Cu(II) immobilized on Fe3O4@APTMS-DFX nanoparticles: An efficient catalyst for the synthesis of 5-substituted 1: H -tetrazoles with cytotoxic activity

Article abstract of DOI:10.1039/c7md00302a

Cu(ii) immobilized on deferasirox loaded amine functionalized magnetic nanoparticles (Cu(ii)/Fe₃O₄@APTMS-DFX) as a novel magnetically recyclable heterogeneous catalyst is able to catalyze the [3 + 2] cycloaddition reactions of various organic nitriles with sodium azide. Using this method, a series of 5-substituted-1H-tetrazoles under mild conditions in DMSO were prepared. The reaction involves mild reaction conditions with efficient transformation capability. The developed catalyst could be easily separated by applying an external magnetic field. Furthermore, it could be recycled for 5 runs with negligible leaching of copper from the surface of the catalyst. The catalyst was characterized by various techniques such as FT-IR, TGA, VSM, SEM-EDX, and ICP-OES. Several derivatives of 1H-tetrazoles were prepared using this catalyst, and their structures were confirmed using different techniques. Then, the synthesized anthraquinones were evaluated for their cytotoxicity against several cell lines including MCF-7, MAD-MD-231, HT-29, HeLa, neuro-2a and L-929. The results obtained from the MTT assay revealed that the 6 derivatives exhibited a high level of cytotoxicity. In order to determine the cytotoxicity mechanism, 2 derivatives with the highest cytotoxic activity were selected, and an apoptosis assay was carried out by flow cytometry, which supported that apoptosis is the major mechanism.

Full text of DOI:10.1039/c7md00302a

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RESEARCH ARTICLE
CuIJII) immobilized on Fe3O4@APTMS-DFX
nanoparticles: an efficient catalyst for the
Cite this: DOI: 10.1039/c7md00302a
synthesis of 5-substituted 1H-tetrazoles with
cytotoxic activity
a
Faezeh Taghavi,a Mostafa Gholizadeh,
Amir Sh. Saljooghi
*
and Mohammad Ramezanib
a
CuIJII) immobilized on deferasirox loaded amine functionalized magnetic nanoparticles (CuIJII)/
Fe3O4@APTMS-DFX) as a novel magnetically recyclable heterogeneous catalyst is able to catalyze the [3 +
2] cycloaddition reactions of various organic nitriles with sodium azide. Using this method, a series of
5-substituted-1H-tetrazoles under mild conditions in DMSO were prepared. The reaction involves mild re-
action conditions with efficient transformation capability. The developed catalyst could be easily separated
by applying an external magnetic field. Furthermore, it could be recycled for 5 runs with negligible leaching
of copper from the surface of the catalyst. The catalyst was characterized by various techniques such as
FT-IR, TGA, VSM, SEM-EDX, and ICP-OES. Several derivatives of 1H-tetrazoles were prepared using this
catalyst, and their structures were confirmed using different techniques. Then, the synthesized anthraqui-
nones were evaluated for their cytotoxicity against several cell lines including MCF-7, MAD-MD-231, HT-
29, HeLa, neuro-2a and L-929. The results obtained from the MTT assay revealed that the 6 derivatives
exhibited a high level of cytotoxicity. In order to determine the cytotoxicity mechanism, 2 derivatives with
the highest cytotoxic activity were selected, and an apoptosis assay was carried out by flow cytometry,
which supported that apoptosis is the major mechanism.
Received 18th June 2017,
Accepted 17th August 2017
DOI: 10.1039/c7md00302a
rsc.li/medchemcomm
cently, various catalytic systems such as Brønsted acids,8
1. Introduction
Lewis acids including BF3·OEt2,9 AlCl3,10 zinc salts,11 copper
Various catalytic methods for the synthesis of tetrazoles as a
class of heterocyclic compounds are currently under immense
focus because of their widespread applications. Tetrazoles are
used in pharmaceuticals as lipophilic spacers,1 photography
and information recording systems,2 catalysis technology and
high energy chemistry.3 Tetrazoles are approximately 10-fold
more lipophilic than the corresponding carboxylates, which is
a useful property for a drug molecule to pass through cell
membranes easily.2 Also, tetrazolic acids are more resistant
than carboxylic acids in many metabolic degradation routes.2
Commonly, 5-substituted 1H-tetrazoles are synthesized
through reaction of nitriles with hydrazoic acid,4 trimethyl-
silyl azide (TMSN3),5 and sodium azide.6,7 Among these
methods, the [3 + 2] cycloaddition reaction of sodium azide
and diverse nitriles is an attractive and common method. Re-
triflates,12 tangstates,13 and AgNO3,14 supported catalysts such
as
SiO2–H3BO3,15
NaHSO4·SiO2,16
FeCl3·SiO2,17
and
Fe3O4@SiO2/salen of CuIJII),18 chitosan derived magnetic ionic
liquids,19 Cu–MCM-41,20 WAIPO5 based microspheres,21
clays,22 Ag NPs,23 metal oxides such as Cu2O24 and ZnO,25
and alloys like Zn/Cu26 and Zn/Al hydrotalcites27 have been
reported for the synthesis of tetrazoles. Although all of these
catalytic systems are useful, a number of existing methods
have some drawbacks such as stringent conditions, expensive
reagents, toxic and expensive metal catalysts, long reaction
times, low yields of final products, difficulty in the separation
and recovery of the catalyst and tedious work-ups.20 In recent
years, heterogeneous catalytic systems have received consider-
able attention because of their easy separation, recyclability of
catalysts and eco-friendly conditions. Recently, magnetic
nanomaterials with potential applications in catalysis, bio-
medicine, biotechnology, materials science and target drug
delivery were reported.28 Magnetic nanoparticles (MNPs) were
used as efficient supports for homogeneous catalysts instead
of porous materials in organic chemistry.29 MNP supported
catalysts can be easily removed and recycled from the reaction
a Department of Chemistry, Faculty of science, Ferdowsi University of Mashhad, P. O.
Box 91775-1436, Mashhad, Iran. E-mail: m_gholizadeh@um.ac.ir;
Tel: +98 513880 5527
b Pharmaceutical Research Center, School of Pharmacy, Mashhad University of
Medical Sciences, P. O. Box 91775-1365, Mashhad, Iran
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mixture using an external magnet, which is a faster method
in comparison with filtration and centrifugation methods,
and the catalyst loss can be minimized during the separation.
Also, it can increase the purity of the products and moreover
improve the operation costs.30 In order to control the size,
shape and magnetic properties of the hybrid materials, the
core–shell structures of MNPs have been reported.31 SiO2
coated magnetic nanoparticles were prepared and modified
with various desirable functional groups for a range of appli-
cations such as metal nanoparticle loading for high catalytic
activity.32 Herein, our research group would like to report the
preparation of CuIJII) immobilized on Fe3o4@APTMS-DFX as a
novel heterogeneous catalyst for the preparation of
5-substituted 1H-tetrazoles.
2.2. Synthesis of 4-[3,5-bisIJ2-hydroxyphenyl)-1,2,4-triazol-1-yl]
benzoic acid (deferasirox)
2-(2-hydroxyphenyl)-4H-3,1-benzoxazin-4-one was synthesized
according to a previously reported procedure with some
modifications.33,34 4-Hydrazinobenzoic acid (11.5 mmol, 1.75
g) and Et3N (11.5 mmol, 1.16 g) were dissolved in boiling
EtOH (80 mL). Then, 2-(2-hydroxyphenyl)-4H-3,1-benzoxazin-4-
one (10.45 mmol, 2.50 g) was added to the clear solution,
and the reaction mixture was refluxed for an additional 2 h.
After the completion of the reaction (monitored by TLC), the
solution was cooled down to room temperature, and water
was added until the first sign of precipitation was observed.
The mixture was then concentrated to a total volume of 50%
under reduced pressure, and an aqueous 6 M HCl solution
(40 mL) was added. The resulting solid was filtered, washed
with water and dried for 24 h in a vacuum. Yellow powder;
(3.11 g, yield = 80%); m.p. 264–266 °C; IR (KBr) ν: 3317, 2540,
1680 (CO), 1607, 1517, 1495, 1431, 1351, 1221, 988, 752
2. Experimental
2.1. Chemicals and apparatus
cm−1; H NMR (C3H6O-d6 400 MHz): δ 7.00 (s, 1H), 7.01–7.04
All solvents were purchased from Merck Co. Triethylamine
(NEt3), 4-hydrazino-benzoic acid, and 2-(2-hydroxyphenyl)-4H-
3,1-benzoxazin-4-one were purchased from Merck without fur-
1
(m, 3H), 7.39 (m, 2H), 7.48 (d, 1H), 7.53 (d, 2H), 8.15 (d, 2H),
8.19 (d, 1H) 10.00 (s, OH), 10.78 (s, OH) ppm; 13C NMR
(C3H6O-d6, 75 MHz): δ 113.7, 113.9, 116.6 (CH), 117.0 (CH),
119.5 (CH), 119.8 (CH), 124.0 (2 CH), 126.9 (CH), 130.4 (2
CH), 130.5, 130.7 (CH), 131.4 (CH), 132.6, 141.9 (CH), 152.1,
155.6, 156.4, 160.4, 165.7 (CO) ppm; M.S. (70 eV) m/z (%):
374 (M+); anal. calc. for C21H15N3O4: C, 67.56%; H, 4.05%; N,
11.25%. Found: C, 67.76%; H, 3.85%; N, 11.14%.
ther
carbodiimide
purification.
1-Ethyl-3-[3-dimethylaminopropyl]
(EDC) and 7-hydroxy-
hydrochloride
benzotriazole hydrate (HOBt) were purchased from Fluka
(Germany). RPMI-1640 medium, Dulbecco's modified Eagle's
medium (DMEM) and fetal bovine serum (FBS) were pur-
chased from GIBCO (Gaithersburg, USA). Penicillin and strep-
tomycin were purchased from Biochrom AG (Berlin, Ger-
many).
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-
2.3. Synthesis of Fe3O4 NPS
diphenyltetrazolium bromide, a yellow tetrazole) was pur-
chased from Sigma Co., Ltd. Cisplatin was purchased from
Sigma Aldrich. Benzonitrile derivatives, sodium azide,
CuIJOAC)2 and (3-aminopropyl) trimethoxysilane (APTMS) were
purchased from Sigma Aldrich. NMR spectra were recorded
on Avance Bruker-400 MHz spectrometers. All chemical shifts
in NMR experiments are reported as ppm and were
referenced to residual solvent. Chemical shifts are reported
in parts per million, and the signals are denoted as s (sin-
glet), br (broad), d (doublet) and m (multiplet). FT-IR spectra
were recorded on an AVATAR-370-FTIR Thermo Nicolet in-
strument. All mass spectra were scanned using a Varian Mat
CH-7 instrument at 70 eV. The reactions were monitored by
TLC using silica gel plates, and the products were identified
by comparing their spectra and physical data with those of
the authentic samples. Melting points were measured using
Electrothermal 9100 apparatus. Scanning electron microscopy
(SEM) images were taken using a Zeiss LEO 1450 VP/35Kv in-
strument (Germany). Thermogravimetric analysis (TGA) was
The magnetite nanoparticles (Fe3O4 MNPs) were prepared via
a previously reported chemical co-precipitation technique
with ferric and ferrous ions in alkaline solution, with some
modifications.35,36 FeCl2·4H2O (9.25 mmol) and FeCl3·6H2O
(15.8 mmol) were dissolved in deionized water (150 mL) un-
der an Ar atmosphere at room temperature. An NH4OH solu-
tion (25%, 50 mL) was then added dropwise (drop rate = 1
mL min−1) to the stirring mixture at room temperature to
reach a reaction pH of about 11. The resulting black disper-
sion was continuously stirred for 1 h at room temperature
and then collected using an external magnet.
2.4. Surface modification of Fe3O4 MNP by APTMS
(MNP@APTMS)
The Fe3O4 magnetic nanoparticles were modified with APTMS
(MNP@APTMS) according to the previously reported proce-
dure by Lui et al.37 The MNPs were dispersed in 100 mL of
methanol/toluene (volume ratio of 1 : 1) as solvent and then
sonicated for 30 min and heated up to 95 °C until 50 mL of
the solvent was evaporated. Thereafter, methanol (50 mL)
was added. The operation was repeated three times to ensure
that the solution was completely anhydrous. Subsequently,
the volume of the reaction mixture was fixed at 100 mL by
the addition of methanol followed by the addition of APTMS
and stirring for a certain time (30 or 70 °C under N2). The
product was washed several times with ethanol and distilled
carried out using
a thermogravimetric analyzer TGA-50
(Shimadzu Japan) instruments. Elemental analysis was car-
ried out using a CHNS (O) Analyzer Model FLASH EA 1112 se-
ries (Thermo Finnigan, Italy). Magnetization values were
obtained using a vibrating sample magnetometer (VSM, 7400
Lake Shore, America). Inductively coupled plasma optical
emission spectrometry (ICP-OES) was carried out using a
Varian, VISTA-PRO, CCD (Australia).
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water by magnetic decantation and dried under vacuum. The
product was washed with toluene, and Soxhlet extraction was
performed for 24 h to remove unreacted APTMS and finally,
dried at 40–50 °C for 6 h.
over anhydrous sodium sulfate, and concentrated to give the
crude solid 5-phenyl-1H-tetrazole. The crude product was
recrystallized in n-hexane/ethyl acetate. The spectral data for
selected products are presented as follows:
5-Phenyl-1H-tetrazole (Table 2, entry 1). White solid; mp
214–216 °C (Lit.39 214–216 °C); FT-IR (KBr): νmax/cm−1 3129,
3054, 2981, 2910, 2701, 2609, 2545, 2481, 1866, 1608, 1563,
1485, 1465, 1409,1290,1258, 1163, 1056, 992, 787, 726,
687 498; H NMR (400 MHz, DMSO-d6, ppm) δ 7.60–7.64 (m,
3H, Ph), 8.02–8.07 (m, 2H, Ph).
2.5. The functionalization of the APTMS modified MNPs with
deferasirox
Deferasirox (4.2 mmol) was dissolved in 100 ml of toluene,
which was then added to 1.1 g of the APTMS-modified Fe3O4
MNPs in the presence of EDC (50 mg)/HOBt (40 mg) to acti-
vate the carboxylate groups of deferasirox for amide bond for-
mation.38 This mixture was refluxed under stirring for 24 h
and was then filtered and washed with 100 ml of dry toluene
(Soxhlet extraction) for 24 h. The solid product was dried at
room temperature under vacuum. The prepared catalyst was
abbreviated as Fe3O4@APTMS-DFX.
1
5-(4-Chlorophenyl)-1H-tetrazole (Table 2, entry 2). White
solid, mp 261–262 °C (Lit.41 261–263 °C); FT-IR (KBr): νmax
/
cm−1 3092, 3060, 3007, 2978, 2907, 2851, 2725, 2622, 2537,
2471, 1609, 1564, 1486, 1435, 1160, 1096, 1053, 1020, 990,
1
833, 745, 508; H NMR (400 MHz, DMSO-d6, ppm) δ 7.68 (d, J
= 8.4 Hz, 2H, Ph), 8.05 (d, J = 8.8 Hz, 2H, Ph).
5-(4-Boromophenyl)-1H-tetrazole (Table 2, entry 4). White
solid, mp 264–265 °C (Lit.40 265 °C); FT-IR (KBr): νmax/cm−1
3117, 3089, 3003, 2900, 2846, 2757, 2730, 2639, 2553, 2479,
2308, 2222, 2128, 1604, 1558, 1482, 1430, 1276, 1156, 1119,
2.6. Preparation of CuIJII) immobilized Fe3O4@APTMS-DFX
Fe3O4@APTMS-DFX (1 g) was mixed with Cu (OAc)2·H2O (0.6
mmol, 0.11 g) and absolute EtOH (5 mL) and stirred at room
temperature under an Ar atmosphere for 4 h. The resulting
suspension was separated using an external magnet and then
dried in an oven at 60 °C for 16 h.
1
1078, 1053, 1017, 990, 874, 829, 743, 691, 502, 452; H NMR
(400 MHz, DMSO-d6, ppm) δ 7.83 (d, J = 12 Hz, 2H, Ph), 8.01
(d, J = 12, 2H, Ph).
5-(3,5-Dimethoxyphenyl)-1H-tetrazole (Table 2, entry 7).
White solid, mp 204–205 °C (Lit.42 204–206 °C). FT-IR (KBr):
νmax/cm−1 3129, 3064, 3011, 2975, 2941, 2843, 2757, 2712,
2634, 1605, 1562, 1480, 1430, 1287, 1208, 1162, 1167, 1054,
827, 747; 1H NMR (400 MHz, DMSO-d6, ppm) δ 3.84 (s, 6H,
–OMe), 6.72 (t, J = 2 Hz, 1H, Ph), 7.21 (d, J = 2 Hz, 2H, Ph),
16.91(1H, br s, NH).
4-(1H-Tetrazol-5-yl) phenol (Table 2, entry 10). White solid,
mp 234–236 °C (Lit.43 234–235 °C). FT-IR (KBr): νmax/cm−1
3252, 3101, 3066, 3019, 3000–2200, 1615, 1599, 1511, 1466,
1413, 1282, 832, 752, 514; 1H NMR (400 MHz, DMSO-d6,
ppm) d 6.97 (d, J = 8.4 Hz, 2H, Ph), 7.87 (d, J = 8.8 Hz, 2H,
Ph), 10.20 (2H, br s, OH).
2.7. Typical procedure for the preparation of 5-phenyl-1H-
tetrazoles
Sodium azide (0.0650 g, 1 mmol), benzonitrile (0.1031 g, 1
mmol), and the catalyst (0.03 g) were mixed and stirred in
DMSO (5 mL). The reaction temperature was raised up to 120
°C for 1 h. The progress of the reaction was monitored by thin-
layer chromatography (TLC) in ethyl acetate:n-hexane. After the
completion of the reaction, the catalyst was removed using an
external magnet, and the reaction mixture was treated with
HCl (4 N, 10 mL) and EtOAc (10 mL). The resultant organic
layer was separated, washed with distilled water (2 × 10), dried
Table 1 Optimization of the model reaction over the CuIJII)/Fe3O4@APTMS-DFX nanocatalyst
Entry
Molar ratio (nitrile : NaN3)
Catalyst (g)
Solvent
Temperature (°C)
Time (h)
Isolated yield (%)
1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1.5
1 : 1
__
0.03
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
__
DMF
H2O
CH3CN
EtOH
DMSO
DMSO
120
120
120
120
120
120
110
100
130
130
120
120
120
120
120
120
120
24
1
9
3
24
1
1
1
1
1
1
1
1
1
1
1
1
30
__
2a
3
0.001 (0.1 mol%)
0.02 (1.5 mol%)
0.03 (2.5 mol%)
0.03 (2.5 mol%)
0.03 (2.5 mol%)
0.03 (2.5 mol%)
0.03 (2.5 mol%)
0.05 (4 mol%)
0.03 (2.5 mol%)
0.03 (2.5 mol%)
0.03 (2.5 mol%)
0.03 (2.5 mol%)
0.03 (2.5 mol%)
0.03 (2.5 mol%)
0.03 (2.5 mol%)
35
80
40
98
80
70
98
98
25
65
Trace
30
70
98
75
4
5b
6
7
8
9
10
11
12
13
14
15
16
17c
a
b
c
The reaction was performed in the presence of Fe3O4@APTMS-DFX. The reaction was carried out in the presence of IV. The reaction was
accomplished in the presence of CuIJOAC)2·H2O.
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Table 2 Scope of the CuIJII)/Fe3O4@APTMS-DFX promoted tetrazole synthesis of various substrates
Entry
1
Substrate
Product
Time (h)
1
Isolated yield (%)
98
2
3
1.5
1.5
95
93
4
5
6
2.5
1
92
98
90
2.5
7
4
85
8
9
3.5
4
83
80
10
5
80
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Table 2 (continued)
Entry
11
Substrate
Product
Time (h)
2
Isolated yield (%)
80
12
13
14
1
95
93
88
1.5
4
15
5
75
16
17
6
5
74
77
5-(Thiophen-2-yl)-1H-tetrazole (Table 2, entry 11). White
Fe3O4 MNPs were modified with APTMS, and then deferasirox
was conjugated to the surface primary amine groups of the
modified MNPs via amidation reaction. Finally, CuIJII) ions
were anchored onto the deferasirox moiety as a chelating
group present on the surface of the catalyst to obtain CuIJII)/
Fe3O4@APTMS-DFX (Scheme 1). The final catalyst was thor-
oughly characterized by various spectroscopy and microscopy
methods, such as FT-IR, TGA, SEM-EDS and ICP-OES
analyses.
solid, mp 206–207 °C (Lit.44 205–207 °C). FT-IR (KBr): νmax
/
cm−1 3109, 3074, 2974, 2891, 2780, 2722, 2628, 2569, 2500,
2456, 1830, 1595, 1503, 1411, 1233, 1139, 1046, 962, 853, 740,
1
719; H NMR (400 MHz, DMSO-d6, ppm) δ 7.29–7.32 (m, 1H,
Thiophen), 7.82 (dd, J = 6, 1 Hz, 1H, thiophen) 7.90 (dd, 1
Hz, 1H, thiophen).
2-(1H-Tetrazol-5-yl) pyridine (Table 2, entry 13). White
solid, mp 212–214 °C (Lit.41 210–213 °C). FT-IR (KBr): δmax
/
cm−1 3088, 3060, 2959, 2929, 2864, 2737, 2692, 2622, 2582,
1728, 1602, 1557, 1483, 1449, 1405, 1284, 1158, 1068, 1024,
1
3.1. Characterization of CuIJII)/Fe3O4@APTMS-DFX
955, 795, 743, 726, 703, 637, 496; H NMR (400 MHz, DMSO-
d6, ppm) δ 7.65 (s, 1H, Py), 8.10 (s, 1H, Py), 8.24 (d, J = 6.4
Hz, 1H, Py), 8.81 (s, 1H, Py).
Fig. 1 shows the FT-IR spectrum of the as-synthesized CuIJII)/
Fe3O4@APTMS-DFX. Fig. 1(a) shows a broad peak around 580
cm−1 attributed to the stretching vibration of Fe–O bonds for
the uncoated Fe3O4 MNPs.45 Fig. 1(b) for Fe3O4@APTMS
shows the characteristic peak of Fe3O4, which was shifted to
597 cm−1. The peaks at 1030 and 935 cm−1 were attributed to
the stretching vibration of the Si–O bonds. C–N stretching
3. Results and discussion
The present study has reported the synthesis of CuIJII)
immobilized on Fe3O4@APTMS-DFX. In the first step, the
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Scheme 1 Preparation procedure of the CuIJII) immobilized on Fe3O4@APTMS-DFX.
vibration and N–H bending vibration peaks appeared at 1150
and 1550 cm−1, repectively.46 The peaks around 2910 cm−1
and 3430 cm−1 could be attributed to the stretching vibra-
tions of CH2 and NH2 groups, respectively. The FT-IR spec-
trum of Fe3O4@APTMS-DFX (Fig. 1(c)) indicates new peaks at
1462, 1605, and 1683 cm−1, which are due to the stretching
vibration of the aromatic CC and CN of deferasirox. Also,
the stretching vibration of the amide carbonyl groups
appeared at 1710 cm−1. C–H stretching of aromatic systems
and propyl groups was observed at 2921–3100 cm−1. The
broad absorption peak at 3435 cm−1 could be attributed to
the O–H groups of deferasirox and the N–H stretching band
of the amide groups. Fig. 1(d) shows the absorption band at
450 cm−1, which was attributed to the Cu–O vibration of
CuIJII)/Fe3O4@APTMS-DFX. All the observed peaks revealed
that the surface of the Fe3O4 NPs was successfully modified
with organic moieties and CuIJII) ions were loaded onto the
modified surface of the catalyst, which were in agreement
with the results reported in the literature.47
To investigate the thermal stability of the catalyst,
thermogravimetric analysis was performed. Fig. 2(a) shows
the typical TGA curve of MNP@APTMS. The initial weight
loss up to 250 °C was probably due to the removal of surface
hydroxyl groups and/or surface adsorbed water molecules.
The weight loss at 250–500 °C could be attributed mainly to
the evaporation and subsequent decomposition of surface
bonded APTMS. The Fe3O4 MNPs can transform to Fe2O3 and
the weight would increase when the temperature is above 500
°C. As shown in Fig. 2(b), the weight loss of Fe3O4@APTMS-
Fig. 1 FTIR spectra of (a) the magnetic nanoparticles (MNPs), (b)
3-(aminopropyl)trimethoxysilane modified MNPs (MNP@APTMS), (c)
deferasirox anchored on MNP@APTMS (MNP@APTMS-DFX) and (d)
CuIJII)/Fe3O4@APTMS-DFX.
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Fig. 4 Magnetization curve of (a) bare Fe3O4 and (b) Fe3O4@APTMS-
DFX. (c) Photograph of the separation of magnetic nanoparticles with
an external magnet.
Fig. 2 TGA curve of (a) MNP@APTMS and (b) MNP@APTMS-DFX.
DFX is much higher than that of Fe3O4@APTMS in the tem-
perature range of 250–500 °C, which confirms the modifica-
tion of the Fe3O4 surfaces with organic DFX and APTMS
Species.
The morphology of the Fe3O4 nanoparticles before and af-
ter deferasirox loading was studied by scanning electron
microscopy (SEM). As shown in Fig. 3(a) and (b), the nano-
particles had a small size with a uniform spherical shape.
The energy dispersive spectrum (EDS) revealed the presence
of Fe, O, C, Si, N and Cu elements (Fig. 3c). These results
confirmed that the magnetic nanoparticles were modified
using CuIJII)/APTMS-DFX species. The ICP-OES analysis of the
catalyst resulted in a value of 5.23 wt% of Cu.
The magnetic properties of the prepared MNPs were inves-
tigated via vibrating sample magnetometry (VSM). The mag-
netic hysteresis loops of Fe3O4 and Fe3O4@APTMS-DFX are
shown in Fig. 4. The saturation magnetization (Ms) values at
room temperature were 64.9 emu g−1 and 35.1 emu g−1, re-
spectively. The Ms value of Fe3O4@APTMS-DFX was much
lower than that of the Fe3O4 MNPs due to the silica coating
and DFX loading, providing a smaller magnetic moment per
unit mass than that of ferromagnetic core regions and lead-
ing to the decreased Ms value. As shown in Fig. 4c, a dark
homogeneous dispersion was observed in the absence of an
external magnetic field, while in the presence of a magnet,
black nanoparticles were adsorbed on the wall of the tube
resulting in a transparent solution.
The efficiency of the synthesized CuIJII)-DFX-APTMS@Fe3O4
nanocatalyst was investigated for the synthesis of tetrazoles.
As a model reaction, the condensation of sodium azide and
benzonitrile was carried out in the presence of the as-
synthesized catalyst. For the optimization of the reaction con-
ditions, various parameters such as the reaction temperature,
catalyst amount and solvent were investigated as shown in
Table 1. The main product was obtained only in 30% yield
when the reaction was carried out without the catalyst in
DMSO as a solvent at 120 °C (Table 1, entry 1). For the reac-
tion performed in the presence of Fe3O4@APTMS-DFX as the
catalyst, no desired product was formed (Table 1, entry 2).
Fig. 3 SEM images of (a) MNP@APTMS and (b) Fe3O4@APTMS-DFX
and (c) EDS spectrum of CuIJII)/Fe3O4@APTMS-DFX.
Scheme 2 Plausible mechanism for the synthesis of 5-substituted-
1H-tetrazoles.
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Table 3 Comparison of various catalysts in the synthesis of 5-phenyl-1H-tetrazole
Entry
Catalysts
Solvent
Temperature (°C)
Time (h)
Yield (%)
TON
Ref.
1
2
3
4
5
6
7
8
9
Imidazole-based zwitterionic-type
Mesoporous ZnS
Cu–MCM-41
Zn/Al-HT
Amberlyst-15
Nano–ZnO
CuFe2O4
ZnO nanoflake
CuIJII)/Fe3O4@APTMS-DFX
—
120
120
140
120
85
120
120
125
120
12
36
9
12
12
14
12
14
1
84
96
92
84
92
72
82
87
98
—
2.15
—
—
—
1.54
2.05
1.77
39.7
48
49, 55
50
51
52
53, 55
54, 55
55
DMF
DMF
DMF
DMSO
DMF
DMF
DMF
DMSO
This work
Under the same reaction conditions, 98% yield was achieved
after 1 h in the presence of 0.03 g of CuIJII)/Fe3O4@APTMS-
DFX nanocatalyst (Table 1, entry 6). Different solvents such
as DMF, H2O, CH3CN, and EtOH were tested in the presence
of 0.03 g catalyst at the reflux temperature (Table 1, entries
12–15). Based on these results, DMSO was chosen as the suit-
able solvent for the synthesis of tetrazoles. In the following
study, the reaction was carried out in DMSO as a solvent at
120 °C in the presence of different catalyst amounts (0.02 g,
0.03 g, and 0.05 g). With increasing the catalyst amount up
to 0.05 g, no significant increase in the yield of main product
was observed (Table 1, entry 10). Therefore, 0.03 g of the cata-
lyst and DMSO as the solvent at 120 °C were chosen as the
optimized conditions.
The versatility of the optimized conditions for the synthesis
of tetrazole derivatives was also investigated (Table 2). The syn-
thesis of various benzonitrile derivatives and sodium azide in
DMSO at 120 °C in the presence of 0.03 g of the catalyst was
studied. Both electron-withdrawing and electron-donating
substituted aromatic benzonitriles were converted to the desired
products in excellent yields (Table 2 entries 1–14). Based on
these results, aromatic nitriles with electron withdrawing groups
(Table 2, entries 2–6, 14) reacted faster than the electron donat-
ing analogs (Table 2, entries 7–10). Additionally, heteroaromatic
nitriles were also converted to the corresponding tetrazoles in
high yields (Table 2, entries 11–13). The cycloaddition reactions
of aliphatic nitriles proceeded more slowly than the cycloaddi-
tion reactions of aromatic nitriles due to the ineffectiveness of ni-
triles with electron donating alkyl groups (Table 2, entries 15–17).
The plausible mechanistic pathways for the CuIJII)/
Fe3O4@APTMS-DFX catalyzed tetrazole synthesis reaction are
shown in Scheme 2. In the first step, the coordination of the
nitrogen atoms of nitriles with CuIJII) forms compound I
which facilitates the cyclization step and enhances the
electrophilic properties of nitriles. Then, it can react with so-
dium azide via [3 + 2] cycloaddition reaction to form interme-
diate II. Acidic work up gives III and IV. The equilibrium
leads to the formation of the more stable tautomer IV
(5-substituted-1H-tetrazole). The efficiency of our method was
compared with that using previously reported heterogeneous
catalysts in the literature (Table 3). According to these results,
all previously reported protocols suffer from long reaction
times to obtain appropriate yields, expensive and toxic
metals, and tedious purification of products. The CuIJII)/
Fe3O4@APTMS-DFX nanocatalyst gave higher yield than the
other heterogeneous catalysts in shorter reaction time. The
recyclability of the CuIJII)/Fe3O4@APTMS-DFX nanocatalyst was
studied in the model reaction. The results showed that the
catalyst had good recyclability with easy recovery and was
used for at least 5 consecutive runs without a significant de-
crease in the product yield (Fig. 5).
4. Biological studies
4.1. Cell culture methods
Human breast cancer cells MDA-MD-231 (ATCC HTB-26)and
MCF-7 (ATCC HTB-22), human cervix epithelial carcinoma
HeLa (ATCC CCL-2), human colon cancer cell line HT-29
(ATCC HTB-38), mouse neuroblastoma cell line neuro-2a
(ATCC CCL-131), and mouse fibroblast L-929 cell line (ATCC
CCL-1) were obtained from the American Type Culture Collec-
tion (ATCC; Manassas, VA, USA) and cultured at 37 °C in a
humidified atmosphere of 5% CO2 in air. HeLa cells were cul-
tured in Dulbecco's Modified Eagle's Medium (DMEM) with
0.1 mM nonessential amino acids, 2 mM L-glutamine, 1.0
mM sodium pyruvate and 5% fetal bovine serum, at 37 °C in
an atmosphere of 5% CO2. The cells were plated into 96-well
sterile plates at a density of 1 × 104 cells per well in 100 μL of
the medium and incubated for 24 h. MDA-MD-231, HT-29,
MCF-7 and neuro-2a were cultured in DMEM containing 10%
fetal bovine serum, 100 units per ml of penicillin and 100 μg
ml−1 of streptomycin. L-929 cells were cultured in RPMI-1640
Fig. 5 Recyclability of CuIJII)/Fe3O4@APTMS-DFX nanocatalyst.
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Table 4 The IC50 values in various cell lines for the synthesized compounds
IC50 SD (μM)a
Compound
MCF-7
MDA-MB-231
HT-29
HeLa
Neuro-2a
L-929
1
2
4
7
19.8 3.23
46.2 5.7
5.77 1.85
29.6 2.32
47.2 4.70
4.29 1.98
39.6 3.29
15.08 4.27
23.5 4.71
18.3 2.83
20.0 2.86
56.4 5.36
66.8 5.03
85.3 6.41
21.6 4.55
19.5 3.46
43.2 5.57
35.2 4.7
84.2 9.29
>100
>100
95.3 9.70
87.1 7.53
98.9 6.80
>100
>100
>100
>100
>100
>100
>100
0.8 0.2
58.1 5.23
54.3 4.68
65.2 7.88
16.9 2.61
5.86 1.47
65.1 5.35
45.5 4.11
57.9 5.53
39.1 4.51
0.47 0.13
9
11
Cisplatin
a
IC50 > 100 μM is considered to be inactive.
medium containing 10% fetal bovine serum, 100 units per
was evaluated by the measurement of the absorbance at 570
nm using a STAT FAX-2100 microplate reader (Awareness Tech-
nology, Palm City, FL, USA). The cell viability was calculated by
dividing the absorbance of each well by that of the control
wells (cells treated with medium containing 1% DMSO).
Each experiment was repeated at least three times and
each point was determined in at least three replicates. All the
synthesized compounds exhibited remarkable cytotoxic activ-
ity against cancer cell lines as shown in Table 4. In some
cases, their IC50 values were lower than those of cisplatin as
a popular anti-cancer drug indicating better anti-tumor ef-
fects. An interesting issue came into sight was that among
the investigated compounds, two derivatives, 7 and 11,
displayed the best results in terms of cytotoxicity. It seems
that the presence of methoxy groups play a decisive role in
the exhibition of cytotoxic effects. Cytotoxic activity was si-
multaneously measured for mouse fibroblast normal cell line
(L-929) as the control. As shown in Table 4, compounds 1, 2,
4, 7, 9 and 11 displayed less cytotoxic activity than those
against cancer cell lines making them appropriate candidates
for anti-cancer drugs. A notable point is that in the case of
cisplatin, the IC50 value against normal cell L-929 was so low
that it was impossible to make a distinction between normal
and cancer cells.
ml of penicillin and 100 μg ml−1 of streptomycin.
4.2. Cytotoxicity assay in cancer cell lines
Synthesized compounds were screened for their cytotoxic ac-
tivity against human breast cancer cells (MDA-MD-231), hu-
man cervix epithelial carcinoma (HeLa), human colon cancer
cell line (HT-29), human breast cancer cells (MCF-7), mouse
neuroblastoma cell line (neuro-2a), and mouse fibroblast
L-929 cell line. Cisplatin was used as a comparative positive
control. Cell viability was evaluated by using a colorimetric
method based on the tetrazolium salt MTT ([3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]), which
is reduced by living cells to yield purple formazan crystals.
The cells were seeded in 96-well plates at a density of 2–5 ×
104 cells of MDA-MD-231, MCF-7, HeLa, HT-29, neuro-2a and
L-929 per well in 200 μL of culture medium and left overnight
for optimal adherence. After careful removal of the medium,
200 μL of a dilution series of the compounds in fresh me-
dium were added, and incubation was performed at 37 °C in
5% CO2 for 72 h. Compounds 1, 2, 4, 7, 9 and 11 were first
solubilized in DMSO, diluted in medium and added to the
cells in the final concentrations between 20 nM and 200 μM.
The percentage of DMSO in cell culture medium did not ex-
ceed 0.3%. Cisplatin was first solubilized in saline and then
added at the same concentrations used for other compounds.
At the end of the incubation period, the medium was re-
moved and the cells were incubated with 200 μL of MTT solu-
tion (500 μg ml−1). After 3–4 h at 37 °C in 5% CO2, the me-
dium was removed and the purple formazan crystals were
dissolved in 200 μL of DMSO by shaking. The cell viability
The IC50 values in all cell lines were measured for the syn-
thesized compounds as well as for cisplatin as a comparative
control. The measurements were carried out after 72 h of in-
cubation using the concentrations of the compounds in the
range between 20 nM and 200 μM. The determined values of
IC50 for the compounds spanned between 4.29 and up to 100
μM, while those found for cisplatin as the comparative
Fig. 6 Flow cytometric results after the exposure of MDA-MB-231
cancer cells to compound 7 and cisplatin. Four areas in the diagrams
represent four different cell states: necrotic cells (Q1), late apoptotic
or necrotic cells (Q2), living cells (Q3) and apoptotic cells (Q4).
Fig. 7 Flow cytometric results after the exposure of MDA-MB-231
cancer cells to compound 11 and cisplatin. Four areas in the diagrams
represent four different cell states: necrotic cells (Q1), late apoptotic
or necrotic cells (Q2), living cells (Q3) and apoptotic cells (Q4).
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Table 5 Percentage of the cell death by either apoptosis or necrosis pathways observed by flow cytometry for compound 7
Treatment
% Vital cells
% Apoptotic cells
% Late apoptotic/necrotic cells
% Necrotic cells
Control
Cisplatin
Compound 7
81.13
33.74
19.75
8.68
33.65
44.91
9.45
31.63
34.54
0.74
0.98
0.80
Table 6 Percentages of the cell death by either apoptosis or necrosis pathways observed by flow cytometry for compound 11
Treatment
% Vital cells
% Apoptotic cells
% Late apoptotic/necrotic cells
% Necrotic cells
Control
Cisplatin
Compound 11
77.5
34.1
24.2
17.8
30.8
37.6
3.6
27.3
31.7
1.1
7.8
6.5
standard ranged between 0.47 and up to 100 μM (Table 4).
Our results indicate that the highest toxicity was observed in
the MDA-MB-231 cell line.
and the results are shown in Fig. 6 and 7. Four areas of the
diagrams stand for necrotic cells (Q1, low Annexin V-FITC
and high PI signal, left square on the top), late apoptotic or
necrotic cells (Q2, high Annexin V-FITC and high PI signal,
right square on the top), live cells (Q3, low Annexin V-FITC
and low PI signal, left square at the bottom), and apoptotic
cells (Q4, high Annexin V-FITC and low PI signal, right
square at the bottom), respectively. As shown in Fig. 5 and 6
and Tables 5 and 6, compounds 7 and 11 could induce apo-
ptosis in MDA-MB-231 cancer cells. However, the pro-
apoptotic properties need further investigation to better un-
derstand the precise mechanism of action of the tested
compounds.
4.3. Apoptosis assay for compounds 7 and 11 by flow
cytometry
In order to study the mechanism of cell toxicity of com-
pounds 1, 2, 4, 7, 9 and 11 (necrosis or apoptosis), flow cy-
tometry analysis was performed on the synthesized com-
pounds and cisplatin as a reference. These compounds were
incubated for 24 h at a concentration close to the IC50 value,
Compound 7 showed a high population of apoptotic cells
(79.45%), nearly 1.2-fold higher than cisplatin (65.28%) at
the same concentration (Fig. 6 and Table 5). Besides,
according to Fig. 7 and Table 6, compound 11 exhibited a
high population of apoptotic cells (69.3%), nearly 1.2 fold
higher than cisplatin (58.1%) at the same concentration
against the MDA-MB-231 cancer cells.
The cell death induced by the synthesized compounds was
also confirmed to be apoptotic using the TUNEL assay of ex-
posed 3'-OH termini of DNA with dUTP-FITC. As shown in
the confocal laser scanning microscopy images in Fig. 8,
compound-treated MDA-MB-231 breast cancer cells, exam-
ined for dUTP-FITC incorporation (green fluorescence) and
propidium iodide counterstaining (red fluorescence),
exhibited many apoptotic yellow nuclei (superimposed green
and red fluorescence) at 24 h after treatment.
The results demonstrated that the newly synthesized com-
pounds could induce apoptosis in the MDA-MB-231 cancer
cell line. However, the pro-apoptotic activity needs further in-
vestigation to better understand the precise mechanism of
action of these compounds. Animal study is needed before
they could be recommended for clinical trials.
Fig. 8 Compounds 7 and 11 induce apoptosis in the human breast
(MDA-MB-231) cancer cell line. The MDA-MB-231 breast cancer (A and
A' for 7 and B and B' for 11) cells were incubated with 10 μM of 7 and
11 for 24 h, fixed, permeabilized and visualized for DNA degradation in
a TUNEL assay using dUTP-labeling. Red fluorescence: nuclei stained
with propidium iodide. Green or yellow (i.e., superimposed red and
green) fluorescence: apoptotic nuclei containing fragmented DNA.
When compared with the controls, treated with 0.3% DMSO (A and B),
several cells incubated with compounds 7 and 11 (A' and B') exhibited
apoptotic nuclei.56,57
5. Conclusion
In summary, an efficient catalyst has been developed using
CuIJII) immobilized on Fe3O4@APTMS-DFX for the preparation
of 5-substituted 1H-tetrazoles derivatives as important biologi-
cally active heterocyclic compounds from nitriles and sodium
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azide. The CuIJII)/Fe3O4@APTMS-DFX nanocatalyst catalyzed
the [3 + 2] cycloaddition reaction of aromatic nitriles and so-
dium azide giving tetrazoles in excellent to good yields, with
short reaction times and easy work up. The developed nano-
catalyst can be easily separated using an external magnet and
reused for at least 5 times without the loss of its catalytic ac-
tivity. The results demonstrated that the newly synthesized
compounds could induce apoptosis in the MDA-MB-231 can-
cer cell line. But the pro-apoptotic activity needs further in-
vestigation to better understand the precise mechanism of
action of these compounds, followed by animal study, before
they could be recommended for human clinical trials.
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study (Grant No: 3/41256) by Ferdowsi University of Mashhad
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Buali Research Institute, School of Pharmacy, Mashhad Uni-
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