Design and synthesis of three Fe(III) mixed-ligand complexes: Exploration of their biological and phenoxazinone synthase-like activities

Article abstract of DOI:10.1016/j.ica.2020.119443

New Fe(III) complexes with mixed ligands based on 1-{(E)-[(4-methylphenyl)imino]methyl}-2-naphthol (HN) as primary ligand and secondary co-ligand of O-hydroxy quinolone (HQ), 2-(1H-benzimidazol-2-yl)phenol (HB) and 2-(4,5-diphenyl-1H-imidazol-2-yl)phenol

Full text of DOI:10.1016/j.ica.2020.119443

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Research paper
Design and Synthesis of Three Fe(III) Mixed-ligand Complexes: Exploration of
Their Biological and Phenoxazinone Synthase-Like Activities
Mohammed Ismael, Abdel-Mawgoud M. Abdel-Mawgoud, Mostafa K. Rabia,
Aly Abdou
PII:
S0020-1693(19)31609-3
https://doi.org/10.1016/j.ica.2020.119443
ICA 119443
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Inorganica Chimica Acta
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22 December 2019
11 January 2020
Please cite this article as: M. Ismael, A.M. Abdel-Mawgoud, M.K. Rabia, A. Abdou, Design and Synthesis of Three
Fe(III) Mixed-ligand Complexes: Exploration of Their Biological and Phenoxazinone Synthase-Like Activities,
Inorganica Chimica Acta (2020), doi: https://doi.org/10.1016/j.ica.2020.119443
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Design and Synthesis of Three Fe(III) Mixed-ligand Complexes: Exploration of Their
Biological and Phenoxazinone Synthase-Like Activities
Mohammed Ismael*, Abdel-Mawgoud M. Abdel-Mawgoud, Mostafa K. Rabia and Aly Abdou
Chemistry Department, Faculty of Science, Sohag University, Sohag 82524, Egypt
Abstract
New
Fe(III)
complexes
with
mixed
ligands
based
on
1-{(E)-[(4-
methylphenyl)imino]methyl}-2-naphthol (HN) as primary ligand and secondary co-ligand of O-
hydroxy quinolone (HQ), 2-(1H-benzimidazol-2-yl)phenol (HB) and 2-(4,5-diphenyl-1H-imidazol-
2-yl)phenol (HI) had been isolated and characterized. The isolated complexes had 1:1:1 ratio for
Fe(III):ligand:co-ligand with one chloride and one water molecule coordinated to the Fe-centre,
suggesting octahedral structure around the Fe-center with the formula [Fe(Ligand)(co-
ligand)(Cl)(H2O)]. Theoretical calculations using density functional theory by B3LYP with
LANL2DZ basis set had been done for two possible orientations of the ligand moieties around the
Fe-center, to find out the most reliable coordination modes. Calculations include geometry
optimization, molecular orbital description, and energy evaluation of trans- and cis- coordination
modes of the chloride and water around the Fe-center. In-vitro antibacterial and antifungal
properties of the isolated complexes have also been examined against pathogenic bacteria, E. coli
(G-), B. cereus (G+), and A. Fumigatus fungi, by using broth microdilution and disc diffusion
methods. The target compounds exhibited considerable growth inhibition against selected
pathogenic microorganisms as antimicrobial candidates with low minimum inhibitory
concentration (MIC). The complexes showed biomimetic phenoxazinone synthase-like activity for
the aerial coupling oxidation of o-aminophenol (OAP) to phenoxazine-2-one (Phz), in acetonitrile
(ACN) solution, with high turnover number (Kcat). Structure Activity Relationship (SAR) model
was derived, using Multi-Linear Regression analysis, by correlation the practical biological (MIC)
data with calculated chemical descriptors.
Keywords
Fe(III) mixed-ligand complexes, phenol-based Schiff base, imidazoles, in-vitro antimicrobial
activity, DFT calculations, phenoxazinone synthase-like activity, Structure activity relationship.

1. Introduction
Transition metal based Schiff-base complexes play a key role in the field of biological
aspects, and organic catalysis[1,2]. Design and synthesis of transition metal complexes incorporating
a combination of Schiff-base ligand with N-containing heterocyclic ring are of great interest in
view of the possibility of increases the variety in chemical structure and obtaining diverse
structures with intriguing specificities.
In biology; imidazoles, benzimidazoles, and 2-hydroxy quinoline derivatives can be termed
‘Master Key’[3] as it is the main important core in many categories of therapeutic reagents, e.g.
anticancer[4],
antimicrobial[5],
anti-inflammatory[6],
anticonvulsant,
anti-diabetic[7],
antihypertensive[8], anti-tubercular[9], antiviral[10] and anti-parasitic reagents[11], anti-oxidants,
proton pump inhibitors, anti-coagulants, hormone modulators, CNS stimulants as well as
depressants and lipid level modulators, etc. [3]. Therefore, introductions of imidazole,
benzimidazole, 2-hydroxy quinoline as secondary co-ligand will significantly enhance the
biological properties.
On the other hand, Phenoxazinone synthase, a multicopper metalloenzyme, plays a crucial
role in the biosynthesis of actinomycin D through the oxidative coupling of a wide range of 2-
aminophenols (OAPs) to phenoxazinone. Actinomycin D is one among the most potent
antineoplastic agents, which is most effective against Wilms tumor, gestational choriocarcinoma,
Ewing’s Sarcoma, testicular cancer and ovarian cancer for some extent. This is due to the
intercalation of phenoxazinone chromophore with DNA base-pairs followed by blocking the
creation of RNA[12]. Research study on the catalytic oxidation of OAP to 2-amino-3H-phenoxazine-3-
one (Phz) by metal complexes has considerable attraction for providing important information about
bioinorganic reactions and mechanistic aspects in the organic transformation which may open a new
avenue in molecular science.
In the present framework, three Fe(III) ternary complexes involving a Schiff base as the
primary ligand, and N-containing heterocyclic ring as the secondary one, had been prepared and
characterized. Additionally, antifungal, antibacterial properties, and catalytic activity towards the
oxidative coupling of OAP had been investigated. Furthermore, density functional theory (DFT)
calculations had been performed to ascertain the 3D structure of the complexes, and correlate
experimental biological (MIC) and catalytic (Kcat) activity with the estimated chemical descriptors
for SAR model generation.

2. Experimental
2.1. Reagents
Chemicals used in synthesis of ligands and their metal complexes including benzil
(ADWIC, 99.0 %), 2-hydroxy-benzaldhyde (ADWIC, 99.0 %), p-toluidine (ADWIC, 99.0 %), 2-
hydroxy-1-naphthaldehyde (Sigma-Aldrich, 98.0 %), O-phenylene-diamine (Sigma-Aldrich, 98.0
%), salicylic acid (ADWIC, 99.0 %), 8-hydroxyquinloine (Sigma-Aldrich, 98.0 %), 2-aminophenol
(Sigma-Aldrich, 98.0 %), Iron(III) chloride hexahydrate (Sigma-Aldrich, 98.0 %), were used as
received without further purification.
2.2. Physical Measurements
JENWAY model 6405 UV-Vis. spectrophotometer, BRUKER Avance 400 instrument,
BRUKER FT-IR model 8101 in the region 4000-400 cm-1, JENWAY conductivity meter,
Bartington Susceptibility instrument model 4320, and BRUKER D8-ADVANCE, were used for
the absorbance, NMR, FT-IR, Conductivity, Magnetic susceptibility, and powder-XRD,
measurements respectively.
2.3. Synthesis of the ligands
Synthetic procedure of the subject ligands, HN, HB and HI ligands are listed in the
Supplementary information.
2.4. Synthesis of the Fe(III) ternary complexes
An aqueous solution of the Iron(III) Chloride (2.0 mmol, 0.54 g in 10.0 H2O) was added
drop wise to a mixture of the phenol-based Schiff base ligand [HN (2.0 mmol, 0.522 g) in 8.0 ml
ethanol] and the N-containing heterocyclic co-ligand [HQ (2.0 mmol, 0.290 g), HB (2.0 mmol,
0.420 g) or HI (2.0 mmol, 0.624 g) in 8.0 ml ethanol]. The reaction mixture was refluxed with
o
stirring for about 8.0 h at 80 C. Afterwards, the mixture was lifted overnight for slow solvent
evaporation. The resulting product was collected, washed by water-ethanol (3:7) and finally
purified, Scheme (1).

Scheme (1): Synthesis of the Fe(III) ternary complexes.
FeNQ complex: [Fe(L)(Q)(H2O)Cl].2H2O:
0
Color, dark brown, m.p. >300 C, yield, 80%. Anal. Found (calc.) for (C27H22ClFeN2O3. 2H2O,
549.8 g/mol) (%): C, 58.06 (58.98), H, 4.10 (4.77), N, 5.57 (5.10), Cl, 6.89 (6.45), Fe, 10.45
(10.16). FT-IR, (υ, cm-1): 3415 (H2O), 3189 (-NH), 1555 (-CH=N), 1510 (-C=N), 1284 (C-O), 560
(M-O), 426 (M-N). UV-vis. (10-3 M acetonitrile solution, (λmax, nm)): 360, 490 (n→π*, LMCT).
Molar conductivity (10-3 M ethanol solution, (µv, Ω-1.cm2.mol-1)): 15.8 (Non-electrolyte nature).
Magnetic moment (µeff, B.M): 1.91.
FeNB complex: [Fe(L)(B)(H2O)Cl].2H2O:
0
Color, dark brown, m.p. >300 C, yield, 85%. Anal. Found (calc.) for (C31H25ClFeN3O3.2H2O,
614.9 g/mol) (%): C, 59.82 (60.55), H, 4.01 (4.75), N, 7.13 (6.83), Cl, 5.04 (5.77), Fe, 9.85 (9.08).
FT-IR, (υ, cm-1): 3430 (H2O), 3140 (-NH), 1580 (-CH=N), 1512 (-C=N), 1273 (C-O), 520 (M-O),
436 (M-N). UV-vis. (10-3 M acetonitrile solution, (λmax, nm)): 320, 460 (n→π*, LMCT). Molar
conductivity (10-3 M ethanol solution, (µv, Ω-1.cm2.mol-1)): 14.0 (Non-electrolyte nature). Magnetic
moment (µeff, B.M): 1.93.

FeNI complex: [Fe(L)(I)(H2O)Cl].3H2O:
Color, dark brown, m.p. >300 0C, yield, 85%. Anal. Found (calc.) for (C39H31ClFeN3O3. 3H2O, 735
g/mol) (%): C, 63.06 (63.73), H, 4.48 (5.07), N, 5.16 (5.72), Cl, 5.14 (4.82), Fe, 7.18 (7.60). FT-IR,
(υ, cm-1): 3420 (H2O), 3122 (-NH), 1585 (-CH=N), 1520 (-C=N), 1287 (C-O), 535 (M-O), 422 (M-
N). UV-vis. (10-3 M acetonitrile solution, (λmax, nm)): 340, 470 (n→π*, LMCT). Molar
conductivity (10-3 M ethanol solution, (µv, Ω-1.cm2.mol-1)): 12.5 (Non-electrolyte nature). Magnetic
moment (µeff, B.M): 1.97.
2.5. Density Functional Theory (DFT) Calculations
Geometry optimizations of the subject compounds in the gas phase were carried out at a
gradient corrected DFT using hybrid correlation functional (B3LYP) combined with LANL2DZ
basis set with effective core potential (ECP)[13], implemented in Gaussian 09 software[14]. The
optimized structures and molecular frontier orbitals were visualized with GaussView 5.0 software.
To investigate the chemical reactivity of the optimized complexes, chemical descriptor
parameters had been calculated including: energy gap ΔE = ELUMO - EHOMO, electronegativity χ = -
½ (EHOMO + ELUMO), electronic chemical potential  = ½ (EHOMO + ELUMO) = -χ, global chemical
hardness η = ½ (ELUMO - EHOMO), global softness  = 1/2η, global electrophilicity index ω = 2/2η,
and nucleophilicity index Nu = 1/ω, where EHOMO and ELUMO are the energies of highest occupied
molecular orbital and the lowest unoccupied molecular orbital, respectively[15].
2.6. In-vitro exploration of antimicrobial activity
Antimicrobial activity of the free ligands and their Fe(III) complexes were in-vitro screened
against pathogenic bacteria, E. coli (G-), B. cereus (G+), and A. fumigatus fungi. Broth
microdilution[16] and disc diffusion methods[17,18] were applied for measuring the minimum
inhibitory concentration (MIC, μg/mL) and the inhibition zone (IZ, mm), respectively.
The Petri dishes were filled by nutrient agar using Pour plate's protocol. Then, the nutrient
agar medium was seeded with tested microorganisms (E. coli (G–), B. cereus (G+) and A.
fumigates) in Petri dishes. Uniformed dices (5.0 mm diameter) of the subject compound with 100
μg/mL concentration were placed on the culture plates and incubated at 37 °C for 24 h. The
antimicrobial activity was determined by measuring the diameter of the inhibition zone (mm).
2.7. Exploration of the Phenoxazinone synthase mimicking activity

To explore the ability of the Fe(III) complexes for coupling oxidation of OAP to Phz; 2.8
mL of 0.01 mol.dm-3 solution of substrate OAP was treated separately with 0.1 mL of 1 x 10-5
mol.dm-3 catalyst solution Fe(III) complex, and the volume was made to 3.0 mL by using
acetonitrile, then a wavelength scan of the solution was recorded at a regular time interval of ten
minutes, under aerobic conditions at room temperature (25 oC). The increase in the absorbance as a
function of time around ≈ 430 nm, characteristic of the Phz, confirmed the phenoxazinone synthase
mimicking activity.
2.7.1. Kinetics of Phenoxazinone synthase mimicking activity
In order to get further insight into the kinetics of penoxazinone synthase mimicking activity
of the Fe(III) complexes; 0.1 mL of 1×10-5 mol.dm-3 of each catalyst Fe(III) complex was added to
a series volumes of 1.8, 2.0, 2.2, 2.4, 2.6 and 2.8 mL of 0.01 mol.dm-3 OAP substrate. All these
solutions were then made to 3.0 mL by using acetonitrile solvent, under aerobic conditions at room
temperature. Raw data was taken as absorbance (A) at a regular time interval of ten minutes for a
period of two hours. The product concentrations [Phz] at several reaction times were calculated
from Beer-Lambert’s law, A430nm = εPhz  L  [Phz], where A430 nm is the Phz absorbance, εPhz is the
molar extinction coefficient (1.093×104 cm-1 mol-1.dm3) [19], L is the length of the quartz cuvette (L
= 1 cm) and [Phz] is the molar concentration of Phz (mol.dm-3). The change in [Phz] is plotted
against time (t, h). The initial rate values for the particular catalyst, at each substrate concentration
were evaluated from the maximum slope (V0 = d[Phz]/dt, mol.dm-3.h-1).
The initial rate values V0 and product concentration [Phz] were used on the basis of
Michaelis-Menten kinetics, Lineweaver-Burk, Hanes equation and Eadie-Hofstee approach of
enzymatic kinetics, to evaluate several kinetic parameters, including maximum reaction velocity
(Vmax, mol.dm-3.h-1), Michaelis constant (KM, mol.dm-3), turnover number (Kcat, h-1) and specificity
constant (kcat/ KM, dm3.mol-1.h-1). The turnover number (Kcat, h-1) was obtained by dividing the
maximum reaction velocity (Vmax) by the concentration of the complex.
2.8. Structure Activity Relationship (SAR)
As biological or catalytic activity ranking cannot be determined by only one parameter, it
depends on a combination of parameters, thus, there is an urgent need to derive SAR model to
correlate the biological or catalytic activity of the subject compounds with their calculated
chemical descriptors and find the most effective description model[20]. SAR statistical model was
constructed by correlating the practical biological (MIC) or catalytic (Kcat) of the subject

compounds with their theoretical chemical descriptors (EHOMO, ELUMO, ΔE, χ, µ, η, σ, ω and Nu),
using multiple linear regressions (MLR).
Kcat = constant + a1 EHOMO + a2 ELUMO + a3 ∆E + a4 χ + a5 µ + a6 η+ a7 σ + a8 ω + a9 Nu
MIC = constant + b1 EHOMO + b2 ELUMO + b3 ∆E + b4 χ + b5 µ + b6 η+ b7 σ + b8 ω + b9 Nu
(1)
(2)
3. Results and Discussion
3.1. Structure elucidation of the prepared ligands
Structure elucidation of the subject HN, HB and HI ligands is listed in the Supplementary
information.
3.2. Structure elucidation of the prepared ternary complexes
3.2.1. Complex stoichiometry
Spectrophotometric study was carried out to determine the Fe(III):Ligand:co-ligand molar
ratio of the target complexes [21], Supplementary information, Fig. (S4).
According to these results a ratio of 1:1:1 (Fe : Ligand : co-ligand) had been identified for all
complexes suggesting the following reaction:
Fe3+ (aqs)+ HLigand + Hco-ligand = [Fe(HLigand)(Hco-ligand)]3+
To find out the manner in which ligands bind to the metal ion, a FT-IR spectrum was
analyzed.
3.2.2. Ligand binding mode with metal ion
IR spectra of the complexes were compared with that of bare ligands to determine the
coordination sites that may be involved in chelation, Supplementary information, Figs. (S5, S6 and
S7).
The azomethine group υ(-CH=N) stretching vibration of the HN ligand appeared at 1610
cm-1, was shifted to lower wave numbers at 1551, 1585 and 1585 cm-1, for FeNQ, FeNB and FeNI,
respectively, which gave good indication for the participation of -CH=N in chelation with the
metal ion [22,23]
.
The υ(C-O) appeared at 1250, 1234, 1248 and 1245 cm-1, for the free HN, HQ, HB and HI
ligands, respectively, was shifted to higher frequencies after complexation to appear at 1284, 1273
and 1287 cm-1 for FeNQ, FeNB and FeNI, respectively[24]. In addition, the phenolic υ(OH)
vibration of the free HN, HQ, HB and HI ligands appeared at 3323, 3370, 3311 and 3191 cm-1,
respectively, was disappeared upon complexation[15]. These confirm the participation of the
phenolic (-OH) of the HN, HQ, HB and HI ligands in coordination with metal ion after de-

protonation. The broad band above 3400 cm-1, had been assigned to υ(OH) of coordinated water
molecules.
The υ(-C=N) of the quinoline, benzimidazole and imidazole moiety appeared at ≈ 1588,
1574 and 1588 cm-1 in the bare in HQ, HB and HI co-ligands, respectively, which shifted to lower
wave numbers upon coordination with the metal ion to appear at 1480, 1522 and 1545 cm-1 for
FeNQ, FeNB and FeNI, respectively. This shift gave evidence for the participation of the (-C=N)
of co-ligands in chelation with the metal ion.
Furthermore, new bands are found in the IR spectra of the complexes in the regions 525-
565 and 420-453, which are assigned to υ(M-O) and υ(M-N), respectively[25].
These data indicate that the HN ligand participated as mono-negatively bi-dentate ligand
through the azomethine nitrogen (-CH=N) and phenolic oxygen after de-protonation. While, the
HQ, HB and HI co-ligands participated as mono-negatively bi-dentate co-ligand through the (-
C=N) nitrogen and phenolic oxygen after de-protonation. Therefore, the formed complexes would
be formulated as; [Fe(ligand)(co-ligand)(H2O)n]+.
3.2.3. Conductivity measurements
The isolated complexes were insoluble in water but soluble in common organic solvents,
e.g. ethanol, acetone, DMF and DMSO. The molar conductivity of the isolated complexes in
ethanol was relatively low, indicating the non-electrolytic nature of the isolated complexes.
Moreover, according to the non-electrolytic nature of the Fe(III) complexes, it would be
inferred that chloride ion coordinated to Fe(III) ion to satisfy the valence. The presence of Cl- ion
inside the coordination sphere of Fe(III) complexes was confirmed by reaction with AgNO3, as
precipitation of AgCl after acid dissociation of the subject complexes using conc. HNO3.
Therefore, the formed complexes would be formulated as; [Fe(ligand)(co-ligand)(H2O)n(Cl)].
3.2.4. Coordinated and non-coordinated water molecules
The number of coordinated and non-coordinated water molecules in the Fe(III) complexes
was determined through the thermal analysis of the complexes[26] undergo drying at ≈ 100-110 ºC,
followed by heating at ≈ 200-210 ºC, the weight loss was evaluated after each step.
The percentage observed (calculated) weight loss in the first step was 6.69 (6.45), 6.17
(5.85), and 7.04 (7.35) %, for FeNQ, NiNB, and CuNI, respectively, suggested the elimination of
two non-coordinated "water of hydration" molecules in the case of FeNQ, and FeNB, while three
molecules in the case of FeNI complex.

In the next step (weight loss), the percentage observed (calculated) was 3.98 (3.63), 3.73
(3.11), and 2.04 (2.64) %, for FeNQ, FeNB, and FeNI, respectively, suggested the elimination of
one coordinated water molecule in all the complexes. Therefore, the formed complexes would be
formulated
as
[Fe(L)(Q)(Cl)(H2O)].2H2O,
[Fe(L)(B)(Cl)(H2O)].2H2O
and
[Fe(L)(I)(Cl)(H2O)].3H2O, for FeNQ, FeNB and FeNI complexes, respectively.
3.2.5. Effective magnetic moment and geometry of the Fe(III) complex
The structure geometry of the Fe(III) complexes would be elucidated from the magnetic
susceptibility measurements. The susceptibility per gram atom of a paramagnetic metal ion in a
particular compound was determined by measuring the molar susceptibility of the compound and
applying diamagnetic corrections. The diamagnetic corrections would be determined by various
methods, however Pascal's constants are more often used to calculate the corrections[27]. The
measured gram magnetic susceptibility, Xg, of the complexes was utilized to calculate effective
magnetic moment, eff = 2.83  (((Xg  Mwt) – (diamagnetic correction))  T)0.5, in Bohr
Magneton, where T is the absolute temperature (K)[17].
The measured µeff of FeNQ, FeNB, and FeNI complexes at room temperature were 1.907,
1.929 and 1.971 B.M, respectively, which would be attributed to one unpaired electron back to
d2sp3 hybridization, suggesting a low spin octahedral geometry.
3.2.6. Electronic spectra
The electronic absorption spectra of Fe(III) complexes was recorded at room temperature
using acetonitrile as solvent and were compared with that of bare ligands, Supplementary
information, Figs. (S8, S9 and S10).
The free ligands exhibit absorption bands at (230, 310 & 380) for HN, at (260 & 350) for
HQ, at (235 & 320) for HB and at (210 & 315) for HI. These bands can be attributed to π → π*
and n → π* transition in the ligand moiety.
The observed shoulder spectral features of the Fe(III) complexes at 360, 320 and 340 ꢀnm,
for FeNQ, FeNB and FeNI, respectively, would be assigned to n→π*. Additionally, the broad band
observed at 490, 460 and 470ꢀnm, for FeNQ, FeNB and FeNI, respectively, would be assigned to
Cl-Fe charge transfer transition[28].
3.2.7. Powder X-ray diffraction studies
Though the synthesized metal complexes were soluble in some polar organic solvents like
DMSO and DMF, crystals that are suitable for single crystal studies were not obtained. In order to

test the degree of crystallinity of the synthesized metal complexes, we obtained the powder X-ray
diffraction pattern of Fe(III) complexes. The powder X-ray diffraction pattern of the complexes
was scanned in the range 10-800 (2θ) at wave length 1.54 Å. The diffractogram and associated data
depict the 2θ value for each peak, relative intensity and inter-planar spacing (d-values). In all the
complexes, the trend of the curves decreases from maximum to minimum intensity indicating the
amorphous nature of the complexes in the present metal–ligand formation.
Powder X-ray diffraction pattern of Fe(III) complex, Fig. (1), showed 17, 14 and 20
reflections for FeNQ, FeNB and FeNI complexes, respectively, with maxima at 2θ = 22.275°,
24.992° and 31.525°, corresponding to d-value 3.98 Å, 3.56 Å and 2.83 Å, respectively. The inter-
planar spacing (d) had been calculated by using Bragg’s equation; d = n/2sin. The calculated
inter-planar d-spacing together with relative intensities with respect to most intense peak had been
recorded and depicted in Supplementary information, Tables. (S1, S2 and S3).
For the Fe(III) complexes unit cell, calculations had been done for cubic symmetry from the
entire peaks and h2+ k2+ l2 values were determined. The observed inter-planar d-spacing values
have been compared with the calculated ones and it was found to be in good agreement,
Supplementary information, Tables. (S1, S2 and S3). The h2+ k2+ l2 values of FeNQ and FeNB
complexes showed the presence of forbidden number 7, while FeNI complex showed the presence
of forbidden number 15 and 23. These indicate that the complexes may belong to hexagonal or
tetragonal systems[24,29]
.
The particle size of these complexes was estimated from their PXRD patterns based on the
highest intensity value compared to the other peaks using the well-known De-bye–Scherrer
formula, D(the apparent particle size of the grains) = k/cos; where; K is a constant (0.94 for Cu
grid), λ is the X-ray wavelength used (1.5406 Å), θ is half the scattering angle (the Bragg
diffraction angle), and β is the full-width at half-maximum (FWHM) of the X-ray diffraction line
(additional peak broadening). The crystallite size of the FeNQ, FeNB and FeNI complexes was
found to be 58.27, 23.61 and 204.41 nm, respectively, suggesting that the complexes are in a nano
crystalline phase, Supplementary information, Table. (S4).
3.3. DFT calculations
3.3.1. Geometry optimization of the complexes
The Fe(III) mixed-ligand complexes were formulated as [Fe(Ligand)(co-ligand)(Cl)(H2O)],
in which the chloride and water molecules can be oriented in trans or cis binding mode to each
other around the Fe(III) center, Fig. (2a,b). In the trans binding mode, the bulky organic ligands
were in the same plane and are close to each other with high steric hindrance, Fig. (2a). While in

the optimized cis binding mode of the Fe(III) complexes, the bulky organic ligands were oriented
perpendicular to each other around the Fe center , Fig. (2b), this orientation decreases the steric
hindrance. The calculated donor-acceptor bond lengths and bond angle for the cis- binding mode,
are listed in Table (1).
Table (1): Calculated selected bond lengths (Å) and bond angels (°) of the Fe(III) complexes.
bond length (Å)
FeNQ
bond angel (°)
Fe-NHN 1.96
Fe-NHQ 2.00
Fe-OHN 1.86
Fe-OHQ 1.90
NHN-Fe-NHQ
NHN-Fe-OHQ
NHN-Fe-OHN
NHN-Fe-Cl
NHN-Fe-H2O
NHQ-Fe-OHQ
NHQ-Fe-OHN
NHQ-Fe-Cl
175.59
92.57
92.61
89.34
93.74
84.26
84.72
94.40
NHQ-Fe-H2O
OHQ-Fe-H2O
OHQ-Fe-OHN
OHQ-Fe-Cl
OHN-Fe-Cl
OHN-Fe- H2O
Cl-Fe- H2O
89.13
86.52
96.36
167.87
95.52
172.91
81.40
Fe-Cl
2.37
Fe-OH2 2.03
FeNB
Fe-NHN 1.98
NHN-Fe-NHB
NHN-Fe-OHB
NHN-Fe-OHN
NHN-Fe-Cl
NHN-Fe-H2O
NHB-Fe-OHB
NHB-Fe-OHN
NHB-Fe-Cl
177.72
88.57
92.01
85.13
93.57
90.39
86.10
96.33
NHB-Fe-H2O
OHB-Fe-H2O
OHB-Fe-OHN
OHB-Fe-Cl
OHN-Fe-Cl
OHN-Fe- H2O
Cl-Fe- H2O
88.44
88.93
96.43
166.10
96.16
172.37
79.14
Fe-NHB
2.01
Fe-OHN 1.85
Fe-OHB
Fe-Cl
1.90
2.41
Fe-OH2 2.03
FeNI
Fe-NHN 2.00
NHN-Fe-NHI
NHN-Fe-OHI
NHN-Fe-OHN
NHN-Fe-Cl
NHN-Fe-H2O
NHI-Fe-OHI
NHI-Fe-OHN
NHI-Fe-Cl
177.96
84.81
91.34
85.49
95.43
93.17
177.96
84.81
NHI-Fe-H2O
OHI-Fe-H2O
OHI-Fe-OHN
OHI-Fe-Cl
OHN-Fe-Cl
OHN-Fe- H2O
Cl-Fe- H2O
84.23
88.49
94.18
164.40
98.24
172.93
80.30
Fe-NHI
2.01
Fe-OHN 1.87
Fe-OHI
Fe-Cl
1.88
2.39
Fe-OH2 2.05
Energetically, cis- binding mode was found to be more stable than trans- mode for all
complexes. The calculated energy difference was -7.76, -0.67 and -4.78 kcal/mol for FeNQ, FeNB
and FeNI, respectively.
In order to perform a comparative study of the stability of the studied complexes, the
Binding Energy (BE) or the complexation energy was calculated as follow[30]
Ebinding = Ecomplex – (EMetal + Eprimary-ligand(HN) + ESecondary co-ligand + ECl +EH2O
)
The more negative binding energy corresponds to the most stable complex. From the
calculated values one can notice that all complexes are more stable than their corresponding

ligands. And the order of the complexes stability is FeNQ (-439.56) > FeNB (-427.64) > FeNI (-
418.61).
3.3.2. Frontier molecular orbital analysis
The HOMO as an electron donor, represents the ability to donate an electron, while, LUMO
as an electron acceptor, represents the ability to obtain an electron. Both HOMO and LUMO are
the main orbitals take part in chemical stability[31]. Whereas, the negative values of EHOMO and
ELUMO point to the stability of the molecule. The larger HOMO energy suggested that the molecule
is a decent electron donor and it proposed that its electron donation character is vital for the
formation of a charge-transfer complex among the molecule and biological target. On opposing,
the low HOMO energy values indicated that the molecule donating ability is weaker. The HOMO
and LUMO energies of ligands and their complexes calculated by the DFT method are listed in
Table (2). Besides, the pictorial representation of their HOMO-LUMO distribution and their
respective positive and negative regions is shown in Fig. (3). Calculations of molecular orbital
configuration indicated that HOMO and LUMO were delocalized through the whole molecule
supporting the aromaticity among the phenyl skeleton. HOMO and LUMO orbitals for the complex
structures were also delocalized all over the molecule, Fig. (3).
Table (2): Calculated HOMO, LUMO, energy gap (ΔE), electronegativity (χ), Chemical hardness
(η), Softness (σ), chemical potentials (μ), electrophilicity index (ω), and nucleophilicity
(Nu), by eV unit, of the Fe(III) complexes.
HOMO LUMO ΔE
η
σ
μ
χ
ω
Nu
FeNQ -5.45
FeNB -5.42
FeNI -5.32
-2.43
-2.51
-2.49
3.03 1.51 0.33 -3.94 3.94 5.13 0.20
2.91 1.46 0.34 -3.97 3.97 5.40 0.19
2.83 1.42 0.35 -3.90 3.90 5.38 0.19
The HOMO-LUMO energy gap is correlated with various biological aspects like
antibacterial, antioxidant and DNA binding aspects [32]. Selected quantum chemical parameters had
been derived from EHOMO, ELUMO, and used to evaluate the chemical reactivity of the complexes.
Specifically, ΔE, χ, µ, η, σ, ω, and Nu, descriptors had been calculated and summarized in Table
(2).
Other important parameter ELUMO is related with electron accepting from appropriate
molecule. If its energy is low, the molecule can accept electrons from the appropriate molecule and
this result shows that chemical reactivity increases with decreasing of ELUMO. Chemical reactivity

ranking should be given as follow: FeNB>FeNI>FeNQ. Accordingly, FeNB is the most reactive in
gas phase.
Energy gap between LUMO and HOMO is important to determine the reactivity ranking
due to the electron mobility. Smaller value of this parameter means that molecule is more reactive,
thus, chemical reactivity increases with decreasing of ∆E values. According to ∆E, the reactivity
ranking should be as follow: FeNI>FeNB>FeNQ. Accordingly, investigated FeNI is the most
reactive in gas phase.
The other important parameters to explain the chemical reactivity ranking are hardness and
softness. The biological activity tendencies of complexes towards appropriate molecules can be
discussed with the hard–soft–acid–base (HSAB) approximation. The rule is that hard acids prefer
to coordinate to hard bases and soft acids to soft bases. Biological structures such as the cell,
enzyme, etc. are soft. Therefore, Soft complexes can interact easily with biological molecules than
hard molecules. So, biological activity increased with the increasing softness and decreasing
hardness values. The order of chemical hardness is FeNQ > FeNB > FeNI suggesting that FeNI is
the most reactive.
Chemical potential is other important parameters to determine the chemical reactivity
ranking. Chemical potential is directly proportional with Gibbs free energy and these parameters
can be related with spontaneity. The chemical reactivity increases with decreasing of chemical
potential, thus chemical reactivity ranking should be as follow: FeNB>FeNQ>FeNI, but the
difference is insignificant.
Other important parameter is electrophilicity and nucleophilicity indexes. The
electrophilicity index implies the ability of the electron accepting while nucleophilicity index
displays the ability of electron donating. The chemical reactivity ranking as electrophile is FeNQ >
FeNI > FeNB and vise-versa as nucleophile.
It can be noticed that reactivity cannot be determined through one descriptor, rather than it
depends on a combination of these mentioned parameters. Thus, there is an urgent need to derive
SAR model to correlate the biological activity of the investigated compounds with the calculated
chemical descriptors and find the most effective description.
3.4. In-vitro antimicrobial Screening
The subject complexes exhibited high enhancement in their antimicrobial activity than the
free ligands as can be seen in Table (3) and Fig. (4). The increased in activity of the Fe(III) mixed-
ligand complexes than their free ligands, would be explained on the basis of chelation
theory[33,34,35]. This theory states that chelation reduces the polarity of the metal ion by the partial

sharing of its positive charge with donor groups and possible π-electron delocalization over the
whole ring. This leads to increasing lipophilic character of the complex and penetration of the
complex through the lipid layer of cell membrane. The complex may block the binding sites of
microorganisms; consequently it disturbs the metabolism pathways and respiration process in the
cell, and thus blocks the synthesis of proteins, which restricts further growth of the organism and
resulting in the extinction of microorganisms.
Table (3): antimicrobial activities in terms of Diameter of inhibition zone in mm and minimum
inhibition concentration (MIC) of HL and its complexes at 100 µg/ml.
Anti-bacteria activity
B. cereus (G+)
Anti-fungi activity
Aspergillus fumigatus
E. coli (G-)
IZ
MIC ( µg/ml )
MICpr MICcalc
IZ
MIC ( µg/ml )
MICpr MICcalc
IZ
MIC ( µg/ml )
MICpr MICcalc
(mm)
6.30
7.10
7.60
6.90
14.50
16.20
16.20
(mm)
6.20
7.30
8.00
6.80
15.30
17.70
17.00
(mm)
6.70
8.20
8.30
7.40
16.40
18.70
17.90
HN
HB
HI
HQ
NFeQ
NFeB
NFeI
75.50
54.50
57.50
65.50
25.50
22.50
23.50
75.48
54.48
57.48
65.48
25.48
22.48
23.48
77.50
53.50
56.50
65.50
27.50
24.50
25.50
77.53
53.53
56.53
65.53
27.52
24.52
25.52
67.50
56.50
54.50
65.50
26.50
23.50
24.50
67.49
56.48
54.49
65.49
26.49
23.49
24.49
Current investigated complexes showed better MIC values comparing with previously
reported transition metal complexes against E. coli [36,37] and B. cereus[38,15].
3.5. Phenoxazinone synthase mimicking activity
Spectrophotometric scan for the coupling oxidation of OAP to Phz in the presence of
Fe(III) complexes revealed a gradual increase in intensity of the absorption band at 430 nm,
confirms the phenoxazinone synthase like activity, Fig. (5).
3.5.1. Kinetic investigation through several enzyme kinetic plots
The Michaelis-Menten model is the simplest and best-known approaches to catalyst
kinetics. The Michaelis-Menten equation relates the reaction velocity to substrate concentration for
a system, as follow;
K1
K2
Catayst-Substrate Intermediate
Product
( P )
Catalst
( C )
Substrate
( C-S )
+
( S )
K-1

Biochemical reactions involving a single substrate are often assumed to follow Michaelis-
Menten kinetics, V = (Vmax / (KM + [S]))[S], which is an important tool for treating this type of
saturation rate dependency on substrate concentration. Where, Vmax (mol.dm-3 h-1) represents the
maximum rate achieved by the system, at saturating substrate concentration. KM (mol.dm-3) is the
Michaelis constant and it is equal to the substrate concentration at which the reaction rate is half of
Vmax. Michaelis-Menten model is used to relate the rate of enzymatic reactions (V) to the
concentration of substrate ([S]), Supplementary information, Figs. (S11.a, S12.a and S13.a).
Linearization of Michaelis-Menten equation produces a double reciprocal Lineweaver-Burk
plot, (1/V) = (1/Vmax) + ((KM/Vmax)(1/[S])), Supplementary information, Figs. (S11.b, S12.b and
S13.b), that is used to analyze a variety of kinetic parameters, e.g. Vmax and KM.
On the other hand, rearrangement of Michaelis-Menten equation gives Hanes equation,
([S]/V) = KM/Vmax + (1/Vmax)[S]. In this case, the ratio of the substrate concentration (S) to the
reaction rate (V) is plotted against substrate concentration ([S]), Supplementary information, Figs.
(S11.c, S12.c and S13.c).
Eadie-Hofstee equation, V = Vmax + KM(V/[S]), is sometimes used in biochemistry for a
graphical representation of enzyme kinetics. Here reaction rate (V) is plotted as a function of the
ratio between reaction rate (V) and substrate concentration ([S]), Supplementary information, Figs.
(S11.d, S12.d and S13.d).
All these enzyme kinetic plots were used to evaluate several kinetic parameters, including
turnover number (Kcat, h-1) and specificity constant (kcat/KM , mol-1.dm3.h-1), Table (4). kcat was
defined as the maximum number of catalytic conversions of substrate molecules per unit time that
a single catalytic site will execute for a specific enzyme concentration. kcat/KM is a measure of how
efficiently a catalyst converts substrates into products. This ratio is a useful index for measuring
the substrate specificity of catalyst. The higher kcat/KM, the more the enzyme prefers that substrate.
Table (4): Kinetic parameters for the oxidation of OAPH catalyzed by the synthesized Fe(III)
complexes in acetonitrile.
Kcat (h-1)
Kcat (practical) Kcat (calculated)
Vmax
KM
kcat/ KM
(mol.dm-3 h-1)
(mol.dm-3) (mol-1.dm3.h-1)
NFeQ 6.98 x 10-4
NFeB 5.73 x 10-4
NFeI 5.25 x 10-4
5.81 x 104
4.13 x 104
209.53
171.82
157.58
209.52
171.81
157.57
3.61 x 10-3
4.16 x 10-3
3.74 x 10-3 4.22 x 104
3.5.2. Mechanistic pathway of catalytic activity

Based on the literature survey[39,40,41,42,43,44,45,46,47], a tentative catalytic cycle for the
formation of the phenoxazinone chromophore was shown in Scheme (2). Initially, OAP forms
adduct with complexes replacing the monodentate chloride ligand. This results in the formation of
OAP radical by the reaction with molecular dioxygen regenerating complexes. In the next step, the
OAP radical may be oxidized to o-benzoquinone monoamine, which, in turn, may be converted to
Phz by the reaction with dioxygen and OAP [19,48]
.
O
H
N
H2
Cl-
+ H+
N
H2
O
Cl
N
O
N
O
Fe
Fe
O
N
O
O
H2
N
O
H2
Complex-Substrate Intermediate
Complex
1/2
+ H+ + Cl-
O2
.
O
O
H2
1/2
+
2
NH2
[O]
O
O
N
H
+ O2
O
N
O
H2
N
H
NH2
Scheme (2): Tentative mechanistic pathway for the coupling oxidation of OAP to Phz.

3.5.3. Comparative study
Transition metal complexes were wildly reported as being influential in the catalytic
conversion of OAP to Phz[49]. A comparison was made between our synthetized complexes with
other reported transition metal complexes. Noticeable differences in Kcat were observed, Table (5).
Kcat of the recent Fe(III) complexes was found to be high comparing with other complexes
indicating that the synthesized Fe(III) complexes will be an effective catalyst for OAP oxidation
reaction.
Table (5): A comparison of turnover number (Kcat) values of some complexes available in the
literature for the conversion of o-aminophenol to the corresponding phenoxazinone.
Kcat (h-1) Reference
Complex
[Cu(L)2](ClO4)2
1
2
360.00
209.53
196.18
171.82
157.58
138.62
137.00
64.07
56.00
54.00
33.26
33.12
32.36
30.09
27.32
26.32
23.04
22.39
21.40
17.00
13.75
13.68
11.48
9.66
[50]
This work
[41]
This work
This work
[51]
[52]
[51]
[53]
[54]
[55]
[55]
[41]
[56]
[57]
[58]
[56]
[59]
[50]
[50]
[60]
[61]
[49]
[58]
[58]
[61]
FeNQ
3 [FeLCl]2
4 FeNB
5 FeNI
6 [Mn(HL)2](ClO4)2
7 [FeCl2(L5)]
8 [Mn(HL)(N(CN)2)(H2O)2](ClO4)
9 [Fe(L1)Cl3]
10 [Co(L)(N3)2]
11 [Co(L2)(N3)3]
12 [Co(L3)(N3)3]
13 [{FeL(4,4′-byp)ClO4}]n
14 [Co2(L6)2(μ-O2)](ClO4)4·2CH3CN
15 [Mn(L2)Cl2]
16 [Mn(L4)Cl2] H2O
17 [Co(L2)2(μ ‐O2)](ClO4)4
18 [Mn(Br4Cat)(Br3pyCat)(py)2]
19 [Ni(L)2](ClO4)2
20 [Zn(L)2](ClO4)2
21 [Co2(amp)2(μ-imp)2Cl2]Cl22H2O
22 [Co(L1)Cl(H2O)]ClH2O
23 [LCo(L′)2]ClO4
24 [Mn(L2)Cl2]
25 [Mn(L3)Cl2]
8.20
7.38
6.37
4.10
26 [Co(L1)(NCS2)]
27 [Co(L4)(H2O)](ClO4)2
28 [Co(L2)(Cl)2]
[56]
[51]
[56]
29 [Co(L3)(CH3CN)](ClO4)2
3.36
30 [Mn(6′Me2indH)(H2O)2(CH3CN)](ClO4)2 2.93
[62]
3.6. Structure-activity relationship (SAR)

Biological or catalytic activity of molecules can be evaluated through experimental and
computational correlation[63,64,65]
.
The aim of the present SAR analysis was to correlate the experimental biological or
catalytic activity of the investigated compounds with their quantum chemical descriptors and find
the most effective description model. The multiple linear regression (MLR) was applied to develop
a best relationship between the dependent variable, Kcat or MIC, with independent variables
(chemical descriptors, EHOMO, ELUMO, ΔE, η, σ, µ, ω, and Nu).
The theoretical correlation formula was derived for phenoxazinone synthase mimicking activity
(equation (3)), E. coli (G-) (equation (4)), B. cereus (G+) (equation (5)), and A. fumigates (equation
(6)).
Kcat = - 86.63 – 165.30 EHOMO – 118.06 ω
(3)
MIC = -1092.78 –5661.18EHOMO –6048.33ELUMO +1702.78σ +11135.10µ -247.00ω +776.55Nu (4)
MIC = -997.92 –5861.27EHOMO –6262.79ELUMO +1610.21σ +11554.00µ -248.91ω +707.86Nu (5)
MIC = -824.71 –4310.36EHOMO –4549.71ELUMO +1461.73σ +8439.13µ -193.87ω +364.20Nu (6)
To investigation the validity of derived formula, predicted MIC and Kcat values were
compared with actual values, Tables (5 and 6.), which, showed excellent agreement.
Additionally, a cross validation squared coefficient, Q2 = 1 – (∑(Propertypred.
–
Propertyexp.)2/ ∑(Propertyexp. – Propertymean.)2), was calculated for evaluate the prediction power of
the proposed model. Where; Propertypred., Propertyexp., and Propertymean, are the predicted,
experimental and mean values of the target property, MIC and Kcat.
Q2 was found to be 0.99, 1.00, 1.00, and 1.00, for the investigated compounds against,
phenoxazinone synthase mimicking activity, E. coli (G-), B. cereus (G+), and A. fumigatus,
respectively, which indicated excellent prediction power of the proposed SAR model, Tables (4
and 5).
4. Conclusion
Three Fe(III) mixed-ligand complexes with a phenol-based Schiff base as the primary
ligand, and N-containing heterocyclic ring as the secondary one had been synthesized and
characterized. DFT calculations were performed to confirm the proposed geometry and estimate
the electronic structure properties. The results indicated that the complexes adapted a distorted
octahedral geometry. The in-vitro antimicrobial activity showed that the Fe(III) complexes
exhibited higher inhibition against selected tested microorganisms compared to the free ligands.
Furthermore, the complexes were found to be active towards phenoxazinone synthase mimicking

activity. The mimicking activity was assessed by following conventional Michaelis-Menten
enzymatic kinetics. Kinetic studies of the catalytic cycles had been performed in detail using a
variety of enzyme kinetics plots to evaluate different kinetic parameters, including the specificity
constant. In addition, SAR model showed an excellent correlation between the experimental
biological (MIC) and catalytic activity (Kcat) with the calculated quantum chemical descriptors.
References
Declaration of interests
☒ The authors declare that there is no Conflict of Interest of the results of the manuscript with any
other third party.
Declaration of interests
☒The authors declare that they have no known competing financialinterestsor personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as
potential competing interests:
Design and Synthesis of Three Fe(III) Mixed-ligand Complexes: Exploration of Their
Biological and Phenoxazinone Synthase Like Activities
Mohammed Ismael*, Abdel-Mawgoud M. Abdel-Mawgoud, Mostafa K. Rabia and Aly Abdou
Chemistry Department, Faculty of Science, Sohag University, Sohag 82524, Egypt

Graphical Abstract
Design and Synthesis of Three Fe(III) Mixed-ligand Complexes: Exploration of Their
Biological and Phenoxazinone Synthase Like Activities
Mohammed Ismael*, Abdel-Mawgoud M. Abdel-Mawgoud, Mostafa K. Rabia and Aly Abdou
Chemistry Department, Faculty of Science, Sohag University, Sohag 82524, Egypt
Highlights
• Three Fe(III) mixed-ligand complexes involving phenol-based Schiff-base ligand and N-
containing heterocyclic ring have been synthesized and characterized.
• DFT calculations were performed to provide deep understanding of the complexes properties.
• The complexes showed a promising antibacterial and antifungal activity

• Phenoxazinone synthase mimicking activity was explored.
• Structure activity relationship (SAR) for these complexes had been derived.
List of figures
Fig. (1): Powder XRD spectrum of (a) FeNQ, (b) FeNB, and (c) FeNI, complexes.
Fig. (2): Optimized structures of (a) FeNQ, (b) FeNB, and (c) FeNI complexes.
Fig. (3): Calculated HOMO and LUMO of (a) FeNQ, (b) FeNB, and (c) FeNI complexes.
Fig. (4): (a) Antimicrobial activity of the free ligands and their Fe(III) complexes in term of
inhibition zone diameter (IZ, mm) at 100 µg/ml, inhibition zone diameter of the Fe(III)
complexes against (b) E. coli (G-), (c) B. cereus (G-) and (d) Aspergillus fumigates.
Fig. (5): Time resolved UV-Vis spectral profiles indicating the increment of Phz peak upon
addition of 12  10-2 M OAP to 10-5 M of (a) FeNQ, (b) FeNB and (c) FeNI complexes.

80
70
60
50
40
30
FeNQ complex
10
20
30
40
50
60
70
80
2 Theta
a)
FeNB complex
100
90
80
70
60
50
40
10
20
30
40
50
60
70
80
2 Theta
(b)
130
120
110
100
90
FeNI complex
80
70
60
50
40
30
10
20
30
40
50
60
70
80
2 Theta
(c)
Fig. (1): Powder XRD spectrum of (a) FeNQ, (b) FeNB, and (c) FeNI, complexes.

(a). Trans-FeNQ complex
(b). Trans-FeNB complex
(c). Trans-FeNI complex
ETotal = - 951073.84 Kcal/mol
ETotal = - 1082200.64 Kcal/mol
ETotal = - 1275725.51 Kcal/mol
(d). Cis-FeNQ complex
(e). Cis-FeNB complex
(f). Cis-FeNI complex
ETotal = - 951081.60 Kcal/mol
ETotal = - 1082201.32 Kcal/mol
ETotal = - 1275730.288 Kcal/mol
Fig. (2): Optimized structures of (a) FeNQ, (b) FeNB, and (c) FeNI complexes.

(a). FeNQ complex
(b). FeNB complex
(c). FeNI complex
ELUMO = -2.425 eV
ELUMO = -2.486 eV
ELUMO = -2.510 eV
∆E = 2.831 eV
∆E = 3.027 eV
∆E = 2.911 eV
EHOMO = -5.317 eV
EHOMO = -5.421 eV
EHOMO = -5.452 eV
Fig. (3): Calculated HOMO and LUMO of (a) FeNQ, (b) FeNB, and (c) FeNI complexes.

(a)
(b)
(c)
(d)
Fig. (4): (a) Antimicrobial activity of the free ligands and their Fe(III) complexes in term of
inhibition zone diameter (IZ, mm) at 100 µg/ml, inhibition zone diameter of the Fe(III)
complexes against (b) E. coli (G-), (c) B. cereus (G-) and (d) Aspergillus fumigates.

(a). FeNQ complex
(b). FeNB complex
(c). FeNI complex
Fig. (5): Time resolved UV-Vis spectral profiles indicating the increment of Phz peak upon
addition of 12  10-2 M OAP to 10-5 M of (a) FeNQ, (b) FeNB and (c) FeNI complexes.

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