Synthesis, bioassay, crystal structure and ab initio studies of Erlenmeyer azlactones

Article abstract of DOI:10.1016/j.saa.2012.11.054

Several 4-arylidene-2-phenyl-5(4H)-azlactones have been synthesized via Erlenmeyer method. The synthesized compounds have been characterized on the basis of systematic spectral studies (IR, ¹H NMR, ¹³C NMR, and MS). The compound (4Z)

Full text of DOI:10.1016/j.saa.2012.11.054

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 538–545
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, bioassay, crystal structure and ab initio studies of Erlenmeyer azlactones
a
a
a
b
Mehtab Parveen a, , Akhtar Ali , Sarfaraz Ahmed , Ali Mohammed Malla , Mahboob Alam ,
⇑
P.S. Pereira Silva c, Manuela Ramos Silva c, Dong-Ung Lee b
a Department of Chemistry, Aligarh Muslim University, Aligarh 202-002, India
b Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea
c CEMDRX Physics Department, University of Coimbra, 3004-516 Coimbra, Portugal
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
A series of 4-arylidene-2-phenyl-5(4H)-azlactones have been synthesized, characterized on the basis of
systematic spectral studies and screened for their biological activity. Moreover, the Z-configuration
and stability of compounds was ascertained on the basis of spectroscopy techniques, X-ray studies as well
as DFT calculations.
"
4-Arylidene-2-phenyl-5(4H)-
azlactones were synthesized and
fully characterized.
Antimicrobial and antioxidant
activity.
The structure of 5 & 6 were studied
by X-ray study and compared to DFT
calculations.
"
"
"
"
DFT calculations of two compounds
were suggested the stability of the
Z-conformer.
Crystal packing was stabilized by
H-bond, weak CAHꢀ ꢀ ꢀ
p
and
pꢀ ꢀ ꢀp
interactions were observed.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 May 2012
Received in revised form 10 November 2012
Accepted 18 November 2012
Available online 5 December 2012
Several 4-arylidene-2-phenyl-5(4H)-azlactones have been synthesized via Erlenmeyer method. The syn-
thesized compounds have been characterized on the basis of systematic spectral studies (IR, 1H NMR, 13
C
NMR, and MS). The compound (4Z)-4-(3,5-dimethoxybenzylidene)-2-phenyl-1,3-oxazol-5(4H)-one,
C18H15NO4, (5), crystallizes in the orthorhombic system, space group P212121, with a = 5.6793(3) Å,
b = 15.2038(7) Å, c = 17.6919(10) Å, Mr = 309.31, V = 1527.64(14) Å3, Z = 4 and R = 0.0547. The compound
(4Z)-2-phenyl-4-(3,4,5-trimethoxybenzylidene)-1,3-oxazol-5(4H)-one, C19H17NO5, (6) crystallizes in tri-
clinic geometry with space group P-1, having unit cell parameters a = 7.3814(3) Å, b = 8.1446(3) Å,
Keywords:
Aromatic azlactones
Bioassay
c = 13.9845(5) Å,
a = 86.918(3), b = 83.314(2), c
= 82.462(3), Mr = 339.34, V = 827.16(5) Å3, Z = 2 and
R = 0.0433. The DFT calculations of compounds (5) and (6) have been carried out to ascertain the stability
of Z-conformer. The in vitro antimicrobial activity of all the compounds (1–6) was evaluated by the disk
diffusion method against gram +ve and gram ꢁve microorganism and fungal strains. The MIC of the syn-
thesized compounds was determined by agar well diffusion method in 96-well microtiter plate. All the
synthesized compounds were also screened for their free radical scavenging activity by DPPH method.
Ó 2012 Elsevier B.V. All rights reserved.
(4Z)-4-(3,5-dimethoxybenzylidene)-2-
phenyl-1,3-oxazol-5(4H)-one
Crystal structure
DFT calculation
Introduction
During the past few decades many research papers have been
published in the area of Erlenmeyer synthesis by using different
methods [1–5]. The synthesis of azlactones involves the condensa-
⇑
Corresponding author. Mobile: +91 9897179498.
E-mail address: mehtab.organic2009@gmail.com (M. Parveen).
tion of aromatic or aliphatic aldehydes and hippuric acid with a
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.11.054

M. Parveen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 538–545
539
stoichiometric amount of fused sodium acetate in presence of ace-
tic anhydride as the dehydrating agent, the reaction is called Erlen-
meyer Plöchl reaction [6]. The Erlenmeyer reaction was first
described in 1893 by Friedrich Gustav Carl Emil Erlenmeyer [7]
who reported the condensation of benzaldehyde with N-acetylgly-
cine in the presence of acetic anhydride and sodium acetate. The
Erlenmeyer azlactones are five membered heterocyclic compounds
containing nitrogen and oxygen as hetero atoms. The C–2 and C–4
positions of the azlactones are significant for their various biolog-
ical activities [8].
Azlactones, or 2,4-substituted oxazolin-5-ones, are important
intermediates in the preparation of several fine chemicals, includ-
ing amino acids, [9] peptides, [10] some heterocyclic precursors
[11] as well as biosensors or coupling and photosensitive devices
for proteins [12]. Erlenmeyer azlactone derivatives possess impor-
tant biological activities such as an antimicrobial [13], antitumor
[14], anti-inflammatory [15], anti-HIV [16,17], anticonvulsant
[18] and antihypertensive [19]. They have been used in active site
titrations of enzymes [20]. Recently, some new reagents have been
explored for the synthesis of azlactones, such as Al2O3AH3BPO3
[21], Bi(OAc)3 [22], Bi(OTF)3 [23], and Yb(OTF)3 [24]. Although
these method are suitable, but some of them need elevated tem-
peratures and hence possess difficult in handling.
and filtered, washed with water, air dried and recrystallized from
suitable solvent to yield the representative compounds.
(4Z)-4-(2-methoxybenzylidene)-2-phenyloxazol-5(4H)-one (1)
It was recrystallized from CHCl3AEtOH as bright yellow solid;
Yield: 80%, m.p. 154–55 °C (lit. m.p. 154 °C) [2]; Anal. Calc. for
C
17H13NO3: C, 73.11; H, 4.69; N, 5.02. Found: C, 72.98; H, 4.64;
KBr
N, 4.98. IR
m
cmꢁ1: 1788 (C@O), 1669 (C@N), 1653 (C@C),
max
1248 (CAO Lactone); 1H NMR (400 MHz, CDCl3, d, ppm): 3.78 (s,
3H, CH3), 6.98 (d, 1H, J = 8.2, H-600), 7.18–7.28 (m, 2H, H-400,500),
7.35 (s, 1H, @CH@), 7.40–7.46 (d, 1H, J = 8.4, H-300), 7.48–7.52 (m,
2H, H-30,50), 8.12–8.14 (m, 2H, H-20,60), 8.70 (dd, 1H, J = 7.4 H-40);
13C NMR (100 MHz, CDCl3, d, ppm): 55.8 (CH3), 113.6 (C300),
121.3 (C500), 127.9 (C30&50), 128.7 (C20&60), 129.0 (C600), 131.1
(C100), 132.2 (C40), 133.8 (C10), 135.8 (CH@C), 144.0 (C4), 161.1
(C200), 164.4 (C2), 181.6 (C5); MS (ES+) m/z: 280 (M+H)+.
(4Z)-4-(3-methoxybenzylidene)-2-phenyloxazol-5(4H)-one (2)
Compound (2) was recrystallized from CHCl3AMeOH as yellow
solid; Yield: 80%, m.p. 102–03 °C (lit. m.p. 102–04 °C) [25]; Anal.
In this work, we report the synthesis and the crystal structures
of compounds (4Z)-4-(3,5-dimethoxybenzylidene)-2-phenyl-1,3-
oxazol-5(4H)-one, C18H15NO4, (5), and (4Z)-2-phenyl-4-(3,4,5-tri-
Calc. for C17H13NO3: C, 73.11; H, 4.69; N, 5.02. Found: C, 73.10;
KBr
max
H, 4.67; N, 5.04. IR
m
cmꢁ1: 1795 (C@O), 1665 (C@N), 1652
(C@C), 1249 (CAO Lactone); 1H NMR (400 MHz, CDCl3, d, ppm):
3.80 (s, 3H, CH3), 7.02 (d, 1H, J = 8.4, H-400), 7.16 (s, 1H, ACH@),
7.30 (m, 2H, H-30,50), 7.42 (m, 1H, H-500), 7.52 (d, 1H, J = 8.4, H-
600), 7.80 (s, 1H, H-200), 8.10–8.06 (m, 2H, H-20,60), 8.40 (dd, 1H,
J = 7.4 H-40); 13C NMR (100 MHz, CDCl3, d, ppm): 55.2 (CH3),
115.5 (C200), 117.1 (C400), 122.5 (C600), 127.6 (C30&50), 128.1
(C20&60), 132.2 (C40), 133.1 (C500), 133.4 (C100), 134.2 (C10), 135.8
(ACH@C), 145.3 (C4), 155.1 (C2), 159.3 (C300), 184.5 (C@O); MS
(ES+) m/z: 280 (M+H)+.
methoxybenzylidene)-1,3-oxazol-5(4H)-one,
C19H17NO5 (6), as
determined by single-crystal X-ray analysis. To investigate the ef-
fect of the intermolecular interactions in the conformation of the
molecules we have also performed the optimization of the geome-
tries of the compounds using density functional theory (DFT) cal-
culations. Moreover, the compounds (1–6) have also been
screened for the antimicrobial and antioxidant properties.
Experimental
(4Z)-4-(4-methoxybenzylidene)-2-phenyloxazol-5(4H)-one (3)
Physical measurements
It was recrystallized from CHCl3AMeOH as orange colored so-
lid; Yield: 90%, m.p. 155 °C (lit. m.p. 157 °C) [26]; Anal. Calc. for
All the solvents and chemical were purchased from commercial
sources (Sigma–Aldrich, Merck) and others and used as received or
dried using standard procedures. Melting points were determined
on a Kofler apparatus and uncorrected. Elemental analysis (C, H, N)
were conducted using Carlo Erba analyzer model 1108. The IR
spectra were recorded on KBr pellets with Interspec 2020 (FT-IR)
C
17H13NO3: C, 73.11; H, 4.69; N, 5.02. Found: C, 73.10; H, 4.68;
KBr
N, 5.06. IR
m
cmꢁ1: 1791 (C@O), 1668 (C@N), 1650 (C@C),
max
1245 (CAO Lactone); 1H NMR (400 MHz, DMSO, d, ppm): 3.87 (s,
3H, CH3), 7.10–7.18 (d, 2H, J = 8.2, H-300,500), 7.32 (s, 1H, ACH@),
7.50, (d, 2H, J = 8.2, H-200,600), 7.70–7.75 (m, 2H, H-30,50), 8.10 (dd,
1H, J = 7.4, H-40), 8.30 (d, 2H, J = 7.2, H-20,60); 13C NMR (100 MHz,
DMSO, d, ppm): 55.4 (CH3), 114.5 (C300&500), 126.6 (C200&600),
127.6 (C30&50), 128.0 (C100), 128.5 (C20&60), 132.3 (C40), 133.4
(C10), 135.5 (ACH = C), 142.3 (C400), 144.4 (C4), 162.0 (C2), 182
(C@O); MS (ES+) m/z: 280 (M+H)+.
spectrometer, Spactro Lab UK and its values are given in cmꢁ1
.
The UV spectra were recorded with UV VIS-1800 spectrophotome-
ter (Shimadzu). 1H and 13C NMR spectra were run in CDCl3 on a
Bruker Avance-II 400 MHz and 100 MHz instrument respectively.
TMS was used as an internal standard; J values are given in Hertz.
Mass spectra were recorded on a JEOL D-300 mass spectrometer.
Thin layer chromatography (TLC) glass plates (20 ꢂ 5) were coated
with silica gel (E-Merck G254, 0.5 mm thickness) and exposed to io-
dine vapors to check the purity as well as the progress of the
reaction.
(4Z)-4-(2, 5-dimethoxybenzylidene)-2-phenyloxazol-5(4H)-one (4)
Its recrystallized from CHCl3AMeOH as bright yellow colored
solid; Yield: 80%, m.p. 140–41 °C; Anal. Calc. for C18H15NO4: C,
General method for the preparation of (4Z)-2-phenyloxazol-5(4H)-
ones (1–6)
69.89; H, 4.89; N, 4.53, Found: C, 69.85; H, 4.86; N, 4.54. IR
cmꢁ1: 1795 (C@O), 1651 (C@N), 1570 (C@C), 1274 (CAO Lac-
KBr
m
max
tone); 1H NMR (400 MHz, CDCl3, d, ppm): 3.90–3.93 (s, 6H,
2 ꢂ CH3), 7.02 (m, 2H, H-400,600), 7.51 (t, 2H, J = 7.4, H-30,50), 7.60
(t, 1H, J = 7.2, H-300),7.71 (s, 1H, ACH@), 8.17 (d, 2H, J = 8.2, H-
20,60), 8.46 (m, 1H,H-40); 13C NMR (100 MHz, CDCl3, d, ppm): 56.4
(CH3), 118.3 (C600), 119.1 (C500) 124.1 (C400), 127.2 (C30&50), 128.5
(C20&60), 131.6 (C100), 132.4 (C40), 133.5 (C10), 135.1 (ACH@C),
145.0 (C4), 151.8 (C300), 154.7 (C200), 160.8 (C2), 184.2 (C@O); MS
(ES+) m/z: 310 (M+H)+.
An equimolar mixture of hippuric acid and suitable aldehyde
(15 mmol) in freshly distilled acetic anhydride (10 mL) containing
fused anhydrous sodium acetate (1.2 g) was heated on an oil bath
at 140–150 °C for 2 h and then cooled. Progress of the reaction was
monitored by TLC. After completion, the compounds were filtered,
washed with light petroleum ether (60–80 °C) and air-dried. They
were triturated with cold saturated solution of sodium carbonate

540
M. Parveen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 538–545
(4E)-4-(3, 5-dimethoxybenzylidene)-2-phenyloxazol-5(4H)-one (5)
It recrystallized from CHCl3AEtOH as yellow color crystal; yield:
geometries performing a rotation of 180° around the C3AC12
bound with the aid of the software Ascalaph Designer version
1.8.50 [33].
75%, m.p. 128–30 °C; Anal. Calc. for C18H15NO4: C, 73.11; H, 4.69; N,
The calculation was performed within density functional theory
(DFT) using B3LYP (Becke three–parameter Lee–Yang–Parr) for ex-
change and correlation, which combines the hybrid exchange func-
tional of Becke [34,35] with the correlation functional of Lee, Yang
and Parr [36]. The calculation was performed with the Pople’s ‘tri-
ple split’ 6-31G(d,p) basis set, which includes a set of p-polariza-
tion functions for the H atoms and a set of d-polarization
functions for the C, N and O atoms. Each self-consistent field calcu-
cmꢁ1: 1799 (C@O),
KBr
5.05. Found: C, 73.15; H, 4.88; N, 5.08. IR
m
max
1655 (C@C), 1591 (C@N), 1270 (CAO Lactone); 1H NMR
(400 MHz, CDCl3, d, ppm): 3.88 (s, 6H, 2 ꢂ CH3), 7.17 (s, 1H, ACH@),
7.40 (m, 2H, H-200,600), 7.49 (s, 1H, H-400), 7.53 (m, 2H, H-30,50), 7.62
(t, 1H, J = 7.4,H-40) 8.14 (m, 2H, H-20,60); 13C NMR (100 MHz, CDCl3,
d, ppm): 56.1 (CH3), 107.2 (C200&600), 108.0 (C400), 127.0 (C30&50),
128.3 (C20&60), 132.1 (C40), 133.1 (C10), 135.0 (ACH@C), 138.5
(C100), 144.3 (C4), 161.3 (C300&500), 162.7 (C2), 184.0 (C@O); MS
(ES+) m/z: 310 (M+H)+.
lation was iterated until a
achieved. The final equilibrium geometries at the minimum energy
had
maximum gradient in internal coordinates of 10ꢁ5
Dq
of less than 10ꢁ5 bohrꢁ3 was
a
-
(4Z)-4-(3, 4, 5-trimethoxybenzylidene)-2-phenyloxazol-5(4H)-one (6)
Hartree bohrꢁ1 or Hartree radꢁ1. At the end of these geometry opti-
mizations, Hessian calculation were performed to guarantee that
the final structures correspond to true minima, using the same le-
vel of theory as in the geometry optimizations. A vibrational anal-
ysis was performed for compounds (5) and (6) during the Hessian
calculation to calculate the IR spectra. We also calculated the single
point energy for the optimized structures of the Z- and E-confor-
mations of compounds (5) and (6) using the B3LYP/6-31G(d,p) le-
vel of theory.
It was crystallized from CHCl3AMeOH as yellow color crystal;
Yield: 70%, m.p. 204–05 °C (lit. mp 205 °C) [27]; Anal. Calc. for
C
19H17NO5: C, 67.25; H, 5.05; N, 4.13. Found: C, 67.15; H, 5.18;
KBr
N, 4.08. IR
m
cmꢁ1: 1783 (C@O), 1655 (C@C), 1578 (C@N),
max
1244 (CAO Lactone); 1H NMR (400 MHz, CDCl3, d, ppm): 3.95–
3.97 (s, 9H, 3 ꢂ CH3), 7.17 (s, 1H, ACH@), 7.60 (t, 1H, J = 7.4, H-
40), 7.48–7.53 (m, 4H, H-20,30,50,60), 8.11 (m, 2H, H-200,600); 13C
NMR (100 MHz, CDCl3, d, ppm): 56.6 (CH3), 106.8 (C200&600), 127.4
(C30&50), 128.5 (C20&60), 131.8 (C100), 132.4 (C40), 133.3 (C10)
135.2 (ACH@C), 143.2 (C400), 144.5 (C4), 153.5 (C300&500), 162.9
(C2), 185.0 (C@O); MS (ES+) m/z: 340 (M+H)+.
Bioassay
Antibacterial studies
The in vitro antimicrobial activities of Erlenmeyer azlactones
(1–6) were tested using the bacterial cultures of Pseudomonas aeru-
ginosa (ATCC-9029), Staphylococcus Pyogenes (clinically isolated),
Klebsiella pneumonia (clinically isolated), Methicillin resistant
Staphylococcus aureus (MRSA + Ve), Escherichia coli (ATCC-25922)
bacterial strains by disk diffusion method [37,38]. A standard
inoculums (1–2 ꢂ 107 c.f.u./mL 0.5 McFarland standards) was
introduced on to the surface of sterile agar plates, and a sterile
glass spreader was used for even distribution of the inoculums.
The disks measuring 6 mm in diameter were prepared from What-
Crystal structure determination
A crystal of (5) with a needle shape and having approximate
dimensions of 0.47 ꢂ 0.07 ꢂ 0.05 mm was glued on a glass fiber
and mounted on a Bruker Apex II diffractometer. The same proce-
dure was done for a crystal of (6) with plate habit and approximate
dimensions 0.44 ꢂ 0.29 ꢂ 0.09 mm. Diffraction data were collected
at room temperature 293(2) K using graphite monochromated Mo
Ka (k = 0.71073 Å). Data reduction was performed with APEX II
[28]. Lorentz and polarization corrections were applied. A multi-
scan absorption correction was applied using SADABS [29]. The crys-
tallographic structure was solved by direct methods (SHELXS-97)
[30]. Refinements were carried out with SHELXL-97 package [30].
All refinements were made by full-matrix least-squares on F2, with
anisotropic displacement parameters for all non-hydrogen atoms.
All the hydrogen atoms could be located in a difference Fourier syn-
thesis but were placed at calculated positions and then, included in
the structure factor calculation in a riding model using SHELXL-97
defaults. For compound (5), the final least-squares cycle was based
Table 1
Crystallographic data and structure refinement of compound (5).
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
C18ꢀH15ꢀNꢀO4
309.31
293(2)
0.71073
Orthorhombic
P212121
5.6793(3)
on 2074 observed reflections [I > 2r(I)], 211 variable parameters, 0
b (Å)
15.2038(7)
restraints, converged with R = 0.0547 and wR = 0.0884.
For compound (6), the final least-squares cycle was based on
3654 observed reflections [I > 2r(I)], 229 variable parameters, 0 re-
straints, converged with R = 0.0433 and wR = 0.0961. Additional
information to the structure determination is given in Table 1
and 2 (Supplementary data), respectively. Selected structural
parameters can be seen in Tables 3 and 4. Supplementary data
have been deposited at the Cambridge Crystallographic Data Cen-
tre (CCDC No. 876047 & 876048).
c (Å)
17.6919(10)
90
90
90
1527.64(14)
4
1.345
0.096
a
(deg.)
b (deg.)
c
(deg.)
Volume (Å3)
Z
Calculated density (g/cm3)
Absorption coefficient (mmꢁ1
Extinction coefficient
F(000)
)
0.0102(17)
648
Crystal size (mm)
h range for data collection (deg.)
Index ranges
Reflections collected/unique
Completeness to h = 25.00°
Refinement method
0.47 ꢂ 0.07 ꢂ 0.05
1.77–27.65
–7 < h < 7, ꢁ19 < k < 19, ꢁ23 < l < 22
19930/3561 [R(int) = 0.147]
100%
Ab initio calculations
The geometry optimizations of (5) and (6) were performed
using the Firefly QC package [31], which is partially based on the
GAMESS (US) source code [32], starting from the experimental X-
ray geometry (Z-conformations). We have also performed the
geometry optimization of the corresponding E-conformations of
(5) and (6). The E-conformations were generated from the X-ray
Full-matrix least-squares on F2
2074/0/211
Data/restraints/parameters
Goodness-of-fit on F2
0.965
Final R indices [I > 2
r
(I)]
R1 = 0.0473 wR2 = 0.0814
R1 = 0.1133 wR2 = 0.1642
0.162 and ꢁ0.165
R indices (all data)
Largest diff. peak and hole (e Åꢁ3
)

M. Parveen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 538–545
541
Table 3
Comparison of selected geometrical parameters for (6) as determined by X-ray
diffraction and from DFT geometry optimization (Å,°).
Experimental
DFT
O2AC2
O1AC2
O1AC5
N4AC5
N4AC3
C2AC3
C3AC12
C12AC13
O1AC2AC3
C2AC3AN4
C3AN4AC5
N4AC5AO1
C5AO1AC2
C3AC12AC13
O1AC5AC6AC11
1.195(4)
1.399(5)
1.384(4)
1.279(4)
1.403(4)
1.452(6)
1.341(5)
1.447(6)
105.2(4)
108.3(4)
105.5(4)
116.1(4)
104.9(3)
130.3(4)
ꢁ9.0(6)
1.2007
1.4094
1.3754
1.2964
1.3971
1.4812
1.3583
1.4520
103.98
108.52
105.63
115.98
105.88
130.06
ꢁ0.12
Fig. 1. Antibacterial activity of azlactones (1–6), Diameter of zone of inhibition
(mm), Positive control (standard): Ciprofloxacin and negative control (DMSO)
measured by the Halo Zone Test (Unit, mm). ⁄ Methicillin resistant Staphylococcus
aureus (MRSA + Ve).
Table 4
Comparison of selected geometrical parameters for (6) as determined by X-ray
diffraction and from DFT geometry optimization (Å,°).
Experimental
DFT
used to make a suspension of spore of fungal strain for lawning.
A loopful of particular fungal strain was transferred to 3 mL saline
to get a suspension of corresponding species. Twenty millilitres of
agar media was poured into each petri dish. Excess of suspension
was decanted and the plates were dried by placing in an incubator
at 37 °C for 1 hour. Using an agar punch, wells were made and each
well was labelled. A control was also prepared and maintained at
37 °C for 3–4 days. The fungal activity of each compound was com-
pared with Amphotericin B as positive control (standard drug),
while the disk containing DMSO was used as negative control. Inhi-
bition zones were measured and compared with the controls. The
fungal zones of inhibition values are given in Fig. 2.
The nutrient broth, which contained logarithmic serially two
fold diluted amount of test compound and controls was inoculated
with approximately 1.6 ꢂ 104–6 ꢂ 104 c.f.u./mL. The cultures were
incubated for 48 h at 35 °C and the growth was monitored. To ob-
tain the minimum fungicidal concentration (MFC), 0.1 mL volume
was taken from each tube and spread on agar plates. The number
of c.f.u. was counted after 48 h of incubation at 35 °C. MFC was de-
fined as the lowest drug concentration at which 99.9% of the inocu-
lums were killed. The minimum inhibitory concentration and
minimum fungicidal concentration are given in Table 6.
O2AC2
O1AC2
O1AC5
N4AC5
N4AC3
C2AC3
C3AC12
C12AC13
O1AC2AC3
C2AC3AN4
C3AN4AC5
N4AC5AO1
C5AO1AC2
C3AC12AC13
O1AC5AC6AC11
1.195(2)
1.397(2)
1.384(2)
1.280(2)
1.2028
1.4097
1.3751
1.2961
1.3966
1.4786
1.3605
1.4485
103.98
108.59
105.62
115.94
105.87
130.14
ꢁ0.18
1.394(2)
1.465(3)
1.446(3)
1.446(2)
104.80(17)
108.18(16)
105.97(15)
115.82(17)
105.21(15)
130.5(18)
11.2(3)
man No. 1 filter paper and sterilized by dry heat at 140 °C for 1 h.
Ciprofloxacin was used as positive control (standard drug) while
the disk poured in DMSO was used as negative control. The suscep-
tibility was assessed on the basis of diameter of zone of inhibition
against gram +ve and gram –ve strains of bacteria. Inhibition zones
were measured and compared with the controls. The bactericidal
zones of inhibition values are given in Fig. 1.
Minimum inhibitory concentrations (MICs) were determined by
broth dilution technique. The nutrient broth, which contained log-
arithmic serially two fold diluted amount of test compound and
controls were inoculated with approximately 5 ꢂ 105 c.f.u./mL of
actively dividing bacteria cells. The lowest concentration (highest
dilution) required to arrest the growth of bacteria was regarded
as minimum inhibitory concentration (MIC). To obtain the mini-
mum bacterial concentration (MBC), 0.1 mL volume was taken
from each tube and spread on agar plates. The number of c.f.u.
was counted after 18–24 h of incubation at 35 °C. MBC was defined
as the lowest drug concentration at which 99.9% of the inoculums
were killed. The minimum inhibitory concentration (MIC) and
minimum bactericidal concentration are given in Table 5.
Antioxidant studies
The Erlenmeyer azlactones (1–6) were tested for their antioxi-
dant property by 1,1-diphenylpicrylhydrazyl (DPPH) method
[41–43]. In this procedure drug stock solution (1 mg/mL) was di-
luted to final concentration of 2, 4, 6, 8, 10 and 12 in methanol.
Methanolic DPPH solution (1 mL, 0.3 mmol) was added to 3.0 mL
of drug solution of different concentrations. The tube was kept at
an ambient temperature for 30 min and the absorbance was mea-
sured at 517 nm in UV VIS-1800 spectrophotometer. The scaveng-
ing activity was calculated by following formula:
½%inhibition ¼ ½ðAControl ꢁ ASampleÞ=AControlꢃ ꢂ 100ꢃ
Antifungal studies
where AControl is the absorbance of the L-ascorbic acid (Standard)
and ASample is the absorbance of different compounds.
The antifungal activity was also done by disk diffusion method
[39–40]. For assaying antifungal activity Candida albicans, Aspergil-
lus fumigatus, Penicillium marneffei and Trichophyton mentagro-
phytes were used. Sabourands agar media was prepared by
dissolving peptone (1 g), D-glucose (4 g) and agar (2 g) in distilled
water (100 mL) and adjusting the pH to 5.7. Normal saline was
The methanolic DPPH solution (1 mL, 0.3 mM) was used as con-
trol. The inhibitory concentration (IC50) value represents the con-
centration required to exhibit 50% antioxidant activity (Fig. 3).
The IC50 values were calculated by the linear regression of plots
where the abscissa represented the concentration of the

542
M. Parveen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 538–545
Table 5
MIC and MBC results of azlactones (1–6) positive control Ciprofloxacin.
Compounds
Gram positive bacteria
Gram negative bacteria
S. Pyogenes
MRSA⁄
P. aeruginosa
K. pneumoniae
E. coli
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
1
2
3
4
5
6
25
25
25
12.5
25
50
50
50
25
50
100
25
25
25
12.5
50
50
50
50
25
100
100
25
25
25
12.5
50
100
100
50
25
>100
>100
25
25
25
25
50
50
100
50
50
50
100
100
25
25
25
12.5
50
100
100
50
25
100
100
50
50
50
50
Standard
6.25
12.5
6.25
12.5
12.5
25
6.25
25
6.25
25
MIC (
lg/ml) = minimum inhibitory concentration, i.e the lowest concentration of the compound to inhibit the growth of bacteria completely; MBC (
lg/ml) = minimum
bacterial concentration, i.e., the lowest concentration of the compound for killing the bacteria completely.
100
90
80
70
60
50
40
30
20
10
0
1
2
3
4
5
6
Ascorbic acid
0
2
4
8
10
12
Conc. 6µg/ml
Fig. 2. Antifungal activity of azlactones (1–6) Positive control (Amphotericin B) and
negative control (DMSO) measured by the Halo Zone Test (Unit, mm). Diameter of
zone of inhibition (mm), CA: Candida albicans, AF: Aspergillus fumigatus, TM:
Trichophyton entagrophytes, PM: Penicillium marneffei.
Fig. 3. Antioxidant activity of compounds (1–6).
Table 6
the olefinic proton which is in agreement with the Z-configuration
as reported in the literature [2]. The Z-configuration has less steric
repulsion as compare to E-configuration. The phenyl protons at-
tached at the position 2 of azlactones were found to resonate in
the range of 8.70–7.30 ppm. The other signals of substituted ben-
zylidines attached at the position 4 of azlactones displayed at d
7.48–6.98 for four protons (1), 7.70–7.02 for four protons (2),
7.50–7.10 for four protons (3), 7.60–7.02 for three protons (4),
7.40–7.49 for three protons (5) and a singlet at d 8.11 ppm for
two protons (6). The methoxy groups of benzylidines substituted
at different positions displayed singlet singles in the range of d
3.87–3.97 ppm. 13C NMR spectra provided a firm support for the
formation of compounds and their signals were in good agreement
with proposed structures. All the compounds exhibited signals at d
182–185.0 due to (C@O) while the signals at d 135.0–135.8 ppm
may be attributed to the olefinic carbon. Further support of struc-
tures (1–6) was given by (+)-ESI mass spectroscopy. The mass
spectra of (1), (2), (3), (4), (5) and (6) showed the molecular ion
peak at m/z 280 (M+H)+, m/z 280 (M+H)+, m/z 280 (M+H)+, m/z
310 (M+H)+, 310 (M+H)+ and m/z 340 (M+H)+ respectively. The
complete chemical shifts for synthesized compounds are listed in
experimental part.
MIC and MFC results of azlactones (1–6) positive control Amphotericin B.
Compounds
CA
AF
TM
PM
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
1
2
3
4
5
6
25
50
25
12.5
25
50
100
50
25
50
25
50
25
12.5
25
50
100
50
25
50
25
50
25
12.5
25
100
100
50
25
50
25
50
50
12.5
50
100
100
100
50
100
100
25
50
6.25
100
25
50
12.5
100
25
50
6.25
100
25
50
12.5
Standard
CA; Candida albicans, AF; Aspergillus fumigatus, TM; Trichophyton mentagrophytes,
PM; Penicillium marneffei. MIC ( g/ml) = minimum inhibitory concentration, i.e., the
lowest concentration of the compound to inhibit the growth of fungus completely;
MFC ( g/ml) = minimum fungicidal concentration, i.e., the lowest concentration of
the compounds for killing the fungus completely.
l
l
compounds (lg/mL). Explicitly, IC50 is the average percentage of
antioxidant activity. Results in the form of percent inhibition are
tabulated in the Table 7. The experiments were done in triplicate.
Results and discussion
Chemistry
Crystal structure
The 1H NMR spectra of the compounds (1–6) exhibited a char-
acteristic sharp down field singlet at d 7.16–7.71 attributed to
Compound (5) crystallizes in the orthorhombic system with
space group P212121 Fig. 4. The molecule as a whole is approxi-

M. Parveen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 538–545
543
Table 7
Quantitative screening of antioxidant activity of azlactones (1–6) by DPPH assay method (n = 3).
S. No
Compounds
Absorbance
Absorbance at 517 nm
g/mL
IC50
2
l
4
l
g/mL
6
lg/mL
8
lg/mL
10 lg/mL
1.
2.
Control
1
(Abscontrol
Abssample
(AA%)
)
0.9420 0.04
0.8542 0.02
9.28
0.9420 0.04
0.7265 0.04
22.84
0.9420 0.03
0.6314 0.07
32.94
0.9420 0.04
0.5125 0.05
45.57
0.9420 0.04
0.3919 0.04
58.37
7.78
3.
4.
5.
6.
7.
8.
2
Abssample
(AA%)
Abssample
(AA%)
Abssample
(AA%)
Abssample
(AA%)
Abssample
(AA%)
Abssample
(AA%)
0.8164 0.05
13.29
0.7820 0.05
16.94
0.7883 0.05
16.28
0.8318 0.08
11.66
0.8703 0.02
7.57
0.7694 0.04
18.32
0.6945 0.07
26.24
0.6394 0.06
32.09
0.6619 0.08
29.70
0.7105 0.07
24.54
0.7498 0.04
20.36
0.6168 0.07
34.52
0.5867 0.05
37.21
0.4589 0.04
51.26
0.4720 0.03
49.87
0.6224 0.08
33.89
0.6709 0.07
41.16
0.4336 0.06
53.97
0.4689 0.06
50.20
0.3098 0.04
67.09
0.3412 0.06
63.79
0.4873 0.04
48.24
0.6138 0.07
32.15
0.2878 0.06
69.44
0.3485 0.08
62.96
0.1705 0.03
81.89
0.1800 0.05
80.91
0.3646 0.04
61.27
0.5540 0.03
28.74
0.1208 0.05
87.17
6.94
5.35
5.15
7.45
10.95
4.78
3
4
5
6
Standard
mately planar, with the oxazolone ring making a dihedral angle of
10.3(2)° with the 2-phenyl ring and 2.9(2) with the methoxy-
phenyl ring. This planarity presumably results from the effects of
conjugation. The molecule adopts the Z configuration about the
central olefinic bond C3@C12 bond [1.342(4) Å] and the coplana-
Table 8
H-bond geometry (Å,°) of azlactones (5).
DAH
Hꢀ ꢀ ꢀA
Dꢀ ꢀ ꢀA
DAHꢀ ꢀ ꢀA
C14AH14ꢀ ꢀ ꢀO3i
0.93
0.96
0.93
0.93
2.57
2.43
2.48
2.43
3.441(4)
3.377(4)
2.794(4)
3.062(5)
155
168
100
126
rity and conjugation of the
p-electron systems in the aromatic
C19AH19Bꢀ ꢀ ꢀO2ii
rings are reflected in the CAC bond lengths between the oxazolone
and methoxyphenyl rings. The C3AC12 bond is significantly short-
er than the C12AC13 bond. The methoxy groups lie in the plane of
the methoxy phenyl ring.
C11AH11ꢀ ꢀ ꢀO1 (intra)
C20AH20ꢀ ꢀ ꢀO2 (intra)
(Symmetry codes i: ꢁ1/2 + x, 1/2 ꢁ y, ꢁz; ii: 3/2 + x, 1/2 ꢁ y ꢁ 1/2, ꢁz).
The crystal structure of (5) is determined by rather weak
CAHꢀ ꢀ ꢀO interactions Table 3. The C19AH19Bꢀ ꢀ ꢀO2 interaction
forms helical chains along the a axis with descriptor C(10) accord-
ing to Etter’s graph-set theory [44,45]. The weaker C14AH14ꢀ ꢀ ꢀO3
interaction also delineate helical chains along the a axis with
graph-set C(4). Additionally, there is one intramolecular CAHꢀꢀꢀN
hydrogen bond between an H atom of the methoxyphenyl ring
and the N atom of oxazolone ring and one intramolecular CAHꢀꢀꢀO
hydrogen bond between one H atom of the phenyl ring and the O
atom of the oxazolone ring [Table 8; Fig. 5 (Supplementary data)].
quantum mechanical calculations of the equilibrium geometry of
the free molecule. The DFT calculations closely reproduce the so-
lid-state geometry of the molecule is (Tables 3 and 4). The agree-
ment between the experimental and calculated bond lengths and
valence angles is very good, but the calculated torsion angle
O1AC5AC6AC11 in the free molecule differs by 8.88° from that
of the molecules in the crystal for (5) and 11.38° for (6). The differ-
ences between the calculated and experimental bond lengths are
smaller than 0.0292 and 0.0205 Å for (5) and (6), respectively
(Figs. 8 and 9).
Overall, our data suggest that the supramolecular aggregation
does not play a major role in stabilizing the observed geometries
of (5) and (6), in agreement with the absence of strong inter-
molecular interactions.
The calculated energy difference between the optimized E- and
Z-conformations of compound (5) is 2.21 kcal/mol, with the Z-con-
formation having the lower energy. In compound (6), the Z-confor-
mation is again the more stable, by 2.08 kcal/mol.
The crystal packing is also stabilized by weak CAHꢀ ꢀ ꢀ
p interactions
and interactions.
p p
ꢀ ꢀ ꢀ
Compound (6) crystallizes in the triclinic system with space
group P-1 [Fig. 6 and 7 (Supplementary data)]. The structure of this
compound was originally reported [27]. We obtained this com-
pound from a different synthesis route of that used by Sun and Cui.
Results of the ab initio calculations
In order to gain some insight on the influence of the intermolec-
ular interactions on the molecular geometry we have performed
The calculated IR spectra of (5) and (6) describe the main fea-
tures of the experimental spectra Figs. 9 and 10 (See Supplemen-
tary data).
Antimicrobial study
The in vitro antimicrobial activities of Erlenmeyer azlactones
(1–6) is presented in Figs. 1 and 2 and Tables 5 and 6. The
in vitro study results demonstrated that the compound (4) was
most active among all compounds in terms of antibacterial as well
as antifungal activity. The MIC of compound (4) is 12.5 lg/mL with
zone of inhibition 24.5 0.5 against Escherichia coli (ATCC-8739)
(bacterial strain) while the zone of inhibition was 25.5 0.2 against
Candida albicans (fungal strain). The MIC results for both bacterial
and fungal strains are shown in (Tables 5 and 6). It is concluded
that compound (4) bearing CH3O-group at position 2,4 of the ben-
zene ring is most potent followed by (3), (2), (5), (1) and (6)
compounds.
Fig. 4. Asymmetric unit of the compound (5) with the ellipsoids drawn at the 50%
probability level, with the atomic labelling scheme (Mercury, version 3.0 [46]).

544
M. Parveen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 538–545
Conclusions
The present work reports the synthesis, spectral characteriza-
tion, bioassay of synthesized azlactones obtained from condensa-
tion of aldehydes and hippuric acid. All the compounds showed
substantial antibacterial and antifungal activity against different
strains of bacteria and fungi respectively. The compounds also
exhibited good antioxidant activity by the DPPH method. The
Z-configuration of synthesized Erlenmeyer azlactones was con-
firmed on the basis of spectroscopy techniques, X-ray crystallo-
graphic studies as well as DFT calculations.
Acknowledgments
Authors thank the Chairman, Department of Chemistry for pro-
viding necessary facilities. P. S. Pereira Silva acknowledges the sup-
port by Fundação para a Ciência e a Tecnologia, under the
Scholarship SFRH/BD/38387/2008.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2012.11.054.
Fig. 8. Comparison of the molecular conformation of (5 & 6), as established from
the X-ray study (red) with the optimized geometry (blue) (Software used for
visualization: VMD, version 1.9.1, January 29, 2012 [47]).
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