion (1945). $$\mathrm{Potency}=\{\text{antilog}[(D/B)\times I]\}\times M\times F$$ where F = dilution factor = 1 (same dilution used for standard and test); M = potency of standard = 100; I = log S_H/S_L; D = (U_H + U_L) − (S_H + S_L); B = (U_H − U_L) + (S_H − S_L); U_H = zone of inhibition of the compound at 100 μg/ml; U_L = zone of inhibition of the compound at 50 μg/ml; S_H = zone of inhibition of the standard at 100 μg/ml; S_L = zone of inhibition of the standard at 50 μg/ml.

3.1 Chemistry

The title compounds, 6-bromo-2-[2-(2,6-dichlorophenyl)amino]benzyl-3-substituted benzylideneamino-quinazolin-4(3H)-ones 4a–k and 6-bromo-3-(3-chloro-4-substituted phenyl-2-oxoazetidin-1-yl)-2-[2-(2,6-dichlorophenyl)amino]benzyl-quinazolin-4(3H)-ones 5a–k, have been synthesized according to the described process in Scheme 1. The structures of all the compounds were established on the basis of elemental analyses, IR, ¹H NMR, and ¹³C NMR spectral data. The required benzoxazinone 2 was prepared by the cyclization reaction between 5-bromo anthranilic acid and 2-[(2,6-dichlorophenyl)amino]phenyl acetyl chloride 1 using pyridine. The formation of the product was confirmed by a sharp band at 1740 cm⁻¹ for C⚌O group along with a band at 1153 cm⁻¹ for C–O stretching in the IR spectrum. Compound 2 was converted to quinazolin-4(3H)-one 3, by its condensation reaction with hydrazine hydrate in methanol. The disappearance of the C–O stretching band at 1153 cm⁻¹ and the presence of a sharp C⚌O stretching band at 1684 cm⁻¹ instead of a C⚌O stretching band at 1740 cm⁻¹ confirmed the formation of quinazoli-4(3H)-one. This was further confirmed by ¹³C NMR spectrum, which showed C⚌O and C⚌N signals of quinazolinone at δ 161.2 ppm and δ 164.5 ppm, respectively. When compound 3 was treated with substituted aromatic aldehydes in the presence of glacial acetic acid as a catalyst, Schiff bases 4a–k were formed, which were confirmed by the presence of strong –N⚌CH– stretching vibration of the Schiff bases at around 1630 cm⁻¹ and ¹H NMR spectra showed a singlet at around δ 6.20 ppm due to one proton of the Schiff base. Further cyclization reaction of Schiff bases 4a–k with chloro acetyl chloride in the presence of triethylamine as a catalyst at 0–5 °C gave the desired compounds 2-oxo-azetidinyl-quinazolin-4(3H)-ones 5a–k. IR spectra of compounds 5a–k showed strong stretching vibration at around 1750 cm⁻¹ due to C⚌O group of 2-azetidinone. ¹H NMR spectra of 5a–k showed a doublet at around δ 3.26 ppm and δ 3.37 ppm equivalent to one proton due to N–CH and CH–Cl of 2-azetidinone ring, respectively.

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3.2 Antimicrobial activity

The results of the antibacterial activity are shown in Tables 1 and 2. Schiff base derivatives 4e, 4f, 4g, and 2-azetidinone derivatives 5g and 5h exhibited good activities (50.88–52.88%) against gram-positive bacteria S. aureus while compounds 4g and 5g displayed good activity (60%) against gram-positive bacteria B. subtilis. Schiff bases as well as 2-azetidinones 4f, 4g, 4h, 5f, 5g, and 5h showed good activities (58.24–61.54%) against gram-negative bacteria P. aeruginosa whereas compounds 4g and 5g showed good activities (56.66% and 64.29%, respectively) against gram-negative bacteria E. coli. The remaining compounds possess moderate to poor activities as compared to penicillin-G.

The results of the antifungal activity are shown in Table 3. Schiff bases possess moderate to poor activities (19.75–44.44%) against C. albicans while 2-azetidinone 5f showed good activity (56.03%) against C. albicans. The remaining 2-azetidinone derivatives exhibited moderate to poor activities as compared to amphotericine-B.