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

Article abstract of DOI:10.3109/14756366.2015.1058257

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.

Full text of DOI:10.3109/14756366.2015.1058257

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J Enzyme Inhib Med Chem, Early Online: 1–19
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1058257
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SHORT COMMUNICATION
Monocyclic b-lactam and unexpected oxazinone formation: synthesis,
crystal structure, docking studies and antibacterial evaluation
Babita Aneja1,2, Mohammad Irfan1, Md. Imtaiyaz Hassan3, Amresh Prakash3, Umesh Yadava4,
Constantin G. Daniliuc5, Md. Zafaryab6, M. Moshahid A. Rizvi6, Amir Azam2, and Mohammad Abid1
2
1Medicinal Chemistry Lab, Department of Biosciences, Jamia Millia Islamia (A Central University), Jamia Nagar, New Delhi, India, Department of
Chemistry, Jamia Millia Islamia (A Central University), Jamia Nagar, New Delhi, India, 3Centre for Interdisciplinary Research in Basic Sciences, Jamia
4
Millia Islamia (A Central University), Jamia Nagar, New Delhi, India, Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva
5
6
University, Bronx, NY, USA, Organisch-Chemisches Institut, Westfa¨lische Wilhelm-Universita¨t, Mu¨nster, Germany, and Genome Biology Lab,
Department of Biosciences, Jamia Millia Islamia (A Central University), Jamia Nagar, New Delhi, India
Abstract
Keywords
Novel monocyclic b-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
b-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 mg/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 mg/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, b-lactam
History
Received 20 December 2014
Revised 8 May 2015
Accepted 11 May 2015
Published online 1 July 2015
Introduction
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.
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, b-lactam
has been considered as privileged structure in chemical, pharma-
ceutical and material industries2–8. b-Lactam antibiotics such as
penicillins, cephalosporins, carbapenems and monobactam
(Figure 1) are widely used as chemotherapeutic agents for the
treatment of microbial infections. b-Lactams exert their bacteri-
cidal 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 b-lactam core structure responsible for
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 b-lactams
and in extension to our efforts to explore novel biologically active
molecules19,20, we synthesized novel N-benzyl-3,4-diaryl sub-
stituted 2-azitidinone (b-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 consid-
erable antibacterial activity was assessed by 3-(4,5-dimethylthia-
zol-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
Address for correspondence: Mohammad Abid, Medicinal Chemistry
Lab, Department of Biosciences, Jamia Millia Islamia (A Central
University), Jamia Nagar, New Delhi 110 025, India. Tel: +91
8750295095. Fax: +91 1126980229. E-mail: mabid@jmi.ac.in

2
B. Aneja et al.
J Enzyme Inhib Med Chem, Early Online: 1–19
Figure 1. Structure of commonly used
b-lactam antibiotics.
most abundant PBP and provides an excellent system to study the ethanol–pyridine (10:1) mixture under reflux for 22–25 h.
catalytic mechanism of penicillin-interacting enzymes21. The After cooling, solvent was removed under reduced pressure and
plausible binding modes and mechanism of best compound water was added to the residue. The mixture was cooled in an ice
through molecular docking was then studied against the predicted bath, stirred and the precipitated solid was filtered off, washed
three-dimensional model of PBP-5 from K. pneumoniae, which with water and purified by recrystallization22.
displayed 94% sequence homology with PBP-5 of E. coli, due to
unavailability of X-ray crystal structure of the former.
Benzaldehydeoxime (1a)
Colorless oil, yield: 80%, Rf ¼ 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): ꢁ (cmꢀ1) 3275 (O–H), 1599
Materials and methods
Chemistry
1
(C¼N). H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.17 (s, 1H, CH),
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
7.59 (d, 2H, J ¼ 3.9 Hz, Ar–H), 7.38 (t, 3H, J ¼ 3.3 Hz, Ar–H).
2-Chlorobenzaldehyde oxime (1b)
(TLC) and spots were visualized under UV light. Ninhydrin test White solid, m.p. 72–74 ꢁC, yield: 87%, Rf ¼ 0.52 (ethyl acetate/
was also performed for the detection of imine (Schiff’s base) as pet. ether, 30:70), Anal. (C7H6ClNO) Calc. C 54.04, H 3.89, N
well as b-lactams. The IR spectra of compounds were recorded on 9.00; found: C 54.01, H 3.87, N 8.98%. IR (neat): ꢁ (cmꢀ1) 3245
1
Agilent Cary 630 FT-IR spectrometer (Santa Clara, CA) and only (O–H), 1596 (C¼N). H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.59
major peaks are reported in cmꢀ1. 1H and 13C NMR spectra were (s, 1H, CH), 7.84 (d, 1H, J ¼ 7.2 Hz, Ar–H), 7.41 (d, 1H,
obtained in CDCl3 using tetramethylsilane as an internal standard J ¼ 7.8 Hz, Ar–H), 7.28 (t, 2H, J ¼ 8.4 Hz, Ar–H).
¨
on Bruker Spectrospin DPX-300 spectrometer (Fallanden,
Switzerland) at 300 and 75 MHz, respectively, and on Agilent- Furan-2-carbaldehyde oxime (1c)
NMR-vnmrs-500 spectrometer (Santa Clara, CA) at 500 and
Creamish solid, m.p. 82–84 ꢁC, yield: 82%, Rf ¼ 0.47 (ethyl
125 MHz, respectively. Multiplicities are designated as s (singlet),
d (doublet), t (triplet), m (multiplet). 1H NMR chemical shifts (ꢀ)
values are reported in parts per million (ppm) relative to residual
solvent (CHCl3, s, ꢀ 7.26) and 13C NMR chemical shifts are
reported relative to CDCl3 (t, ꢀ 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
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): ꢁ
1
(cmꢀ1) 3149 (O–H), 1570 (C¼N). H NMR (300 MHz, CDCl3)
(ꢀ, ppm): 9.21 (brs, 1H, OH), 7.48 (d, 2H, J ¼ 11.1 Hz, Ar–H),
7.33 (d, 1H, J ¼ 3.6 Hz, Ar–H), 6.54 (s, 1H, CH).
4-Fluorobenzaldehyde oxime (1d)
LC/MS spectrometer (Santa Clara, CA). Melting points were White crystalline solid, m.p. 86–88 ꢁC, yield: 85%, Rf ¼ 0.56
recorded on a digital Buchi melting point apparatus (M-560) (ethyl acetate/pet. ether, 30:70), Anal. (C7H6FNO) Calc. C 60.43,
(Flawil, Switzerland) and were uncorrected. Elemental analyses H 4.35, N 10.07; found: C 60.41, H 4.32, N 10.08%. IR (neat): ꢁ
1
were performed on Elementar Vario Analyzer (Hanau, Germany). (cmꢀ1) 3253 (O–H), 1599 (C¼N). H NMR (300 MHz, CDCl3)
Purification of the compounds was performed by column (ꢀ, ppm): 8.55 (brs, 1H, OH), 8.13 (s, 1H, CH), 7.56 (t, 2H,
chromatography using silica gel (230–400 mesh size) as station- J ¼ 7.8, 8.7 Hz, Ar–H), 7.08 (t, 2H, J ¼ 7.2, 8.4 Hz, Ar–H).
ary phase with ethyl acetate/petroleum ether as an eluent.
General procedure for the synthesis of amines (2a–d)
General procedure for the synthesis of oximes (1a–d)
To a clear solution of oxime (1.0 equiv) in ethanol (10 mL), a
Oximes were obtained by reacting substituted aldehydes mixture of water and conc. HCl (1:2) was added. The reaction
(1.2 equiv.) with hydroxylamine hydrochloride (1.3 equiv.) in mixture was stirred at room temperature for 15 min then it was

DOI: 10.3109/14756366.2015.1058257
ꢂ-Lactam and unexpected oxazinone formation
3
allowed to cool to 0 ꢁC. Zinc dust (6.0 equiv.) was added slowly (3-Nitrobenzylidene)(phenyl)methylamine (3b)
and the reaction mixture was refluxed for 1 h at 80–90 ꢁC. After
Light yellow solid, m.p. 60–61 ꢁC, yield: 93%, Rf ¼ 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): ꢁ
(cmꢀ1) 1646 (C¼N), 1527 (NO2 asym), 1350 (NO2 sym). 1H NMR
(300 MHz, CDCl3) (ꢀ, ppm): 8.60 (s, 1H, Ar–H), 8.46 (s, 1H,
CH), 8.27 (d, 1H, J ¼ 8.4 Hz, Ar–H), 8.13 (d, 1H, J ¼ 7.5 Hz, Ar–
H), 7.59 (t, 1H, J ¼ 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) (ꢀ, ppm): 159.17,
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%, Rf ¼ 0.32 (methanol/DCM, 10:90), 148.65, 138.55, 137.86, 133.65, 129.62, 128.64, 128.08, 127.30,
Anal. (C7H9N) Calc. C 78.46, H 8.47, N 13.07; found: C 78.43, H 125.14, 123.09, 64.96. ESI-MS (m/z): 241.46 [M + H]+.
8.44, N 13.06%. IR (neat): ꢁ (cmꢀ1) 3372 (NH2). 1H NMR
(300 MHz, CDCl3) (ꢀ, ppm): 7.43–7.24 (m, 5H, Ar–H), 3.88 (4-Nitrobenzylidene)(phenyl)methylamine (3c)
(s, 2H, CH2), 1.57 (s, 2H, NH2).
Red solid, m.p. 31–33 ꢁC, yield: 89%, Rf ¼ 0.53 (ethyl acetate/pet.
ether, 20:80), Anal. (C14H12N2O2) Calc. C 69.99, H 5.03, N
(2-Chlorophenyl)methanamine (2b)
11.66; found: C 69.95, H 5.01, N 11.61%. IR (neat): ꢁ (cmꢀ1
)
Light yellow oil, yield: 72%, Rf ¼ 0.35 (methanol/DCM, 1641 (C¼N), 1518 (NO2 asym), 1343 (NO2 sym). 1H NMR
10:90), Anal. (C7H8ClN) Calc. C 59.38, H 5.69, N 9.89; (300 MHz, CDCl3) (ꢀ, ppm): 8.46 (s, 1H, CH), 8.27 (d, 2H,
found: C 59.35, H 5.67, N 9.86%. IR (neat): ꢁ (cmꢀ1) 3372 J ¼ 8.4 Hz, Ar–H), 7.94 (d, 2H, J ¼ 8.4 Hz, Ar–H), 7.40–7.26 (m,
(NH2). H NMR (300 MHz, CDCl3) (ꢀ, ppm): 7.39–7.36 (m, 2H, 5H, Ar–H), 4.89 (s, 2H, CH2). 13C NMR (75 MHz, CDCl3) (ꢀ,
1
Ar–H), 7.29–7.20 (m, 2H, Ar–H), 3.94 (s, 2H, CH2), 1.64 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)
(s, 2H, NH2).
Furan-2-ylmethanamine (2c)
Light yellow oil, yield: 70%, Rf ¼ 0.36 (methanol/DCM, 10:90), Light yellow oil, yield: 93%, Rf ¼ 0.78 (ethyl acetate/pet. ether,
Anal. (C5H7NO) Calc. C 61.84, H 7.27, N 14.42; found: C 61.81, 20:80), Anal. (C14H12ClN) Calc. C 73.20, H 5.27, N 6.10; found:
H
7.25,
N
14.39%. IR (neat):
ꢁ
(cmꢀ1
)
3372 (NH2). C 73.17, H 5.23, N 6.08%. IR (neat): ꢁ (cmꢀ1) 1639 (C¼N), 754
1
1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 7.37 (s, 1H, Ar–H), 6.33 (C–Cl). H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.85 (s, 1H, CH),
(s, 1H, Ar–H), 6.15 (s, 1H, Ar–H), 3.85 (s, 2H, CH2), 1.63 8.10 (d, 1H, J ¼ 7.5 Hz, Ar–H), 7.39–7.30 (m, 6H, Ar–H), 7.29–
(s, 2H, NH2).
7.24 (m, 2H, Ar–H), 4.87 (s, 2H, CH2). 13C NMR (75 MHz,
CDCl3) (ꢀ, 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-Fluorophenyl)methanamine (2d)
Light yellow oil, yield: 65%, Rf ¼ 0.32 (methanol/DCM, 10:90),
Anal. (C7H8FN) Calc. C 67.18, H 6.44, N 11.19; found: C 67.16,
(4-Chlorobenzylidene)(phenyl)methylamine (3e)
H 6.41, N 11.17%. IR (neat): ꢁ (cmꢀ1) 3372 (NH2). H NMR
1
(300 MHz, CDCl3) (ꢀ, ppm): 7.28 (t, 2H, J ¼ 5.4, 8.1 Hz, Ar–H), Light yellow oil, yield: 94%, Rf ¼ 0.71 (ethyl acetate/pet. ether,
7.02 (t, 2H, J ¼ 8.7, 11.4 Hz, Ar–H), 3.84 (s, 2H, CH2), 1.56 (s, 20:80), Anal. (C14H12ClN) Calc. C 73.20, H 5.27, N 6.10; found:
2H, NH2).
C 73.15, H 5.25, N 6.07%. IR (neat): ꢁ (cmꢀ1) 1646 (C¼N), 735
1
(C–Cl). H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.34 (s, 1H, CH),
General procedure for the synthesis of imines (3a–d)
7.71 (d, 2H, J ¼ 7.8 Hz, Ar–H), 7.38 (d, 2H, J ¼ 7.8 Hz, Ar–H),
7.33–7.25 (m, 5H, Ar–H), 4.81 (s, 2H, CH2). 13C NMR (75 MHz,
CDCl3) (ꢀ, 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]+.
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
(2-Nitrobenzylidene)(2-chlorophenyl)methylamine (3f)
the aqueous layer was extracted with ethyl acetate. The organic Yellow solid, m.p. 82–84 ꢁC, yield: 86%, Rf ¼ 0.52 (ethyl acetate/
phase was washed with brine, dried over anhydrous Na2SO4 and pet. ether, 20:80), Anal. (C14H11ClN2O2) Calc. C 61.21, H 4.04, N
)
concentrated under reduced pressure. The crude product was then 10.20; found: C 61.16, H 4.02, N 10.40%. IR (neat): ꢁ (cmꢀ1
purified by recrystallization with ethanol to afford the desired 1641 (C¼N), 1520 (NO2 asym), 1330 (NO2 sym), 741 (C–Cl).
compounds in good to excellent yields24.
1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.77 (s, 1H, CH), 8.05
(d, 1H, J ¼ 7.5 Hz, Ar–H), 7.97 (d, 1H, J ¼ 7.5 Hz, Ar–H), 7.61 (t,
1 H, J ¼ 6.9, 7.5 Hz, Ar–H), 7.51 (t, 1H, J ¼ 6.9, 7.5 Hz, Ar–H),
7.35 (t, 2H, J ¼ 6.6, 8.1 Hz, Ar–H), 7.19 (d, J ¼ 8.1 Hz, 2H, Ar–
H), 4.91 (s, 2H, CH2). 13C NMR (75 MHz, CDCl3) (ꢀ, 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]+.
(2-Nitrobenzylidene)(phenyl)methylamine (3a)
Reddish brown oil, yield: 90%, Rf ¼ 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): ꢁ (cmꢀ1) 1639
1
(C¼N), 1523 (NO2 asym), 1344 (NO2 sym). H NMR (300 MHz,
CDCl3) (ꢀ, ppm): 8.83 (s, 1H, CH), 8.11 (d, 1H, J ¼ 7.8 Hz, Ar–
H), 8.03 (d, 1H, J ¼ 8.1 Hz, Ar–H), 7.66 (t, 1H, J ¼ 6.9, 7.5 Hz,
Ar–H), 7.56 (t, 1H, J ¼ 7.2, 8.1 Hz, Ar–H), 7.37–7.26 (m, 5H, Ar–
(3-Nitrobenzylidene)(2-chlorophenyl)methylamine (3g)
H), 4.88 (s, 2H, CH2). 13C NMR (75 MHz, CDCl3) (ꢀ, Light yellow solid, m.p. 62–64 ꢁC, yield: 91%, Rf ¼ 0.59 (ethyl
ppm): 159.47, 148.43, 138.23, 136.01, 132.56, 132.12, 129.07, acetate/pet. ether, 20:80), Anal. (C14H11ClN2O2) Calc. C 61.21,
128.86, 128.10, 125.82, 122.65, 64.03. ESI-MS (m/z): 241.38 H 4.04, N 10.20; found: C 61.18, H 4.01, N 10.16%. IR
[M + H]+.
(neat): ꢁ (cmꢀ1) 1650 (C¼N), 1527 (NO2 asym), 1352 (NO2 sym),

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B. Aneja et al.
J Enzyme Inhib Med Chem, Early Online: 1–19
1
752 (C–Cl). H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.64 (s, 1H, J ¼ 7.5 Hz, Ar–H), 7.61 (t, 1H, J ¼ 7.8, 8.1 Hz, Ar–H), 7.42
Ar–H), 8.49 (s, 1H, CH), 8.31 (d, 1H, J ¼ 6.9 Hz, Ar–H), 8.16 (d, (d, 1H, J ¼ 7.8 Hz, Ar–H), 6.39 (t, 1H, J ¼ 1.8, 2.7 Hz, Ar–H),
1H, J ¼ 7.2 Hz, Ar–H), 7.63 (t, 1H, J ¼ 7.2, 8.1 Hz, Ar–H), 7.44 6.33 (d, 1H, J ¼ 3.3 Hz, Ar–H), 4.85 (s, 2H, CH2). 13C NMR
(t, 2H, J ¼ 7.6 Hz, Ar–H), 7.28 (d, 2H, J ¼ 7.6 Hz, Ar–H), 4.98 (s, (75 MHz, CDCl3) (ꢀ, ppm): 160.94, 150.87, 148.16, 142.30,
2H, CH2). 13C NMR (75 MHz, CDCl3) (ꢀ, ppm): 160.08, 148.61, 141.05, 134.87, 129.56, 124.64, 123.67, 110.81, 106.72, 57.34.
137.73, 136.19, 133.72, 133.56, 129.93, 129.66, 129.46, 128.61, ESI-MS (m/z): 231.54 [M + H]+.
127.06, 125.26, 123.07, 61.84. ESI-MS (m/z): 275.54 [M + H]+.
(4-Nitrobenzylidene)(furan-2-yl)methylamine (3m)
(4-Nitrobenzylidene)(2-chlorophenyl)methylamine (3h)
Yellow solid, m.p. 82–84 ꢁC, yield: 89%, Rf ¼ 0.66 (ethyl acetate/
Yellow solid, m.p. 72–74 ꢁC, yield: 84%, Rf ¼ 0.66 (ethyl acetate/ pet. ether, 20:80), Anal. (C12H10N2O3) Calc. C 62.60, H 4.38,
pet. ether, 20:80), Anal. (C14H11ClN2O2) Calc. C 61.21, H 4.04, N N 12.17; found: C 62.56, H 4.31, N 12.14%. IR (neat): ꢁ (cmꢀ1
)
10.20; found: C 61.17, H 4.10, N 10.15%. IR (neat): ꢁ (cmꢀ1
)
1648 (C¼N), 1510 (NO2 asym), 1341 (NO2 sym), 1153 (C–O–C).
1641 (C¼N), 1520 (NO2 asym), 1335 (NO2 sym), 750 (C–Cl). H 1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.42 (s, 1H, CH), 8.29 (d,
NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.50 (s, 1H, CH), 8.30 (d, 2H, 2H, J ¼ 8.7 Hz, Ar–H), 7.95 (d, 2H, J ¼ 8.7 Hz, Ar–H), 7.43 (d,
J ¼ 8.4 Hz, Ar–H), 7.98 (d, 2H, J ¼ 8.7 Hz, Ar–H), 7.44 (t, 2H, 1H, J ¼ 5.4 Hz, Ar–H), 6.39 (t, 1H, J ¼ 1.8 Hz, Ar–H), 6.33 (d,
J ¼ 5.4 Hz, Ar–H), 7.33–7.25 (m, 2H, Ar–H), 4.99 (s, 2H, CH2). 1H, J ¼ 2.7 Hz, Ar–H), 4.87 (s, 2H, CH2). 13C NMR (75 MHz,
13C NMR (75 MHz, CDCl3) (ꢀ, ppm): 160.39, 149.13, 141.47, CDCl3) (ꢀ, ppm): 160.54, 151.40, 149.16, 142.53, 141.37, 129.02,
136.10, 133.55, 129.90, 129.46, 128.98, 128.63, 127.05, 123.88, 123.85, 110.49, 108.06, 57.14. ESI-MS (m/z): 231.49 [M + H]+.
62.06. ESI-MS (m/z): 275.67 [M + H]+.
1
(2-Chlorobenzylidene)(furan-2-yl)methylamine (3n)
(2-Chlorobenzylidene)(2-chlorophenyl)methylamine (3i)
Yellow oil, yield: 82%, Rf ¼ 0.68 (ethyl acetate/pet. ether, 20:80),
Yellow oil, yield: 87%, Rf ¼ 0.68 (ethyl acetate/pet. ether, 20:80), Anal. (C12H10ClNO) Calc. C 65.61, H 4.59, N 6.38; found: C
Anal. (C14H11Cl2N) Calc. C 63.66, H 4.20, N 5.30; found: C 65.58, H 4.60, N 6.34%. IR (neat): ꢁ (cmꢀ1) 1639 (C¼N), 1149
63.61, H 4.17, N 5.26%. IR (neat): ꢁ (cmꢀ1) 1641 (C¼N), 750 (C–O–C), 760 (C–Cl). 1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.82
1
(C–Cl). H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.80 (s, 1H, CH), (s, 1H, CH), 8.07 (d, 1H, J ¼ 7.5 Hz, Ar–H), 7.42–7.36 (m, 3H,
8.05 (d, 1H, J ¼ 6.6 Hz, Ar–H), 7.40–7.23 (m, 4H, Ar–H), 7.21– Ar–H), 7.34–7.28 (m, 1H, Ar–H), 6.37 (t, 1H, J ¼ 1.8, 3.0 Hz, Ar–
7.13 (m, 3H, Ar–H), 4.88 (s, 2H, CH2). 13C NMR (75 MHz, H), 6.30 (d, 1H, J ¼ 3.0 Hz, Ar–H), 4.84 (s, 2H, CH2). 13C NMR
CDCl3) (ꢀ, ppm): 160.82, 137.70, 136.21, 134.87, 133.41, 130.31, (75 MHz, CDCl3) (ꢀ, ppm): 159.85, 152.09, 142.29, 135.31,
129.98, 129.63, 129.31, 128.58, 128.21, 127.01, 126.72, 61.84. 132.97, 131.78, 129.77, 128.49, 127.30, 110.40, 107.57, 57.46.
ESI-MS (m/z): 264.63 [M + H]+.
ESI-MS (m/z): 220.32 [M + H]+.
(4-Chlorobenzylidene)(2-chlorophenyl)methylamine (3j)
(4-Chlorobenzylidene)(furan-2-yl)methylamine (3o)
Light yellow solid, m.p. 48–50 ꢁC, yield: 80%, Rf ¼ 0.71 (ethyl Brown oil, yield: 81%, Rf ¼ 0.61 (ethyl acetate/pet. ether, 20:80),
acetate/pet. ether, 20:80), Anal. (C14H11Cl2N) Calc. C 63.66, H Anal. (C12H10ClNO) Calc. C 65.61, H 4.59, N 6.38; found: C
4.20, N 5.30; found: C 63.58, H 4.15, N 5.28%. IR (neat): ꢁ 65.57, H 4.55, N 6.36%. IR (neat): ꢁ (cmꢀ1) 1641 (C¼N), 1128
(cmꢀ1) 1642 (C¼N), 756 (C–Cl). 1H NMR (300 MHz, CDCl3) (ꢀ, (C–O–C), 750 (C–Cl). 1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.44
ppm): 8.30 (s, 1H, CH), 7.66 (d, 2H, J ¼ 8.4 Hz, Ar–H), 7.33 (d, (s, 1H, CH), 7.71 (d, 2H, J ¼ 6.6 Hz, Ar–H), 7.41–7.18 (m, 3H,
3H, J ¼ 8.4 Hz, Ar–H), 7.22–7.13 (m, 3H, Ar–H), 4.83 (s, 2H, Ar–H), 6.36 (t, 1H, J ¼ 7.5 Hz, Ar–H), 6.29 (d, 1H, J ¼ 7.8 Hz,
CH2). 13C NMR (75 MHz, CDCl3) (ꢀ, ppm): 161.52, 136.88, Ar–H), 4.79 (s, 2H, CH2). 13C NMR (75 MHz, CDCl3) (ꢀ, ppm):
136.74, 134.52, 133.44, 129.74, 129.49, 129.35, 128.93, 128.35, 161.67, 152.04, 142.31, 136.90, 134.38, 129.53, 128.88, 110.41,
126.94, 61.83. ESI-MS (m/z): 264.41 [M + H]+.
107.65, 57.07. ESI-MS (m/z): 220.50 [M + H]+.
(2-Nitrobenzylidene)(furan-2-yl)methylamine (3k)
(2-Nitrobenzylidene)(4-fluorophenyl)methylamine (3p)
Brown solid, m.p. 60–62 ꢁC, yield: 94%, Rf ¼ 0.55 (ethyl acetate/ Brown solid, m.p. 44–45 ꢁC, yield: 95%, Rf ¼ 0.50 (ethyl acetate/
pet. ether, 20:80), Anal. (C12H10N2O3) Calc. C 62.60, H 4.38, N pet. ether, 20:80), Anal. (C14H11FN2O2) Calc. C 65.11, H 4.29, N
)
12.17; found: C 62.61, H 4.27, N 12.16%. IR (neat): ꢁ (cmꢀ1
10.85; found: C 65.10, H 4.30, N 10.80%. IR (neat): ꢁ (cmꢀ1
)
1
1639 (C¼N), 1523 (NO2 asym), 1346 (NO2 sym), 1149 (C–O–C). 1641 (C¼N), 1510 (NO2 asym), 1341 (NO2 sym), 1156 (C–F). H
1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.54 (s, 1H, CH), 8.31 (d, NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.84 (s, 1H, CH), 8.08 (d,
1H, J ¼ 8.1 Hz, Ar–H), 7.98 (d, 1H, J ¼ 7.8 Hz, Ar–H), 7.67 J ¼ 8.1 Hz, 2H, Ar–H), 7.68 (d, 2H, J ¼ 8.4 Hz, Ar–H), 7.34 (s,
(t, 1H, J ¼ 7.8 Hz, Ar–H), 7.51 (t, 1H, J ¼ 8.1 Hz, Ar–H), 7.34 (d, 2H, Ar–H), 7.07 (s, 2H, Ar–H), 4.86 (s, 2H, CH2). 13C NMR
1H, J ¼ 7.1 Hz, Ar–H), 6.42 (t, 1H, J ¼ 3.4 Hz, Ar-–H), 5.89 (d, (75 MHz, CDCl3) (ꢀ, ppm): 160.46, 157.94, 148.88, 134.26,
1H, J ¼ 5.0 Hz, Ar–H), 4.67 (s, 2H, CH2). 13C NMR (75 MHz, 133.57, 131.12, 130.86, 129.84, 124.36, 115.57, 115.28, 64.45.
CDCl3) (ꢀ, ppm): 160.56, 150.98, 148.65, 142.13, 135.23, 132.70, ESI-MS (m/z): 259.65 [M + H]+.
132.22, 130.58, 122.67, 110.62, 106.83, 56.78. ESI-MS (m/z):
231.43 [M + H]+.
(3-Nitrobenzylidene)(4-fluorophenyl)methylamine (3q)
Yellow solid, m.p. 83–84 ꢁC, yield: 85%, Rf ¼ 0.53 (ethyl acetate/
(3-Nitrobenzylidene)(furan-2-yl)methylamine (3l)
pet. ether, 20:80), Anal. (C14H11FN2O2) Calc. C 65.11, H 4.29, N
)
Yellow solid, m.p. 58–60 ꢁC, yield: 92%, Rf ¼ 0.54 (ethyl acetate/ 10.85; found: C 65.07, H 4.21, N 10.80%. IR (neat): ꢁ (cmꢀ1
1
pet. ether, 20:80), Anal. (C12H10N2O3) Calc. C 62.60, H 4.38, N 1641 (C¼N), 1508 (NO2 asym), 1350 (NO2 sym), 1160 (C–F). H
12.17; found: C 62.63, H 4.49, N 12.13%. IR (neat): ꢁ (cmꢀ1
)
NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.62 (s, 1H, Ar–H), 8.47 (s,
1642 (C¼N), 1529 (NO2 asym), 1346 (NO2 sym), 1147 (C–O–C). 1H, CH), 8.30 (d, 1H, J ¼ 8.1 Hz, Ar–H), 8.14 (d, 1H, J ¼ 7.8 Hz,
1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.60 (s, 1H, Ar–H), 8.41 Ar–H), 7.62 (t, 1H, J ¼ 7.8, 8.1 Hz, Ar–H), 7.33 (t, 2H, J ¼ 8.4 Hz,
(s, 1H, CH), 8.29 (d, 1H, J ¼ 8.1 Hz, Ar–H), 8.13 (d, 1H, Ar–H), 7.07 (t, 2H, J ¼ 8.7 Hz, Ar–H), 4.86 (s, 2H, CH2).

DOI: 10.3109/14756366.2015.1058257
ꢂ-Lactam and unexpected oxazinone formation
5
13C NMR (75 MHz, CDCl3) (ꢀ, ppm): 160.46, 159.27, 148.60, 7.61–7.24 (m, 13H, Ar–H), 5.43 (d, 1H, J ¼ 5.7 Hz, azetidinone
137.68, 134.27, 133.67, 129.65, 125.25, 123.07, 115.59, 115.31, ring-Ha), 5.14 (s, 2H, CH2), 4.15 (d, 1H, J ¼ 4.2 Hz, azetidinone
64.16. ESI-MS (m/z): 259.46 [M + H]+.
ring-Hb). 13C NMR (75 MHz, CDCl3) (ꢀ, 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]+.
(4-Nitrobenzylidene)(4-fluorophenyl)methylamine (3r)
Light yellow solid, m.p. 62–64 ꢁC, yield: 90.7%, Rf ¼ 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): ꢁ
1-Benzyl-4-(3-nitrophenyl)-3-phenylazetidin-2-one (4b)
(cmꢀ1) 1644 (C¼N), 1508 (NO2 asym), 1341 (NO2 sym), 1158 (C– Yellow solid, m.p. 98–100 ꢁC, yield: 21%, Rf ¼ 0.21 (ethyl
1
F). H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.40 (s, 1H, CH), 8.21 acetate/pet. ether, 20:80), Anal. (C22H18N2O3) Calc. C 73.73, H
(d, 2H, J ¼ 8.7 Hz, Ar–H), 7.87 (d, 2H, J ¼ 8.7 Hz, Ar–H), 7.27– 5.06, N 7.82; found: C 73.71, H 5.01, N 7.80%. IR (neat): ꢁ
7.20 (m, 2H, Ar–H), 6.98 (t, 2H, J ¼ 8.7 Hz, Ar–H), 4.78 (s, 2H, (cmꢀ1) 1749 (C¼O), 1527 (NO2 asym), 1346 (NO2 sym). 1H NMR
CH2). 13C NMR (75 MHz, CDCl3) (ꢀ, ppm): 159.56, 149.11, (300 MHz, CDCl3) (ꢀ, ppm): 7.93 (s, 1H, Ar–H), 7.82 (s, 1H, Ar–
141.46, 134.20, 129.66, 128.96, 123.89, 115.60, 115.32, 64.39. H), 7.32–7.19 (m, 7H, Ar–H), 7.09–6.97 (m, 5H, Ar–H), 4.98–
ESI-MS (m/z): 257.36 [M ꢀ H]–.
4.93 (m, 3H, azetidinone ring-Ha, CH2), 4.04 (d, 1H, J ¼ 11.1 Hz,
azetidinone ring-Hb). 13C NMR (75 MHz, CDCl3) (ꢀ, 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]+.
(2-Chlorobenzylidene)(4-fluorophenyl)methylamine (3s)
Yellow oil, yield: 87%, Rf ¼ 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): ꢁ (cmꢀ1) 1639 (C¼N), 1158
(C–F), 754 (C–Cl). 1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 8.77 (s,
1-Benzyl-4-(4-nitrophenyl)-3-phenylazetidin-2-one (4c)
1H, CH), 8.01 (d, 1H, J ¼ 7.5 Hz, Ar–H), 7.30–7.19 (m, 5H, Ar– Light yellow solid, m.p. 160–165 ꢁC, yield: 36%, Rf ¼ 0.23 (ethyl
H), 6.97 (t, 2H, J ¼ 8.7 Hz, Ar–H), 4.75 (s, 2H, CH2). 13C NMR acetate/pet. ether, 20:80), Anal. (C22H18N2O3) Calc. C 73.73, H
(75 MHz, CDCl3) (ꢀ, ppm): 160.96, 158.93, 138.88, 134.57, 5.06, N 7.82; found: C 73.69, H 5.04, N 7.78%. IR (neat): ꢁ
133.07, 130.70, 130.62, 129.87, 128.97, 127.64, 113.12, 63.98. (cmꢀ1) 1754 (C¼O), 1510 (NO2 asym), 1344 (NO2 sym). 1H NMR
ESI-MS (m/z): 248.73 [M + H]+.
(300 MHz, CDCl3) (ꢀ, ppm): 7.89 (d, 2H, J ¼ 8.7 Hz, Ar–H), 7.25
(d, 2H, J ¼ 5.1 Hz, Ar–H), 7.14–6.89 (m, 10H, Ar–H), 4.92 (d,
1H, J ¼ 14.7 Hz, azetidinone ring-Ha), 4.85 (s, 2H, CH2), 3.93 (d,
1H, J ¼ 14.7 Hz, azetidinone ring-Hb). 13C NMR (75 MHz,
CDCl3) (ꢀ, 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]+.
(4-Chlorobenzylidene)(4-fluorophenyl)methylamine (3t)
Yellow solid, m.p. 48–50 ꢁC, yield: 95%, Rf ¼ 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 (C¼N),
1214 (C–F), 1093 (C–Cl) cmꢀ1. H NMR (300 MHz, CDCl3) (ꢀ,
1
ppm): 8.27 (s, 1H, CH), 7.64 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.32 (d,
1-Benzyl-4-(2-chlorophenyl)-3-phenylazetidin-2-one (4d)
2H, J ¼ 8.4 Hz, Ar–H), 7.22 (t, 2H, J ¼ 8.1 Hz, Ar–H), 6.96 (t, 2H,
J ¼ 8.4, 8.7 Hz, Ar–H), 4.70 (s, 2H, CH2). 13C NMR (75 MHz, Yellow solid, m.p. 110–112 ꢁC yield: 75%, Rf ¼ 0.53 (ethyl
CDCl3) (ꢀ, ppm): 160.66, 159.13, 138.56, 134.43, 130.86, 130.54, acetate/pet. ether, 20:80), Anal. (C22H18ClNO) Calc. C 75.97, H
129.78, 127.64, 113.12, 63.45. ESI-MS (m/z): 248.45 [M + H]+.
5.22, N 4.03; found: C 75.96, H 5.17, N 4.00%. IR (neat): ꢁ
(cmꢀ1) 1752 (C¼O), 754 (C–Cl). 1H NMR (300 MHz, CDCl3) (ꢀ,
ppm): 7.33 (t, 4H, J ¼ 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 ¼ 4.2 Hz, azetidinone
ring-Ha), 5.02 (s, 2H, CH2), 4.89 (d, 1H, J ¼ 4.2 Hz, azetidinone
ring-Hb). 13C NMR (75 MHz, CDCl3) (ꢀ, 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]+.
General procedure for the synthesis of b-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
1-Benzyl-4-(4-chlorophenyl)-3-phenylazetidin-2-one (4e)
bicarbonate solution. The mixture was extracted with ethyl acetate Light yellow solid, m.p. 138–140 ꢁC, yield: 26%, Rf ¼ 0.33 (ethyl
and the organic layer was washed with brine and dried over acetate/pet. ether, 20:80), Anal. (C22H18ClNO) Calc. C 75.97, H
anhydrous Na2SO4. After concentration under reduced pressure, 5.22, N 4.03; found: C 75.93, H 5.18, N 3.98%. IR (neat): ꢁ
the residue was purified by silica-gel chromatography (230–400 (cmꢀ1) 1754 (C¼O), 758 (C–Cl). 1H NMR (500 MHz, CDCl3) (ꢀ,
mesh) using 30–40% ethyl acetate in petroleum ether. The title ppm): 7.37 (d, J ¼ 4.8 Hz, 2H, Ar–H), 7.34–7.27 (m, 6H, Ar–H),
compounds (4a–g, 4i–s) were obtained in low to good yields25. 7.22–7.18 (m, 6H, Ar–H), 4.95 (d, 1H, J ¼ 9.3 Hz, azetidinone
The 1,3-oxazin-4-one derivatives (4h, 4t) were also obtained ring-Ha), 4.16 (s, 2H, CH2), 3.84 (d, 1H, J ¼ 8.7 Hz, azetidinone
under the same reaction conditions in low to moderate yields ring-Hb). 13C NMR (75 MHz, CDCl3) (ꢀ, ppm): 168.07, 135.35,
ranging from 22% to 41%.
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-Benzyl-4-(2-nitrophenyl)-3-phenylazetidin-2-one (4a)
Brown solid, m.p. 100–102 ꢁC, yield: 64%, Rf ¼ 0.20 (ethyl
1-(2-Chlorobenzyl)-4-(2-nitrophenyl)-3-phenylazetidin-2-one (4f)
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): ꢁ Brown oil, yield: 46%, Rf ¼ 0.26 (ethyl acetate/pet. ether, 20:80),
(cmꢀ1) 1749 (C¼O), 1518 (NO2 asym), 1346 (NO2 sym). 1H NMR Anal. (C22H17ClN2O3) Calc. C 67.26, H 4.36, N 7.13; found: C
(300 MHz, CDCl3) (ꢀ, ppm): 7.91 (d, 1H, J ¼ 8.1 Hz, Ar–H), 67.21, H 4.33, N 7.12%. IR (neat): ꢁ (cmꢀ1) 1756 (C¼O), 1525

6
B. Aneja et al.
J Enzyme Inhib Med Chem, Early Online: 1–19
(NO2 asym), 1344 (NO2 sym), 754 (C–Cl). 1H NMR (300 MHz, 133.20, 129.91, 129.42, 128.91, 128.53, 127.56, 121.91, 110.91,
CDCl3) (ꢀ, ppm): 7.80 (d, 2H, J ¼ 7.8 Hz, Ar–H), 7.40–7.17 (m, 107.89, 52.43, 51.50, 43.30. ESI-MS (m/z): 349.0 [M + H]+.
8H, Ar–H), 7.00–6.64 (m, 3H, Ar–H), 5.37 (d, 1H, J ¼ 6.0 Hz,
azetidinone ring-Ha), 4.99 (d, 1H, J ¼ 6.6 Hz, azetidinone ring- 1-(Furan-2-ylmethyl)-4-(3-nitrophenyl)-3-phenylazetidin-2-
Hb), 4.39 (s, 2H, CH2). 13C NMR (75 MHz, CDCl3) (ꢀ, ppm): one (4l)
169.98, 148.56, 144.39, 137.92, 136.54, 134.75, 133.42, 133.12,
Yellow oil, yield: 44%, Rf ¼ 0.22 (ethyl acetate/pet. ether, 20:80),
132.62, 131.81, 129.51, 129.11, 128.86, 128.37, 128.19, 126.56,
Anal. (C20H16N2O4) Calc. C 68.96, H 4.63, N 8.04; found: C
124.39, 62.76, 57.42, 45.78. ESI-MS (m/z): 393.01 [M + H]+.
68.92, H 4.59, N 8.02%. IR (neat): ꢁ (cmꢀ1) 1754 (C¼O), 1531
(NO2 asym), 1350 (NO2 sym). 1H NMR (300 MHz, CDCl3) (ꢀ,
1-(2-Chlorobenzyl)-4-(3-nitrophenyl)-3-phenylazetidin-2-one (4g)
ppm): 8.22 (d, 2H, J ¼ 7.8 Hz, Ar–H), 8.17 (s, 1H, Ar–H), 7.67–
Brown oil, yield: 35%, Rf ¼ 0.33 (ethyl acetate/pet. ether, 20:80), 7.53 (m, 2H, Ar–H), 7.39–7.24 (m, 5H, Ar–H), 6.27–6.17 (m, 2H,
Anal. (C22H17ClN2O3) Calc. C 67.26, H 4.36, N 7.13; found: C Ar–H), 4.79 (d, 1H, J ¼ 15.9 Hz, azetidinone ring-Ha), 4.57 (s,
67.22, H 4.34, N 7.11%. IR (neat): ꢁ (cmꢀ1) 1771 (C¼O), 1531 2H, CH2), 4.22 (d, 1H, J ¼ 15.6 Hz, azetidinone ring-Hb). 13C
(NO2 asym), 1350 (NO2 sym), 756 (C–Cl). 1H NMR (300 MHz, NMR (75 MHz, CDCl3) (ꢀ, ppm): 168.56, 148.34, 148.09, 142.14,
CDCl3) (ꢀ, ppm): 7.77 (s, 1H, Ar–H), 7.53 (d, 1H, J ¼ 6.6 Hz, Ar– 139.76, 136.42, 129.72, 129.49, 128.26, 127.61, 127.58, 126.87,
H), 7.42–7.04 (m, 11H, Ar–H), 5.15 (d, 1H, J ¼ 15.3 Hz, 122.32, 109.83, 106.89, 56.23, 50.62, 45.42. ESI-MS (m/z): 349.0
azetidinone ring-Ha), 4.37 (d, 1H, J ¼ 15.6 Hz, azetidinone ring- [M + H]+.
Hb), 3.39 (d, 1H, J ¼ 14.7 Hz, CH2), 3.23 (d, 1H, J ¼ 14.7 Hz,
CH2). 13C NMR (75 MHz, CDCl3) (ꢀ, ppm): 169.63, 148.94, 1-(Furan-2-ylmethyl)-4-(4-nitrophenyl)-3-phenylazetidin-2-
145.34, 139.72, 136.41, 134.92, 133.91, 133.67, 132.52, 131.98, one (4m)
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]+.
Brown oil, yield: 24%, Rf ¼ 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): ꢁ (cmꢀ1) 1752 (C¼O), 1518
(NO2 asym), 1344 (NO2 sym). 1H NMR (300 MHz, CDCl3) (ꢀ,
ppm): 7.87 (s, 2H, Ar–H), 7.48–6.90 (m, 8H, Ar–H), 6.16 (d, 2H,
1-(2-Chlorobenzyl)-4-(2-chlorophenyl)-3-phenylazetidin-2-
one (4i)
Yellow oil, yield: 45%, Rf ¼ 0.38 (ethyl acetate/pet. ether, 20:80), J ¼ 26.4 Hz, Ar–H), 4.92 (d, 1H, J ¼ 21.3 Hz, azetidinone ring-
Anal. (C22H17Cl2NO) Calc. C 69.12, H 4.48, N 3.66; found: C Ha), 4.72 (d, 1H, J ¼ 15.3 Hz, azetidinone ring-Hb), 4.20 (s, 2H,
69.08, H 4.50, N 3.62%. IR (neat): ꢁ (cmꢀ1) 1765 (C¼O), 754 CH2). 13C NMR (125 MHz, CDCl3) (ꢀ, ppm): 167.49, 148.24,
(C–Cl). 1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 7.55 (d, 1H, 147.37, 142.80, 131.64, 128.48, 128.43, 128.01, 127.57, 123.21,
J ¼ 7.5 Hz, Ar–H), 7.35 (t, 2H, J ¼ 4.2 Hz, Ar–H), 7.28–7.13 (m, 110.60, 109.29, 61.59, 60.04, 37.97. ESI-MS (m/z): 349.06
7H, Ar–H), 7.10–7.03 (m, 3H, Ar–H), 5.42 (d, 1H, J ¼ 14.4 Hz, [M + H]+.
azetidinone ring-Ha), 4.30 (d, 1H, J ¼ 15.9 Hz, azetidinone ring-
Hb), 3.43 (s, 2H, CH2). 13C NMR (125 MHz, CDCl3) (ꢀ, ppm): 4-(2-Chlorophenyl)-1-(furan-2-ylmethyl)-3-phenylazetidin-2-
168.47, 136.56, 134.07, 131.92, 130.72, 130.45, 130.26, 129.54, one (4n)
129.39, 128.74, 128.26, 128.21, 128.07, 127.39, 127.20, 126.38,
Yellow solid, m.p. 131–133 ꢁC yield: 72%, Rf ¼ 0.45 (ethyl
126.07, 66.54, 57.80, 44.56. ESI-MS (m/z): 382.04 [M + H]+.
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): ꢁ
(cmꢀ1) 1756 (C¼O), 735 (C–Cl). 1H NMR (300 MHz, CDCl3) (ꢀ,
1-(2-Chlorobenzyl)-4-(4-chlorophenyl)-3-phenylazetidin-2-
one (4j)
ppm): 7.39–7.20 (m, 7H, Ar–H), 7.14–7.06 (m, 4H, Ar–H), 6.34
(d, 1H, J ¼ 7.8 Hz, Ar–H), 4.91 (d, 1H, J ¼ 16.5 Hz, azetidinone
ring-Ha), 4.28 (d, 1H, J ¼ 9.6 Hz, azetidinone ring-Hb), 3.46 (s,
2H, CH2). 13C NMR (75 MHz, CDCl3) (ꢀ, 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]+.
Yellow oil, yield: 36%, Rf ¼ 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): ꢁ (cmꢀ1) 1765 (C¼O), 756
(C–Cl). 1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 7.55 (d, 2H,
J ¼ 6.6 Hz, Ar–H), 7.50 (t, 2H, J ¼ 6.0 Hz, Ar–H), 7.38–7.17 (m,
8H, Ar–H), 7.04 (d, 1H, J ¼ 8.4 Hz, Ar–H), 5.29 (d, 1H,
J ¼ 15.6 Hz, azetidinone ring-Ha), 4.56 (s, 2H, CH2), 4.29
(d, 1H, J ¼ 15.0 Hz, azetidinone ring-Hb). 13C NMR (75 MHz, 4-(4-Chlorophenyl)-1-(furan-2-ylmethyl)-3-phenylazetidin-2-
CDCl3) (ꢀ, ppm): 167.56, 139.71, 138.94, 137.67, 136.51, 134.51, one (4o)
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]+.
Light yellow oil, yield: 32%, Rf ¼ 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): ꢁ (cmꢀ1) 1756
(C¼O), 737 (C–Cl) cmꢀ1. 1H NMR (300 MHz, CDCl3) (ꢀ, ppm):
7.36–7.13 (m, 7 H, Ar–H), 7.04 (d, 2H, J ¼ 6.3 Hz, Ar–H), 6.83
(d, 2H, J ¼ 5.1 Hz, Ar–H), 6.26 (d, 1H, J ¼ 3.0 Hz, Ar–H), 4.83 (d,
1H, J ¼ 11.7 Hz, azetidinone ring-Ha), 4.39 (s, 2H, CH2), 4.26 (d,
1H, J ¼ 11.7 Hz, azetidinone ring-Hb). 13C NMR (75 MHz,
CDCl3) (ꢀ, 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-(Furan-2-ylmethyl)-4-(2-nitrophenyl)-3-phenylazetidin-2-
one (4k)
Brown solid, m.p. 122–124 ꢁC, yield: 44%, Rf ¼ 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): ꢁ
(cmꢀ1) 1762 (C¼O), 1525 (NO2 asym), 1344 (NO2 sym). 1H NMR
(300 MHz, CDCl3) (ꢀ, ppm): 7.92 (d, 1H, J ¼ 8.1 Hz, Ar–H), 7.49
(d, 1H, J ¼ 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 ¼ 17.1 Hz, Ar–H), 5.54 (d, 1H,
J ¼ 5.7 Hz, azetidinone ring-Ha), 5.12 (d, 1H, J ¼ 5.7 Hz,
azetidinone ring-Hb), 4.88 (s, 2H, CH2). 13C NMR (75 MHz,
CDCl3) (ꢀ, ppm): 168.42, 147.79, 147.21, 142.10, 136.71, 134.94,
1-(4-Fluorobenzyl)-4-(2-nitrophenyl)-3-phenylazetidin-2-one (4p)
Brown solid, m.p. 146–148 ꢁC yield: 46%, Rf ¼ 0.19 (ethyl
acetate/pet. ether, 20:80), Anal. (C22H17FN2O3) Calc. C 70.20,

DOI: 10.3109/14756366.2015.1058257
ꢂ-Lactam and unexpected oxazinone formation
7
H 4.55, N 7.44; found: C 70.21, H 4.52, N 7.41%. IR (neat): ꢁ (s, 1H), 4.46 (q, 2H, J ¼ 14.7 Hz), 3.44 (d, 1H, J ¼ 9.3 Hz), 3.32
(cmꢀ1) 1752 (C¼O), 1510 (NO2 asym), 1344 (NO2 sym), 1160 (d, 1H, J ¼ 8.7 Hz). 13C NMR (125 MHz, CDCl3) (ꢀ, ppm):
(C–F). 1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 7.89 (d, 1H, 165.61, 148.23, 142.72, 135.13, 133.83, 131.89, 131.40, 130.87,
J ¼ 8.1 Hz, Ar–H), 7.46 (t, 1H, J ¼ 7.8 Hz, Ar–H), 7.36 (d, 130.71, 129.86, 129.57, 128.99, 128.39, 127.81, 127.43, 126.91,
1H, J ¼ 6.9 Hz, Ar–H) 7.34–7.21 (m, 3H, Ar–H), 7.22–7.16 (m, 126.28, 123.24, 115.00, 86.19, 69.31, 45.39, 43.13, 37.48. ESI-
7H, Ar–H), 5.41 (d, 1H, J ¼ 5.7 Hz, azetidinone ring-Ha), 5.06 (d, MS (m/z): 511.2 [M + H]+.
1H, J ¼ 6.0 Hz, azetidinone ring-Hb), 4.12 (s, 2H, CH2). 13C NMR
(125 MHz, CDCl3) (ꢀ, ppm): 169.01, 160.94, 134.96, 130.87, 3-(4-Fluorobenzyl)-6-benzyl-2-(4-chlorophenyl)-5-phenyl-2,3-
130.40, 130.17, 128.95, 128.79, 128.61, 128.51, 128.34, 128.14, dihydro-1,3-oxazin-4-one (4t)
127.49, 115.94, 85.70, 62.42, 57.66, 45.21. ESI-MS (m/z): 377.0
Light yellow solid, m.p. 119–121 ꢁC, yield: 41%, Rf ¼ 0.42 (ethyl
[M + H]+.
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): ꢁ (cmꢀ1
)
1-(4-Fluorobenzyl)-4-(3-nitrophenyl)-3-phenylazetidin-2-one (4q)
1754 (C¼O), 1158 (C–F), 748 (C–Cl) cmꢀ1. 1H NMR (500 MHz,
Brown oil, yield: 42%, Rf ¼ 0.14 (ethyl acetate/pet. ether, 20:80), CDCl3) (ꢀ, ppm): 7.22–6.88 (m, 15H), 6.82 (d, 2H, J ¼ 6.3 Hz),
Anal. (C22H17FN2O3) Calc. C 70.20, H 4.55, N 7.44; found: C 6.07 (s, 1H), 5.22 (d, 1H, J ¼ 15.3 Hz), 3.95 (d, 2H, J ¼ 15.3 Hz),
70.18, H 4.53, N 7.46%. IR (neat): ꢁ (cmꢀ1) 1754 (C¼O), 1531 3.37 (q, 2H, J ¼ 14.7 Hz). 13C NMR (125 MHz, CDCl3) (ꢀ, ppm):
(NO2 asym), 1348 (NO2 sym), 1160 (C–F) cmꢀ1 1H NMR 168.02, 160.93, 135.53, 134.56, 130.95, 129.84, 129.42, 129.01,
.
(300 MHz, CDCl3) (ꢀ, ppm): 8.12 (s, 1H, Ar–H), 7.94 (d, 2H, 128.80, 128.53, 128.26, 127.26, 126.66, 115.64, 86.64, 65.28,
J ¼ 5.4 Hz, Ar–H), 7.84 (d, 2H, J ¼ 4.5 Hz, Ar–H), 7.78–7.72 (m, 46.69, 44.04. ESI-MS (m/z): 482.0 [M ꢀ H]ꢀ, 484.0 [M + H]+.
2H, Ar–H), 7.32 (t, 2H, J ¼ 7.9 Hz, Ar–H), 7.12–7.10 (m, 2H, Ar–
H), 6.75 (d, 2H, J ¼ 5.1 Hz, Ar–H), 5.22 (d, 1H, J ¼ 11.4 Hz, X-ray crystallographic analysis
azetidinone ring-Ha), 4.93 (s, 2H, CH2), 4.88 (d, 1H, J ¼ 10.8 Hz,
The structures of the compounds 4e and 4t were unequivocally
azetidinone ring-Hb). 13C NMR (75 MHz, CDCl3) (ꢀ, ppm):
established by X-ray crystallographic analysis. Single crystal of 4e
167.81, 161.25, 148.18, 137.53, 134.90, 133.22, 132.64, 132.33,
was obtained through the slow evaporation of its EtOAc–DCM
130.91, 129.17, 128.77, 128.33, 127.51, 126.79, 115.64, 65.58,
solution and single crystal of 4t was obtained through the slow
59.10, 47.05. ESI-MS (m/z): 377.0 [M + H]+.
evaporation of its EtOAc–hexane solution. Suitable crystals
(C22H18ClNO, compound 4e; C30H23ClFNO2, compound 4t)
1-(4-Fluorobenzyl)-4-(4-nitrophenyl)-3-phenylazetidin-2-one (4r)
were selected and analyzed on a D8 Venture Dual Source 100
Yellow solid, m.p. 167–169 ꢁC, yield: 22%, Rf ¼ 0.28 (ethyl CMOS diffractometer (Karlsruhe, Germany) equipped with Cu
˚
acetate/pet. ether, 20:80), Anal. (C22H17FN2O3) Calc. C 70.20, H radiation (Cu Ka ¼ 1.54178 A). Intensity data were collected at
4.55, N 7.44; found: C 70.16, H 4.52, N 7.42%. IR (neat): ꢁ 100 K using ꢃ and ! scans. No significant loss in intensities was
(cmꢀ1) 1754 (C¼O), 1508 (NO2 asym), 1344 (NO2 sym), 1160 (C–F). observed during data collection. Multi-scan absorption correc-
1H NMR (300 MHz, CDCl3) (ꢀ, ppm): 7.97 (d, 2H, J ¼ 8.7 Hz, tions were applied to the intensity data empirically using
Ar–H), 7.20–6.95 (m, 12H, Ar–H), 4.96–4.89 (m, 3H, CH2, SADABS26. Data collection, reduction and refinement were
azetidinone ring-Ha), 3.98 (d, 1H, J¼ 14.7 Hz, azetidinone ring-Hb). performed using APEX2 V2014.5-026, SAINT V8.34A27 soft-
13C NMR (75 MHz, CDCl3) (ꢀ, ppm): 167.78, 161.12, 147.50, ware. Crystal structures were solved by direct methods using
142.59, 131.62, 130.49, 130.38, 128.47, 128.20, 127.62, 123.38, SHELXT-201428 and refined with full-matrix least-squares based
116.11, 115.82, 61.58, 59.14, 44.61. ESI-MS (m/z): 376.99 on F2 using SHELXT-201428. All non-hydrogen atoms were
[M + H]+.
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.
1-(4-Fluorobenzyl)-4-(2-chlorophenyl)-3-phenylazetidin-2-
one (4s)
Yellow solid, m.p. 96–98 ꢁC yield: 63%, Rf ¼ 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): ꢁ (cmꢀ1) 1754
(C¼O), 1160 (C–F), 754 (C–Cl). 1H NMR (300 MHz, CDCl3) (ꢀ,
ppm): 7.26 (d, 1H, J ¼ 8.1 Hz, Ar–H), 7.19 (t, 2H, J ¼ 7.7 Hz, Ar–
H), 7.10 (d, 2H, J ¼ 7.7 Hz, Ar–H), 7.05–7.01 (m, 6H, Ar–H),
6.81 (t, 2H, J ¼ 6.1 Hz, Ar–H), 5.37 (s, 2H, CH2), 5.04 (d, 1H,
J ¼ 15.0 Hz, azetidinone ring-Ha), 4.30 (d, 1H, J ¼ 15.0 Hz,
azetidinone ring-Hb). 13C NMR (75 MHz, CDCl3) (ꢀ, 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]+.
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 indica-
tions to predict drug disposition in human body, their pharma-
cological and toxicological effects. Hepato-toxocity level predicts
the probability of drugs to cause liver injuries. The carcinogenic
3-(2-Chlorobenzyl)-6-benzyl-2-(4-nitrophenyl)-5-phenyl-2,3-dihy-
dro-1,3-oxazin-4-one (4h)
Yellow oil, yield: 22%, Rf ¼ 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): ꢁ (cmꢀ1) 1767 (C¼O), 1523
(NO2 asym), 1346 (NO2 sym), 756 (C–Cl). 1H NMR (500 MHz,
CDCl3) (ꢀ, ppm): 8.25 (d, 1H, J ¼ 8.5 Hz), 7.97 (d, 2H,
J ¼ 8.0 Hz), 7.89 (d, 1H, J ¼ 8.5 Hz), 7.35–7.10 (m, 10 H), 6.81
(d, 2H, J ¼ 8.0 Hz), 6.33 (s, 1H), 5.25 (d, 1H, J ¼ 15.5 Hz), 5.07

8
B. Aneja et al.
J Enzyme Inhib Med Chem, Early Online: 1–19
Scheme 1. Synthesis of monocyclic
b-lactams and unexpected 1,3-oxazin-4-ones.
and mutagenic effects of all synthesized compounds were PBS was added to yield a 10% (v/v) erythrocytes/PBS suspension.
analyzed with TOPKAT tool.
The 10% suspension was then diluted 1:10 in PBS. From each
suspension, 100 mL was added in triplicate to 100 mL 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 mL 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:
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 ꢂ 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 mL 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.
ꢀ
ꢁ
A450 of test compound treated sample
ꢀA450 of buffer treated sample
ꢀ
ꢁ
% Hemolysis ¼
A450 of 1% Triton X ꢀ 100 treated sample
ꢀA450 of buffer treated sample
ꢂ100%:
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 mg/mL
streptomycin, HiMedia) at 37 ꢁC in humidified atmosphere of 5%
CO2. The cells were sub-cultured twice in a week.
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,
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 ꢂ 104 cells/well were seeded into 96-well plates

DOI: 10.3109/14756366.2015.1058257
ꢂ-Lactam and unexpected oxazinone formation
9
Table 1. Structural data of b-lactam derivatives (4a–g, 4i–s).
Ph
O
N
Ar
2
Ar
1
.
Compound
Ar1
Ar2
Mol. formula
C22H18N2O3
Mol. wt.
358.39
4a
NO
2
4b
4c
C22H18N2O3
358.39
358.39
NO
2
C22H18N2O3
NO
2
4d
4e
C22H18ClNO
347.84
347.84
CI
C22H18ClNO
Cl
4f
C22H17ClN2O3
392.83
392.83
Cl
Cl
NO
2
4g
C22H17ClN2O3
NO
2
4i
4j
C22H17Cl2NO
382.28
382.28
Cl
Cl
Cl
C22H17Cl2NO
Cl
(continued )

10 B. Aneja et al.
J Enzyme Inhib Med Chem, Early Online: 1–19
Table 1. Continued
Ar2
Compound
Ar1
Mol. formula
C20H16N2O4
Mol. wt.
348.35
4k
4l
NO
2
O
C20H16N2O4
348.35
348.35
O
O
NO
2
4m
C20H16N2O4
NO
2
4n
4o
C20H16ClNO2
337.83
337.83
Cl
O
O
C20H16ClNO2
Cl
4p
4q
4r
C22H17FN2O3
C22H17FN2O3
C22H17FN2O3
376.38
376.38
376.38
NO
2
F
F
F
NO
2
NO
2
4s
C22H17ClFNO
365.83
Cl
F

DOI: 10.3109/14756366.2015.1058257
ꢂ-Lactam and unexpected oxazinone formation 11
Table 2. Structural data of 1,3-oxazin-4-one derivatives (4h, 4t).
Ph
Ar
O
O
2
N
Ph
Ar
1
.
Compound
Ar1
Ar2
Mol. formula
Mol. wt.
510.97
4h
C30H23ClN2O4
Cl
NO
2
4t
C30H23ClFNO2
483.96
F
Cl
Scheme 2. Plausible mechanism for the formation of b-lactam derivatives (4a–g and 4i–s). Step 1. Formation of ketene. Step 2. Cyclization of imine
and ketene.
(200 mL/well) and incubated for 24 h before treatment. The cells Reader (Bio-Rad, Hercules, CA). All assays were carried out in
were then treated with variable concentration (50–700 mg/mL) of triplicate. Percent viability was taken as the relative absorbance of
the compounds. After 48 h of incubation at 37 ꢁC, the medium treated versus control (untreated) cells.
was removed and 20 mL 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
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
reduction of MTT, were solubilized in DMSO (200 mL/well)
and quantification was performed by reading the absorbance at
570 nm after incubation period of 15 min on the iMark Microplate

12 B. Aneja et al.
J Enzyme Inhib Med Chem, Early Online: 1–19
Scheme 3. Plausible mechanism for the formation of 1,3-oxazin-4-ones (4h, 4t).
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 A of each other were clustered
Figure 2. General structure of synthesized b-lactam derivatives.
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.
(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.
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 corres-
ponding 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 quanti-
tative 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
Molecular docking
In order to rationalize the mechanism of PBP-5 inhibition by
b-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

DOI: 10.3109/14756366.2015.1058257
ꢂ-Lactam and unexpected oxazinone formation 13
Figure 3. Stack plot of 13C and DEPT-135
NMR spectrum of compound 4b.
gave the b-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 b-lactams as confirmed by single crystal X-ray analysis of 4t.
All imines (3a–t), b-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 b-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 a-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 b-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 [4ꢄ]-conrotatory cyclization may account for
the formation of 1,3-oxazin-4-one.
Table 3. Crystal data and structure refinement details for 4e and 4t.
4e
4t
Formula
C22H18ClNO
347.82
C30H23ClFNO2
483.94
1.348
MW (g/mol)
dcalcd. (g/cm3)
Crystal size (mm3)
T (K)
1.295
0.40 ꢂ 0.24 ꢂ 0.21
0.44 ꢂ 0.19 ꢂ 0.15
100(2)
1.54178
1456
100(2)
1.54178
˚
ꢅ (A)
F (000)
1008
Monoclinic
P21/c
7.8270(3)
11.6200(5)
26.2346(10)
90
92.314(1)
90
2384.1(2)
4
Crystal system
Space group
Monoclinic
P21/n
22.9921(7)
7.3951(2)
23.2487(7)
90
115.472(1)
90
3568.7(2)
8
˚
a (A)
˚
b (A)
˚
c (A)
ꢆ (ꢁ)
ꢂ (ꢁ)
ꢇ (ꢁ)
3
˚
V (A )
Z
ꢈ range (ꢁ)
2.26–68.23
1.00 to 2y 136.5ꢁ
52 509
3.37–68.23
1.00 to 2y 136.5ꢁ
38 217
Completeness
Collected reflections
Unique reflections; Rint
ꢉ/mmꢀ1
6511 [Rint ¼ 0.0333]
4372 [Rint ¼ 0.0398]
1.952
0.0369
1.717
0.0318
R (Fo), [I 42s(I)]
Rw (Fo2) (all data)
0.0955
1.046
0.391/ꢀ0.205
0.0783
1.050
0.236/ꢀ0.288
GoF (F2ꢀ) 3
˚
Dꢊ [e A
]

14 B. Aneja et al.
J Enzyme Inhib Med Chem, Early Online: 1–19
The structures of all the compounds were in good agreement 1.43–1.64 ppm due to CH2 and NH2 protons, respectively. The
with IR, 1H, 13C NMR, mass spectroscopic and analytical data. IR protons belonging to aromatic ring appeared at their usual
spectra of the oximes (1a–d) showed a sharp band in the range chemical shift values.
1570–1599 cmꢀ1 due to C¼N stretch. In addition to this,
In the IR spectra of imine (Schiff’s base) intermediates (3a–t),
appearance of a broad band accounted for O–H stretch in the a band due to C¼N stretch in the region 1639–1650 cmꢀ1 and
1
region 3149–3275 cmꢀ1. In H NMR spectra, oximes showed a disappearance of bands for NH2 as well as for the CHO group
sharp singlet at 8.05–8.59 ppm due to CH proton. The OH proton provided evidence for their formation. The absorption bands for
appeared as a broad singlet at 8.55–9.21 ppm. The protons of aryl as well as heteroaryl rings occurred at their expected
aromatic ring appeared at expected chemical shift and integral positions. The protons belonging to imine group (CH¼N) showed
values which confirmed the formation of the oximes (1a–d). In a sharp singlet at 8.27–8.85 ppm, while a singlet appeared at
case of amines (2a–d), the appearance of broad band at 4.67–4.99 ppm due to CH2 protons. The peaks for aldehydic
3372 cmꢀ1 due to NH2 stretching and disappearance of bands proton and NH2 protons were absent in 1H NMR spectra of
due to C¼N stretch and O–H stretch provided strong evidence for imines. The 13C NMR spectra further confirmed the formation of
1
the formation of amines from their respective oximes. In the H imines as a characteristic peak corresponding to azomethine
NMR spectra, there appeared sharp singlets at 3.46–3.94 ppm and carbon appeared at 157.78–161.67 ppm, while peaks correspond-
ing to other groups appeared at their expected chemical
Figure 4. ORTEP diagram of compound 4e with atomic labeling scheme
(50% probability level of thermal ellipsoids).
Figure 6. ORTEP diagram of compound 4t 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).

DOI: 10.3109/14756366.2015.1058257
ꢂ-Lactam and unexpected oxazinone formation 15
shift values. In the mass spectra, all imines exhibited molecular 51.56–65.58 ppm, respectively. In the 13C NMR spectra of
ion peaks as the base peak. In the spectral analysis of b-lactam compound 4b, 17 different carbon atoms were observed which
derivatives (4a–g, 4i–s), IR absorption peaks for the imine group were consistent with the proposed structure of the compound.
(CH¼N) disappeared, while strong peaks for the carbonyl group Distortionless enhancement of NMR signals by polarization
appeared in the region 1749–1771 cmꢀ1 indicated the formation transfer (DEPT) is used to discriminate primary, secondary and
1
of b-lactam ring. In H NMR spectra, both protons belonging to tertiary carbon atoms (Figure 3). In the DEPT spectrum of
b-lactam ring appeared as doublets. The proton (Ha) in the compound 4b, peak for CH2 group appeared in negative phase at
vicinity of ring nitrogen was deshielded and appeared at the 45.31 ppm, while peaks for two CH protons of b-lactam ring
chemical shift value in the range 4.79–5.54 ppm, while other appeared in positive phase at 61.42 and 59.06 ppm. The carbons
proton (Hb) near carbonyl group of b-lactam ring was shielded in that are recorded in 13C NMR, but absent in DEPT spectrum, are
comparison to other proton and appeared at 4.28–5.12 ppm quaternary carbons without any attached hydrogens so the
(Figure 2). The formation of b-lactam ring was further confirmed resonances corresponding to three aromatic carbons which
by 13C NMR spectral data in which carbonyl group appeared at appeared at 147.92, 137.51, 134.85 ppm in 13C NMR spectra
chemical shift values 169.98–160.66 ppm and both the tetrahedral were absent from DEPT spectrum confirming their quaternary
carbons (C3 and C4) of b-lactam ring appeared at 47.81–63.37 and nature. Similarly, peak for carbonyl group at 167.64 in 13C NMR
Figure 7. Unit cell in the crystal packing of compound 4t (hydrogen bonding is shown by dotted lines).
Table 4. ADMET and TOPKAT analysis.
ADMET
TOPKAT
Ames Mut.
Compound
Mol. wt.
Sol. level
Alog P
HepTox level
CYP2D6 prob.
PPB level
PSA
WOE
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
358.39
358.39
358.39
347.84
347.84
392.84
392.84
392.84
382.28
382.28
348.35
348.35
348.35
337.83
337.83
376.38
376.38
376.38
365.83
365.83
2
2
2
1
1
2
2
2
1
1
3
3
3
2
2
2
2
2
1
1
5.23
5.22
5.22
6.49
6.48
5.20
5.20
5.19
6.46
6.45
3.40
3.39
3.39
4.66
4.65
4.93
4.92
4.90
6.19
6.18
0
0
0
1
1
0
1
1
0
1
0
0
0
1
1
0
0
0
1
1
0.46
0.43
0.41
0.38
0.38
0.34
0.32
0.34
0.32
0.33
0.46
0.44
0.44
0.45
0.36
0.38
0.38
0.40
0.34
0.34
2
2
2
2
2
2
2
2
2
2
0
0
0
1
1
1
1
1
2
2
69.15
69.15
69.15
24.16
24.16
69.15
69.15
69.15
24.16
24.16
78.08
78.08
78.08
33.09
33.09
69.15
69.15
69.15
24.16
24.16
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
4t
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 probability50.5 is ideal, and level with 0. The probability value for CYP2D650.5 is good value and
2
˚
PPB value comes under level-0 is ideal. For drug like activity, the PSA of compounds ꢃ100 A is ideal.

16 B. Aneja et al.
J Enzyme Inhib Med Chem, Early Online: 1–19
Table 5. In vitro antibacterial activity (MIC and MBC) of b-lactams and
1,3-oxazin-4-one derivatives (in mg/mL).
E. coli (MTCC-739)
K. pneumonia (MTCC-109)
MIC
(mg/mL)
MBC
(mg/mL)
MIC
(mg/mL)
MBC
(mg/mL)
Compound
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
41000
41000
41000
250
41000
41000
41000
250
41000
41000
41000
500
41000
41000
41000
500
41000
41000
41000
41000
41000
41000
41000
41000
41000
41000
41000
62.5
41000
41000
41000
41000
41000
41000
41000
41000
41000
41000
41000
62.5
1000
1000
41000
250
500
41000
500
500
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.
4m
4n
4o
4p
4q
4r
41000
41000
41000
41000
41000
41000
41000
41000
57.8
41000
41000
41000
41000
41000
41000
41000
41000
7.8
41000
41000
41000
41000
41000
41000
41000
41000
15.6
41000
41000
41000
41000
41000
41000
41000
41000
31.2
70.60ꢁ, respectively, while in other molecule the corresponding
angles are 58.95ꢁ, 61.65ꢁ and 64.52ꢁ, 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. . .ꢄ, C¼O. . .ꢄ and C–H. . .Cl
(Figure 5) forming a sheet-like structure.
4s
4t
Ampicillin
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 dimen-
sions 0.44 mm ꢂ 0.19 mm ꢂ 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 A from the least square
plane passing through C3, C2, C1, N1 and C4 atoms, while atom
˚
C4 deviates by 0.680 A 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. . .ꢄ 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).
Pharmacokinetics and in silico toxicity
Figure 8. Hemolytic activity of compounds 4d, 4h, 4k, and 4l on
human RBCs.
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 5500, 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 com-
pounds (4k–r) presented good A log P value ꢃ 5.0. Hepatotoxicity
probability50.5 is unlikely to cause dose-dependent liver injuries
and comes under predicted class-0 (non-toxic), whereas hepato-
toxicity probability 40.5 is liable to cause dose-dependent liver
injuries, i.e. toxic and comes under predicted class-1. The
CYP2D6 probability of all compounds showed 50.5 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.
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 b-lactam ring.
X-ray crystallographic analysis of 4e and 4t
X-ray crystallographic determination of 4e (b-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 ꢂ 0.24 mm
ꢂ 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 A). The angles between planar aromatic rings of
one molecule from the asymmetric unit are 55.41ꢁ, 59.81ꢁ and

DOI: 10.3109/14756366.2015.1058257
ꢂ-Lactam and unexpected oxazinone formation 17
Figure 10. Structure of PBP-5 (K. pneumo-
niae) docked with compound 4l. Polar inter-
actions with active site residues are shown
with black-dot line.
against E. coli. Moreover, compound 4l was found with consid-
erable antibacterial activity having MIC 62.5 mg/mL against
K. pneumoniae. The MBC values were also calculated and
compounds 4d, 4h, 4k and 4l indicated their bactericidal nature.
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 520% hemolysis.
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 mg/mL conc. and none of the
compounds showed any significant cytotoxic effect on CHO cells
up to the conc. of 200 mg/mL. Compound 4l exhibited maximum
cellular viability (100%) at 200 mg/mL concentration. By
increasing the conc. between 300 and 400 mg/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 mg/mL conc.
Figure 11. Pharmacophore feature analysis of compound 4l.
2
˚
For drug activity, the optimum value of PSA ꢃ100 A is well
defined41. The hydrogen bonding and log P are the two main
descriptors, defining the PSA of molecule. All synthesized
2
˚
compounds showed appreciable PSA value ꢃ80 A . TOPKAT
analysis was carried out to predict the probable toxicity of the
compounds. All 20 compounds showed weight of evidence
(WOE) probability ꢃ0.5 and were predicted as non-carcinogen.
Moreover, Ames prediction test evidenced all compounds to be
non-mutagenic.
Antibacterial activity
In silico analysis
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 mg/mL, respectively,
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 A,
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

18 B. Aneja et al.
J Enzyme Inhib Med Chem, Early Online: 1–19
9. Wilke MS, Lovering AL, Strynadka NCJ. b-Lactam antibiotic
resistance: a current structural perspective. Curr Opin Microbiol
2005;8:525–33.
10. Rosenblum SB, Huynh T, Afonso A, et al. Discovery of 1-(4-
fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-hydroxypropyl]-(4S)-
(4-hydroxyphenyl)-2-azetidinone (SCH 58235): a designed, potent,
orally active inhibitor of cholesterol absorption. J Med Chem 1998;
41:973–80.
11. Borthwick AD, Weingarten G, Haley TM, et al. Design and
synthesis of monocyclic beta-lactams as mechanism-based inhibitors
of human cytomegalovirus protease. Bioorg Med Chem Lett 1998;8:
365–70.
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 (b-lactam ring), Arg247 showed polar interaction
with furan ring and nitro group of the ligand and Tyr271 was
involved in ꢄ–ꢄ interaction with m-nitro phenyl (Figure 10).
Pharmacophore feature analysis of compound 4l (Figure 11)
with DS 3.5 showed that carbonyl group of b-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.
´
¨
12. Sperka T, Pitlik J, Bagossi P, Tozser J. Beta-lactam compounds as
apparently uncompetitive inhibitors of HIV-1 protease. Bioorg Med
Chem Lett 2005;15:3086–90.
13. Han WT, Trehan AK, Wright JJ, et al. Azetidin-2-one derivatives as
inhibitors of thrombin. Bioorg Med Chem 1995;3:1123–43.
14. Veinberg G, Shestakova I, Vorona M, et al. Synthesis of antitumor
6-alkylidenepenicillanate sulfones and related 3-alkylidene-2-azeti-
dinones. Bioorg Med Chem Lett 2004;14:147–50.
15. Goel RK, Mahajan MP, Kulkarni SK. Evaluation of anti-hypergly-
cemic activity of some novel monocyclic beta lactams. J Pharm
Pharm Sci 2004;7:80–3.
16. Saturnino C, Fusco B, Saturnino P, et al. Evaluation of analgesic and
anti-inflammatory activity of novel beta-lactam monocyclic com-
pounds. Biol Pharm Bull 2000;23:654–6.
17. Clemente A, Domingos A, Grancho AP, et al. Design, synthesis and
stability of N-acyloxymethyl- and N-aminocarbonyloxymethyl-2-
azetidinones as human leukocyte elastase inhibitors. Bioorg Med
Chem Lett 2001;11:1065–8.
Conclusion
In summary, we have designed a series of novel N-benzyl-3,4-
diaryl substituted 2-azitidinone (b-lactam) derivatives and sur-
prisingly 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.
18. Worthington RJ, Melander C. Overcoming resistance to b-lactam
antibiotics. J Org Chem 2013;78:4207–13.
19. Irfan M, Aneja B, Yadava U, et al. Synthesis, QSAR and
anticandidal evaluation of 1,2,3-triazoles derived from naturally
bioactive scaffolds. Eur J Med Chem 2015;93:246–54.
20. Masood MM, Pillalamarri VK, Irfan M, et al. Diketo acids and their
amino acid/peptidic analogues as promising scaffolds for the
development of bacterial methionine aminopeptidase inhibitors.
RSC Adv 2015;5:34173–83.
21. Nicholas RA, Krings S, Tomberg J, et al. Crystal structure of wild-
type penicillin-binding protein 5 from Escherichia coli. Implications
for deacylation of the acyl-enzyme complex. J Biol Chem 2003;278:
52826–33.
Acknowledgements
The authors are thankful to Prof. S. Chandershekhran, Department of
Organic Chemistry, IISc Bangalore for providing DEPT spectrum.
Declaration of interest
22. Abid M, Husain K, Azam A. Synthesis and antiamoebic activity of
new oxime ether derivatives containing 2-acetylpyridine/2-acetyl-
furan. Bioorg Med Chem Lett 2005;15:4375–9.
23. Moreno E, Plano D, Lamberto I, et al. Sulfur and selenium
derivatives of quinazoline and pyrido[2,3-d]pyrimidine: synthesis
and study of their potential cytotoxic activity in vitro. Eur J Med
Chem 2012;47:283–98.
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.
24. Jarrahpour A, Ebrahimi E, De Clercq E, et al. Synthesis of mono-,
bis-spiro- and dispiro-b-lactams and evaluation of their antimalarial
activities. Tetrahedron 2011;67:8699–704.
25. Bari SS, Bhalla A, Nagpal Y, et al. Synthesis and characterization of
novel trans-3-benzyl/(diphenyl)methyl/naphthyl seleno substituted
monocyclic b-lactams: X-ray structure of trans-1-(40-methoxyphe-
nyl)-3-(diphenyl)methylseleno-4-(40-methoxyphenyl)azetidin-2-one.
J Organomet Chem 2010;695:1979–85.
26. Bruker AXS Inc. SADABS, APEX2. Madison (WI): Bruker AXS
Inc.; 2014.
27. Bruker AXS Inc. SAINT. Madison (WI): Bruker AXS Inc.; 2013.
28. Sheldrick, GM. SHELXT-Integrated space-group and crystal-struc-
ture determination. Acta Crystallogr A Found Crystallogr 2015;71:
3–8.
29. Sheldrick, GM. SHELXTL, version 6.10. Madison (WI): Bruker
AXS Inc.; 2000.
30. Macrae CF, Bruno IJ, Chisholm JA, et al. Mercury CSD 2.0 – new
features for the visualization and investigation of crystal structures.
J Appl Cryst 2008;41:466–70.
31. Accelrys Software Inc. Discovery Studio Modeling Environment
version 3.5.0. San Diego (CA): Accelrys Software Inc.; 2012.
32. NCCLS. Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically, Approved standard. 5th ed. NCCLS
document M7-A5. Wayne (PA): NCCLS; 2006.
References
1. Lohan S, Cameotra SS, Bisht GS. Antibacterial evaluation of
structurally amphipathic, membrane active small cationic peptido-
mimetics: synthesized by incorporating 3-amino benzoic acid as
peptidomimetic element. Eur J Med Chem 2014;83:102–15.
2. Troisi L, Granito C, Pindinelli E. Novel and recent synthesis and
applications of b-lactams. In: Banik BK, ed. Heterocyclic scaffolds
I. Vol. 22. Berlin: Springer; 2010:101–209.
3. Mandal B, Basu B. Synthesis of b-lactams through alkyne–nitrone
cycloadditions. Top Heterocycl Chem 2012;30:85–110.
4. Ojima I, Zuniga ES, Seitz JD. Advances in the use of enantiopure
b-lactams for the synthesis of biologically active compounds of
medicinal interests. Top Heterocycl Chem 2012;30:1–64.
5. Fernndez I, Sierra MA. b-Lactams from Fischer carbene complexes:
scope, limitations, and reaction mechanism. Top Heterocycl Chem
2012;30:65–84.
6. Banik I, Banik BK. Synthesis of b-lactams and their chemical
manipulations via microwave-induced reactions. Top Heterocycl
Chem 2012;30:183–221.
7. Tidwell TT. Preparation of Bis-b-lactams by ketene–imine cyclo-
additions. Top Heterocycl Chem 2012;30:111–45.
8. Turos E. The chemistry and biology of N-thiolated b-lactams. Top
Heterocycl Chem 2012;30:147–81.

DOI: 10.3109/14756366.2015.1058257
ꢂ-Lactam and unexpected oxazinone formation 19
33. Khan A, Ahmad A, Xess I, et al. Anticandidal effect of Ocimum 38. Morris GM, Huey R, Lindstrom W, et al. AutoDock 4 and
sanctum essential oil and its synergy with fluconazole and AutoDockTools 4: automated docking with selective receptor
ketoconazole. Phytomedicine 2010;17:921–5. flexibility. J Comput Chem 2009;30:2785–91.
34. Mehdi SJ, Ahmad A, Irshad M, et al. Cytotoxic effect of carvacrol 39. Fuhrmann J, Rurainski A, Lenhof HP, Neumann D. A new
on human cervical cancer cells. Biol Med 2011;3:307–12.
35. Laskowski RA, MacArthur MW, Moss DS, Thornton JM.
PROCHECK: a program to check the stereochemical quality of 40. Arjona O, Csaky¨ AG, Murcia MC, Plumet J. The Staudinger
Lamarckian genetic algorithm for flexible ligand-receptor docking.
J Comput Chem 2010;31:1911–18.
´
protein structures. J Appl Cryst 1993;26:283–91.
36. Hooft RW, Vriend G, Sander C, Abola EE. Errors in protein
structures. Nature 1996;381:272.
reaction of imines derived from 7-oxanorbornenone: formation of
spiranic oxazinone versus b-lactam rings. Tetrahedron Lett 2002;43:
6405–8.
37. DeLano WL. The PyMOL molecular graphics system. San Carlos 41. Pajouhesh H, Lenz GR. Medicinal chemical properties of successful
(CA): DeLano Scientific LLC; 2003. central nervous system drugs. NeuroRx 2005;2:541–53.
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.