Monocyclic β-lactam and unexpected oxazinone formation: synthesis, crystal structure, docking studies and antibacterial evaluation

Abstract

Novel monocyclic β-lactam derivatives bearing aryl, phenyl and heterocyclic rings were synthesized as possible antibacterial agents. Cyclization of imines (3h, 3t) with phenylacetic acid in the presence of phosphoryl chloride and triethyl amine did not afford the expected β-lactams. Instead, highly substituted 1,3-oxazin-4-ones (4h, 4t) were isolated as the only product and confirmed by single crystal X-ray analysis of 4t. The results of antibacterial activity showed that compound 4l exhibited considerable antibacterial activity with MIC and MBC values of 62.5 µg/mL against Klebsiella pneumoniae. Cytotoxicity assay on Chinese Hamster Ovary (CHO) cell line revealed non-cytotoxic behavior of compounds 4d, 4h, 4k and 4l up to 200 μg/mL conc. Molecular docking was performed for compound 4l with penicillin binding protein-5 to identify the nature of interactions. The results of both in silico and in vitro evaluation provide the basis for compound 4l to be carried as a potential lead molecule in the drug discovery pipeline against bacterial infections.

• 1,3-Oxazin-4-one
• antibacterial
• cytotoxicity
• docking
• β-lactam

Introduction

Bacterial infections pose serious threats to human life and are responsible for morbidity and mortality worldwide. Moreover, the emergence of resistant pathogenic bacterial strains has further limited the treatment options1. Therefore to confront global burden of antibacterial resistance, identification of inhibitors against existing/new targets has emerged as an important strategy in the development of antibacterial agents.

One of the most important heterocyclic scaffolds, β-lactam has been considered as privileged structure in chemical, pharmaceutical and material industries2–8. β-Lactam antibiotics such as penicillins, cephalosporins, carbapenems and monobactam (Figure 1) are widely used as chemotherapeutic agents for the treatment of microbial infections. β-Lactams exert their bactericidal effect by inhibiting transpeptidases or penicillin binding proteins (PBPs) that are crucial for bacterial cell wall synthesis. This results in weakly cross-linked peptidoglycan layer of cell wall leading to the high susceptibility of growing bacteria to cell lysis and death9. The β-lactam core structure responsible for antimicrobial activities is also associated with other interesting biological properties such as cholesterol absorption inhibitors10, human cytomegalovirus protease inhibitors11, anti-HIV12, thrombin inhibitors13, anti-tumor14, anti-hyperglycemic15, anti-inflammatory, analgesic activities16 and serine-dependent enzyme inhibitors17.

Figure 1. Structure of commonly used β-lactam antibiotics.

However, bacteria have built up resistance against these antibiotics due to their wide-spread use, thus rendering them useless for the treatment of these infections18. Therefore, it has lent additional urgency to develop new molecular scaffolds having novel mode of action or to modify existing drug molecules. Considering the large pharmacological importance of β-lactams and in extension to our efforts to explore novel biologically active molecules19^,20, we synthesized novel N-benzyl-3,4-diaryl substituted 2-azitidinone (β-lactam) derivatives (4a–g, 4i–s), while compounds 4h and 4t were isolated as highly substituted 1,3-oxazin-4-one derivatives. In silico pharmacokinetic and toxicity parameters were also assessed for all the synthesized compounds. All the derivatives were evaluated for in vitro antibacterial activity against Escherichia coli and Klebsiella pneumoniae. The cytotoxic behavior of compounds with considerable antibacterial activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Chinese Hamster Ovary (CHO) cell line as well as by hemolytic assay on human red blood cells (hRBCs). In E. coli, PBP-5 is the most abundant PBP and provides an excellent system to study the catalytic mechanism of penicillin-interacting enzymes21. The plausible binding modes and mechanism of best compound through molecular docking was then studied against the predicted three-dimensional model of PBP-5 from K. pneumoniae, which displayed 94% sequence homology with PBP-5 of E. coli, due to unavailability of X-ray crystal structure of the former.

Materials and methods

Chemistry

All the chemicals and solvents (analytical grade) were purchased from commercial sources and used without further purification. Precoated Merck Silica gel 60 F₂₅₄ TLC aluminum sheets (Darmstadt, Germany) were used for thin-layer chromatography (TLC) and spots were visualized under UV light. Ninhydrin test was also performed for the detection of imine (Schiff’s base) as well as β-lactams. The IR spectra of compounds were recorded on Agilent Cary 630 FT-IR spectrometer (Santa Clara, CA) and only major peaks are reported in cm⁻¹. ¹H and ¹³C NMR spectra were obtained in CDCl₃ using tetramethylsilane as an internal standard on Bruker Spectrospin DPX-300 spectrometer (Fällanden, Switzerland) at 300 and 75 MHz, respectively, and on Agilent-NMR-vnmrs-500 spectrometer (Santa Clara, CA) at 500 and 125 MHz, respectively. Multiplicities are designated as s (singlet), d (doublet), t (triplet), m (multiplet). ¹H NMR chemical shifts (δ) values are reported in parts per million (ppm) relative to residual solvent (CHCl₃, s, δ 7.26) and ¹³C NMR chemical shifts are reported relative to CDCl₃ (t, δ 77.23) and coupling constants (J) are given in Hertz (Hz). Mass spectra were obtained on Waters Tandem Quadrupole Detector (TQD) electron spray ionization mass spectrometer (Milford, MA) and Agilent Quadrupole-6150 LC/MS spectrometer (Santa Clara, CA). Melting points were recorded on a digital Buchi melting point apparatus (M-560) (Flawil, Switzerland) and were uncorrected. Elemental analyses were performed on Elementar Vario Analyzer (Hanau, Germany). Purification of the compounds was performed by column chromatography using silica gel (230–400 mesh size) as stationary phase with ethyl acetate/petroleum ether as an eluent.

General procedure for the synthesis of oximes (1a–d)

Oximes were obtained by reacting substituted aldehydes (1.2 equiv.) with hydroxylamine hydrochloride (1.3 equiv.) in ethanol–pyridine (10:1) mixture under reflux for 22–25 h. After cooling, solvent was removed under reduced pressure and water was added to the residue. The mixture was cooled in an ice bath, stirred and the precipitated solid was filtered off, washed with water and purified by recrystallization22.

Benzaldehydeoxime (1a)

Colorless oil, yield: 80%, R_f = 0.50 (ethyl acetate/pet. ether, 30:70), Anal. (C₇H₇NO) Calc. C 69.41, H 5.82, N 11.56; found: C 69.39, H 5.80, N 11.61%. IR (neat): ν (cm⁻¹) 3275 (O–H), 1599 (C=N). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.17 (s, 1H, CH), 7.59 (d, 2H, J = 3.9 Hz, Ar–H), 7.38 (t, 3H, J = 3.3 Hz, Ar–H).

2-Chlorobenzaldehyde oxime (1b)

White solid, m.p. 72–74 °C, yield: 87%, R_f = 0.52 (ethyl acetate/pet. ether, 30:70), Anal. (C₇H₆ClNO) Calc. C 54.04, H 3.89, N 9.00; found: C 54.01, H 3.87, N 8.98%. IR (neat): ν (cm⁻¹) 3245 (O–H), 1596 (C=N). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.59 (s, 1H, CH), 7.84 (d, 1H, J = 7.2 Hz, Ar–H), 7.41 (d, 1H, J = 7.8 Hz, Ar–H), 7.28 (t, 2H, J = 8.4 Hz, Ar–H).

Furan-2-carbaldehyde oxime (1c)

Creamish solid, m.p. 82–84 °C, yield: 82%, R_f = 0.47 (ethyl acetate/pet. ether, 30:70), Anal. (C₅H₅NO₂) Calc. C 54.05, H 4.54 N 12.61, found: C 54.01, H 4.51 N 12.59%. IR (neat): ν (cm⁻¹) 3149 (O–H), 1570 (C=N). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 9.21 (brs, 1H, OH), 7.48 (d, 2H, J = 11.1 Hz, Ar–H), 7.33 (d, 1H, J = 3.6 Hz, Ar–H), 6.54 (s, 1H, CH).

4-Fluorobenzaldehyde oxime (1d)

White crystalline solid, m.p. 86–88 °C, yield: 85%, R_f = 0.56 (ethyl acetate/pet. ether, 30:70), Anal. (C₇H₆FNO) Calc. C 60.43, H 4.35, N 10.07; found: C 60.41, H 4.32, N 10.08%. IR (neat): ν (cm⁻¹) 3253 (O–H), 1599 (C=N). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.55 (brs, 1H, OH), 8.13 (s, 1H, CH), 7.56 (t, 2H, J = 7.8, 8.7 Hz, Ar–H), 7.08 (t, 2H, J = 7.2, 8.4 Hz, Ar–H).

General procedure for the synthesis of amines (2a–d)

To a clear solution of oxime (1.0 equiv) in ethanol (10 mL), a mixture of water and conc. HCl (1:2) was added. The reaction mixture was stirred at room temperature for 15 min then it was allowed to cool to 0 °C. Zinc dust (6.0 equiv.) was added slowly and the reaction mixture was refluxed for 1 h at 80–90 °C. After completion of the reaction as confirmed by TLC analysis, zinc dust was filtered, washed with ethanol and filtrate was concentrated under reduced pressure to yield hydrochloride salt of amine (2a–d). The hydrochloride salt was treated with dried Amberlyst A-21 to afford the free amine23.

Phenylmethanamine (2a)

Light yellow oil, yield: 75%, R_f = 0.32 (methanol/DCM, 10:90), Anal. (C₇H₉N) Calc. C 78.46, H 8.47, N 13.07; found: C 78.43, H 8.44, N 13.06%. IR (neat): ν (cm⁻¹) 3372 (NH₂). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.43–7.24 (m, 5H, Ar–H), 3.88 (s, 2H, CH₂), 1.57 (s, 2H, NH₂).

(2-Chlorophenyl)methanamine (2b)

Light yellow oil, yield: 72%, R_f = 0.35 (methanol/DCM, 10:90), Anal. (C₇H₈ClN) Calc. C 59.38, H 5.69, N 9.89; found: C 59.35, H 5.67, N 9.86%. IR (neat): ν (cm⁻¹) 3372 (NH₂). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.39–7.36 (m, 2H, Ar–H), 7.29–7.20 (m, 2H, Ar–H), 3.94 (s, 2H, CH₂), 1.64 (s, 2H, NH₂).

Furan-2-ylmethanamine (2c)

Light yellow oil, yield: 70%, R_f = 0.36 (methanol/DCM, 10:90), Anal. (C₅H₇NO) Calc. C 61.84, H 7.27, N 14.42; found: C 61.81, H 7.25, N 14.39%. IR (neat): ν (cm⁻¹) 3372 (NH₂). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.37 (s, 1H, Ar–H), 6.33 (s, 1H, Ar–H), 6.15 (s, 1H, Ar–H), 3.85 (s, 2H, CH₂), 1.63 (s, 2H, NH₂).

(4-Fluorophenyl)methanamine (2d)

Light yellow oil, yield: 65%, R_f = 0.32 (methanol/DCM, 10:90), Anal. (C₇H₈FN) Calc. C 67.18, H 6.44, N 11.19; found: C 67.16, H 6.41, N 11.17%. IR (neat): ν (cm⁻¹) 3372 (NH₂). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.28 (t, 2H, J = 5.4, 8.1 Hz, Ar–H), 7.02 (t, 2H, J = 8.7, 11.4 Hz, Ar–H), 3.84 (s, 2H, CH₂), 1.56 (s, 2H, NH₂).

General procedure for the synthesis of imines (3a–d)

To an ethanolic solution of substituted aryl aldehyde (1.0 equiv.), equimolar amount of amine (2a–d) was added. The reaction mixture was stirred at room temperature for 1–2 h. After completion of the reaction, the solvent was distilled off under reduced pressure. To this residue, ice-cold water was added and the aqueous layer was extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was then purified by recrystallization with ethanol to afford the desired compounds in good to excellent yields24.

(2-Nitrobenzylidene)(phenyl)methylamine (3a)

Reddish brown oil, yield: 90%, R_f = 0.50 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₂N₂O₂) Calc. C 69.99, H 5.03, N 11.66; found: C 69.87, H 4.98, N 11.59%. IR (neat): ν (cm⁻¹) 1639 (C=N), 1523 (NO_{2 asym}), 1344 (NO_{2 sym}). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.83 (s, 1H, CH), 8.11 (d, 1H, J = 7.8 Hz, Ar–H), 8.03 (d, 1H, J = 8.1 Hz, Ar–H), 7.66 (t, 1H, J = 6.9, 7.5 Hz, Ar–H), 7.56 (t, 1H, J = 7.2, 8.1 Hz, Ar–H), 7.37–7.26 (m, 5H, Ar–H), 4.88 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 159.47, 148.43, 138.23, 136.01, 132.56, 132.12, 129.07, 128.86, 128.10, 125.82, 122.65, 64.03. ESI-MS (m/z): 241.38 [M + H]⁺.

(3-Nitrobenzylidene)(phenyl)methylamine (3b)

Light yellow solid, m.p. 60–61 °C, yield: 93%, R_f = 0.48 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₂N₂O₂) Calc. C 69.99, H 5.03, N 11.66; found: C 69.98, H 4.98, N 11.68%. IR (neat): ν (cm⁻¹) 1646 (C=N), 1527 (NO_{2 asym}), 1350 (NO_{2 sym}). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.60 (s, 1H, Ar–H), 8.46 (s, 1H, CH), 8.27 (d, 1H, J = 8.4 Hz, Ar–H), 8.13 (d, 1H, J = 7.5 Hz, Ar–H), 7.59 (t, 1H, J = 7.8, 8.1 Hz, Ar–H), 7.37–7.26 (m, 5H, Ar–H), 4.88 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 159.17, 148.65, 138.55, 137.86, 133.65, 129.62, 128.64, 128.08, 127.30, 125.14, 123.09, 64.96. ESI-MS (m/z): 241.46 [M + H]⁺.

(4-Nitrobenzylidene)(phenyl)methylamine (3c)

Red solid, m.p. 31–33 °C, yield: 89%, R_f = 0.53 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₂N₂O₂) Calc. C 69.99, H 5.03, N 11.66; found: C 69.95, H 5.01, N 11.61%. IR (neat): ν (cm⁻¹) 1641 (C=N), 1518 (NO_{2 asym}), 1343 (NO_{2 sym}). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.46 (s, 1H, CH), 8.27 (d, 2H, J = 8.4 Hz, Ar–H), 7.94 (d, 2H, J = 8.4 Hz, Ar–H), 7.40–7.26 (m, 5H, Ar–H), 4.89 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 159.45, 149.12, 141.63, 138.48, 128.94, 128.66, 128.08, 127.32, 123.87, 65.20. ESI-MS (m/z): 241.50 [M + H]⁺.

(2-Chlorobenzylidene)(phenyl)methylamine (3d)

Light yellow oil, yield: 93%, R_f = 0.78 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₂ClN) Calc. C 73.20, H 5.27, N 6.10; found: C 73.17, H 5.23, N 6.08%. IR (neat): ν (cm⁻¹) 1639 (C=N), 754 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.85 (s, 1H, CH), 8.10 (d, 1H, J = 7.5 Hz, Ar–H), 7.39–7.30 (m, 6H, Ar–H), 7.29–7.24 (m, 2H, Ar–H), 4.87 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 157.78, 148.93, 138.50, 133.50, 131.24, 130.75, 129.90, 128.63, 128.15, 127.26, 124.32, 65.27. ESI-MS (m/z): 230.53 [M + H]⁺.

(4-Chlorobenzylidene)(phenyl)methylamine (3e)

Light yellow oil, yield: 94%, R_f = 0.71 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₂ClN) Calc. C 73.20, H 5.27, N 6.10; found: C 73.15, H 5.25, N 6.07%. IR (neat): ν (cm⁻¹) 1646 (C=N), 735 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.34 (s, 1H, CH), 7.71 (d, 2H, J = 7.8 Hz, Ar–H), 7.38 (d, 2H, J = 7.8 Hz, Ar–H), 7.33–7.25 (m, 5H, Ar–H), 4.81 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.56, 139.05, 136.73, 134.65, 129.47, 128.90, 128.56, 128.01, 127.10, 65.00. ESI-MS (m/z): 230.49 [M + H]⁺.

(2-Nitrobenzylidene)(2-chlorophenyl)methylamine (3f)

Yellow solid, m.p. 82–84 °C, yield: 86%, R_f = 0.52 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₁ClN₂O₂) Calc. C 61.21, H 4.04, N 10.20; found: C 61.16, H 4.02, N 10.40%. IR (neat): ν (cm⁻¹) 1641 (C=N), 1520 (NO_{2 asym}), 1330 (NO_{2 sym}), 741 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.77 (s, 1H, CH), 8.05 (d, 1H, J = 7.5 Hz, Ar–H), 7.97 (d, 1H, J = 7.5 Hz, Ar–H), 7.61 (t, 1 H, J = 6.9, 7.5 Hz, Ar–H), 7.51 (t, 1H, J = 6.9, 7.5 Hz, Ar–H), 7.35 (t, 2H, J = 6.6, 8.1 Hz, Ar–H), 7.19 (d, J = 8.1 Hz, 2H, Ar–H), 4.91 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 158.77, 148.88, 136.21, 133.65, 133.54, 131.16, 130.86, 129.96, 129.88, 129.48, 128.58, 127.01, 124.35, 62.14. ESI-MS (m/z): 275.31 [M + H]⁺.

(3-Nitrobenzylidene)(2-chlorophenyl)methylamine (3g)

Light yellow solid, m.p. 62–64 °C, yield: 91%, R_f = 0.59 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₁ClN₂O₂) Calc. C 61.21, H 4.04, N 10.20; found: C 61.18, H 4.01, N 10.16%. IR (neat): ν (cm⁻¹) 1650 (C=N), 1527 (NO_{2 asym}), 1352 (NO_{2 sym}), 752 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.64 (s, 1H, Ar–H), 8.49 (s, 1H, CH), 8.31 (d, 1H, J = 6.9 Hz, Ar–H), 8.16 (d, 1H, J = 7.2 Hz, Ar–H), 7.63 (t, 1H, J = 7.2, 8.1 Hz, Ar–H), 7.44 (t, 2H, J = 7.6 Hz, Ar–H), 7.28 (d, 2H, J = 7.6 Hz, Ar–H), 4.98 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.08, 148.61, 137.73, 136.19, 133.72, 133.56, 129.93, 129.66, 129.46, 128.61, 127.06, 125.26, 123.07, 61.84. ESI-MS (m/z): 275.54 [M + H]⁺.

(4-Nitrobenzylidene)(2-chlorophenyl)methylamine (3h)

Yellow solid, m.p. 72–74 °C, yield: 84%, R_f = 0.66 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₁ClN₂O₂) Calc. C 61.21, H 4.04, N 10.20; found: C 61.17, H 4.10, N 10.15%. IR (neat): ν (cm⁻¹) 1641 (C=N), 1520 (NO_{2 asym}), 1335 (NO_{2 sym}), 750 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.50 (s, 1H, CH), 8.30 (d, 2H, J = 8.4 Hz, Ar–H), 7.98 (d, 2H, J = 8.7 Hz, Ar–H), 7.44 (t, 2H, J = 5.4 Hz, Ar–H), 7.33–7.25 (m, 2H, Ar–H), 4.99 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.39, 149.13, 141.47, 136.10, 133.55, 129.90, 129.46, 128.98, 128.63, 127.05, 123.88, 62.06. ESI-MS (m/z): 275.67 [M + H]⁺.

(2-Chlorobenzylidene)(2-chlorophenyl)methylamine (3i)

Yellow oil, yield: 87%, R_f = 0.68 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₁Cl₂N) Calc. C 63.66, H 4.20, N 5.30; found: C 63.61, H 4.17, N 5.26%. IR (neat): ν (cm⁻¹) 1641 (C=N), 750 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.80 (s, 1H, CH), 8.05 (d, 1H, J = 6.6 Hz, Ar–H), 7.40–7.23 (m, 4H, Ar–H), 7.21–7.13 (m, 3H, Ar–H), 4.88 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.82, 137.70, 136.21, 134.87, 133.41, 130.31, 129.98, 129.63, 129.31, 128.58, 128.21, 127.01, 126.72, 61.84. ESI-MS (m/z): 264.63 [M + H]⁺.

(4-Chlorobenzylidene)(2-chlorophenyl)methylamine (3j)

Light yellow solid, m.p. 48–50 °C, yield: 80%, R_f = 0.71 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₁Cl₂N) Calc. C 63.66, H 4.20, N 5.30; found: C 63.58, H 4.15, N 5.28%. IR (neat): ν (cm⁻¹) 1642 (C=N), 756 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.30 (s, 1H, CH), 7.66 (d, 2H, J = 8.4 Hz, Ar–H), 7.33 (d, 3H, J = 8.4 Hz, Ar–H), 7.22–7.13 (m, 3H, Ar–H), 4.83 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 161.52, 136.88, 136.74, 134.52, 133.44, 129.74, 129.49, 129.35, 128.93, 128.35, 126.94, 61.83. ESI-MS (m/z): 264.41 [M + H]⁺.

(2-Nitrobenzylidene)(furan-2-yl)methylamine (3k)

Brown solid, m.p. 60–62 °C, yield: 94%, R_f = 0.55 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₂H₁₀N₂O₃) Calc. C 62.60, H 4.38, N 12.17; found: C 62.61, H 4.27, N 12.16%. IR (neat): ν (cm⁻¹) 1639 (C=N), 1523 (NO_{2 asym}), 1346 (NO_{2 sym}), 1149 (C–O–C). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.54 (s, 1H, CH), 8.31 (d, 1H, J = 8.1 Hz, Ar–H), 7.98 (d, 1H, J = 7.8 Hz, Ar–H), 7.67 (t, 1H, J = 7.8 Hz, Ar–H), 7.51 (t, 1H, J = 8.1 Hz, Ar–H), 7.34 (d, 1H, J = 7.1 Hz, Ar–H), 6.42 (t, 1H, J = 3.4 Hz, Ar-–H), 5.89 (d, 1H, J = 5.0 Hz, Ar–H), 4.67 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.56, 150.98, 148.65, 142.13, 135.23, 132.70, 132.22, 130.58, 122.67, 110.62, 106.83, 56.78. ESI-MS (m/z): 231.43 [M + H]⁺.

(3-Nitrobenzylidene)(furan-2-yl)methylamine (3l)

Yellow solid, m.p. 58–60 °C, yield: 92%, R_f = 0.54 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₂H₁₀N₂O₃) Calc. C 62.60, H 4.38, N 12.17; found: C 62.63, H 4.49, N 12.13%. IR (neat): ν (cm⁻¹) 1642 (C=N), 1529 (NO_{2 asym}), 1346 (NO_{2 sym}), 1147 (C–O–C). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.60 (s, 1H, Ar–H), 8.41 (s, 1H, CH), 8.29 (d, 1H, J = 8.1 Hz, Ar–H), 8.13 (d, 1H, J = 7.5 Hz, Ar–H), 7.61 (t, 1H, J = 7.8, 8.1 Hz, Ar–H), 7.42 (d, 1H, J = 7.8 Hz, Ar–H), 6.39 (t, 1H, J = 1.8, 2.7 Hz, Ar–H), 6.33 (d, 1H, J = 3.3 Hz, Ar–H), 4.85 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.94, 150.87, 148.16, 142.30, 141.05, 134.87, 129.56, 124.64, 123.67, 110.81, 106.72, 57.34. ESI-MS (m/z): 231.54 [M + H]⁺.

(4-Nitrobenzylidene)(furan-2-yl)methylamine (3m)

Yellow solid, m.p. 82–84 °C, yield: 89%, R_f = 0.66 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₂H₁₀N₂O₃) Calc. C 62.60, H 4.38, N 12.17; found: C 62.56, H 4.31, N 12.14%. IR (neat): ν (cm⁻¹) 1648 (C=N), 1510 (NO_{2 asym}), 1341 (NO_{2 sym}), 1153 (C–O–C). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.42 (s, 1H, CH), 8.29 (d, 2H, J = 8.7 Hz, Ar–H), 7.95 (d, 2H, J = 8.7 Hz, Ar–H), 7.43 (d, 1H, J = 5.4 Hz, Ar–H), 6.39 (t, 1H, J = 1.8 Hz, Ar–H), 6.33 (d, 1H, J = 2.7 Hz, Ar–H), 4.87 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.54, 151.40, 149.16, 142.53, 141.37, 129.02, 123.85, 110.49, 108.06, 57.14. ESI-MS (m/z): 231.49 [M + H]⁺.

(2-Chlorobenzylidene)(furan-2-yl)methylamine (3n)

Yellow oil, yield: 82%, R_f = 0.68 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₂H₁₀ClNO) Calc. C 65.61, H 4.59, N 6.38; found: C 65.58, H 4.60, N 6.34%. IR (neat): ν (cm⁻¹) 1639 (C=N), 1149 (C–O–C), 760 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.82 (s, 1H, CH), 8.07 (d, 1H, J = 7.5 Hz, Ar–H), 7.42–7.36 (m, 3H, Ar–H), 7.34–7.28 (m, 1H, Ar–H), 6.37 (t, 1H, J = 1.8, 3.0 Hz, Ar–H), 6.30 (d, 1H, J = 3.0 Hz, Ar–H), 4.84 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 159.85, 152.09, 142.29, 135.31, 132.97, 131.78, 129.77, 128.49, 127.30, 110.40, 107.57, 57.46. ESI-MS (m/z): 220.32 [M + H]⁺.

(4-Chlorobenzylidene)(furan-2-yl)methylamine (3o)

Brown oil, yield: 81%, R_f = 0.61 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₂H₁₀ClNO) Calc. C 65.61, H 4.59, N 6.38; found: C 65.57, H 4.55, N 6.36%. IR (neat): ν (cm⁻¹) 1641 (C=N), 1128 (C–O–C), 750 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.44 (s, 1H, CH), 7.71 (d, 2H, J = 6.6 Hz, Ar–H), 7.41–7.18 (m, 3H, Ar–H), 6.36 (t, 1H, J = 7.5 Hz, Ar–H), 6.29 (d, 1H, J = 7.8 Hz, Ar–H), 4.79 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 161.67, 152.04, 142.31, 136.90, 134.38, 129.53, 128.88, 110.41, 107.65, 57.07. ESI-MS (m/z): 220.50 [M + H]⁺.

(2-Nitrobenzylidene)(4-fluorophenyl)methylamine (3p)

Brown solid, m.p. 44–45 °C, yield: 95%, R_f = 0.50 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₁FN₂O₂) Calc. C 65.11, H 4.29, N 10.85; found: C 65.10, H 4.30, N 10.80%. IR (neat): ν (cm⁻¹) 1641 (C=N), 1510 (NO_{2 asym}), 1341 (NO_{2 sym}), 1156 (C–F). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.84 (s, 1H, CH), 8.08 (d, J = 8.1 Hz, 2H, Ar–H), 7.68 (d, 2H, J = 8.4 Hz, Ar–H), 7.34 (s, 2H, Ar–H), 7.07 (s, 2H, Ar–H), 4.86 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.46, 157.94, 148.88, 134.26, 133.57, 131.12, 130.86, 129.84, 124.36, 115.57, 115.28, 64.45. ESI-MS (m/z): 259.65 [M + H]⁺.

(3-Nitrobenzylidene)(4-fluorophenyl)methylamine (3q)

Yellow solid, m.p. 83–84 °C, yield: 85%, R_f = 0.53 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₁FN₂O₂) Calc. C 65.11, H 4.29, N 10.85; found: C 65.07, H 4.21, N 10.80%. IR (neat): ν (cm⁻¹) 1641 (C=N), 1508 (NO_{2 asym}), 1350 (NO_{2 sym}), 1160 (C–F). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.62 (s, 1H, Ar–H), 8.47 (s, 1H, CH), 8.30 (d, 1H, J = 8.1 Hz, Ar–H), 8.14 (d, 1H, J = 7.8 Hz, Ar–H), 7.62 (t, 1H, J = 7.8, 8.1 Hz, Ar–H), 7.33 (t, 2H, J = 8.4 Hz, Ar–H), 7.07 (t, 2H, J = 8.7 Hz, Ar–H), 4.86 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.46, 159.27, 148.60, 137.68, 134.27, 133.67, 129.65, 125.25, 123.07, 115.59, 115.31, 64.16. ESI-MS (m/z): 259.46 [M + H]⁺.

(4-Nitrobenzylidene)(4-fluorophenyl)methylamine (3r)

Light yellow solid, m.p. 62–64 °C, yield: 90.7%, R_f = 0.61 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₁FN₂O₂) Calc. C 65.11, H 4.29, N 10.85; found: C 65.06, H 4.23, N 10.81%. IR (neat): ν (cm⁻¹) 1644 (C=N), 1508 (NO_{2 asym}), 1341 (NO_{2 sym}), 1158 (C–F). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.40 (s, 1H, CH), 8.21 (d, 2H, J = 8.7 Hz, Ar–H), 7.87 (d, 2H, J = 8.7 Hz, Ar–H), 7.27–7.20 (m, 2H, Ar–H), 6.98 (t, 2H, J = 8.7 Hz, Ar–H), 4.78 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 159.56, 149.11, 141.46, 134.20, 129.66, 128.96, 123.89, 115.60, 115.32, 64.39. ESI-MS (m/z): 257.36 [M − H]^–.

(2-Chlorobenzylidene)(4-fluorophenyl)methylamine (3s)

Yellow oil, yield: 87%, R_f = 0.77 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₁ClFN) Calc. C 67.89, H 4.48, N 5.65; found: C 67.85, H 4.45, N 5.61%. IR (neat): ν (cm⁻¹) 1639 (C=N), 1158 (C–F), 754 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.77 (s, 1H, CH), 8.01 (d, 1H, J = 7.5 Hz, Ar–H), 7.30–7.19 (m, 5H, Ar–H), 6.97 (t, 2H, J = 8.7 Hz, Ar–H), 4.75 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.96, 158.93, 138.88, 134.57, 133.07, 130.70, 130.62, 129.87, 128.97, 127.64, 113.12, 63.98. ESI-MS (m/z): 248.73 [M + H]⁺.

(4-Chlorobenzylidene)(4-fluorophenyl)methylamine (3t)

Yellow solid, m.p. 48–50 °C, yield: 95%, R_f = 0.74 (ethyl acetate/pet. ether, 20:80), Anal. (C₁₄H₁₁ClFN) Calc. C 67.89, H 4.48, N 5.65; found: C 67.82, H 4.43, N 5.63%. IR (neat): 1646 (C=N), 1214 (C–F), 1093 (C–Cl) cm⁻¹. ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.27 (s, 1H, CH), 7.64 (d, 2H, J = 8.1 Hz, Ar–H), 7.32 (d, 2H, J = 8.4 Hz, Ar–H), 7.22 (t, 2H, J = 8.1 Hz, Ar–H), 6.96 (t, 2H, J = 8.4, 8.7 Hz, Ar–H), 4.70 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 160.66, 159.13, 138.56, 134.43, 130.86, 130.54, 129.78, 127.64, 113.12, 63.45. ESI-MS (m/z): 248.45 [M + H]⁺.

General procedure for the synthesis of β-lactams (4a–g, 4i–s) and 1,3-oxazin-4-one derivatives (4h, 4t)

To a refluxing suspension of imine (1.0 equiv.), phenylacetic acid (1.5 equiv.) and triethylamine (4.0 equiv.) in toluene (10 mL) was added dropwise, a solution of phosphoryl chloride (1.1 equiv.) in toluene with constant stirring under inert atmosphere. The reaction was refluxed overnight at 110 °C. When the reaction was completed as monitored by TLC, the reaction mixture was cooled to room temperature and neutralized with sat. sodium bicarbonate solution. The mixture was extracted with ethyl acetate and the organic layer was washed with brine and dried over anhydrous Na₂SO₄. After concentration under reduced pressure, the residue was purified by silica-gel chromatography (230–400 mesh) using 30–40% ethyl acetate in petroleum ether. The title compounds (4a–g, 4i–s) were obtained in low to good yields25. The 1,3-oxazin-4-one derivatives (4h, 4t) were also obtained under the same reaction conditions in low to moderate yields ranging from 22% to 41%.

1-Benzyl-4-(2-nitrophenyl)-3-phenylazetidin-2-one (4a)

Brown solid, m.p. 100–102 °C, yield: 64%, R_f = 0.20 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₈N₂O₃) Calc. C 73.73, H 5.06, N 7.82; found: C 73.69, H 5.02, N 7.78%. IR (neat): ν (cm⁻¹) 1749 (C=O), 1518 (NO_{2 asym}), 1346 (NO_{2 sym}). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.91 (d, 1H, J = 8.1 Hz, Ar–H), 7.61–7.24 (m, 13H, Ar–H), 5.43 (d, 1H, J = 5.7 Hz, azetidinone ring-H_a), 5.14 (s, 2H, CH₂), 4.15 (d, 1H, J = 4.2 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 169.06, 147.74, 134.83, 133.26, 132.42, 131.79, 130.91, 129.07, 128.74, 128.62, 128.53, 128.21, 128.12, 127.44, 125.24, 62.37, 57.65, 45.93. ESI-MS (m/z): 359.04 [M + H]⁺.

1-Benzyl-4-(3-nitrophenyl)-3-phenylazetidin-2-one (4b)

Yellow solid, m.p. 98–100 °C, yield: 21%, R_f = 0.21 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₈N₂O₃) Calc. C 73.73, H 5.06, N 7.82; found: C 73.71, H 5.01, N 7.80%. IR (neat): ν (cm⁻¹) 1749 (C=O), 1527 (NO_{2 asym}), 1346 (NO_{2 sym}). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.93 (s, 1H, Ar–H), 7.82 (s, 1H, Ar–H), 7.32–7.19 (m, 7H, Ar–H), 7.09–6.97 (m, 5H, Ar–H), 4.98–4.93 (m, 3H, azetidinone ring-H_a, CH₂), 4.04 (d, 1H, J = 11.1 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 167.64, 147.92, 137.51, 134.85, 133.25, 131.81, 129.11, 128.66, 128.56, 128.45, 128.14, 127.45, 122.91, 122.38, 61.45, 59.08, 45.33. ESI-MS (m/z): 357.0 [M − H]⁻, 359.0 [M + H]⁺.

1-Benzyl-4-(4-nitrophenyl)-3-phenylazetidin-2-one (4c)

Light yellow solid, m.p. 160–165 °C, yield: 36%, R_f = 0.23 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₈N₂O₃) Calc. C 73.73, H 5.06, N 7.82; found: C 73.69, H 5.04, N 7.78%. IR (neat): ν (cm⁻¹) 1754 (C=O), 1510 (NO_{2 asym}), 1344 (NO_{2 sym}). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.89 (d, 2H, J = 8.7 Hz, Ar–H), 7.25 (d, 2H, J = 5.1 Hz, Ar–H), 7.14–6.89 (m, 10H, Ar–H), 4.92 (d, 1H, J = 14.7 Hz, azetidinone ring-H_a), 4.85 (s, 2H, CH₂), 3.93 (d, 1H, J = 14.7 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 167.59, 147.39, 142.77, 134.82, 131.74, 129.01, 128.68, 128.49, 128.44, 128.22, 128.17, 127.56, 123.33, 61.51, 59.12, 45.34. ESI-MS (m/z): 359.57 [M + H]⁺.

1-Benzyl-4-(2-chlorophenyl)-3-phenylazetidin-2-one (4d)

Yellow solid, m.p. 110–112 °C yield: 75%, R_f = 0.53 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₈ClNO) Calc. C 75.97, H 5.22, N 4.03; found: C 75.96, H 5.17, N 4.00%. IR (neat): ν (cm⁻¹) 1752 (C=O), 754 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.33 (t, 4H, J = 6 Hz, Ar–H), 7.26–7.25 (m, 2H, Ar–H), 7.13–7.04 (m, 8H, Ar–H), 5.24 (d, 1H, J = 4.2 Hz, azetidinone ring-H_a), 5.02 (s, 2H, CH₂), 4.89 (d, 1H, J = 4.2 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 168.59, 135.27, 133.22, 132.96, 132.15, 129.45, 129.03, 128.85, 128.71, 128.30, 128.07, 128.01, 127.95, 127.25, 126.38, 61.09, 57.14, 45.47. ESI-MS (m/z): 348.07 [M + H]⁺.

1-Benzyl-4-(4-chlorophenyl)-3-phenylazetidin-2-one (4e)

Light yellow solid, m.p. 138–140 °C, yield: 26%, R_f = 0.33 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₈ClNO) Calc. C 75.97, H 5.22, N 4.03; found: C 75.93, H 5.18, N 3.98%. IR (neat): ν (cm⁻¹) 1754 (C=O), 758 (C–Cl). ¹H NMR (500 MHz, CDCl₃) (δ, ppm): 7.37 (d, J = 4.8 Hz, 2H, Ar–H), 7.34–7.27 (m, 6H, Ar–H), 7.22–7.18 (m, 6H, Ar–H), 4.95 (d, 1H, J = 9.3 Hz, azetidinone ring-H_a), 4.16 (s, 2H, CH₂), 3.84 (d, 1H, J = 8.7 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 168.07, 135.35, 134.69, 134.54, 130.96, 129.36, 128.98, 128.89, 128.60, 127.92, 127.88, 127.78, 127.34, 65.29, 62.51, 44.77. ESI-MS (m/z): 348.0 [M + H]⁺.

1-(2-Chlorobenzyl)-4-(2-nitrophenyl)-3-phenylazetidin-2-one (4f)

Brown oil, yield: 46%, R_f = 0.26 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₇ClN₂O₃) Calc. C 67.26, H 4.36, N 7.13; found: C 67.21, H 4.33, N 7.12%. IR (neat): ν (cm⁻¹) 1756 (C=O), 1525 (NO_{2 asym}), 1344 (NO_{2 sym}), 754 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.80 (d, 2H, J = 7.8 Hz, Ar–H), 7.40–7.17 (m, 8H, Ar–H), 7.00–6.64 (m, 3H, Ar–H), 5.37 (d, 1H, J = 6.0 Hz, azetidinone ring-H_a), 4.99 (d, 1H, J = 6.6 Hz, azetidinone ring-H_b), 4.39 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 169.98, 148.56, 144.39, 137.92, 136.54, 134.75, 133.42, 133.12, 132.62, 131.81, 129.51, 129.11, 128.86, 128.37, 128.19, 126.56, 124.39, 62.76, 57.42, 45.78. ESI-MS (m/z): 393.01 [M + H]⁺.

1-(2-Chlorobenzyl)-4-(3-nitrophenyl)-3-phenylazetidin-2-one (4g)

Brown oil, yield: 35%, R_f = 0.33 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₇ClN₂O₃) Calc. C 67.26, H 4.36, N 7.13; found: C 67.22, H 4.34, N 7.11%. IR (neat): ν (cm⁻¹) 1771 (C=O), 1531 (NO_{2 asym}), 1350 (NO_{2 sym}), 756 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.77 (s, 1H, Ar–H), 7.53 (d, 1H, J = 6.6 Hz, Ar–H), 7.42–7.04 (m, 11H, Ar–H), 5.15 (d, 1H, J = 15.3 Hz, azetidinone ring-H_a), 4.37 (d, 1H, J = 15.6 Hz, azetidinone ring-H_b), 3.39 (d, 1H, J = 14.7 Hz, CH₂), 3.23 (d, 1H, J = 14.7 Hz, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 169.63, 148.94, 145.34, 139.72, 136.41, 134.92, 133.91, 133.67, 132.52, 131.98, 129.71, 129.01, 128.56, 128.30, 128.09, 127.56, 125.39, 62.87, 57.49, 45.78. ESI-MS (m/z): 393.31 [M + H]⁺.

1-(2-Chlorobenzyl)-4-(2-chlorophenyl)-3-phenylazetidin-2-one (4i)

Yellow oil, yield: 45%, R_f = 0.38 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₇Cl₂NO) Calc. C 69.12, H 4.48, N 3.66; found: C 69.08, H 4.50, N 3.62%. IR (neat): ν (cm⁻¹) 1765 (C=O), 754 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.55 (d, 1H, J = 7.5 Hz, Ar–H), 7.35 (t, 2H, J = 4.2 Hz, Ar–H), 7.28–7.13 (m, 7H, Ar–H), 7.10–7.03 (m, 3H, Ar–H), 5.42 (d, 1H, J = 14.4 Hz, azetidinone ring-H_a), 4.30 (d, 1H, J = 15.9 Hz, azetidinone ring-H_b), 3.43 (s, 2H, CH₂). ¹³C NMR (125 MHz, CDCl₃) (δ, ppm): 168.47, 136.56, 134.07, 131.92, 130.72, 130.45, 130.26, 129.54, 129.39, 128.74, 128.26, 128.21, 128.07, 127.39, 127.20, 126.38, 126.07, 66.54, 57.80, 44.56. ESI-MS (m/z): 382.04 [M + H]⁺.

1-(2-Chlorobenzyl)-4-(4-chlorophenyl)-3-phenylazetidin-2-one (4j)

Yellow oil, yield: 36%, R_f = 0.45 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₇Cl₂NO) Calc. C 69.12, H 4.48, N 3.66, found: C 69.10, H 4.42, N 3.65%. IR (neat): ν (cm⁻¹) 1765 (C=O), 756 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.55 (d, 2H, J = 6.6 Hz, Ar–H), 7.50 (t, 2H, J = 6.0 Hz, Ar–H), 7.38–7.17 (m, 8H, Ar–H), 7.04 (d, 1H, J = 8.4 Hz, Ar–H), 5.29 (d, 1H, J = 15.6 Hz, azetidinone ring-H_a), 4.56 (s, 2H, CH₂), 4.29 (d, 1H, J = 15.0 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 167.56, 139.71, 138.94, 137.67, 136.51, 134.51, 133.89, 129.81, 129.32, 128.91, 128.43, 128.23, 127.56, 126.59, 124.91, 62.43, 57.49, 45.30. ESI-MS (m/z): 382.34 [M + H]⁺.

1-(Furan-2-ylmethyl)-4-(2-nitrophenyl)-3-phenylazetidin-2-one (4k)

Brown solid, m.p. 122–124 °C, yield: 44%, R_f = 0.30 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₀H₁₆N₂O₄) Calc. C 68.96, H 4.63, N 8.04; found: C 68.94, H 4.60, N 7.99%. IR (neat): ν (cm⁻¹) 1762 (C=O), 1525 (NO_{2 asym}), 1344 (NO_{2 sym}). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.92 (d, 1H, J = 8.1 Hz, Ar–H), 7.49 (d, 1H, J = 6.9 Hz, Ar–H), 7.38–7.28 (m, 5H, Ar–H), 7.08–6.97 (m, 4H, Ar–H), 6.29 (d, 1H, J = 17.1 Hz, Ar–H), 5.54 (d, 1H, J = 5.7 Hz, azetidinone ring-H_a), 5.12 (d, 1H, J = 5.7 Hz, azetidinone ring-H_b), 4.88 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 168.42, 147.79, 147.21, 142.10, 136.71, 134.94, 133.20, 129.91, 129.42, 128.91, 128.53, 127.56, 121.91, 110.91, 107.89, 52.43, 51.50, 43.30. ESI-MS (m/z): 349.0 [M + H]⁺.

1-(Furan-2-ylmethyl)-4-(3-nitrophenyl)-3-phenylazetidin-2-one (4l)

Yellow oil, yield: 44%, R_f = 0.22 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₀H₁₆N₂O₄) Calc. C 68.96, H 4.63, N 8.04; found: C 68.92, H 4.59, N 8.02%. IR (neat): ν (cm⁻¹) 1754 (C=O), 1531 (NO_{2 asym}), 1350 (NO_{2 sym}). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.22 (d, 2H, J = 7.8 Hz, Ar–H), 8.17 (s, 1H, Ar–H), 7.67–7.53 (m, 2H, Ar–H), 7.39–7.24 (m, 5H, Ar–H), 6.27–6.17 (m, 2H, Ar–H), 4.79 (d, 1H, J = 15.9 Hz, azetidinone ring-H_a), 4.57 (s, 2H, CH₂), 4.22 (d, 1H, J = 15.6 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 168.56, 148.34, 148.09, 142.14, 139.76, 136.42, 129.72, 129.49, 128.26, 127.61, 127.58, 126.87, 122.32, 109.83, 106.89, 56.23, 50.62, 45.42. ESI-MS (m/z): 349.0 [M + H]⁺.

1-(Furan-2-ylmethyl)-4-(4-nitrophenyl)-3-phenylazetidin-2-one (4m)

Brown oil, yield: 24%, R_f = 0.29 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₀H₁₆N₂O₄) Calc. C 68.96, H 4.63, N 8.04; found: C 68.94, H 4.61, N 8.01%. IR (neat): ν (cm⁻¹) 1752 (C=O), 1518 (NO_{2 asym}), 1344 (NO_{2 sym}). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.87 (s, 2H, Ar–H), 7.48–6.90 (m, 8H, Ar–H), 6.16 (d, 2H, J = 26.4 Hz, Ar–H), 4.92 (d, 1H, J = 21.3 Hz, azetidinone ring-H_a), 4.72 (d, 1H, J = 15.3 Hz, azetidinone ring-H_b), 4.20 (s, 2H, CH₂). ¹³C NMR (125 MHz, CDCl₃) (δ, ppm): 167.49, 148.24, 147.37, 142.80, 131.64, 128.48, 128.43, 128.01, 127.57, 123.21, 110.60, 109.29, 61.59, 60.04, 37.97. ESI-MS (m/z): 349.06 [M + H]⁺.

4-(2-Chlorophenyl)-1-(furan-2-ylmethyl)-3-phenylazetidin-2-one (4n)

Yellow solid, m.p. 131–133 °C yield: 72%, R_f = 0.45 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₀H₁₆ClNO₂) Calc. C 71.11, H 4.77, N 4.15; found: C 71.08, H 4.74, N 4.14%. IR (neat): ν (cm⁻¹) 1756 (C=O), 735 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.39–7.20 (m, 7H, Ar–H), 7.14–7.06 (m, 4H, Ar–H), 6.34 (d, 1H, J = 7.8 Hz, Ar–H), 4.91 (d, 1H, J = 16.5 Hz, azetidinone ring-H_a), 4.28 (d, 1H, J = 9.6 Hz, azetidinone ring-H_b), 3.46 (s, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 168.78, 147.91, 142.25, 138.41, 136.53, 133.94, 129.83, 129.31, 128.56, 127.79, 127.56, 126.93, 125.91, 110.87, 107.76, 51.56, 51.61, 42.70. ESI-MS (m/z): 338.26 [M + H]⁺.

4-(4-Chlorophenyl)-1-(furan-2-ylmethyl)-3-phenylazetidin-2-one (4o)

Light yellow oil, yield: 32%, R_f = 0.44 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₀H₁₆ClNO₂) Calc. C 71.11, H 4.77, N 4.15; found: C 71.10, H 4.78, N 4.12%. IR (neat): ν (cm⁻¹) 1756 (C=O), 737 (C–Cl) cm⁻¹. ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.36–7.13 (m, 7 H, Ar–H), 7.04 (d, 2H, J = 6.3 Hz, Ar–H), 6.83 (d, 2H, J = 5.1 Hz, Ar–H), 6.26 (d, 1H, J = 3.0 Hz, Ar–H), 4.83 (d, 1H, J = 11.7 Hz, azetidinone ring-H_a), 4.39 (s, 2H, CH₂), 4.26 (d, 1H, J = 11.7 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 168.14, 148.70, 142.79, 135.25, 130.99, 129.34, 128.90, 128.53, 128.27, 127.90, 127.46, 126.72, 110.56, 65.49, 63.37, 37.59. ESI-MS (m/z): 338.09 [M + H]⁺.

1-(4-Fluorobenzyl)-4-(2-nitrophenyl)-3-phenylazetidin-2-one (4p)

Brown solid, m.p. 146–148 °C yield: 46%, R_f = 0.19 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₇FN₂O₃) Calc. C 70.20, H 4.55, N 7.44; found: C 70.21, H 4.52, N 7.41%. IR (neat): ν (cm⁻¹) 1752 (C=O), 1510 (NO_{2 asym}), 1344 (NO_{2 sym}), 1160 (C–F). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.89 (d, 1H, J = 8.1 Hz, Ar–H), 7.46 (t, 1H, J = 7.8 Hz, Ar–H), 7.36 (d, 1H, J = 6.9 Hz, Ar–H) 7.34–7.21 (m, 3H, Ar–H), 7.22–7.16 (m, 7H, Ar–H), 5.41 (d, 1H, J = 5.7 Hz, azetidinone ring-H_a), 5.06 (d, 1H, J = 6.0 Hz, azetidinone ring-H_b), 4.12 (s, 2H, CH₂). ¹³C NMR (125 MHz, CDCl₃) (δ, ppm): 169.01, 160.94, 134.96, 130.87, 130.40, 130.17, 128.95, 128.79, 128.61, 128.51, 128.34, 128.14, 127.49, 115.94, 85.70, 62.42, 57.66, 45.21. ESI-MS (m/z): 377.0 [M + H]⁺.

1-(4-Fluorobenzyl)-4-(3-nitrophenyl)-3-phenylazetidin-2-one (4q)

Brown oil, yield: 42%, R_f = 0.14 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₇FN₂O₃) Calc. C 70.20, H 4.55, N 7.44; found: C 70.18, H 4.53, N 7.46%. IR (neat): ν (cm⁻¹) 1754 (C=O), 1531 (NO_{2 asym}), 1348 (NO_{2 sym}), 1160 (C–F) cm⁻¹. ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 8.12 (s, 1H, Ar–H), 7.94 (d, 2H, J = 5.4 Hz, Ar–H), 7.84 (d, 2H, J = 4.5 Hz, Ar–H), 7.78–7.72 (m, 2H, Ar–H), 7.32 (t, 2H, J = 7.9 Hz, Ar–H), 7.12–7.10 (m, 2H, Ar–H), 6.75 (d, 2H, J = 5.1 Hz, Ar–H), 5.22 (d, 1H, J = 11.4 Hz, azetidinone ring-H_a), 4.93 (s, 2H, CH₂), 4.88 (d, 1H, J = 10.8 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 167.81, 161.25, 148.18, 137.53, 134.90, 133.22, 132.64, 132.33, 130.91, 129.17, 128.77, 128.33, 127.51, 126.79, 115.64, 65.58, 59.10, 47.05. ESI-MS (m/z): 377.0 [M + H]⁺.

1-(4-Fluorobenzyl)-4-(4-nitrophenyl)-3-phenylazetidin-2-one (4r)

Yellow solid, m.p. 167–169 °C, yield: 22%, R_f = 0.28 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₇FN₂O₃) Calc. C 70.20, H 4.55, N 7.44; found: C 70.16, H 4.52, N 7.42%. IR (neat): ν (cm⁻¹) 1754 (C=O), 1508 (NO_{2 asym}), 1344 (NO_{2 sym}), 1160 (C–F). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.97 (d, 2H, J = 8.7 Hz, Ar–H), 7.20–6.95 (m, 12H, Ar–H), 4.96–4.89 (m, 3H, CH_2, azetidinone ring-H_a), 3.98 (d, 1H, J = 14.7 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 167.78, 161.12, 147.50, 142.59, 131.62, 130.49, 130.38, 128.47, 128.20, 127.62, 123.38, 116.11, 115.82, 61.58, 59.14, 44.61. ESI-MS (m/z): 376.99 [M + H]⁺.

1-(4-Fluorobenzyl)-4-(2-chlorophenyl)-3-phenylazetidin-2-one (4s)

Yellow solid, m.p. 96–98 °C yield: 63%, R_f = 0.37 (ethyl acetate/pet. ether, 20:80), Anal. (C₂₂H₁₇ClFNO) Calc. C 72.23, H 4.68, N 3.83; found: C 72.21, H 4.65, N 3.81%. IR (neat): ν (cm⁻¹) 1754 (C=O), 1160 (C–F), 754 (C–Cl). ¹H NMR (300 MHz, CDCl₃) (δ, ppm): 7.26 (d, 1H, J = 8.1 Hz, Ar–H), 7.19 (t, 2H, J = 7.7 Hz, Ar–H), 7.10 (d, 2H, J = 7.7 Hz, Ar–H), 7.05–7.01 (m, 6H, Ar–H), 6.81 (t, 2H, J = 6.1 Hz, Ar–H), 5.37 (s, 2H, CH₂), 5.04 (d, 1H, J = 15.0 Hz, azetidinone ring-H_a), 4.30 (d, 1H, J = 15.0 Hz, azetidinone ring-H_b). ¹³C NMR (75 MHz, CDCl₃) (δ, ppm): 169.21, 161.23, 140.62, 138.22, 133.42, 132.78, 130.27, 129.69, 129.38, 128.57, 128.32, 127.54, 127.31, 126.38, 115.23, 54.58, 53.24, 48.61. ESI-MS (m/z): 366.02 [M + H]⁺.

3-(2-Chlorobenzyl)-6-benzyl-2-(4-nitrophenyl)-5-phenyl-2,3-dihydro-1,3-oxazin-4-one (4h)

Yellow oil, yield: 22%, R_f = 0.40 (ethyl acetate/pet. ether, 20:80), Anal. (C₃₀H₂₃ClN₂O₄) Calc. C 70.52, H 4.54, N 5.48; found: C 70.49, H 4.51, N 5.44%. IR (neat): ν (cm⁻¹) 1767 (C=O), 1523 (NO_{2 asym}), 1346 (NO_{2 sym}), 756 (C–Cl). ¹H NMR (500 MHz, CDCl₃) (δ, ppm): 8.25 (d, 1H, J = 8.5 Hz), 7.97 (d, 2H, J = 8.0 Hz), 7.89 (d, 1H, J = 8.5 Hz), 7.35–7.10 (m, 10 H), 6.81 (d, 2H, J = 8.0 Hz), 6.33 (s, 1H), 5.25 (d, 1H, J = 15.5 Hz), 5.07 (s, 1H), 4.46 (q, 2H, J = 14.7 Hz), 3.44 (d, 1H, J = 9.3 Hz), 3.32 (d, 1H, J = 8.7 Hz). ¹³C NMR (125 MHz, CDCl₃) (δ, ppm): 165.61, 148.23, 142.72, 135.13, 133.83, 131.89, 131.40, 130.87, 130.71, 129.86, 129.57, 128.99, 128.39, 127.81, 127.43, 126.91, 126.28, 123.24, 115.00, 86.19, 69.31, 45.39, 43.13, 37.48. ESI-MS (m/z): 511.2 [M + H]⁺.

3-(4-Fluorobenzyl)-6-benzyl-2-(4-chlorophenyl)-5-phenyl-2,3-dihydro-1,3-oxazin-4-one (4t)

Light yellow solid, m.p. 119–121 °C, yield: 41%, R_f = 0.42 (ethyl acetate/pet. ether, 20:80), Anal. (C₃₀H₂₃ClFNO₂) Calc. C 74.45, H 4.79, N 2.89; found: C 74.41, H 4.76, N 2.85%. IR (neat): ν (cm⁻¹) 1754 (C=O), 1158 (C–F), 748 (C–Cl) cm⁻¹. ¹H NMR (500 MHz, CDCl₃) (δ, ppm): 7.22–6.88 (m, 15H), 6.82 (d, 2H, J = 6.3 Hz), 6.07 (s, 1H), 5.22 (d, 1H, J = 15.3 Hz), 3.95 (d, 2H, J = 15.3 Hz), 3.37 (q, 2H, J = 14.7 Hz). ¹³C NMR (125 MHz, CDCl₃) (δ, ppm): 168.02, 160.93, 135.53, 134.56, 130.95, 129.84, 129.42, 129.01, 128.80, 128.53, 128.26, 127.26, 126.66, 115.64, 86.64, 65.28, 46.69, 44.04. ESI-MS (m/z): 482.0 [M − H]⁻, 484.0 [M + H]⁺.

X-ray crystallographic analysis

The structures of the compounds 4e and 4t were unequivocally established by X-ray crystallographic analysis. Single crystal of 4e was obtained through the slow evaporation of its EtOAc–DCM solution and single crystal of 4t was obtained through the slow evaporation of its EtOAc–hexane solution. Suitable crystals (C₂₂H₁₈ClNO, compound 4e; C₃₀H₂₃ClFNO_2, compound 4t) were selected and analyzed on a D8 Venture Dual Source 100 CMOS diffractometer (Karlsruhe, Germany) equipped with Cu radiation (Cu Kα = 1.54178 Å). Intensity data were collected at 100 K using φ and ω scans. No significant loss in intensities was observed during data collection. Multi-scan absorption corrections were applied to the intensity data empirically using SADABS26. Data collection, reduction and refinement were performed using APEX2 V2014.5-026, SAINT V8.34A27 software. Crystal structures were solved by direct methods using SHELXT-201428 and refined with full-matrix least-squares based on F² using SHELXT-201428. All non-hydrogen atoms were refined anisotropically. Hydrogens were first located in the Fourier difference map, then positioned geometrically and allowed to ride on their respective parent atoms. The molecular graphics and crystallographic illustrations were prepared using XP (Bruker AXS, 2000)29 and MERCURY30.

Pharmacokinetics and in silico toxicity

The pharmacokinetic properties and probable toxicity of all synthesized compounds were analyzed with ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity properties) and TOPKAT (TOxicity Prediction by Komputer Assisted Technology) tools of Discovery Studio (DS) 3.531. The movement of drug molecules, from the site of administration into the bloodstream, depends on physicochemical properties of compounds. Aqueous solubility (log S), lipophilicity (clog P), polar surface area (PSA) and molecular weight (MW) are important parameters which define absorption, movement and action of drug molecule. Plasma–protein binding prediction tool is used to measure the degree to which drug molecule binds to plasma proteins of blood and affects diffusion of drug from blood to the site of action (cells/tissues). CYPs (cytochrome P450) belong to the family of biotransformation enzymes that play crucial role in drug metabolism. This analysis provides indications to predict drug disposition in human body, their pharmacological and toxicological effects. Hepato-toxocity level predicts the probability of drugs to cause liver injuries. The carcinogenic and mutagenic effects of all synthesized compounds were analyzed with TOPKAT tool.

Antibacterial activity

All the synthesized compounds (4a–t) were evaluated for their in vitro antibacterial potential against E. coli (MTCC 739) and K. pneumoniae (MTCC 109). Ampicillin was used as a positive control. The minimum inhibitory and bactericidal concentrations (MICs and MBCs) were determined using microbroth dilution 96-well microtitre plates method32. All the compounds were dissolved in DMSO and serially diluted in broth medium to achieve the final concentration of DMSO less than 4%. The nutrient broth, which contained logarithmic serially two fold diluted amount of test compounds and control were inoculated with approximately 5 × 10⁵ cells/mL of actively dividing bacterial cells and incubated for 24 h at 37 °C. After incubation period each well was analyzed for the presence or absence of visual growth of bacterial cells. The lowest concentration of the tested compound at which no visible growth occurs represents its MIC value. Furthermore, 100 µL of aliquots were drawn from the wells in which no bacterial growth occurs and spread on nutrient agar plates. The inoculated plates were incubated at 37 °C for 24 h. The MBC was determined as the lowest concentration of the compound that resulted in the 99.9% killing of the inoculum. To achieve precision, all assays were carried out in triplicate.

Hemolytic assay

The toxicity of the active compounds was checked by hemolytic assay on hRBCs33. Human erythrocytes from healthy individuals were collected in tubes containing EDTA as anti-coagulant. The erythrocytes were harvested by centrifugation for 10 min at 2000 rpm, 20 °C and washed three times in PBS. To the pellet, PBS was added to yield a 10% (v/v) erythrocytes/PBS suspension. The 10% suspension was then diluted 1:10 in PBS. From each suspension, 100 µL was added in triplicate to 100 µL of a different dilution series of test compounds in the same buffer in microcentrifuge tubes. Total hemolysis was achieved with 1% Triton X-100. The tubes were incubated for 1 h at 37 °C and then centrifuged for 10 min at 2000 rpm and room temperature. From the supernatant fluid, 150 µL was transferred to a flat-bottomed microtiter plate (Tarson, Kolkata, India), and the absorbance was measured spectrophotometrically at 450 nm. The hemolysis percentage was calculated by the following equation:

Cytotoxicity studies (MTT assay)

MTT, Dulbecco’s modified Eagle’s medium (DMEM), 0.25% trypsin, 0.02% EDTA mixture were purchased from HiMedia (Mumbai, India) and fetal bovine serum (FBS) was obtained from Gibco (Grand Island, NY). CHO cell line was procured from National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured and maintained as monolayer in DMEM containing 10% FBS and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin, HiMedia) at 37 °C in humidified atmosphere of 5% CO₂. The cells were sub-cultured twice in a week.

In vitro cytotoxicity of the compounds (4d, 4h, 4k and 4l) was evaluated by the standard MTT assay34 on the CHO cell line. Cell count of 1 × 10⁴ cells/well were seeded into 96-well plates (200 µL/well) and incubated for 24 h before treatment. The cells were then treated with variable concentration (50–700 µg/mL) of the compounds. After 48 h of incubation at 37 °C, the medium was removed and 20 μL of MTT at a concentration of 5 mg/mL in PBS was added to each well. The plates were further incubated at 37 °C for 4 h. Formazan crystals, formed by mitochondrial reduction of MTT, were solubilized in DMSO (200 μL/well) and quantification was performed by reading the absorbance at 570 nm after incubation period of 15 min on the iMark Microplate Reader (Bio-Rad, Hercules, CA). All assays were carried out in triplicate. Percent viability was taken as the relative absorbance of treated versus control (untreated) cells.

Homology modeling

The primary sequence of PBP-5 (d-alanyl-d-alanine carboxypeptidase) from K. pneumoniae was obtained from Expasy server (UniProt ID: W9BNN9). PDB-BLAST for PBP-5 (K. pneumoniae) showed 94% sequence homology and 85% query coverage with PBP-5 (PDB ID: 1NZO; E. coli) and selected as a suitable template to model the protein. This template was aligned and structurally conserved regions were calculated. MODELLER 9.9 (http://www.salilab.org/modeller/) was used to predict the three-dimensional model for PBP-5 (K. pneumoniae). The overall stereochemical quality of predicted model was assessed on SAVS server35^,36. The root mean square deviation between the main chain atoms of models and the respective templates was calculated by structural superimpositions of predicted structures on their respective templates using PyMOL37.

Molecular docking

In order to rationalize the mechanism of PBP-5 inhibition by β-lactams, molecular docking was carried out for the selected compound using Auto Dock 4.238. Lamarckian Genetic Algorithm was used to carry out the docking analysis39. For binding energy in the docking step, the Van der Waals interaction representing as a Lennard-Jones 12–6 dispersion/repulsion, the hydrogen bonding as a directional 12–10 term, and the Coulombic electrostatic potential for charges were used. The docking simulations end with multiple runs and cluster analysis of ligands were performed with their corresponding docked energy. Docking solutions with ligand all-atom RMSDs within 2.0 Å of each other were clustered together and ranked by the lowest energy representative. Finally, the obtained top-posed docking conformations were subjected to post-docking energy minimization on DS 3.531. The resultant structure files were analyzed using PyMOL visualization programs37.

Results and discussion

Chemistry

Synthetic approaches for the preparation of title compounds (4a–t) and their intermediates are depicted in Scheme 1. In the reaction sequence, the oximation of readily available aryl aldehydes with hydroxyl amine hydrochloride in ethanol/pyridine (10:1) mixture at refluxing temperature lead to the oximes (1a–d), which were reduced in the presence of Zn/HCl to give corresponding amines (2a–d). The imine intermediates (3a–t) were obtained by the reaction of amines (2a–d) with aromatic aldehydes substituted by electron withdrawing groups in quantitative yields. Finally, Staudinger synthesis, the formal (2 + 2) cycloaddition of imine (3a–g, 3i–s) and ketene derived from phenylacetic acid in the presence of triethyl amine (Et₃N) and phosphoryl chloride (POCl₃) in toluene at refluxing temperature gave the β-lactam derivatives (4a–g, 4i–s) in low to good yields. Surprisingly, after overnight stirring at reflux temperature, 4h, 4t were obtained as unexpected 1,3-oxazin-4-one derivatives instead of β-lactams as confirmed by single crystal X-ray analysis of 4t. All imines (3a–t), β-lactam derivatives (4a–g, 4i–s) (Table 1) and 1,3-oxazin-4-one derivatives (4h, 4t) (Table 2) were well characterized by spectroscopic and elemental analyses. Single crystal structure of one of the β-lactams (4e) was also recorded. The probable mechanism for the synthesis of ketene and its (2 + 2) cycloaddition with imines (3a–g, 3i–s) is shown in Scheme 2. Phenylacetic acid on the treatment with POCl₃ gives intermediate I which on nucleophillic attack by chloride ion affords phenylacetyl chloride. Triethylamine abstracts α-proton from phenylacetyl chloride to form ketene II via intermediate (E)-1-chloro-2-phenylethenolate which exists in dynamic equilibrium with its tautomeric form (Step 1). Ketene undergoes heat induced [2 + 2] cycloaddition with imine (3a–g, 3i–s) to form β-lactam ring (Step 2). To rationalize the unexpected cyclization, a possible mechanism40 is shown in Scheme 3. The 1,3-oxazin-4-one formation takes place by initial acylation of imines followed by attack of chloride ion to the iminium ion (I) gives intermediate (II). Enolization followed by C-acylation by phenylacetyl chloride gives intermediate (IV). This leads to the formation of 1,3-oxazin-4-one via intramolecular displacement of chloride. Another possible pathway is C-acylation of iminium ion (III) followed by enolization and [4π]-conrotatory cyclization may account for the formation of 1,3-oxazin-4-one.

Scheme 1. Synthesis of monocyclic β-lactams and unexpected 1,3-oxazin-4-ones.

Scheme 2. Plausible mechanism for the formation of β-lactam derivatives (4a–g and 4i–s). Step 1. Formation of ketene. Step 2. Cyclization of imine and ketene.

Scheme 3. Plausible mechanism for the formation of 1,3-oxazin-4-ones (4h, 4t).

Table 1. Structural data of β-lactam derivatives (4a–g, 4i–s)..

Compound	Ar1	Ar2	Mol. formula	Mol. wt.
4a			C22H18N2O3	358.39
4b			C22H18N2O3	358.39
4c			C22H18N2O3	358.39
4d			C22H18ClNO	347.84
4e			C22H18ClNO	347.84
4f			C22H17ClN2O3	392.83
4g			C22H17ClN2O3	392.83
4i			C22H17Cl2NO	382.28
4j			C22H17Cl2NO	382.28
4k			C20H16N2O4	348.35
4l			C20H16N2O4	348.35
4m			C20H16N2O4	348.35
4n			C20H16ClNO2	337.83
4o			C20H16ClNO2	337.83
4p			C22H17FN2O3	376.38
4q			C22H17FN2O3	376.38
4r			C22H17FN2O3	376.38
4s			C22H17ClFNO	365.83

Table 2. Structural data of 1,3-oxazin-4-one derivatives (4h, 4t)..

Compound	Ar1	Ar2	Mol. formula	Mol. wt.
4h			C30H23ClN2O4	510.97
4t			C30H23ClFNO2	483.96

The structures of all the compounds were in good agreement with IR, ¹H, ¹³C NMR, mass spectroscopic and analytical data. IR spectra of the oximes (1a–d) showed a sharp band in the range 1570–1599 cm⁻¹ due to C=N stretch. In addition to this, appearance of a broad band accounted for O–H stretch in the region 3149–3275 cm^−1. In ¹H NMR spectra, oximes showed a sharp singlet at 8.05–8.59 ppm due to CH proton. The OH proton appeared as a broad singlet at 8.55–9.21 ppm. The protons of aromatic ring appeared at expected chemical shift and integral values which confirmed the formation of the oximes (1a–d). In case of amines (2a–d), the appearance of broad band at 3372 cm⁻¹ due to NH₂ stretching and disappearance of bands due to C=N stretch and O–H stretch provided strong evidence for the formation of amines from their respective oximes. In the ¹H NMR spectra, there appeared sharp singlets at 3.46–3.94 ppm and 1.43–1.64 ppm due to CH₂ and NH₂ protons, respectively. The protons belonging to aromatic ring appeared at their usual chemical shift values.

In the IR spectra of imine (Schiff’s base) intermediates (3a–t), a band due to C=N stretch in the region 1639–1650 cm⁻¹ and disappearance of bands for NH₂ as well as for the CHO group provided evidence for their formation. The absorption bands for aryl as well as heteroaryl rings occurred at their expected positions. The protons belonging to imine group (CH=N) showed a sharp singlet at 8.27–8.85 ppm, while a singlet appeared at 4.67–4.99 ppm due to CH₂ protons. The peaks for aldehydic proton and NH₂ protons were absent in ¹H NMR spectra of imines. The ¹³C NMR spectra further confirmed the formation of imines as a characteristic peak corresponding to azomethine carbon appeared at 157.78–161.67 ppm, while peaks corresponding to other groups appeared at their expected chemical shift values. In the mass spectra, all imines exhibited molecular ion peaks as the base peak. In the spectral analysis of β-lactam derivatives (4a–g, 4i–s), IR absorption peaks for the imine group (CH=N) disappeared, while strong peaks for the carbonyl group appeared in the region 1749–1771 cm⁻¹ indicated the formation of β-lactam ring. In ¹H NMR spectra, both protons belonging to β-lactam ring appeared as doublets. The proton (H_a) in the vicinity of ring nitrogen was deshielded and appeared at the chemical shift value in the range 4.79–5.54 ppm, while other proton (H_b) near carbonyl group of β-lactam ring was shielded in comparison to other proton and appeared at 4.28–5.12 ppm (Figure 2). The formation of β-lactam ring was further confirmed by ¹³C NMR spectral data in which carbonyl group appeared at chemical shift values 169.98–160.66 ppm and both the tetrahedral carbons (C₃ and C₄) of β-lactam ring appeared at 47.81–63.37 and 51.56–65.58 ppm, respectively. In the ¹³C NMR spectra of compound 4b, 17 different carbon atoms were observed which were consistent with the proposed structure of the compound. Distortionless enhancement of NMR signals by polarization transfer (DEPT) is used to discriminate primary, secondary and tertiary carbon atoms (Figure 3). In the DEPT spectrum of compound 4b, peak for CH₂ group appeared in negative phase at 45.31 ppm, while peaks for two CH protons of β-lactam ring appeared in positive phase at 61.42 and 59.06 ppm. The carbons that are recorded in ¹³C NMR, but absent in DEPT spectrum, are quaternary carbons without any attached hydrogens so the resonances corresponding to three aromatic carbons which appeared at 147.92, 137.51, 134.85 ppm in ¹³C NMR spectra were absent from DEPT spectrum confirming their quaternary nature. Similarly, peak for carbonyl group at 167.64 in ¹³C NMR spectra was also absent in the DEPT spectrum of 4b. The mass spectra showed molecular ion peaks which provided confirmatory evidence for the formation of β-lactam ring.

Figure 2. General structure of synthesized β-lactam derivatives.

Figure 3. Stack plot of ¹³C and DEPT-135 NMR spectrum of compound 4b.

X-ray crystallographic analysis of 4e and 4t

X-ray crystallographic determination of 4e (β-lactam) and 4t (1,3-oxazinone) was performed to support the structural analysis data. The data collection and structure refinement details are described in Table 3. A colorless prism-like crystal of compound 4e (C₂₂H₁₈ClNO), approximate dimensions 0.40 mm × 0.24 mm × 0.21 mm, was used for the X-ray crystallographic analysis. The compound 4e crystallized with two independent molecules in asymmetric unit having slightly different conformations (Figure 4). Benzyl, chlorophenyl and phenyl rings are planar, while four membered azetidine-2-one ring is nearly planar (deviations of atoms from least square plane are within the range 0.27–0.33 Å). The angles between planar aromatic rings of one molecule from the asymmetric unit are 55.41°, 59.81° and 70.60°, respectively, while in other molecule the corresponding angles are 58.95°, 61.65° and 64.52°, respectively. The unit cell contains four asymmetric units, i.e. eight molecules which are connected by weak intermolecular non-covalent interactions including C–H…O, C–H…π, C=O…π and C–H…Cl (Figure 5) forming a sheet-like structure.

Figure 4. ORTEP diagram of compound 4e with atomic labeling scheme (50% probability level of thermal ellipsoids).

Figure 5. Unit cell in the crystal packing of compound 4e (hydrogen bonding is shown by dotted lines).

Table 3. Crystal data and structure refinement details for 4e and 4t.

	4e	4t
Formula	C22H18ClNO	C30H23ClFNO2
MW (g/mol)	347.82	483.94
dcalcd. (g/cm^3)	1.295	1.348
Crystal size (mm^3)	0.40 × 0.24 × 0.21	0.44 × 0.19 × 0.15
T (K)	100(2)	100(2)
λ (Å)	1.54178	1.54178
F (000)	1456	1008
Crystal system	Monoclinic	Monoclinic
Space group	P21/n	P21/c
a (Å)	22.9921(7)	7.8270(3)
b (Å)	7.3951(2)	11.6200(5)
c (Å)	23.2487(7)	26.2346(10)
α (°)	90	90
β (°)	115.472(1)	92.314(1)
γ (°)	90	90
V (Å^3)	3568.7(2)	2384.1(2)
Z	8	4
θ range (°)	2.26–68.23	3.37–68.23
Completeness	1.00 to 2θ 136.5°	1.00 to 2θ 136.5°
Collected reflections	52 509	38 217
Unique reflections; Rint	6511 [Rint = 0.0333]	4372 [Rint = 0.0398]
μ/mm^−1	1.952	1.717
R (Fo), [I  > 2σ(I)]	0.0369	0.0318
Rw () (all data)	0.0955	0.0783
GoF (F^2)	1.046	1.050
Δρ [e Å^−3]	0.391/−0.205	0.236/−0.288

Figure 6 shows the XP diagram of compound 4t with 50% probability level of thermal ellipsoids. A colorless prism-like specimen of compound 4t (C₃₀H₂₃ClFNO₂), approximate dimensions 0.44 mm × 0.19 mm × 0.15 mm, was used for the X-ray crystallographic analysis. Molecular structure contains four planar six membered rings, connected with a central non-planar six member ring “A”. The central ring “A” adopts nearly boat conformation. Atom O2 deviates by 0.500 Å from the least square plane passing through C3, C2, C1, N1 and C4 atoms, while atom C4 deviates by 0.680 Å from the least square plane passing through O2, C3, C2, C1 and N1. Crystal packing exhibits four molecules, forming two pairs of similar conformations within the unit cell. There exist weak intermolecular C–H…π and C–H…O hydrogen bonding interactions between each pair of molecules of the unit cell and also between the molecules at different symmetry positions (Figure 7).

Figure 6. ORTEP diagram of compound 4t with atomic labeling scheme (50% probability level of thermal ellipsoids).

Figure 7. Unit cell in the crystal packing of compound 4t (hydrogen bonding is shown by dotted lines).

Pharmacokinetics and in silico toxicity

These are important parameters for the development of bioactive leads into effective therapeutic agents. Therefore, a computational study was performed for ADMET and TOPKAT predictions of all the derivatives (4a–t) and the results are listed in Table 4. According to ADMET assessment, compounds 4k, 4l and 4m exhibited good solubility with level 3, and rest compounds fall under level 2 and 1 showed moderate to low solubility, respectively. The MW of all compounds is <500, and is ideally good to qualify as a drug molecule. In pharmaceutical sciences, log P is the reliable method to check the accessibility of drug candidate in biological system. Of 20 compounds, eight compounds (4k–r) presented good A log P value ≤ 5.0. Hepatotoxicity probability <0.5 is unlikely to cause dose-dependent liver injuries and comes under predicted class-0 (non-toxic), whereas hepatotoxicity probability >0.5 is liable to cause dose-dependent liver injuries, i.e. toxic and comes under predicted class-1. The CYP2D6 probability of all compounds showed <0.5 values, which demonstrated that all compounds are non-inhibitor of CYP2D6 enzyme. For good druggability, plasma protein binding (PPB) level-0 is ideal. Compounds 4k, 4l and 4m have displayed better PPB activity and come under level-0. The PSA is dependent on the conformation and possible internal hydrogen bonding that corresponds to the single low-energy conformer of the molecule. For drug activity, the optimum value of PSA ≤100 Ų is well defined41. The hydrogen bonding and log P are the two main descriptors, defining the PSA of molecule. All synthesized compounds showed appreciable PSA value ≤80 Ų. TOPKAT analysis was carried out to predict the probable toxicity of the compounds. All 20 compounds showed weight of evidence (WOE) probability ≤0.5 and were predicted as non-carcinogen. Moreover, Ames prediction test evidenced all compounds to be non-mutagenic.

Table 4. ADMET and TOPKAT analysis.

	ADMET							TOPKAT
Compound	Mol. wt.	Sol. level	Alog P	HepTox level	CYP2D6 prob.	PPB level	PSA	Ames Mut.	WOE
4a	358.39	2	5.23	0	0.46	2	69.15	NM	NC
4b	358.39	2	5.22	0	0.43	2	69.15	NM	NC
4c	358.39	2	5.22	0	0.41	2	69.15	NM	NC
4d	347.84	1	6.49	1	0.38	2	24.16	NM	NC
4e	347.84	1	6.48	1	0.38	2	24.16	NM	NC
4f	392.84	2	5.20	0	0.34	2	69.15	NM	NC
4g	392.84	2	5.20	1	0.32	2	69.15	NM	NC
4h	392.84	2	5.19	1	0.34	2	69.15	NM	NC
4i	382.28	1	6.46	0	0.32	2	24.16	NM	NC
4j	382.28	1	6.45	1	0.33	2	24.16	NM	NC
4k	348.35	3	3.40	0	0.46	0	78.08	NM	NC
4l	348.35	3	3.39	0	0.44	0	78.08	NM	NC
4m	348.35	3	3.39	0	0.44	0	78.08	NM	NC
4n	337.83	2	4.66	1	0.45	1	33.09	NM	NC
4o	337.83	2	4.65	1	0.36	1	33.09	NM	NC
4p	376.38	2	4.93	0	0.38	1	69.15	NM	NC
4q	376.38	2	4.92	0	0.38	1	69.15	NM	NC
4r	376.38	2	4.90	0	0.40	1	69.15	NM	NC
4s	365.83	1	6.19	1	0.34	2	24.16	NM	NC
4t	365.83	1	6.18	1	0.34	2	24.16	NM	NC

Sol., (solubility) level; Alog P, lipophilicity; HTL, hepatotoxicity level; CYP2D6, cytochrome P450 2D6 enzyme; Ames Mut, Ames mutagen prediction; WOE_Prediction, weight of evidence; NM, non-mutagen; NC, non-carcinogen. Ideal value of solubility level is 3, Alog P value should not be greater than 5.0. Similarly, hepatotoxicity probability <0.5 is ideal, and level with 0. The probability value for CYP2D6 <0.5 is good value and PPB value comes under level-0 is ideal. For drug like activity, the PSA of compounds ≤100 Ų is ideal.

Antibacterial activity

In order to explore the potential of all the derivatives (4a–t) as a possible scaffold for bacterial inhibitors, they were evaluated against E. coli (MTCC 739) and K. pneumoniae (MTCC 109) and ampicillin was used as a reference drug. All the experiments were carried out in triplicate at each concentration level and repeated thrice. The results are summarized in Table 5. Among all the derivatives, 4d, 4h, 4k and 4l displayed moderate activity with MIC values of 250, 250, 250 and 500 µg/mL, respectively, against E. coli. Moreover, compound 4l was found with considerable antibacterial activity having MIC 62.5 µg/mL against K. pneumoniae. The MBC values were also calculated and compounds 4d, 4h, 4k and 4l indicated their bactericidal nature.

Table 5. In vitro antibacterial activity (MIC and MBC) of β-lactams and 1,3-oxazin-4-one derivatives (in µg/mL).

	E. coli (MTCC-739)		K. pneumonia (MTCC-109)
Compound	MIC (µg/mL)	MBC (µg/mL)	MIC (µg/mL)	MBC (µg/mL)
4a	>1000	>1000	>1000	>1000
4b	>1000	>1000	>1000	>1000
4c	>1000	>1000	>1000	>1000
4d	250	500	>1000	>1000
4e	>1000	>1000	>1000	>1000
4f	>1000	>1000	>1000	>1000
4g	>1000	>1000	>1000	>1000
4h	250	500	>1000	>1000
4i	1000	1000	>1000	>1000
4j	>1000	>1000	>1000	>1000
4k	250	500	>1000	>1000
4l	500	500	62.5	62.5
4m	>1000	>1000	>1000	>1000
4n	>1000	>1000	>1000	>1000
4o	>1000	>1000	>1000	>1000
4p	>1000	>1000	>1000	>1000
4q	>1000	>1000	>1000	>1000
4r	>1000	>1000	>1000	>1000
4s	>1000	>1000	>1000	>1000
4t	>1000	>1000	>1000	>1000
Ampicillin	<7.8	7.8	15.6	31.2

Hemolytic assay

On the basis of in vitro results, compounds 4d, 4h, 4k and 4l were selected to check their toxic effect on hRBCs by hemolytic assay. At 1.0 mg/mL, all the compounds showed more than 50% hemolysis which indicated their moderate toxicity. Compounds 4d, 4h, 4k and 4l showed 14.65%, 11.15%, 21.53% and 15.77% hemolysis, respectively, at 0.25 mg/mL concentration (Figure 8). At their respective MIC concentrations against E. coli (MTCC 739) and K. pneumoniae (MTCC 109), compound 4l was found fairly less toxic with <20% hemolysis.

Figure 8. Hemolytic activity of compounds 4d, 4h, 4k, and 4l on human RBCs.

Cytotoxicity by MTT assay

To examine toxic effect of compounds 4d, 4h, 4k, and 4l on normal cell growth, MTT assay was performed on CHO cell line. The cell viability (%) observed with continuous exposure for 48 h has been detailed in Figure 9. The cytotoxicity of all the compounds was found to be concentration dependent. It is evident from the results that 4d, 4h, 4k, and 4l displayed almost 100% cell viability between 50 and 100 μg/mL conc. and none of the compounds showed any significant cytotoxic effect on CHO cells up to the conc. of 200 μg/mL. Compound 4l exhibited maximum cellular viability (100%) at 200 μg/mL concentration. By increasing the conc. between 300 and 400 μg/mL, the compounds showed more than 50% cell viability. However, at higher conc. there was sharp decrease in cells viability which showed the toxic effect of the compounds. These results clearly indicate the non-toxic nature of these compounds up to 200 μg/mL conc.

Figure 9. Cytotoxic effect of the compounds (4d, 4h, 4k and 4l) through cell viability assay after 48 h of the treatment on CHO cells.

In silico analysis

The stereochemical quality of the modeled co-ordinates of PBP-5 on SAVS server exhibited that 98.0% is present in the most favored regions (A, B, L) of the Ramachandran plot (PROCHECK). All atom structural superimposition of model with template (PDB ID: 1NZO) showed RMSD value 0.3 Å, indicating appreciable quality of the model. Molecular docking of all synthesized compounds was carried with AutoDock 4.2, and results showed that all compounds are almost spatially fitted in the active site. Compound 4l displayed best binding affinity with the homology model of PBP-5 of K. pneumoniae. Active site residues Asn161 was involved in polar interaction with oxygen of carbonyl group (β-lactam ring), Arg247 showed polar interaction with furan ring and nitro group of the ligand and Tyr271 was involved in π–π interaction with m-nitro phenyl (Figure 10).

Figure 10. Structure of PBP-5 (K. pneumoniae) docked with compound 4l. Polar interactions with active site residues are shown with black-dot line.

Pharmacophore feature analysis of compound 4l (Figure 11) with DS 3.5 showed that carbonyl group of β-lactam ring and nitro group of phenyl ring may act as a hydrogen bond acceptor and phenyl at C₃ and m-nitro phenyl at C₄ are involved in ring aromatic interaction, whereas furan ring is associated with hydrophobic feature.

Figure 11. Pharmacophore feature analysis of compound 4l.

Conclusion

In summary, we have designed a series of novel N-benzyl-3,4-diaryl substituted 2-azitidinone (β-lactam) derivatives and surprisingly discovered the formation of two unexpected 1,3-oxazin-4-one derivatives (4h, 4t) confirmed by X-ray analysis. The pharmacokinetic properties and probable toxicity data also advocate them as possible drug molecules. Compounds 4d, 4h, 4k and 4l showed moderate to fair antibacterial potential. In vitro cytotoxicity as well as hemolysis results revealed non-toxic nature of these compounds. Compound 4l displayed considerably good binding affinity with the predicted three dimensional model of PBP-5 of K. pneumoniae. To conclude it, 4l can be carried forward as a lead molecule in the drug discovery pipeline against bacterial infections. With this lead in hand, we intend to exploit it further to study the exact mechanism of inhibition and in vivo studies in the near future.

Supplementary materials online only

CCDC1022181–1022182 contains the supplementary crystallographic data for 4t and 4e reported in this article. This data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/. Supplementary data includes X-ray crystallographic analysis of 4e and 4t.

Acknowledgements

The authors are thankful to Prof. S. Chandershekhran, Department of Organic Chemistry, IISc Bangalore for providing DEPT spectrum.

Declaration of interest

Authors have no conflict of interest. Mohammad Abid gratefully acknowledges the Young Scientist Award from Science & Engineering Research Board (Grant No. SR/FT/LS-03/2011), Govt. of India, New Delhi, India. U.Y. acknowledges UGC for “Raman Research Fellowship Award”. B.A. and M.I. would like to acknowledge UGC for non-NET fellowship.