The solvent-free synthesis of polysubstituted pyrroles by a reusable copper Schiff base complex immobilized on silica coated Fe3O4, and DNA binding study of one resulting derivative as a potential anticancer drug

Article abstract of DOI:10.1002/aoc.4754

We demonstrate herein the synthesis of a new copper Schiff base complex immobilized on silica-coated Fe₃O₄ nanoparticles. The structure and composition of this magnetic nanocatalyst were analyzed using Fourier transform infrared (FT-IR), X-ray powder diffraction (XRD), vibrating sample magnetometry (VSM), scanning electron microscopy (SEM), energy dispersive X-ray (EDX) and inductively coupled plasma atomic emission spectroscopy (ICP-AES). This nanocomposite was found to be an efficient nanocatalyst for the synthesis of polysubstituted pyrrole derivatives and the products were isolated with high turnover number (TON) and high to excellent yields. Among the new synthesized polysubstituted pyrrole derivatives, we explored the first computational and experimental binding study of methyl 1-benzyl-4-(furan-2-yl)-2-methyl-1H-pyrrole-3-carboxylate (SP-10) with calf thymus deoxyribonucleic acid (ct-DNA), suggesting their application as potential anticancer activity. In addition, the binding modes of SP-10 with DNA and human serum albumin (HSA) were verified by molecular docking technique.

Full text of DOI:10.1002/aoc.4754

Received: 29 August 2018
DOI: 10.1002/aoc.4754
Revised: 1 November 2018
Accepted: 14 November 2018
F U L L P A P E R
The solvent‐free synthesis of polysubstituted pyrroles by a
reusable copper Schiff base complex immobilized on silica
coated Fe3O4, and DNA binding study of one resulting
derivative as a potential anticancer drug
Jamshid Rakhtshah1 | Behrooz Shaabani1
Neda Hosseinpour Moghadam2
| Sadegh Salehzadeh2
|
1 Department of Inorganic Chemistry,
We demonstrate herein the synthesis of a new copper Schiff base complex
immobilized on silica‐coated Fe3O4 nanoparticles. The structure and composi-
tion of this magnetic nanocatalyst were analyzed using Fourier transform
infrared (FT‐IR), X‐ray powder diffraction (XRD), vibrating sample magnetom-
etry (VSM), scanning electron microscopy (SEM), energy dispersive X‐ray
(EDX) and inductively coupled plasma atomic emission spectroscopy (ICP‐
AES). This nanocomposite was found to be an efficient nanocatalyst for the
synthesis of polysubstituted pyrrole derivatives and the products were isolated
with high turnover number (TON) and high to excellent yields. Among the
new synthesized polysubstituted pyrrole derivatives, we explored the first com-
putational and experimental binding study of methyl 1‐benzyl‐4‐(furan‐2‐yl)‐2‐
methyl‐1H‐pyrrole‐3‐carboxylate (SP‐10) with calf thymus deoxyribonucleic
acid (ct‐DNA), suggesting their application as potential anticancer activity. In
addition, the binding modes of SP‐10 with DNA and human serum albumin
(HSA) were verified by molecular docking technique.
Faculty of Chemistry, Tabriz University,
Tabriz, Iran
2 Faculty of Chemistry, Bu‐Ali Sina
University, Hamedan 6517838683, Iran
Correspondence
Behrooz Shaabani, Department of
Inorganic Chemistry, Faculty of
Chemistry, Tabriz University, Tabriz, Iran.
Email: shaabani.b@gmail.com
KEYWORDS
DNA binding, immobilized complex, molecular docking, multicomponent reaction, Polysubstituted
pyrroles
1 | INTRODUCTION
The goal of scientists has lately shifted to alleviate envi-
ronmental issues; thus, catalyst recovery and reusability
Transition metal Schiff base complexes have shown
high catalytic activities because of the central metal
ions in these complexes which easily lend and take
electrons from other compounds. The interest in the
Schiff base complexes has grown over the past
decades.[1–3] Both homogeneous and heterogeneous
catalysts of Schiff base complex are important and pow-
erful in the oxidation of organic compounds by various
oxygen atom donors.[4–7]
have become important aspects of catalytic systems that
embrace the principles of green chemistry.[8,9] The major
disadvantages of homogeneous catalysts are the difficult
separation and regeneration of the expensive catalyst from
the catalyst‐product mixture at the end of the process.
Therefore, to overcome this problem, heterogenization of
homogeneous catalysts may combine the ease of catalyst
separation with the selectivity of the homogeneous coun-
terparts. Immobilization of the soluble catalyst onto
Appl Organometal Chem. 2018;e4754.
wileyonlinelibrary.com/journal/aoc
© 2018 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.4754

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RAKHTSHAH ET AL.
various insoluble supports, especially porous materials
with high surface areas through the formation of a cova-
lent bond, has been adopted because of easy separation,
easy recyclability, thermal stability and long catalytic life-
time.[10–14] Recently, iron oxide magnetic nanoparticles
have been found out to be the kind of novel functional
materials which are abundant, cheap, environmental
friendly and non‐toxic.[9] In contrast, immobilized cata-
lysts on Fe3O4 can be magnetically recoverable from the
reaction mixture by an external magnet. Synthesis, charac-
terization and surface modification of nanoparticles with
desired properties have attracted growing attention in
recent researches.[10–18]
pyrroles at room temperature under solvent free condition
has been described (Scheme 1). Also, due to the diverse
pharmaceutical properties of the compounds synthesized
here, the DNA binding affinity of methyl 1‐benzyl‐4‐
(furan‐2‐yl)‐2‐methyl‐1H‐pyrrole‐3‐carboxylate (SP‐10) as
a new derivative was investigated by using molecular
docking simulations as well as UV–vis spectroscopy and
viscometry method.
2 | EXPERIMENTAL
2.1 | Materials and characterization
Pyrroles and their derivatives are extensively found
as general core units for various biologically and phar-
maceutically active compounds in numerous natural
products.[19] There are a number of approaches described
in the literature for the synthesis of polysubstituted
pyrroles. The preparation of pyrroles can be carried out
by four component condensation of aldehydes, amines,
ethyl acetoacetate and nitromethane in the presence of
various catalysts such as FeCl3,[20] gluconic acid,[21] nano
copper oxide,[22] nickel ferrite nanoparticles,[23] β‐
Cyclodextrin,[24] nano‐CoFe2O4 supported molybde-
num,[25] NiCl2,[26] heterogenized tungsten complex,[27]
ionic liquid[28] and copper complex immobilized on
Fe3O4.[29] However, some protocols suffer from certain
drawbacks such as high reaction temperature, low yields,
prolonged reaction time, tedious workup procedure, low
recovery and reusability of the catalyst.
Because of the reasons described above and also as a
part of our ongoing research program on the synthesis
and characterization of immobilized Schiff base complexes
catalysts and their application in multicomponent reac-
tions,[30–33] here, we report an efficient procedure for syn-
thesizing a recoverable and reusable copper Schiff base
complex immobilized through a covalent bond linkage
on silica‐coated magnetic nanoparticles. The successful
application of the aforementioned catalyst in the four
component reactions for the synthesis of polysubstituted
techniques
All chemicals were purchased from Merck, Fluka or
Aldrich and used without any further purification. The
particle size and morphology were investigated by SEM
using FESEM‐TESCAN MIRA3. FT‐IR measurements
were performed using the KBr disc on a Perkin–Elmer
FT‐IR spectrometer 17259. The XRD patterns of samples
were recorded on a D8 Advanced diffractometer (Bruker
AXS Inc., 40 kV, 30 mA) X‐ray diffractometer. Scans were
taken with a 2θ and step size of 0.02 from 20–80° and a
counting time of 1.0 s using a Cu Kα radiation source
(λ = 1.542 Å) to assess the crystallinity. VSM measure-
ment was recorded by a magnetometer (VSM‐4 inch;
Daghigh Meghnatis Kashan, Kashan, Iran) at room tem-
perature. The copper content of the catalyst was deter-
mined using an ICP‐AES instrument (HORIBA Jobin
Yvon, Longjumeau Cedex, France). UV–vis spectroscopy
was conducted by UV Win T80 spectrophotometer.
The procedure for synthesis of copper Schiff base
complex supported on Fe3O4 nanoparticles includes four
steps, schematically illustrated in Scheme 2.
2.2 | General procedure for the synthesis
of copper Schiff base complex (CuSB)
2‐Formylpyridine (0.5 g) was added to a magnetically stir-
ring solution of 4‐aminophenol (0.5 g) in ethanol (40 ml).
After refluxing for 3 hr, the mixture was cooled to room
temperature. The resulting crystals were separated by
filtration, washed with ethanol and dried under vacuum.
Then, an ethanol (10 ml) solution of copper acetate
(1.81 g, 10 mmol) was added dropwise to an ethanol
solution (10 ml) of prepared Schiff base ligand (1.98 g,
10 mmol) and the mixture was refluxed for 2 hr. The
resulting crystalline solid was separated and filtered off,
then washed with ethanol (3 × 5 ml) to give copper Schiff
base complex (Yield: 85%).
SCHEME 1 The synthesis of pyrrole derivatives using copper
complex supported on functionalized Fe3O4 magnetite
nanoparticles

RAKHTSHAH ET AL.
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SCHEME 2 The sequence of events in
the preparation of Fe3O4@SiO2@CuSB
and washed with ethanol and also distilled water for
three times. The solid was dried at 60 °C in oven
for 12 hr.
2.3 | Synthesis of immobilized copper
Schiff base complex on magnetic iron oxide
nanoparticles (Fe3O4@SiO2@CuSB)
Firstly, preparation of naked Fe3O4 was carried out
according to the previously reported method.[34] A mix-
ture of FeCl3.6H2O (2 mmol, 7.8 g) and FeCl2.4H2O
(1 mmol, 2.9 g) was dissolved in 110 ml deionized water
at 80 °C, then after stirring for 10 min, a 25% NH4OH
solution (90 ml) was added quickly to the resulting solu-
tion in one portion with vigorous mechanical stirring.
After cooling to room temperature, the black precipitates
were collected using an external magnet. The magnetic
nanoparticles were washed with deionized water and a
solution of NaCl (10%wt) through magnetic decantation.
Secondly, silica coated Fe3O4 nanoparticles (Fe3O4@SiO2)
were prepared by Stober method[35] with slight modifica-
tion. The obtained magnetic nanoparticles (MNPs) (1 g)
were dispersed in 220 ml ethanol/water (volume ratio,
10:1) solution by sonication for 10 min, and then 4 ml
of tetraethylorthosilicate (TEOS) was added dropwise
to the mixture. After mechanical stirring in 60 °C
under N2 atmosphere for 6 hr, the silica coated nanopar-
ticles were collected using a magnet and washed with
ethanol to remove remnant of compound. Thirdly,
Fe3O4@SiO2 (1 g) was added to the solution of 3‐
chloropropyltrimethoxysilane (CPTMS) (2 ml) in ethanol
(50 ml) and the resultant mixture was kept at 80 °C under
reflux and N2 atmosphere for 12 hr. Practically, the etha-
nol solution deleted the excess amount of Si‐linker,
providing a better and faster decantation of suspended
Fe3O4 nanoparticles in ethanol, when an external magnet
was used. Finally, chloropropyl coated nanoparticles
(0.5 g), triethylamine and synthesized copper Schiff base
complex (0.5 g) were added to ethanol (50 ml).
The resulting mixture was stirred for 3 hr and then the
precipitates were collected using an external magnet
2.4 | General procedure for the synthesis
of polysubstituted pyrrole derivatives (SP)
To a 50 ml round‐bottomed flask reactor were added
various amines (1 mmol), different aldehydes (1 mmol),
ethyl acetoacetate (1 mmol), nitromethane (1 mmol)
and Fe3O4@SiO2@CuSB as catalyst (6 mg) sequentially.
The suspension was stirred vigorously at room tempera-
ture followed by an appropriate time of stirring in
solvent‐free condition. The progress and completion of the
reaction was monitored by thin layer chromatography
(TLC) (n‐hexane/ethyl acetate (5:3)). After the reaction
completion, the mixture was diluted with dichloromethane
(3 ml) and stirred at high temperature. Finally, the catalyst
was separated by magnetic decantation from resulting mix-
ture. After evaporation of the solvent, the crude product
was recrystallized with ethanol to give a pure product. All
1
new compounds were characterized by IR, H and 13C
NMR spectroscopy (supporting information).
2.5 | Molecular docking
The structure of methyl 1‐benzyl‐4‐(furan‐2‐yl)‐2‐methyl‐
1H‐pyrrole‐3‐carboxylate (SP‐10) was optimized at DFT
(BP86) level of theory.[36,37] The def2‐TZVP[38] basis set
was employed for all atoms, and the structure was opti-
mized without symmetry restrictions. All calculations
were performed using the Gaussian 03 set of programs.[39]
The optimized structure of SP‐10 was used for the molec-
ular docking calculations (Figure 1). The crystal struc-
tures of all the pdb DNA and HSA used in molecular

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RAKHTSHAH ET AL.
2.6 | UV–visible absorption spectral
measurements
The concentration of drug candidate was fixed at
5 × 10−5 M and the ct‐DNA concentration varied from 0
to 9.6 × 10−5 M. Control samples consisted of only ct‐
DNA solution. The spectra were recorded in the range
of 220–440 nm at room temperature.
2.7 | Viscosity measurements
FIGURE 1 The optimized structure of methyl 1‐benzyl‐4‐(furan‐
2‐yl)‐2‐methyl‐1H‐pyrrole‐3‐carboxylate (SP‐10)
Viscometric measurements were carried out using a
viscometer which was kept at 25 0.2 °C by a constant
temperature bath. The solution of (DNA + SP‐10)
was prepared in Tris–HCl buffer and the flow times of
ct‐DNA alone and its mixtures with different ratios of
SP‐10 to ct‐DNA through the capillary were then
measured in three replicates using a digital stopwatch
with an accuracy of 0.02 s. The data were presented
as (η/η0)1/3 versus the ratio of the concentration of
compound to that of DNA, where η and η0 are the
viscosity of DNA in the presence and absence of our
derivative, respectively.
docking were extracted from Protein Data Bank (http://
www.rcsb.org/pdb/home/home.do) and listed in Table 1.
The binding interactions of SP‐10 with all of the pdb
DNA structures and HSA were simulated by molecular
docking method using AutoDock 4.0 program.[40] All of
the water molecules were removed from all pdb struc-
tures. The polar hydrogen atoms were added to all of
the pdb structures. The grid maps of dimensions
60 × 60 × 60 A° and 126 × 126 × 126 A° with a grid‐point
spacing of 0.375 A° were created respectively for five DNA
structures and HSA to ensure an appropriate size for X‐
accessible space. Also the values of the centers of grid
boxes are shown in Table 1. Other miscellaneous param-
eters were assigned to the default values given by the
Autodock program. Finally, we obtained the dominating
configuration of the binding complex of SP‐10 with five
DNA structures and HSA with minimum binding free
energy (ΔG).
2.8 | Circular dichroism (CD) studies
The CD measurements were recorded on a JASCO (J‐810)
spectropolarimeter, using a 1.0 cm quartz cell by keeping
the concentration of DNA at 8 × 10−5 M and various con-
centrations of SP‐10.
TABLE 1 Five DNA sequences and HSA used for molecular dockinga
ID
PDB ID
Sequences
Unit Cell constants
Centers of grid boxes
1
1BNA
D (CGCGAATTCGCG)2
a = 24.87 Å, b = 40.39 Å, c = 66.20 Å
14.719, 20.979, 8.824
α = 90 °, β = 90 °, γ = 90 °
2
3
1D32
D (CGCG)2
a = 16.88 Å, b = 26.88 Å, c = 82.60 Å
α = 90 °, β = 90 °, γ = 90 °
26.699, 13.011, 10.343
15.259, 21.297, 75.998
1DNE
D (CGCGATATCGCG)2
a = 25.48 Å, b = 41.26 Å, c = 66.88 Å
α = 90 °, β = 90 °, γ = 90 °
4
5
1K2J
D (CGTACG)2
D (CGCG)2
No data
0.218, 1.887, 7.463
1ZNA
a = 31.27 Å, b = 64.67 Å, c = 19.50 Å
18.079, −8.217, 8.853
α = 90 °, β = 90 °, γ = 90 °
6
1AO6
HSA
a = 59.68 Å, b = 96.98 Å, c = 59.72 Å
29.535, 31.826, 23.5
α = 91.07 °, β = 103.50 °, γ = 75.08 °
aThese data were extracted from Protein Data Bank (http://www.rcsb.org/pdb/home/home.do).

RAKHTSHAH ET AL.
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obtained materials had similar diffraction peaks at
2θ = 30, 35, 43, 53, 62 and 74°, which could be indexed
to an inverse spinel ferrite of Fe3O4 (JCPDS file number
89–3854).[43] Eight peaks in XRD patterns correspond to
Miller indices value (hkl) of (220), (311), (400), (422),
(511) and (440), respectively.[32] In addition, no specific
peaks due to any impurities were observed and the
copper species is highly dispersed on nanoparticles.
Figure 3b presents almost the same feature as the one
shown in Figure 3a, which is attributed to the fact that
surface modification of the nanoparticles does not
lead to their phase change. According to the Scherrer
equation[44] based on the full width at half‐maximum
(FWHM) of main diffraction peak, the mean crystallite
size of synthesized nanocrystals is estimated to be about
30 nm. Also, the XRD pattern of reused catalyst
(Figure 3c) shows that the Fe3O4 phase has not been
changed during the catalytic reaction.
In order to investigate the morphology of the
Fe3O4@SiO2@CuSB, SEM images of the magnetic nano-
particles were provided and the images are presented in
Figure 4. As it is clear, the copper complex immobilized
on Fe3O4 exhibits a cluster of aggregated spherical
particles. The EDS pattern of elements in nanomaterial
confirms the presence of Fe, O, Si, N, C, Cl and Cu
species in the structure of catalyst.
The superparamagnetic property of Fe3O4 and
Fe3O4@SiO2@CuSB was investigated by vibrating sample
magnetometer (VSM) at room temperature. As shown
from the loops in Figure 5, the saturation magnetization
(MS) of naked magnetic nanoparticles and immobilized
complex on magnetic nanoparticles are 48.85 and
40.27 emu g−1, respectively. The decrease in the magneti-
zation value of the composite in comparison with Fe3O4
NPs is due to the immobilizing of silica layer and Schiff
base complex of copper on Fe3O4 nanoparticles through
their bond formation via their hydroxyl functional
groups.[45] Nonetheless, the synthesized catalysts in this
study had a sufficient superparamagnetic behavior and
3 | RESULTS AND DISCUSSION
3.1 | Characterization of copper Schiff
base complex immobilized on silica‐coated
iron oxide nanoparticles as a
heterogeneous catalyst
All the evidence from Fourier transform infrared (FT‐IR)
spectroscopy, X‐ray diffraction (XRD), field emission
scanning electron microscopy (FE‐SEM), energy‐
dispersive X‐ray spectroscopy (EDS), inductively coupled
plasma (ICP) and vibrating sample magnetometry
(VSM) analyses support the successful grafting of copper
Schiff base complex onto the surface of silica coated mag-
netic nanoparticles.
FT‐IR spectra of the Fe3O4, Fe3O4@SiO2,
Fe3O4@SiO2@Cl, Fe3O4@SiO2@CuSB and Cu Schiff base
complex are shown in Figures 2a, 2b, 2c, 2d and 2e, respec-
tively. The strong absorption signal observed at 570 cm−1
is attributed to the Fe‐O bond stretching vibration. The
broad band at 3000–3500 cm−1 is referred to the absorp-
tion of –OH stretching of surface hydroxyl groups.[41]
The introduction of silica layer to the surface of naked
Fe3O4 is confirmed by the sharp band at 1078 cm−1
assigned to the Si‐O‐Si anti‐symmetric stretching vibra-
tion.[42] The synthesized Fe3O4@SiO2@Cl possesses the
main peak at 2840 to 2918 cm−1 which indicates the pres-
ence of the anchored propyl group of CPTMS on magnetic
NPs by C–H stretching vibrations.[41] Reaction of CPTMS
coated nanoparticles with copper Schiff base produces
Fe3O4@SiO2@CuSB in which the presence of imine
stretching band is asserted with 1628 cm−1 bands in
Figure 2(d) spectra. Also, in IR spectra of neat copper
Schiff base, the vibrational stretching frequency of C=N
bond appears at 1628 cm−1.[30]
The structure and phase purity of the Fe3O4 (3a) and
Fe3O4@SiO2@CuSB (3b) were examined by XRD. The
FIGURE
2
FT‐IR spectra of Fe3O4 (a), Fe3O4@SiO2 (b),
Fe3O4@SiO2@Cl (c), Fe3O4@SiO2@CuSB (d), neat copper Schiff
base complex (e)
FIGURE
3
Powder
XRD
patterns
of
Fe3O4
(a),
Fe3O4@SiO2@CuSB (b) and reused Fe3O4@SiO2@CuSB

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RAKHTSHAH ET AL.
FIGURE 4 FE‐SEM micrograph with the scale bar of 500 nm and EDX spectrum of Fe3O4@SiO2@CuSB
thus, they still could be readily separated from reaction
mixture with the aid of an external magnet.
In the absence of nanocatalyst, the reaction did not pro-
ceed with good yield even after a long reaction time
(Table 2, entries 1–2). It was found that at a higher
amount, the catalyst performed in shorter reaction times
(Table 2, entries 1–5). By screening different amounts of
the catalyst (2, 4, 6, 8 mg) at room temperature, pyrroles
were afforded with 45%, 75%, 94%, and 94% isolated
yields, respectively. Therefore, the best yield is observed
Also, in order to determine the amount of copper
content of the catalyst, ICP analysis was performed and
it was found to be 0.34 mmol g−1
.
3.2 | Application of copper Schiff base
complex immobilized on silica coated iron
oxide nanoparticles as a heterogeneous
catalyst
TABLE 2 The effect of amount of the catalyst and temperature
on the four‐component reaction under solvent‐free conditionsa
Polysubstituted pyrroles have widespread uses due to
their significant biological activity. Thus, our studies were
directed towards the possibility of applying magnetic
nanoparticles to the design of an efficient, recyclable
and magnetically recoverable heterogeneous catalyst for
the synthesis of polysubstituted pyrroles under mild reac-
tion conditions. For initial optimization of the reaction
conditions and the identification of the best amount of
the catalyst at different temperatures, aniline, benzalde-
hyde, ethyl acetoacetate and nitromethane were chosen
as model substrates. In order to show the role of the cat-
alyst, similar reactions in the absence and also in the
presence of different amounts of catalyst were examined.
Reaction
temperature
(°C)b
Amount of
Entry catalyst (mg)
Reaction
time (min)
Yieldc
(%)
1
—
—
2
r.t.
100
r.t.
100
r.t.
100
r.t.
50
45
45
45
45
45
45
20
20
20
20
20
20
30
30
45
50
75
77
94
94
94
94
94
94
2
3
4
2
5
4
6
4
7
6
8
6
9
6
75
10
11
12
6
100
r.t.
100
8
8
aReaction conditions: benzaldehyde (1 mmol), aniline (1 mmol), ethyl
acetoacetate (1 mmol) and nitromethane (1 mmol).
bThis is the temperature of oil bath and not inside the reaction medium.
cIsolated yield.
FIGURE
5
Magnetic hysteresis curves measured at room
temperature for Fe3O4 (a) and Fe3O4@SiO2@CuSB (b)

RAKHTSHAH ET AL.
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TABLE 3 Solvent effect on the four‐component reactiona at room temperature using 6 mg catalyst
Solvent‐free
H2O
C2H5OH
CH3CN
CH2Cl2
Toluene
n‐Hexane
Yield (%)b
94
20
35
40
70
45
60
40
50
35
30
50
35
60
Reaction time (min)
aBenzaldehyde (1 mmol), aniline (1 mmol), ethyl acetoacetate (1 mmol) and nitromethane (1 mmol) under room temperature;
bIsolated yield.
in the presence of just 6 mg nanocatalyst, and the higher
amounts of catalyst than this does not improve the yield
(Table 2, entries 11–12). We next investigated the effect
of different temperatures (25, 50, 75 and 100 °C) on the
reaction rate as well as the yields of products. The best
results were obtained at room temperature and higher
temperatures did not improve the yield or reaction rate
in the same amount of catalyst (Table 2, entries 7–10).
Then, the effect of various solvents and solvent‐free
conditions on the model reaction was screened. As shown
in Table 3., we found that using solvent‐free condition,
compared with other solvents, in the presence of magne-
tite nanoparticles, the desired product with high yield
and shorter reaction time will be obtained.
comparison of their spectroscopic data with those
reported in the literature.
To compare the catalytic activity of the new catalyst
with previously known ones, we evaluated the catalytic
activity of these catalysts in the model reaction under
different conditions. Table 5 shows that our catalyst has
efficiently catalyzed the model reaction to give the
desired product in excellent yield and relatively shorter
reaction time among all catalysts.
For investigations on the recyclability of heteroge-
neous system, the lifetime of the catalyst was studied
(Figure 6). After the first use of nanocatalyst in the model
reaction to give the desired product in quantitative yield,
ethyl acetate was added to the reaction mixture and
stirred and heated to separate the product. More than
99% of the catalyst could simply be recovered by using a
magnet near to the reaction medium, and the catalyst
was magnetically separated and washed thoroughly with
ethanol. A new reaction was then performed with fresh
reactants under the same conditions. Surprisingly, the
recovered catalyst at each reaction was reused without
significant deactivation even after ten times reuse of
catalyst. We believe that this is also the possible reason
for the high reusability of the catalyst presented herein.
The amount of leached copper in the final product was
measured by the ICP technique. No Cu metal was
detected after completion of the reaction which confirms
the fact that Schiff base provides enough binding sites on
the surface of silica coated magnetic nanoparticles and
serves as a preventing metal leaching, and enabling effi-
cient catalyst recycling.
To survey the efficiency of the procedure, we studied
the catalytic performance of the immobilized copper
Schiff base complex on magnetic nanoparticles in one‐
pot four‐component condensation of different amines,
various aromatic aldehydes, ethyl acetoacetate and
nitromethane for the preparation of corresponding
polysubstituted pyrrole derivatives. From the experi-
ments mentioned above, the optimum condition (6 mg
of the catalyst under solvent‐free conditions at room
temperature) was chosen. The reaction can tolerate a
wide range of aromatic aldehydes bearing halogens, het-
erocyclic, electron‐donating and electron withdrawing
substituents in the ortho, meta and para positions. Rep-
resentative results are summarized in Table 4. All of
these reactions proceed rapidly and they are completed
in the range of 20–40 min. Aldehydes with electron‐
donating groups are less active than those with
electron‐withdrawing groups in the para‐positions. All
products were prepared in the short reaction time and
in high to excellent yields. Furthermore, we calculated
turnover numbers (TONs) and turnover frequencies
(TOFs) (according to the ICP results, 6 mg of catalyst is
equivalent to 0.204 mol% of catalyst) states on the
effectiveness of these catalysts. The results are summa-
rized in Table 4. The new products were characterized
3.3 | Molecular docking study of SP‐10 as a
potential anticancer activity
Computational molecular docking is a valuable method
for predicting the stable structure of receptor‐ligand com-
plex for better diagnosis of the interaction details in the
drug discovery process.[46,47] This technique is often used
1
by IR, H NMR and 13CNMR spectroscopy, and also by

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TABLE 5 Comparison of the Fe3O4@SiO2@CuSB catalytic activity with those of reported catalystsa
Catalyst
Solv.
Temp (°C)
Time (min)
Yieldb (%)
R1
R2
[Ref.]
[19]
Amberlyst‐15
‐
‐
‐
‐
‐
‐
‐
r.t.
30
80
80
78
85
78
96
90
94
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
[20]
[22]
[27]
[26]
[23]
[29]
FeCl3
Reflux
100–105
Reflux
r.t.
420
720
270
480
240
25
Nano Copper oxide
Heterogenized Tungsten Complex
Nickel (II) chloride hexahydrate
Nickel ferrite nanoparticles
Fe3O4@SiO2@SBCu
Fe3O4@SiO2@CuSB
100
r.t.
r.t.
20
‐
aModel reaction: benzaldehyde, aniline, ethyl acetoacetate and nitromethane;
bIsolated yield.
as a virtual searching tool in the early stages of drug
design and development. In the present work, in order
to find out the preferred location of SP‐10 on DNA and
HSA, molecular docking studies were carried out using
Autodock 4.0. Furthermore, to determine the preferential
binding sites of SP‐10 on different types of DNA, the SP‐
10 was docked into various types of rigid DNA and the
results were listed in Table 5 (Figure 7.). The values of
electrostatic forces between SP‐10 and DNAs is much
lower than the sum of van der Waals energy, hydrogen
bonding energy and desolvation free energy in the bind-
ing process, indicating that the main binding mode
between SP‐10 and DNAs is not electrostatic.
In order to obtain a deep insight into the SP‐10 bind-
ing mode with HSA, computational molecular docking
has been employed and the possible conformations of
the protein‐(SP‐10) complex were calculated. Among the
possible conformers, the conformer with the lowest bind-
ing free energy was used for further analyses. From the
crystal structure of human serum albumin (HSA), it can
be concluded that HSA is a heart‐shaped protein
consisting of a single polypeptide chain of 585 amino acid
residues.[48] Each of the structurally similar α‐helix
domains (I–III) has two subdomains (A and B), with six
α‐helices in subdomain
A and four α‐helices in
subdomain B.[49] Figure. 8 shows the most possible bind-
ing mode between SP‐10 and HSA and clearly indicates
TABLE 6 The various energies and the hydrogen bonding interactions in the formation process of DNAs‐(SP‐10) and HSA‐(SP‐10)
complexes
ΔG0
kcal
mol−1
ΔE1(b)
kcal
mol−1
ΔE2(c)
kcal
mol−1
Hydrogen bonding
Met
(a)
PDB
ID
DNA
Bond length (Å)
Binding site
Major groove
Intercalation
Minor groove
Major groove
1BNA
1D32
1DNE
1K2J
−6.55
−6.74
−7.75
−6.29
−8.04
−8.23
−9.24
−7.78
+0.02
−0.11
−0.09
−0.04
No hydrogen bonds formed
No hydrogen bonds formed
No hydrogen bonds formed
G6:H22 (Chain A)
G8:H3 (Chain B)
O2
C5 = O1
1.914
1.701
1ZNA
104D
1AO6
−6.65
−6.04
−7.94
−8.14
−7.53
−9.43
−0.23
+0.02
−0.12
G2:H22 (Chain A)
O2
O2
O2
2.223
2.034
1.828
Major groove
Major groove
IIA
C15:H42 (Chain B)
VAL482(NH) (Chain A)
(a) ΔG0 is the binding free energy changes in the binding process. (b) ΔE1 denotes intermolecular interaction energy, which is a sum of van der Waals energy,
hydrogen bonding energy, desolvation free energy and electrostatic energy. (c) ΔE2 is the electrostatic energy

12 of 15
RAKHTSHAH ET AL.
FIGURE
6
Reusability of Fe3O4@SiO2@CuSB as
a
heterogeneous catalyst
FIGURE 9 UV spectra of SP‐10 (1 × 10−5 M) in the presence of
increasing amounts of ct‐DNA. The arrow shows the changes
½DNAꢀ
upon increasing amounts of ct‐DNA. Inset in Fig: plot of
vs.[DNA]
ðεA − εf Þ
3.4 | DNA‐binding studies of SP‐10 as a
potential anticancer activity
Recently investigation on the anti‐cancer properties of
substances has been widely considered. Therefore, study-
ing the mechanism of interaction of substances with
DNA is very important.[51,52] In this section, to gain a
better understanding of the interaction between new
polysubstituted pyrroles and DNA, we used UV–Visible
spectroscopy, circular dichroism spectroscopy and viscos-
ity measurements. The investigation on the interaction of
a new compound with DNA is very important for the
design of new drugs.[53,54] Generally, the mechanism of
interactions between compounds and DNA includes a
series of interactions, such as hydrogen bonding, van
der Waals interactions, π‐stacking interactions of aro-
matic heterocyclic groups between base pairs and electro-
static interactions.[55,56] The most common and effective
technique used to study the interaction between DNA
and compounds is the UV–Vis absorption spectroscopy.
FIGURE 7 Molecular docking of the interaction of 1D32 as a
type of DNA with SP‐10
that SP‐10 is located in the site I subdomain IIA, and was
stabilized by the formation of a hydrogen bond with Val
482 in the system. Moreover, SP‐10 was in the locality
of some hydrophobic residues of HSA (site I subdomain
IB),[50] which suggested the existence of hydrophobic
interaction between it and HSA. This means that hydro-
gen bonding and hydrophobic interactions play an impor-
tant role in the stability of HSA‐(SP‐10) complex.
FIGURE 8 Molecular docking of the interaction of HSA with SP‐10

RAKHTSHAH ET AL.
13 of 15
The existence and the possible mode of interaction
between compounds and DNA will be determined by
the changes in the absorption intensity and the position
of the spectrum bands of the compounds in UV–Vis
absorption spectroscopy.[55,57] Figure
9 shows the
UV–Vis absorption spectra of SP‐10 (5 × 10−5 M) in the
absence and presence of different concentrations of
ct‐DNA (0.26–2.3 × 10−5 M). The band centered at
241 nm exhibits a hypochromism with a moderate red
shift. These results indicate that SP‐10 molecules bind to
ct‐DNA by intercalating of their aromatic chromophore
between DNA base pairs.[58,59] The binding constant of
the SP‐10 with ct‐DNA, Kb, is calculated by the ratio of
½DNAꢀ
FIGURE 11 CD‐spectra of the ct‐DNA in the presence of
different concentrations of SP‐10. Conditions: c
(DNA) = 8 × 10−5 M; c (SP‐10) (1 × 10−5 M): 0.0, 0.9, 1.9, 2.9 and
pH = 7.4
slope to the y intercept in plots
versus [DNA]
ðεA − εf Þ
(Insets in Figure 9), according to the equation[60]
:
the insertion of aromatic chromophore of SP‐10 between
DNA base pairs.
½DNAꢀ
½DNAꢀ
1
¼
þ
ðεA − εf Þ ðεb − εf Þ Kbðεb − εf Þ
In order to further study, the interaction mode of SP‐10
with DNA and change of CD spectra of DNA in the present
of SP‐10 was investigated. Generally, CD spectroscopy is a
useful technique in diagnosing changes in DNA morphol-
ogy during drug–DNA interactions. The CD spectrum of
ct‐DNA exhibits a negative band at 245 nm due to the
right‐handed helicity of B‐DNA form and a positive band
at 275 nm due to base stacking which are quite sensitive
to the mode of binding between small molecules and
DNA.[63] The results of CD studies indicated by addition
of SP‐10 to a solution containing ct‐DNA, CD spectra of
ct‐DNA in the UV region decrease in the positive and
negative bands due to a transition from a more B‐like to
a more A‐like structure (Figure 11).[64]
where [DNA] is the concentration of DNA in base pairs,
εA = Aobsd/[SP‐10], εf = the extinction coefficient for the
free SP‐10 and εb = the extinction coefficient for the SP‐
10 in the fully bound form. The Kb value for interaction
between SP‐10 and ct‐DNA was 3 × 104 M−1
.
The viscosity measurement study was carried out to
achieve further support for the binding mode of SP‐10
with ct‐DNA. In general, the classic intercalation of dif-
ferent intercalators between the DNA base pairs causes
an increase in DNA length and subsequently increases
DNA viscosity.[61,62] Within this context, the viscosity of
a ct‐DNA solution was monitored in the presence of
different concentration of SP‐10 and the results show a
considerable increase in the viscosity of ct‐DNA upon
addition of the SP‐10 (Figure 10). Such a behavior may
reveal the existence of intercalation binding mode
between DNA and the SP‐10 which may take place via
4 | CONCLUSION
In summary, an efficient, recoverable and reusable cop-
per Schiff base complex immobilized on magnetic iron
oxide nanoparticles was synthesized and the structural,
surface, morphological and magnetic properties of
resulting nanoparticles were evaluated. Covalent
functionalization of complex onto the magnetic nanopar-
ticles is successfully achieved by a multiple synthetic
procedure. The catalyst also pairs the advantages of
heterogeneous and homogeneous based systems, which
makes it a promising material for catalytic reactions.
Also, we have shown that our catalyst is an efficient,
stable and strongly active nanocatalyst in one‐pot
four‐component coupling reaction in the synthesis of
pyrrole derivatives. The important features of this method
include the use of cheap reagents, high yields, short
reaction time, operational simplicity, and high efficiency
FIGURE 10 Effect of increasing concentration of SP‐10 on the
relative viscosity of ct‐DNA at 25 °C

14 of 15
RAKHTSHAH ET AL.
[14] B. Qi, X. H. Lu, S. Y. Fang, J. Lei, Y. L. Dong, D. Zhou, Q. H.
of the catalyst under mild conditions such as solvent free
medium and room temperature. In all the reactions, turn-
over numbers (TON) ranging from 392 to 460 were
observed. The catalyst was easily separated by a magnet
and the recovered catalyst was reused for at least 10
reaction cycles without any significant loss of activity.
We have also studied the binding of ct‐DNA with the
one of synthesized compounds here (SP‐10). The different
instrumental methods (absorption and viscosity measure-
ments) suggested that the SP‐10 binds to DNA via interca-
lation binding mode and also CD spectroscopy study
inducing conformation structural changes of DNA duplex
in present of SP‐10. In addition, the binding affinity of
SP‐10 towards both ct‐DNA and HSA was also investi-
gated with molecular docking.
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ACKNOWLEDGMENTS
[24] K. Konkala, R. Chowrasia, P. S. Manjari, N. L. C. Domingues,
We thank University of Tabriz and Iran National Science
Foundation (INSF) (Grant Number: 95014554) for finan-
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Behrooz Shaabani https://orcid.org/0000-0001-5576-4604
Sadegh Salehzadeh https://orcid.org/0000-0003-2840-1896
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How to cite this article: Rakhtshah J, Shaabani
B, Salehzadeh S, Hosseinpour Moghadam N. The
solvent‐free synthesis of polysubstituted pyrroles by
a reusable copper Schiff base complex immobilized
on silica coated Fe3O4, and DNA binding study of
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