Semi-synthesis, structural modification and biological evaluation of 5-arylbenzofuran neolignans

Abstract

5-Arylbenzofuran neolignans, a newfound class of natural products, were reported to possess several kinds of pharmacological activities. To solve the lack of natural sources and promote the research of 5-arylbenzofuran neolignans in all fields, an available semi-synthesis methodology of 5-arylbenzofuran neolignans was developed, and a detailed structural modification was conducted. In the meantime, a one-pot process of Waker-type cyclization and Wacker-type oxidation was developed. To explore the potential of 5-arylbenzofuran neolignans as bioactive substances, 5-arylbenzofuran neolignans and their derivatives were evaluated for their cytotoxicity. As a result, a preliminary structure–activity relationship was obtained. Most derivatives revealed low cytotoxic effects suggesting that they were relatively safer than the natural 5-arylbenzofuran neolignan. Several derivatives showed high cytotoxicities which were found to be closely associated with apoptosis-inducing. The selectivity assay for cytotoxicity showed tumor cells were more sensitive to the promising compounds than normal cells.

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Introduction

Neolignans are a large class of natural products, which consist of two propylbenzene (C₆C₃) units.¹ Benzofuran neolignans, a significant kind of neolignans, have drawn great interest due to their special skeleton and pharmacological activities. So far, the research of benzofuran neolignans has mainly focused on 2-arylbenzofuran neolignans (Fig. 1). Lots of 2-arylbenzofuran neolignans, such as ailanthoidol,² eupomatenoid 7,³ licarin A⁴ and acuminatin,⁵ have been proved to possess excellent biological activities.

Fig. 1 The comparison of 5-arylbenzofuran neolignans with 2-arylbenzofuran neolignans, and the semi-synthesis methodology and structural modification of 5-arylbenzofuran neolignans.

In recent years, some 5-arylbenzofuran neolignans (Fig. 1) have come into sight with favorable anti-inflammatory,^6,7 anti-microbial,⁸ and anti-influenza activities.⁹ Due to the difference in the bonding position of the two aromatic rings, 5-arylbenzofuran neolignans may have quite different biological effects compared to 2-arylbenzofuran neolignans. However, the low content in natural products and the difficulty of chemical synthesis have blocked the research and development of 5-arylbenzofuran neolignans.

In order to solve the problem of a lack of a natural source of 5-arylbenzofuran neolignans, we developed a semi-synthesis methodology utilizing honokiol, a high-yield natural product whose total synthesis methodologies are various, as the starting material (Fig. 1). To promote the study of 5-arylbenzofuran neolignans in all fields, we made structural modification and investigated their cytotoxicity. In addition, inspired by the achievements of 2-arylbenzofuran neolignans in tumor therapy,¹⁰ 5-arylbenzofuran neolignan derivatives with high cytotoxicities were further evaluated for their anti-proliferative effects and mechanisms.

Results and discussion

Chemistry

It is of great difficulty to totally synthesize 5-arylbenzofuran neolignans because the two propylbenzene units are directly connected by benzenes. Nonetheless we found there are a number of natural products containing the structure of 2-allyl-4-arylphenol which could be intramolecularly cyclized to afford 5-arylbenzofuran neolignans. In this paper, honokiol, a high-yield natural product with the structure of 2-allyl-4-arylphenol, was selected as the starting material (Scheme 1).

Scheme 1 Reagents and conditions: (a) PdCl₂, NaOAc, O₂, DMA/H₂O, 60 °C; (b) H₂SO₄, DCE, 50 °C; (c) NaBH₄, NiCl₂·6H₂O, EtOH, r.t.; (d) Pd/C, H₂, MeOH, reflux; (e) CH₃I, KOH, THF, r.t.

5-Arylbenzofuran neolignan 1, which can only be extracted from natural product before,⁶ was synthesized starting from honokiol by Wacker-type intramolecular cyclization¹¹ in a high yield. For investigating the influence by furan ring, dihydrofuran derivative 2 was prepared through acid catalytic cyclization from honokiol in the presence of concentrated sulfuric acid.¹² To discuss the effects of phenolic hydroxyl and allyl group, we synthesized 3–5 by the method depicted in Scheme 1. Compound 3 and 4 were obtained by hydrogenation reduction of 1 using NaBH₄ (ref. 13) or H₂ (ref. 14) as hydrogen source respectively, while compound 5 was generated via the etherification of 1.

In order to study the influences by different substituents on benzene ring, compound 7–26 with diverse substituents were designed and prepared. Among them, compounds 6–10 and 20–24 were synthesized from 1 according to the method described in Scheme 2. The bromination reaction of 1 provided compound 7 using NBS as the brominating reagent¹⁵ and the nitration of 1 prepared compound 8 by classic procedure. Compound 10 was synthesized via reduction reaction with zinc powder as a catalyst from 9, which was prepared by nitrosation from 1. Compounds 20–24 were obtained through Mannich reaction of 1 with formalin and appropriate secondary amines.¹⁶ And compound 6 was generated by hydrogenation reduction of 20 catalyzed by palladium–carbon.

Scheme 2 Reagents and conditions: (a) NBS, MeCN, r.t.; (b) HNO₃, HOAc, CH₂Cl₂, 0 °C; (c) NaNO₂, concentrated hydrochloric acid (36–38%), MeCN/H₂O, r.t.; (d) secondary amines, HCHO(37%), MeOH, 60 °C; (e) Zn, NH₄Cl, HOAc, EtOH/H₂O, r.t.; (f) Pd/C, H₂, MeOH, reflux.

Compounds 11–19, 25 and 26 were prepared from 10 according to the approach presented in Scheme 3. Compound 11 was produced via methylation of 10. Acylamides 12–14 were obtained from 10 by acylation reaction with acetic anhydride, acryloyl chloride or chloroacetyl chloride, respectively. Aminoacetamides 15–18 were synthesized through reaction between compound 14 and appropriate secondary amines.¹⁷ Lactams 25 and 26 were prepared by treatment of 10 with chloroacetyl chloride¹⁸ or oxalyl chloride¹⁹ in the presence of 4-dimethylaminopyridine and triethylamine in THF, respectively.

Scheme 3 Reagents and conditions: (a) CH₃I, NaOAc, DMF, r.t.; (b) acetic anhydride, K₂CO₃, ethyl acetate, r.t.; (c) acryloyl chloride, K₂CO₃, CH₂Cl₂, r.t.; (d) chloroacetyl chloride or oxalyl chloride, DMAP, NEt₃, THF, r.t. to reflux; (e) benzaldehyde, acetic acid, EtOH, r.t.; (f) chloroacetyl chloride, K₂CO₃, CH₂Cl₂, r.t.; (g) secondary amines, NEt₃, THF, r.t.

For further investigation on the influences of side chain, groups with large polarity or steric hindrance were introduced, and compounds 27–32 were synthesized via the route outlined in Scheme 4. Compound 27 was generated from 1 by Wacker oxidation adopting the condition reported previously.¹¹ The condensation reaction of 27 with appropriate primary amines prepared compounds 28–30,^20–22 and the reduction of 29 and 30 afforded compounds 31 and 32, respectively.²³

Scheme 4 Reagents and conditions: (a) PdCl₂, O₂, DMA/H₂O, 60 °C; (b) R–NH₂, EtOH, r.t.; (c) NaBH₄, EtOH, r.t.

In the meantime, a one-pot method of Waker-type cyclization and Wacker-type oxidation was developed. The synthetic process was supplied in the Experimental section, and the discussion was provided in the ESI.†

Cytotoxicity and SAR analysis

The cytotoxicity of 5-arylbenzofuran neolignans (1–32) were evaluated via MTT assay against A549 (human lung carcinoma), K562 (human myelogenous leukemia) and HepG2 (human hepatocellular liver carcinoma) tumor cell lines described with IC₅₀ (concentration required for 50% inhibition). For a clear view, the IC₅₀ values of tested compounds were presented in Tables 1–3 according to their structural differences. It's found that most derivatives showed no cytotoxic effects against three tumor cell lines (IC₅₀ > 128 μM) which suggested they were relatively safer than the natural 5-arylbenzofuran neolignan. Several compounds, such as 23, 26 and 30, displayed obviously higher cytotoxicity than the natural 5-arylbenzofuran neolignan 1. In addition, the A549 and K562 cells were more susceptive to 5-arylbenzofuran neolignans than HepG2 and the structural modifications caused appreciable impact on cytotoxicity.

Table 1 IC₅₀ values of compounds 1–6 against HepG2, A549 and K562 cell lines

Compd.	IC50 (μM)
	A549	K562	HepG2
1	71.5 ± 1.94	67.8 ± 8.3	107.2 ± 12.7
2	115.5 ± 2.0	126.2 ± 1.5	125.6 ± 2.2
3	61.9 ± 2.7	73.9 ± 7.3	88.2 ± 8.0
4	96.1 ± 7.0	72.8 ± 8.9	121.6 ± 2.5
5	>128	>128	>128
6	84.9 ± 7.8	102.3 ± 15.5	>128

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Table 2 IC₅₀ values of compound 7–26 against HepG2, A549 and K562 cell lines

Compd.	R	IC50 (μM)
		A549	K562	HepG2
7	Br	>128	>128	>128
8	NO2	>128	>128	>128
9	NO	>128	>128	>128
10	NH2	>128	>128	>128
11	N(CH3)2	>128	>128	>128
12	NHCOCH3	88.2 ± 3.4	80.0 ± 5.1	87.4 ± 6.1
13		53.0 ± 5.7	34.9 ± 1.2	63.8 ± 3.1
14		65.0 ± 2.0	58.5 ± 0.5	>128
15		>128	>128	>128
16		>128	>128	>128
17		75.4 ± 2.2	61.2 ± 5.6	98.5 ± 7.8
18		>128	>128	>128
19		>128	>128	>128
20		>128	87.78 ± 13.9	>128
21		>128	>128	>128
22		>128	>128	>128
23		25.1 ± 4.4	29.6 ± 2.5	36.8 ± 0.2
24		>128	>128	>128
25		53.4 ± 5.6	41.8 ± 5.2	>128
26		32.4 ± 1.7	18.0 ± 2.6	75.4 ± 0.3

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Table 3 IC₅₀ values of compound 27–32 against HepG2, A549 and K562 cell lines

Compd.	R^1 or R^2	IC50 (μM)
		A549	K562	HepG2
a 5-FU: 5-fluorouracil.b PTX: paclitaxel.
27	O	>128	>128	>128
28	N–OH	125.1 ± 12.2	>128	>128
29		64.7 ± 7.4	31.8 ± 4.4	92.1 ± 6.2
30		22.0 ± 2.1	29.0 ± 1.8	26.5 ± 3.5
31		103.8 ± 5.5	48.1 ± 3.4	80.9 ± 1.5
32		97.4 ± 8.0	66.8 ± 0.7	106.1 ± 13.6
5-FU ^a		34.3 ± 2.1	81.9 ± 7.4	>128
PTX ^b		21.0 ± 3.0	4.6 ± 0.7	16.5 ± 2.2

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As shown in Table 1, 5-arylbenzofuran 1 and 3 displayed higher cytotoxicity than 5-aryldihydrbenzofuran 2, 4 and 6. The reduction of allyl group made little difference to activity when comparing compound 1 and 3 with compound 2 and 4. Phenolic hydroxyl was essential to the cytotoxicity as compound 5 possessing very low cytotoxicity against three tumor cells. In view of the above results, further research was carried out around the substructure of 5-(phenol-2-yl)benzofuran due to its comparatively good anti-proliferative activity.

In order to discuss the influences by substituents on benzene ring, compounds 7–26 were evaluated for the cytotoxicities and the results were displayed in Table 2. Compounds with simple substituents such as bromo, nitro, nitroso, amino and dimethylamino (7–11) showed no cytotoxicity. Acylamides (12–14) seemed to have equipotent cytotoxicity compared to the natural product 1 while aminoacetamides (15–18) showed lower cytotoxicity except substituted by 4-methylpiperazine (17). Similar rule appeared in mannich bases (20–24), that is the mannich base of 4-methylpiperazine (23) exhibited the highest cytotoxic effect which is much higher than the natural compound 1. Different from compound 5, lactams 25 and 26 showed relatively high cytotoxicity even though neither of their hydroxyl group was free. As a consequence, the structural modifications around phenolic hydroxyl group would have obvious impacts on cytotoxicity.

For further investigation on the modification of side chain, compounds 27–32 were synthesized and their cytotoxicities were exhibited in Table 3. As a result, compounds 29–32 presented higher cytotoxicity than 27 and 28, while 30 showed the highest cytotoxicity. For this position, the introduction of groups with non-polarity or large steric effect may enhance the cytotoxicity.

In addition, the selectivity for cytotoxicity was further studied by testing the cytotoxicity against human umbilical vein endothelial cell (HUVEC). As shown in Table 4, the selectivities of promising compounds 23 and 30 were 2.02 and 2.50 respectively, which were higher than the positive control paclitaxel. As already reported, some human normal cells were more sensitive to paclitaxel than tumor cells,²⁴ and it's also a common problem for traditional anticancer drugs.²⁵ The promising compounds 23 and 30 seemed to possess certain advantage in the respect.

Table 4 The selectivity of promising compounds

Compd.	IC50 (μM)		Selectivity^a
	A549	HUVEC
a Selectivity is defined as IC50 (HUVEC)/IC50 (A549).b PTX: paclitaxel.
1	71.5 ± 1.94	134.4 ± 18.8	1.88
23	25.1 ± 4.4	50.8 ± 5.7	2.02
30	22.0 ± 2.1	55.1 ± 6.2	2.50
PTX^b	21.0 ± 3.0	8.6 ± 1.6	0.41

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Morphological analysis

Microscopic observations were used to investigate the changes of cellular morphology and prove the anti-proliferative activities of compounds with high cytotoxicity. As displayed in Fig. 2A, no apparent changes of cell morphology were observed after HepG2 cells were treated with 20 μM of compound 1 for 24 h. However, HepG2 cells treated with compounds 23 and 30 displayed some morphological changes, such as cell shrinkage, rounding, loss of contact with neighbouring cells and detachment from plate. And the compound 30 had a greater effect on cell morphologies than that of compound 23, which was consistent with their cytotoxicities.

Fig. 2 (A) Cellular morphologies of HepG2 cells treated with different compounds (1, 23, 30) for 24 h. (B) Images of Hoechst 33258 stained HepG2 cells which were treated with 20 μM of different compounds (1, 23, 30) for 24 h. The arrows indicated the apoptotic cell. Original magnification is 400×.

Apoptosis assessment by Hoechst 33258 staining

In order to observe the changes of the nuclear morphology, the HepG2 cells were stained by Hoechst 33258 and the results were exhibited in Fig. 2B. In the control group it's showed slightly blue and homogeneous fluorescence, and there was no obvious changes of fluorescence in the cells treated with compound 1 at the concentration of 20 μM. However, in the groups treated with 23 or 30, there was a lot of enhanced fluorescence which means karyopyknosis and chromatin condensation in apoptotic cells. It's suggested that compounds 23 and 30 could induce apoptosis of HepG2.

Apoptosis assessment by flow cytometry analysis

To clearly analyze the apoptosis-inducing effects of the modified compounds, the apoptosis ratios induced by compound 1, 23 and 30 in HepG2 cells were evaluated by flow cytometry analysis (Fig. 3). The result showed that compound 1 have little influence on the apoptosis of HepG2 compared with the control group. Compounds 23 and 30 caused more apoptosis than compound 1 at the same concentration, and it was consistent with the results of the Hoechst 33258 staining assay.

Fig. 3 Apoptosis effect of compounds 1, 23 and 30 at 20 μM in HepG2 cells for 24 h. The cells were stained with Annexin V-FITC/P and followed by flow cytometry analysis.

mRNA expression in HepG2 cells

Furthermore, for studying the mechanism of apoptosis-inducing caused by modified compounds, the expression levels of mRNAs related to the mitochondria-mediated apoptosis pathway were analyzed by qRT-PCR assay. The mRNA expression levels of caspase-3, caspase-9, Bax, Bcl-2, p53 and p21 in HepG2 cells were displayed in Fig. 4. Caspase-3 and caspase-9 contributes a major role in mitochondria-mediated apoptotic pathway.²⁶ And in the HepG2 cells treated with compound 23, the mRNA expression level of caspase-3 and caspase-9 was about twice the level of control group (Fig. 4A), while the mRNA expression level of caspase-9 and caspase-3 have no change treated with compound 1 and the caspase-3 expression increased treated with compound 30.

Fig. 4 mRNA expression levels of (A) caspase-3, caspase-9, (B) Bax, Bcl-2, (C) p53 and p21 in HepG2 cells treated with 20 μM of different compounds (1, 23, 30) for 24 h. *P < 0.05, **P < 0.01.

The activation of caspase-dependent apoptosis pathway could be regulated by Bcl-2 family members such as Bax and Bcl-2.²⁷ In Fig. 4B, the Bax/Bcl-2 ratio of compound 23 treated HepG2 cells increased obviously, which always resulted in the activation of caspases and led to apoptosis.²⁸ However, it made no difference for Bax/Bcl-2 ratio after treated with compound 1 and 30.

In the previous studies, p53 and its downstream gene p21 were also found able to cause apoptosis via the regulation of Bax/Bcl-2 (ref. 29 and 30) and the activation of caspases.³¹ In addition, p53 and p21 played a key role in cell cycle regulation.³² As shown in Fig. 4C, the mRNA expression level of p53 and p21 in the HepG2 cells treated with compound 23 were about 2 times higher than the control.

In summary, the compound 23 induces apoptosis of HepG2 cells through the regulation of caspase-dependent p53-dependent and Bax/Bcl-2 pathway. However, compound 30 also induce the expression of caspase-3, but had no effect on Bax/Bcl-2 activation pathway. It is suggested that compound 30 induces apoptosis mainly by other signal pathways. Therefore, different structural modifications of 5-arylbenzofuran neolignan would induce apoptosis by regulation of different signal pathways.

Experimental section

General methods and materials

All the reagents were commercially available and used without further purification. Melting points were determined on an X-4 binocular microscope melting point apparatus. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AV-400 spectrometer, using tetramethylsilane (TMS) as the internal standard and chemical shifts (δ) were expressed in ppm. Mass spectra were obtained by an Agilent 1100 series LC-MS. Elemental analyses were performed on a Vario EL III (Germany) instrument.

Synthesis of title compounds

4-Allyl-2-(2-methylbenzofuran-5-yl)phenol (1). A mixture of honokiol (10 mmol), NaOAc (1.0 mmol) and PdCl₂ (0.15 mmol) in the co-solvent of DMA and H₂O (35 mL, v/v = 6 : 1) was stirred under O₂ (0.8 Mpa) at 60 °C for 16 h. After cooling, the mixture was diluted with water and extracted with ethyl acetate. The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 1 as light yellow oil (86%). ¹H NMR (400 MHz, CDCl₃), δ 7.53 (d, J = 1.5 Hz, 1H, benzofuran 4 H), 7.50 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.26 (dd, J = 8.4, 1.8 Hz, 1H, benzofuran 6 H), 7.10–7.07 (m, 2H, C₆H₃ 3,5 H), 6.94 (d, 1H, J = 8.8 Hz, C₆H₃ 6 H), 6.41 (s, 1H, benzofuran 3 H), 6.04–5.93 (m, 1H, CH₂CH̲=CH₂), 5.21 (s, 1H, OH), 5.09 (dd, J = 23.1, 5.9 Hz, 2H, CH₂CH=CH̲₂), 3.36 (d, J = 6.7 Hz, 2H, CH̲₂CH=CH₂), 2.49 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃), δ 156.58, 154.44, 151.10, 138.08, 132.29, 131.79, 130.83, 130.21, 129.00, 128.75, 124.43, 120.87, 115.98, 115.75, 111.34, 102.93, 39.61, 14.19. LC-MS, m/z: 265.1 [M + H]⁺. Elemental anal (%) calcd for C₁₈H₁₆O₂: C 81.79, H 6.10; found: C 81.85, H 6.08.

4-Allyl-2-(2-methyl-2,3-dihydrobenzofuran-5-yl)phenol (2). To a stirred solution of honokiol (10 mmol) in 1,2-dichloroethane (40 mL) was added concentrated sulfuric acid (98%, 0.2 mL) dropwise at room temperature. Then the mixture was heated to 50 °C and stirred for 24 h. After cooling, the solvent was evaporated under reduced pressure and the residue was then purified by silica gel chromatography to afford 2 as light yellow oil (12%). ¹H NMR (400 MHz, CDCl₃) δ 7.22 (s, 1H, Ar–H), 7.17 (d, J = 8.1 Hz, 1H, Ar–H), 7.03–7.01 (m, 2H, Ar–H), 6.87 (d, J = 8.1 Hz, 1H, Ar–H), 6.82 (d, J = 8.1 Hz, 1H, Ar–H), 5.96 (dt, J = 16.6, 6.9 Hz, 1H, CH₂CH̲=CH₂), 5.26 (s, 1H, OH), 5.11–5.01 (m, 2H, CH₂CH=CH̲₂), 4.99–4.91 (m, 1H, CH₃CH̲CH₂), 3.36–3.29 (m, 3H, CH̲₂CH=CH₂ and CH₃CHCH̲₂), 2.83 (dd, J = 15.5, 7.6 Hz, 1H, CH₃CHCH̲₂), 1.47 (d, J = 6.2 Hz, 3H, CH̲₃CHCH₂); ¹³C NMR (101 MHz, CDCl₃) δ 159.36, 150.92, 137.88, 132.18, 130.38, 129.15, 128.96, 128.67, 128.33, 128.27, 125.95, 115.64, 115.60, 109.88, 80.11, 39.48, 37.10, 21.85. LC-MS, m/z: 267.2 [M + H]⁺. Elemental anal (%) calcd for C₁₈H₁₈O₂: C 81.17, H 6.81; found: C 81.20, H 6.83.

2-(2-Methylbenzofuran-5-yl)-4-propylphenol (3). To a stirred solution of 1 (2.0 mmol) and NiCl₂·6H₂O (2.0 mmol) in EtOH (10 mL) was added NaBH₄ (10 mmol) in five portions over 15 min and kept stirring for 5 min at room temperature. Then the reaction was quenched with dilute hydrochloric acid and extracted with CH₂Cl₂. The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 3 as light yellow oil (81%). ¹H NMR (400 MHz, CDCl₃) δ 7.53 (s, 1H, benzofuran 4 H), 7.48 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.26 (d, J = 8.4 Hz, 1H, benzofuran 6 H), 7.08–7.04 (m, 2H, C₆H₃ 4,6 H), 6.90 (d, J = 7.9 Hz, 1H, C₆H₃ 3 H), 6.39 (s, 1H, benzofuran 3 H), 5.17 (s, 1H, OH), 2.55 (t, J = 7.6 Hz, 2H, CH̲₂CH₂CH₃), 2.47 (s, 3H, CH₃), 1.69–1.59 (m, 2H, CH₂CH̲₂CH₃), 0.95 (t, J = 7.3 Hz, 3H, CH₂CH₂CH̲₃); ¹³C NMR (101 MHz, CDCl₃) δ 156.60, 154.34, 150.53, 134.86, 131.60, 130.44, 130.17, 128.77, 128.22, 124.24, 120.67, 115.41, 111.36, 102.73, 37.27, 24.85, 14.17, 13.90. LC-MS, m/z: 267.0 [M + H]⁺. Elemental anal (%) calcd for C₁₈H₁₈O₂: C 81.17, H 6.81; found: C 81.22, H 6.79.

2-(2-Methyl-2,3-dihydrobenzofuran-5-yl)-4-propylphenol (4). A mixture of 1 (5.0 mmol) and Pd/C (10%, 0.25 mmol) in MeOH (20 mL) was refluxed under H₂ (1.5 MPa) for 5 h. After cooling, the mixture was filtered and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 4 as light yellow oil (93%). ¹H NMR (400 MHz, CDCl₃) δ 7.24 (s, 1H, Ar–H), 7.19 (d, J = 8.1 Hz, 1H, Ar–H), 7.04–6.98 (m, 2H, Ar–H), 6.86 (d, J = 8.5 Hz, 1H, Ar–H), 6.83 (d, J = 8.1 Hz, 1H, Ar–H), 5.28 (s, 1H, OH), 5.01–4.92 (m, 1H, CH₃CH̲CH₂), 3.34 (dd, J = 15.5, 8.8 Hz, 1H, CH₃CHCH̲₂), 2.85 (dd, J = 15.6, 7.7 Hz, 1H, CH₃CHCH̲₂), 2.53 (t, J = 7.6 Hz, 2H, CH₃CH₂CH̲₂), 1.67–1.58 (m, 2H, CH₃CH̲₂CH₂), 1.49 (d, J = 6.2 Hz, 3H, CH̲₃CHCH₂), 0.94 (t, J = 7.3 Hz, 3H, CH̲₃CH₂CH₂); ¹³C NMR (101 MHz, CDCl₃) δ 159.29, 150.56, 134.80, 130.22, 129.35, 128.94, 128.47, 128.25, 127.99, 125.93, 115.39, 109.81, 80.06, 37.25, 37.11, 24.82, 21.83, 13.88. LC-MS, m/z: 269.1 [M + H]⁺. Elemental anal (%) calcd for C₁₈H₂₀O₂: C 80.56, H 7.51; found: C 80.52, H 7.50.

5-(5-Allyl-2-methoxyphenyl)-2-methylbenzofuran (5). To a stirred solution of 1 (1.0 mmol) and CH₃I (3.0 mmol) in DMF (5 mL) was added KOH (2 mmol). After 3 h at room temperature, the mixture was diluted with water and extracted with ethyl acetate. The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 5 as light yellow oil (71%). ¹H NMR (400 MHz, CDCl₃) δ 7.58 (s, 1H, benzofuran 4 H), 7.41 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.35 (d, J = 8.5 Hz, 1H, benzofuran 6 H), 7.16 (d, J = 1.8 Hz, 1H, C₆H₃ 6 H), 7.12 (d, J = 8.3 Hz, 1H, C₆H₃ 4 H), 6.91 (d, J = 8.3 Hz, 1H, C₆H₃ 3 H), 6.37 (s, 1H, benzofuran 3 H), 5.99 (ddt, J = 16.8, 10.0, 6.7 Hz, 1H, CH₂CH̲=CH₂), 5.13–5.03 (m, 2H, CH₂CH=CH̲₂), 3.78 (s, 3H, OCH₃), 3.38 (d, J = 6.7 Hz, 2H, CH̲₂CH=CH₂), 2.45 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 155.69, 154.97, 154.02, 137.82, 133.01, 132.26, 131.52, 131.21, 129.10, 128.06, 124.98, 121.04, 115.59, 111.37, 110.01, 102.78, 55.78, 39.46, 14.15. LC-MS, m/z: 279.0 [M + H]⁺. Elemental anal (%) calcd for C₁₉H₁₈O₂: C 81.99, H 6.52; found: C 81.96, H 6.50.

2-Methyl-6-(2-methyl-2,3-dihydrobenzofuran-5-yl)-4-propylphenol (6). A mixture of 20 (2.0 mmol) and Pd/C (10%, 0.1 mmol) in MeOH (10 mL) was refluxed under H₂ (1.5 Mpa) for 5 h. After cooling, the mixture was filtered and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 6 as light yellow oil (88%). ¹H NMR (400 MHz, CDCl₃) δ 7.22 (s, 1H, dihydrobenzofuran 4 H), 7.17 (d, J = 8.1 Hz, 1H, dihydrobenzofuran 6 H), 6.91 (s, 1H, Ar–H), 6.86–6.81 (m, 2H, Ar–H), 5.12 (s, 1H, OH), 5.02–4.93 (m, 1H, CH₃CH̲CH₂), 3.35 (dd, J = 15.5, 8.8 Hz, 1H, CH₃CHCH̲₂), 2.86 (dd, J = 15.5, 7.6 Hz, 1H, CH₃CHCH̲₂), 2.50 (t, J = 7.6 Hz, 2H, CH₃CH₂CH̲₂), 2.27 (s, 3H, CH₃), 1.66–1.57 (m, 2H, CH₃CH̲₂CH₂), 1.50 (d, J = 6.1 Hz, 3H, CH̲₃CHCH₂), 0.94 (t, J = 7.2 Hz, 3H, CH̲₃CH₂CH₂); ¹³C NMR (101 MHz, CDCl₃) δ 159.35, 148.66, 134.17, 130.08, 129.42, 128.95, 128.40, 127.64, 127.50, 125.97, 124.06, 109.90, 80.04, 37.27, 37.10, 24.86, 21.82, 16.25, 13.93. LC-MS, m/z: 283.1 [M + H]⁺. Elemental anal (%) calcd for C₁₉H₂₂O₂: C 80.82, H 7.85; found: C 80.87, H 7.84.

4-Allyl-2-bromo-6-(2-methylbenzofuran-5-yl)phenol (7). To a stirred solution of 1 (1.0 mmol) in MeCN (10 mL) was added NBS (1.0 mmol) in five portions over 1 h and kept stirring for 15 min at room temperature. Then the mixture was diluted with water and extracted with CH₂Cl₂. The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 7 as light yellow oil (30%). ¹H NMR (400 MHz, CDCl₃) δ 7.58 (s, 1H, benzofuran 4 H), 7.46 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.33–7.29 (m, 2H, Ar–H), 7.08 (s, 1H, Ar–H), 6.40 (s, 1H, benzofuran 3 H), 6.01–5.89 (m, 1H, CH₂CH̲=CH₂), 5.59 (s, 1H, OH), 5.15–5.06 (m, 2H, CH₂CH=CH̲₂), 3.34 (d, J = 6.5 Hz, 2H, CH̲₂CH=CH₂), 2.48 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 156.37, 154.37, 147.64, 137.00, 133.38, 131.53, 131.07, 130.58, 129.90, 129.64, 124.33, 120.77, 116.26, 110.74, 110.57, 102.73, 39.07, 14.14. LC-MS, m/z: 342.9 [M + H]⁺. Elemental anal (%) calcd for C₁₈H₁₅O₂Br: C 62.99, H 4.41; found: C 63.03, H 4.44.

4-Allyl-2-nitro-6-(2-methylbenzofuran-5-yl)phenol (8). To a stirred solution of 1 (1.0 mmol) in the co-solvent of CH₂Cl₂ and acetic acid (20 mL, v/v = 1 : 1) was added a solution of concentrated nitric acid (65–68%, 4 mmol) in 5 mL CH₂Cl₂ dropwise over 30 min in an ice bath and kept stirring for 30 min at room temperature. Then the reaction was quenched with water and extracted with CH₂Cl₂. The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 8 as yellow oil (58%). ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H, Ar–H), 7.62 (s, 1H, benzofuran 4 H), 7.49 (s, 1H, Ar–H), 7.46 (d, J = 8.5 Hz, 1H, benzofuran 7 H), 7.35 (d, J = 8.4 Hz, 1H, benzofuran 6 H), 6.41 (s, 1H, benzofuran 3 H), 5.95 (dt, J = 16.6, 7.0 Hz, 1H, CH₂CH̲=CH₂), 5.17–5.10 (m, 2H, CH₂CH=CH̲₂), 3.40 (d, J = 6.6 Hz, 2H, CH̲₂CH=CH₂), 2.48 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 156.34, 154.50, 151.41, 139.41, 136.04, 133.88, 133.54, 131.78, 130.30, 129.38, 124.59, 123.08, 121.08, 117.17, 110.43, 102.74, 38.96, 14.14. LC-MS, m/z: 310.0 [M + H]⁺. Elemental anal (%) calcd for C₁₈H₁₅NO₄: C 69.89, H 4.89, N 4.53; found: C 69.94, H 4.90, N 4.51.

4-Allyl-2-nitroso-6-(2-methylbenzofuran-5-yl)phenol (9). To a stirred solution of 1 (10 mmol) and NaNO₂ (15 mmol) in the co-solvent of MeCN and H₂O (30 mL, v/v = 5 : 1) was added concentrated hydrochloric acid (36–38%, 3 mL) over 1 h in an ice bath and kept stirring for 30 min at room temperature. Then the reaction was quenched with water and extracted with CH₂Cl₂. The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 9 as a yellow powder (77%). Mp 58–60 °C. ¹H NMR (400 MHz, CDCl₃) δ 11.04 (s, 1H, OH), 7.93 (d, J = 2.2 Hz, 1H, Ar–H), 7.63 (d, J = 1.5 Hz, 1H, benzofuran 4 H), 7.50 (d, J = 2.2 Hz, 1H, Ar–H), 7.47 (d, J = 8.5 Hz, 1H, benzofuran 7 H), 7.36 (dd, J = 8.5, 1.8 Hz, 1H, benzofuran 6 H), 6.41 (s, 1H, benzofuran 3 H), 5.96 (ddt, J = 17.2, 10.7, 6.7 Hz, 1H, CH₂CH̲=CH₂), 5.18–5.11 (m, 2H, CH₂CH=CH̲₂), 3.41 (d, J = 6.6 Hz, 2H, CH̲₂CH=CH₂), 2.48 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 156.34, 154.51, 151.41, 139.42, 136.07, 133.88, 133.53, 131.78, 130.31, 129.38, 124.61, 123.07, 121.10, 117.17, 110.43, 102.75, 38.96, 14.15. LC-MS, m/z: 294.2 [M + H]⁺. Elemental anal (%) calcd for C₁₈H₁₅NO₃: C 73.71, H 5.15, N 4.78; found: C 73.66, H 5.17, N 4.74.

4-Allyl-2-amino-6-(2-methylbenzofuran-5-yl)phenol (10). To a stirred solution of 9 (5.0 mmol) and NH₄Cl (1.0 mmol) in the co-solvent of EtOH, H₂O and acetic acid (62.5 mL, v/v/v = 20 : 4 : 1) was added zinc dust (25 mmol) in five portions over 30 min at room temperature. Then the mixture was filtered, diluted with water and extracted with ethyl acetate. The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 10 as a brown powder (73%). Mp 80–82 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.56 (s, 1H, benzofuran 4 H), 7.46 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.29 (d, J = 8.5 Hz, 1H, benzofuran 6 H), 6.58 (s, 1H, Ar–H), 6.46 (s, 1H, Ar–H), 6.31 (s, 1H, benzofuran 3 H), 5.98–5.87 (m, 1H, CH₂CH̲=CH₂), 5.07 (d, J = 17.0 Hz, 1H, CH₂CH=CH̲₂), 5.00 (d, J = 10.0 Hz, 1H, CH₂CH=CH̲₂), 4.72 (br, 1H), 3.20 (d, J = 6.7 Hz, 2H, CH̲₂CH=CH₂), 2.45 (s, 3H, CH₃); ¹³C NMR (101 MHz, DMSO-d₆) δ 155.97, 153.57, 138.97, 138.87, 138.82, 134.47, 131.91, 130.42, 129.11, 125.16, 121.06, 118.96, 115.56, 114.34, 110.29, 103.34, 14.26. LC-MS, m/z: 280.0 [M + H]⁺. Elemental anal (%) calcd for C₁₈H₁₇NO₂: C 77.40, H 6.13, N 5.01; found: C 77.46, H 6.17, N 4.99.

4-Allyl-2-(dimethylamino)-6-(2-methylbenzofuran-5-yl)-phenol (11). To a solution of 10 (1.0 mmol) and CH₃I (3 mmol) in DMF (5 mL) was added NaOAc (1.2 mmol). After reacting for 3 h at room temperature, the mixture was diluted with water and extracted with ethyl acetate. The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 11 as red oil (71%). ¹H NMR (400 MHz, CDCl₃) δ 7.69 (s, 1H, benzofuran 4 H), 7.47–7.41 (m, 2H, benzofuran 6,7 H), 7.01 (s, 1H, Ar–H), 6.99 (s, 1H, Ar–H), 6.39 (s, 1H, benzofuran 3 H), 5.99 (dt, J = 16.6, 7.4 Hz, 1H, CH₂CH̲=CH₂), 5.14–5.04 (m, 2H, CH₂CH=CH̲₂), 3.36 (d, J = 6.5 Hz, 2H, CH̲₂CH=CH₂), 2.71 (s, 6H, 2× CH₃), 2.46 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 155.74, 154.08, 146.85, 140.53, 137.84, 132.59, 131.16, 129.28, 127.61, 127.28, 124.51, 120.69, 119.38, 115.63, 110.23, 102.83, 45.25, 39.86, 14.14. LC-MS, m/z: 308.0 [M + H]⁺. Elemental anal (%) calcd for C₂₀H₂₁NO₂: C 78.15, H 6.89, N 4.56; found: C 78.11, H 6.87, N 4.58.

N-(5-Allyl-2-hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-acetamide (12). To a mixture of 10 (1.0 mmol) and K₂CO₃ (1.5 mmol) in ethyl acetate (5 mL) was added acetic anhydride (2 mmol) dropwise over 10 min at room temperature. Then the reaction mixture was washed with hot water and the aqueous layer was extracted with ethyl acetate. The combined organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 12 as a brown powder (88%). Mp 70–72 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.85 (br, 1H), 7.55 (s, 1H, benzofuran 4 H), 7.45 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.40 (s, 1H, Ar–H), 7.29 (d, J = 8.3 Hz, 1H, benzofuran 6 H), 6.94 (s, 1H, Ar–H), 6.38 (s, 1H, benzofuran 3 H), 5.94 (dt, J = 16.5, 6.8 Hz, 1H, CH₂CH̲=CH₂), 5.11–5.02 (m, 2H, CH₂CH=CH̲₂), 3.32 (d, J = 6.3 Hz, 2H, CH̲₂CH=CH₂), 2.46 (s, 3H, CH₃), 2.20 (s, 3H, COCH₃); ¹³C NMR (101 MHz, CDCl₃) δ 169.77, 156.29, 154.26, 142.30, 137.50, 132.17, 131.91, 130.85, 129.72, 127.22, 126.25, 124.47, 120.87, 120.63, 115.84, 110.80, 102.74, 39.51, 24.19, 14.14. LC-MS, m/z: 322.1 [M + H]⁺. Elemental anal (%) calcd for C₂₀H₁₉NO₃: C 74.75, H 5.96, N 4.36; found: C 74.77, H 5.96, N 4.39.

N-(5-Allyl-2-hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-acrylamide (13). To a stirred mixture of 10 (1.0 mmol) and K₂CO₃ (1.5 mmol) in CH₂Cl₂ (10 mL) was added acryloyl chloride (1.0 mmol) in CH₂Cl₂ (1.0 mL) dropwise over 10 min at room temperature. Then the reaction was quenched with water and extracted with CH₂Cl₂. The combined organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 13 as a yellow powder (46%). Mp 64–66 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1H), 7.58 (s, 1H, Ar–H), 7.55 (d, J = 1.4 Hz, 1H, benzofuran 4 H), 7.46 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.29 (dd, J = 8.4, 1.7 Hz, 1H, benzofuran 6 H), 7.15 (s, 1H), 6.94 (s, 1H, Ar–H), 6.44 (d, J = 16.2 Hz, 1H, COCH=CH̲₂), 6.39 (s, 1H, benzofuran 3 H), 6.31 (dd, J = 16.8, 10.1 Hz, 1H, COCH̲=CH₂), 5.95 (ddt, J = 16.8, 10.0, 6.7 Hz, 1H, CH₂CH̲=CH₂), 5.77 (d, J = 9.9 Hz, 1H, COCH=CH̲₂), 5.12–5.02 (m, 2H, CH₂CH=CH̲₂), 3.33 (d, J = 6.7 Hz, 2H, CH̲₂CH=CH₂), 2.47 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 164.33, 156.35, 154.29, 142.17, 137.45, 132.29, 131.71, 130.51, 129.80, 128.45, 127.21, 126.03, 124.39, 120.83, 120.64, 115.87, 110.90, 102.74, 39.56, 14.14. LC-MS, m/z: 334.0 [M + H]⁺. Elemental anal (%) calcd for C₂₁H₁₉NO₃: C 75.66, H 5.74, N 4.20; found: C 75.60, H 5.76, N 4.19.

N-(5-Allyl-2-hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-2-chloroacetamide (14). The title compound was prepared from 10 and chloroacetyl chloride in a similar procedure described for 13. Purification by silica gel chromatography afforded 14 as a white powder (81%). Mp 118–120 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.81 (brs, 1H), 7.84 (s, 1H, Ar–H), 7.53 (s, 1H, benzofuran 4 H), 7.49 (d, J = 8.3 Hz, 1H, benzofuran 7 H), 7.27 (d, J = 6.3 Hz, 1H, benzofuran 6 H), 6.94 (s, 1H, Ar–H), 6.41 (s, 1H, benzofuran 3 H), 6.15 (br, 1H), 5.98 (dt, J = 16.6, 6.7 Hz, 1H, CH₂CH̲=CH₂), 5.14–5.05 (m, 2H, CH₂CH=CH̲₂), 4.22 (s, 2H, COCH₂), 3.37 (d, J = 6.3 Hz, 2H, CH̲₂CH=CH₂), 2.49 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 164.31, 156.72, 154.42, 141.21, 137.44, 132.57, 131.00, 130.13, 129.48, 126.82, 125.19, 124.13, 120.66, 119.98, 115.94, 111.34, 102.70, 43.01, 39.67, 14.19. LC-MS, m/z: 356.0 [M + H]⁺. Elemental anal (%) calcd for C₂₀H₁₈NO₃Cl: C 67.51, H 5.10, N 3.94; found: C 67.55, H 5.11, N 3.91.

N-(5-Allyl-2-hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-2-(piperidin-1-yl)acetamide (15). A solution of 14 (0.5 mmol), piperidine (1.5 mmol) and triethylamine (1.0 mmol) in THF (5 mL) was stirred at room temperature for 8 h. Then the mixture was concentrated under reduced pressure and purified by silica gel chromatography to afford 15 as a brown powder (48%). Mp 125–127 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.95 (s, 1H), 8.80 (br, 1H), 7.92 (s, 1H, Ar–H), 7.61 (s, 1H, benzofuran 4 H), 7.52 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.32 (d, J = 8.4 Hz, 1H, benzofuran 6 H), 6.83 (s, 1H, Ar–H), 6.61 (s, 1H, benzofuran 3 H), 5.96 (dt, J = 16.7, 6.8 Hz, 1H, CH₂CH̲=CH₂), 5.10 (d, J = 17.0 Hz, 1H, CH₂CH=CH̲₂), 5.04 (d, J = 9.9 Hz, 1H, CH₂CH=CH̲₂), 3.32 (d, J = 6.6 Hz, 2H, CH̲₂CH=CH₂), 3.09 (s, 2H, COCH₂), 2.51 (s, 3H, CH₃), 2.47 (br, 4H, piperidine 2,6 H), 1.58 (br, 4H, piperidine 3,5 H), 1.41 (s, 2H, piperidine 4 H); ¹³C NMR (101 MHz, DMSO-d₆) δ 169.15, 156.27, 153.86, 141.70, 138.41, 133.24, 132.13, 131.27, 129.36, 129.23, 125.81, 125.10, 121.23, 119.03, 116.07, 110.66, 103.36, 62.81, 54.63, 39.64, 26.19, 23.81, 14.25. LC-MS, m/z: 405.1 [M + H]⁺. Elemental anal (%) calcd for C₂₅H₂₈N₂O₃: C 74.23, H 6.98, N 6.93; found: C 74.27, H 7.00, N 6.96.

N-(5-Allyl-2-hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-2-morpholinoacetamide (16). The title compound was prepared from 14 and morpholine in a similar procedure described for 15. Purification by silica gel chromatography afforded 16 as a light purple powder (85%). Mp 147–149 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.91 (s, 1H), 8.82 (s, 1H), 7.85 (s, 1H, Ar–H), 7.62 (s, 1H, benzofuran 4 H), 7.52 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.33 (d, J = 8.4 Hz, 1H, benzofuran 6 H), 6.86 (s, 1H, Ar–H), 6.61 (s, 1H, benzofuran 3 H), 5.96 (dt, J = 16.6, 6.8 Hz, 1H, CH₂CH̲=CH₂), 5.11 (d, J = 17.0 Hz, 1H, CH₂CH=CH̲₂), 5.05 (d, J = 10.0 Hz, 1H, CH₂CH=CH̲₂), 3.66 (br, 4H, morpholine 2,6 H), 3.33 (d, J = 6.6 Hz, 2H, CH̲₂CH=CH₂), 3.18 (s, 2H, COCH₂), 2.56 (br, 4H, morpholine 3,5 H), 2.47 (s, 3H, CH₃); ¹³C NMR (101 MHz, DMSO-d₆) δ 168.66, 156.29, 153.86, 142.04, 138.38, 133.23, 132.13, 131.37, 129.35, 128.99, 126.11, 125.11, 121.24, 119.54, 116.10, 110.65, 103.36, 66.77, 62.18, 53.63, 39.59, 14.24. LC-MS, m/z: 407.1 [M + H]⁺. Elemental anal (%) calcd for C₂₄H₂₆N₂O₄: C 70.92, H 6.45, N 6.89; found: C 70.90, H 6.49, N 6.86.

N-(5-Allyl-2-hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-2-(4-methylpiperazin-1-yl)acetamide (17). The title compound was prepared from 14 and 4-methylpiperazine in a similar procedure described for 15. Purification by silica gel chromatography afforded 17 as a brown powder (76%). Mp 101–103 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.86 (s, 1H), 8.82 (brs, 1H), 7.89 (s, 1H, Ar–H), 7.60 (s, 1H, benzofuran 4 H), 7.51 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.31 (d, J = 8.4 Hz, 1H, benzofuran 6 H), 6.82 (s, 1H, C₆H₂, Ar–H), 6.61 (s, 1H, benzofuran 3 H), 5.95 (dt, J = 16.7, 6.9 Hz, 1H, CH₂CH̲=CH₂), 5.09 (d, J = 17.0 Hz, 1H, CH₂CH=CH̲₂), 5.03 (d, J = 10.0 Hz, 1H, CH₂CH=CH̲₂), 3.31 (d, J = 6.6 Hz, 2H, CH̲₂CH=CH₂), 3.14 (s, 2H, COCH₂), 2.54 (br, 4H, piperazine-H), 2.47 (s, 3H, CH₃), 2.40 (brs, 4H, piperazine-H), 2.17 (s, 3H, NCH₃); ¹³C NMR (101 MHz, DMSO-d₆) δ 168.79, 156.27, 153.86, 141.81, 138.42, 133.26, 132.07, 131.29, 129.35, 129.16, 125.89, 125.12, 121.25, 119.15, 116.07, 110.65, 103.37, 61.93, 55.19, 53.16, 46.09, 39.62, 14.26. LC-MS, m/z: 420.2 [M + H]⁺. Elemental anal (%) calcd for C₂₅H₂₉N₃O₃: C 71.57, H 6.97, N 10.02; found: C 71.55, H 6.99, N 10.05.

N-(5-Allyl-2-hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-2-(4-phenylpiperazin-1-yl)acetamide (18). The title compound was prepared from 14 and 4-phenylpiperazine in a similar procedure described for 15. Purification by silica gel chromatography afforded 18 as a light purple powder (79%). Mp 136–138 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.98 (s, 1H), 8.81 (s, 1H), 7.92 (s, 1H, Ar–H), 7.60 (s, 1H, benzofuran 4 H), 7.50 (d, J = 8.4 Hz, 1H, benzofuran 7 H), 7.31 (d, J = 8.4 Hz, 1H, benzofuran 6 H), 7.24–7.17 (m, 2H, C₆H₅ 3,5 H), 6.94 (d, J = 7.9 Hz, 2H, C₆H₅ 2,6 H), 6.84 (s, 1H, Ar–H), 6.81–6.75 (m, 1H, C₆H₅ 4 H), 6.58 (s, 1H, benzofuran 3 H), 5.96 (dt, J = 16.7, 6.6 Hz, 1H, CH₂CH̲=CH₂), 5.10 (d, J = 16.7 Hz, 1H, CH₂CH=CH̲₂), 5.04 (d, J = 9.9 Hz, 1H, CH₂CH=CH̲₂), 3.32 (d, J = 6.6 Hz, 2H, CH̲₂CH=CH₂), 3.23 (s, 2H, COCH₂), 3.20 (br, 4H, piperazine-H), 2.70 (br, 4H, piperazin-H), 2.45 (s, 3H, CH₃); ¹³C NMR (101 MHz, DMSO-d₆) δ 168.72, 156.26, 153.87, 151.43, 141.86, 138.42, 133.22, 132.11, 131.23, 129.39, 129.13, 125.94, 125.10, 121.24, 119.43, 119.19, 116.09, 115.94, 110.66, 103.37, 61.84, 53.19, 48.92, 39.65, 14.26. LC-MS, m/z: 482.1 [M + H]⁺. Elemental anal (%) calcd for C₃₀H₃₁N₃O₃: C 74.82, H 6.49, N 8.73; found: C 74.88, H 6.50, N 8.75.

(E)-4-Allyl-2-(benzylideneamino)-6-(2-methylbenzofuran-5-yl)phenol (19). A mixture of 10 (1.0 mmol), benzaldehyde (1.0 mmol) and acetic acid (0.1 mL) in EtOH (10 mL) was stirred at room temperature for 30 min. The reaction was left overnight until precipitation. The precipitate was separated by filtration, washed with EtOH and dried to afford 19 as a yellow powder (68%). Mp 91–93 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.86 (s, 1H, N=CH), 8.51 (br, 1H, OH), 8.19–8.09 (m, 2H, Ar–H), 7.71 (s, 1H, benzofuran 4 H), 7.55–7.49 (m, 4H, Ar–H), 7.44 (d, J = 8.6 Hz, 1H, benzofuran 6 H), 7.20 (s, 1H, Ar–H), 7.07 (s, 1H, Ar–H), 6.61 (s, 1H, benzofuran 3 H), 6.03 (dt, J = 16.8, 6.8 Hz, 1H, CH₂CH̲=CH₂), 5.14 (d, J = 17.0 Hz, 1H, CH₂CH=CH̲₂), 5.06 (d, J = 9.8 Hz, 1H, CH₂CH=CH̲₂), 3.38 (d, J = 6.6 Hz, 2H, CH̲₂CH=CH₂), 2.46 (s, 3H, CH₃); ¹³C NMR (101 MHz, DMSO-d₆) δ 158.93, 156.21, 153.71, 147.78, 138.53, 137.44, 136.70, 133.26, 131.87, 131.15, 129.70, 129.16, 129.12, 128.95, 125.12, 121.13, 116.46, 116.10, 110.38, 103.35, 39.47, 14.27. LC-MS, m/z: 368.0 [M + H]⁺. Elemental anal (%) calcd for C₂₅H₂₁NO₂: C 81.72, H 5.76, N 3.81; found: C 81.67, H 5.78, N 3.84.

4-Allyl-2-((dimethylamino)methyl)-6-(2-methylbenzofuran-5-yl)phenol (20). A solution of 1 (2.0 mmol), dimethylamine (aqueous solution, 40%, 6.0 mmol) and formalin (37%, 6.0 mmol) in MeOH (5.0 mL) was stirred at 60 °C for 6 h. Then the mixture was concentrated under reduced pressure and purified by silica gel chromatography to afford 20 as light yellow oil (53%). ¹H NMR (400 MHz, CDCl₃) δ 7.67 (s, 1H, benzofuran 4 H), 7.47–7.39 (m, 2H, benzofuran 6,7 H), 7.11 (s, 1H, Ar–H), 6.78 (s, 1H, Ar–H), 6.37 (s, 1H, benzofuran 3 H), 6.05–5.92 (m, 1H, CH₂CH̲=CH₂), 5.14–5.02 (m, 2H, CH₂CH=CH̲₂), 3.68 (s, 2H, ArCH̲₂N), 3.33 (d, J = 6.5 Hz, 2H, CH̲₂CH=CH₂), 2.45 (s, 3H, CH₃), 2.33 (s, 6H, N(CH₃)₂); ¹³C NMR (101 MHz, CDCl₃) δ 155.52, 153.97, 153.39, 138.04, 133.03, 130.20, 130.18, 129.19, 129.14, 127.40, 124.83, 122.06, 120.92, 115.44, 110.01, 102.84, 62.97, 44.35, 39.53, 14.15. LC-MS, m/z: 322.1 [M + H]⁺. Elemental anal (%) calcd for C₂₁H₂₃NO₂: C 78.47, H 7.21, N 4.36; found: C 78.42, H 7.19, N 4.38.

4-Allyl-2-(2-methylbenzofuran-5-yl)-6-(piperidin-1-ylmethyl)phenol (21). The title compound was prepared from 1 and piperidine in a similar procedure described for 20. Purification by silica gel chromatography afforded 21 as light yellow oil (81%). ¹H NMR (400 MHz, CDCl₃) δ 7.66 (s, 1H, benzofuran 4 H), 7.44–7.41 (m, 2H, benzofuran 6,7 H), 7.09 (s, 1H, Ar–H), 6.78 (s, 1H, Ar–H), 6.38 (s, 1H, benzofuran 3 H), 5.98 (dt, J = 16.3, 8.0 Hz, 1H, CH₂CH̲=CH₂), 5.12–5.02 (m, 2H, CH₂CH=CH̲₂), 3.70 (s, 2H, ArCH̲₂N), 3.33 (d, J = 6.5 Hz, 2H, CH̲₂CH=CH₂), 2.52 (br, 4H, piperidine 2,6 H), 2.46 (s, 3H, CH₃), 1.61 (br, 4H, piperidine 3,5 H), 1.47 (br, 2H, piperidine 4 H); ¹³C NMR (101 MHz, CDCl₃) δ 155.51, 153.95, 153.40, 138.05, 133.18, 130.13, 129.21, 129.15, 127.61, 127.55, 124.80, 121.87, 120.87, 115.40, 110.05, 102.85, 62.43, 53.87, 39.51, 25.66, 23.97, 14.14. LC-MS, m/z: 362.1 [M + H]⁺. Elemental anal (%) calcd for C₂₄H₂₇NO₂: C 79.74, H 7.53, N 3.87; found: C 79.79, H 7.54, N 3.88.

4-Allyl-2-(2-methylbenzofuran-5-yl)-6-(morpholinomethyl)-phenol (22). The title compound was prepared from 1 and morpholin in a similar procedure described for 20. Purification by silica gel chromatography afforded 22 as colourless oil (58%). ¹H NMR (400 MHz, CDCl₃) δ 7.65 (s, 1H, benzofuran 4 H), 7.45–7.40 (m, 2H, benzofuran 6,7 H), 7.12 (s, 1H, Ar–H), 6.81 (s, 1H, Ar–H), 6.38 (s, 1H, benzofuran 3 H), 5.97 (dt, J = 16.7, 6.8 Hz, 1H, CH₂CH̲=CH₂), 5.13–5.03 (m, 2H, CH₂CH=CH̲₂), 3.74 (s, 2H, ArCH̲₂N), 3.72 (br, 4H, morpholine 2,6 H), 3.33 (d, J = 6.6 Hz, 2H, CH̲₂CH=CH₂), 2.59 (br, 4H, morpholine 3,5 H), 2.45 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 155.65, 154.01, 152.83, 137.90, 132.92, 130.63, 130.57, 129.41, 129.20, 127.93, 124.77, 120.91, 120.88, 115.56, 110.11, 102.83, 66.69, 62.17, 52.90, 39.48, 14.15. LC-MS, m/z: 364.1 [M + H]⁺. Elemental anal (%) calcd for C₂₃H₂₅NO₃: C 76.01, H 6.93, N 3.85; found: C 75.97, H 6.94, N 3.82.

4-Allyl-2-(2-methylbenzofuran-5-yl)-6-((4-methylpiperazin-1-yl)methyl)phenol (23). The title compound was prepared from 1 and 4-methylpiperazine in a similar procedure described for 20. Purification by silica gel chromatography afforded 23 as light yellow oil (68%). ¹H NMR (400 MHz, CDCl₃) δ 7.65 (s, 1H, benzofuran 4 H), 7.45–7.39 (m, 2H, benzofuran 6,7 H), 7.10 (s, 1H, Ar–H), 6.80 (s, 1H, Ar–H), 6.38 (s, 1H, benzofuran 3 H), 5.97 (dt, J = 16.6, 7.7 Hz, 1H, CH₂CH̲=CH₂), 5.29 (br, 1H, OH), 5.12–5.02 (m, 2H, CH₂CH=CH̲₂), 3.75 (s, 2H, ArCH̲₂N), 3.33 (d, J = 6.6 Hz, 2H, CH̲₂CH=CH₂), 2.62 (br, 8H, piperazine-H), 2.46 (s, 3H, NCH₃), 2.29 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 155.59, 153.97, 152.98, 137.91, 133.02, 130.50, 130.41, 129.31, 129.16, 127.78, 124.77, 121.32, 120.86, 115.52, 110.06, 102.82, 61.61, 54.51, 52.10, 45.52, 39.48, 14.14. LC-MS, m/z: 377.1 [M + H]⁺. Elemental anal (%) calcd for C₂₄H₂₈N₂O₂: C 76.56, H 7.50, N 7.44; found: C 76.55, H 7.47, N 7.48.

4-Allyl-2-(2-methylbenzofuran-5-yl)-6-((4-phenylpiperazin-1-yl)methyl)phenol (24). The title compound was prepared from 1 and 4-phenylpiperazine in a similar procedure described for 20. Purification by silica gel chromatography afforded 24 as a white powder (55%). Mp 50–52 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.67 (s, 1H, benzofuran 4 H), 7.45–7.40 (m, 2H, Ar–H), 7.27–7.21 (m, 2H, Ar–H), 7.23 (s, 1H, Ar–H), 6.92–6.81 (m, 4H, Ar–H), 6.37 (s, 1H, benzofuran 3 H), 5.98 (dt, J = 16.4, 6.7 Hz, 1H, CH₂CH̲=CH₂), 5.14–5.03 (m, 2H, CH₂CH=CH̲₂), 3.79 (s, 2H, ArCH̲₂N), 3.34 (d, J = 6.1 Hz, 2H, CH̲₂CH=CH₂), 3.20 (br, 4H, piperazine-H), 2.74 (br, 4H, piperazine-H), 2.44 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 155.64, 154.00, 152.99, 150.97, 137.95, 132.98, 130.58, 130.52, 129.41, 129.21, 127.87, 124.81, 121.30, 120.92, 120.26, 116.47, 115.60, 110.13, 102.87, 61.79, 52.55, 49.18, 39.54, 14.20. LC-MS, m/z: 439.1 [M + H]⁺. Elemental anal (%) calcd for C₂₉H₃₀N₂O₂: C 79.42, H 6.90, N 6.39; found: C 79.48, H 6.91, N 6.42.

6-Allyl-8-(2-methylbenzofuran-5-yl)-2H-benzo[b][1,4]-oxazin-3(4H)-one (25). To a stirred solution of 10 (1.0 mmol), triethylamine (2.5 mmol) and 4-dimethylaminopyridine (0.2 mmol) in THF (5 mL) was added chloroacetyl chloride (1.0 mmol) in CH₂Cl₂ (1.0 mL) dropwise over 10 min at room temperature. Then the reaction was heated to reflux and stirred for 12 h. After cooling, the mixture was concentrated under reduced pressure and purified by silica gel chromatography to afford 25 as a white powder (44%). Mp 169–171 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.72 (s, 1H, NH), 7.62 (s, 1H, benzofuran 4 H), 7.51 (d, J = 8.5 Hz, 1H, benzofuran 7 H), 7.33 (d, J = 8.5 Hz, 1H, benzofuran 6 H), 6.84 (s, 1H, Ar–H), 6.73 (s, 1H, Ar–H), 6.60 (s, 1H, benzofuran 3 H), 5.95 (ddt, J = 16.5, 9.5, 6.7 Hz, 1H, CH₂CH̲=CH₂), 5.12 (d, J = 17.2 Hz, 1H, CH₂CH=CH̲₂), 5.06 (d, J = 9.9 Hz, 1H, CH₂CH=CH̲₂), 4.54 (s, 2H, OCH₂), 3.33 (d, J = 6.7 Hz, 1H, CH̲₂CH=CH₂), 2.46 (s, 3H, CH₃); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.89, 156.43, 153.93, 139.24, 138.07, 134.40, 131.93, 130.32, 129.25, 128.52, 125.01, 124.86, 121.14, 116.42, 115.21, 110.55, 103.32, 67.32, 39.31, 14.25. LC-MS, m/z: 320.0 [M + H]⁺. Elemental anal (%) calcd for C₂₀H₁₇NO₃: C 75.22, H 5.37, N 4.39; found: C 75.18, H 5.34, N 4.42.

6-Allyl-8-(2-methylbenzofuran-5-yl)-2H-benzo[b][1,4]-oxazine-2,3(4H)-dione (26). To a stirred solution of 10 (1.0 mmol) and triethylamine (2.5 mmol) in CH₂Cl₂ (10 mL) was added oxalyl chloride (1.5 mmol) in CH₂Cl₂ (1.0 mL) dropwise over 10 min at room temperature. Then the reaction was quenched with water and extracted with CH₂Cl₂. The combined organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 26 as a brown powder (42%). Mp 160–162 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.57 (br, 1H, NH), 7.87 (s, 1H, benzofuran 4 H), 7.58 (d, J = 8.5 Hz, 1H, benzofuran 7 H), 7.48 (d, J = 8.5 Hz, 1H, benzofuran 6 H), 7.16 (s, 1H, Ar–H), 6.86 (s, 1H, Ar–H), 6.43 (s, 1H, benzofuran 3 H), 6.04–5.93 (m, 1H, CH₂CH̲=CH₂), 5.17–5.10 (m, 2H, CH₂CH=CH̲₂), 3.45 (d, J = 6.6 Hz, 2H, CH̲₂CH=CH₂), 2.49 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 156.34, 154.63, 139.49, 137.06, 136.76, 130.12, 129.70, 129.18, 124.66, 123.56, 122.68, 120.16, 116.35, 110.81, 108.82, 102.90, 40.16, 14.15. LC-MS, m/z: 334.1 [M + H]⁺. Elemental anal (%) calcd for C₂₀H₁₅NO₄: C 72.06, H 4.54, N 4.20; found: C 72.11, H 4.56, N 4.15.

1-(4-Hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)propan-2-one (27). A mixture of 1 (10 mmol) and PdCl₂ (0.15 mmol) in the co-solvent of DMA and H₂O (40 mL, v/v = 4 : 1) was stirred under O₂ (0.8 Mpa) at 60 °C for 16 h. After cooling, the mixture was diluted with water and extracted with ethyl acetate. The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 27 as a white powder (64%). Mp 118–120 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.53–7.48 (m, 2H, benzofuran 4,7 H), 7.26–7.24 (m, 1H, benzofuran 6 H), 7.10–7.07 (m, 2H, Ar–H), 6.97 (d, 1H, J = 7.8 Hz, Ar–H), 6.41 (s, 1H, benzofuran 3 H), 5.35 (br, 1H, OH), 3.66 (s, 2H, CH₂), 2.49 (s, 3H, CH₃), 2.18 (s, 3H, COCH₃); ¹³C NMR (101 MHz, CDCl₃) δ 190.98, 158.27, 157.11, 154.69, 132.77, 131.21, 130.51, 130.05, 129.53, 129.28, 123.93, 120.69, 116.34, 111.78, 102.69, 50.17, 29.23, 14.17. LC-MS, m/z: 280.9 [M + H]⁺. Elemental anal (%) calcd for C₁₈H₁₆O₃: C 77.12, H 5.75; found: C 77.07, H 5.79.

(E/Z)-1-(4-Hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-propan-2-one oxime (28). A mixture of 27 (2.0 mmol), hydroxylamine hydrochloride (3.0 mmol) and NaOAc (3.0 mmol) in EtOH (20 mL) was stirred and refluxed for 30 min. After cooling, the mixture was diluted with water, shocked and placed until precipitation. Then the precipitate was separated by filtration, washed with water and dried to afford 28 as a white powder (95%). Mp 38–40 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.52 (s, 1H, benzofuran 4 H), 7.49 (d, J = 7.8 Hz, 1H, benzofuran 7 H), 7.27–7.24 (m, 1H, benzofuran 6 H), 7.13–7.09 (m, 2H, Ar–H), 6.98–6.90 (m, 1H, Ar–H), 6.41 (s, 1H, benzofuran 3 H), 5.07 (br, 1H, OH), 3.71 (s, 1H, N–OH), 3.52–3.44 (m, 2H, CH₂), 2.49 (s, 3H, CH₃), 1.86 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 158.53, 156.47, 154.30, 151.67, 131.50, 131.22, 129.99, 129.23, 128.97, 128.56, 124.34, 120.78, 116.13, 111.13, 102.80, 41.21, 34.09, 19.64, 14.13, 13.45. LC-MS, m/z: 296.1 [M + H]⁺. Elemental anal (%) calcd for C₁₈H₁₇NO₃: C 73.20, H 5.80, N 4.74; found: C 73.17, H 5.79, N 4.70.

(E/Z)-2-(1-(4-Hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-propan-2-ylidene)hydrazine-1-carbothioamide (29). A solution of 27 (1.0 mmol), thiosemicarbazide (4.0 mmol) and acetic acid (0.2 mL) in EtOH (20 mL) was stirred and refluxed for 1 h. After cooling, the mixture was diluted with water, shocked and placed until precipitation. Then the precipitate was separated by filtration, washed with water and dried to afford 29 as a light yellow powder (57%). Mp 97–99 °C ¹H NMR (400 MHz, Acetone-d₆) δ 8.96 (brs, 1H), 8.01 (brs, 1H), 7.58–6.75 (m, 8H), 6.37 (s, 1H, benzofuran 3 H), 3.72–3.34 (m, 2H, CH₂), 2.31 (s, 3H, CH₃), 1.94–1.77 (m, 3H, CH₃); ¹³C NMR (101 MHz, acetone-d₆) δ 180.10, 155.88, 153.92, 152.99, 152.21, 133.13, 131.51, 129.23, 129.04, 128.68, 128.35, 124.86, 120.95, 116.27, 109.86, 102.75, 43.98, 14.51, 13.11. LC-MS, m/z: 354.0 [M + H]⁺. Elemental anal (%) calcd for C₁₉H₁₉N₃O₃S: C 64.57, H 5.42, N 11.89; found: C 64.61, H 5.44, N 11.85.

(E/Z)-N′-(1-(4-Hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)propan-2-ylidene)benzohydrazide (30). A solution of 27 (0.5 mmol), benzoylhydrazide (1.0 mmol) and acetic acid (0.2 mL) in EtOH (20 mL) was stirred and refluxed for 1 h. After cooling, the mixture was diluted with water, shocked and placed until precipitation. Then the precipitate was separated by filtration, washed with water and dried to afford 30 as a light yellow powder (90%). Mp 99–101 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.87–7.73 (m, 2H, Ar–H), 7.53–7.44 (m, 5H, Ar–H), 7.26–6.93 (m, 4H, Ar–H), 6.40 (s, 1H, benzofuran 3 H), 5.56 (br, 1H, OH), 3.78–3.62 (m, 2H, CH₂), 2.48 (s, 3H, CH₃), 2.18 (s, 1H, CH₃), 1.88 (s, 2H, CH₃); ¹³C NMR (101 MHz, acetone-d₆) δ 164.42, 156.78, 155.00, 154.08, 149.27, 135.21, 134.23, 133.62, 132.46, 131.36, 130.25, 129.95, 129.71, 129.41, 128.56, 127.66, 125.89, 121.97, 117.63, 117.36, 110.80, 103.76, 45.19, 15.91, 14.10. LC-MS, m/z: 399.2 [M + H]⁺. Elemental anal (%) calcd for C₂₅H₂₂N₂O₃: C 75.36, H 5.57, N 7.03; found: C 75.30, H 5.54, N 6.99.

2-(1-(4-Hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-propan-2-yl)hydrazine-1-carbothioamide (31). To a stirred solution of 29 (0.5 mmol) in EtOH (5.0 mL) was added NaBH₄ (5.0 mmol) in five portions over 1 h and kept stirring for 1 h at room temperature. Then the reaction was quenched with dilute hydrochloric acid and extracted with CH₂Cl₂. The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was then purified by silica gel chromatography to afford 31 as a white powder (31%). Mp 81–83 °C ¹H NMR (400 MHz, DMSO-d₆) δ 9.30 (brs, 1H), 8.76 (brs, 1H), 7.68 (brs, 1H), 7.65 (d, J = 1.0 Hz, 1H, benzofuran 4 H), 7.47 (d, J = 8.5 Hz, 1H, benzofuran 7 H), 7.38 (dd, J = 8.5, 1.3 Hz, 1H, benzofuran 6 H), 7.17 (br, 1H), 7.08 (d, J = 1.5 Hz, 1H, C₆H₃ 2 H), 6.95 (dd, J = 8.2, 1.6 Hz, 1H, C₆H₃ 6 H), 6.88 (d, J = 8.1 Hz, 1H, C₆H₃ 5 H), 6.58 (s, 1H, benzofuran 3 H), 4.88 (br, 1H), 3.08–2.96 (m, 1H, CH), 2.72 (dd, J = 13.2, 4.7 Hz, 1H, CH₂), 2.44 (s, 3H, CH₃), 2.32 (dd, J = 13.1, 8.7 Hz, 1H, CH₂), 0.89 (d, J = 6.1 Hz, 3H, CH₃); ¹³C NMR (101 MHz, DMSO-d₆) δ 181.88, 156.04, 153.55, 152.87, 133.71, 131.81, 130.12, 129.16, 129.06, 128.36, 125.17, 121.10, 116.32, 110.23, 103.30, 57.09, 17.93, 14.24. LC-MS, m/z: 356.0 [M + H]⁺. Elemental anal (%) calcd for C₁₉H₂₁N₃O₂S: C 64.20, H 5.96, N 11.82; found: C 64.23, H 6.00, N 11.78.

N′-(1-(4-Hydroxy-3-(2-methylbenzofuran-5-yl)phenyl)-propan-2-yl)benzohydrazide (32). The title compound was prepared from 30 in a similar procedure described for 31. Purification by silica gel chromatography afforded 32 as a white powder (47%). Mp 188–190 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J = 7.5 Hz, C₆H₅ 2,6 H), 7.49–7.45 (m, 3H, Ar–H), 7.43–7.38 (m, 2H, benzofuran 4,7 H), 7.22 (d, J = 8.5 Hz, 1H, benzofuran 6 H), 7.15–7.11 (m, 2H, Ar–H), 6.95 (d, J = 8.1 Hz, 1H, Ar–H), 6.39 (s, 1H, benzofuran 3 H), 5.26 (br, 2H), 3.45–3.35 (m, 1H, CH), 2.81 (dd, J = 12.7, 6.7 Hz, 1H, CH₂), 2.67 (dd, J = 13.0, 6.6 Hz, 1H, CH₂), 2.48 (s, 3H, CH₃), 1.16 (d, J = 6.0 Hz, 3H, CH₃); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.06, 156.07, 153.55, 152.74, 133.72, 133.58, 131.92, 131.76, 130.19, 129.28, 129.07, 128.80, 128.24, 127.59, 125.19, 121.12, 116.21, 110.24, 103.31, 56.95, 40.80, 18.68, 14.22. LC-MS, m/z: 401.2 [M + H]⁺. Elemental anal (%) calcd for C₂₅H₂₄N₂O₃: C 74.98, H 6.04, N 7.00; found: C 75.03, H 6.00, N 7.02.

Biological assays

Cell culture. The A549, K562, HepG2 and HUVEC cells were obtained from Institute of Cell Biology, Chinese Academy of Science (Shanghai China) and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 U mL⁻¹ penicillin–streptomycin at 37 °C in incubator with 5% CO₂/95% air atmosphere.

Cytotoxicity assay. The cytotoxicity of all synthesized compounds were evaluated by typical MTT assay or MTS assay according to our previous method.³³ The IC₅₀ (the concentration required for 50% inhibition) was calculated by the Logit method.

Morphological analysis. The changes of cellular morphology were observed under an inverted fluorescence microscope. The HepG2 cells of experimental groups were treated with 20 μM of different compounds (1, 23 and 30) for 24 h, and the control group was treated with DMSO.

Hoechst 33258 staining. After treated with 20 μM of different compounds (1, 23 and 30) for 24 h, the cells were fixed with stationary liquid (24% glacial acetic, 72% methyl alcohol, 4% paraformaldehyde). Then the HepG2 cells were rinsed three times with PBS, each time for 5 min, and then stained with a solution of Hoechst 33258 (2 μg mL⁻¹) for 15 min at room temperature, and then washed with PBS again for three time, each time for 5 min. Subsequently, the stained nuclei were observed under an inverted fluorescence microscope. The control group was treated with DMSO.

Flow cytometry analysis. The extent of apoptosis was quantitatively measured by an Annexin V-FITC/PI dual staining assay with annexin V-FITC/PI apoptosis detection Kit (Solarbio, CA1020). Briefly, HepG2 Cells were treated with 20 μM of different compounds (1, 23 and 30) for 24 h. Then detached and adherent cells were centrifuged at 1500 × g for 5 min, and then rinsed with ice-cold PBS for two times. Adjust the density of HepG2 cells to 5 × 10⁵ cells per mL. And the cells were subsequently stained with binding buffer for 10 min at 4 °C, and labeled with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide. Sample were analyzed using a flow cytometer (BD Biosciences).

qRT-PCR assay. Total RNA was isolated from the HepG2 cells (6 × 6 cm, 2 × 10⁶/well) by using Trizol according to manufacturer's instructions and digested using DNase I (Takara) to remove the genomic DNA. The first-strand cDNA was synthesized using a cDNA synthesis kit (Fermentas, K1622). The mRNA expression levels of Bax, Bcl-2, caspase-3, caspase-9, p53 and p21 were measured by qRT-PCR in the 96-well plates with the real-time detection PCR system (Bio-Rad, Shanghai, China) with SYBR Premix ExTaqII (Takara) and the gene β-actin was used as an endogenous control. The primers used for the qPCR analysis were as follows: Bcl-2 forward, 5′-CAAAGGTGGATCAGATTCAAG-3′; Bcl-2 reverse, 5′-GGTGAGCATTATCACCCAGAA-3′; Bax forward, 5′-TGGCAGCAGTGACAGCAGCG-3′; Bax reverse, 5′-TACGGAGGTGGAGTGGGTGT-3′; caspase-3 forward, 5′-AGAACACTGAAAACTCAGTGGATTC-3′; caspase-3 reverse, 5′-TCCATTTTATAACTG-TTGTCCAGGG-3′; caspase-9 forward, 5′-AGTCTATTTTATTATGGGCTCG-3′; caspase-9 reverse, 5′-TGGATGTTTATGTCACCTTTTC-3′; p21 forward, 5′-ATGGAGAACACTGAAAACTC-3′; p21 reverse, 5′-TGTGAGCATGGAAACAATAC-3′; p53 forward, 5′-ACTCCCATTCTTCCACCTTTG-3′, p53 reverse, 5′-CCCTGTTGCTGTAGCCATATT-3′; β-actin forward, 5′-CCATAAACGATGCCGGA-3′; β-actin reverse, 5′-CACCACCCATAGAATCAAGA-3′.

Conclusions

In conclusion, a semi-synthesis methodology of 5-arylbenzofuran neolignans was developed. A series of 5-arylbenzofuran neolignan derivatives were synthesized and evaluated for their cytotoxicity in vitro against three tumor cell lines (HepG2, A549 and K562). Most derivatives revealed low cytotoxic effects and several compounds such as 23, 26 and 30 displayed obviously better cytotoxicity than the natural 5-arylbenzofuran neolignan 1. A preliminary structure–activity relationship was obtained, which showed that the modification of benzofuran, phenolic hydroxyl, allyl and the substituent on benzene ring made different effects on the cytotoxicity. For further understanding the anti-proliferative activities and mechanism of 5-arylbenzofuran neolignans, a series of biological assays were done around these compounds. As a result, compounds 23 and 30 could induce the apoptosis of HepG2 cells through regulation of different signal pathways. The selectivity assay for cytotoxicity showed tumor cells were more sensitive to the promising compounds than normal cells. In addition, a one-pot method of Waker-type cyclization and Wacker-type oxidation was developed, which may promote the synthesis of benzofuran ketones and related structures.

It's the first time for detailed research of 5-arylbenzofuran neolignans including semi-synthesis methodology, structural modification, cytotoxicity, structure–activity relationship and anti-proliferation mechanism after they were isolated from plants. The methods and conclusions in this paper would promote the research on 5-arylbenzofuran neolignan and its derivatives in the cancer therapy and other fields.

Conflicts of interest

The authors declare no competing interests.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21442014).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: ¹H NMR, ¹³C NMR. See DOI: 10.1039/c8ra04773a

‡ These authors contributed equally to this work.

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