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Design, Synthesis, and Biological Evaluation of Novel Benzofuran Derivatives Bearing N-Aryl Piperazine Moiety

1
School of Traditional Chinese Medicine, Yunnan University of Traditional Chinese Medicine, Kunming 650500, China
2
Central Laboratory, The No. 1 Affiliated Hospital of Yunnan University of Traditional Chinese Medicine, Kunming 650021, China
3
Key Laboratory of Medicinal Chemistry for Natural Resource Education Ministry, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China
*
Authors to whom correspondence should be addressed.
Academic Editor: Derek J. McPhee
Molecules 2016, 21(12), 1684; https://doi.org/10.3390/molecules21121684
Received: 6 October 2016 / Revised: 27 November 2016 / Accepted: 1 December 2016 / Published: 9 December 2016
(This article belongs to the Section Medicinal Chemistry)

Abstract

A series of novel hybrid compounds between benzofuran and N-aryl piperazine have been synthesized and screened in vitro for anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages and for anticancer activity against three human tumor cell lines. The results demonstrated that derivative 16 not only had inhibitory effect on the generation of NO (IC50 = 5.28 μM), but also showed satisfactory and selective cytotoxic activity against human lung cancer line (A549) and gastric cancer cell (SGC7901) (IC50 = 0.12 μM and 2.75 μM, respectively), which was identified as the most potent anti-inflammatory and anti-tumor agent in this study.
Keywords: benzofuran; N-aryl piperazine moiety; anti-inflammatory activity; anticancer activity benzofuran; N-aryl piperazine moiety; anti-inflammatory activity; anticancer activity

1. Introduction

Natural and synthetic benzofuran derivatives are a class of important organic compounds with a broad range of biological activities, such as antioxidant, anti-inflammatory, antibacterial, antitumor, and so on [1,2,3,4,5,6,7,8,9,10,11]. Recently, benzofuran derivatives have attracted considerable interest for their versatile properties in chemistry and pharmacology. In addition, 2-benzoylbenzofuran compounds have been identified to show potent cytotoxic activities, as exemplified in Scheme 1. 2-benzoylphenyl benzofuran compound (A) [12] and 2-(2,4-dimethoxybenzoyl)-phenyl benzofuran derivative (B) [13] showed potent anticancer activities. Furthermore, synthetic 2-benzylbenzofurane-imidazole hybrid (C) [14] and 2-(4-imidazoyl benzoyl)benzofuran (D) [15] were attractive with excellent anti-inflammatory and cytotoxic activities (Scheme 1).
Nitrogen heterocycles are an important class of compounds having versatile biological activities, which are used in drug design and synthesis, generally as active units [16,17]. Piperazine—one of the most biological active moieties—represents a series of important organic compounds that make up the core structures in medicines and have been widely used in the development of drug molecular design [18,19].
In previous work, we reported that benzofuran compounds containing N-heterocyclic moieties displayed potent cytotoxic activities [20], and the hybrids between chalcone and N-aryl piperazine bearing acetophenone showed potent anti-inflammatory and anticancer activities [21,22]. In addition, the acetophenone substituent was vital for modulating cytotoxic activities.
Based on these results, in the present research, we have designed and synthesized novel hybrids towards the recombination of benzofuran and N-aryl piperazine (Scheme 2). In order to study the structure–activity relationship (SAR) of hybrid compounds, various R-X were selected, including α-bromoacetophenone, benzyl bromide, and alkyl bromide. The potent in vitro anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages and anticancer activities of compounds against a panel of human cancer cell lines (A549, Hela, and SGC7901) were evaluated, respectively.

2. Results

2.1. Chemistry

The general synthetic route used to synthesize hybrid compounds is outlined in Scheme 3. Treatment of commercial 5-diethylaminosalicylaldehyde (1) with 2-bromo-4′-fluoro acetophenone (2) gave the 2-phenzoylbenzofuran compound (3) in the presence of K2CO3 in refluxing acetone. Then, the key benzofuran–piperazine intermediate (4) was prepared by substitution with piperazine from compound (3) in the presence of K2CO3 at 110 °C in DMF. With the desired intermediate (4) in hand, we began the synthesis of amines by treatment of compound (4) with several commercially available R-X. To get further insight into the structure–activity relationship, the tertiary amines (525) were prepared with excellent yields by the reaction of intermediate (4) with various R-X. To compare the biological activities of substituents of the NH group of piperazine ring, we prepared the title compounds by substitution of α-bromoacetophenone, benzyl bromide, alkyl bromide, and hetero aromatic bromide. Comparative data for novel hybrid compounds with respect to structures and yield are provided in Table 1. All of the synthesized compounds were characterized by 1H-NMR and 13C-NMR, and some representative compounds were characterized by high-resolution mass spectrometry (HRMS) analysis. The spectra of title compounds were in Supplementary Materials.

2.2. Biological Evaluation

2.2.1. Anti-Inflammatory Activity

RAW 264.7 cells are widely used to establish inflammatory models in vitro. In this work, we investigated the anti-inflammatory activity of synthetic compounds in LPS-induced RAW 264.7 on the generation of NO. The results of the title hybrids are summarized in Table 2.
As shown in Table 2, the substituents of the NH group of the piperazine ring have an obvious influence on anti-inflammatory activities. To our delight, benzofuran–piperazine compounds 16, 18, and 22 displayed good anti-inflammatory activity on the generation of NO (IC50 < 10 μM). Especially, compound 16 was found to be the most potent anti-inflammatory agent (IC50 = 5.28 μM). In addition, compounds 5, 9, and 24 showed potent anti-inflammatory activity (IC50 < 20 μM). However, hetero aromatic compound 25 displayed no activity compared to others. Notably, except for the mentioned hybrids, most of the derivatives had little or no inhibitory effects on the release of NO (IC50 > 20 μM). On the other hand, we could find that the substituents of the NH group of the piperazine ring played an important role. Overall, keto- substituents contributed to better activity, alkyl and aryl substituents led to weaker activity, and the pyridyl substituent had no effect on anti-inflammatory activity (keto- > alkyl ≈ aryl > pyridyl). So, in future research, we will focus on the keto- substituents.

2.2.2. Anticancer Activity

The anticancer activities of novel synthesized derivatives were evaluated against human lung cancer cell line (A549), human cervical carcinoma (Hela), and human gastric carcinoma (SGC7901) by MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide] assay, using cisplatin (DDP) as the reference drug. The anti-tumor results for the hybrids are summarized in Table 3.
As shown in Table 3, the structures of the hybrid compounds have an obvious influence on cytotoxic activities. There were three series of substituents of the piperazine ring, including keto-, alkyl, and aryl. In general, derivatives bearing keto- substituent (1624) were most active, displaying similar or better cytotoxic activity in vitro compared to cisplatin (DDP). However, hetero aromatic compound 25 had weak cytotoxic activity against Hela, and alkyl-substituted compounds showed no activity, except for hybrid 9 (IC50 = 19.27 μM against A549). Furthermore, the electron withdrawing group or halide substituent at position 4 of the benzene ring of the acetophenone moiety could be more sensitive to cytotoxic activity, such as 4-F (19), 4-Cl (20), and 4-CN (23). Compound 16 showed the most potent cytotoxic activity and pronounced selectivity against A549 (IC50 = 0.12 μM) and SGC7901 (IC50 = 2.75 μM). In addition, some aryl-substituted compounds displayed good inhibitory activity. For example, hybrids 11 and 12 had selective anti-tumor activity against cancer cells (IC50 = 8.57–16.27 μM).
The biological results suggested that the existence of a keto- substituent played an important role in the anti-inflammatory and anticancer activity of compounds. In all synthesized derivatives, hybrid 16 had better inhibitory effect on the generation of NO and showed more potent cytotoxic activity against A549 and SGC7901, and could be identified as the most potent anti-inflammatory and anti-tumor agent among those studied. The structure–activity relationship (SAR) results are summarized in Scheme 4.
Overall, although only a few compounds were found to exhibit excellent anti-inflammatory and antitumor activities, we could study the tendency of benzofuran–piperazine compounds and conduct further medicine chemistry research following the results.

3. Materials and Methods

3.1. General Information

Starting materials were commercially available and analytically pure. Melting points were measured on a YANACO microscopic melting point meter and were uncorrected. 1H-NMR and 13C-NMR spectra were recorded on Bruker AV 300 and Bruker AV 400 spectrometers (Bruker, Karlsruhe, Germany) using TMS as internal standard and CDCl3 as solvent, respectively. Thin layer chromatographic (TLC) analysis was carried out on silica gel plates GF254. High-resolution mass spectra were performed on an ESI Q-TOF MS spectrometer (Agilent, Santa Clara, CA, USA).

3.2. Chemistry

3.2.1. General Procedure

General procedure for the synthesis of compound 3: To a solution of acetone (50 mL), 5-diethylamino salicylaldehyde (1.93 g, 10 mmol) and 2-bromo-4′-fluoroacetophenone (2.17 g, 10 mmol), was added K2CO3 (2.76 g, 20 mmol) and left to react for 4 h in reflux. The reaction was poured into 100 mL cold water. After stirring for 10 min, the mixture was filtered, and the filtrate was concentrated in vacuo and dried to afford a yellow solid.
General procedure for the preparation of compound 4: To a stirred solution of compound 3 (1.56 g, 5 mmol) and K2CO3 (1.38 g, 10 mmol) in dried DMF (20 mL), piperazine (0.86 g, 10 mmol) was added, and the reaction mixture was stirred for 12 h at 110 °C. After completion of the reaction as indicated by TLC, the reaction was quenched by the addition of CHCl3 (50 mL) and washed with water (3 × 20 mL). The organic layer was dried by anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography to afford a brown solid.
General procedure for the preparation of hybrid derivatives 57: To a stirred solution of compound 4 (0.5 mmol) in dried DMF (5 mL), NaH (0.06 g, 60% in oil) was added, and the mixture was stirred at 0 °C for 1 h. Then, R-X (1 mmol) was added, and after completion of the reaction as indicated by TLC, the reaction was quenched by the addition of H2O (30 mL) and was extracted with DCM (3 × 10 mL). The organic layer was dried by anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography (1% MeOH/DCM) to afford products.
General procedure for the preparation of derivatives 814, 25: To a stirred solution of compound 4 (0.5 mmol) and Cs2CO3 (0.5 g) in dried DCM (15 mL), R-X (0.6 mmol) was added, and the reaction mixture was stirred for 12 h at room temperature (r.t.). After completion of the reaction as indicated by TLC, the mixture was filtered off. Then, the organic layer was concentrated in vacuo and purified by column chromatography (1% MeOH/DCM) to afford products.
General procedure for the preparation of derivatives 1524: To a stirred solution of compound 4 (0.5 mmol) and K2CO3 (0.2 g) in dried DCM (10 mL), R-X (0.6 mmol) was added, and the reaction mixture was stirred for 12 h at r.t. After completion of the reaction as indicated by TLC, the reaction was quenched by the addition of 5% NaOH (20 mL) and was extracted with DCM (3 × 10 mL). The organic layer was dried using anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography (1% MeOH/DCM) to afford products.

3.2.2. The Character of All Compounds

Compound 4: Brown solid; 1H-NMR (300 MHz, CDCl3) δ: 8.02 (d, J = 9.0 Hz, 2H), 7.48 (d, J = 8.7 Hz, 1H), 7.38 (s, 1H), 6.95 (d, J = 9.0 Hz, 2H), 6.72–6.78 (m, 2H), 3.46 (q, J = 7.2 Hz, 4H), 3.35 (t, J = 4.8 Hz, 4H), 3.05 (t, J = 4.8 Hz, 4H), 1.90 (s, 1H), 1.23 (t, J = 6.9 Hz, 6H); 13C-NMR (75 MHz, CDCl3) δ: 181.94, 158.99, 154.37, 151.09, 149.06, 131.51, 128.06, 123.48, 116.76, 116.51, 113.71, 110.93, 93.16, 48.70, 45.99, 45.14, 12.63; HRMS (ESI-TOF): m/z calcd for C23H27N3O2Na [M + Na]+ 400.1995, found 400.1995.
Compound 5: Pale yellow solid; 1H-NMR (400 MHz, CDCl3) δ: 8.02 (d, J = 8.9 Hz, 2H), 7.47 (d, J = 8.8 Hz, 1H), 7.37 (s, 1H), 6.94 (d, J = 8.9 Hz, 2H), 6.78 (s, 1H), 6.73–6.75 (dd, J = 2.2 Hz, 2.2 Hz, 1H), 3.37–3.43 (m, 8H), 2.58 (t, J = 5.2 Hz, 4H), 2.35 (s, 3H), 1.22 (t, J = 7.0 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 181.86, 158.99, 153.94, 151.22, 149.10, 131.53, 128.10, 123.45, 116.59, 113.75, 111.01, 93.27, 54.90, 47.56, 46.23, 45.14, 12.64.
Compound 6: Yellow solid; 1H-NMR (400 MHz, CDCl3) δ: 8.01 (d, J = 9.0 Hz, 2H), 7.46 (d, J = 8.8 Hz, 1H), 7.37 (s, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.77 (s, 1H), 6.72–6.75 (dd, J = 2.2 Hz, 2.2 Hz, 1H), 3.38–3.44 (m, 8H), 2.63 (t, J = 5.1 Hz, 4H), 2.51 (q, J = 7.2 Hz, 2H), 1.22 (t, J = 7.0 Hz, 6H), 1.15 (t, J = 7.2 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ: 181.84, 158.95, 153.90, 151.11, 149.05, 131.48, 128.01, 123.43, 116.66, 116.50, 113.67, 110.95, 93.16, 52.55, 52.43, 47.46, 45.09, 12.60, 11.97.
Compound 7: Pale brown solid; 1H-NMR (400 MHz, CDCl3) δ: 8.01 (d, J = 9.0 Hz, 2H), 7.45 (d, J = 8.8 Hz, 1H), 7.36 (s, 1H), 6.91 (d, J = 9.0 Hz, 2H), 6.76 (s, 1H), 6.71–6.73 (dd, J = 2.2 Hz, 2.2 Hz, 1H), 3.43 (q, J = 7.1 Hz, 4H), 3.37 (t, J = 5.2 Hz, 4H), 2.57 (t, J = 5.0 Hz, 4H), 2.538 (t, J = 7.6 Hz, 2H), 1.25–1.30 (m, 32H), 1.20 (t, J = 7.0 Hz, 6H), 0.88 (t, J = 6.6 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ: 181.71, 158.90, 153.96, 151.20, 149.00, 131.46, 127.86, 123.37, 116.53, 116.44, 113.53, 110.92, 93.21, 58.83, 53.03, 47.52, 45.06, 31.99, 29.76, 29.69, 29.67, 29.42, 27.64, 26.95, 22.75, 14.17, 12.58; HRMS (ESI-TOF): m/z calcd for C41H63N3O2 [M + H]+ 630.4993, found 630.4983.
Compound 8: Yellow solid; 1H-NMR (300 MHz, CDCl3) δ: 8.02 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.7 Hz, 1H), 7.38 (s, 1H), 6.95 (d, J = 8.7 Hz, 2H), 6.72–6.78 (m, 2H), 5.83–5.97 (m, 1H), 5.26 (d, J = 15.0 Hz, 1H), 5.21 (d, J = 8.1 Hz, 1H), 3.37–3.46 (m, 8H), 3.08 (d, J = 6.6 Hz, 2H), 2.63 (t, J = 5.1 Hz, 4H), 1.23 (t, J = 6.9 Hz, 6H); 13C-NMR (75 MHz, CDCl3) δ:181.94, 158.98, 153.98, 151.11, 149.05, 134.75, 131.53, 128.03, 123.47, 118.60, 116.74, 116.51, 113.70, 110.93, 93.17, 61.86, 52.85, 47.57, 45.14, 12.63; HRMS (ESI-TOF): m/z calcd for C26H31N3O2Na [M + Na]+ 440.2308, found 440.2307.
Compound 9: Pale yellow solid; 1H-NMR (400 MHz, CDCl3) δ: 8.02 (d, J = 9.0 Hz, 2H), 7.47 (d, J = 8.8 Hz, 1H), 7.37 (s, 1H), 6.94 (d, J = 9.0 Hz, 2H), 6.77 (s, 1H), 6.72–6.75 (dd, J = 2.2 Hz, 2.2 Hz, 1H), 3.36–3.43 (m, 10H), 2.73 (t, J = 5.1 Hz, 4H), 2.28 (s, 1H), 1.22 (t, J = 7.1 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 181.81, 158.97, 153.86, 151.19, 149.09, 131.50, 128.16, 123.43, 116.60, 116.55, 113.80, 110.99, 93.23, 78.47, 73.65, 51.62, 47.54, 47.00, 45.11, 12.62; HRMS (ESI-TOF): m/z calcd for C26H29N3O2 [M + H]+ 416.2333, found 416.2337.
Compound 10: Yellow solid; 1H-NMR (400 MHz, CDCl3) δ: 8.01 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.8 Hz, 1H), 7.25–7.37 (m, 6H), 6.91 (d, J = 8.5 Hz, 2H), 6.78 (s, 1H), 6.74 (d, J = 6.7 Hz, 1H), 3.55 (s, 2H), 3.43 (q, J = 8.8 Hz, 4H), 3.36 (t, J = 4.7 Hz, 4H), 2.60 (t, J = 4.4 Hz, 4H), 1.21 (t, J = 6.9 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 181.79, 158.91, 153.97, 151.12, 149.00, 137.88, 131.47, 129.23, 128.39, 127.84, 127.29, 123.41, 116.58, 116.49, 113.57, 110.91, 93.14, 63.07, 52.81, 47.51, 45.07, 12.59.
Compound 11: Pale red solid; 1H-NMR (300 MHz, CDCl3) δ: 8.00 (d, J = 8.7 Hz, 2H), 7.62 (d, J = 8.1 Hz, 2H), 7.44–7.48 (m, 3H), 7.36 (s, 1H), 6.92 (d, J = 9.0 Hz, 2H), 6.76 (s, 1H), 6.71–6.76 (dd, J = 2.1 Hz, 2.1 Hz, 1H), 3.58 (s, 2H), 3.33–3.44 (m, 8H), 2.59 (t, J = 4.8 Hz, 4H), 1.22 (t, J = 6.9 Hz, 6H); 13C-NMR (75 MHz, CDCl3) δ: 181.78, 158.93, 153.83, 150.99, 149.01, 143.91, 132.25, 131.45, 129.56, 128.04, 123.44, 119.00, 116.73, 116.40, 113.68, 111.05, 93.02, 62.39, 52.86, 47.51, 45.07, 12.57.
Compound 12: Brown solid; 1H-NMR (400 MHz, CDCl3) δ: 8.02 (d, J = 9.0 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.48 (d, J = 3.1 Hz, 2H), 7.46 (d, J = 3.9 Hz, 1H), 7.38 (s, 1H), 6.91 (d, J = 9.0 Hz, 2H), 6.78 (s, 1H), 6.72–6.75 (dd, J = 2.2 Hz, 2.2 Hz, 1H), 3.58 (s, 2H), 3.43 (q, J = 7.0 Hz, 4H), 3.36 (t, J = 4.9 Hz, 4H), 2.58 (t, J = 5.0 Hz, 4H), 1.21 (t, J = 7.0 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 181.68, 158.90, 153.87, 151.08, 149.01, 142.32, 131.43, 129.24, 127.92, 125.32, 125.29, 125.25, 125.22, 123.40, 116.57, 116.44, 113.60, 110.91, 93.06, 62.34, 52.78, 47.46, 45.02, 12.53; HRMS (ESI-TOF): m/z calcd for C31H32F3N3O2 [M + H]+ 536.2519, found 536.2526.
Compound 13: Pale yellow solid; 1H-NMR (400 MHz, CDCl3) δ: 8.01 (d, J = 9.0 Hz, 2H), 7.46 (d, J = 8.8 Hz, 1H), 7.37 (s, 1H), 7.19- 7.22 (m, 1H), 7.03–7.17 (m, 2H), 8.01 (d, J = 9.0 Hz, 2H), 6.77 (s, 1H), 6.72–6.74 (dd, J = 2.2 Hz, 2.2 Hz, 1H), 3.46 (s, 2H), 3.43 (q, J = 7.1 Hz, 4H), 3.35 (t, J = 4.9 Hz, 4H), 2.56 (t, J = 5.0 Hz, 4H), 1.21 (t, J = 7.1 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 181.67, 158.88, 153.84, 151.05, 148.99, 135.27, 135.22, 135.18, 131.41, 127.89, 124.76, 124.72, 124.70, 124.66, 123.39, 117.69, 117.52, 117.02, 116.85, 116.57, 116.42, 113.58, 110.90, 93.04, 61.76, 52.66, 47.45, 45.01, 12.53; HRMS (ESI-TOF): m/z calcd for C30H32F2N3O2 [M + H]+ 504.2457, found 504.2453.
Compound 14: Pale brown solid; 1H-NMR (400 MHz, CDCl3) δ: 8.20 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 9.0 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.8 Hz, 1H), 7.37 (s, 1H), 6.93 (d, J = 9.0 Hz, 2H), 6.77 (s, 1H), 6.73–6.76 (dd, J = 2.2 Hz, 2.2 Hz, 1H), 3.64 (s, 2H), 3.36–3.45 (m, 8H), 2.62 (t, J = 5.1 Hz, 4H), 1.23 (t, J = 7.0 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 181.79, 158.99, 153.88, 151.18, 149.13, 147.41, 146.08, 131.52, 129.59, 128.20, 123.71, 123.45, 116.63, 116.54, 113.77, 111.03, 93.21, 62.17, 52.97, 47.63, 45.12, 12.63.
Compound 15: Yellow solid; 1H-NMR (300 MHz, CDCl3) δ: 8.01 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 8.7 Hz, 1H), 7.37 (s, 1H), 6.94 (d, J = 8.7 Hz, 2H), 6.77 (s, 1H), 6.75 (d, J = 9.0 Hz, 1H), 4.24 (q, J = 6.9 Hz, 2H), 3.45 (q, J = 6.9 Hz, 8H), 3.27 (s, 2H), 2.76 (t, J = 5.1 Hz, 4H), 1.31 (t, J = 7.2 Hz, 3H), 1.22 (t, J = 7.2 Hz, 6H); 13C-NMR (75 MHz, CDCl3) δ: 181.91, 170.21, 158.97, 153.85, 151.06, 149.04, 131.50, 128.15, 123.47, 116.77, 113.79, 110.92, 93.12, 60.88, 59.46, 52.75, 47.47, 45.12, 14.37, 12.61; HRMS (ESI-TOF): m/z calcd for C27H34N3O4 [M + H]+ 464.2543, found 464.2545.
Compound 16: Pale red solid; 1H-NMR (400 MHz, CDCl3) δ: 7.99 (d, J = 8.9 Hz, 2H), 7.45 (d, J = 8.8 Hz, 1H), 7.35 (s, 1H), 6.90 (d, J = 9.0 Hz, 2H), 6.75 (s, 1H), 6.70–6.73 (dd, J = 2.2 Hz, 2.2 Hz, 1H), 3.36–3.40 (m, 8H), 3.23 (s, 2H), 2.62 (t, J = 5.0 Hz, 4H), 2.14 (s, 3H), 1.19 (t, J = 7.0 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 206.03, 181.69, 158.87, 153.75, 151.00, 148.98, 131.39, 128.00, 123.38, 116.60, 116.39, 113.65, 110.89, 93.02, 67.93, 52.98, 47.33, 45.00, 27.81, 12.52; HRMS (ESI-TOF): m/z calcd for C26H31N3O3 [M + H]+ 434.2438, found 434.2444.
Compound 17: Pale yellow solid; 1H-NMR (300 MHz, CDCl3) δ: 8.03 (d, J = 9.0 Hz, 4H), 7.59 (d, J = 7.2 Hz, 1H), 7.45–7.50 (m, 3H), 7.38 (s, 1H), 6.96 (d, J = 9.0 Hz, 2H), 6.78 (s, 1H), 6.73–6.77 (dd, J = 2.1 Hz, 2.1 Hz, 1H), 3.90 (s, 2H), 3.39–3.48 (m, 8H), 2.80 (t, J = 4.8 Hz, 4H), 1.24 (t, J = 7.2 Hz, 6H); 13C-NMR (75 MHz, CDCl3) δ: 196.18, 181.89, 158.96, 153.88, 151.10, 149.05, 136.01, 133.52, 131.50, 128.73, 128.20, 123.45, 116.72, 116.50, 113.76, 110.94, 93.16, 64.36, 53.26, 47.50, 45.10, 12.61; HRMS (ESI-TOF): m/z calcd for C31H34N3O3 [M + H]+ 496.2595, found 496.2598.
Compound 18: Pale red solid; 1H-NMR (300 MHz, CDCl3) δ: 8.59 (s, 1H), 7.97–8.09 (m, 4H), 7.93 (t, J = 8.7 Hz, 2H), 7.54–7.64 (m, 2H), 7.49 (d, J = 8.7 Hz, 1H), 7.39 (s, 1H), 6.97 (d, J = 8.7 Hz, 2H), 6.78 (s, 1H), 6.73–6.77 (dd, J = 2.1 Hz, 2.1 Hz, 1H), 4.04 (s, 2H), 3.40–3.50 (m, 8H), 2.85 (t, J = 5.1 Hz, 4H), 1.24 (t, J = 6.9 Hz, 6H); 13C-NMR (75 MHz, CDCl3) δ: 196.21, 182.00, 159.02, 153.94, 149.11, 135.87, 132.60, 131.56, 129.96, 129.76, 128.80, 128.65, 128.21, 127.97, 127.03, 123.96, 123.49, 116.75, 116.57, 113.83, 110.99, 93.24, 64.56, 53.38, 47.60, 45.16, 12.66; HRMS (ESI-TOF): m/z calcd for C35H35N3O3Na [M + Na]+ 568.2570, found 568.2569.
Compound 19: Orange solid; 1H-NMR (300 MHz, CDCl3) δ: 8.04–8.08 (m, 2H), 8.01 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 8.7 Hz, 1H), 7.37 (s, 1H), 7.16 (t, J = 8.4 Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H), 6.77 (s, 1H), 6.72–6.75 (dd, J = 2.1 Hz, 2.1 Hz, 1H), 3.84 (s, 2H), 3.38–3.45 (m, 8H), 2.77 (t, J = 5.1 Hz, 4H), 1.22 (t, J = 6.9 Hz, 6H); 13C-NMR (75 MHz, CDCl3) δ: 194.77, 181.92, 159.00, 153.86, 151.05, 149.08, 132.41, 131.51, 131.10, 130.97, 128.19, 123.49, 116.82, 116.49, 116.01, 115.72, 113.81, 110.95, 93.12, 64.51, 53.26, 47.51, 45.12, 12.62.
Compound 20: Pale brown solid; 1H-NMR (300 MHz, CDCl3) δ: 8.01 (d, J = 9.3 Hz, 2H), 7.98 (d, J = 9.0 Hz, 2H), 7.47 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.37 (s, 1H), 6.94 (d, J = 9.0 Hz, 2H), 6.77 (s, 1H), 6.72–6.77 (dd, J = 2.1 Hz, 2.1 Hz, 1H), 3.83 (s, 2H), 3.43 (t, J = 7.2 Hz, 8H), 2.76 (t, J = 4.8 Hz, 4H), 1.22 (t, J = 7.2 Hz, 6H); 13C-NMR (75 MHz, CDCl3) δ: 195.17, 181.86, 158.96, 153.82, 151.01, 149.04, 139.94, 134.21, 131.48, 129.75, 129.03, 128.15, 123.47, 116.79, 116.44, 113.78, 110.92, 93.07, 64.50, 53.22, 47.47, 45.10, 12.60; HRMS (ESI-TOF): m/z calcd for C31H32N3O3NaCl [M + Na]+ 552.2024, found 552.2023.
Compound 21: Pale green solid; 1H-NMR (300 MHz, CDCl3) δ: 8.03 (d, J = 8.7 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.7 Hz, 1H), 7.38 (s, 1H), 6.96 (d, J = 9.0 Hz, 2H), 6.78 (s, 1H), 6.73–6.77 (dd, J = 2.1 Hz, 2.1 Hz, 1H), 3.84 (s, 2H), 3.44 (t, J = 7.2 Hz, 8H), 2.78 (t, J = 5.1 Hz, 4H), 1.24 (t, J = 6.9 Hz, 6H); HRMS (ESI-TOF): m/z calcd for C31H32BrN3O3 [M + H]+ 574.1700, found 574.1699.
Compound 22: Orange solid; 1H-NMR (400 MHz, CDCl3) δ: 8.00 (d, J = 8.9 Hz, 2H), 7.99 (d, J = 8.8 Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.36 (s, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.75 (s, 1H), 6.73 (d, J = 8.8 Hz, 1H), 3.83 (s, 3H), 3.82 (s, 2H), 3.37–3.43 (m, 8H), 2.76 (t, J = 5.0 Hz, 4H), 1.20 (t, J = 7.0 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 194.62, 181.78, 163.78, 158.92, 153.85, 151.08, 149.05, 131.46, 130.52, 129.05, 128.00, 123.42, 116.66, 116.48, 113.83, 113.69, 110.96, 93.12, 64.02, 55.53, 53.14, 47.37, 45.05, 12.57; HRMS (ESI-TOF): m/z calcd for C32H36N3O4 [(M + H)]+ 526.2700, found 526.2700.
Compound 23: Pale yellow solid; 1H-NMR (400 MHz, CDCl3) δ: 8.11 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.9 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.8 Hz, 1H), 7.36 (s, 1H), 6.92 (d, J = 9.0 Hz, 2H), 6.72–6.75 (m, 2H), 3.86 (s, 2H), 3.44 (q, J = 7.1 Hz, 8H), 2.76 (t, J = 4.9 Hz, 4H), 1.21 (t, J = 7.0 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 195.18, 181.85, 159.01, 153.77, 151.06, 149.15, 138.84, 132.57, 131.51, 128.84, 128.30, 123.49, 117.96, 116.83, 116.74, 116.50, 113.87, 111.05, 93.14, 64.70, 53.15, 47.48, 45.11, 12.61; HRMS (ESI-TOF): m/z calcd for C32H33N4O3 [M + H]+ 521.2547, found 521.2547.
Compound 24: Pale green solid; 1H-NMR (400 MHz, CDCl3) δ: 8.04 (q, J = 8.6 Hz, 2H), 7.93 (q, J = 7.9 Hz, 2H), 7.49 (q, J = 8.7 Hz, 1H), 7.40 (d, J = 15.3 Hz, 1H), 7.28 (q, J = 7.7 Hz, 2H), 6.95 (q, J = 8.6 Hz, 2H), 6.79 (d, J = 13.3 Hz, 1H), 6.73 (d, J = 8.9 Hz, 1H), 3.88 (d, J = 15.4 Hz, 2H), 3.39–3.44 (m, 8H), 2.79 (t, J = 4.1 Hz, 4H), 2.42 (t, J = 6.2 Hz, 3H), 1.16–1.24 (m, 6H); 13C-NMR (100 MHz, CDCl3) δ: 195.75, 181.80, 158.95, 153.88, 151.15, 149.08, 144.33, 133.55, 131.49, 129.37, 128.30, 128.05, 123.42, 116.62, 116.52, 113.72, 110.98, 93.17, 64.15, 53.17, 47.43, 45.07, 21.74, 12.59.
Compound 25: Brown solid; 1H-NMR (400 MHz, CDCl3) δ: 8.56 (d, J = 6.0 Hz, 2H), 8.01 (d, J = 8.9 Hz, 2H), 7.46 (d, J = 8.8 Hz, 1H), 7.37 (s, 1H), 7.31 (d, J = 5.9 Hz, 1H), 6.93 (d, J = 9.0 Hz, 2H), 6.77 (s, 1H), 6.75 (d, J = 8.8 Hz, 1H), 3.56 (s, 2H), 3.36–3.44 (m, 8H), 2.61 (t, J = 5.0 Hz, 4H), 1.22 (t, J = 7.0 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 181.81, 158.98, 153.89, 151.15, 150.57, 150.01, 149.09, 147.36, 131.50, 128.15, 123.92, 123.44, 121.35, 116.64, 116.52, 114.50, 113.74, 110.98, 93.19, 61.76, 52.97, 47.60, 45.11, 12.62; HRMS (ESI-TOF): m/z calcd for C29H33N4O2 [M + H]+ 469.2598, found 469.2601.

3.3. Biological Activity Experiments

3.3.1. Anti-Inflammatory Activity

Murine RAW264.7 macrophages were plated in 96-well plate at a density of 1 × 105 cells/well and stimulated with 1 μg/mL LPS in the presence or absence of various concentrations of compound for 24 h. The production of NO was determined by assaying culture supernatant for NO2−. Supernatant (100 μL) was mixed with an equal volume of Griess reagent at r.t. for 10 min. Absorbance was measured at 540 nm in a microplate reader.

3.3.2. Antitumor Activity

The assay was carried out using the method described previously. About 1 × 104 cell/well were seeded into 96-well microtiter plates. At twenty-four hours post-seeding, cells were treated with vehicle control or various concentrations of samples for 48 h. Twenty microliters of MTT solution (5 mg/mL) was added to each well, and the tumor cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 air for 4 h. Upon removal of MTT/medium, 150 μL of DMSO was added to each well, and the plate was agitated at oscillator for 5 min to dissolve the MTT-formazan. The assay plate was read at a wavelength of 570 nm using a microplate reader.

4. Conclusions

In summary, a series of novel hybrid compounds between benzofuran and N-aryl piperazine have been synthesized and screened in vitro for anti-inflammatory and anticancer activity. The results demonstrated that derivative 16 not only had an inhibitory effect on the generation of NO (IC50 = 5.28 μM), but also displayed good cytotoxic activity against A549 and SGC7901 (IC50 = 0.12 μM and 2.75 μM, respectively), which was considered to be the most potent anti-inflammatory and anti-tumor agent in this study. Further research is currently underway, and the results will be reported in due course.

Supplementary Materials

The 1H-NMR, 13C-NMR and HRMS spectra are available online at: https://www.mdpi.com/1420-3049/21/12/1684/s1.

Acknowledgments

This work was financially supported by the Natural Science Foundation of China (81460624, 81560620), the Application Basic Research Program of Yunnan Province (2014FZ078, 2014FZ087) and the Yunnan Province doctoral newcomer Award (YN2015015). We acknowledge the Key Discipline of Pharmacy and Yi medicine (Yunnan University of Traditional Chinese Medicine, China) for support on research.

Author Contributions

Yulu Ma and Zewei Mao designed and carried out the experiments and wrote the paper; Xi Zheng assisted in experiment; Hui Gao analyzed the data; Chunping Wan supervised and directed the biological assays; Gaoxiong Rao supervised the whole experiment and provided technical guidance. All authors have read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of the compounds 325 are available from the authors.
Scheme 1. Structures of biological benzofuran agents.
Scheme 1. Structures of biological benzofuran agents.
Molecules 21 01684 sch001
Scheme 2. Designed strategy of benzofuran hybrids.
Scheme 2. Designed strategy of benzofuran hybrids.
Molecules 21 01684 sch002
Scheme 3. Synthetic routes of hybrid compounds.
Scheme 3. Synthetic routes of hybrid compounds.
Molecules 21 01684 sch003
Scheme 4. Structure–activity relationship of hybrid compounds.
Scheme 4. Structure–activity relationship of hybrid compounds.
Molecules 21 01684 sch004
Table 1. Structures and yields of compounds 525.
Table 1. Structures and yields of compounds 525.
Molecules 21 01684 i001
CompoundRm.p. (°C)Yields (%) a
5 Molecules 21 01684 i002171–17351
6 Molecules 21 01684 i003171–17357
7 Molecules 21 01684 i004185–18668
8 Molecules 21 01684 i005174–17683
9 Molecules 21 01684 i006176–17881
10 Molecules 21 01684 i007179–18183
11 Molecules 21 01684 i008186–18879
12 Molecules 21 01684 i009180–18276
13 Molecules 21 01684 i010178–18082
14 Molecules 21 01684 i011187–18981
15 Molecules 21 01684 i012156–15891
16 Molecules 21 01684 i013161–16386
17 Molecules 21 01684 i014188–19082
18 Molecules 21 01684 i015202–20470
19 Molecules 21 01684 i016192–19384
20 Molecules 21 01684 i017194–19675
21 Molecules 21 01684 i018198–20083
22 Molecules 21 01684 i019181–18385
23 Molecules 21 01684 i020204–20677
24 Molecules 21 01684 i021184–18682
25 Molecules 21 01684 i022135–13786
a Yields represent isolated yields.
Table 2. Anti-inflammatory activities of compounds.
Table 2. Anti-inflammatory activities of compounds.
CompoundNO Generation (IC50, μM) aCompoundNO Generation (IC50, μM) a
514.12165.28
634.241725.40
7>40186.53
8>4019>40
918.5220>40
10>4021>40
11>40229.13
12>402323.56
1323.062418.37
1420.2725>40
1531.68
a Values represent the concentration required to produce 50% inhibition of the response.
Table 3. In vitro cytotoxic activities of title compounds.
Table 3. In vitro cytotoxic activities of title compounds.
CompoundCell Lines (IC50, μM) a
A549HelaSGC7901
5>40>4027.24
6>4032.53>40
7>40>40>40
8>40>40>40
919.27>40>40
10>40>40>40
1116.148.57>40
12>4025.1416.27
13>40>4025.04
14>4033.24>40
15>4022.3630.43
160.1226.322.75
1727.82>4015.41
1819.34>4023.92
196.2518.7136.23
208.1128.74>40
2123.2215.35>40
2226.07>40>40
2334.1312.687.45
24>4027.58>40
25>4026.22>40
DDP11.5420.5212.44
a Cytotoxicity as IC50 values for each cell line, the concentration of compound that inhibits 50% of the cell growth measured by MTT assay. DDP: cisplatin.
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