Synthesis of novel pyrazole derivatives and their tumor cell growth inhibitory activity

Article abstract of DOI:10.3390/molecules24020279

To find novel antitumor agents, a series of 1H-benzofuro[3,2-c]pyrazole derivatives 4a-e were designed and synthesized. The treatment of 6-methoxybenzofuran-3(2H)-one 3 with LiHMDS in anhydrous tetrahydrofuran (THF) followed by reaction with 3-substitued phenyl isothiocyanate gave the thioamide intermediates, which underwent condensation with hydrazine monohydrate in dioxane/EtOH (1:1) to provide the benzofuropyrazole derivatives 4a–e as well as the unexpected pyrazole derivatives 5a–e. In tumor cell growth inhibitory assay, all the benzofuropyrazole derivatives were not active against the breast tumor MCF-7 cell, only 4a was highly active and more potent than ABT-751 against the leukemia K562 (GI50 = 0.26 μM) and lung tumor A549 cells (GI50 = 0.19 μM), while other benzofuropyrazoles showed very weak inhibitory activity. In contrast, the pyrazoles 5a-e were in general more potent than the benzofuropyrazoles 4a–e. Compound 5a exhibited a similar tendency to that of 4a with high potency against K562 and A549 cells but weak effects on MCF-7 cell. Both pyrazoles 5b and 5e exhibited high inhibitory activities against K562, MCF-7 and A549 cells. The most active compound 5b was much more potent than ABT-751 against K562 and A549 cells with GI50 values of 0.021 and 0.69 M, respectively. Moreover, 5b was identified as a novel tubulin polymerization inhibitor with an IC50 of 7.30 M.

Full text of DOI:10.3390/molecules24020279

Article
Synthesis of Novel Pyrazole Derivatives and Their
Tumor Cell Growth Inhibitory Activity
Ying-Jie Cui, Long-Qian Tang, Cheng-Mei Zhang and Zhao-Peng Liu*
Institute of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of
Pharmaceutical Sciences, Shandong University, Jinan 250012, China; yingjiecui@sina.cn (Y.-J.C.),
lqtang.student@sina.com (L.-Q.T.), zhangcm@sdu.edu.cn (C.-M.Z.)
*
Correspondence:Correspondence: liuzhaop@sdu.edu.cn; Tel. +86-531-8838-2006; Fax: +86-531-8838-2548
Academic Editor: Carla Boga and Gabriele Micheletti
Received: 31 October 2018; Accepted: 10 January 2019; Published: 13 January 2019
Abstract: To find novel antitumor agents, a series of 1H-benzofuro[3,2-c]pyrazole derivatives 4a-e
were designed and synthesized. The treatment of 6-methoxybenzofuran-3(2H)-one 3 with LiHMDS
in anhydrous tetrahydrofuran (THF) followed by reaction with 3-substitued phenyl isothiocyanate
gave the thioamide intermediates, which underwent condensation with hydrazine monohydrate in
dioxane/EtOH (1:1) to provide the benzofuropyrazole derivatives 4a–e as well as the unexpected
pyrazole derivatives 5a–e. In tumor cell growth inhibitory assay, all the benzofuropyrazole
derivatives were not active against the breast tumor MCF-7 cell, only 4a was highly active and more
potent than ABT-751 against the leukemia K562 (GI50 = 0.26 μM) and lung tumor A549 cells (GI50
=
0
.19 μM), while other benzofuropyrazoles showed very weak inhibitory activity. In contrast, the
pyrazoles 5a-e were in general more potent than the benzofuropyrazoles 4a–e. Compound 5a
exhibited a similar tendency to that of 4a with high potency against K562 and A549 cells but weak
effects on MCF-7 cell. Both pyrazoles 5b and 5e exhibited high inhibitory activities against K562,
MCF-7 and A549 cells. The most active compound 5b was much more potent than ABT-751 against
K562 and A549 cells with GI50 values of 0.021 and 0.69 M, respectively. Moreover, 5b was identified
as a novel tubulin polymerization inhibitor with an IC50 of 7.30 M.
Keywords: privileged structure; pyrazoles; tubulin inhibitors; antitumor; antitumor agents
1
. Introduction
Privileged structures are defined as molecular frameworks that are able to provide useful
ligands for multiple types of receptors or enzymes through proper structural modifications. In
combination with their favorable drug-like properties, privileged structures or scaffolds are widely
used in rational drug design to find new lead compounds or drug candidates [1–3]. Pyrazole
derivatives represent one of the most active classes of compounds that possess a wide spectrum of
biological activities, including antibacterial and antifungal [4,5], antitumor [6,7], anti-inflammatory
and analgesic [8,9], antitubercular [10], antiviral [11,12], anti-Alzheimer's [13,14], α-glucosidase
inhibitory [15], anti-diabetic [16], antileishmanial [17,18], anti-malarial [19], radioimaging [20],
acaricidal and insecticidal [21,22] activities. As a privileged scaffold, pyrazole has been recently
widely used in the design of anticancer agents for a multiple of tumor targets [23].
N-(2-((4-Hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide (ABT-751, 1,
Figure 1) is an orally available sulfonamide tubulin inhibitor under clinical investigations for the
treatment of cancers [24,25]. On its X-ray crystal structures with tubulin, ABT-751 interacted with all
the three pockets of tubulin at the colchicine binding site [26]. Based on the binding mode of 1 with
tubulin and the pyrazole pharmacophore, our group designed and synthesized a series of
Molecules 2019, 24, 279; doi:10.3390/molecules24020279
www.mdpi.com/journal/molecules

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indenopyrazoles as potential tubulin polymerization inhibitors targeting the colchicine binding site
27]. The indenopyrazole analogue 2 (Figure 1) was found to compete with colchicine in binding to
[
the tubulin colchicine site and inhibit the polymerization of tubulin. In vitro, 2 displayed nanomolar
potency against a variety of tumor cell lines, arrested tumor cells in G2/M phase through the
regulation of cell cycle-related proteins, and induced tumor cell apoptosis through the activation of
caspase pathways. Furthermore, 2 was effective for multidrug resistance tumor cells and inhibited
phosphatase and tensin homolog (PTEN) phosphorylation and PTEN/Akt/NF-кB signaling [28]. In
vivo, 2 demonstrated its potency in non-small cell lung cancer (NSCLC) and vincristine-resistance
human oral epidermoid carcinoma cell (KB/V) xenograft models without obvious side effects [27,28].
In this study, we replaced the indenopyrazole core with the 1H-benzofuro[3,2-c]pyrazole framework
and designed a series of the 1H-benzofuro[3,2-c]pyrazole derivatives 4a–e. In the preparation of 4a–
e, the partial cleavage of the furan ring was observed and a series of 5-methoxy-2-(3-(phenylamino)-
1
H-pyrazol-5-yl)phenol derivatives 5a–e were isolated. We reported here the synthesis and
preliminary results of their tumor cell growth inhibitory activity as well as the identification of
pyrazole 5b as a novel tubulin polymerization inhibitor.
Figure 1. Tubulin inhibitors 1 and 2.
2
. Results and Discussion
2
.1. Chemistry
The synthetic route towards the benzofuropyrazole derivatives was shown in Scheme 1. The 6-
methoxybenzofuran-3‐(2H)-one 3 was prepared according to the reported method in three steps [29–
1] (please refer to the supplementary materials). The Hoesch reaction of resorcinol with
chloroacetonitrile in the presence of anhydrous ZnCl and HCl gas generated an imine intermediate,
which upon hydrolysis, provided 2-chloro-1-(2,4-dihydroxyphenyl)ethanone in 94% yield.
Treatment of the chloromethyl ketone with a mild base, CH COONa, gave the cyclized 6-
hydroxybenzofuran-3-(2H)-one (54%). Methylation of 6-hydroxybenzofuran-3-(2H)-one with Me SO
3
2
3
2
4
produced the 6-methoxybenzofuran-3-(2H)-one 3 in 86% yield. After the deprotonation of the α-
proton of carbonyls in 3 with lithium hexamethyldisilazide (LiHMDS), the resulting enolates were
reacted with 3-substitued phenyl isothiocyanates to give the thioamide intermediates, which
underwent condensation with hydrazine monohydrate in dioxane/EtOH (1:1) to form the
benzofuropyrazole derivatives 4a–e in 11% to 30% yield. In this process, the partial cleavage of the
furan ring occurred, a series of 5-methoxy-2-(3-(phenylamino)-1H-pyrazol-5-yl)phenol derivatives
5
5
a–e were also isolated in 13% to 31% yield. The structures of benzofuropyrazoles 4a–e and pyrazoles
1
13
a–e were determined by H nuclear magnetic resonance (NMR), C-NMR and electrospray
1
ionization mass spectrometry (ESI-MS). In the H NMR spectrum of benzofuropyrazole 4c, the
pyrazole 1-NH appeared at 11.91 ppm, the aniline NH at 8.05 as a singlet, the amide NH at 8.28 (q, J
=
4.6 Hz) in corresponding with the N-methyl at 2.77 (d, J = 4.6 Hz). The seven protons at the two
1
phenyl rings appeared at 6.42 to 7.82 ppm. In comparison with 4c, in the H NMR spectrum of
pyrazole 5c, there were two additional peaks at 10.23 ppm for the phenol OH, 6.24 ppm for the
pyrazole 4-H, supporting its estimated structure.

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Scheme 1. Synthesis of benzofuropyrazole and pyrazole derivatives.
2
.2. Tumor Cell Growth Inhibitory Activity
All the synthesized compounds were evaluated for their tumor cell growth inhibitory activity
against human breast cancer MCF-7 cell, human erythroleukemia K562 cell and human lung cancer
A549 cell by the conventional MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide) assay. ABT-751 was used as positive control.
As shown in Table 1, the breast tumor MCF-7 cell was not sensitive to the benzofuropyrazole
derivatives 4a–e. For the K562 and A549 cells, only 4a exhibited high potency and was more potent
than ABT-71 with the GI50 of 0.26 and 0.19 μM, respectively, while other benzofuropyrazoles showed
moderate or weak activity. It seems the substitution of the ethoxy at the aniline ring with the electron-
withdrawing ester, amide, and cyano groups was not tolerated among the benzofuropyrazole series.
In contrast, all the three tumor cell lines were sensitive to the pyrazole analogues 5a–e. The methyl
ester 5b was the most active that inhibited the K562, MCF-7, and A549 cell growth with GI50 values
of 0.021, 1.7 and 0.69 μM, respectively. Both compounds 5a and 5b were highly active against the
K562 and A549 cells, and were 5- to 35-fold more potent than ABT-751. The cyano derivative 5e was
also highly potent against the three tumor cell lines, although it showed slightly less potency than
ABT-751. Unlike the benzofuropyrazole derivatives, the substitution of the ethoxy at the aniline ring
with an electron-withdrawing ester, amide, and cyano group in the pyrazole series was well
tolerated, and even preferred, indicating that the benzofuropyrazole derivatives 4a–e and the
pyrazoles 5a–e might involve different mechanisms of action.
Table 1. Tumor cell growth inhibitory activity of 4a–e and 5a–e.
GI50 (M)
Compound
R
K562
MCF-7
A549
4
a
OCH
COOCH
CONHCH
2
CH
3
0.26 ± 0.04
5.46 ± 1.04
5.11 ± 0.31
9.01 ± 1.81
13.53 ± 0.41
0.046 ± 0.007 16.72 ± 2.6 0.92 ± 0.17
0.021 ± 0.004 1.7 ± 0.43 0.69 ± 0.18
7.33 ± 1.004 7.78 ± 0.87 9.46 ± 2.03
14.77 ± 2.62 5.8 ± 0.202 10.9 ± 0.99
1.45 ± 0.047 2.27 ± 0.34 3.24 ± 0.99
0.74 ± 0.078 0.88 ± 0.24 4.58 ± 0.04
>20
>20
>20
>20
0.19 ± 0.08
>20
15.11 ± 2.18
10.08 ± 2.21
17.01 ± 2.76
4
b
3
4
c
3
4
d
CONH
CN
2
4
5
e
a
>20
OCH
COOCH
CONHCH
CONH
CN
2
CH
3
5
b
3
5
c
3
5
d
2
5
e
ABT-751
2
.3. In Vitro Tubulin Polymerization Inhibitory Activity
The pyrazole derivative 5b showed the best tumor cell growth inhibitory activity among all the
tested compounds. To investigate whether 5b was a tubulin inhibitor, the tubulin polymerization
inhibition assay was carried out. At 37 °C, tubulin will polymerize into microtubules, which is
followed by the observed fluorescence enhancement due to the incorporation of a fluorescent

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reporter into microtubules as polymerization occurs [32]. As shown in Figure 2, 5b inhibited the
tubulin polymerization in a concentration-dependent way with a calculated IC50 of 7.30 μM.
Therefore, 5b may be a good lead for further structural modification to find more potent tubulin
inhibitors based on the privileged pyrazole structure.
Figure 2. Effect of 5b on tubulin polymerization in vitro. Purified tubulin protein at 2 mg/mL in a reaction buffer
was incubated at 37 °C in the presence of 1% dimethyl sulfoxide (DMSO), test compound 5b at 1.25, 2.5, 5, or 10
μM or colchicine at 20 μM. The fluorescence intensity was measured every 60 second for 60 min and is presented
as increases in the polymerized microtubule.
3
. Materials and Methods
3
.1. General Chemical Experimental Procedures
Melting points were determined on an X-6 micromelting point apparatus (Beijing Tech. Co.,
1
13
Ltd.). H- and C-NMR spectra were recorded on Bruker-400 NMR or Bruker-600 NMR
spectrometers. All spectra were recorded at room temperature for DMSO or CDCl solutions. ESI-MS
3
was performed on an API 4000 instrument. Thin-layer chromatography (TLC) was performed on
silica gel GF254 plates. Silica gel GF254 and silica gel (200−300 mesh) from Qingdao Haiyang
Chemical Company were used for TLC and column chromatography, respectively. All reagents were
commercially available and were used as purchased without further purification. All reactions
involving oxygen- or moisture sensitive compounds were carried out under a dry N atmosphere.
2
Unless otherwise noted, reagents were added by syringe. Tetrahydrofuran (THF) was distilled from
sodium/benzophenone immediately prior to use.
3
-((6-Methoxy-1H-benzofuro[3,2-c]pyrazol-3-yl)amino)ethoxybenzene
(4a)
and
3-((5-(2-hydroxy-4-
methoxyphenyl)-1H-pyrazol-3-yl)amino)ethoxybenzene (5a) A solution of 6-methoxybenzofuran-3-(2H)-
one 3 (150 mg, 0.9 mmol) in anhydrous THF (5 mL) was cooled to −78 °C under nitrogen atmosphere.
LiHMDS (1.09 mL, 1.1 mmol, 1.0 M THF solution) was added dropwise. The mixture was stirred at
−
78 °C for 2 h and then warmed to −45 °C in 45 min. After a solution of the 1-ethoxy-3-
isothiocyanatobenzene (164 mg, 1.0 mmol) in anhydrous THF (3 mL) was added, the resulting
mixture was stirred at room temperature overnight. Water (30 mL) was added, and the mixture was
extracted with EtOAc (3 × 30 mL). The organic layer was washed with brine, dried over anhydrous
Na
2
SO
4
. After filtration and evaporation, flash column chromatography on silica gel (hexane/EtOAc
=
15:1) gave the resulting thioamide intermediate, which was dissolved in 1,4-dioxane (3 mL) and
ethanol (3 mL). Hydrazine hydrate (0.46 mL, 7.3 mmol) was added dropwise. The mixture was heated
to 50 °C and stirred for 24 h. Water (40 mL) was added, and the mixture was extracted with EtOAc
(
3 × 40 mL). The organic layer was washed with brine, and dried over anhydrous Na
filtration and evaporation, the residue was purified by column chromatography on silica gel
hexane/EtOAc = 4:1) to give 4a (32 mg, 11%) and 5a (38 mg, 13%).
2
SO . After
4
(

Molecules 2019, 24, 279
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1
4
a: Brown solid, m.p.: 62–64 °C. H-NMR (CDCl
3
) δ 7.32 (d, J = 8.6 Hz, 1H, Ar-H), 7.08 (t, J = 8.1
Hz, 1H, Ar-H), 6.56 (d, J = 2.4 Hz, 1H, Ar-H), 6.46 (dd, J = 2.4, 8.6 Hz, 1H, Ar-H), 6.36 (d, J = 8.2 Hz,
1
H, Ar-H), 6.25 (d, J = 8.1 Hz, 1H, Ar-H), 6.21 (s, 1H, Ar-H), 5.09 (s, 1H, 3-NH), 3.96 (q, J = 6.9 Hz, 2H,
1
3
OCH
2
), 3.81 (s, 3H, 6’-OCH
3
), 1.37 (t, J = 6.9 Hz, 3H, OCH
2
CH
3
). C-NMR (CDCl ) δ 161.6, 160.3, 156.6,
3
1
46.0, 137.8, 130.2, 129.6, 127.9, 125.8, 111.5, 109.5, 107.0, 105.1, 103.7, 101.0, 63.3, 55.3, 14.9. MS (ESI)
+
calcd. for C18
H
19
N
3
O3
[M + NH
4] : 341.1, found: 341.4.
1
5
a: Brown solid, m.p.: 82–84 °C. H-NMR (CDCl
3
) δ 7.39 (d, J = 8.6 Hz, 1H, Ar-H), 7.17 (t, J = 8.0
Hz, 1H, Ar-H), 6.57–6.49 (m, 5H, Ar-H), 6.22 (s, 1H, 4’-H), 5.79 (s, 1H, 3-NH), 4.01 (q, J = 6.9 Hz, 2H,
1
3
1
-OCH
2
), 3.79 (s, 3H, 4’’-OCH
3
), 1.41 (t, J = 6.9 Hz, 3H, 1-CH
3
). C-NMR (CDCl ) δ 160.7, 160.2, 157.0,
3
1
C
43.9, 130.4, 127.6, 109.8, 108.7, 107.1, 106.5, 102.8, 101.7, 90.4, 63.3, 55.3, 15.4. MS (ESI) calcd. for
+
18
H
19
N
3
O3
[M + H] : 326.1, found: 326.4.
Methyl 3-((6-methoxy-1H-benzofuro[3,2-c]pyrazol-3-yl)amino)benzoate (4b) and methyl 3-((5-(2-hydroxy-4-
methoxyphenyl)-1H-pyrazol-3-yl)amino)benzoate (5b) According to the procedures described for the
synthesis of 4a and 5a, compounds 4b and 5b were prepared from 3 (100 mg, 0.6 mmol), LiHMDS
(
0.7 mL, 0.7 mmol), methyl 3-isothiocyanatobenzoate (135 mg, 0.7 mmol) and hydrazine hydrate (0.3
mL, 4.8 mmol). The crude residue was purified by column chromatography on silica gel
(hexane/EtOAc = 3:1) to give 4b (36 mg, 18%) and 5b (33 mg, 16%).
1
4
b: Brown solid, m.p.: 103–105 °C. H-NMR (DMSO-d
6
) δ 11.96 (s, 1H, 1’-NH), 8.19 (s, 1H, 3-NH),
8
.13 (s, 1H, Ar-H), 7.61 (s, 1H, Ar-H), 7.43 (d, J = 8.3 Hz, 1H, Ar-H), 7.33 (d, J = 4.3 Hz, 2H, Ar-H), 6.50
), 3.74 (s, 3H, OCH ).
(
dd, J = 1.9, 8.5 Hz, Ar-H), 6.42 (d, J = 2.0 Hz, 1H, Ar-H), 3.84 (s, 3H, COOCH
3
3
1
3
C-NMR (DMSO-d
6
) δ 167.2, 161.1, 156.9, 145.9, 144.6, 134.1, 130.6, 129.6, 128.3, 119.8, 119.2, 115.6,
+
1
10.4, 109.1, 106.2, 103.6, 55.5, 52.5. MS (ESI) calcd. for C18
H
15
N
3
O4
[M + NH
4
] : 355.1, found: 355.5.
1
5
b: Yellow solid, m.p.: 185–186 °C. H-NMR (DMSO-d
6) δ 12.01 (s, 1H, 1’-NH), 10.24 (s, 1H, 2’’-
OH), 8.67 (s, 1H, 3-NH), 8.13 (s, 1H, Ar-H), 7.57 (s, 1H, Ar-H), 7.53 (d, J = 6.8 Hz, 1H, Ar-H), 7.31 (s,
2
H, Ar-H), 6.51 (s, 1H, Ar-H), 6.50 (dd, J = 1.9, 6.9 Hz, 1H, Ar-H), 6.23 (s, 1H, 4’-H), 3.84 (s, 3H,
1
3
COOCH
3
), 3.74 (s, 3H, OCH
3
). C-NMR (DMSO-d
6
) δ 167.2, 160.3, 155.8, 151.6, 144.8, 139.8, 130.7,
[M +
1
29.4, 128.4, 119.7, 118.8, 115.5, 110.0, 105.8, 102.1, 93.0, 55.5, 52.2. MS (ESI) calcd. for C18
H
17N
3O4
+
H] : 340.1, found: 340.4.
3
-((6-Methoxy-1H-benzofuro[3,2-c]pyrazol-3-yl)amino)-N-methylbenzamide (4c) and 3-((5-(2-hydroxy-4-
methoxyphenyl)-1H-pyrazol-3-yl)amino)-N-methylbenzamide (5c) According to the procedures described
for the synthesis of 4a and 5a, compounds 4c and 5c were prepared from 3 (100 mg, 0.6 mmol),
LiHMDS (0.7 mL, 0.7 mmol), 3-isothiocyanato-N-methylbenzamide (134 mg, 0.7 mmol) and
hydrazine hydrate (0.3 mL, 4.8 mmol). The crude residue was purified by column chromatography
on silica gel (hexane/EtOAc = 1:1) to give 4c (30 mg, 15%) and 5c (33 mg, 16%).
1
4
c: Brown solid, m.p.: 180–182 °C. H-NMR (DMSO-d
6
) δ 11.91 (s, 1H, 1’-NH), 8.28 (q, J = 4.6 Hz,
1
H, CONH), 8.05 (s, 1H, 3-NH), 7.82 (s, 1H, Ar-H), 7.55 (s, 1H, Ar-H), 7.43 (d, J = 5.7 Hz, 1H, Ar-H),
7
.27 (t, J = 7.8 Hz, 1H, Ar-H), 7.15 (d, J = 7.6 Hz, 1H, Ar-H), 6.49 (dd, J = 2.6, 8.6 Hz, 1H, Ar-H), 6.42 (d,
1
3
J = 2.6 Hz, 1H, Ar-H), 3.74 (s, 3H, OCH
3), 2.77 (d, J = 4.6 Hz, 3H, N-CH
3
). C-NMR (DMSO-d ) δ 167.8,
6
1
2
61.1, 156.9, 146.1, 144.5, 135.9, 134.2, 129.1, 128.3, 117.4, 116.7, 114.5, 110.5, 109.1, 106.1, 103.6, 55.5,
+
6.7. MS (ESI) calcd. for C18
H
16
N
4
O3
[M + NH
4] : 354.1, found: 354.4.
1
5
c: Brown solid, m.p.: 184–186 °C. H-NMR (DMSO-d
6
) δ 11.94 (s, 1H, 1’-NH), 10.23 (s, 1H, OH),
8
.52 (q, J = 3.6 Hz, 1H, CONH), 8.25 (s, 1H, 3-NH), 7.81 (s, 1H, Ar-H), 7.52 (s, 2H, Ar-H), 7.25 (s, 1H,
Ar-H), 7.11 (s, 1H, Ar-H), 6.51 (s, 1H, Ar-H), 6.49 (dd, J = 1.6, 6.9 Hz, 1H, Ar-H), 6.24 (s, 1H, 4’-H), 3.74
1
3
(s, 3H, OCH
3
), 2.76 (d, J = 3.6 Hz, 3H, N-CH
3
). C-NMR (DMSO-d
6
) δ 170.0, 160.3, 155.8, 151.8, 144.6,
1
C
39.7, 136.1, 129.0, 128.4, 117.6, 116.4, 114.3, 110.2, 105.8, 102.1, 93.0, 55.5, 26.7. MS (ESI) calcd. for
+
18
H
18
N
4
O3 [M + H] : 339.1, found: 339.1.
3
-((6-Methoxy-1H-benzofuro[3,2-c]pyrazol-3-yl)amino)benzamide
(4d)
and
3-((5-(2-hydroxy-4-
methoxyphenyl)-1H-pyrazol-3-yl)amino)benzamide (5d) According to the procedures described for the
synthesis of 4a and 5b, compounds 4d and 5d were prepared from 3 (100 mg, 0.6 mmol), LiHMDS

Molecules 2019, 24, 279
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(
4
=
0.7 mL, 0.7 mmol), 3-isothiocyanatobenzamide (124 mg, 0.7 mmol) and hydrazine hydrate (0.3 mL,
.8 mmol). The crude residue was purified by column chromatography on silica gel (hexane/EtOAc
1:3) to give 4d (59 mg, 30%) and 5d (61 mg, 31%).
1
4
d: Brown solid, m.p.: 172–174 °C. H-NMR (DMSO-d
6
) δ 12.02 (s, 1H, 1’-NH), 8.12 (s, 1H, 3-NH),
, Ar-
) δ 169.0,
7
.82 (s, 2H, Ar-H), 7.52 (s, 1H, Ar-H), 7.43 (d, J = 2.9 Hz, 1H, Ar-H), 7.26–7.21 (m, 3H, 1-CONH
H), 6.51 (d, J = 5.7 Hz, 1H, Ar-H), 6.44 (s, 1H, Ar-H), 3.74 (s, 3H, OCH
2
1
3
3
). C-NMR (DMSO-d
6
1
61.0, 156.9, 146.1, 144.5, 135.6, 134.2, 129.0, 128.3, 117.7, 117.2, 114.8, 110.5, 109.2, 106.1, 103.6, 55.5.
+
MS (ESI) calcd. for C17
H
14N
4
O3
[M + NH
4
] : 340.1, found: 340.1.
1
5
d: Brown solid, m.p.: 128–130 °C. H-NMR (DMSO-d
6
) δ 11.84 (s, 1H, 1’-NH), 8.56 (s, 1H, 3-NH),
7
3
.83 (s, 1H, Ar-H), 7.75 (s, 1H, Ar-H), 7.54 (d, J = 6.8 Hz, 1H, Ar-H), 7.43 (s, 1H, Ar-H), 7.26-7.21 (m,
H, 1-CONH , Ar-H), 6.51 (s, 1H, Ar-H), 6.49 (d, J = 6.8 Hz, 1H, Ar-H), 6.29 (s, 1H, 4’-H), 3.74 (s, 3H,
2
1
3
OCH
5
3
). C-NMR (DMSO-d
6
) δ 169.1, 160.3, 144.5, 135.8, 129.1, 128.3, 117.8, 114.6, 110.3, 105.8, 102.1,
+
5.5. MS (ESI) calcd. for C17
H
16
N
4
O3
[M + H] : 325.1, found: 325.1.
3
-((6-Methoxy-1H-benzofuro[3,2-c]pyrazol-3-yl)amino)benzonitrile
(4e)
and
3-((5-(2-hydroxy-4-
methoxyphenyl)-1H-pyrazol-3-yl)amino)benzonitrile (5e) According to the procedures described for the
synthesis of 4a and 5b, compounds 4e and 5e were prepared from 3 (100 mg, 0.6 mmol), LiHMDS
(
4
=
0.7 mL, 0.7 mmol), 3-isothiocyanatobenzonitrile (112 mg, 0.7 mmol) and hydrazine hydrate (0.3 mL,
.8 mmol). The crude residue was purified by column chromatography on silica gel (hexane/EtOAc
1:4) to give 4e (31 mg, 17%) and 5e (33 mg, 18%).
1
4
e: White solid, m.p.: 200–202 °C. H-NMR (DMSO-d
6
) δ 12.01 (s, 1H, 1’-NH), 8.39 (s, 1H, 3-NH),
7
.91( s, 1H, Ar-H), 7.53(d, J = 6.9 Hz, 1H, Ar-H), 7.43-7.38 (m, 2H, Ar-H), 7.15 (d, J = 7.4 Hz, 1H, Ar-
1
3
H), 6.51 (dd, J = 2.5, 8.6 Hz, 1H, Ar-H), 6.44 (d, J = 2.5 Hz, 1H, Ar-H), 3.75 (s, 3H, OCH ). C-NMR
3
(
1
DMSO-d
6
) δ 161.1, 156.9, 145.2, 145.0, 134.0, 130.5, 128.5, 121.7, 120.0, 119.9, 117.2, 112.0, 110.3, 109.7,
+
06.2, 103.6, 55.5. MS (ESI) calcd. for C17
H
12
N
4
O2
[M + NH
e: White solid, m.p.: 166–168 °C. H-NMR (DMSO-d
.93 (s, 1H, 3-NH), 7.99 (s, 1H, Ar-H), 7.55 (s, 1H, Ar-H), 7.53 (s, 1H, Ar-H), 7.38 (s, 1H, Ar-H), 7.12 (d,
4] : 322.1, found: 322.1.
1
5
6
) δ 12.05 (s, 1H, 1’-NH), 10.28 (s, 1H, OH),
8
J = 4.4 Hz, 1H, Ar-H), 6.53 (s, 1H, Ar-H), 6.51 (dd, J = 1.3, 5.7 Hz, 1H, Ar-H), 6.25 (s, 1H, 4’-H), 3.78 (s,
1
3
3
1
H, OCH
3
). C-NMR (DMSO-d
6
) δ 160.4, 155.8, 151.2, 145.0, 140.0, 130.4, 128.5, 121.3, 120.0, 117.3,
+
12.0, 109.8, 105.8, 102.1, 92.8, 55.5. MS (ESI) calcd. for C17
H
14
N
4
O2
[M + H] : 307.1, found: 307.3.
3
.2. MTT Assay
The human tumor cell lines, were grown in Roswell Park Memorial Institute (RPMI) 1640
medium and supplemented with 10% foetal bovine serum in the 37 °C in an atmosphere containing
5
2
% CO . All the synthesized compounds were assayed by conventional 3-(4,5-dimethyl-2-thiazolyl)-
2
,5-diphenyl-2H-tetrazolium bromide (MTT) method. In brief, the exponentially growing cells were
3
seeded into 96-well cell plates at a density of 4−4.5 × 10 cells per well and allowed to adhere
overnight. Cells were incubated with various concentrations of the test compounds for 72 h. Then 20
μL of MTT (2.5 mg/mL) was added, the cells were incubated at 37 °C for another 4 h. The reduced
MTT crystals were dissolved in DMSO, and the absorbance was measured at 570 nm by a microplate
spectrophotometer. The growth in inhibitory effects of each compound were expressed as GI50 values,
which represent the molar drug concentrations required to cause 50% tumor cell growth inhibition.
3
.3. In Vitro Tubulin Polymerization Inhibition Assay
The fluorescence-based in vitro tubulin polymerization assay was performed using the Tubulin
Polymerization Assay Kit (BK011P, Cytoskeleton, USA) according to the manual. The tubulin
reaction mix contained 2 mg/mL porcine brain tubulin (>99% pure), 2 mM MgCl , 0.5 mM ethylene
glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM guanosine triphosphate
GTP), and 15% glycerol. First, a 96-well plate was incubated with 5 μL of inhibitors in different
concentrations at 37 °C for 1 min. Then 50 μL of the tubulin reaction mix was added. Immediately,
2
(

Molecules 2019, 24, 279
7 of 9
the increase in fluorescence was monitored by excitation at 355 nm and emission at 460 nm in a
multimode reader.
4
. Conclusions
In the synthesis of 1H-benzofuro[3,2-c]pyrazole derivatives 4a–e, the furan ring-opening was
observed, and a series of pyrazole derivatives 5a–e were identified. In the tumor cell growth
inhibitory assay, only 4a was highly active towards the K562 and A549 cells, while other
benzofuropyrazole derivatives were not active or showed weak activity. In general, the pyrazoles 5a-
e were more potent than the corresponding benzofuropyrazole derivatives. Compound 5a exhibited
a similar tendency to that of 4a with high potency against K562 and A549 cells but weak effects on
MCF-7 cells. Both pyrazoles 5b and 5e exhibited high inhibitory activities against K562, MCF-7 and
A549 cells. The most active compound 5b was 5- to 35-fold more potent than ABT-751 in the inhibition
of A549 and K562 cells. In addition, 5b inhibited tubulin polymerization inhibition with an IC50 of
7
.30 M. These results indicated that 5b was a novel tubulin polymerization inhibitor and it may be
a good lead for the discovery of novel pyrazoles as potent anticancer agents.
Supplementary Materials: Supplementary materials are available online.
Author Contributions: Conceptualization, Y.-J.C. and Z.-P.L.; methodology, Y.-J.C.; validation, Y.-J. C., L.-Q.T.,
C.-M.Z. and Z.-P.L; formal analysis, L.-Q.T.; investigation, Y.-J.C., L.-Q.T. and C.-M.Z.; resources, C.-M.Z.; data
curation, Y.-J.C.; writing—original draft preparation, Y.-J.C.; writing—review and editing, Z.-P.L.; visualization,
Y.-J.C.; supervision, Z.-P.L.; project administration, Z.-P.L.; funding acquisition, Z.-P.L.
Funding: This work was partially supported by the National Natural Science Foundation of China (NSFC, Grant
No. 81573275) and the key research and development program of Shandong province (2017CXGC1401).
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
THF
Tetrahydrofuran
MCF‐7
K562
A549
Breast cancer cell
Human erythroleukemia cell
Lung cancer cell
NSCLC
KB/V
Non‐small cell lung cancer
Vincristine‐resistance human oral epidermoid carcinoma cell
Lithium bis(trimethylsilyl)amide
Phosphatase and tensin homolog
Dimethyl sulfoxide
Roswell park memorial institute
Ethylene glycol‐bis(2‐aminoethylether)‐N,N,N‘,N’‐tetraacetic acid
Guanosine triphosphate
LiHMDS
PTEN
DMSO
RPMI
EGTA
GTP

Molecules 2019, 24, 279
8 of 9
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(