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Article

Synthesis of Novel Pyrazole Derivatives and Their Tumor Cell Growth Inhibitory Activity

by
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
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(2), 279; https://doi.org/10.3390/molecules24020279
Submission received: 31 October 2018 / Revised: 6 January 2019 / Accepted: 10 January 2019 / Published: 13 January 2019
(This article belongs to the Special Issue Design and Synthesis of Organic Molecules as Antineoplastic Agents)
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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 μM, respectively. Moreover, 5b was identified as a novel tubulin polymerization inhibitor with an IC50 of 7.30 μM.

Graphical Abstract

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 [1,2,3]. 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 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)-1H-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.

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 [29,30,31] (please refer to the Supplementary Materials). The Hoesch reaction of resorcinol with chloroacetonitrile in the presence of anhydrous ZnCl2 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, CH3COONa, gave the cyclized 6-hydroxybenzofuran-3-(2H)-one (54%). Methylation of 6-hydroxybenzofuran-3-(2H)-one with Me2SO4 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 5a–e were also isolated in 13% to 31% yield. The structures of benzofuropyrazoles 4a–e and pyrazoles 5a–e were determined by 1H nuclear magnetic resonance (NMR), 13C-NMR and electrospray ionization mass spectrometry (ESI-MS). In the 1H 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 phenyl rings appeared at 6.42 to 7.82 ppm. In comparison with 4c, in the 1H 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.

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.

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 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.

3. Materials and Methods

3.1. General Chemical Experimental Procedures

Melting points were determined on an X-6 micromelting point apparatus (Beijing Tech. Co., Ltd.). 1H- and 13C-NMR spectra were recorded on Bruker-400 NMR or Bruker-600 NMR spectrometers. All spectra were recorded at room temperature for DMSO or CDCl3 solutions. ESI-MS 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 N2 atmosphere. 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 Na2SO4. 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 Na2SO4. After 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%).
4a: Brown solid, m.p.: 62–64 °C. 1H-NMR (CDCl3) 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, 1H, 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, OCH2), 3.81 (s, 3H, 6’-OCH3), 1.37 (t, J = 6.9 Hz, 3H, OCH2CH3). 13C-NMR (CDCl3) δ 161.6, 160.3, 156.6, 146.0, 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 C18H19N3O3 [M + NH4]+: 341.1, found: 341.4.
5a: Brown solid, m.p.: 82–84 °C. 1H-NMR (CDCl3) 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-OCH2), 3.79 (s, 3H, 4’’-OCH3), 1.41 (t, J = 6.9 Hz, 3H, 1-CH3). 13C-NMR (CDCl3) δ 160.7, 160.2, 157.0, 143.9, 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 C18H19N3O3 [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%).
4b: Brown solid, m.p.: 103–105 °C. 1H-NMR (DMSO-d6) 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 (dd, J = 1.9, 8.5 Hz, Ar-H), 6.42 (d, J = 2.0 Hz, 1H, Ar-H), 3.84 (s, 3H, COOCH3), 3.74 (s, 3H, OCH3). 13C-NMR (DMSO-d6) 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, 110.4, 109.1, 106.2, 103.6, 55.5, 52.5. MS (ESI) calcd. for C18H15N3O4 [M + NH4]+: 355.1, found: 355.5.
5b: Yellow solid, m.p.: 185–186 °C. 1H-NMR (DMSO-d6) 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, 2H, 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, COOCH3), 3.74 (s, 3H, OCH3). 13C-NMR (DMSO-d6) δ 167.2, 160.3, 155.8, 151.6, 144.8, 139.8, 130.7, 129.4, 128.4, 119.7, 118.8, 115.5, 110.0, 105.8, 102.1, 93.0, 55.5, 52.2. MS (ESI) calcd. for C18H17N3O4 [M + 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%).
4c: Brown solid, m.p.: 180–182 °C. 1H-NMR (DMSO-d6) δ 11.91 (s, 1H, 1’-NH), 8.28 (q, J = 4.6 Hz, 1H, 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, J = 2.6 Hz, 1H, Ar-H), 3.74 (s, 3H, OCH3), 2.77 (d, J = 4.6 Hz, 3H, N-CH3). 13C-NMR (DMSO-d6) δ 167.8, 161.1, 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, 26.7. MS (ESI) calcd. for C18H16N4O3 [M + NH4]+: 354.1, found: 354.4.
5c: Brown solid, m.p.: 184–186 °C. 1H-NMR (DMSO-d6) 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 (s, 3H, OCH3), 2.76 (d, J = 3.6 Hz, 3H, N-CH3). 13C-NMR (DMSO-d6) δ 170.0, 160.3, 155.8, 151.8, 144.6, 139.7, 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 C18H18N4O3 [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 (0.7 mL, 0.7 mmol), 3-isothiocyanatobenzamide (124 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 = 1:3) to give 4d (59 mg, 30%) and 5d (61 mg, 31%).
4d: Brown solid, m.p.: 172–174 °C. 1H-NMR (DMSO-d6) δ 12.02 (s, 1H, 1’-NH), 8.12 (s, 1H, 3-NH), 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-CONH2, Ar-H), 6.51 (d, J = 5.7 Hz, 1H, Ar-H), 6.44 (s, 1H, Ar-H), 3.74 (s, 3H, OCH3). 13C-NMR (DMSO-d6) δ 169.0, 161.0, 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 C17H14N4O3 [M + NH4]+: 340.1, found: 340.1.
5d: Brown solid, m.p.: 128–130 °C. 1H-NMR (DMSO-d6) δ 11.84 (s, 1H, 1’-NH), 8.56 (s, 1H, 3-NH), 7.83 (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, 3H, 1-CONH2, 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, OCH3). 13C-NMR (DMSO-d6) δ 169.1, 160.3, 144.5, 135.8, 129.1, 128.3, 117.8, 114.6, 110.3, 105.8, 102.1, 55.5. MS (ESI) calcd. for C17H16N4O3 [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 (0.7 mL, 0.7 mmol), 3-isothiocyanatobenzonitrile (112 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 = 1:4) to give 4e (31 mg, 17%) and 5e (33 mg, 18%).
4e: White solid, m.p.: 200–202 °C. 1H-NMR (DMSO-d6) 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-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, OCH3). 13C-NMR (DMSO-d6) 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, 106.2, 103.6, 55.5. MS (ESI) calcd. for C17H12N4O2 [M + NH4]+: 322.1, found: 322.1.
5e: White solid, m.p.: 166–168 °C. 1H-NMR (DMSO-d6) δ 12.05 (s, 1H, 1’-NH), 10.28 (s, 1H, OH), 8.93 (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, 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, 3H, OCH3). 13C-NMR (DMSO-d6) δ 160.4, 155.8, 151.2, 145.0, 140.0, 130.4, 128.5, 121.3, 120.0, 117.3, 112.0, 109.8, 105.8, 102.1, 92.8, 55.5. MS (ESI) calcd. for C17H14N4O2 [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% CO2. 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 seeded into 96-well cell plates at a density of 4−4.5 × 103 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 MgCl2, 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, 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 μM. 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

THFTetrahydrofuran
MCF-7Breast cancer cell
K562Human erythroleukemia cell
A549Lung cancer cell
NSCLCNon-small cell lung cancer
KB/VVincristine-resistance human oral epidermoid carcinoma cell
LiHMDSLithium bis(trimethylsilyl)amide
PTENPhosphatase and tensin homolog
DMSODimethyl sulfoxide
RPMIRoswell park memorial institute
EGTAEthylene glycol-bis(2-aminoethylether)-N,N,N‘,N’-tetraacetic acid
GTPGuanosine triphosphate

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Sample Availability: Samples of the compounds 4a–e and 5a–e are available from the authors.
Molecules 24 00279 g001 550
Figure 1. Tubulin inhibitors 1 and 2.
Figure 1. Tubulin inhibitors 1 and 2.
Molecules 24 00279 g001
Molecules 24 00279 sch001 550
Scheme 1. Synthesis of benzofuropyrazole and pyrazole derivatives.
Scheme 1. Synthesis of benzofuropyrazole and pyrazole derivatives.
Molecules 24 00279 sch001
Molecules 24 00279 g002 550
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 s for 60 min and is presented as increases in the polymerized microtubule.
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 s for 60 min and is presented as increases in the polymerized microtubule.
Molecules 24 00279 g002
Table
Table 1. Tumor cell growth inhibitory activity of 4a–e and 5a–e.
Table 1. Tumor cell growth inhibitory activity of 4a–e and 5a–e.
GI50 (μM)
CompoundRK562MCF-7A549
4aOCH2CH30.26 ± 0.04>200.19 ± 0.08
4bCOOCH35.46 ± 1.04>20>20
4cCONHCH35.11 ± 0.31>2015.11 ± 2.18
4dCONH29.01 ± 1.81>2010.08 ± 2.21
4eCN13.53 ± 0.41>2017.01 ± 2.76
5aOCH2CH30.046 ± 0.00716.72 ± 2.60.92 ± 0.17
5bCOOCH30.021 ± 0.0041.7 ± 0.430.69 ± 0.18
5cCONHCH37.33 ± 1.0047.78 ± 0.879.46 ± 2.03
5dCONH214.77 ± 2.625.8 ± 0.20210.9 ± 0.99
5eCN1.45 ± 0.0472.27 ± 0.343.24 ± 0.99
ABT-751 0.74 ± 0.0780.88 ± 0.244.58 ± 0.04

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MDPI and ACS Style

Cui, Y.-J.; Tang, L.-Q.; Zhang, C.-M.; Liu, Z.-P. Synthesis of Novel Pyrazole Derivatives and Their Tumor Cell Growth Inhibitory Activity. Molecules 2019, 24, 279. https://doi.org/10.3390/molecules24020279

AMA Style

Cui Y-J, Tang L-Q, Zhang C-M, Liu Z-P. Synthesis of Novel Pyrazole Derivatives and Their Tumor Cell Growth Inhibitory Activity. Molecules. 2019; 24(2):279. https://doi.org/10.3390/molecules24020279

Chicago/Turabian Style

Cui, Ying-Jie, Long-Qian Tang, Cheng-Mei Zhang, and Zhao-Peng Liu. 2019. "Synthesis of Novel Pyrazole Derivatives and Their Tumor Cell Growth Inhibitory Activity" Molecules 24, no. 2: 279. https://doi.org/10.3390/molecules24020279

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