English

• Matrine was mainly isolated from kuh-seng, a member of the quetiapine oxazine organism family, and it was primarily found in Sophora flavescens Ait. In recent decades, matrine and its analogues have received considerable attention around the world due to their ample occurrence, accessible structural modifications, effortless synthesis and multifarious biological activities. Furthermore, it was also reported that matrine exhibited anti-tumor effects by inhibiting cell proliferation and inducing apoptosis of cancer cells from cervical cancer, leukemia, gastric cancer, hepatocellular carcinoma, breast cancer, and lung cancer. Although these small-molecule inhibitors have provided some relief to patients suffering from pain and inflammation, they have shortcomings, like higher toxicity.^{[1, 2]} Therefore, investigation of new matrine derivatives might be still academically desirable.

  According to the literatures, ^[3~8] modification of matrine generally resulted in 13-monosubstituted matrine derivatives, 14-monosubstituted matrine derivatives, some matrine derivatives from carbonyl transformation, and sophora acid derivatives where the lactam ring was amputated.

  It was reported that introduction of a pharmacophore group at N16 could improve the matrine's antitumor activity, ^[9] and the substituents could be the alkyl, acyl, and benzyl groups. The expression of HSC70 mRNA of hepatocellular carcinoma cells transfected with hepatitis B virus (HBV) was extensively studied using the real-time polymerase chain reaction (RT-PCR), and it indicated that the sophora acid derivatives of CH₂PhOCH₃-p showed superior anti-HBV and hepatitis C virus (HCV) activities, and the electron-withdrawing substituent at N16 would be beneficial to the biological activities.

  Many studies showed that 2(5H)-furanone derivatives had good pharmacological activities, ^[10~20] such as anti- tumor, antibacterial, anti-virus, and anti-inflammation properties. The common structure feature of the 2(5H)- furanone derivatives is the α, β-unsaturated-δ-lactone motif. Exploration of the stereoselective preparation of the 5-aloxy-3, 4-dihali-2(5H)-furanone was generally realized in the Lewis acid-promoted conditions. We suspect that the specific biological activity and Lewis acid structure of furanone are likely to enhance the biological activity of matrine. Herein, the first synthesis of matrine derivatives containing furanone was described. As shown in Scheme 1, matrine derivative 5 was synthesized from the γ-substituted butenolide moiety 4 and sophora acid ester 3 through a C—N coupling reaction. Then, the antitumor activity against human liver cancer cell lines SMMC-7721 in vitro of these derivatives was investigated.

  Scheme 1

  

  Scheme 1.  Retrosynthetic analysis of 5a~5f

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  2.   Results and discussion

  According to the reported procedure, It could be envisioned that 5-(S)-alkane-3, 4-dibromo-2(5H)-furan (4)^{[21, 22]} could be synthesized from furfural and bromine through the two steps conversions of oxidation and etherification, and the single diastereomer might be afforded from multiple recrystallization processes (Scheme 2). Ester 3^[23] could be easily accessed from sophora acid sodium 2-1 by an esterification manipulation, and the sodium salt 2-1 would be conveniently produced from matrine by cleavage of the lactam ring of the matrine.

  Scheme 2

  

  Scheme 2.  General synthetic procedure of furanone derivatives 5a~5f

  Reagents and conditions: (a) Br₂/H₂O (V:V = 1:4), 0 ℃ to reflux, 30 min; (b) vacuum distillation; (c) R²OH, H⁺, benzene, reflux, 30~40 h; (d) recry- stallization; (e) NaOH, H₂O, reflux, 12 h; (f) SOCl₂, R¹OH, 0 ℃ to reflux, 5 h; (g) compound 4, DCM, NEt₃, 40 ℃, 24 h.

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  Thus, synthesis of matrine derivatives started from ring- opening of the matrine's lactam ring, which was depicted in Scheme 2. Treatment of matrine with 18.75 equiv. of aqueous NaOH at refluxing temperature for 12 h gave the crude sophora acid sodium 2-1, and it was used for the next step without purification. Esterification of sophora acid sodium 2-1 with methanol and ethanol was carried out under the common conditions. The corresponding esters 3a, 3b were synthesized by two steps from the matrine in 83% yield (3a) and 60% yield (3b) respectively.

  Furfural and liquid bromide were used for synthesis of the furanone unit 4. Treatment of furfural with 5.4 equiv. of liquid bromide in H₂O at refluxing temperature for 30 min led to the formation of 5-hydroxyl-3, 4-dibromo- 2(5H)-furanone (1-2). The compound 1-2 was subjected for etherification reaction using different alcohols in dry benzene, affording the enantio-enriched furanone 4.^{[21, 22, 23]}

  Then nucleophilic substitution of 5-hydroxyl-3, 4-di- bromo-2(5H)-furanone with different sophora acid esters (3a, 3b) in the presence of catalytic triethylamine in dichloromethane (DCM) afforded the corresponding matrine derivatives containing a furanone skeleton in good yields (Scheme 2).

  The newly synthesized matrine derivatives 5a~5f were selected for the preliminary anticancer activity test, and the marine, vincristine (VCR) and cytosine arabinoside (ARA) were employed for the positive control experiments. The nine compounds were screened for in vitro anticancer activity against human liver cell lines (SMMC-7721), and the values of IC₅₀ were carried out using Curve Expert software. The results were summarized in Table 1, and to our delight, all matrine derivatives 5a~5f have better anticancer activity compared with matrine and cytosine arabinoside (ARA) in vitro. More specifically, 5a (IC₅₀＝0.0004 μmol/L), 5b (IC₅₀＝0.0006 μmol/L) and 5e (IC₅₀＝0.0006 μmol/L) displayed markedly higher inhibitory activity on human liver cancer cell lines (SMMC-7721) than VCR (IC₅₀＝0.1284 μmol/L), ARA (IC₅₀＝0.4578 μmol/L) and the parent compound matrine (IC₅₀＝0.9018 μmol/L).

  Table 1

  

  Table 1.  In vitro anticancer activities against SMMC-7721 cell lines of compounds 5a~5f, matrine, VCR and ARA

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  Compound	IC50/(μmol•L－1)
  	24 h	48 h	72 h
  5a	0.0004	—	—
  5b	0.0006	—	—
  5e	0.0006	—	—
  5c	0.2391	0.2238	0.2136
  5d	0.1634	0.1482	0.1465
  5f	0.2163	0.1360	0.1062
  Matrine	0.9018	—	—
  VCR	0.1284	—	—
  ARA	0.4578	—	—

  As shown in Table 2, the compounds 5a~5f displayed higher anti-proliferative activities at different concentrations than that of matrine, VCR and ARA, and it was clear that the inhibitory rate increased with the increased concentration.

  Table 2

  

  Table 2.  Inhibition rate of compounds 5a~5f, matrine, VCR and ARA on SMCC-7721 cells in different concentrations

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  Compd.	Concentration/(µg•mL－1)	Inhibition ratea/%
  		24 h	48 h	72 h
  55a	1	92.25±1	—	—
  	5	94.32±1	—	—
  	10	96.09±1	—	—
  	50	95.48±1	—	—
  	100	96.09±1	—	—
  55b	1	81.54±5	—	—
  	5	84.51±1	—	—
  	10	89.50±2	—	—
  	50	91.82±2	—	—
  	100	92.22±1	—	—
  5e	1	81.36±2	—	—
  	5	86.72±3	—	—
  	10	96.42±0	—	—
  	50	96.42±0	—	—
  	100	97.25±0	—	—
  5c	1	2.65±1	0.96±2	7.84±6
  	5	4.79±5	5.68±3	14.87±4
  	10	9.06±7	11.35±6	25.34±1
  	50	23.50±4	30.48±8	39.32±2
  	100	41.71±9	48.35±6	51.27±1
  5d	1	3.50±3	2.50±3	7.54±4
  	5	3.42±7	6.64±3	13.31±2
  	10	16.32±10	21.00±5	27.08±3
  	50	31.79±8	44.95±10	48.47±4
  	100	53.16±4	60.05±4	67.16±2
  5f	1	2.82±3	4.26±1	6.74±5
  	5	5.38±3	5.45±6	13.86±2
  	10	11.97±10	14.30±6	27.25±5
  	50	21.71±7	43.59±9	50.47±4
  	100	46.58±7	52.38±5	63.43±1
  Matrine	1	6.93±2	—	—
  	5	8.21±1	—	—
  	10	15.14±4	—	—
  	50	16.72±4	—	—
  	100	27.48±2	—	—
  VCR	1	3.13±2	—	—
  	5	14.19±8	—	—
  	10	24.36±3	—	—
  	50	25.45±2	—	—
  	100	32.61±4	—	—
  ARA	1	5.80±2	—	—
  	5	9.71±6	—	—
  	10	18.67±4	—	—
  	50	28.84±4	—	—
  	100	31.85±4	—	—
  aValues represent the means of three separate experiments with SD less than 10%.

  In the experiment of morphological change (at the concentration of 1 µg/mL), the SMMC-7721 cells were observed under an inverted microscope after treated with the drug for 24 h. As shown in Figure 1, nuclei cleave into debris and produce apoptotic bodies after treated with compounds 5a, 5b and 5e, while the SMMC-7721 cells wall is still had intact cell membranes in the blank control group (Figure 1A).

  Figure 1

  

  Figure 1.  Morphological changes of SMCC-7721 cells induced by 5a, 5b and 5e for 24 h

  (A) Normal SMMC-7721 cells; (B) induced by 5a; (C) induced by 5b; (D) induced by 5e

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  3.   Conclusions

  In conclusion, we have developed an efficient synthesis of matrine derivatives containing a furanone skeleton. The anticancer activity against human liver cancer cell lines SMMC-7721 has been evaluated. All of the tested compounds exhibited good anticancer activities. Compound 5a possessed best inhibitory activity against SMMC-7721 cell lines with the IC₅₀ value of 0.0004 μmol/L at 24 h, showing markedly higher inhibitory activity in vitro than matrine (0.9018 μmol/L), cytosine arabinoside (0.4578 μmol/L), and vincristine (0.1284 μmol/L). The important feature of this novel method is the improving bioactivity of the matrine against hepatocellular carcinoma. Further mechanistic studies and the extension of the scope of these reactions are currently under way in our group.

  4.   Experimental section

  4.1   Apparatus reagents and chemicals

  ¹H NMR spectra were recorded on a Bruker AVIII-400 spectrometer. Chemical shifts were calibrated with CDCl₃. ¹³C NMR spectra were obtained by using the same NMR spectrometer and were calibrated with CDCl₃. Infrared (IR) spectra of compounds dispersed in potassium bromide were recorded using a Spectrum Two spectrometer. GF254 thin layer chromatography silica gel and 2000~300 column chromatography silica gel were used and obtained from Qingdao Marine chemical plant. All starting materials and reagents were purchased from commercially available sources and used without further purification, unless otherwise indicated. Compounds 4a~4c were synthesized according to the references [21~23].

  4.2   General procedure for sophora acid ester

  Matrine (4.2 g, 0.016 mol) was added to a stirred solution of NaOH (11.9 g, 0.3 mol) in 34 mL of H₂O. The mixture was heated to reflux for 12 h, and then it was cooled to room temperature. A white solid was observed to precipitate, and after filtration and distillation, the sodium matrine crude product was obtained. The crude salt was used for the next step without any purification. Approximately 1.5 mL of thionyl chloride was added dropwise to a stirred solution of 15 mL of alcohol at 0 ℃. Then the mixture was kept at room temperature for 1 h, a 10 mL of alcohol solution of sodium matronate (1 g, 3.73 mmol) was added after stirring for 40 min at room temperature. Then, the solution was heated to reflux and stirred for an additional 4 h. The solvent was then removed in vacuo, and 15 mL of chloroform was added into the residual reaction mixture along with 1.0 g of sodium bicarbonate solid. The solution was stirred at room temperature for about 30 min. The precipitate was then filtered, and the solvent was removed in vacuo to give 3.5 g of compound 3a, which was a white solid. Yield 83%, m.p. 110~115 ℃ (lit.^[24] m.p. 113~119 ℃); ¹H NMR (CDCl₃, 400 MHz) δ: 3.67 (s, 3H), 3.59~3.67 (m, 1H), 3.44 (t, J＝12.6 Hz, 1H), 3.07 (dd, J＝12.0, 4.4 Hz, 1H), 2.80 (t, J＝13.2 Hz, 2H), 2.34~2.43 (m, 2H), 2.17 (s, 1H), 1.89~2.04 (m, 6H), 1.78~1.86 (m, 2H), 1.54~1.67 (m, 2H), 1.45 (d, J＝11.7 Hz, 4H); ¹³C NMR (CDCl₃, 100 MHz) δ: 173.79, 62.30, 57.08, 56.99, 53.25, 51.82, 44.23, 37.90, 33.49, 32.87, 29.48, 27.15, 26.00, 20.74, 20.50, 20.06.

  Sophora acid ethyl ester (3b): Yield 60%, a white solid. m.p. 113~117 ℃ (lit.^[24] m.p. 115~119 ℃); ¹H NMR (CDCl₃, 400 MHz) δ: 4.11 (q, J＝7.2 Hz, 2H), 3.84~3.57 (m, 1H), 3.44 (t, J＝12.1 Hz, 1H), 3.08 (d, J＝11.5 Hz, 1H), 2.96~2.66 (m, 1H), 2.39 (s, 3H), 2.19 (s, 1H), 1.90 (q, J＝34.7, 32.5 Hz, 6H), 1.74~1.35 (m, 5H), 1.28~1.21 (m, 4H).

  4.3   Procedure for the synthesis of the matrine derivatives 5

  5-Menthyl-3, 4-dibromine-2(5H)-furanone (4a)^{[21, 22]} (0.177 g, 0.45 mmol) was added into a stirred solution of sophora acid methyl ester 3a (0.1 g, 0.4 mmol) in dry dichloromethane (DCM, 10 mL) and dry triethylanmine (0.06 mL, 0.45 mmol) at 40 ℃. After the reaction finished [monitored by thin layer chromatography (TLC)], the reaction mixture was extracted with DCM, washed with saturated sodium bicarbonate solution (8.0 mL×2) and saturated salt solution (8.0 mL×2). The organic phase was separated, dried over MgSO₄, and the solvent was removed in vacuo. The residue was chromatographed on silica gel [V(DCM):V(MeOH)＝60:1→20:1] to give 0.15 g of methyl 3-(2-((S)-4-bromo-2-(((1S, 2R, 5S)-2-isopropyl-5-methylcyclohexyl)oxy)-5-oxo-2, 5-dihydrofuran-3-yl)decahydro-1H, 4H-pyrido[3, 2, 1-ij][1, 6]naphthyridin-1-yl)propanoate (5a) as a brownish-yellow sticky liquid, yield 65%. $[\alpha ]_{\rm{D}}^{{\rm{25}}}$－3 (c 1.00, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz) δ: 6.89 (s, 1H), 4.70~4.75 (m, 1H), 4.08 (s, 1H), 3.63 (s, 3H), 3.38~3.42 (m, 1H), 3.36 (dd, J＝18.6, 8.4 Hz, 1H), 2.68 (d, J＝9.6 Hz, 2H), 2.32 (s, 1H), 2.05 (d, J＝12.0 Hz, 1H), 1.75~1.96 (m, 4H), 1.50~1.67 (m, 8H), 1.40~1.46 (m, 8H), 0.99~1.05 (m, 2H), 0.83~0.97 (m, 7H), 0.74 (d, J＝7.2 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz) δ: 174.18, 164.35, 161.93, 136.66, 118.29, 62.96, 56.63, 53.97, 53.53, 51.49, 47.06, 46.10, 40.29, 39.98, 34.87, 34.26, 33.89, 31.69, 31.51, 28.90, 28.35, 26.06, 23.34, 21.79, 21.13, 20.91, 16.21; IR (KBr) ν: 2953, 2870, 2761, 2694, 1736, 1647, 1608, 1447, 1369, 1298, 1200, 995, 736 cm^－1; HRMS calcd for C₂₉H₄₉N₃O₅Br [M+NH₄]⁺ 598.2856, found 598.2773.

  Methyl 3-(2-((2S)-4-bromo-5-oxo-2-(((2R, 4S)-1, 7, 7-trimethylbicyclo[2.2.1]heptan-2-yl)oxy)-2, 5-dihydrofuran-3-yl)decahydro-1H, 4H-pyrido[3, 2, 1-ij][1, 6]naphthyridin-1-yl)propanoate (5b): Yield 50%, yellow sticky liquid. $[\alpha ]_{\rm{D}}^{{\rm{25}}}$－1 (c 1.00, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz) δ: 6.89 (s, 1H), 4.70~4.75 (m, 1H), 4.08 (s, 1H), 3.63 (s, 3H), 3.38~3.42 (m, 1H), 3.36 (dd, J＝18.6, 8.4Hz, 1H), 2.68 (d, J＝9.6 Hz, 2H), 2.32 (s, 1H), 2.05 (d, J＝12.0 Hz, 1H), 1.75~1.96 (m, 4H), 1.50~1.67 (m, 8H), 1.40~1.46 (m, 8H), 0.99~1.05 (m, 2H), 0.83~0.97 (m, 7H), 0.74 (d, J＝7.2 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz) δ: 174.18, 164.35, 161.93, 136.66, 118.29, 62.96, 56.63, 53.97, 53.53, 51.49, 47.06, 46.10, 40.29, 39.98, 34.87, 34.26, 33.89, 31.69, 31.51, 28.90, 28.35, 26.06, 23.34, 21.79, 21.13, 20.91, 16.21; IR (KBr) ν: 2953, 2870, 2761, 2694, 1736, 1647, 1608, 1447, 1369, 1298, 1200, 995, 736 cm^－1; HRMS calcd for C₂₉H₄₇N₃O₅Br [M+NH₄]⁺ 596.2699, found 596.2616.

  Methyl 3-(2-(4-bromo-2-methoxy-5-oxo-2, 5-dihydro-furan-3-yl)decahydro-1H, 4H-pyrido[3, 2, 1-ij][1, 6]naphthy-ridin-1-yl)propanoate (5c): Yield 70%, yellow sticky liquid. $[\alpha ]_{\rm{D}}^{{\rm{25}}}$－1 (c 1.00, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz) δ: 6.98 (s, 1H), 4.11 (s, 1H), 3.76 (s, 3H), 3.60 (s, 3H), 2.66 (s, 2H), 2.31 (s, 2H), 1.87~1.99 (m, 3H), 1.36~1.64 (m, 13H); ¹³C NMR (CDCl₃, 100MHz) δ: 173.98, 164.27, 162.50, 137.97, 116.79, 62.51, 56.40, 53.42, 51.42, 45.18, 40.13, 34.19, 33.72, 32.06, 29.68, 29.33, 29.00, 28.21, 22.67, 21.63, 20.96, 14.13; IR (KBr) ν: 2926. 1735, 1635, 1494, 1451, 1050, 824, 606 cm^－1; HRMS calcd for C₂₀H₃₃N₃O₅Br [M+NH₄]⁺ 474.1520, found 474.1497.

  Ethyl 3-(2-((S)-4-bromo-2-(((1S, 2R, 5S)-2-isopropyl-5-methylcyclohexyl)oxy)-5-oxo-2, 5-dihydrofuran-3-yl)-decahydro-1H, 4H-pyrido[3, 2, 1-ij][1, 6]naphthyridin-1-yl)propanoate (5d): Yield 40%, brownish-yellow sticky liquid. $[\alpha ]_{\rm{D}}^{{\rm{20}}}$－0.012 (c 1.00, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ: 6.85 (s, 1H), 4.68 (t, J＝11.0 Hz, 1H), 4.04 (d, J＝8.9 Hz, 3H), 3.63~3.16 (m, 2H), 2.80~2.45 (m, 2H), 2.25 (s, 3H), 2.09~1.77 (m, 6H), 1.39 (q, J＝26.6, 25.7 Hz, 20H), 1.17 (d, J＝8.3 Hz, 5H), 0.83 (td, J＝55.7, 53.0, 9.1 Hz, 15H); ¹³C NMR (100 MHz, CDCl₃) δ: 173.48, 164.11, 161.74, 136.40, 119.07, 118.10, 99.92, 76.78, 62.75, 60.11, 56.42, 53.78, 53.41, 46.85, 45.88, 40.10, 39.73, 34.68, 34.07, 33.93, 31.31, 29.58, 28.71, 28.12, 25.86, 23.14, 21.88, 21.61, 20.96, 20.72, 16.02, 14.17; IR (KBr) ν: 3944. 3691, 3054, 2986, 2928, 2856, 2685, 2410, 2305, 1727, 1629, 1422, 1261, 1098, 1011, 896, 755, 722, 455 cm^－1; HRMS calcd for C₃₀H₅₁BrN₃O₅ [M+NH₄]⁺ 612.2910, found 612.2908.

  Ethyl 3-(2-((2S)-4-bromo-5-oxo-2-(((2R, 4S)-1, 7, 7- trimethylbicyclo[2.2.1]heptan-2-yl)oxy)-2, 5-dihydrofuran-3-yl)decahydro-1H, 4H-pyrido[3, 2, 1-ij][1, 6]naphthyridin-1-yl)propanoate (5e): Yield 50%, brownish-yellow sticky liquid. $[\alpha ]_{\rm{D}}^{{\rm{20}}}$－0.004 (c 1.00, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz) δ: 7.08~6.85 (m, 1H), 4.93 (dt, J＝14.5, 9.1 Hz, 1H), 4.09 (h, J＝6.7, 6.1 Hz, 3H), 3.51~3.25 (m, 2H), 3.06 (dq, J＝69.2, 9.7, 8.3 Hz, 1H), 2.69 (d, J＝10.8 Hz, 2H), 2.46~2.15 (m, 3H), 2.12~1.85 (m, 3H), 1.84~1.42 (m, 5H), 1.43~0.97 (m, 6H), 0.97~0.68 (m, 11H); ¹³C NMR (100 MHz, CDCl₃) δ: 173.79, 164.40, 162.40, 136.91, 135.72, 119.08, 118.02, 83.16, 82.63, 62.93, 60.18, 56.58, 54.00, 49.12, 47.94, 46.11, 44.87, 39.90, 36.21, 34.13, 31.63, 29.74, 28.85, 27.97, 27.09, 21.68, 21.13, 19.72, 18.90, 14.32, 13.49; IR (KBr) ν: 3944. 3691, 3054, 2986, 2962, 2521, 2410, 2305, 1730, 1605, 1550, 1422, 1260, 1097, 1013, 896, 752 cm^－1; HRMS calcd for C₃₀H₄₉BrN₃O₅ [M+ NH₄]⁺ 610.2775, found 610.2754.

  Ethyl3-(2-(4-bromo-2-methoxy-5-oxo-2, 5-dihydrofuran-3-yl)decahydro-1H, 4H-pyrido[3, 2, 1-ij][1, 6]naphthyridin-1-yl)propanoate (5f): Yield 70%, yellow sticky liquid. $[\alpha ]_{\rm{D}}^{{\rm{20}}}$－0.011 (c 1.00, CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz) δ: 7.00 (s, 1H), 4.10 (qt, J＝7.3, 4.4 Hz, 3H), 3.80 (dd, J＝5.9, 1.8 Hz, 3H), 3.51~3.22 (m, 1H), 2.68 (d, J＝10.9 Hz, 2H), 2.32 (q, J＝9.3, 8.5 Hz, 2H), 2.08~1.83 (m, 2H), 1.77 (t, J＝10.4 Hz, 1H), 1.71~1.31 (m, 6H), 1.23 (td, J＝7.1, 2.1 Hz, 4H), 1.20~1.01 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ: 173.58, 164.27, 162.58, 137.92, 116.80, 62.58, 60.19, 56.44, 53.45, 45.24, 40.17, 34.24, 34.04, 32.14, 29.68, 29.07, 28.24, 21.69, 20.98, 14.26; IR (KBr) ν: 3943, 3053, 2985, 2685, 2304, 1729, 1628, 1421, 1275, 1261, 1016, 895, 764, 749, 408 cm^－1; HRMS calcd for C₂₁H₃₅BrN₃O₅ [M+NH₄]⁺ 488.1669, found 488.1653.

  4.4   Pharmacology [human liver cell lines (SMMC-7721)]

  Cells (1×10⁴ in 100 μL) were seeded on 96 plates in triplicate. Following a 24 h culture at 37 ℃, the medium was replaced with fresh medium at various concentrations (1, 5, 10, 50, 100 μg/mL) of compounds 5a~5f, matrine, vincristine (VCR) and cytosine arabinoside (ARA) in a final volume of 100 μL. At the same time, the drug-free medium negative control well and the solvent control well were set with the same volume of dimethyl sulfoxide (DMSO). Cells were respectively incubated at 37 ℃ for 24, 48 and 72 h. Then, 10 μL of 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT) [2 mg/mL in a phosphate buffer solution (PBS)] was added to each well, incubated for an additional 4 h, centrifuged at 1000 r/min for 10 min, and then the medium was removed. MTT formazan precipitates were dissolved in 150 μL of DMSO, shaken mechanically for 10 min and then read immediately at 568 nm using a plate reader (Multiskan MK3, Thermo Fisher Scientific, USA).

  Cell inhibition rate (%)＝[A₅₆₈(negative control well)－A₅₆₈(dosing well)]/A₅₆₈(negative control well)×100%

  Supporting Information    ¹H NMR, ¹³C NMR and HRMS spectra of compounds 5a~5f. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn.

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  Scheme 1  Retrosynthetic analysis of 5a~5f

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  Scheme 2  General synthetic procedure of furanone derivatives 5a~5f

  Reagents and conditions: (a) Br₂/H₂O (V:V = 1:4), 0 ℃ to reflux, 30 min; (b) vacuum distillation; (c) R²OH, H⁺, benzene, reflux, 30~40 h; (d) recry- stallization; (e) NaOH, H₂O, reflux, 12 h; (f) SOCl₂, R¹OH, 0 ℃ to reflux, 5 h; (g) compound 4, DCM, NEt₃, 40 ℃, 24 h.

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  Figure 1  Morphological changes of SMCC-7721 cells induced by 5a, 5b and 5e for 24 h

  (A) Normal SMMC-7721 cells; (B) induced by 5a; (C) induced by 5b; (D) induced by 5e

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  Table 1.  In vitro anticancer activities against SMMC-7721 cell lines of compounds 5a~5f, matrine, VCR and ARA

  Compound	IC50/(μmol•L－1)
  	24 h	48 h	72 h
  5a	0.0004	—	—
  5b	0.0006	—	—
  5e	0.0006	—	—
  5c	0.2391	0.2238	0.2136
  5d	0.1634	0.1482	0.1465
  5f	0.2163	0.1360	0.1062
  Matrine	0.9018	—	—
  VCR	0.1284	—	—
  ARA	0.4578	—	—

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  Table 2.  Inhibition rate of compounds 5a~5f, matrine, VCR and ARA on SMCC-7721 cells in different concentrations

  Compd.	Concentration/(µg•mL－1)	Inhibition ratea/%
  		24 h	48 h	72 h
  55a	1	92.25±1	—	—
  	5	94.32±1	—	—
  	10	96.09±1	—	—
  	50	95.48±1	—	—
  	100	96.09±1	—	—
  55b	1	81.54±5	—	—
  	5	84.51±1	—	—
  	10	89.50±2	—	—
  	50	91.82±2	—	—
  	100	92.22±1	—	—
  5e	1	81.36±2	—	—
  	5	86.72±3	—	—
  	10	96.42±0	—	—
  	50	96.42±0	—	—
  	100	97.25±0	—	—
  5c	1	2.65±1	0.96±2	7.84±6
  	5	4.79±5	5.68±3	14.87±4
  	10	9.06±7	11.35±6	25.34±1
  	50	23.50±4	30.48±8	39.32±2
  	100	41.71±9	48.35±6	51.27±1
  5d	1	3.50±3	2.50±3	7.54±4
  	5	3.42±7	6.64±3	13.31±2
  	10	16.32±10	21.00±5	27.08±3
  	50	31.79±8	44.95±10	48.47±4
  	100	53.16±4	60.05±4	67.16±2
  5f	1	2.82±3	4.26±1	6.74±5
  	5	5.38±3	5.45±6	13.86±2
  	10	11.97±10	14.30±6	27.25±5
  	50	21.71±7	43.59±9	50.47±4
  	100	46.58±7	52.38±5	63.43±1
  Matrine	1	6.93±2	—	—
  	5	8.21±1	—	—
  	10	15.14±4	—	—
  	50	16.72±4	—	—
  	100	27.48±2	—	—
  VCR	1	3.13±2	—	—
  	5	14.19±8	—	—
  	10	24.36±3	—	—
  	50	25.45±2	—	—
  	100	32.61±4	—	—
  ARA	1	5.80±2	—	—
  	5	9.71±6	—	—
  	10	18.67±4	—	—
  	50	28.84±4	—	—
  	100	31.85±4	—	—
  aValues represent the means of three separate experiments with SD less than 10%.

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