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Dec 9, 2016 - towards the recombination of benzofuran and N-aryl piperazine .... General procedure for the synthesis of compound 3: To a solution of acetone ...
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Design, Synthesis, and Biological Evaluation of Novel Benzofuran Derivatives Bearing N-Aryl Piperazine Moiety Yulu Ma 3 , Xi Zheng 2 , Hui Gao 1, *, Chunping Wan 2, *, Gaoxiong Rao 1 and Zewei Mao 1, * 1 2 3

*

School of Traditional Chinese Medicine, Yunnan University of Traditional Chinese Medicine, Kunming 650500, China; [email protected] Central Laboratory, The No. 1 Affiliated Hospital of Yunnan University of Traditional Chinese Medicine, Kunming 650021, China; [email protected] Key Laboratory of Medicinal Chemistry for Natural Resource Education Ministry, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China; [email protected] Correspondence: [email protected] (H.G.); [email protected] (C.W.); [email protected] (Z.M.); Tel.: +86-871-6591-8232 (H.G. & Z.M.)

Academic Editor: Derek J. McPhee Received: 6 October 2016; Accepted: 1 December 2016; Published: 9 December 2016

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

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 [1–11]. 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.

Molecules 2016, 21, 1684; doi:10.3390/molecules21121684

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Scheme agents. Scheme 1. 1. Structures Structures of of biological biological benzofuran benzofuran agents. Scheme 1. Structures of biological benzofuran agents.

Based on these results, in the present research, we have designed and synthesized novel hybrids Based Based on on these these results, results, in in the the present present research, research, we we have have designed designed and and synthesized synthesized novel novel hybrids hybrids towards the recombination of benzofuran and N-aryl piperazine (Scheme 2). In order to study the towards towards the the recombination recombination of of benzofuran benzofuran and and N-aryl N-aryl piperazine piperazine (Scheme (Scheme 2). 2). In order to to study study the the structure–activity relationship (SAR) of hybrid compounds, various R-X were selected, including structure–activity structure–activity relationship relationship (SAR) (SAR) of of hybrid hybrid compounds, compounds, various various R-X R-X were were selected, selected, including including α-bromoacetophenone, benzyl bromide, and alkyl bromide. The potent in vitro anti-inflammatory α-bromoacetophenone, α-bromoacetophenone, benzyl bromide, and alkyl bromide. bromide. The potent in vitro vitro anti-inflammatory anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages and anticancer activities of activity lipopolysaccharide (LPS)-stimulated (LPS)-stimulatedRAW-264.7 RAW-264.7macrophages macrophagesand andanticancer anticancer activities activity in lipopolysaccharide activities of compounds against a panel of human cancer cell lines (A549, Hela, and SGC7901) were evaluated, of compounds against a panel of human cancer cell lines (A549, Hela, and SGC7901) were compounds against a panel of human cancer cell lines (A549, Hela, and SGC7901) were evaluated, respectively. evaluated, respectively. respectively.

Scheme 2. Designed strategy of benzofuran hybrids. Scheme 2. 2. Designed Designed strategy strategy of of benzofuran benzofuran hybrids. hybrids. Scheme

2. Results 2. Results Results 2. 2.1. Chemistry 2.1. Chemistry Chemistry 2.1. The general synthetic route used to synthesize hybrid compounds is outlined in Scheme 3. The general general synthetic synthetic route route used used to to synthesize synthesize hybrid hybrid compounds compounds is is outlined outlined in in Scheme Scheme 3. The 3. Treatment of commercial 5-diethylaminosalicylaldehyde (1) with 2-bromo-4′-fluoro acetophenone 0 Treatment of commercial 5-diethylaminosalicylaldehyde (1) with 2-bromo-4′-fluoro acetophenone Treatment of commercial 5-diethylaminosalicylaldehyde (1)presence with 2-bromo-4 -fluoro acetophenone (2) (2) gave the 2-phenzoylbenzofuran compound (3) in the of K2CO 3 in refluxing acetone. (2) gave the 2-phenzoylbenzofuran compound (3)presence in the presence of K 2CO3 in refluxing acetone. gave the 2-phenzoylbenzofuran compound (3) in the of K CO in refluxing acetone. Then, the 2 3 substitution with piperazine Then, the key benzofuran–piperazine intermediate (4) was prepared by Then, the key benzofuran–piperazine intermediate (4) was prepared by substitution with piperazine key benzofuran–piperazine intermediate (4) was prepared by substitution with piperazine from from compound (3) in the presence of K2CO 3 at 110 °C in DMF. With the desired intermediate (4) in ◦C from compound (3) in the presence of 3Kat 2CO 3 at 110 in DMF. With the desired intermediate (4) in compound (3) in the presence of K CO 110 in °C DMF. With the desired intermediate (4) in hand, 2 hand, we began the synthesis of amines by treatment of compound (4) with several commercially hand, we the began the synthesis of by amines by treatment of compound (4) with several commercially we began of amines treatment of compound (4) with several available available R-X. synthesis To get further insight into the structure–activity relationship, thecommercially tertiary amines (5–25) available R-X. To get further insight into the structure–activity relationship, the tertiary amines (5–25) R-X. To get further into the by structure–activity relationship,(4) the tertiary amines (5–25) were were prepared with insight excellent yields the reaction of intermediate with various R-X. To compare were prepared with excellent yields by the reaction of intermediate (4) with various R-X. To compare 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 the biological biological activities activities of of substituents substituents of of the the NH NH group group of of piperazine piperazine ring, ring, we prepared prepared the title title the compounds by substitution of α-bromoacetophenone, benzyl bromide, alkyl we bromide, andthe hetero compounds by substitution of α-bromoacetophenone, benzyl bromide, alkyl bromide, and hetero compounds by substitution of α-bromoacetophenone, bromide, bromide, and hetero aromatic bromide. Comparative data for novel hybrid benzyl compounds withalkyl respect to structures and aromatic bromide. bromide. Comparative Comparative data for novel novel hybrid hybrid compounds compounds with with respect respect to to structures structures and aromatic data for and yield are provided in Table 1. All of the synthesized compounds were characterized by 1H-NMR 1H-NMR 1 yield are provided in Table 1. All of the synthesized compounds were characterized by yield provided Table 1. All of the synthesized were characterized by H-NMR and 13are C-NMR, and in some representative compounds compounds were characterized by high-resolution mass 13C-NMR, and some representative compounds were characterized by high-resolution mass and 13 and C-NMR, and some representative compounds were characterized by high-resolution mass spectrometry (HRMS) analysis. The spectra of title compounds were in Supplementary Materials. spectrometry (HRMS) (HRMS) analysis. analysis. The The spectra spectra of of title title compounds compoundswere werein inSupplementary SupplementaryMaterials. Materials. spectrometry

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Molecules 2016, 21, 1684 Molecules 2016, 21, 1684 TableTable 1. Structures compounds 1. Structuresand and yields yields ofof compounds 5–25.5–25. Molecules 2016, 21, 1684 Molecules 2016, 21, 1684 Molecules 2016, 21, 1684 Table 1. Structures and yields of compounds 5–25. Table 1. Structures and yields of compounds 5–25. Molecules 2016, 21, 1684 Table 1. Structures and yields of compounds 5–25. Molecules 2016, 21, 1684 Table 1. Structures and yields of compounds 5–25. Table 1. Structures and yields of compounds 5–25. Molecules 2016, 21, 1684 Molecules 2016, 21, 1684 Table 1. Structures and yields of compounds 5–25. Molecules 2016, 21, 1684 Table 1. Structures and yields of compounds 5–25. Molecules 2016, 21, 1684 Table 1. Structures and yields of compounds 5–25. Molecules 2016, 21, 1684 Table 1. Structures and yields of compounds 5–25. Molecules 2016, 21, 1684 Compound Table 1. Structures compounds R and yields ofm.p. (°C) 5–25. Yields (%) a a Table 1. Structures and yields ofm.p. compounds Molecules 2016, 21, 1684 Compound R (◦ C) 5–25. Yields 5 Table 1. Structures 51(%) aa (%) of171–173 compounds 5–25. R and yields m.p. (°C) Yields Molecules 2016, 21, 1684 Compound Compound R m.p. (°C) Yields (%) Table 1. Structures of compounds 5–25. a Molecules 2016, 21, 1684 Compound R and yields m.p. (°C) Yields 171–173 57(%) 171–173 51 a 5 556 Table 1. Structures 171–173 of compounds 5–25. R and yields m.p. (°C) Yields (%)51 Molecules 2016, 21, 1684 Compound 171–173 51(%) a Compound R m.p. (°C) Yields 5 171–173 51 Table 1. Structures and yields of compounds 5–25. Molecules 2016, 21, 1684 185–186 68(%) a 657 171–173 57 Compound R m.p. (°C) Yields 171–173 51 57 57 yields of compounds 5–25. 51 Molecules 2016, 21, 1684 6 566 Table 1. Structures 171–173 171–173 57 R and m.p. (°C) Yields 174–176 83(%) aa 7658 Table 1. Structures 185–186 68 Molecules 2016, 21, 1684 Compound and yields m.p. of compounds 5–25. 51 171–173 57 7 Table 1. Structures 185–186 68 Compound R (°C) Yields (%) 6 171–173 57 and yields of compounds 5–25. Molecules 2016, 21, 1684 Compound 185–186 68 a 171–173 51 7 5879 185–186 176–178 81(%)68 R m.p. (°C) Yields

174–176 83 yields m.p. of compounds 5–25. 171–173 57(%) a 76 Table 1. Structures 185–186 68 174–176 83 171–173 51 Compound R and (°C) Yields 756885 Table 1. Structures 185–186 68 174–176 83 171–173 57 and yields of compounds 5–25. a 171–173 51 9 176–178 81 Compound R and yields m.p. (°C) Yields 185–186 68 85710Table 1. Structures 174–176 83 179–181 83(%)83 8 174–176 9 176–178 81 6 171–173 57 of compounds 5–25. 51 879 174–176 83 Compound R m.p. (°C) Yields (%) a 176–178 81 185–186 68 6 171–173 57 5 171–173 51 174–176 98 176–178 81 Compound R m.p. (°C) Yields (%) a 10 179–181 83 185–186 68 171–173 57 97865 176–178 81 171–173 51 (%)81 a 179–181 9 10 176–178 174–176 83 Compound R m.p. (°C) Yields 765911 185–186 68 10 179–181 83 186–188 79 171–173 57 176–178 81 51(%) a 876 174–176 83 Compound R m.p. (°C) Yields 185–186 68 10 179–181 83 171–173 57 9875 176–178 81 171–173 51 174–176 10 179–181 83 185–186 68 11 186–188 79 Compound R m.p. (°C) Yields 171–173 57(%) aa 176–178 81 59876 171–173 51 174–176 83 186–188 79 10 179–181 10 11 179–181 185–186 68 Compound R m.p. (°C) Yields 171–173 57 11 186–188 79 9857612 176–178 81 180–182 76(%)83 174–176 10 179–181 83 171–173 51 185–186 68 Compound R m.p. (°C) Yields (%) a 11 186–188 79 9 6 176–178 81 171–173 57 8 174–176 10 179–181 83 11 186–188 79 171–173 51 185–186 68(%) a Compound R m.p. (°C) Yields 12 180–182 76 96587 176–178 81 171–173 57 174–176 10 179–181 83 12 180–182 76 11 186–188 79 5 171–173 51 769 185–186 68 Compound R m.p. (°C) Yields (%) a 176–178 81 12 180–182 76 171–173 57 10 179–181 83 174–176 178–180 82(%)79 171–173 51 186–188 79 a 11 11 186–188 759813 185–186 68 Compound R m.p. (°C) Yields 12 180–182 76 176–178 81 6 171–173 57 10 179–181 8 174–176 83 11 5 186–188 79 171–173 51 12 180–182 76 185–186 68 176–178 81 679 171–173 57 13 178–180 82 10 179–181 11 186–188 79 8 174–176 83 5 171–173 51 12 180–182 76 13 178–180 82 185–186 68 9786 176–178 81 171–173 57 10 179–181 83 11 186–188 79 13 178–180 82 174–176 83 12 180–182 76 187–189 81 714 185–186 68 176–178 81 171–173 57 13 178–180 82 10 179–181 83 11 186–188 79 8697 174–176 12 180–182 76 1213 180–182 76 185–186 68 178–180 82 9 176–178 81 11 186–188 79 10 179–181 83 8 174–176 14 12 187–189 81 180–182 76 185–186 68 13 178–180 82 14 187–189 81 97815 176–178 11 186–188 79 10 179–181 83 12 180–182 76 174–176 156–158 91 14 187–189 81 13 178–180 82 176–178 81 11 186–188 79 12 180–182 76 10 179–181 89 174–176 83 14 187–189 81 13 178–180 82 9 176–178 187–189 81 11 186–188 79 12 180–182 76 82 15 156–158 91 10 179–181 83 13 178–180 82 1314 178–180 15 156–158 91 16 161–163 86 9 176–178 81 14 187–189 12 180–182 76 10 179–181 83 11 186–188 79 15 156–158 91 13 178–180 82 14 187–189 81 10 179–181 83 12 180–182 76 11 186–188 79 15 156–158 91 13 178–180 82 16 161–163 86 15 156–158 91 14 187–189 81 12 180–182 76 10 179–181 83 11 186–188 79 16 161–163 86 13 178–180 82 14 187–189 81 16 15 161–163 86 156–158 91 17 188–190 82 11 186–188 79 12 180–182 76 13 178–180 82 16 161–163 86 14 187–189 81 15 156–158 91 1416 187–189 11 186–188 79 12 180–182 76 161–163 86 13 178–180 82 81 17 188–190 82 15 156–158 91 14 187–189 81 11 186–188 79 12 180–182 76 16 161–163 86 17 188–190 13 178–180 82 14 187–189 81 15 156–158 91 17 188–190 82 12 180–182 76 16 161–163 86 13 178–180 82 15 14 156–158 91 187–189 81 17 188–190 82 18 202–204 70 12 180–182 76 16 161–163 86 17 188–190 82 13 178–180 15 156–158 91 14 187–189 81 91 15 16 156–158 161–163 86 12 180–182 76 17 188–190 13 178–180 82 15 156–158 91 18 202–204 70 14 187–189 81 16 161–163 86 18 202–204 70 17 13 188–190 82 178–180 15 156–158 91 14 187–189 81 18 202–204 70 16 161–163 86 17 188–190 82 13 178–180 15 156–158 91 18 202–204 70 14 187–189 81 19 192–193 84 86 16 161–163 86 1618 161–163 17 188–190 82 202–204 70 13 178–180 15 156–158 91 14 187–189 81 16 161–163 86 17 188–190 82 18 202–204 70 15 156–158 91 19 14 192–193 84 187–189 81 16 161–163 86 17 188–190 82 18 202–204 70 19 192–193 84 15 156–158 91 14 187–189 81 19 192–193 84 16 161–163 86 17 188–190 82 18 202–204 70 15 156–158 91 14 187–189 81 19 192–193 84 16 161–163 86 17 188–190 82 20 194–196 75 82 18 1719 188–190 202–204 70 192–193 84 15 156–158 91 16 161–163 86 17 188–190 82 18 202–204 70 19 192–193 84 15 156–158 91 20 194–196 75 16 161–163 86 17 188–190 82 18 202–204 70 19 192–193 84 20 194–196 75 15 156–158 91 16 161–163 86 18 202–204 70 20 194–196 75 17 188–190 82 19 192–193 84 20 194–196 75 16 161–163 86 18 202–204 70 19 192–193 84 17 188–190 82 21 198–200 83 20 194–196 75 16 161–163 86 18 202–204 70 70 19 192–193 84 18 17 202–204 188–190 82 20 194–196 75 18 202–204 70 17 188–190 82 21 19 198–200 83 192–193 84 20 194–196 75 21 198–200 83 19 192–193 84 17 188–190 82 18 202–204 70 21 198–200 83 20 194–196 75 19 192–193 84 17 188–190 82 18 202–204 70 21 198–200 83 20 194–196 75 22 181–183 85 21 198–200 83 19 192–193 84 18 202–204 70 20 194–196 75 21 198–200 83 84 18 202–204 70 19 192–193 84 192–193 19 21 20 194–196 75 22 181–183 85 198–200 83 18 202–204 70 22 181–183 85 19 192–193 84 20 194–196 75 22 181–183 85 21 198–200 83 18 202–204 70 19 192–193 84 20 194–196 75 22 181–183 85 21 198–200 83 23 204–206 77 22 181–183 85 19 192–193 84 20 194–196 75 21 198–200 83 19 192–193 84 22 181–183 85 20 194–196 75 23 204–206 77 21 198–200 83 22 19 181–183 85 192–193 84 75 23 204–206 77 20 194–196 75 20 22 194–196 21 198–200 83 23 204–206 77 181–183 85 19 192–193 84 20 194–196 75 21 198–200 83 23 204–206 77 24 184–186 82 22 181–183 85 23 204–206 77 20 194–196 75 21 198–200 83 22 181–183 85 20 194–196 75 23 204–206 77 21 198–200 83 24 184–186 82 22 181–183 85 23 20 204–206 77 194–196 75 24 184–186 82 21 198–200 83 22 181–183 85 24 184–186 82 23 204–206 77 20 194–196 75 25 135–137 86 83 21 198–200 83 24 184–186 82 2124 198–200 22 181–183 85 23 204–206 77 184–186 82 21 198–200 83 22 181–183 85 25 135–137 86 23 204–206 77 24 184–186 82 a 21 198–200 83 Yields represent isolated yields. 25 135–137 86 22 181–183 85 23 204–206 77 24 184–186 82 25 135–137 86 21 198–200 83 22 181–183 85 23 204–206 77 a Yields represent isolated 25 135–137 86 24 184–186 82 yields. 21 198–200 83 a 25 135–137 86 22 181–183 85 23 204–206 77 Yields represent isolated yields. 24 184–186 82 a Yields represent isolated yields. 25 135–137 86 22 181–183 85 23 204–206 77 85 22 25 181–183 24 184–186 82 a Yields represent isolated yields. 135–137 86 a Yields 22 181–183 85 23 204–206 77 represent isolated yields. 24 184–186 82 25 135–137 86 a Yields represent isolated 22 181–183 85 24 184–186 82 yields. 23 204–206 77 25 135–137 86 a Yields represent isolated 22 181–183 85 24 184–186 82 yields. 23 204–206 77 25 135–137 86 a Yields represent isolated yields. 24 184–186 82 23 204–206 77 25 135–137 86 a Yields represent isolated yields. 23 204–206 77 184–186 82 23 24 204–206 77 25 135–137 86 a Yields represent isolated yields. 23 204–206 77 24 184–186 82 25 135–137 86 a Yields represent isolated yields. 23 204–206 77 24 184–186 82 25 135–137 86 a Yields represent isolated yields. 24 184–186 82 a Yields represent isolated 25 135–137 86 yields. 24 184–186 82 a Yields represent isolated 135–137 86 yields. 24 184–186 82 82 24 25 184–186 a Yields represent isolated 25 135–137 86 yields. 24 184–186 82 25 135–137 86 a Yields represent isolated yields. 25 135–137 86 a Yields represent isolated yields. 25 135–137 86 a Yields represent isolated yields. 135–137 86 a Yields represent isolated 25 25 135–137 86 yields. a

a

a

Yields represent isolated yields. Yields represent isolated yields.

Yields represent isolated yields.

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Scheme Scheme 3. 3. Synthetic Synthetic routes routes of of hybrid hybrid compounds. compounds.

2.2. 2.2. Biological Biological Evaluation Evaluation 2.2.1. 2.2.1. Anti-Inflammatory Anti-Inflammatory Activity Activity RAW models in in vitro. In this work, we RAW 264.7 264.7 cells cells are are widely widelyused usedtotoestablish establishinflammatory inflammatory models vitro. In this work, investigated the anti-inflammatory activity of synthetic compounds in LPS-induced RAW 264.7 we investigated the anti-inflammatory activity of synthetic compounds in LPS-induced RAW 264.7 on on the of NO. NO. The The results results of of the the title title hybrids hybrids are are summarized summarized in in Table Table2. 2. the generation generation of Table 2. Anti-inflammatory activities of compounds. Table 2.

Compound NO Generation (IC50, μM) a a Compound NO Generation (IC50, μM)a a Compound NO Generation (IC50 , µM) Compound NO Generation (IC50 , µM) 5 16 14.12 5.28 5 14.12 16 5.28 6 17 34.24 25.40 6 34.24 17 25.40 7 7 1818 >40>40 6.53 6.53 8 8 1919 >40>40 >40 >40 18.52 >40 9 9 2020 18.52 >40 >40 21 >40 10 10 21 >40 >40 11 >40 22 9.13 11 12 2223 >40>40 9.13 23.56 12 13 23 >40 23.56 23.06 24 18.37 20.27 >40 13 14 2425 23.06 18.37 15 31.68 14 25 20.27 >40 a Values represent the concentration required to produce 50% inhibition of the response. 15 31.68 a

Values represent the concentration required to produce 50% inhibition of the response.

As shown in Table 2, the substituents of the NH group of the piperazine ring have an obvious influence on anti-inflammatory activities. To benzofuran–piperazine compounds 16, 18, As shown in Table 2, the substituents ofour the delight, NH group of the piperazine ring have an obvious and 22 displayed good anti-inflammatory on the generation of NO (IC50 < compounds 10 µM). Especially, influence on anti-inflammatory activities. activity To our delight, benzofuran–piperazine 16, 18, compound 16 wasgood found to be the most potent anti-inflammatory agent (IC(IC addition, and 22 displayed anti-inflammatory activity on the generation of NO < 10µM). μM).In Especially, 50 =505.28 compounds165,was 9, and 24 showed anti-inflammatory activity (IC50(IC < 50 20=µM). However, hetero compound found to be thepotent most potent anti-inflammatory agent 5.28 μM). In addition, aromatic compound no activity compared to others. except the mentioned compounds 5, 9, and25 24displayed showed potent anti-inflammatory activityNotably, (IC50 < 20 μM).for However, hetero hybrids, most of the derivatives had or no inhibitory the release of NO > 20 µM). aromatic compound 25 displayed nolittle activity compared toeffects others.on Notably, except for (IC the50mentioned On the other hand, we could find that the substituents of the on NH ofofthe ring hybrids, most of the derivatives had little or no inhibitory effects thegroup release NOpiperazine (IC50 > 20 μM). played an important ketosubstituentsofcontributed to better alkyl and aryl On the other hand, werole. couldOverall, find that the substituents the NH group of the activity, piperazine ring played substituents weakerketoactivity, and the pyridyl substituent no effect anti-inflammatory an important led role.toOverall, substituents contributed to betterhad activity, alkylon and aryl substituents activity (keto- activity, > alkyl ≈and arylthe > pyridyl). So, in future research, weon will focus on the ketosubstituents. led to weaker pyridyl substituent had no effect anti-inflammatory activity (keto> alkyl ≈ aryl > pyridyl). So, in future research, we will focus on the keto- substituents. 2.2.2. Anticancer Activity

2.2.2.The Anticancer Activity anticancer activities of novel synthesized derivatives were evaluated against human lung cancer cell line (A549), humanofcervical carcinoma (Hela), and human gastric carcinoma (SGC7901) The anticancer activities novel synthesized derivatives were evaluated against human lung by MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide] assay, using cisplatin cancer cell line (A549), human cervical carcinoma (Hela), and human gastric carcinoma (SGC7901) (DDP) as[3-(4, the reference drug. The anti-tumor results for the hybrids are summarized Tablecisplatin 3. by MTT 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide] assay,inusing (DDP) as the reference drug. The anti-tumor results for the hybrids are summarized in Table 3.

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Table 3. In vitro cytotoxic activities of title compounds. Compound 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 DDP

Cell Lines (IC50 , µM) a A549

Hela

SGC7901

>40 >40 >40 >40 19.27 >40 16.14 >40 >40 >40 >40 0.12 27.82 19.34 6.25 8.11 23.22 26.07 34.13 >40 >40 11.54

>40 32.53 >40 >40 >40 >40 8.57 25.14 >40 33.24 22.36 26.32 >40 >40 18.71 28.74 15.35 >40 12.68 27.58 26.22 20.52

27.24 >40 >40 >40 >40 >40 >40 16.27 25.04 >40 30.43 2.75 15.41 23.92 36.23 >40 >40 >40 7.45 >40 >40 12.44

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.

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 (16–24) 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.

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Scheme 4. Structure–activity relationship of hybrid compounds.

Scheme 4. Structure–activity relationship of hybrid compounds. 3. Materials and Methods

3. Materials and Methods

3.1. General Information

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

Starting materials commercially available andBruker analytically pure. Melting points were 13C-NMR spectra were were recorded on Bruker AV 300 and AV 400 spectrometers (Bruker, 1 H-NMR and Germany) microscopic using TMS as internal standard CDCl3and as solvent, Thin layer measuredKarlsruhe, on a YANACO melting pointand meter wererespectively. uncorrected. 13 C-NMR spectra chromatographic (TLC) analysis was carried out on Bruker silica gelAV plates 254. High-resolution mass were recorded on Bruker AV 300 and 400GF spectrometers (Bruker, Karlsruhe, spectra were performed on an ESI Q-TOF MS spectrometer (Agilent, Santa Clara, CA, USA). 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 3.2. Chemistry spectra were performed on an ESI Q-TOF MS spectrometer (Agilent, Santa Clara, CA, USA). 3.2.1. General Procedure

3.2. Chemistry 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 3.2.1. General Procedure 100 mL cold water. After stirring for 10 min, the mixture was filtered, and the filtrate was concentrated

General procedure forafford thea synthesis in vacuo and dried to yellow solid. of compound 3: To a solution of acetone (50 mL), General procedure for the preparation compound To a stirred 0solution of compound 3 (2.17 g, 5-diethylamino salicylaldehyde (1.93 g, 10 of mmol) and4: 2-bromo-4 -fluoroacetophenone (1.56 g, 5 mmol) and K2CO3 (1.38 g, 10 mmol) in dried DMF (20 mL), piperazine (0.86 g, 10 mmol) 10 mmol), was added K2 CO3 (2.76 g, 20 mmol) and left to react for 4 h in reflux. The reaction was added, and the reaction mixture was stirred for 12 h at 110 °C. After completion of the reaction was poured into 100 by mL cold stirringby forthe10addition min, the mixture was filtered, and the filtrate as indicated TLC, thewater. reactionAfter was quenched of CHCl 3 (50 mL) and washed with was concentrated in vacuo and dried to afford a yellow solid. water (3 × 20 mL). The organic layer was dried by anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography afford a brown solid. General procedure for the preparationto of compound 4: To a stirred solution of compound 3 General procedure for the preparation of hybrid derivatives 5–7: To a stirred solution of (1.56 g, 5 mmol) and K2 CO3 (1.38 g, 10 mmol) in dried DMF (20 mL), piperazine (0.86 g, 10 mmol) compound 4 (0.5 mmol) in dried DMF (5 mL), NaH (0.06 g, 60% in ◦oil) was added, and the mixture was added, and theatreaction was(1stirred for added, 12 h atand 110 After completion of the was stirred 0 °C for 1mixture h. Then, R-X mmol) was afterC.completion of the reaction as reaction as indicated by TLC, the the reaction quenched addition andwith washed with indicated by TLC, reactionwas was quenched by by the the addition of H2O of (30CHCl mL) and wasmL) extracted 3 (50 (3 × 10The mL).organic The organic layer wasdried dried by sodium sulfate, concentrated in vacuo, in vacuo, water (3 ×DCM 20 mL). layer was byanhydrous anhydrous sodium sulfate, concentrated and purified by column chromatography (1% MeOH/DCM) to afford products. and purified by column chromatography to afford a brown solid. General procedure for the preparation of derivatives 8–14, 25: To a stirred solution of General procedure the preparation ofg)hybrid derivatives 5–7: To(0.6 a stirred solution compound 4 compound 4 (0.5for mmol) and Cs2CO3 (0.5 in dried DCM (15 mL), R-X mmol) was added,ofand (0.5 mmol)the inreaction dried DMF (5 mL), NaH (0.06 g, 60% in oil) was added, and the mixture was mixture was stirred for 12 h at room temperature (r.t.). After completion of the reactionstirred at 0 ◦ C for 1 h.asThen, indicated the mixture was filtered Then, the organic layer wasreaction concentrated in vacuo by TLC, R-Xby(1TLC, mmol) was added, andoff. after completion of the as indicated and purified by column chromatography (1% MeOH/DCM) to afford products. the reaction was quenched by the addition of H2 O (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 8–14, 25: To a stirred solution of compound 4 (0.5 mmol) and Cs2 CO3 (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 15–24: To a stirred solution of compound 4 (0.5 mmol) and K2 CO3 (0.2 g) in dried DCM (10 mL), R-X (0.6 mmol) was added, and the reaction

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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; 1 H-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); 13 C-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 C23 H27 N3 O2 Na [M + Na]+ 400.1995, found 400.1995. Compound 5: Pale yellow solid; 1 H-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); 13 C-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; 1 H-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); 13 C-NMR (100 MHz, CDCl ) δ: 181.84, 158.95, 153.90, 151.11, 149.05, 131.48, 128.01, 123.43, 116.66, 3 116.50, 113.67, 110.95, 93.16, 52.55, 52.43, 47.46, 45.09, 12.60, 11.97. Compound 7: Pale brown solid; 1 H-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); 13 C-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 C41 H63 N3 O2 [M + H]+ 630.4993, found 630.4983. Compound 8: Yellow solid; 1 H-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); 13 C-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 C26 H31 N3 O2 Na [M + Na]+ 440.2308, found 440.2307. Compound 9: Pale yellow solid; 1 H-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); 13 C-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 C26 H29 N3 O2 [M + H]+ 416.2333, found 416.2337. Compound 10: Yellow solid; 1 H-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); 13 C-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; 1 H-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,

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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); 13 C-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; 1 H-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); 13 C-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 C31 H32 F3 N3 O2 [M + H]+ 536.2519, found 536.2526. Compound 13: Pale yellow solid; 1 H-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); 13 C-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 C30 H32 F2 N3 O2 [M + H]+ 504.2457, found 504.2453. Compound 14: Pale brown solid; 1 H-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); 13 C-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; 1 H-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); 13 C-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 C27 H34 N3 O4 [M + H]+ 464.2543, found 464.2545. Compound 16: Pale red solid; 1 H-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); 13 C-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 C26 H31 N3 O3 [M + H]+ 434.2438, found 434.2444. Compound 17: Pale yellow solid; 1 H-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); 13 C-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 C31 H34 N3 O3 [M + H]+ 496.2595, found 496.2598. Compound 18: Pale red solid; 1 H-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); 13 C-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 C35 H35 N3 O3 Na [M + Na]+ 568.2570, found 568.2569.

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Compound 19: Orange solid; 1 H-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); 13 C-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; 1 H-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); 13 C-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 C31 H32 N3 O3 NaCl [M + Na]+ 552.2024, found 552.2023. Compound 21: Pale green solid; 1 H-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 C31 H32 BrN3 O3 [M + H]+ 574.1700, found 574.1699. Compound 22: Orange solid; 1 H-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); 13 C-NMR (100 MHz, CDCl ) δ: 194.62, 181.78, 163.78, 158.92, 153.85, 151.08, 149.05, 131.46, 130.52, 3 129.05, 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 C32 H36 N3 O4 [(M + H)]+ 526.2700, found 526.2700. Compound 23: Pale yellow solid; 1 H-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); 13 C-NMR (100 MHz, CDCl ) δ: 195.18, 181.85, 159.01, 153.77, 151.06, 149.15, 138.84, 132.57, 131.51, 3 128.84, 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 C32 H33 N4 O3 [M + H]+ 521.2547, found 521.2547. Compound 24: Pale green solid; 1 H-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); 13 C-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; 1 H-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); 13 C-NMR (100 MHz, CDCl ) δ: 181.81, 158.98, 153.89, 151.15, 150.57, 150.01, 149.09, 147.36, 131.50, 3 128.15, 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 C29 H33 N4 O2 [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

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(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 1 H-NMR, mdpi.com/1420-3049/21/12/1684/s1.

13 C-NMR

and HRMS spectra are available online at: http://www.

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 3–25 are available from the authors. © 2016 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/).