Non-covalent supported of L-proline on Graphene Oxide/Fe3O4 Nanocomposite: A novel, highly efficient and superparamagnetically separable catalyst for the synthesis of bis-pyrazole derivatives

Article abstract of DOI:10.3390/molecules23020330

A superparamagnetic graphene oxide/Fe₃O₄/L-proline nano hybrid that was obtained from the non-covalent immobilization of L-proline on graphene oxide/Fe₃O₄ nanocomposite was used as a new magnetically separable c

Full text of DOI:10.3390/molecules23020330

molecules
Article
Non-Covalent Supported of L-Proline on Graphene
Oxide/Fe3O4 Nanocomposite: A Novel, Highly
Efficient and Superparamagnetically Separable
Catalyst for the Synthesis of Bis-Pyrazole Derivatives
Mosadegh Keshavarz 1, Amanollah Zarei Ahmady 2,3, ID , Luigi Vaccaro 4
*
and Maryam Kardani 5
ID
1
Department of Gas and Petroleum, Yasouj University, Gachsaran 75918-74831, Iran;
chem.mosadegh@gmail.com
2
3
4
5
Nanotechnology Research Center, Ahvaz Jundishapur University of Medical Sciences,
Ahvaz 14536-33143, Iran
Department of Medicinal Chemistry, School of Pharmacy, Ahvaz Jundishapur University of Medical
Sciences, Ahvaz 14536-33143, Iran
Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy;
luigi.vaccaro@unipg.it
Marine Pharmaceutical Science Research Center, Ahvaz JundiShapur University of Medical sciences,
Ahvaz 14536-33143, Iran; maryam138967@yahoo.com
*
Correspondence: zarei-a@ajums.ac.ir; Tel.: +98-613-373-8378
Received: 1 December 2017; Accepted: 14 January 2018; Published: 5 February 2018
Abstract: A superparamagnetic graphene oxide/Fe3O4/L-proline nano hybrid that was
obtained from the non-covalent immobilization of L-proline on graphene oxide/Fe3O4
nanocomposite was used as a new magnetically separable catalyst for the efficient synthesis of
4,40-(arylmethylene)bis(1H-pyrazol-5-ol) derivatives. The prepared heterogeneous catalyst was
characterized using FTIR, TGA, DTG, XRD, TEM, SEM, and elemental analysis techniques. Short
reaction times (5–15 min), excellent yields (87–98%), and simple experimental procedure with an easy
work-up are some of the advantages of the introduced catalyst.
Keywords: L-proline; nanocomposite; graphene oxide; heterogeneous catalyst; magnetically
separable catalyst
1. Introduction
In recent years, carbocatalysis has attracted much attention, as it is cheap and can be easily obtained.
Catalysts based on carbon constituents, such as activated carbon and carbon nanotubes, have also been
used as catalyst supports [
class of carbocatalysts, as well as catalyst supports in organic synthesis [
as a favorable support due to its inherent properties, such as high chemically stability, large surface
area, and good availability [ ]. Most notably, GO has functional groups, mainly including epoxide,
1]. More recently, graphene and graphene oxide (GO) are emerging as a novel
2,3]. GO has been proved
4
hydroxyl, and carboxyl on the edge, top, and bottom surface of each sheet that even after sufficient
reduction, cannot be completely removed which makes GO a suitable support for metal and metal
oxide nanoparticles [5]. Similar to carbon nanotubes, these functional groups can offer a platform for
various chemical reactions. Recently, GO owing its surface decorated myriad oxygenated functions and
conductivity with very high surface area has been used in sensors, nanoelectronics, contaminant removal,
drug delivery, as metal-free catalysts or as supports for immobilizing active species for facilitating
synthetic transformations. Among the various uses of GO, assembling other inorganic materials, like
Molecules 2018, 23, 330; doi:10.3390/molecules23020330
www.mdpi.com/journal/molecules

Molecules 2018, 23, 330
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metal or metal oxide nanoparticles/nanocrystals on GO to fabricate composites or hybrids is an area of
great potential [ ]. Moreover, integrating GO with inorganic nanoparticles allows for the properties of
6–9
the nanocomposite to be engineered for specific applications. In addition, the existence of the abundant
oxygen functional groups affords GO sheets with great ability for the loading of organic molecules via
covalent or non-covalent methods, simplifying the development of a broad new class of materials with
improved properties, and even introducing new functionalities to GO sheet [10–12]. These catalysts have
showed high catalytic activity and could be reused for several cycles.
Metal NPs can endow their unique properties to the resulting nanoassemblies derived from
hybrid systems composed of polymer–nanoparticle or inorganic material–nanoparticle arrangements.
The structure and functionalities of the host support can control the spatial distribution of nanoparticles.
Therefore, the most prominent problem, which is the tendency of nanoparticles to agglomerate due
to their high surface energy, can be avoided [13]. For example, various organic materials, such as
dendrimers [14], polymers brushes [15], graphene sheets [16], as well as inorganic materials [17
have been utilized as carriers for the immobilization of metal NPs.
–21],
In recent years, amino acids, especially L-proline and proline-derivatives, have attracted much
attention as promising catalysts for essential transformations in the fine chemical and pharmaceutical
industries [22–24]. L-proline amino acid has been used more and more as catalyst, and is commercially
available at low cost, but often employed at high catalyst loading as high as 30 mol % [25]. This
unpleasant point is the main reason to take efforts for improving or modifying its catalytic activity
through its immobilization on suitable supports, if and only the immobilization approach does not
require the use of synthetic methodologies more expensive than proline itself [26].
When considering the above mentioned argument, we recently introduced a new facile and cheap
methodology for the preparation of L-prolinate-amberlite adduct by using ion-pair immobilization
of L-proline on the surface of commercially available amberlite IRA-900OH as a heterogeneous and
recoverable organocatalyst for the efficient synthesis of 2-amino-4H-chromenes [27], spiroindoles [28],
0
3,3 -diaryloxindoles [29], bis-pyrazoles [30], 4H-pyrano[2,3-c] pyrazole derivatives [31], and biscoumarin
derivatives [32].
Herein, we wish to introduce the L-proline on the GO/Fe3O4 nanocomposite through the
non-covalent immobilization via hydrogen bonding interaction between the L-proline and the
GO/Fe3O4 to prepare the GO/Fe3O4/L-proline nano hybrid. This technique gives robustness to
the catalytic system, and, on the other hand, lets the L-proline organocatalyst to be flexible, mobile,
and free on the surface of the GO/Fe3O4 support at the same time. Moreover, this method can enhance
the thermal stability of the L-proline organocatalyst. Finally, this catalyst can be recovered simply
by applying an external magnet. Such advantages are characteristic properties of homogeneous and
heterogeneous catalysts, which have been included in GO/Fe3O4/L-proline hybrid. The prepared
heterogeneous catalyst was used as an efficient and magnetically recoverable organocatalyst for the
one-pot pseudo three-component synthesis of bis-pyrazoles in ethanol.
2. Results and Discussion
2.1. Catalyst Preparation
The concise method for the preparation of GO/Fe3O4/L-proline is illustrated in Scheme 1.
GO and GO/Fe3O4 were prepared according to the modified Hummer’s method [33,34] and Song’s
method [35], respectively. Then, a mixture of the achieved GO/Fe3O4 nano hybrid and pristine
L-proline was sonicated in deionized water for 0.5 h and further stirred at room temperature for
24 h. Hydrogen-bonding interaction between hydroxyl, epoxy and carboxyl groups on the GO sheet
in GO/Fe3O4 nano hybrid with carboxyl and secondary amine groups of L-proline is the driving
force for L-proline binding to the GO/Fe3O4 nano hybrid, which has been presented in Scheme 1.
The catalyst has been characterized by Fourier transform infrared (FT-IR), XRD, TGA, DTG, TEM,
SEM, and elemental analysis.

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Scheme 1. Preparation pathway of graphene oxide (GO)/Fe3O4/L-proline nano hybrid.
2.2. Characterization of the Catalyst
2.2.1. IR Spectra
The non-covalent immobilization of L-proline on the surface of GO/Fe3O4 nano hybrid can be
confirmed by characterizing the pure GO, pristine L-proline, and GO/Fe3O4/L-proline nano hybrid
using FT-IR spectroscopy, as shown in Figure 1. The FT-IR spectrum of GO displays distinguishing
stretching vibration band−s1of carboxylic groups at round 3390, 1735, and 1224 cm−1, and stretching
vibration band at 1050 cm denotes to epoxy C-O groups (Figure 1a). The FT-IR spectrum of pristine
L-proline shows typical stretching frequencies, including N-H asymmetric stretching at 3055 cm−1
and carboxylate (COO−) asymmetric and symmetric stretching at 1622 and 1380 cm−1, respectively
(Figure 1c). These bands are appeared as new peaks in the FT-IR spectrum of GO/Fe3O4/L-proline
nano hybrid when compared to the FT-IR spectrum of pure GO sheet (Figure 1a,b). The carboxylate
(COO−) asymmetric and symmetric stretching of L-proline are presented in GO/Fe3O4/L-proline
nano hybrid spectrum and seemed to move to lower positions at 1615 and 1375 cm−1, respectively
(Figure 1b). The –Fe–O– stretching peak of GO/Fe3O4/L-proline appeared at 588 cm−1. Furthermore,
the band at 3056 cm−1 corresponding to the asymmetric stretching vibration of the N-H group
in L-proline is around 2800 cm−1 in FT-IR spectrum of GO/Fe3O4/L-proline. On the other hand,
the wavenumbers of the characteristic bands of GO have also been decreased to lower locations and
the individual band at 1735 cm−1 related to the carboxyl group in the spectrum of GO sheet vanished
after L-proline was loaded. All of these results from the comparison of FT-IR spectra propose that
L-proline organocatalyst has been successfully and effectively loaded on the surface of GO/Fe3O4
nano hybrid through hydrogen bonding interaction between carboxylic and secondary amine groups
of L-proline with hydroxyl, carboxyl, and epoxy groups on the GO sheets of GO/Fe3O4 nano hybrid.

Molecules 2018, 23, 330
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Figure 1. Fourier transform infrared (FT-IR) spectrum of GO (a), GO/Fe3O4/L-pro (b), and pristine
L-proline (c).
2.2.2. TGA and DTG Analysis
Thermogravimetric analysis (TGA) and differential thermal analysis (DTG) associated to the
decomposition profiles of the pristine L-proline and GO/Fe3O4/L-proline nano hybrid under air
atmosphere deliver additional proof for the immobilization of L-proline on the surfaces of GO/Fe3O4
nanocomposite (Figures 2 and 3). The pristine L-proline shows two distinct steps of weight loss in
the combined TG–DTG curves (Figure 2). The first weight loss centers around 60 ◦C, which is due
to a loss of water. The second large weight loss assigned to the successive cleavage of the L-proline
existed at 215–250 ◦C. The weight loss extends up to ca. 600 ◦C until the L-proline is almost entirely
decomposed under air flow. Figure 3 illustrates the TG–DTG curves of GO/Fe3O4/L-proline nano
hybrid. The first step (weight loss = ca. 10 wt %) before 100 ◦C is referred to the elimination of the
surface-adsorbed water and interlayer water molecules. The second step contains the weight loss
located at 125 ◦C and the trailed weight loss at 220 ◦C. The two weight loss peaks are assigned to the
successive pyrolysis of the labile oxygen-containing functional groups (weight loss = ca. 20 wt %). This
content of the highly rich oxygenated species is the main cause for the efficient loading of L-proline
on the surface of GO/Fe3O4 nanocomposite. The third step, in the range of 310–750 ◦C (ca. 25 wt %),
is reliable to refer to the successive cleavage of the L-proline moiety [36], since the weight loss in
this range is nearly equal to the content of L-proline moiety calculated from the elemental analysis
(24.5 wt %). The non-removable residue belongs to the remaining carbon and Fe3O4 nanoparticles.
The increased decomposition temperature of the pristine L-proline from 215–250 ◦C to 310–750 ◦C
range, when loaded on GO/Fe3O4 nano composite, suggests that the guest/host interaction was well
done through the oxygen-containing functional groups of the GO sheet in GO/Fe3O4 support with
carboxylic and secondary amine groups of L-proline (Scheme 1). This observation is also in accordance
with the carboxylate asymmetric and symmetric stretching vibration of L-proline, which has been
shifted to lower positions in FT-IR spectrum of GO/Fe3O4/L-proline (Figure 1b). Moreover, this
increased thermal stability clearly confirms that the L-proline is successfully loaded on the GO/Fe3O4
support by forming the non-covalent hydrogen bond, rather than physical adsorption because this
remarkable thermal stability could not be gained by physical adsorption. The high loading of L-proline
on the surface of GO/Fe3O4 support (25 wt %), together with the unique non-covalent hydrogen
binding behaviors between L-proline and GO/Fe3O4 support, makes the GO/Fe3O4/L-proline nano
hybrid an efficient and stable catalyst in the reaction system.

Molecules 2018, 23, 330
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Figure 2. TGA (A) and DTG (B) curves of pristine L-proline.
Figure 3. TGA (A) and DTG (B) curves of GO/Fe3O4/L-pro hybrid.
2.2.3. XRD
Comparison of the XRD pattern of the synthesized GO with the XRD pattern of pure graghite
indicated that the interlayer spacing between GO sheets is much more than graghite sheets (more than
two times). Oxidation leads to the abundant formation of hydroxy, epoxy and carboxyl groups on
the edge, top, and bottom surface the each sheet and increasing of the interlayer spacing between the
GO sheets. This created interlayer spacing is a good opportunity for both Fe3O4 nanoparticles and
L-proline organocatalyst to occupy interlayers and interact with GO functional groups, which is the
cause that the loading amount of immobilized L-proline on GO is more than on graghite. The main
peaks of pristine L-proline (denoted as *, 2
of GO/Fe3O4 support and the XRD pattern of GO/Fe3O4/L-proline hybrid is shown in Figure 4b.
The main peaks (denoted as #, 2 ) in Figure 4c at 2
= 30.1◦, 35.6◦, 43.6◦, 53.6◦, 57.5◦, and 62.8◦
θ) are shown in Figure 4a. Figure 4c displays the XRD pattern
θ
θ
show the characteristics of Fe3O4 (JCPDS No. 19-629). The main intense diffraction peaks of pristine
L-proline based on the standard spectrum are observed in the XRD pattern of GO/Fe3O4/L-proline
hybrid due to the presence of the L-proline on the GO/Fe3O4 support (Figure 4a,b). The size of Fe3O4
nanoparticles was determined from X-ray line broadening using the Debye–Scherrer formula to be
25 nm.

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Figure 4. XRD patterns of pristine L-proline (a), GO/Fe3O4/L-pro (b), and GO/Fe3O4 (c). Symbles #
and * refer to Fe3O4 NPs and L-proline patterns respectively.
2.2.4. TEM and SEM
SEM was used to see the morphology of GO nanoplatelets. images of the prepared GO
nanoplatelets show crumpled thin layers with wrinkles and folds on the surface of GO (Figure 5).
The morphology of GO/Fe3O4/L-proline and the reused GO/Fe3O4/L-proline after eight runs was
determined by TEM. As Figure 6 shows, Fe3O4 nanoparticles are chemically deposited on GO;
although, some Fe3O4 aggregation is detected. The results of TEM confirmed that the nano-sized
organic–inorganic hybrid material was prepared. The morphology of the reused catalyst did not show
any significant change even after eight reaction cycles, which proved its robustness (Figure 7).
Size data of Fe3O4 nanoparticles were achieved by the counting of almost 500 particles from the
TEM images of GO/Fe3O4/L-pro catalyst. Particle size and size distribution of Fe3O4 nanoparticles
are represented in Figure 8. The number-frequency histogram illustrates the frequency of existence
versus the size range of Fe3O4 nanoparticles. As it can be seen from Figure 8, the size distribution of
the particles is twisted to the larger end of the particle-size scale. The mean size of Fe3O4 nanoparticles
was obtained to be 24 nm with a standard deviation of 12.2.

Molecules 2018, 23, 330
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Figure 5. SEM image of synthesized graphene oxide.
Figure 6. TEM image of fresh GO/Fe3O4/L-pro nano hybrid.
Figure 7. TEM image of reused GO/Fe3O4/L-pro nano hybrid after eight runs.

Molecules 2018, 23, 330
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Figure 8. Number-frequency histogram of Fe3O4 nanoparticles in GO/Fe3O4/L-pro hybrid.
2.3. Optimization of the Reaction Conditions
After the preparation and characterization of GO/Fe3O4/L-proline catalyst, its catalytic
act0ivity was investigated in a one-pot pseudo three-component reaction for the synthesis of
4,4 -(arylmethylene)bis(1H-pyrazol-5-ol) derivatives. To search for the optimal conditions, the reaction
of benzaldehyde and two equivalents of 3-methyl-1-phenyl-2-pyrazolin-5-one was selected as the
model reaction to examine the effect of GO/Fe3O4/L-proline catalyst (0.01–0.1 g), under a variety of
conditions (Table 1).
The present optimization studies revealed that the best result was achieved by caring out the
reaction in the presence of 0.05 g of GO/Fe3O4/L-proline under the reflux condition in ethanol
(entry 10). The use of larger amounts of the catalyst (0.1 g, entry 11) did not improve the yield, while
decreasing its amount led to decreased yield (Table 1, entries 8, 9).
Table 1. Investigation of catalytic activity of GO/Fe3O4/L-pro for the synthesis of -bis-pyrazole (3a
)
under various Conditions.
◦
Entry
Conditions
Temperature ( C)
GO/Fe3O4/L-pro (g)
Time (min)
Yield (%) a
1
2
3
4
5
6
7
8
9
neat
CH2Cl2
CH3CN
THF
DMF
H2O/DMF
H2O
CH3CH2OH
CH3CH2OH
CH3CH2OH
CH3CH2OH
100
reflux
reflux
65
100
100
0.02
0.02
0.02
0.02
0.02
0.02
0.02
-
40
45
40
45
45
20
25
120
20
10
10
60
65
55
60
65
75
80
40
92
98
95
reflux
reflux
reflux
reflux
reflux
a
0.02
0.05
0.1
10
11
Yield refer to isolated and pure product.

Molecules 2018, 23, 330
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Using the optimized reaction conditions, the efficiency of this approach was explored for the
0
synthesis of a wide variety of 4,4 -(arylmethylene)bis(1H-pyrazol-5-ol) derivatives (Scheme 2, Table 2).
All of the reactions delivered excellent product yields and accommodated a wide range of aromatic
aldehydes bearing both electron-donating and electron-withdrawing substituents (3a–r).
Scheme 2. Synthesis of bis-pyrazole derivatives catalyzed by GO/Fe3O4/L-proline nano hybrid.
Table 2. Synthesis of bis-pyrazole derivatives (3a–r) catalyzed by GO/Fe3O4/L-proline.
◦
m.p. ( C)
Entry
Ar
Product
Time (min)
Yield a (%)
Reported
Found
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
C6H5-
4-Me-C6H4-
4-Cl-C6H4-
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
10
13
8
10
10
5
98
94
93
91
94
96
90
92
87
90
89
95
89
93
87
91
94
89
171–172
202–204
213–215
236–237
227–229
224–226
149–150
152–153
165–168
195–197
201–203
210–212
230–231
180–182
132–134
172–174
198–200
173–175
170–172
203–205
213–215
233–235
227–229
225–227
152–154
153–155
169–170
194–196
205–207
210–212
232–234
180–182
132–134
173–175
198–200
170–173
2-Cl-C6H4-
2,4-(Cl)2-C6H3-
4-NO2-C6H4-
3-NO2-C6H4-
4-OH-C6H4-
3-OH-C6H4-
3,4-(MeO)2-C6H3-
4-MeS-C6H4-
4-CN-C6H4-
2-OH-C6H4-
4-F-C6H4-
8
15
14
7
15
15
15
13
12
10
8
4-iPr-C6H4-
4-MeO-C6H4-
2-Br-C6H4-
3-Br-C6H4-
5
a
Isolated yields.
The recovery of a catalyst is highly preferred for a greener process. For this purpose, the reusability
of GO/Fe3O4/L-proline was studied for eight consecutive cycles (fresh + seven cycles) for the synthesis
of 4,40-(phenylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol) (3a). From Figure 9, it can be
observed that GO/Fe3O4/L-proline catalyst can be reused up to eight runs without the need to
reload, and the yield difference between the first and eighth runs is only 5%, indicating that the catalyst
efficiency is almost completely maintained during eight consecutive runs. The nitrogen content of
the fresh and reused catalyst was measured using the elemental analysis and the comparison of the
nitrogen contents suggested that the catalyst lost only 5% of its nitrogen content after eight runs. This
is solid proof for the very low leaching account of L-proline organocatalyst from GO/Fe3O4/L-proline
catalyst into the reaction mixture during eight runs, and also, confirms that the catalytic ability of
GO/Fe3O4/L-proline has almost completely remained stable after eight runs, which is in agreement
with the recyclability study. The TEM image of the recovered catalyst after eight runs did not reveal
any significant change in the morphology of the reused catalyst, which proved its robustness while
maintaining catalytic activity (Figure 7). Superparamagnetic properties and simple recovery of the

Molecules 2018, 23, 330
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catalyst are shown in Figure 9. GO/Fe3O4/L-proline catalyst can be recovered from the reaction
mixture simply by applying an external magnet and its superparamagnetic properties remain constant
even after eight runs.
Ph
H3C
CH3
N
H3C
O
EtOH, reflux
2
N
N
+
N
O
Ph
H
N
N
L
GO/Fe3O4/ -pro
8 runs
Ph
HO HO
Ph
Ph
98
98
100
80
96
95
95
93
93
93
Yield(%)
Time(min)
60
40
20
0
10
15
10
15
20
25
15
20
1
2
3
4
5
6
7
8
No. of Runs
Figure 9. Recycling of GO/Fe3O4/L-pro nano hybrid for the synthesis of 3a.
Figure 10 shows the powerful superparamagnetic property of fresh GO/Fe3O4/L-pro by using
an external magnet. For more detailed study, magnetic properties of fresh GO/Fe3O4/L-pro and the
reused GO/Fe3O4/L-pro after eight runs were investigated using VSM (Figure 11). The achieved
results illustrated that the catalyst samples have appropriate property for magnetic actuations, but
there were some decrease in the values of saturation magnetization after eight consecutive runs, which
possibly originated from the decrease of loading amount of Fe3O4 nanoparticles on the GO surface
during using under eight repeated heating condition (Figure 11a,b).
Figure 10. Powerful superparamagnetic property and simple recovery of GO/Fe3O4/L-proline catalyst
using an external magnet.

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Figure 11. The magnetization curve of fresh GO/Fe3O4/L-pro (a) and reused GO/Fe3O4/L-pro after
eight recycling (b).
It is worth noting that the introduced GO/Fe3O4/L-proline nano hybrid is a collection of three
most applied catalysts, including GO, Fe3O4 nanoparticles, and pristine L-proline organocatalyst. Each
of these catalysts has been separately used for several organic transformations. Hence, we became
encouraged to compare the catalytic efficiency of GO/Fe3O4/L-proline nano hybrid with each of
its component catalysts for the preparation of 4,40-(arylmethylene)bis(1H-pyrazol-5-ol) derivatives.
The reaction of benzaldehyde and two equivalents of 3-methyl-1-phenyl-2-pyrazolin-5-one was
selected again as the model reaction (Table 3). On the base of the CHN analysis and TG (disscused
above), the loading amount of L-proline in 0.05 g of GO/Fe3O4/L-proline nano hybrid is 0.01 g.
To compare the catalytic activity of pristine L-proline with L-proline on GO/Fe3O4 nanocomposite,
the equivalent amount of pristine L-proline (0.01 g) was also used as catalyst (entry 1 vs. 4).
Table 3. Catalytic activity of GO/Fe3O4/L-proline nano hybrid in comparison with pure GO, Fe3O4
nanoparticles, prestine L-proline, and GO/Fe3O4 nanocomposite.
Entry
Catalyst
Time (min)
Yield (%)
1
2
3
4
5
GO/Fe3O4/L-pro (0.05 g)
GO (0.05 g)
Fe3O4 nanoparticle (0.05 g)
L-Proline (0.01 g)
10
120
90
45
40
98
70
80
90
89
GO/Fe3O4 (0.05 g)
The results show that the L-proline immobilized on GO/Fe3O4 nanocomposite has better catalytic
activity than pristine L-proline with the same loading amount of L-proline. This observation confirms
that the catalytic activity of L-proline is improved when is immobilized on GO/Fe3O4 nanocomposite.
Moreover, GO/Fe3O4/L-proline is a more powerful catalyst than its other component catalysts
regarding the reaction time and the yield of the product (entry
1 vs. 2, 3 and 5). Finally, to show

Molecules 2018, 23, 330
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the efficiency of this method in comparison with other reported procedures, we selected the reaction
of benzaldehyde and two equivalents of 3-methyl-1-phenyl-2-pyrazolin-5-one for the synthesis of
4,40-(phenylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol) (3a) as a representative model. This
comparison is shown in Table 4. It is clear from the data that our method has shorter reaction times
and provides higher yields of the products.
Table 4. Comparison of the catalytic efficiency of GO/Fe3O4/L-pro with various catalysts reported for
the synthesis of 3a.
Entry
Catalyst
Condition
Time (min)
Yield (%)
Ref.
1
2
3
4
5
6
7
8
9
10
11
12
13
GO/Fe3O4/L-pro (0.05 g)
[Amb]L-prolinate (10 mol %)
NiFe2O4@SiO2–H3PW12O40
Nano–SiO2/HClO4
EtOH/reflux
EtOH/reflux
EtOH/reflux
H2O/reflux
EtOH/reflux
10
11
15
20
30
11
240
10
30
75
60
18
120
98
97
91
94
90
89
96
98
92
80
86
90
80
This work
[30]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
SBNPTT
◦
[Pyridine–SO3H]Cl (1 mol %)
Phosphomolybdic acid
AP-SiO2 (30 mol %)
Solvent-free /50 C
EtOH/r.t.
CH3CN/r.t.
PEG-SO3H (1.5 mol %)
LiOH·H2O (10 mol %)
[Dsim]AlCl4 (1 mol %)
SASPSPE ( 0.1 g)
H2O/reflux
◦
H2O/90 C
◦
Solvent-free/50 C
[45]
[46]
[47]
EtOH/reflux
EtOH/reflux
SBSSA (18 mol %)
3. Experimental
All of the chemicals were purchased from Merck and Aldrich. GO was prepared using modified
Hummers method from flake graphite (Merck Company, Darmstadt, Germany). The reaction
progresses were monitored by thin layer chromatography (TLC; silica-gel 60 F254, n-hexane: AcOEt).
The 1H-NMR spectra were obtained on a Bruker Instrument DPX-400 Avance 2 model spectrometer
(Bruker, Billerica, MA, USA). The vario El CHNS (made in Germany) was used for elemental analysis.
Scanning electron micrographs (SEM) were obtained using a Cambridge S-360 instrument with
an accelerating voltage of 20 kV (Cambridge, Austin, TX, USA). The powder X-ray diffraction (XRD)
pattern was obtained by a Bruker AXS (D8, Avance) (Avance, San Antonio, TX, USA) instrument
employing the reflection Bragg–Brentano geometry with CuKa radiation. A continuous scan mode was
◦
◦
used to collect 2
θ
from 5 to 40 . Fourier transform infrared (FT-IR) spectra were obtained as potassium
−1
bromide pellets in the range 400–4000 cm using an AVATAR 370 Thermo Nicolet spectrophotometer.
The thermogravimetric and differential thermogravimetric (TG–DTG) analysis was performed on
Netzsch STA449c (Kimia Sanat Ara, Tehran, Iran). The sample weight was ca. 10 mg and was heated
from room temperature up to 600 ◦C with 10 ◦C/min using alumina sample holders. The structure of
the products was confirmed on the basis of IR, NMR spectroscopic data, and elemental analysis.
3.1. General Procedure for the Preparation of GO
A flask containing graphite (1 g) and NaNO3 (0.75 g) were placed in an ice-water bath. H2SO4
(75 mL) was added with stirring, and then KMnO4 (4.5 g) was slowly added during about 1 h. After
strongly stirring for 3 days at room temperature, 5% H2SO4 (140 mL) aqueous solution was slowly
added over for about 1 h with stirring, keeping the temperature at 98 ◦C. Then, the temperature was
◦
reduced to 60 C, 3 mL of H2O2 (30 wt % aqueous solution) was added, and the mixture was stirred for
2 h at room temperature. The prepared GO was suspended in pure water to give a brown dispersion,
which was subjected to dialysis to absolutely remove the remaining salts and acids. The resultant
purified GO powders were collected by centrifugation and air-dried [33,34].

Molecules 2018, 23, 330
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3.2. Preparation of GO/Fe3O4 Nanocomposite
The GO/Fe3O4 nanocomposite was prepared by the chemical co-precipitation method similar to
that of described above [35]. GO (200 mg) was dispersed into deionized water (250 mL) under
ultrasonication for 30 min. FeCl3 (1.0 mmol) was added into the aqueous dispersion solution.
◦
The mixture was stirred overnight at 65 C. Then, NH3
·
H2O solution (30 mL, 25–28%) and FeSO4
·
7H2O
◦
(0.5 mmol) were added, respectively. After the prepared mixture was stirred for additional 2 h at 65 C,
the precipitate was separated by an external permanent magnet, and it was washed with distilled
water until neutralization and dried under vacuum.
3.3. Preparation of GO/Fe3O4/L-Proline Nano Hybrid
The mixture of the dried GO/Fe3O4 (1 g) and L-proline (0.5 g) was sonicated in deionized water
for 0.5 h and further stirred at room temperature for 24 h. The prepared GO/Fe3O4/L-proline nano
hybrid was separated by an external permanent magnet and dried under vacuum. The prepared
catalyst was characterized using FTIR, TGA, DTG, XRD, TEM, and elemental analysis techniques.
Based on TGA and elemental analysis the L-proline content of GO/Fe3O4/L-proline hybrid was
obtained at 24–25 wt % (0.25 g of L-proline per 1 g of GO/Fe3O4/L-proline hybrid).
3.4. General Procedure for the GO/Fe3O4/L-Proline Nanohybrid Catalyzed Synthesis of
4,40-(Arylmethylene)bis(1H-pyrazol-5-ol) Derivatives
A mixture of 3-methyl-1-phenyl-2-pyrazoline-5-one (2 mmol), aromatic aldehyde (1 mmol), were
placed together in a round-bottom flask containing 5 mL of EtOH. Subsequently, GO/Fe3O4/L-proline
nanohybrid catalyst (0.05 g) was added to the mixture. The suspension was magnetically stirred
at reflux condition for sufficient time according to Table 2. After the completion of the reaction as
followed by TLC (n-hexane:ethyl acetate; 3:1), the catalyst was separated by an external permanent
magnet. The recovered catalyst was washed with acetone, dried, and stored for other similar repeated
runs.0The remaining mixture was filtered and cooled to room temperature to afford the pure crystals
of 4,4 -(arylmethylene)bis(1H-pyrazol-5-ol) derivatives. The products are known compounds and their
IR, NMR spectroscopy data, and melting points are compared with the reported values [30,37–47].
4. Conclusions
In summary, the introduced GO/Fe3O4/L-proline nano hybrid was prepared via hydrogen
bonding interaction between the L-proline and GO/Fe3O4 nanocomposite. This technique gives
robustness to the catalytic system; on the other hand, lets the L-proline organocatalyst to be
flexible, mobile, and free on the surface of the GO/Fe3O4 support. Moreover, this method
improves the thermal stability of the L-proline organocatalyst. The results showed that immobilized
L-proline on GO/Fe3O4 nanocomposite has better catalytic activity than pristine L-prolin at the
same wight ratios. GO/Fe3O4/L-proline catalyst can be recovered simply by applying an external
magnet and reused at least for six runs for the one-pot pseudo three-component synthesis of
4,40-(arylmethylene)bis(1H-pyrazol-5-ol) derivatives.
Acknowledgments: This work was financially supported by grant number: N-9504 from Vice-Chancellor for
Research Affairs of Ahvaz Jundishapur University of Medical Sciences.
Author Contributions: Amanollah Zarei Ahmady, Mosadegh Keshavarz and Luigi Vaccaro conceived, designed
and performed the experiments and analyzed the data; Maryam Kardani wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 3a–3r are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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