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

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.

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 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 4ae, 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.

Chemistry
The synthetic route towards the benzofuropyrazole derivatives was shown in Scheme 1. The 6methoxybenzofuran-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 6hydroxybenzofuran-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 αproton 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 1 H nuclear magnetic resonance (NMR), 13 C-NMR and electrospray ionization mass spectrometry (ESI-MS). In the 1 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 phenyl rings appeared at 6.42 to 7.82 ppm. In comparison with 4c, in the 1 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. B 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. 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 4ae, 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.
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 electronwithdrawing 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.

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 Scheme 1. Synthesis of benzofuropyrazole and pyrazole derivatives.
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 GI 50 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 GI 50 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.

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 IC 50 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. 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.

General Chemical Experimental Procedures
Melting points were determined on an X-6 micromelting point apparatus (Beijing Tech. Co., Ltd.). 1 H-and 13 C-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.

General Chemical Experimental Procedures
Melting points were determined on an X-6 micromelting point apparatus (Beijing Tech. Co., Ltd.). 1 H-and 13 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 3 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 N 2 atmosphere. Unless otherwise noted, reagents were added by syringe. Tetrahydrofuran (THF) was distilled from sodium/benzophenone immediately prior to use.

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% CO 2 . 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 × 10 3 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 GI 50 values, which represent the molar drug concentrations required to cause 50% tumor cell growth inhibition.

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 2 , 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.

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 IC 50 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.

Conflicts of Interest:
The authors declare no conflict of interest.