English

• 1.   Introduction

  Berberine is an isoquinoline alkaloids isolated from the roots, rhizomes, and stem barks of Hydrastis canadensis (Goldenseal), Coptis chinensis (Coptis or goldenthread) and Berberis aquifolium (Oregon grape).^[1~3] Honokiol, a natural hydroxylated biphenol compound, isolated from the root bark of Magnolia officinalis.^{[4, 5]} Quercetin, one of the most abundant bioflavonoid, is major in the human diet and Chinese herbal medicine.^[6] Previous data showed that honokiol, quercetin and berberine were extensively employed in traditional Chinese medicine for the treatment of inflammations, cancers, microbial and hyperlipoidemia.^[7~10]

  From a review of recent advances in structural modifications of berberine, a variety of berberine derivatives have been synthesized successfully. In 2017, Enkhtaivan et al.^[11] designed and synthesized a series of derivatives at the 13 position of berberine, and the results indicated that derivatives inhibited various strains of influenza virus by blocking of viral neuraminidase (NA) subunit. In the same year, Mistry et al.^[12] linked benzothiazole moieties with berberine through a pentyl side chain. The results revealed that compounds bearing methoxy or cyano functional groups were highly antioxidant potency. Additionally, honokiol and quercetin derivatives have also been synthesized. In 2011, Ma et al.^[13] synthesized 47 honokiol derivatives with preferable antiangiogenic and antitumor activities. In 2013, Lin^[14] synthesized three honokiol ether derivatives, the anti-inflammatory activity showed that the activity was improved after methylation of honokiol; In 2010, Mattarei et al.^[15] selectively synthesized 7-O- methylquercetin derivatives and 7-O-(4-quercetin) derivatives. And then, Zhou et al.^[16] synthesized acetylated quercetin derivatives with quercetin in the presence of acetic anhydride and sodium acetate.

  In our previous studies, ^[17] we synthesized a novel triazine ring compound from berberine hydrochloride and metformin (Figure 1), and then investigated its potential pharmacological activity. The preliminary study showed that the compound performed remarkable anti-inflamma- tory activity by inhibiting the release of cyclooxygenase-2 (COX-2) and reducing the production of nitric oxide (NO) and human prostaglandin E2 (PGE2). In addition, the compound had a stimulatory effect on the insulin secretion from INS-1 cells.

  Figure 1

  

  Figure 1.  Structures of berberine derivative

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  However, these reports on honokiol, quercetin and berberine derivatives depicted individual structural transformations, while there are no reports on honokiol, quercetin and berberine combined in the formation of hydrocarbon-linked structures. In this study, we report that these natural product skeletons can be used to develop novel agents with anti-tumor effects. Thus, three novel compounds 1~3 (Figure 2) from honokiol, quercetin and berberine were synthesized. Furthermore, the previous reaction conditions were optimized, the structures of the target products were characterized by ¹H NMR, ¹³C NMR, 2D NMR, HRMS, IR and UV, and the activities of against HepG2 cells in vitro were investigated by MTT assay.

  Figure 2

  

  Figure 2.  Structures of compounds 1~3.

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  2.   Results and discussion

  2.1   Chemistry

  The synthetic strategy for preparing compounds 1~3 is outlined in Scheme 1. Initially, according to the studies of Cao, Nechepurenko and Zhang, ^[18~20] a pintsch reaction in berberine hydrochloride (4) gave the product berberrubine hydrochloride (5) under the high temperature environment in N, N-dimethylformamide (DMF). Then the hydroxyl radical of berberrubine hydrochloride was alkylated by 1, 2-dibromoethane or 1, 4-dibromobutane in acetonitrile at 80 ℃ and gave intermediates 7a or 7b with high yield (85%) and high purity (90%). Finally, intermediate 7a was alkylated by honokiol (8) to get the final product 1 and 2. Similarly, intermediate 7b was alkylated by quercetin (9) to get the final product 3.

  Scheme 1

  

  Scheme 1.  Synthetic route of the target compounds

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  In order to further improve the yields of the final products, various conditions of these reactions were studied including temperatures, bases, solvents and time. Initially, compounds 1 and 2 were obtained at 25 ℃ with anhydrous potassium carbonate as base, acetonitrile as a solvent. Compound 3 was obtained at 80 ℃ with triethylamine (Et₃N) as base, acetonitrile as a solvent. After the reaction conditions were selected (Tables 1, 2), the final yield of compound 1 was 35.2% and compound 2 was 25.5% with anhydrous acetonitrile as solvent and anhydrous potassium carbonate as base at 80 ℃ for 8 h. Similarly, DMF as solvent, triethylamine as base, 120 ℃ reacted 90 min, the highest yield of compound 3 was 26.3%.

  Table 1

  

  Table 1.  Effects of reaction conditions on the synthesis of thetarget compounds 1 and 2^a

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  Entry	Solvent	Base	Temp./℃	Time/h	Yield/%
  					1	2
  1	CH3CN	K2CO3	25	8	22.1	13.2
  2	CH3CN	K2CO3	60	8	29.2	18.5
  3	CH3CN	K2CO3	80	8	35.2	25.5
  4	CH3CN	K2CO3	82	8	31.2	22.5
  5	DMF	K2CO3	80	8	30.4	19.8
  6	Isopropanol	K2CO3	80	8	11.7	7.7
  7	CH3CN	Na2CO3	80	8	30.5	21.5
  8	CH3CN	Et3N	80	8	30.1	21.4
  9	CH3CN	NaOH	80	8	20.5	16.4
  10	CH3CN	K2CO3	80	2	10.7	5.6
  11	CH3CN	K2CO3	80	4	15.6	10.8
  12	CH3CN	K2CO3	80	6	20.2	15.7
  13	CH3CN	K2CO3	80	8	35.2	25.5
  14	CH3CN	K2CO3	80	12	21.2	12.5
  a Unless otherwise specified, the reactions to produce compounds 1 and 2 were performed with 8 (5.64 mmol), 7a (2.63 mmol), base (36.23 mmol), and TBAB (2.48 mmol) in 30 mL of solvent.

  Table 2

  

  Table 2.  Effects of reaction conditions on the synthesis of the target compound 3^a

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  Entry	Solvent	Base	Temp./℃	Time/h	Yield/%
  1	DMF	Et3N	120	90	26.3
  2	DMF	NaOH	120	90	16.8
  3	DMF	K2CO3	120	90	20.2
  4	MeOH	Et3N	65	90	NRb
  5	CH3CN	Et3N	80	90	6.4
  6	DMF	Et3N	80	90	8.4
  7	DMF	Et3N	100	90	10.6
  8	DMF	Et3N	140	90	20.8
  9	DMF	Et3N	120	30	11.6
  10	DMF	Et3N	120	60	12.9
  11	DMF	Et3N	120	120	19.3
  a Unless otherwise specified, the reactions to produce compounds 3 were performed with 9 (1.656 mmol), 7b (2.068 mmol), and base (25.23 mmol) in 30 mL of solvent. b NR: The reaction did not occur.

  2.2   Characterization of compounds

  High-resolution electrospray ionization mass spectrometry (HRESIMS) of compound 1 showed an [M]⁺ ion at m/z 642.2786 (calcd for C₄₁H₄₀O₆N, 642.2788), consistent with the molecular formula C₄₁H₄₀O₆N. Compound 1 was obtained as yellow acicular solid. The fourier transform infrared (FT-IR, KBr) spectrum of 1 suggested the presence of a hydroxyl group (3399.89 cm^－1), benzene rings (1600.63, 1503.24 cm^－1). ¹H NMR spectra of compound 1 suggested 26 resonance absorption peak signals including 1 methyl group, 11 methylene groups and 14 methine groups. ¹³C NMR and distortionless enhancement by polarization transfer (135°DEPT) spectra of compound 1 confirmed 41 carbon atoms including 1 methyl signal, 11 methylene signals, 14 methine signals and 15 quaternary carbon atoms. ¹H NMR spectra showed visibly the singe peak of H-16'''. A detailed examination of 2D-NMR spectra could confirm the five ring of structure. The heteronuclear multiple bond correlation (HMBC) spectrum of H-16'''/C-10'''; H-12'''/C-10''', C-8a; H-11'''/C-12a, C-9''', and the ¹H-¹H correlation spectroscopy (COSY) correla- tions of H-12'''/ H-11''', clearly showing the locations of the benzene ring A. According to HMBC of H-8'''/C-12a, C-9'''; H-13'''/C-8a, C-12''' could confirmed the pyridine ring B. Then the ¹H-¹H COSY of H-5'''/H-6''' and the HMBC of H-6'''/C-8''', C-14''' could confirmed the dihydropyridine ring C. The HMBC of H-4'''/C-5''', C-14a; H-1'''/C-4a, C-14'''; H-15'''/C-2''', C-3''' could confirmed the benzene ring D. The HMBC of H-6''/C-2'', C-7''; H-3''/C-1'', C-5''; H-4''/C-2'', C-7''; H-8''/C-5'' and the ¹H-¹H COSY of H-3''/H-4'' could confirmed the benzene ring E. The HMBC of H-2'/C-4', C-7', C-1''; H-6'/C-2', C-4', C-1''; H-5'/C-3', C-1' and the ¹H-¹H COSY of H-5' /H-6' could confirmed the benzene ring F. Finally, according to the HMBC of H-1/C-9''', H-4/C-2'' and the ¹H-¹H COSY of H-1/H-2, H-2/H-3, H-3/H-4 could confirmed that the 9''' position of ring A and the 2" position of ring E are linked through a butyl side chain.

  Compound 2 was isomers of compound 1, [M]⁺ ion at m/z 642.2786 (calcd for C₄₁H₄₀O₆N 642.2787). A detailed comparison of the ¹³C NMR data and 2D-NMR spectra of 2 and 1 showed that the structure of 2 was similar to the structure of 1. The only difference was that the 9''' position of ring A and the 4' position of ring F are linked through a butyl side chain, as confirmed by HMBC of H-1/C-9'''; H-4/C-4'.

  The molecular formula of 3 was established as C₃₆H₂₈O₁₁N⁺ by its HRESIMS m/z 650.1661 [M]⁺ (calcd For C₃₆H₂₈O₁₁N 650.1667). The fourier transform infrared (FT-IR, KBr) spectrum of 3 suggested the presence of a hydroxyl group (3425.92 cm^－1), double-bond (1644.98 cm^－1), benzene rings (1600.63, 1503.24, 1473.35 cm^－1). The ¹H NMR spectra of compound 3 suggested 17 resonance absorption peak signals including 1 methyl group, 5 methylene groups and 11 methine groups. ¹³C NMR and 135°DEPT spectra confirmed 36 carbon atoms including 1 methyl signal, 5 methylene signals, 11 methine signals and 19 quaternary carbon atoms. Similarly the ABCD ring in the structure 3 could be determined by HMBC spectrum, ¹H-¹H COSY spectrum and HSQC spectrum. Then according to the HMBC of H-6'/C-10', C-8'; H-8'/C-10', C-6' and the high δ of C-5', C-7', C-9' could confirm the benzene ring F. The HMBC of H-2''/C-4'', C-6''; H-6''/C-2'', C-4''; H-5''/C-3'', C-1'', the ¹H-¹H COSY of H-6''/H-5'' and the high δ of C-3'', C-4'' could confirm the benzene ring G. Finally, according to the HMBC of H-1/C-9''', H-2/C-3' and the ¹H-¹H COSY of H-1/H-2, the 9''' position of ring A and the 3' position of ring E are linked through a ethyl side chain.

  2.3   Biological evaluation

  In order to evaluate the activities of compounds 1~3 against HepG2 cell in vitro. The MTT method was applied to measure the cell inhibition of the compounds on HepG2 cell. The unexpected results revealed that the inhibitory effects of compound 1 (IC₅₀＝7.858 mmol/L), 2 (IC₅₀＝9.509 mmol/L) and 3 (IC₅₀＝203.080 mmol/L) on HepG2 cell were superior to honokiol (IC₅₀＝87.414 mmol/L), berberine hydrochloride (IC₅₀＝359.494 mmol/L) and quercetin (IC₅₀＝261.752 mmol/L), furthermore the inhibitory effects of compounds 1 and 2 on HepG2 cell were similar efficacy with positive drug cisplatin (CDDP, IC₅₀＝11.113 mmol/L). Overall, product 1~3 exerted increased potential in both anti-tumor bioassays compared to that of the analog parents berberine, honokiol and quercetin themselves.

  3.   Conclusions

  In summary, above three-step reaction of pyrolysis and alkylation, we have synthesized three novel products 1~3, which combined two pharmacophores from honokiol, quercetin and berberine. Meanwhile the yields of the three compounds were investigated under various experimental conditions, and the optimal reaction conditions were selected. In addition, the anti-tumor activities of products 1~3 against HepG2 cells were evaluated in vitro for the first time, and compounds 1 and 2 showed remarkable anti-tumor effects against HepG2 cells compared with cisplatin. They are expected to become potential anti-liver cancer drugs. Moreover, based on the method of this study, various pharmacophoric groups were well-tolerated under the optimized reaction conditions. Thus, they may serve as drug leads in pharmaceutical chemistry.

  Table 3

  

  Table 3.  IC₅₀ values of target compounds against HepG2cells^a

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  Compound	IC50/(mmol·L－1)
  CDDP	11.113±1.235
  Honokiol	87.414±2.357
  Berberine hydrochloride	359.494±1.423
  Quercetin	261.752±2.065
  Compound 1	7.858±2.057
  Compound 2	9.509±1.128
  Compound 3	203.080±1.856
  a P < 0.01 compared to CDDP

  Figure 3

  

  Figure 3.  IC₅₀ values of target compounds against HepG2cells

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  4.   Experimental

  4.1   General experimental procedures

  FT-IR spectra were measured on a Jasco FT-IR-4600 microscopic spectrometer (KBr pellets. Jasco Corporation, Tokyo, Japan). UV spectra were recorded on a Jasco V-550 UV/Vis spectrophotometer (Jasco Corporation, Tokyo, Japan). Melting points were taken on a Beijing Tech X-6 microscopic melting point apparatus (Beijing Tech Corporation, Beijing, China). The 1D- and 2D-NMR spectra were determined by a Bruker-AVANCEIII- 010601AM-500 spectrometer (Bruker Corporation, Karlsruhe, Germany) using TMS (Tetramethylsilane) as an internal standard. HRESIMS data in the positive ion mode were obtained on a Waters UPLC/Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA).

  4.2   Synthesis and purification of compounds 1~3

  4.2.1   Synthesis of compound 5

  The berberine hydrochloride (3.71 g, 10 mmol) and DMF (20 mL) were added into a 50 mL of three-necked flask and refluxed for 2 h. The mixture was poured into 30 mL of ice water and crystallized at 4 ℃, and a red precipitate generated. Finally a red solid compound 5 was got by filtered (2.78 g, 8.63 mmol, purity 94%).

  9-Hydroxy-10-methoxy-5, 6-dihydro-[1, 3]dioxolo[4, 5- g]isoquinolino[3, 2-a]isoquinolin-7-ium chloride (5): Yield 75%. Red crystal, m.p. 243~245 ℃ (Lit.^[20] m.p. 280.2~281.5 ℃); ¹H NMR (500 MHz, DMSO-d₆) δ: 9.91 (s, 1H, H-8), 8.83 (s, 1H, H-13), 8.08 (d, J＝9.0 Hz, 1H, H-11), 7.78 (s, 1H, H-1), 7.69 (d, J＝9.0 Hz, 1H, H-12), 7.06 (s, 1H, H-4), 6.16 (s, 2H, H-15), 4.89 (t, J＝6.5 Hz, 2H, H-6), 4.03 (s, 3H, H-16), 3.18 (t, J＝6.5 Hz, 2H, H-5); ¹³C NMR (125 MHz, DMS0-d₆) δ: 149.57, 147.61, 145.77, 145.34, 143.83, 136.55, 132.39, 130.46, 125.47, 120.63, 119.79, 117.96, 117.60, 108.39, 105.32, 102.00, 57.02, 54.87, 26.45; ESI-MS m/z: 322.1002 (M⁺).

  4.2.2   Synthesis of compound 7a

  Titled compound was prepared using reported method.^[20] Compound 5 (3.57 g, 10 mmol) in DMF (100 mL) was heated to 40 ℃, and 1, 4-dibromobutane (30 mL, 250 mmol) was dropwise added. Then the temperature of mixture was raised to 80~85 ℃ for 8 h. After cooling to 40 ℃, 95% ethanol (10 mL) and ethyl acetate (30 mL) was added, and the mixture was stirred for 3 h to obtain product 7a as a yellow solid (4.6 g, 9.37 mmol, purity 90%).

  9-(4-Bromobutoxy)-10-methoxy-5, 6-dihydro-[1, 3]diox- olo[4, 5-g]isoquinolino[3, 2-a]isoquinolin-7-ium chloride (7a): Yield 85%. Yellow solid, m.p. 246~248 ℃; ¹H NMR (500 MHz, DMSO-d₆) δ: 4.31 (t, J＝6.5 Hz, 2H, H-1), 2.01~2.09 (m, 2H, H-2), 2.10~2.12 (m, 2H, H-3), 3.68 (t, J＝6.5 Hz, 2H, H-4), 7.94 (s, 1H, H-1′), 7.09 (s, 1H, H-4′), 3.21 (t, J＝6.5 Hz, 2H, H-5′), 4.96 (t, J＝6.5 Hz, 2H, H-6′), 9.78 (s, 1H, H-8′), 8.20 (d, J＝9.5 Hz, 1H, H-11′), 7.99 (d, J＝9.5 Hz, 1H, H-12′), 8.95 (s, 1H, H-13′), 6.17 (s, 2H, H-15′), 4.06 (3H, s, H-16′); ¹³C NMR (125 MHz, DMSO-d₆) δ: 73.37 (C-1), 28.21 (C-2), 28.87 (C-3), 34.98 (C-4), 105.41 (C-1′), 149.80 (C-2′), 147.66 (C-3′), 121.6 (C-4a), 108.41 (C-4′), 26.31 (C-5′), 55.30 (C-6′), 120.42 (C-8a), 145.28 (C-8′), 142.68 (C-9′), 150.38 (C-10′), 126.60 (C-11′), 123.43 (C-12′), 132.97 (C-12a), 120.21 (C-13′), 137.42 (C-14′), 130.67 (C-14a), 102.08 (C-15′), 57.05 (C-16′); ESI- MS m/z: 456.0805 (M⁺).

  4.2.3   Synthesis of compound 7b

  Compound 5 (3.57 g, 10 mmol) in DMF (100 mL) was heated to 40 ℃, and then 1, 2-dibromoethane (22 mL, 250 mmol) was dropwise added. Then the temperature of mixture was raised to 80~85 ℃ for 8 h. After cooling to 40 ℃, 95% ethanol (10mL) and ethyl acetate (30 mL) was added, and the mixture was stirred for 3 h to obtain product 7b as a yellow solid (4.4 g, 9.50 mmol, purity 90%).

  9-(2-Bromoethoxy)-10-methoxy-5, 6-dihydro-[1, 3]diox- olo[4, 5-g]isoquinolino[3, 2-a]isoquinolin-7-ium (7b): Yield 85%. Yellow solid, m.p. 282~286 ℃; ¹H NMR (500 MHz, DMSO-d₆) δ: 4.61 (t, J＝6.0 Hz, 2H, H-1), 3.98 (t, J＝6.0 Hz, 2H, H-2), 7.80 (s, 1H, H-1′), 7.10 (s, 1H, H-4′), 3.21 (t, J＝6.5 Hz, 2H, H-5′), 4.94 (t, J＝6.5 Hz, 2H, H-6′), 9.87 (s, 1H, H-8′), 8.20 (d, J＝9.0 Hz, 1H, H-11′), 8.02 (d, J＝9.0 Hz, 1H, H-12′), 8.97 (s, 1H, H-13′), 6.18 (s, 2H, H-15′), 4.07 (s, 3H, H-16′); ¹³C NMR (125 MHz, DMSO-d₆) δ: 73.29 (C-1), 31.87 (C-2), 105.43 (C-1′), 149.85 (C-2′), 147.49 (C-3′), 121.51 (C-4a), 108.43 (C-4′), 26.33 (C-5′), 57.08 (C-6′), 120.22 (C-8a), 145.34 (C-8′), 141.71 (C-9′), 150.28 (C-10′), 126.57 (C-11′), 123.88 (C-12′), 132.97 (C-12a), 120.49 (C-13′), 137.52 (C-14′), 130.67 (C-14a), 102.10 (C-15′), 55.43 (C-16′); ESI-MS m/z: 428.0492 (M⁺).

  4.2.4   Synthesis of compounds 1and 2

  Compound 7a (1.2 g, 2.63 mmol), anhydrous acetonitrile (30 mL), honokiol (1.5 g, 5.64 mmol), anhydrous potassium carbonate (5 g, 36.23 mmol), tetrabutylammonium bromide (0.8 g, 2.48 mmol) were added to three-necked flask, stirred at 80 ℃ for 8 h. The mixture was filtered and the residue was washed by DCM (20 mL). The filtrate was evaporated to dryness and recrystallized from ethyl acetate (20 mL) to get the crude product as an orange-yellow solid. Compound 1 (0.65 g, 0.96 mmol, purity 97%) and 2 (0.46 g, 0.68 mmol, purity 97%) were purified by silica gel chromatography [V(CH₂Cl₂):V(CH₃OH)＝10:1 to 4:1 eluting until no compound was detected in the eluent].

  9-(4-((3', 5-Diallyl-4'-hydroxy-[1, 1'-biphenyl]-2-yl)oxy)-butoxy)-10-methoxy-5, 6-dihydro-[1, 3]dioxolo[4, 5-g]iso- quinolino[3, 2-a]isoquinolin-7-ium chloride (1): Yield 35.2%. Yellow solid, m.p. 232~235 ℃; UV-vis (MeOH) λ_max: 206, 225, 264 nm; ¹H NMR (500 MHz, MeOD-d₄) δ: 4.39 (t, J＝5.5 Hz, 2H, H-1), 2.01~2.03 (m, 2H, H-2), 1.99~2.00 (m, 2H, H-3), 4.08 (t, J＝5.5 Hz, 2H, H-4), 7.16 (d, J＝2.0 Hz, 1H, H-2′), 6.96 (d, J＝8.5 Hz, 1H, H-3′′), 7.04 (t, J＝3.0 Hz, 1H, H-4′′), 6.59 (d, J＝8.5 Hz, 1H, H-5′), 7.02 (t, J＝2.5 Hz, 1H, H-6′), 7.00 (d, J＝2.0 Hz, 1H, H-6′′), 3.22 (d, J＝6.5 Hz, 2H, H-7′), 3.29 (d, J＝6.5 Hz, 2H, H-7′′), 5.90~5.93 (m, 1H, H-8′), 5.94~5.97 (m, 1H, H-8′′), 4.91~4.93 (m, 2H, H-9′), 4.94~5.01 (m, 2H, H-9′′), 7.64 (s, 1H, H-1′′′), 6.93 (s, 1H, H-4′′′), 3.13 (t, J＝5.5 Hz, 2H, H-5′′′), 4.71 (t, J＝5.5 Hz, 2H, H-6′′′), 9.49 (1H, s, H-8′′′), 8.01 (d, J＝9.0 Hz, 1H, H-11′′′), 7.95 (d, J＝9.0 Hz, 1H, H-12′′′), 8.64 (s, 1H, H-13′′′), 6.11 (s, 2H, H-15′′′), 4.01 (3H, s, H-16′′′); ¹³C NMR (125 MHz, MeOD-d₄) δ: 75.69 (C-1), 27.38 (C-2), 28.49 (C-3), 69.72 (C-4), 131.26 (C-1′), 132.27 (C-1′′), 132.39 (C-2′), 155.68 (C-2′′), 126.99 (C-3′), 129.23 (C-3′′), 155.21 (C-4′), 115.48 (C-4′′), 114.19 (C-5′), 133.98 (C-5′′), 129.04 (C-6′), 131.83 (C-6′′), 35.39 (C-7′), 40.53 (C-7′′), 138.56 (C-8′), 139.41 (C-8′′), 115.66 (C-9′), 115.81(C-9′′), 106.69 (C-1′′′), 153.00 (C-2′′′), 150.08 (C-3′′′), 121.95 (C-4a), 109.51 (C-4′′′), 28.30 (C-5′′′), 57.32 (C-6′′′), 123.62 (C-8a), 146.08 (C-8′′′), 144.91 (C-9′′′), 152.32 (C-10′′′), 128.09 (C-11′′′), 124.43 (C-12′′′), 135.29 (C-12a), 121.72 (C-13′′′), 139.60 (C-14′′′), 131.82 (C-14a), 103.83 (C-15′′′), 57.69 (C-16′′′); IR (KBr) ν: 3399.89, 2913.91, 1600.63, 1503.24, 1355.71, 1337.39, 1269.90, 1228.43, 1098.26, 1035.59 cm^－1; HRMS (ESI) calcd for C₄₁H₄₀O₆N 642.2788, found 642.2786.

  9-(4-((3, 5'-Diallyl-2'-hydroxy-[1, 1'-biphenyl]-4-yl)oxy)-butoxy)-10-methoxy-5, 6-dihydro-[1, 3]dioxolo[4, 5-g]iso- quinolino[3, 2-a]isoquinolin-7-ium chloride (2): Yield 25.5%. Yellow solid, m.p. 233~235 ℃; UV-vis (MeOH) λ_max: 225, 263, 350, 433 nm; ¹H NMR (500 MHz, MeOD-d₄) δ: 4.57 (t, J＝5.5 Hz, 2H, H-1), 2.06~2.09 (m, 2H, H-2), 2.11~2.13 (m, 2H, H-3), 4.15 (t, J＝5.5 Hz, 2H, H-4), 7.29 (d, J＝2.2 Hz, 1H, H-2′), 6.86 (d, 1H, J＝1.5 Hz, H-3′′), 6.89 (d, J＝2.0 Hz, 1H, H-4′′), 6.76 (d, J＝8.0 Hz, 1H, H-5′), 7.24 (t, J＝2.0 Hz, 1H, H-6′), 6.90 (d, J＝2.1 Hz, 1H, H-6′′), 3.24 (d, J＝7 Hz, 2H, H-7′), 3.29 (d, J＝6.5 Hz, 2H, H-7′′), 5.90~5.94 (m, 1H, H-8′), 5.94~5.99 (m, 1H, H-8′′), 4.92~4.94 (m, 2H, H-9′), 4.98~5.00 (m, 2H, H-9′′), 7.49 (s, 1H, H-1′′′), 6.85 (s, 1H, H-4′′′), 3.13 (t, J＝6.0 Hz, 2H, H-5′′′), 4.75 (t, J＝6.0 Hz, 2H, H-6′′′), 9.54 (s, 1H, H-8′′′), 8.08 (d, J＝9.0 Hz, 1H, H-11′′′), 7.54 (d, J＝9.0 Hz, 1H, H-12′′′), 8.56 (s, 1H, H-13′′′), 6.07 (s, 2H, H-15′′′), 4.07 (s, 3H, H-16′′′); ¹³C NMR (125 MHz, MeOD-d₄) δ: 75.51 (C-1), 27.38 (C-2), 28.28 (C-3), 68.74 (C-4), 129.24 (C-1′), 132.53 (C-1′′), 129.24 (C-2′), 170.39 (C-2′′), 129.37 (C-3′), 111.95 (C-3′′), 156.67 (C-4′), 129.37 (C-4′′), 117.12 (C-5′), 132.57(C-5′′), 131.83 (C-6′), 131.47 (C-6′′), 35.67 (C-7′), 40.59 (C-7′′), 138.53 (C-8′), 139.69 (C-8′′), 115.62 (C-9′), 115.76(C-9′′), 106.60 (C-1′′′), 151.73 (C-2′′′), 149.94 (C-3′′′), 121.83 (C-4a), 109.43 (C-4′′′), 28.13 (C-5′′′), 57.25 (C-6′′′), 123.53 (C-8a), 146.00 (C-8′′′), 144.64 (C-9′′′), 152.24 (C-10′′′), 128.05 (C-11′′′), 124.28 (C-12′′′), 135.24 (C-12a), 121.69(C-13′′′), 135.24 (C-14′′′), 131.98 (C-14a), 103.75 (C-15′′′), 57.77 (C-16′′′); IR (KBr) ν: 3396.99, 29260.66, 1727.91, 1636.30, 1600.63, 1506.13, 1480.1, 1397.17, 1364.39, 1337.39, 1269.90, 1228.43 cm^－1; HRMS (ESI) calcd for C₄₁H₄₀O₆N 642.2787, found 642.2789.

  4.2.5   Synthesis of compound 3

  Quercetin (0.5 g, 1.656 mmol), DMF (20 mL), triethylamine (3.5 mL, 25.23 mmol) and compound 7b (0.885 g, 2.068 mmol) were added to a 100 mL of three neck flask. The mixture was heated to 120 ℃ for 1.5 h. The reaction solution was poured into ice water, 0.1 mol/L diluted HCl solution (5~7 mL) was adjusted to pH 3~4, and then filtered after 24 h. The residue was refluxed 3 times with 30 ml of 0.1 mol/L dilute hydrochloric acid at 60 ℃. Finally the residue was purified by silica gel column chromatography [V(CH₂Cl₂):V(CH₃OH)＝10:1 to 5:1 eluting until no compound was detected in the eluent] to obtain a yellow solid compound 3 (0.34 g, 0.50 mmol, purity 90%).

  9-(2-((2-(3, 4-Dihydroxyphenyl)-5, 7-dihydroxy-4-oxo- 4H-chromen-3-yl)oxy)ethoxy)-10-methoxy-5, 6-dihydro-[1, 3]dioxolo[4, 5-g]isoquinolino[3, 2-a]isoquinolin-7-ium chloride (3): Yield 26.3%. Yellow solid, m.p. 262~265 ℃; UV-vis (MeOH) λ_max: 202, 224, 264, 348, 420 nm; ¹H NMR (500 MHz, DMSO-d₆) δ: 4.66 (s, 2H, H-1), 4.36 (s, 2H, H-2), 7.44 (dd, J＝8.5, 2.1 Hz, 1H, H-2′′), 6.78 (d, J＝8.5 Hz, 1H, H-5′′), 6.16 (s, 1H, H-6′), 7.55 (d, J＝2.0 Hz, 1H, H-6′′), 6.38 (s, 1H, H-8′), 7.71 (s, 1H, H-1′′′), 7.01 (s, 1H, H-4′′′), 3.15 (t, J＝6.0 Hz, 2H, H-5′′′), 4.76 (t, J＝5.8 Hz, 2H, H-6′′′), 9.85 (s, 1H, H-8′′′), 7.94 (d, J＝9.0 Hz, 1H, H-11′′′), 7.86 (s, 1H, H-12′′′), 8.79 (s, 1H, H-13′′′), 6.12 (s, 2H, H-15′′′), 4.00 (s, 3H, H-16′′′); ¹³C NMR (125 MHz, DMSO-d₆) δ: 74.98 (C-1), 74.12 (C-2), 122.68 (C-1′′), 149.67 (C-2′), 122.72 (C-2′′), 139.35 (C-3′), 150.55 (C-3′′), 179.80 (C-4′), 158.28 (C-4′′), 161.78 (C-5′), 117.46 (C-5′′), 100.58 (C-6′), 117.46 (C-6′′), 163.07 (C-7′), 95.56 (C-8′), 157.84 (C-9′), 106.06 (C-10′), 107.38 (C-1′′′), 151.64 (C-2′′′), 128.46 (C-3′′′), 146.98 (C-4a), 110.24 (C-4′′′), 28.30 (C-5′′′), 57.33 (C-6′′′), 122.32 (C-8a), 147.50 (C-8′′′), 144.50 (C-9′′′), 151.81 (C-10′′′), 125.14 (C-11′′′), 123.50 (C-12′′′), 134.87 (C-12a), 121.99 (C-13′′′), 138.40 (C-14′′′), 132.34 (C-14a), 103.98 (C-15′′′), 58.82 (C-16′′′). IR (KBr) ν: 3425.92, 2920.66, 1644.98, 2849.31, 1644.98, 1600.63, 1503.24, 1473.35, 1391.39, 1355.71, 1302.68, 1267.00 cm^－1; HRMS (ESI) calcd for C₃₆H₂₈O₁₁N 650.1667; found 650.1661.

  4.3   Cellular proliferation assay

  The cytotoxic activity of the synthesized compounds was tested against human hepatocellular liver car-cinoma cell line (HepG2). And HepG2 cells were cultured in DMEM medium and supplemented with 10% fetal bovine serum (FBS, Gibco Laboratories, Grand Island, NY, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37 ℃ with 5% CO₂. The stock samples of the compounds were diluted with medium to desired concentrations. The final concentration of DMSO in each sample did not exceed 1% (V/V).

  The cells were seeded in 96-well plates of 1×10⁴ cells/well in fresh medium in 96-well microtiter plastic plates at 37 ℃ for 24 h under 5% CO₂ using a water- jacketed carbon dioxide incubator. Then, the cells were treated with various concentrations of compounds 1~3 (1, 5, 10, 20, 40, 80, 160, 320 μg/mL) and cisplatin (2, 4, 8, 16 μg/mL) with a volume of 100 μL in sextuplicates. After 48 h of treatment, 20 μL of MTT (5 mg/mL) was added to each well, and the plates were incubated for 4 h at 37 ℃. Then, DMSO (100 μL/well) was added. The optical density of lysate at 490 nm was measured by an enzyme immunoassay instrument (SpectraMax Plus 384, Molecular Devices, Sunnyvale, CA, USA).

  Supporting Information The ¹H NMR、¹³C NMR、2D NMR、UV、IR、HRMS spectra of all compounds. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn/.

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• 

  Figure 1  Structures of berberine derivative

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  Figure 2  Structures of compounds 1~3.

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  Scheme 1  Synthetic route of the target compounds

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  Figure 3  IC₅₀ values of target compounds against HepG2cells

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  Table 1.  Effects of reaction conditions on the synthesis of thetarget compounds 1 and 2^a

  Entry	Solvent	Base	Temp./℃	Time/h	Yield/%
  					1	2
  1	CH3CN	K2CO3	25	8	22.1	13.2
  2	CH3CN	K2CO3	60	8	29.2	18.5
  3	CH3CN	K2CO3	80	8	35.2	25.5
  4	CH3CN	K2CO3	82	8	31.2	22.5
  5	DMF	K2CO3	80	8	30.4	19.8
  6	Isopropanol	K2CO3	80	8	11.7	7.7
  7	CH3CN	Na2CO3	80	8	30.5	21.5
  8	CH3CN	Et3N	80	8	30.1	21.4
  9	CH3CN	NaOH	80	8	20.5	16.4
  10	CH3CN	K2CO3	80	2	10.7	5.6
  11	CH3CN	K2CO3	80	4	15.6	10.8
  12	CH3CN	K2CO3	80	6	20.2	15.7
  13	CH3CN	K2CO3	80	8	35.2	25.5
  14	CH3CN	K2CO3	80	12	21.2	12.5
  a Unless otherwise specified, the reactions to produce compounds 1 and 2 were performed with 8 (5.64 mmol), 7a (2.63 mmol), base (36.23 mmol), and TBAB (2.48 mmol) in 30 mL of solvent.

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  Table 2.  Effects of reaction conditions on the synthesis of the target compound 3^a

  Entry	Solvent	Base	Temp./℃	Time/h	Yield/%
  1	DMF	Et3N	120	90	26.3
  2	DMF	NaOH	120	90	16.8
  3	DMF	K2CO3	120	90	20.2
  4	MeOH	Et3N	65	90	NRb
  5	CH3CN	Et3N	80	90	6.4
  6	DMF	Et3N	80	90	8.4
  7	DMF	Et3N	100	90	10.6
  8	DMF	Et3N	140	90	20.8
  9	DMF	Et3N	120	30	11.6
  10	DMF	Et3N	120	60	12.9
  11	DMF	Et3N	120	120	19.3
  a Unless otherwise specified, the reactions to produce compounds 3 were performed with 9 (1.656 mmol), 7b (2.068 mmol), and base (25.23 mmol) in 30 mL of solvent. b NR: The reaction did not occur.

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  Table 3.  IC₅₀ values of target compounds against HepG2cells^a

  Compound	IC50/(mmol·L－1)
  CDDP	11.113±1.235
  Honokiol	87.414±2.357
  Berberine hydrochloride	359.494±1.423
  Quercetin	261.752±2.065
  Compound 1	7.858±2.057
  Compound 2	9.509±1.128
  Compound 3	203.080±1.856
  a P < 0.01 compared to CDDP

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•