Synthesis, spectral, DFT calculations and antibacterial studies of Fe(III) complexes of new fluorescent Schiff bases derived from imidazo[4',5':3,4]benzo[1,2-c]isoxazole

Article abstract of DOI:10.1002/aoc.4178

New fluorescent heterocyclic ligands were synthesized by the reaction of 8-(4-chlorophenyl)-3-alkyl-3H-imidazo[4',5':3,4]benzo [1,2-c]isoxazol-5-amine with p-hydroxybenzaldehyde and p-chlorobenzaldehyde in good yields. The coordination ability of the ligands with Fe³⁺ ion was examined in an aqueous metanolic solution. Schiff base ligands and their metal complexes were characterized by elemental analyses, IR, UV–vis, mass, and NMR spectra. The optical properties of the compounds were investigated and the results showed that the fluorescence of all compounds is intense and their obtained emission quantum yields are around 0.15 – 0.53. Optimized geometries and assignment of the IR bands and NMR chemical shifts of the new complexes were also computed by using density functional theory (DFT) methods. The DFT-calculated vibrational wavenumbers and NMR chemical shifts are in good agreement with the experimental values, confirming suitability of the optimized geometries for Fe(III) complexes. Also, the 3D-distribution map for HOMO and LUMO of the compounds were obtained. The new compounds showed potent antibacterial activity and their antibacterial activity (MIC) against Gram-positive and Gram-negative bacterial species were also determined. Results of antibacterial test revealed that coordination of ligands to Fe(III) leads to improvement in the antibacterial activity.

Full text of DOI:10.1002/aoc.4178

Received: 29 June 2017
DOI: 10.1002/aoc.4178
Revised: 19 October 2017
Accepted: 19 October 2017
F U L L P A P E R
Synthesis, spectral, DFT calculations and antibacterial
studies of Fe(III) complexes of new fluorescent Schiff bases
derived from imidazo[4',5':3,4]benzo[1,2‐c]isoxazole
Shirin Ramezani | Mehdi Pordel
| Abolghasem Davoodnia
Department of Chemistry, Mashhad
Branch, Islamic Azad University,
Mashhad, Iran
New fluorescent heterocyclic ligands were synthesized by the reaction of 8‐(4‐
chlorophenyl)‐3‐alkyl‐3H‐imidazo[4',5':3,4]benzo [1,2‐c]isoxazol‐5‐amine with
p‐hydroxybenzaldehyde and p‐chlorobenzaldehyde in good yields. The coordi-
nation ability of the ligands with Fe3+ ion was examined in an aqueous metanolic
solution. Schiff base ligands and their metal complexes were characterized by
elemental analyses, IR, UV–vis, mass, and NMR spectra. The optical properties
of the compounds were investigated and the results showed that the fluorescence
of all compounds is intense and their obtained emission quantum yields are
around 0.15 – 0.53. Optimized geometries and assignment of the IR bands and
NMR chemical shifts of the new complexes were also computed by using density
functional theory (DFT) methods. The DFT‐calculated vibrational wavenumbers
and NMR chemical shifts are in good agreement with the experimental values,
confirming suitability of the optimized geometries for Fe(III) complexes. Also,
the 3D‐distribution map for HOMO and LUMO of the compounds were
obtained. The new compounds showed potent antibacterial activity and their
antibacterial activity (MIC) against Gram‐positive and Gram‐negative bacterial
species were also determined. Results of antibacterial test revealed that coordina-
tion of ligands to Fe(III) leads to improvement in the antibacterial activity.
Correspondence
Mehdi Pordel, Department of Chemistry,
Mashhad Branch, Islamic Azad
University, Mashhad, Iran.
Email: mehdipordel58@mshdiau.ac.ir
KEYWORDS
antibacterial activity, bidentate ligand, density functional theory, Fe(III) complex, Schiff base
1 | INTRODUCTION
activity.[9–11] The stabilities and coordination chemistry of
Fe(III) with bidentate ligands[12,13] have also resulted from
One of the most interesting areas of coordination chemis-
try is the interaction of transition metal ions with biolog-
ical molecules. There are many metal complexes that can
use as drugs and chemotherapeutic agents.[1]
Iron is one of the essential mineral that can function as
regulators, activators, transmitters, and controllers of vari-
ous enzymatic reactions.[2,3] In hence, the iron complexes
have received considerable attention owing to their
effective biological importance such as antibacterial[4,5]
antifungal[6] antivirus[7] antiproliferative[8] and anticancer
their efficacy as oral iron chelating agents[14,15] and as
agents for the treatment of iron overload conditions.[16–18]
Benzo[1,2‐c]isoxazoles are an important class of hetero-
cyclic pharmaceuticals and bioactive compounds which
are prescribed as antipsychotic risperidone[19] and anti‐
HIV drugs[20] and play a key role in many organic reac-
tions.[21] Isoxazole‐metal complexes are often postulated
as intermediates in reactions of considerable synthetic util-
ity, for example the reductive ring opening of isoxazoles.
Several isoxazole‐metal complexes have been reported
Appl Organometal Chem. 2017;e4178.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
1 of 11
https://doi.org/10.1002/aoc.4178

2 of 11
RAMEZANI ET AL.
and well characterized. In a review of the literature of
isoxazole‐metal complexes,[22] the binding characteristics
of the isoxazoles in the complexes have been examined,
and some tentative conclusions regarding the regularity
of isoxazole complexation behavior have been discussed.
On the other hand, Schiff bases are an important class
of organic ligands having considerable biological proper-
ties.[23–25] Schiff bases have many advantages between
ligands in the coordination chemistry. They are the most
versatile studied ligands in coordination chemistry
because of their structural varieties and very unique char-
acteristics. These findings promoted us to the synthesis
and characterization of two new fluorescent heterocyclic
Schiff‐base ligands derived from 2‐8‐(4‐chlorophenyl)‐3‐
alkyl‐3H‐imidazo[4',5':3,4]benzo [1,2‐c]isoxazol‐5‐amine
and their Fe(III) complexes. In addition, antibacterial
activities of the new ligands and complexes against gram
positive and negative bacterial species were studied.
2.2 | Computational methods
All of the calculations have been performed using the
DFT method with the B3LYP functional[31] as imple-
mented in the Gaussian 03 program package.[32] The 6‐
311+G(d,p) basis sets were employed except for the Fe
atom where the LANL2DZ basis sets were used with
considering its effective core potential. Geometry of the
Fe complex was fully optimized, which was confirmed
to have no imaginary frequency of the Hessian. Geometry
optimization and frequency calculation simulate the
properties in the gas/solution phases.
The fully‐optimized geometries were confirmed to
have no imaginary frequency of the Hessian.
The solute‐solvent interactions have been investigated
using one of the self‐consistent reaction field methods, i.e.,
the sophisticated Polarizable Continuum Model (PCM).[33]
2.3 | General procedure for the synthesis
of 7a,b from 5a,b
2 | EXPERIMENTAL
Aldehydes 6a,b (1 mmol) were added to a solution of com-
pounds 5a,b (0.34 g, 1 mmol) in EtOH (15 ml). The reac-
tion mixture was heated under reflux for 5 hours. The
solvent was removed under reduced pressure and the yel-
low product was filtered and washed with EtOH to give
Schiff bases (7a,b). More purification was achieved by
crystallization from suitable solvent such as acetone.
(E)‐4‐(((8‐(4‐chlorophenyl)‐3‐propyl‐3H‐imidazo[4',5':3,
4] benzo[1,2‐c]isoxazol‐5‐yl) imino) methyl)phenol (7a, L1)
2.1 | Equipment and materials
Melting points were measured on an Electrothermaltype‐
9100 melting‐point apparatus. The FT‐IR (as KBr discs)
spectra were obtained on a Tensor 27 spectrometer and
only noteworthy absorptions are listed. The 13C NMR
(100 MHz), 1H NMR (400 MHz) and NOESY spectra were
recorded on a Bruker Avance DRX‐400 spectrometer.
Chemical shifts are reported in ppm downfield from TMS
as internal standard; coupling constant J is given in Hz.
The mass spectrum was recorded on a Varian Mat, CH‐7
at 70 eV and ESI mass spectrum was measured using a
Waters Micromass ZQ spectrometer. Elemental analysis
was performed on a Thermo Finnigan Flash EA microana-
lyzer. Absorption and fluorescence spectra were recorded
on Varian 50‐bio UV‐Visible spectrophotometer and
Varian Cary Eclipse spectrofluorophotometer. UV–vis
and fluorescence scans were recorded from 200 to
1000 nm. Percentage of the Fe+3 ion was obtained by using
a Hitachi 2‐2000 atomic absorption spectrophotometer.
The microorganisms Bacillus subtilis ATCC 6633,
Pseudomonas aeruginosa ATCC 27853 and Escherichia
coli ATCC 25922 were purchased from Pasteur Institute
of Iran and S. aureus methicillin resistant was isolated
from different specimens which were referred to the
Microbiological Laboratory of Ghaem Hospital of Medical
University of Mashhad, Iran and its methicillin resistance
was tested according to the NCCLS guidelines.[26] All
solvents were dried according to standard procedures.
Compounds 1,[27] 3,[28] 4[29] and 5a,b[30] were obtained
according to the published methods. Other reagents were
commercially available.
1
was obtained as a yellow powder. m.p.: 149‐154 °C. H
NMR (CDCl3): δ 0.91 (t, J = 7.5 Hz, 3H, CH3), 1.80–1.91
(m, 2H, CH2), 4.37 (t, J = 7.5 Hz, 2H, NCH2), 6.95 (d, J
= 9.0 Hz, 2H, Ar H), 7.69 (s, 1H, Ar H), 7.73 (d, J =
9.0 Hz, 2H, Ar H), 7.87 (d, J = 9.0 Hz, 2H, Ar H), 8.31
(s, 1H, Ar H), 8.95 (d, J = 9.0 Hz, 2H, Ar H), 9.08 (s, 1H,
CH), 10.37 (br s, 1H, OH) ppm; 13C NMR (CDCl3): δ
16.1, 28.7, 51.4, 114.3, 114.7, 121.0, 131.5, 132.8, 133.7,
134.5, 135.1, 135.3, 136.1, 140.0, 140.6, 146.5, 159.9,
165.9, 166.1, 166.9 ppm. IR (KBr): 3348 cm‐1 (OH),
1645 cm‐1 (CH=N). MS (m/z) 432 (M++2). Anal. Calcd
for C24H19ClN4O2 (430.9): C, 66.90; H, 4.44; N, 13.00.
Found: C, 66.78; H, 4.41; N, 12.82.
(E)‐3‐Butyl‐N‐(4‐chlorobenzylidene)‐8‐(4‐chlorophenyl)
‐3H‐imidazo[4',5':3,4]benzo[1,2‐c] isoxazol‐5‐amine (7b,
L2) was obtained as a yellow powder. m.p: 181–184 °C;
1
yield: 75%. H NMR (CDCl3): δ 0.92 (t, J = 7.5 Hz, 3H,
CH3), 1.24–1.37 (m, 2H, CH2), 1.75–1.82 (m, 2H, CH2),
4.31 (t, J = 7.5 Hz, 2H, NCH2), 7.62 (d, J = 9.0 Hz, 2H,
Ar H), 7.69 (d, J = 9.0 Hz, 2H, Ar H), 7.75 (s, 1H, Ar H),
7.98 (d, J = 9.0 Hz, 2H, Ar H), 8.30 (s, 1H, Ar H), 8.88
(d, J = 9.0 Hz, 2H, Ar H), 9.25 (s, 1H, CH=N) ppm; 13C
NMR (CDCl3): δ 13.9, 19.8, 32.6, 44.9, 109.4,112. 6,

RAMEZANI ET AL.
3 of 11
126.6, 129.0, 129.5, 129.7, 130.0, 130.6, 131.5, 131.7, 134.1,
135.4, 135.6, 136,6, 142.1, 154.7, 161.2, 161.2 ppm. IR
(KBr): 1643 cm‐1 (CH=N). MS (m/z) 466 (M++4). Anal.
Calcd for C25H20Cl2N4O (463.4): C, 64.80; H, 4.35; N,
12.09. Found: C, 64.31; H, 4.32; N, 12.01.
led to the formation of 3‐alkyl‐8‐(4‐chlorophenyl)‐5‐
nitro‐3H‐imidazo[4',5':3,4]benzo[1,2‐c]isoxazoles 4a,b in
good yield.[29] Reduction of compounds 4a,b in EtOH by
SnCl2, gave the 8‐(4‐chlorophenyl)‐3‐alkyl‐3H‐imidazo
[4',5':3,4]benzo[1,2‐c]isoxazol‐5‐amines (5a,b) in high
yields. Finally, new heterocyclic Schiff‐bases 7a,b were
obtained by the reaction of amines 5a,b with aldehydes
6a,b in good yields (Scheme 1).
2.4 | General procedure for the synthesis
of complexes 8a,b from ligands 7a,b
The structural assignments of compounds 7a,b were
determined based on the analytical and spectral data. For
example, in the 1H NMR spectrum of compound 7a there
is an exchangeable peak at δ 10.37 ppm assignable to OH
group proton. Also, there are four doublet signals (δ =
6.95, 7.73, 7.78, 8.95 ppm) and two singlet signals (δ = 7.69
and 8.31 ppm) attributed to ten protons of aromatic rings.
Also, there is a singlet signal (δ = 9.08) assignable to the
imine CH proton. In addition, 20 different carbon atom sig-
nals are observed in the 13C NMR spectrum of compound
7a. Moreover, the IR spectrum of compound 7a in KBr
revealed a broad absorption band at 3348 cm‐1 assignable
to OH group. All this evidence taken in conjunction with
molecularionpeakatm/z432[M+2]+ supportthestructure
of Schiff‐base 7a. In addition, the data from NOESY experi-
ment showed a cross‐peak between the H‐4 proton (δH 7.69,
s, CH‐benzene) and the imine CH proton (δH 9.08),
confirming the E configuration of the product structure
(Scheme 2). Furthermore, as can be seen in Scheme S1
(Supplementary Data), there are a massive cross‐peak
between the H‐4 proton and the N‐3 alkyl group (δH 4.37
(t, J = 7.2 Hz, NCH2) and interactions between proton of
imidazole ring (δH 8.31, s, CH‐imidazole) and CH2 protons
of propyl group (δH 4.37 (t, J = 7.2 Hz, NCH2).
To the yellow solution of ligand 7a,b (467 mmol) in aque-
ous metanolic solution (20 ml, MeOH, H2O, 10:90) iron
(III) chloride (0.476, 294 mmol) was added, resulting in
color change to deep green. The reaction was carried out
for another 6 h in room temperature. The complex was
isolated by evaporation of the solvent and washed with
cold MeOH and then H2O.
[Fe(L1)2] Cl3.2(H2O) (8a): was obtained as a dark green
1
powder. mp > 300 °C (decomp). H NMR (DMSO‐d6): δ
0.93 (t, J = 7.5 Hz, 6H, CH3), 1.78–1.89 (m, 4H, CH2), 4.31
(t, J = 7.5 Hz, 4H, NCH2), 7.01 (d, J = 9.0 Hz, 4H, Ar H),
7.65–7.85 (m, 10H, Ar H), 8.33 (s, 2H, Ar H), 8.98 (d, J =
9.0 Hz, 4H, Ar H), 9.16 (s, 2H, CH), 10.75 (br s, 2H, OH).
IR (KBr): 3382 cm‐1 (OH), ESI‐MS (+) m/z (%): 917
[Fe(L2)2]3+. Anal. Calcd for C48H42Cl5FeN8O6 (1060.0):
C, 54.39; H, 3.99; N, 10.57; Fe, 5.27. Found: C, 54.01; H,
3.93; N, 11.04; Fe, 4.68.
[Fe(L2)2] Cl3.2(H2O) (8b): was obtained as a dark
1
green powder. mp > 300 °C (decomp). H NMR (DMSO‐
d6): δ 0.85 (t, J = 7.0 Hz, 6H, CH3), 1.20–1.23 (m, 4H,
CH2), 1.69–1.72 (m, 4H, CH2), 4.25 (t, J =7.0 Hz, 4H,
NCH2), 7.66 (d, J = 9.0 Hz, 4H, Ar H), 7.71 (s, 2H, Ar
H), 7.74 (d, J = 9.0 Hz, 4H, Ar H), 7.95 (d, J = 9.0 Hz,
4H, Ar H), 8.33 (s, 2H, Ar H), 8.89 (d, J = 9.0 Hz, 4H,
Ar °H), 9.12 (s, 2H, CH=N) ppm; IR (KBr): 3435, cm‐1
(OH), ESI‐MS (+) m/z (%): 982 [Fe(L1)2]3+. Anal. Calcd
for C50H44Cl7FeN8O4 (1110.9): C, 53.38; H, 3.94; N, 9.96;
Fe, 4.96. Found: C, 52.90; H, 3.89; N, 10.36; Fe, 4.46.
The coordination ability of Schiff‐bases 7a,b with Fe3+
ion was examined in an aqueous metanolic solution. The
elemental analysis results (Experimental section) and the
stoichiometry of the deep green complexes obtained by
Job's method (Figures S1 and S2, Supplementary Data),[34]
proposed the [Fe(L)2] Cl3.2(H2O) formulae for the com-
plexes (Scheme 3). Electrospray ionisation mass spec-
trometry (ESI‐MS) of the complexes 8a,b showed
molecular ion peak at m/z 917 ([Fe(L1)2]3+) and m/z
982 ([Fe(L2)2]3+) which strongly confirmed that molar
ratio between Fe (III) ions and ligands in the complexes
is 1:2. Moreover, basic molecular ion peak at m/z 159
(C8H5N3O) can be related to imidazo[4',5':3,4]benzo[1,2‐
c]isoxazole scaffold in ligands and complexes.
3 | RESULTS AND DISCUSSION
3.1 | Synthesis and structure of the new
ligands 7a,b and complexes 8a,b
In order to the synthesis of new heterocyclic Schiff‐base
ligands, the commercially available 5‐nitro‐1H‐benzimid-
azole was alkylated with 1‐bromopropane and 1‐
bromobutane in KOH and DMF to give 1‐alkyl‐5‐nitro‐
1H‐benzimidazole (1a,b).[27] 3‐Alkyl‐8‐(4‐chlorophenyl)‐
3H‐imidazo [4',5':3,4]benzo[1,2‐c]isoxazoles (3a,b) were
prepared from the reaction of 1‐alkyl‐5‐nitro‐1H‐benz-
imidazole 1a,b with (4‐chlorophenyl)acetonitrile (2) in
basic MeOH solution.[28] Regioselective nitration of 3a,b
using a mixture of sulfuric acid and potassium nitrate
3.2 | Photophysical properties of the new
ligands and complexes
Compounds 7a,b, and Iron complexes 8a,b were spec-
trally characterized by UV‐Vis and fluorescence spectros-
copy in the wavelength range of 200–1000 nm.

4 of 11
RAMEZANI ET AL.
SCHEME 1 Synthesis of the new ligands 7a,b
coefficient (ε) were calculated as the slope of the plot
of absorbance vs concentration. As depicted in Figure 1,
the spectra of complexes have an absorption maximum
at 750 nm at which the ligand has no absorbance. An
efficient charge transfer of electron from p‐orbital on
ligand to Fe (III) d‐orbital can be considered as the main
reason for the color of the complexes described as
Ligand to‐Metal charge transfer (LMCT).[35] Also,
Schiff‐base ligands 7a,b, and Iron complexes 8a,b pro-
duced fluorescence at concentration 1 × 10–5 M in
MeOH (Figure 2). The fluorescence quantum yield of
the compounds was determined via comparison
methods, using fluorescein as a standard sample in
0.1 M NaOH and MeOH solution.[36] The used value of
the fluorescein emission quantum yield is 0.79 and the
obtained emission quantum yields of the new com-
pounds are around 0.15 – 0.53. As can be seen from
Table 1, extinction coefficient (ε) in Schiff‐base 7b, fluo-
rescence intensity and the emission quantum yield in
Schiff‐base 7a were the biggest values.
SCHEME 2 NOESY spectrum of compound 7a
The absorption and fluorescence emission spectra of
the ligands 7a,b and iron (III) complexes 8a,b are shown
in Figures 1 and 2, respectively. Also, numerical spectral
data are presented in Table 1. Values of extinction
3.3 | DFT calculations
According to our experimental results and reported litera-
tures,[37] an octahedral geometry was proposed for the

RAMEZANI ET AL.
5 of 11
SCHEME 3 Synthesis of Fe(III) complexes 8a,b
TABLE 1 Spectroscopic data for the new compounds 7a,b and 8a,
b at 298 K
Dye
7a
7b
8a
8b
λabs (nm)a
440
360
750
750
‐3
‐1
ε × 10 [(mol L‐1)
6.00
6.25
4.30
3.00
cm‐1]
b
λflu (nm)c
600
0.53
595
0.32
550
0.24
550
0.15
d
ΦF
aWavelengths of maximum absorbance (λabs).
bExtinction coefficient.
cWavelengths of fluorescence emission (λflu) with excitation at 400 nm.
dFluorescence quantum yield.
frontier orbitals in the UV‐visible absorption spectra of
Schiff‐bases 7a,b and iron complexes 8a,b. Geometry of
the complex 8b was optimized in both of the gas phase
and the PCM model, where the methanol was the used
solvent. The optimized geometry of the ligands 7a,b can
be found in Figure 3. The optimized geometry of the com-
plex 8b with labeling of its atoms is also depicted in
Figure 4. Some of the calculated structural parameters of
the Fe(III) complex are collected in Table 2.
FIGURE 1 The absorption spectra of the ligands 7a,b and Fe(III)
complexes 8a,b in MeOH solution (2 × 10‐4 M)
In the optimized geometry of the complex 8b, the
ligand 7b acts as a bidentate ligand, coordinates to the
Fe(III) via nitrogen atom of the imine group (–N=CH)
and nitrogen atom of the isoxazole ring.
Except of the butyl group, the ligands 7b are planar.
The aromatic rings of the ligand are in a same plane. Also,
both of the ligands are in a same plane forming a square
plane of the octahedral complex. Two other positions of
the complex are filled by two H2O molecules, which are
Trans to each other. The H2O ligands are perpendicular
to the square plane of the complex. The Fe‐O and Fe‐N
Lengths bonds are listed in Table 2.
FIGURE 2 The fluorescence emission spectra of the ligands 7a,b
and Fe(III) complexes 8a,b in MeOH solution (1 × 10‐5 M)
3.3.1 | NMR spectra
The DFT computed 1H NMR chemical shifts (δ) of Fe(III)
complex 8b are listed in Table 3 together with the
iron complexes 8a,b. We performed DFT calculations at
the B3LYP/6‐311+G(d,p) level to gain a deeper insight
into the geometries and role of HOMO and LUMO

6 of 11
RAMEZANI ET AL.
FIGURE 3 The optimized geometry of the ligands 7a,b
FIGURE 4 The optimized geometry of
the iron(III) complex 8b
experimental values for comparison. The atoms are num-
bered as in Figure 4.
As seen in Table 3, the DFT‐calculated NMR chemical
shifts are in good agreement with the experimental
values, confirming suitability of the optimized geometries
for Fe(III) complex 8b.
frequencies is gathered in Table 4. There is good agree-
ment between the experimental and DFT‐calculated fre-
quencies of the high spin complex, confirming validity of
the optimized geometry as a proper structure for the
complex 8b.
The 3D‐distribution map for the highest‐occupied‐
molecular orbital (HOMO) and the lowest‐unoccupied‐
molecular orbital (LUMO) of the ligands 7a,b and the
complex 8b are shown in Figure 5. As seen, the HOMO
orbital of the ligands is localized on the benzimidazole
and isoxazole rings. But the LUMO orbital is mainly local-
ized on the benzene ring and its substitutions. Since, in
3.3.2 | Vibrational spectroscopy
The vibrational modes of Fe complex 8b were also
analyzed by comparing the experimental and DFT‐com-
puted IR spectra. Assignment of the selected‐vibrational

RAMEZANI ET AL.
7 of 11
TABLE 2 Selected structural parameters of Fe(III) complex 8b
Bond
Bond length (A0)
Angle
(°)
Dihedral angle
(°)
Fe‐N1
2.076
N1‐Fe‐N5
163.54
N1‐N5‐N4‐N8
27.598
12.668
70.171
‐95.595
177.499
93.484
‐100.750
‐40.017
‐6.741
‐34.899
‐0.256
‐0.327
0.894
Fe‐N4
2.729
2.124
2.364
2.203
2.152
1.398
1.360
1.401
1.425
1.430
1.446
1.410
1.415
1.305
1.333
1.363
1.323
1.364
1.469
N1‐Fe‐N4
N5‐Fe‐N8
N1‐Fe‐N8
N4‐Fe‐N5
N1‐Fe‐O4
N5‐Fe‐O3
N1‐Fe‐O3
N5‐Fe‐O4
O3‐Fe‐O4
Fe‐N5‐C27
N5‐C27‐C28
C3‐N1‐Fe
Fe‐O1‐N1
C3‐N1‐O1
Fe‐O3‐N1
C3‐N1‐O1
N1‐O1‐C7
C1‐C6‐N3
C1‐N2‐C8
70.055
75.787
107.593
112.01
100.325
78.28
N1‐N5‐N4‐Fe
O2‐N5‐Fe‐O3
O2‐N5‐Fe‐O4
O2‐N5‐Fe‐N8
C27‐N5‐Fe‐O4
C27‐N5‐Fe‐O3
C27‐C28‐N8‐O4
C28‐N8‐Fe‐N5
C28‐N8‐N1‐Fe
O1‐C7‐C2‐C3
O1‐C7‐C12‐C13
C7‐C2‐C1‐N2
C6‐C1‐N2‐C8
N2‐C8‐ N3‐C6
C2‐C7‐C12‐C14
N5‐C27‐C26‐C31
C1‐C6‐N3‐C8
C3‐C4‐C5‐C6
N3‐C9‐C10‐C11
Fe‐N5
Fe‐N8
Fe‐O3
Fe‐O4
N5‐O2
O2‐C31
C31‐C26
C26‐C27
C27‐C28
C31‐C36
C36‐C38
C28 –N8
N8‐C42
C27‐N5
C25‐N6
N6‐C32
C32‐N7
N7‐C33
85.27
95.834
164.76
116.093
123.00
123.09
120.3
‐0.030
‐0.683
‐0.683
‐0.695
0.600
123.09
45.533
105.52
110.66
104.81
104.656
2.178
178.514
1
TABLE 3 DFT calculated and experimental H NMR chemical shifts of Fe(III) complex 8b in DMSO solution, δ [ppm]
Chemical shift
Chemical shift
Atomic
number
Atomic
number
Cal.
Exp.
Cal.
Exp.
H30
H12
H19
H28
H10
H18
9.28
9.12
H31
H20
H22
H24
H36
7.49
7.66
9.06
8.18
7.80
7.77
7.54
8.89
8.33
7.95
7.74
7.71
4.10
1.75
1.32
0.97
4.24
1.69–1.71
1.20–1.23
0.85
the ligands 7a,b, electron transition from the HOMO
orbital to the LUMO orbital is π → π* transition. On the
other hand, the HOMO and LUMO frontier orbitals of
the complex 8b species are mainly localized on the
isoxazole ring and Fe atom, respectively. It implies that
electron transition from the HOMO orbital to the LUMO
orbital is Ligand to‐Metal charge transfer (LMCT).[35]
The energy difference between the HOMO and LUMO
frontier orbitals is one of the important characteristics of
molecules, which has a determining role in such cases
as electric properties, electronic spectra and photochemi-
cal reactions. Energy separation between the HOMO
and LUMO (Δε = εLUMO – εHOMO) of 7a, 7b and 8b is
3.22 eV (385 nm), 3.29 eV (377 nm) and 1.75 eV
(708 nm), compared with the experimental values of
440, 360 and 750 nm, respectively.
3.4 | Antibacterial studies
The antibacterial activity of the ligands 7a,b and complexes
8a,b was tested against a panel of strains of Gram negative
bacterial (Pseudomonas aeruginosa (ATCC 27853) and
Escherichia coli, (ATCC 25922)) and Gram positive
(Staphylococcuse aureus methicillin resistant S. aureus

8 of 11
RAMEZANI ET AL.
TABLE 4 Selected experimental and calculated IR vibrational frequencies (cm‐1) of Fe(III) complex 8b
Experimental frequency Calculated frequency Intensity (km.mol‐1) Vibrational assignment
504 (w)
549 (w)
834(s)
513
562
833
64
υ
υ
υ
sym(Fe‐N)
186
97
asym(Fe‐N)
sym (C‐cl) of the benzene rings involving the –cl substituent
958(w)
905
931
163
1135
δwagging of the –CH2 moieties
Breathing of the aromatic rings
1016 (m)
1045 (s)
970, 972
1014
183, 206
108
υ(N1‐O1, N4‐O2)+ υ(C‐C) aliphatic
υ(C4‐N7, C18‐N8)
1043
1054, 1062
935
1085, 1273
υ(C29‐N3, C33‐N6)+ υ(C‐cl) + υ(C‐O)
υ(C‐cl) + υasym(C2‐O1‐N1, C16‐O2‐N4)
1091 (s)
1082
1107
145
33
υsym(C‐C) aliphatic
δip(aromatic hydrogens)
1187 (m, sh)
2007
1113
420
652
υ(C4‐N7, C18‐N8, C2‐O1, C16‐O2)
υ(C2‐C9, C16‐C23)
1211 (m)
1263(m)
1214
203
υ(C2‐O1, C16‐O2)
1235
1274
658
118
υ(C29‐N3, C33‐N6)
υasym(C7‐N2‐C8, C21‐N5‐C22)
1318 (m)
1375 (s)
1449 (vs)
1323
1402
2745
1531
υ(C29‐N3, C33‐N6)
υ(C=C, C=N) of the aromatic rings
1404
1433
1452
833
86
175
υ(C=C, C=N) of the aromatic rings
δoscissoring of the methyl groups
δoscissoring of the –CH2 moieties
1492 (vs, sh)
1564(vs)
1475
1489
83
68
υasym(C‐C) of the benzene rings involving the –cl substituent
1532
1555
1574
1989
3995
1087
υ(C=C, C=N) of the aromatic rings
1630(m)
2870 (w)
2902(m)
2917(m)
2962 (w)
1627
49
υasym (C=N) of the imine
2898, 2982
2887
62,13
57
υsym(C‐H) of the –CH2 moieties
υsym(C‐H) of the methyl groups
υasym(C‐H) of the –CH2 moieties
2883‐2952
7‐47
2958 ‐2997
13‐38
υ(C‐H) aromatic
3024
79
υ(C8‐H2, C22‐H8)
3435 (m,br)
3381
3432
127
9
υ
υ
sym(O‐H) of the H2O ligands
asym(O‐H) of the H2O ligands
Abbreviation: op, out‐of‐plane; ip, in‐plane; w, weak; m, medium; s, strong; vs, very strong; br, broad; sh, shoulder.
(MRSA) clinical isolated and Bacillus subtilis (ATCC 6633))
species (Table 5) using broth microdilution method as pre-
viously described.[38] Comparison with Ampicillin as a
standard was done. The lowest concentration of the anti-
bacterial agent that prevents growth of the test organism,
as detected by lack of visual turbidity (matching the nega-
tive growth control), is designated the minimum inhibitory
concentration (MIC). Experimental details of the tests can
be found in our earlier study.[39,40]
are less than those of Ampicillin. Coordination of ligands
7a,b to Fe(III) leads to improvement in the antibacterial
activity. This can be explained by Tweedy's chelation
theory,[41] which explicated that the lipophilicity of the
uncoordinated ligand can be changed by reducing of the
polarizability of the Mn+ ion via the L→M donation, and
the possible electron delocalization over the metal
complexes. Also, the results revealed that the complex
8a with R= Pr and Ar= 4‐OHC6H4 groups, displayed
greater antibacterial activity against Gram‐negative
bacteria than did the well‐known antibacterial agent
Ampicillin (Table 5).
As seen in Table 5, compounds 7a,b inhibit the
metabolic growth of the tested Gram positive and negative
bacteria to the same extent, but the inhibitions percent

RAMEZANI ET AL.
9 of 11
FIGURE 5 The HOMO (down) and LUMO (up) frontier orbitals of the ligands 7a,b and complex 8b
TABLE 5 Antibacterial activity (MIC, μg mL‐1) of reference and compounds 7a,b and 8a,b
Compds.
7a
S.a. (MRSA)
B.s. (ATCC 6633)
P.a. (ATCC 27853)
E.c. (ATCC 25922)
90
85
85
80
7b
100
25
100
15
95
20
95
5
8a
8b
40
35
35
15
8
Ampicillin
62
0.50
125
spectral properties are in good agreement with the exper-
imental values, confirming suitability of the optimized
geometries for Fe(III) complexes. Moreover, results from
the antimicrobial screening tests show that new com-
pounds are effective against standard strains of Gram‐neg-
ative growth inhibitors. An improvement in the
antibacterial activity is observed upon the coordination
to the Fe(III) ion.
4 | CONCLUSION
In summary, we have synthesized two new fluorescent
heterocyclic Schiff base ligands from the reaction of 8‐(4‐
chlorophenyl)‐3‐alkyl‐3H‐imidazo[4',5':3,4]benzo [1,2‐c]
isoxazol‐5‐amine with p‐hydroxybenzaldehyde and p‐
chlorobenzaldehyde. Results from NOESY experiment of
the ligands confirmed the E configuration of Schiff‐bases.
Coordination of the ligands with Fe(III) cation led to the
formation of deep green complexes in high yields. The
structures of the complexes have been confirmed by spec-
tral, analytical data and Job's method. Schiff‐base ligands
and iron complexes were spectrally characterized by UV‐
Vis and fluorescence spectroscopy. Studies of optical prop-
erties showed that the intensity of fluorescence emission
of all compounds is high and their obtained emission
quantum yields are around 0.15 – 0.53. Optimized geome-
tries and assignment of the IR bands and NMR chemical
shifts of the new complexes were also computed by DFT
methods. The results revealed that the DFT‐calculated
Such ligands would appear to offer a suitable template
for the speciation of iron in different samples and further
studies are under way to this end in our laboratory.
ACKNOWLEDGMENT
We would like to express our sincere gratitude to
Research Office, Mashhad Branch, Islamic Azad
University, Mashhad‐Iran, for financial support of this
work. We must also acknowledge Maryam Jajarmi
(Khorasan Razavi Rural Water and Wastewater Co.) for
her assistance in antibacterial studies.

10 of 11
RAMEZANI ET AL.
[21] M. A. Kalwat, Z. Huang, C. Wichaidit, K. McGlynn, S. Earnest,
C. Savoia, E. M. Dioum, J. W. Schneider, M. R. Hutchison, M.
H. Cobb, ACS Chem. Biol. 2016, 11, 1128.
ORCID
Mehdi Pordel http://orcid.org/0000-0003-3445-8792
[22] M. S. Munsey, N. R. Natale, Coord. Chem. Rev. 1991, 109, 251.
[23] Ö. Güngör, P. Gürkan, J. Mol. Struct. 2014, 1074, 62.
REFERENCES
[1] C. J. Dhanaraj, J. Johnson, Appl. Organomet. Chem. 2016, 30, 860.
[24] J. A. Makawana, J. Sun, H. ‐L. Zhu, Bioorg. Med. Chem. Lett.
2013, 23, 6264.
[2] S. H. Bae, M. S. Seo, Y. M. Lee, K. B. Cho, W. S. Kim, W. Nam,
Angew. Chem. Int. Ed. 2016, 55, 8027.
[25] E. L. de Araújo, H. F. Barbosa, E. R. Dockal, É. T. Cavalheiro,
Int. J. Biol. Macromolec. 2017, 95, 168.
[3] (a) L. C. M. Castro, H. Li, J. ‐B. Sortais, C. Darcel, Green Chem. 2015,
17, 2283. (b) K. Natte, W. Li, S. Zhou, H. Neumann, X.‐F. W.
Tetrahedron, Lett. 2015, 56, 1118. (c) B. M. Peterson, M. E. Herried,
R. L. Neve, R. W. McGaff, Dalton Trans. 2014, 48, 17899.
[26] D. Sweeney, A. Stoneburner, D. L. Shinabarger, F. F. Arhin, A.
Belley, G. Moeck, C. M. Pillar, J. Antimicrob. Chemother. 2016,
72, 622.
[4] U. Onyenze, I. E. Otuokere, J. C. Igwe, Research J. Science and
Tech. 2016, 8, 215.
[27] P. N. Preston, In The Chemistry of Heterocyclic Compounds,
Part 1, 40, John Wiley & Sons 87.
[5] D. Mandal, S. Raha, T. Rakshit, A. De Das, K. C. Ghosh, P.
[28] M. Rahimizadeh, M. Pordel, M. Bakavoli, Z. Bakhtiarpoor, A.
Mitra, J. Environ. Sociobiol. 2016, 1, 15.
Orafaie, Monatsh. Chem. 2009, 140, 633.
[6] A. K. Hijazi, Z. A. Taha, A. M. Ajlouni, W. M. Al‐Momani, T. S.
Ababneh, H. M. Alshare, F. E. Kuehn, Applied Org.metallic
Chem. 2017, 5, 31.
[29] (a) S. Ramezani, M. Pordel, S. Beyramabadi, J. Fluoresc. 2016,
26, 513. (b) M. Rezazadeh, M. Pordel, A. Davoodnia, S. Saberi,
Chem. Hetero. Comp. 2015, 51, 918.
[7] A. N. Alaghaz, M. E. Zayed, S. A. Alharbi, J. Mol. Struct. 2015,
1084, 36.
[30] Z. Agheli, M. Pordel, S. A. Beyramabadi, J. Mol. Struct. 2017,
1130, 55.
[8] T. Fukushima, E. Taniguchi, H. Yamada, K. Kato, A. Shimizu,
Y. Nishiguchi, M. Onozato, H. Ichiba, Y. Azuma, BioMetals
2015, 28, 669.
[31] C. Lee, W. Yang, R. G. Parr, Phys. Rev. B. 1988, 37, 785.
[32] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, J. A. Montgomery Jr., T. Vreven, K. N.
Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V.
Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A.
Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R.
Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian,
J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,
R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli,
J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P.
Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A.
D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck,
K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G.
Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A.
Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T.
Keith, M. A. Al‐Laham, C. Y. Peng, A. Nanayakkara, M.
Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong,
C. Gonzalez, J. A. Pople, Gaussian 03, Revision C.02, Gaussian,
Inc., Wallingford CT 2004.
[9] Y. Gou, J. Wang, S. Chen, Z. Zhang, Y. Zhang, W. Zhang, F.
Yang, Eur. J. Med. Chem. 2016, 123, 354.
[10] K. Saleem, W. A. Wani, A. Haque, M. N. Lone, M. F. Hsieh, M.
A. Jairajpuri, I. Ali, Future med. chem. 2013, 5, 135.
[11] D. Murtinho, Z. N. Rocha, A. S. Pires, R. P. Jiménez, A. M.
Abrantes, M. Laranjo, A. C. Mamede, J. E. Casalta‐Lopes, M. F.
Botelho, A. A. Pais, S. C. Nunes, Appl. Org. metal. Chem. 2015,
29, 425.
[12] M. Sova, R. Frlan, S. Gobec, G. Stavber, Z. Časar, Appl.
Organomet. Chem. 2015, 29, 528.
[13] M. Y. Khan, J. Zhou, X. Chen, A. Khan, H. Mudassir, Z. Xue,
S. W. Lee, S. K. Noh, Polymer 2016, 90, 309.
[14] M. Chieppa, V. Galleggiante, G. Serino, M. Massaro, A. Santino,
Curr. Pharm. Des. 2017, 23, 2289.
[15] M. Kono, K. Saigo, S. Yamamoto, K. Shirai, S. Iwamoto, T.
Uematsu, T. Takahashi, S. Imoto, M. Hashimoto, Y. Minami,
A. Wada, Clin. Exp. Pharmacol. Physiol. 2016, 43, 915.
[33] J. Tomasi, R. Cammi, J. Comput. Chem. 1995, 16, 1449.
[34] W. C. Vosburgh, G. R. Copper, J. Am. Chem. Soc. 1941, 63, 437.
[35] S. Saureu, C. de Graaf, Phys. Chem. Chem. Phys. 2016, 18, 1233.
[36] J. Q. Umberger, V. K. LaMer, J. Am. Chem. Soc. 1945, 67, 1099.
[16] S. M. El Sayed, A. Abou‐Taleb, H. S. Mahmoud, H. Baghdadi, R. A.
Maria, N. S. Ahmed, M. M. Nabo, Med. hypotheses 2014, 83, 238.
[17] R. F. Júnior, V. B. Marques, D. O. Nunes, I. Stefanon, L. dos
Santos, Toxicol. Lett. 2017, 279, 43.
[37] C. Preti, G. Tosi, M. Massacesi, G. Ponticelli, Spectrochim. Acta
1976, 32, 1779.
[18] G. Y. Oudit, P. H. Backx, Can. J. Cardiol. 2016, 32, 938.
[19] M. A. Barmade, P. R. Murumkar, M. Kumar Sharma, M. Ram
[38] N. Tezer, N. Karakus, J. Mol. Model. 2009, 15, 223.
Yadav, Curr. Top. Med. Chem. 2016, 16, 2863.
[20] M. Madni, S. Hameed, M. N. Ahmed, M. N. Tahir, N. A. Al‐
Masoudi, C. Pannecouque, Med. Chem. Res. 2017, 26, 2653.
[39] M. Pordel, S. Ramezani, M. Jajarmi, M. Sokhanvar, Russ. J.
Bioorg. Chem. 2016, 42, 106.

RAMEZANI ET AL.
11 of 11
[40] M. Pordel, A. Abdollahi, B. Razavi, Russ. J. Bioorg. Chem. 2013,
39, 240.
How to cite this article: Ramezani S, Pordel M,
Davoodnia A. Synthesis, spectral, DFT calculations
and antibacterial studies of Fe(III) complexes of
new fluorescent Schiff bases derived from
imidazo[4',5':3,4]benzo[1,2‐c]isoxazole. Appl
Organometal Chem. 2017;e4178. https://doi.org/
10.1002/aoc.4178
[41] B. Tweedy, Phytopathalogy 1964, 55, 910.
SUPPORTING INFORMATION
Additional Supporting Information may be found online
in the supporting information tab for this article.