Iron oxide magnetic nanoparticles supported on amino propyl-functionalized KCC-1 as robust recyclable catalyst for one pot and green synthesis of tetrahydrodipyrazolopyridines and cytotoxicity evaluation

Article abstract of DOI:10.1002/aoc.5440

In this study, paramagnetic dendritic fibrous nano-silica functionalized by aminopropyltriethoxysilan (Fe₃O₄@KCC-1-nPr-NH₂) was synthesized using a novel hydrothermal protocol and used as a highly efficient, recyclable and

Full text of DOI:10.1002/aoc.5440

Received: 7 October 2019
DOI: 10.1002/aoc.5440
Revised: 25 November 2019
Accepted: 8 December 2019
F U L L P A P E R
Iron oxide magnetic nanoparticles supported on
amino propyl-functionalized KCC-1 as robust
recyclable catalyst for one pot and green synthesis of
tetrahydrodipyrazolopyridines and cytotoxicity evaluation
Sajjad Azizi | Jafar Soleymani | Mohammad Hasanzadeh
Pharmaceutical Analysis Research Center
In this study, paramagnetic dendritic fibrous nano-silica functionalized by
aminopropyltriethoxysilan (Fe3O4@KCC-1-nPr-NH2) was synthesized using a
novel hydrothermal protocol and used as a highly efficient, recyclable and
and Faculty of Pharmacy, Tabriz
University of Medical Sciences, Tabriz,
Iran
heterogeneous nanocatalyst for the synthesis of
a wide range of
Correspondence
Mohammad Hasanzadeh, Pharmaceutical
Analysis Research Center and Faculty of
Pharmacy, Tabriz University of Medical
Sciences, Tabriz, Iran.
tetrahydrodipyrazolopyridines derivatives (5a-5 m). The influence of different
reaction parameters, such as the effects of solvent, temperature, time and con-
centration of catalyst for the synthesis of tetrahydrodipyrazolopyridine
(2a) were studied. This catalyst could be reused for ten consecutive recycles
without any considerable loss in its catalytic activity. This novel synthesis
method offers some advantages including short reaction time, high yield and
simple work-up procedure. Finally, the newly synthesized tetrahydro-
dipyrazolopyridines derivatives (5a-5f) were characterized by 1H and 13C
NMR, IR and CHN.
Email: mhmmd_hasanzadeh@yahoo.com;
hasanzadehm@tbzmed.ac.ir
K E Y W O R D S
bioactive compounds, fibrous nano-silica, magnetic nanoparticle, nano-catalyst,
tetrahydrodipyrazolopyridines
1 | INTRODUCTION
conditions. Therefore, looking for simple and efficient
methods for the synthesis of pyrazolopyridines is essen-
Pyrazolopyridines represent a well-known scaffold in var-
ious bioactive compounds and have numerous pharma-
cological properties. These compounds exhibit important
tial. For these reasons, Multicomponent reactions
(MCRs) are specially well suited for variety-oriented syn-
thesis.[15,16] Thus; the synthesis of pyrazolopyridines by
the multicomponent reactions with a suitable catalyst
could enhance their efficiency from ecological points
of view.
Several kinds of catalysts have been used to advance
the reactions using MCRs (multicomponent reactions),
such as acetic acid,[17] carbonaceous material (C-
SO3H),[18] p- TSA[19] and L-Proline.[20] In recent years,
the use of nanocatalysts has increased quickly and caused
in the advancement of several active and capable
biological
properties,
including
antibacterial,[1,2]
antimicrobial,[3,4] antifungal,[5] antitumor,[6] anti-virus,[7]
anti-Leishmania,[8]
inhibitors,[9] B-Raf
HIF
1-αprolyl
hydroxylase
V600E3
inhibitors,[10] protein kinase
inhibitors,[11] PDE4B inhibitors,[12] dopaminergic proper-
ties[13] and cancer cell growth inhabitation activities.[14]
The main problems for the synthesis of pyrazolopyridine
compounds are long reaction times, utilize of non-
reusable and toxic catalyst and use of particular
Appl Organometal Chem. 2020;e5440.
wileyonlinelibrary.com/journal/aoc
© 2020 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.5440

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AZIZI ET AL.
nanocatalysts for various procedures.[21–24] These mate-
rials have several advantages over formal catalysts, such
as excellent activity and high stability. In addition, metal
nanoparticles with a superior support provides a large
area for the discovery of novel, highly active
nanocatalysts for significant and challenging reactions,
which also propose the more advantage of recyclability.
Moreover, surface functionalized mesoporous systems
have appeared as one of the most significant research
areas in the concerning of advanced functional materials.
Particularly, Polshettiwar et al reported fibrous nano-
silica (KCC-1), with the high surface area (typically
>700 m2/g), broad pore size distribution, large pore
sizes,[25,26] ease of surface modification, low density, sta-
bility and low toxicity with good biocompatibility.[9,11,12]
This Dendritic fibrous nanosilica showed special activi-
ties in vast fields such as heterogeneous catalysis,[27] gas
capture, solar energy harvesting, energy storage,[28] medi-
cal diagnosis, targeting of drugs,[7,29–31] DNA adsorption
[26] drug delivery applications,[32] bio sensing [33] and CO2
Mitigation.[34]
In this study, we reported the use of Fe3O4@KCC-
1-nPr-NH2 as an advanced novel nanocatalystis for the
synthesis of tetrahydrodipyrazolopyridine (5a-m) com-
pounds by multicomponent reaction of aromatic alde-
hydes, hydrazinehydrate (or phenylhydrazinehydrate),
ethyl acetoacetate (or diethylmalonate) and ammonium
acetate under reflux conditions in ethanol as a solvent
(Scheme 1). Finally, we investigated the anti-cancer
activity of novel synthesized pyrazolopyridines scaffold
(5a-5f) by MTT assay.
2 | EXPERIMENTAL
2.1 | Materials and methods
All of materials and solvents were purchased from
Merck, Sigma Aldrich and Fluka in high purity and used
without further purification. Melting points were mea-
sured in open capillariesusing an Electrothermal MEL-
TEMP apparatus (model 9200). FTIR spectra were
obtained with a Bruker Tensor 27 spectrometer; υ in
cm−1 in the transmission mode in spectroscopic grade
KBr tablets for all the powders. The H and 13C NMR
1
spectra were observed with a Bruker Spectrospin Avance
400 spectrometer operating at 400 MHz and 100 MHz
respectively. Elemental analyses for C, H, and N were
performed using a Heraeus CHN–O-Rapid. X-ray diffrac-
tion (XRD) patterns of KCC-1 based materials were
recorded by Siemens D 5000 X-Ray diffractometer (Texas,
USA) with a Cu Kα anode (λ = 1.54 Aꢀ) operating at
40 kV and 100 mA. The Scanning Electron Microscopy
(SEM) images and Energy Dispersive X-Ray (EDX) were
recorded with FEG-SEM MIRA3 TESCAN, Czech Repub-
lic) at 1000 kV. Transmission Electron Microscopy (TEM)
analysis was conducted on a Carl Zeiss LEO 906 electron
microscope operated at 100 kV (Oberkochen, Germany).
Brunauer–Emmett–Teller (BET) was recorded by Micro-
meritics NOVA 2000 (Florida, USA) apparatus at 77 K
using nitrogen as the adsorption gas. The particle size dis-
tribution and zeta potential values were determined
using Malvern particle size analyzer (Malvern, UK). The
purity determination of the products and reaction moni-
toring were accomplished by TLC on silica gel poly gram
SILG/UV 254 plates. MCF 7 and HT-29 were purchased
from the national cell bank of Iran (NCBI) where is
located in the Pasteur (Tehran, Iran).
In the present work, Fe3O4@KCC-1-npr-NH2 was
applied as a novel catalyst for synthesis of sulfonamide
derivatives. The use of this novel dendritic nanocatalyst
has some important advantages including produce the
products in excellent yields, simple recovery and reusabil-
ity of catalyst for ten times without significant decrease
in activity and easy separation by external magnet. Also
this method has short reaction time and simple work-up
procedure. According to the obtained results, proposed
method gave the product in high yield in comparison
with other reports.
2.2 | General procedure for the synthesis
of Fe3O4 magnetic nanoparticles
The synthesis procedure of hydrophilic nanoparticles
Fe3O4 is explained as follows:
SCHEME 1 Synthesis of tetrahydrodipyrazolopyridines (5a-m) in the presence of KCC-1-NH2- Fe3O4@KCC-1-npr-NH2 NPs

AZIZI ET AL.
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Briefly, FeCl3.6H2O (3.25 g) was dispersed along with
sodium acetate (NaAc, 6.0 g) and trisodium citrate (1.3 g)
in the ethylene glycol (100 ml) with stirring. The collected
purified by washing with ethanol and toluene several
times and dried at 80 C. Scheme 2 showes synthesize
procedure of Fe3O4@ KCC-1-npr-NH2 as an advanced
nanocatalyst.
ꢀ
yellow solution was transferred in
a Teflon-lined
stainless-steel autoclave with 200 ml capacity and the
heating process of autoclave was continued about 10 hr at
200 ꢀC. After cooling at room temperature the black prod-
uct was rinsed several times with water and ethanol.[35]
2.5 | General procedures for the
synthesize of tetrahydrodipyrazolopyridine
(5a-m)
2.3 | General procedure for the synthesis
of Fe3O4@KCC-1 magnetic nanoparticles
A mixture of ethyl acetoacetate (or diethylmalonate)
(2 mmol) (1) and hydrazine hydrate (or phenyl hydra-
zine) (2.0 mmol) (2) and Fe3O4@ KCC-1-NH2 (0.1 mg)
in EtOH (3 ml) was magnetically stirred for 5 min at
25 ꢀC followed by addition of aromatic aldehyde
(1.0 mmol) (3) and ammonium acetate (4.0 mmol) (4).
The reaction mixture was heated under reflux conditions
At first stage the mixture of 0.1 g of the Fe3O4 and 1.8 g
of urea in 15 ml of water were sonicated about
30 minutes. Then cetyl trimethyl ammonium bromide
(CTAB) 1.0 g, n-butyl alcohol 1.0 g and cyclohexane 20 g
were added respectively and stirred for 30 minutes. At
this stage tetraethyl orthosilicate (TEOS) (1.00 g) was
added drop wise and stirred with mechanical stirrer for
24 hr. at 70 ꢀC. Then, the product was washed with etha-
nol and water a few times for further process and dried
in vacuum at 80 ꢀC for 12 hr and finally calcined at
550 ꢀC for 5 hr in air to remove CTAB as template.
ꢀ
for 5–30 min and then cooled to 25 C. Then, the mix-
ture monitored by TLC and after completion of the reac-
tion, the catalyst Fe3O4@ KCC-1-NH2 MNPs were
separated by strong external magnet. The obtained pre-
cipitate was washed with EtOH to afford the pure prod-
uct and then dried well under vacuum pump. As can be
seen, the synthesis of derivative (5f) shows different
chemical structure (scheme 3).
2.4 | General procedure for the synthesis
of nanocatalyst (Fe3O4@ KCC-1-npr-NH2)
2.6 | Cell culture and biological tests
2.6.1 | General cell culture procedure
For this purpose, Fe3O4@KCC-1 (0.2 g) added in toluene
(100 ml) and sonicated for 30 min. Then, (3-aminopropyl)
triethoxysilane (APTES) (500 μL) were added in the mix-
ture and the solution was refluxed for 24 hr under N2.
After that, the product was centrifuged, and was further
The MCF 7 and HT 29 cancer cells were cultured in Ros-
well Park Memorial Institute 1640 (RPMI) media
SCHEME 2 Schematic for the synthesis of Fe3O4@KCC-1-npr-NH2
SCHEME 3 Balance of tetrahydrodipyrazolopyridines (5f)

4 of 10
AZIZI ET AL.
supplemented with 10% FBS and 1% penicillin and strep-
tomycin at 37 C under 5% CO2. The cells were washed
Si-OH groups that could grows from the center to outside.
The TEM images revealed the porous, fibrous and den-
dritics form of the magnetic nanomaterials which the
fibrous system is as a result of using the CTAB for the
MNPs design while the fibrous-sphere reveals the forma-
tion of KCC-1 based MNPs. The size of the Fe3O4 MNPs
is about 15 nm. As can be seen in Figure 1 (a and b), the
Fe3O4@KCC-1-NH2 MNPs had a constant particle size
range of 20 nm. EDX results indicate the atomic structure
of the produced compound that the KCC-1 is composed
only with Si and O. Though, the carbon is arising from
the SEM grid and CTAB as a pattern agent. Moreover,
functionalization of KCC-1 with APTES, the weight per-
cent of N, O and C are increased which confirmed suc-
cessful functionalization of KCC-1 by n-pr-NH2
(Figure S1).
The powder X-ray diffraction patterns of
Fe3O4@KCC-1-NH2 are shown in Figure S2. The XRD
pattern of Fe3O4@KCC-1-NH2 NPs was performed from
5.0ꢀ (2θ) to 100.0ꢀ (2θ) to investigate the crystallinity of
the produced nanomaterial in order to obtain additional
information about their molecule structures. As can be
observed, sample possess the diffraction peaks at (220),
(311), (400), (422), (511) and (440), which are in similarity
with the data for a standard Fe3O4 sample, as reported in
the work. The wide peak between 20ꢀ and 30ꢀ is related to
the amorphous silica and proved the effective coating of
the silica on Fe3O4 core. It is important to point out that
the XRD templates of the Fe3O4@KCC-1-NH2 NPs is sim-
ilar to the fibrous mesoporous silica with Fe3O4 core.[37]
The N2 adsorption–desorption isotherms of Fe3O4 @
KCC-1-NH2 are shown in (Figure S3 (c)). The BET and
BJH analyses of the Fe3O4 @ KCC-1-NH2 MNPs were
used to determine the porous essence of the synthesized
nanocatalyst Figure 3S. (c). The specific surface area and
porosity of the nanomaterials were determined using the
adsorption isotherm and calculated by BET. Also, BJH
technique was used to evaluate the pore volume of the
KCC-1, KCC-1-NH2 and Fe3O4 @ KCC-1-NH2 (Figure 3S.
ꢀ
with phosphate buffer solution (PBS) to remove the
excess RPMI and then the cells were treated with
ꢀ
trypsin–EDTA solution and incubated for 5 min at 37 C
under 5% CO2 to separate the cell from the flask bottom
surface. Then, the separated cancer cells were transferred
to a tube and centrifuged to wash out excess trypsin–
EDTA. Finally, the separated cells were resuspended to
the fresh RPMI medium.
2.6.2 | MTT assay
Cell viability of MCF 7and HT 29 cancer cells were tested
by adding of 1.0 × 104 cells per each well a 96-well tex-
ture plate. Afterwards, the cancer cells were incubated
for about 24 hr to grow and cover at least 40% of each
well. Then, the wells were treated with different concen-
trations of the 1,4-dihydropyridines and incubated for
48 hr. Next, the wells were washed with PBS buffer solu-
tion, and subsequently 20 μL of MTT (2.5 mg.ml−1
)
reagent and 180 μL RPMI refresh media were added to
ꢀ
each well and incubated for 4 hr at 37 C under 5% CO2.
Afterwards, the RPMI media was replaced by 200 μL of
DMSO and incubated for 0.5 rh. Finally, the absorbance
of the wells was measured at 570 nm.[36]
3 | RESULTS AND DISCUSSION
The synthesis of Fe3O4@ KCC-1-NH2 magnetic
nanocatalyst involved several stages (Scheme 3). To inves-
tigate of the morphology of Fe3O4@KCC-1-NH2 MNPs,
the FESEM images were recorded (Figure 1a). Moreover,
the structure and size of the Fe3O4@ KCC-1-NH2 MNPs
were evaluated utilizing transmission electron micros-
copy (TEM). As shown in Figure 1b, the uniform fibers of
the magnetic KCC-1 with high surface area have several
FIGURE 1 FE-SEM (a) and TEM
(b) images of Fe3O4@KCC-1-npr-NH2

AZIZI ET AL.
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TABLE 1 The effect of solvent and temperature for the
synthesis of tetrahydrodipyrazolo pyridine
(a-c)). The surface area of KCC-1, KCC-1-NH2 and
Fe3O4@ KCC-1-npr-NH2 was observed about 617, 367
and 87 m2 g−1, and also the average pore size is 5.8 nm.
The pore volumes, pore size, and surface area of KCC-1,
KCC-1-NH2 and Fe3O4 @ KCC-1-NH2 are clearly proved
by the reported results.[33,38,39]
Zeta potentials of Fe3O4 @ KCC-1-NH2 was checked
at pH 7.5 to control the surface charge to determine the
possible surface modification. The zeta potential of the
Fe3O4@KCC-1@NH2 shows positive charges which ver-
ify the anchoring amine and Fe3O4 groups on the surface
of the fibrous system, respectively.[39] Also, DLS result of
the Fe3O4@KCC-1-NH2 approves again the successful
functionalization of KCC-1 with npr-NH2 and Fe3O4
witch the first peak related to Fe3O4 and the second peak
related to KCC-NH2 (Figure S4).
After characterization of Fe3O4@ KCC-1-npr-NH2 the
catalytical performance of this nanomatrial was tested for
the synthesis of tetrahydrodipyrazolopyridines. In order
to optimize the multicomponent reaction conditions and
obtain well catalytic activity, the synthesis of tetra-
hydrodipyrazolopyridine was used as a model, and inves-
tigated under different reaction parameters including
amount of the catalyst, time, temperature, and solvent
type. Initially, the effect of solvent on the synthesis of tet-
Entry
1
Solvent
EtOH
Temp. (ꢀC)
r.t.
Yield (%)
71
98
50
76
14
18
21
33
40
32
42
20
44
33
39
60
52
2
EtOH
reflux
r.t.
3
H2O
4
H2O
reflux
r.t.
5
Cyclohexane
n-Hexane
CCl4
6
r.t.
7
r.t.
8
THF
r.t.
9
CH3CN
CH2Cl2
CHCl3
DMF
r.t.
10
11
12
13
14
15
16
17
r.t.
r.t.
r.t.
EtOAc
Toluene
DMSO
Methanol
i-PrOH
r.t.
r.t.
r.t.
r.t.
r.t.
Reaction conditions: ethyl acetoacetate (or diethylmalonate) (2 mmol),
hydrazine hydrate (or phenyl hydrazine) (2.0 mmol), aromatic aldehyde
(1.0 mmol) and ammonium acetate (4.0 mmol), Fe3O4@ KCC-1-npr-NH2
(0.1 gm), EtOH (5 ml), under reflux conditions for 30 min. Isolated yield (%).
rahydrodipyrazolopyridine
compounds using the
Fe3O4@KCC-1-npr-NH2 as a nanocatalyst was investi-
gated. It is obtained that; the solvent type does affect the
performance of the nanocatalyst. For example, cyclohex-
ane, n-Hexane and CCl4, which are non-polar solvents,
gave tetrahydrodipyrazolopyridine in a lower yield than
other solvents (Table 1, entry 5–7). Also, some aprotic
polar solvents including THF, CH3CN, CH2Cl2, CHCl3,
DMF, EtOAc, toluene and DMSO lead to low efficiency
(Table 1, entry 8–15). But, the protic solvents improved
reaction performance. Methanol and iPrOH gave
tetrahydrodipyrazolo pyridine in average yields (Table 1,
entries 16 and 17). In contrast, the utilize of water caused
in an increased yield of 76%, while the yield was consid-
erably increased up to 98% when ethanol was used as an
organic solvent in the presence of Fe3O4@KCC-1-NH2
MNPs. Also, it is found that conventional heating under
reflux conditions in ethanol as a solvent (Table 1) in
5–30 min in the presence 0.0001 g of Fe3O4@KCC-1-NH2
is optimum condition for the synthesize of
tetrahydrodipyrazolo pyridines. The role of temperature
in the synthesis of tetrahydrodipyrazolo pyridine in the
presence of Fe3O4@KCC-1-NH2 MNPs as the catalyst
was evaluated, also. In this case, the tetrahydrodipyrazolo
pyrꢀidines were obtained with excellent isolated yield at
76 C and results clearly indicated that reaction comple-
tion is related to reaction temperature. The optimum
temperature for this reaction was at 76 ꢀC. The high
temperatures lead to alterations in the efficiency of the
reaction (Table 1, entry 1 and 2). Finally, the amount of
nanocatalyst on the reaction efficiently was tested.
According to obtained results, the variation in the
Fe3O4@KCC-1-NH2 MNPs amount had a key role on the
reaction efficiency. The optimum amount of
Fe3O4@KCC-1-NH2 MNP was 0.1 mg, which obtained
the desired product in 98% yields (Figure 2). It is impor-
tant to point out that, we could achieve to excellent yields
of tetrahydrodipyrazolo pyridine synthesize using this
nanocatalyst, in 5–30 min (Figure 3).
To further investigate the efficiency of the
nanocatalyst, we compared the catalytic performance of
the newly nanocatalyst with different control experiment
and the results are shown in Table 2. Originally, a stan-
dard reaction was accomplished using KCC-1, KCC-
1-NH2, Fe3O4 and KCC-1-Fe3O4; and the results con-
firmed that the desired product was not formed (Table 2,
entries 1 and 4) after 1 hr of reaction time in any
amount. When, Fe3O4@KCC-1-NH2 was used as the
nanocatalyst, a reaction was performed and completed
(Table 2, entries 5).
Ultimately, the reaction conditions were optimized,
and to carry out this approach, we specially evaluated
this
methodology
utilizing
phenyl
hydrazine

6 of 10
AZIZI ET AL.
(or hydrazine hydrate), ethyl acetoacetate (or diethyl-
malonate), ammonium acetate, and a variety of different
substituted aromatic aldehydes in the presence of
Fe3O4@KCC-1-NH2 MNP in ethanol under reflux condi-
tions. As can be seen in Table 3, the type of substituents
on the aromatic ring and electronic effects did not show
extremely evident effects in terms of yields under the
reaction conditions. Aromatic aldehydes containing
electron-donating groups or electron-withdrawing and
even without these groups wereused and reacted well to
afford the desired tetrahydrodipyrazolo pyridines in
excellent yields with high purity.
It is undeniable that for a catalytic process, the recov-
ery and reuse of catalyst materials are highly preferable.
In this regard, the recyclability of the Fe3O4@KCC-
1-NH2 MNP was investigated using the model reaction of
phenyl hydrazine (or hydrazine hydrate), ethyl
acetoacetate (or diethylmalonate), aromatic aldehydes
and ammonium acetate under identical reaction condi-
tions. After the completion of reaction, the recovered cat-
alyst from the reaction mixture was washed with
chloroform and dried at room temperature and reused
for subsequent reactions. It is obviousthat the heteroge-
neous and magnetic property of the Fe3O4@KCC-1-NH2
MNP facilitates the effective recovery of the nanocatalyst
from the reaction mixture by external magnet during the
work-up procedure so that the catalyst could be recycled
and reused up to ten consecutive trialswithout remark-
able loss of its catalytic activity (Figure 4a and 4b) after
nine tests. Thus, these results indicated that the
nanocatalyst was stable and could tolerate the mul-
ticomponent reaction conditions.
FIGURE 2 Optimization of the conditions for synthesis of
tetrahydrodipyrazolo pyridines: ethyl acetoacetate
(or diethylmalonate) (2 mmol), hydrazine hydrate (or phenyl
hydrazine) (2.0 mmol), aromatic aldehyde (1.0 mmol) and
ammonium acetate (4.0 mmol), Fe3O4@ KCC-1-npr-NH2 (0.1 mg),
EtOH (5 ml), under reflux conditions, 1 hr
FIGURE 3 Effect of time on yield of tetrahydrodipyrazolo
pyridines: ethyl acetoacetate (or diethylmalonate) (2 mmol),
hydrazine hydrate (or phenyl hydrazine) (2.0 mmol), aromatic
aldehyde (1.0 mmol) and ammonium acetate (4.0 mmol),
Fe3O4@KCC-1-npr-NH2 (0.1 gm), EtOH (5 ml), under reflux
conditions for 30 min
In this research, we also synthesized six new
tetrahydrodipyrazolo pyridine derivatives according to
the explained method in Scheme 1. Spectroscopic data:
3.1 | 4-(1H-indol-3-yl)-3,5-dimethyl-
1,4,7,8-tetrahydrodipyrazolo[3,4-b:40,30-e]
pyridine (5a, C17H16N6)
TABLE 2 Influence of different nanocatalysts for the synthesis
of tetrahydrodipyrazol pyridine
Orange solid; m.p.: >300 ꢀC; FT-IR (KBr): νmax/cm−1
3250 (NH), 3058, 2917, 2827, 2761, 2394, 2280, 1804,
1750, 1680, 1510, 1475, 1345, 1240, 1160, 1005, 984, 751;
1H NMR (400 MHz, DMSO-d6): δ = 2.24 (s, 6H, CH3),
4.94 (s, 1H, CH, Aliphatic), 7.26–7.28 (m, 2H, Ar),
7.55–7.58 (m, 2H, Ar), 7.92 (s, 1H, Ar), 9.75 (s, 1H, NH),
10.81 (s, 1H, NH), 11.03 (s, 1H, NH), 12.77 (s, 1H, NH)
ppm; 13C NMR (100 MHz, DMSO-d6): δ = 10.31 (CH3),
13.01 (CH3), 23.08 (CH), 111.9, 112.7, 118.4, 118. 8, 121.6,
123.1, 135.1, 136.3, 136.9, 149.4, 166.3 ppm. Anal. Calcd
for C17H16N6. Calcd. C, 67.09; H, 5.30; N, 27.61.
Found, C, 66.99; H, 5.27; N, 27.69%.
Entry
Catalyst
Yield (%)
1
2
3
4
5
KCC-1
-
KCC-1-npr-NH2
Fe3O4
-
-
Fe3O4@KCC-1
Fe3O4@KCC-1-npr-NH2
-
98
Reaction conditions: ethyl acetoacetate (or diethylmalonate) (2 mmol),
hydrazine hydrate (or phenyl hydrazine) (2.0 mmol), aromatic aldehyde
(1.0 mmol) and ammonium acetate (4.0 mmol), Fe3O4@ KCC-1-npr-NH2
(0.1 gm), EtOH (5 ml), under reflux conditions for 30 min. Isolated yield (%).

AZIZI ET AL.
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TABLE 3 Synthesis of tetrahydrodipyrazolo pyridines in the presence of the Fe3O4@KCC-1-npr-NH2 catalyst in ethanol
MP (ꢀC)
Entry
Product No.
R
R0
H
R"
Product
Time (min)
Yield (%)
Found
>300
Reported
1
5a
5b
5c
Me
Me
Me
Me
Me
OEt
Me
Me
Me
Me
Me
Me
Me
1H-indole
1,10-biphenyl
1H-indole
1,10-biphenyl
4-BrC6H4
1,10-biphenyl
Ph
30
30
30
30
20
2
90
92
95
93
96
98
92
92
91
97
87
94
90
-
2
H
280–282
245–247
193–195
>300
-
3
Ph
Ph
Ph
Ph
H
-
4
5d
5e
-
5
-
6
5f
196–197
238–239
151–152
260–261
>300
-
7
5 g
5 h
5i
30
30
30
30
30
30
30
237–239[40]
251–253[41]
258–260[40]
>300[40]
181–183[40]
245–247[41]
269–271[40]
8
H
4-ClC6H4
4-FC6H4
9
H
10
11
12
13
5j
H
4-NO2C6H4
4-MeOC6H4
4-MeC6H4
4-OHC6H4
5 k
5 l
5 m
H
182–184
246–247
269–270
H
H
Reaction conditions: ethyl acetoacetate (or diethylmalonate) (2 mmol), hydrazine hydrate (or phenyl hydrazine) (2.0 mmol), aromatic aldehyde (1.0 mmol) and
ammonium acetate (4.0 mmol), Fe3O4@ KCC-1-npr-NH2 (0.1 gm), EtOH (5 ml), under reflux conditions for 2–30 min. Isolated yield (%).
FIGURE 4 (a) Reusability of
Fe3O4@ KCC-1-npr-NH2 MNPCs
and (b) separation of the
nanocatalyst by external magnet
3.2 | 4-([1,10-biphenyl]-4-yl)-3,5-dimethyl-
1,4,7,8-tetrahydrodipyrazolo[3,4-b:40,30-e]
pyridine (5b, C21H19N5)
3.3 | 4-(1H-indol-3-yl)-3,5-dimethyl-
1,7-diphenyl-
1,4,7,8-tetrahydrodipyrazolo[3,4-b:40,30-e]
pyridine (5c, C29H24N6)
White solid; m.p.: 280–282 C; FT-IR (KBr): νmax/cm−1
ꢀ
Orange solid; m.p.: 245–247 C; FT-IR (KBr): νmax/cm−1
ꢀ
3590, 3500 (NH), 3378, 3050, 2907, 2850, 2214, 1980,
1650, 1600, 1570, 1505, 1460, 1405, 1370, 1230, 1005,
954, 812; 1H NMR (400 MHz, DMSO-d6): δ = 2.18 (s, 6H,
CH3), 4.84 (s, 1H, CH, Aliphatic), 7.22 (d, J = 8.01 Hz,
2H, PH), 7.31 (t, J = 7.22 Hz,1H, PH), 7.42 (t,
J = 7.58 Hz, 2H, PH), 7.50 (d, J = 8.10 Hz, 2H, PH), 7.60
(d, J = 7.50 Hz, 2H, PH), 11.51 (b, 3H, NH) ppm; 13C
NMR (100 MHz, DMSO-d6): δ = 10.37 (CH3), 32.47 (CH),
104.21, 126.1, 126.4, 127.0, 128.0, 128.8, 137.4, 139.9,
140.2, 142.7, 161.2 ppm. Anal. Calcd for C21H19N5. Cal-
cd. C, 73.88; H, 5.61; N, 20.51. Found, C, 74.00; H,
5.58; N, 20.48%.
3750, 3500 (NH), 3200, 3007, 2950, 2904, 2870, 2300,
2280, 1690, 1646, 1600, 1475, 1390, 1376, 1358, 1200,
1103, 1002, 850, 795; 1H NMR (400 MHz, DMSO-d6):
δ = 2.41 (s, 6H, CH3), 4.12 (s, 1H, CH, Aliphatic),
7.16–7.69 (10H, Ar), 8.00–8.16 (6H, Ar), 9.8 (s, 1H, NH),
12.6 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6):
δ = 13.0 (CH3), 28.47 (CH), 112.2, 112.9, 118.0, 118.
3, 118.6, 122.1, 123.5, 123.9, 128.1, 128.6, 136.4, 137.2,
138.2, 138.9, 150.9, 162.8 ppm. Anal. Calcd for C29H24N6.
Calcd. C, 76.29; H, 5.30; N, 18.41. Found, C, 76.20; H,
5.25; N, 18.45%.

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AZIZI ET AL.
3.4 | 4-([1,10-biphenyl]-4-yl)-3,5-dimethyl-
1,7-diphenyl-
3.7 | Biological studies
1,4,7,8-tetrahydrodipyrazolo[3,4-b:40,30-e]
pyridine (5d, C33H27N5)
After
implementation
the
synthesis
of
tetrahydrodipyrazolopyridines noted in Table 3, the pre-
pared novel tetrahydrodipyrazolopyridines (5a-5f) were
tested for in vitro cytotoxicity analyses against two differ-
ent cancer cell lines; human breast adenocarcinoma
(MCF 7) and human colon adenocarcinoma (HT 29)
using the MTT assay.[36] Also doxorubicin, a well-known
anticancer agent, was used as the positive control in our
study. The antiproliferaty efficacy records are presented
as IC50 values described as the concentration of the prod-
uct that reduces cell proliferation at 50% (Table 4). The
IC50 values for cell lines were determined after at least
three individual experiments. Cell survival was
ascertained by MTT assay as explained in methods and
materials subsection.
White solid; m.p.: 193–195 C; FT-IR (KBr): νmax/cm−1
ꢀ
3760, 3400 (NH), 2960, 2850, 2280, 2904, 2870, 2300,
1
2280, 1800, 1686, 1530, 1475, 1290, 1100, 1001, 853; H
NMR (400 MHz, DMSO-d6): δ = 2.35 (s, 6H, CH3), 5.01
(s, 1H, CH, Aliphatic), 7.25–7.73 (m, 19 H, Ar), 14.04 (s,
1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ = 11.61
(CH3), 28.14 (CH), 120.5, 125.5, 126.5, 127.7, 128.8,
142.13, 146.2, 150.3 ppm. Anal. Calcd for C33H27N5. Cal-
cd. C, 80.30; H, 5.51; N, 14.19. Found, C, 81.01; H,
5.47; N, 14.21%.
3.5 | 4-(4-bromophenyl)-3,5-dimethyl-
1,7-diphenyl-
1,4,7,8-tetrahydrodipyrazolo[3,4-b:40,30-e]
pyridine (5e, C27H22BrN5)
3.8 | Cytotoxicity evaluation and the
structure activity relationships study
Yellow solid; m.p.: >300 ꢀC; 3760, FT-IR (KBr):
Novel synthetic tetrahydrodipyrazolopyridines (5a-f)
were investigated on cytotoxicity effects and some of
them showed interesting results. The results proved that
derivatives 5a, 5c and 5 f possessed considerable activity
against two cell lines (Table 4). The compound 5e
exhibited promising activities against MCF7 cell line,
while compound 5b against HT 29 cell line. During this
bioevaluation, 5c, 5 f and 5a have been recognized as the
most active products against MCF-7 and HT-29 cell lines,
respectively. It has been clear that the tetrahydro-
dipyrazolopyridines containing indole (5c and 5a) and
hydroxide (5f) moieties showed impressive cytotoxic
activity also these mentions compounds were found to be
more active among all synthetic compounds. In our
study, all the synthetic compounds were found to have
cytotoxic properties and any of these compounds were
ν
max/cm−1 3400 (NH), 2962, 2900, 2380, 2304, 1800, 1686,
1
1530, 1475, 1160, 880, 1001, 720, 660 (C-Br); H NMR
(400 MHz, DMSO-d6): δ = 2.35 (s, 6H, CH3), 5.13 (s, 1H,
CH, Aliphatic), 7.24 (t, J = 6.72 Hz, 2H, Ar), 7.44 (t,
J = 7.42 Hz,4H, Ar), 7.52 (d, J = 8.21 Hz, 2H, Ar), 7.71 (d,
J = 7.72 Hz, 4H, Ar), 8.17 (d, J = 8.30 Hz, 2H, Ar), 13.88
(s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6):
δ = 11.56 (CH3), 33.20 (CH), 103.8, 120.5, 123.3, 125.6,
128.5, 128.9,137.1, 145.9, 146.2, 150.3 ppm. Anal. Calcd
for C27H22BrN5. Calcd. C, 65.33; H, 4.47; Br, 16.10; N,
14.11. Found, C, 65.25; H, 4.45; Br, 16.17; N, 14.08%.
3.6 | 4-([1,10-biphenyl]-4-yl)-1,7-diphenyl-
1,4,7,8-tetrahydrodipyrazolo[3,4-b:40,30-e]
pyridine-3,5-diol (5f, C31H23N5O2)
TABLE 4 IC50 (μM) of the synthesized compounds against
different cancer cell lines
White solid; m.p.: 196–197 C; FT-IR (KBr): νmax/cm−1
ꢀ
3720, 3500 (NH), 3995 (OH), 3370, 3180, 3004, 2900,
2806, 2390, 2300, 2260, 1880, 1701, 1670, 1560, 1501,
1450, 1400, 1210, 1140, 1100, 900, 820, 800, 756; 1H NMR
(400 MHz, DMSO-d6): δ = 4.18 (s, 1H, CH, Aliphatic),
6.76 (t, J = 7.06, 1H, Ar), 7.12 (d, J = 7.8, 2H, Ar), 7.24 (t,
J = 7.5, 2H, Ar), 7.35 (t, J = 7.1, 1H, Ar), 7.46 (t, J = 7.5,
2H, Ar), 7,68–7.75 (m, 6H, Ar), 7.92 (s, 1H, OH), 10.43 (s,
1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ =112.0,
118.8, 126.1, 126.4, 126.8, 127.4, 128.9, 129.1, 135.0, 135.9,
139.3, 139.6, 145.2 ppm. Anal. Calcd for C35H31N5O2. Cal-
cd. C, 74.83; H, 4.66; N, 14.08; O, 6.43 Found, C, 73.99; H,
4.63; N, 14.12; O, 6.39%.
IC50 values (in μM)
Entry
Sample Code
MFC 7
HT 29
8
1
5a
8
2
5b
50
75
3
5c
2.5
2.5
12
4
5d
10
5
5e
5f
60
35
6
5
4.5
0.49
STD
Doxorubicin
2.4 0.12

AZIZI ET AL.
9 of 10
FIGURE 5 Graphical presentation of the
cytotoxic activity of the
tetrahydrodipyrazolopyridines
not inactive. In addition, the existence of a heterocyclic
moiety (5c and 5a) enhanced the cytotoxic properties
against MCF-7 and HT-29 cell lines. However, a
tetrahydrodipyrazolopyridines containing an aromatic
ring without an electron-withdrawing or even electron-
donating group (as for example, in 5b and 5d except 5 f)
was found to have less cytotoxic activities. Although the
compound with bromide (5e) moiety was also low active
against HT-29 cell line. The cytotoxic activity of all the
terahydrodipyrazolopyridines has been graphically
exhibited in Figure 5.
CONFLICT OF INTEREST
No potential conflict of interest was reported by the
authors.
ORCID
Mohammad Hasanzadeh https://orcid.org/0000-0003-
4918-1239
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
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Hasanzadeh M. Iron oxide magnetic nanoparticles
supported on amino propyl-functionalized KCC-1
as robust recyclable catalyst for one pot and green
synthesis of tetrahydrodipyrazolopyridines and
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