Design and Synthesis of Novel Butenolide Derivatives and Inducing Apoptosis in Gastric Cancer Cells

1. Introduction

The development of novel therapeutic drugs from medicinal plants has been one of the most important resource to drug development. Natural butenolides, such as aspulvione, ^[1] uncinine^[2] and so on including a similar structures BH3I-1^[3] and obaloclax^[4](Figure 1), have a wide range of biological activities, such as antibacterial, antiviral, anticancer, antimalarial, antibiotic and phospholipase A2 inhibition etc.^[5]

Figure 1

Figure 1. Structures of BH3I-1, obaloclax and selected natural butenolides

下载: 全尺寸图片幻灯片

In connection with previous studies concerning the synthesis and bioactivities of butenolide, ^{[6, 7]} we are interested in discovery novel anticancer compounds based on the natural product uncinine. Gastric cancer (GC) is a highly aggressive malignant tumor, ^[8] especially in East Asia, such as China, South Korea and Japan. Its high mortality rate prompts the urgent need for novel therapeutic agents. Focusing on GC, our group designed and synthesized novel butenolides and assayed their anti tumor activities. Most of synthetic compounds showed significant anti proliferation activity against gastric cancer cells. Among of them, compound 9l was discovered with good and selective inhibition of proliferation on cancer cells by triggering apoptotic cell death. At the molecular level, compound 9l induced apoptosis in gastric cancer cells through dependent caspase activation.

2. Results and discussion

2.1 Chemistry

Uncinine (6) as a natural bioactive butenolide, could be synthesized according to literature procedures that is the only one work about its synthesis so far.^[2] However the method is deficient in low yield and many steps. In order to get more uncinine (6) and its analogues to research, a new route for preparation of Uncinine was designed and carried out via 4 steps with 30.4% yield shown in Scheme 1. Bromolactone 3 was obtained from the commercially available tulipalin (1) by bromination reaction followed by regioselective elimination. The bromination reaction was done in the present of Me₃PhN⁺Br₃^－ (1.2 equiv.) at room temperature. The crude product 2 was treated with 5 equiv. of LiBr/Li₂CO₃ in the present of TBAB in CH₃CN. By the above two steps reaction, compound 3 was obtained with a yield of 76%. Compounds 4 and 5 were isolated as a mixture after completion of a nucleophilic substitution reaction of bromolactone 3 and ethyl 4-aminobutanoate hydrochloride. The mixture was stirred in acetone and methanol for 72 h at room temperature and purified by silicon chromatography to give Uncinine (6) with yield of 40% as a white solid.

Scheme 1

Scheme 1. Synthesis of compound 6

Reagents and conditions: (a) Me₃PhNBr₃, dioxane, r.t., 10 h; (b) LiBr/Li₂CO₃, TBAB, CH₃CN, r.t., 20 h; (c) ethyl 4-aminobutanoate hydrochloride, Et₃N, dry THF, 0 ℃~r.t., 26 h; (d) acetone, CH₃OH, r.t., 72 h

下载: 全尺寸图片幻灯片

Based on the intermediate 3, 3-(alkylamino)methyl)-5-(propan-2-ylidene)furan-2(5H)-ones (8a~8g) were synthesized to investigate the structure activity relationship (SAR) of the pyrolidin-2-ones moiety in the Uncinine (6) by keeping the lactone and isopropyl group (Scheme 2). Different alkenes were introduced to C-5 of 7a to replace the isopropanylidene group to prepare 5-benzylidene-3-(morpholinomethyl)furan-2(5H)-ones (9a~9o) and compound 9p in Scheme 3. The configurations of double bonds on C-5 of compounds 9a~9o were cis-double bonds according to NMR and the previous works.^{[9, 10]}

Scheme 2

Scheme 2. Synthesis of compound 8

Reagents and conditions: (a) various substituted amine, K₂CO₃/ Et₃N, THF, r.t., 2 h; (b) Acetone, CH₃OH, reflux, 1 h

下载: 全尺寸图片幻灯片

Scheme 3

Scheme 3. Synthesis of compound 9

Reagents and conditions: (a) 5-methylfuran-2-carbaldehyde, ethylenediamine, CH₃OH, reflux; (b) cyclohexanone, ethylenediamine, CH₃OH, reflux; (c) substitued benzaldehyde, ethylenediamine, CH₃OH, reflux

下载: 全尺寸图片幻灯片

Among of synthetic compounds, compound 9l showed potent anticancer effect in gastric cancer MGC803 and less cytotoxicity in normal gastric epithelial cell line (GES1). Compound 9l has a 4-isopropyl phenyl group in its structure. So two another compounds with 4-isopropyl phenyl methyl indene indol-2-ones were synthesized (Scheme 4).

Scheme 4

Scheme 4. Synthesis of compounds 11 and 12

Reagents and conditions: (a) 4-butylbenzaldehyde, ethylenediamine, CH₃OH, reflux; (b) 4-bromomethyl-tetrahy-dro-2H-pyran, DMF, NaH, 0 ℃~r.t.

下载: 全尺寸图片幻灯片

2.2 Biology

2.2.1 Evaluation of antiproliferation on MGC803

Anti-proliferation of compounds 8a~8g is evaluated in vitro on MGC803. The results are reported in Table 1. Just compound 8a shows inhibition on the tested cancer cells with IC₅₀ of 42.7 μmol/L. Uncinine (6) and compounds 8b~8g show no significant anti-proliferative activity on MGC803. Based on the above results, compounds 9a~9p are synthesized and assayed to optimize the structure of compound 8a. The anti-proliferation of compounds 9a~9p are listed in Table 1, and most of them give better anti-proliferation than compound 8a. Compounds 9a1~9a3 with furan have no anti-proliferation on MGC803 cell. Among them compound 9l is the best one with IC₅₀ of 2.9 μmol/L. Compounds 11 and 12 with similar substituted phenyl group can inhibit proliferation of MGC803 with IC₅₀ of 7.5 and 12.3 μmol/L which are less than compound 9l. Therefore compound 9l is chosen to evaluate further.

Table 1

Table 1. Antiproliferative activity of synthetic compounds on MGC803

下载: 导出CSV

Compd.	IC₅₀^a/(μmol•L^－1)
8a	42.7±3.1
8b	> 128
8c	> 128
8d	> 128
8e	> 128
8f	> 128
8g	119.8±3.5
5-Fu^b	7.1±1.3
9a1	> 50
9a2	44.4±1.6
9a3	> 128
9b	23.1±2.9
9c	18.6±2.5
9d	10.9±1.7
9e	23.2±2.9
Uncinine	> 128
9f	28.1±3.1
9g	14.7±2.1
9h	13.3±2.1
9i	26.3±2.7
9j	19.3±2.2
9k	20.4±1.2
9l	2.9±0.1
9m	5.6±0.8
9n	17.1±1.2
9o	26.7±1.4
9p	23.0±1.4
11	7.5±0.9
12	12.4±1.1
^a MTT assay: All data are mean values from triplicate experiments. ^b Positive control: fluorouracil.

The SAR of synthetic compounds for anti-proliferation on MGC803 suggests that morpholine aminomethyl group on carbon-3 of butenolide is beneficial to the bioactivity (such as compound 8a). As for the substitution on carbon-5 butenolide, phenyl groups on the structures of compounds 9b~9q, play more important role than the furan heterocyclic groups on the structures of compounds 9a1~9a3. Among the substituted groups on the phenyl group for compounds 9b~9q, hydrophilic groups on carbon-4 of phenyl are better than others, such as compounds 9l and 9m.

2.2.2 Caspase-dependent apoptosis was induced by compound 9l in MGC803 cells

Compound 9l was proved hypotoxicity in normal human gastric epithelial cell line (GES1) and showed a selectivity to gastric cancer cells (MGC803, SGC7901 and HGC27) comparing with GES1 cells (Figure 2A). Based on the above results, compound 9l was prioritized to perform further experiment for evaluating its anti-cancer potential in gastric cancer.

Figure 2

Figure 2. Apoptosis induced by compound 9l in MGC803 cells

(A) The cytotoxicity of compound 9l by MTT assay. Cells were treated with indicated concentrations for 24 h. **P < 0.01 vs. untreated group. (B) MGC803 cells were incubated with compound 9l (20 μmol/L) for 24 h, and then stained with DAPI. The stained nuclei were observed under a fluorescent microscope (magnification, ×200). (C) A dose-dependent induction of apoptosis by compound 9l was demonstrated in MGC803 cells by flow cytometric analysis of annexin V/PI stain assay. (D) The effects of compound 9l-induced (20 μmol/L) protein changes were detected by Western blot analysis for 24 h in MGC803 cells. (E) The effects of caspase inhibitor Z-VAD-fmk (100 μmol/L) on compound 9l-induced (20 μmol/L) apoptosis of MGC803 cells were shown through DAPI solution (magnification, ×200). (F) Annexin V/PI stained cells showed the effects of caspase inhibitor Z-VAD-fmk (100 μmol/L) on compound 9l-induced (20 μmol/Lol/L) apoptosis of MGC803 cells

下载: 全尺寸图片幻灯片

Morphological changes of MGC803 cells were determined by use of 4, 6-diamidino-2-phenylindole (DAPI) staining, as shown in Figure 2B. An increasing number of cells with chromatin condensation were observed in compound 9l-treated MGC803 cells in a dose-dependent manner. Chromatin condensation was not observed in the untreated control cells, suggesting that compound 9l-induced cell death is related to apoptosis. We evaluated apoptotic induction in MGC803 by compound 9l with annexin V and propidium iodide (PI) staining. It was found that the number of annexin V-positive cells showed a dose-dependent increase from 8.6% to 49.5% in MGC803 cells, whereas treatment of control cells only resulted in a 5.2% increase in annexin V-positive stained cells (Figure 2C). These data suggested that apoptosis was induced by compound 9l in MGC803 cells. The apoptosis-related proteins were deteted by wester blot analysis in cells treated with compound 9l. Caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP-1) proteins were down regulated, whereas cleaved caspase-9 protein was up regulated by compound 9l compared with control MGC803 cells (Figure 2D). To further evaluate the significance of caspase activation in compound 9l-induced apoptosis, z-VAD-fmk (100 μmol/L), an irreversible pan-caspase inhibitor, was used to pretreat MGC803 cells for 1 h, followed by treatment with 20 μmol/L compound 9l for 24 h. Morphological changes of MGC803 cells were then determined by DAPI staining, as shown in Figure 2E. A significant number of cells with chromatin condensation were observed in cells treated with compound 9l, and these features were almost invisible in cells pretreated with z-VAD-fmk (100 μmol/L) followed by treatment with 20 μmol/L compound 9l. In Figure 2F, compound 9l-induced apoptosis was reduced from 47.8% to 17.2% by pretreatment with z-VAD-fmk (100 μmol/L), according to flow cytometry analysis.

The activity of caspases is the molecular hallmark of apoptosis and is a controlling factor in the apoptotic cascade.^{[11, 12]} Thus, caspase activity is considered in compound 9l-induced apoptosis. Caspase-9 is the initiating enzyme of the apoptotic pathway, and caspase-3 is the key executioner caspase and is activated through cleavage of caspase-9.^[13] The activity of these factors can be indirectly measured at the protein expression by Western blot analysis. Fulda et al.^[14] found that resveratrol-induced apoptosis was triggered by the activation of caspases and reversed by pretreatment with the caspase inhibitor Z-VAD-fmk. In this study, the results showed that the expressions of caspase-3 and caspase-9 were decreased by increasing compound 9l concentrations. Compound 9l induced apoptosis in gastric cancer cells through dependent caspase activation.

3. Conclusions

In summary, based on the active natural products butenolide, an effective anti cancer agent compound 9l in gastric cancer was found. The results suggested that apoptosis was involved in compound 9l-induced gastric cancer cell death. The potential mechanisms of compound 9l have resulted from the induction of apoptosis as evidenced by partially dependent caspase activation. It deserves further investigation into it utility for prevention and treatment of gastric cancer.

4. Experimental

4.1 Chemistry

All solvents and reagents were purchased from commercial sources, generally with no need of drying and/or purification unless necessary. Melting points were determined on a Beijing Fukai X-5 apparatus and were uncorrected. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker DPX-400 spectrometer at 400 and 100 MHz with TMS as internal standard. HRMS was recorded on a Q-Tof Micro HRMS of waters.

4.1.1 Synthesis of 3-(bromomethyl)furan-2(5H)-one (3)

Thimethylphenylammonium tribromide (Me₃PhN⁺Br₃^－) (13.8 g, 36.7 mmol) was added to the solution of α-methylene-γ-butene lactone (3 g, 30.6 mmol) in dioxane (90 mL). The reaction mixture was stirred for 10 h at room temperature. The reaction was monitored by TLC until all α-methylene-γ-butene lactone was consumed. Upon completion, the reaction mixture was diluted with 250 mL of diethyl ether and filtered. The filtrate was concentrated under reduced pressure. Without purification, the above crude product, Li₂CO₃ (11.1 g, 150 mmol), LiBr (15.6 g, 150 mmol) and tetrabutylammonium bromide (0.966 g, 3 mmol) were suspended in CH₃CN (90 mL). The mixture was stirred at 85 ℃ for 20 h. The reaction was monitored by TLC. After filtering and removing the spare solvent under vacuum, the residue was dissolved in 300 mL of diethyl ether and washed with saturated salt water (150 mL). The organic phase was dried over anhydrous Na₂SO₄ and then concentrated. The concentrate was purified by column chromatography (petroleum ether/acetic ether, V:V＝5:1) to afford the product 3.^[15] Yellow oil, yield 4.09 g (76%). ¹H NMR (400 MHz, CDCl₃) δ: 7.55 (s, 1H), 4.89 (d, J＝1.7 Hz, 2H), 4.13 (d, J＝1.4 Hz, 2H).

4.1.2 Synthesis of compounds 4 and 5

Compound 3 (0.5 g, 2.82 mmol) in dry tetrahydrofuran (THF) (5 mL) was added dropwise in four portions to a solution of ethyl-γ-aminobutyrate hydrochloride (0.72 g, 7.23 mmol) and triethylamine (0.86 g, 8.74 mmol) in dry THF (10 mL) at 0 ℃ over 2 h. The reaction mixture was then warmed to room temperature, stirred for another 24 h. After the reaction completion, the solid was filtered off, and the solvent was removed on a rotary evaporator. The crude product was mixture of compounds 4 and 5, which were used directly to the next step.

4.1.3 Synthesis of uncinine (6)

The mixture of 4, 5 (0.576 g) and acetone (5 mL) was added to a solution of methyl alcohol (10 mL), and the reaction mixture was stirred at room temperature for 72 hours. The reaction system was filtered and the solvent was evaporated in vacuo. The reaction mixture was then diluted with diethyl ether (30 mL×3), and washed with H₂O (50 mL). The organic phase was dried over anhydrous Na₂SO₄, and the solvent was removed. The crude product was purified by column chromatography on silica gel [V(petroleum ether):V(acetone)＝6:1] to afford the product 6^[2] as a white crystalline substance (0.249 g, white solid). Yield 40%. ¹H NMR (400MHz, CDCl₃) δ: 7.49 (s, 1H), 4.19 (s, 2H), 3.48 (m, 2H), 2.40 (m, 2H), 2.08~2.01 (m, 2H), 2.00 (s, 3H), 1.92 (s, 3H).

4.1.4 Synthesis of compounds 7a~7e

Nitrogen compounds (3 mmol) were diluted with 1 mL of THF and then slowly dropped into the THF solution (1 mL) of compound 3 (400 mg, 2.27 mmol) with a lot of white solid precipitated. The reaction mixture was stirred at room temperature. After completion of the reaction, the reaction system was filtered, concentrated under vacuum and crystallized from ethyl acetate to yield the pure products 7a~7e, which directly used for the next reaction.

4.1.5 General procedure for the synthesis of uncinine analogues 8a~8e

The corresponding compounds 7a~7e were dissolved in methanol and then refluxed with appropriate acetone at 65 ℃ for 1 h. The reaction was monitored by TLC. After the reaction completed, the reaction mixture was evaporated in vacuo and purified by column chromatography to afford the pure product 8a~8e.

3-(Morpholinomethyl)-5-(propan-2-ylidene)furan-2(5H)-one (8a): Light yellow solid, yield 60%. ¹H NMR (400 MHz, CDCl₃) δ: 6.10 (s, 1H), 3.76~3.67 (m, 4H), 3.44 (s, 2H), 2.55~2.46 (m, 4H), 2.04 (s, 3H), 2.05 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ: 144.8, 134.6, 129.9, 127.6, 122.5, 113.4, 39.9, 29.7, 20.4, 18.7. HRMS (ESI) calcd for C₁₂H₁₈NO₃ [M+H]⁺ 224.1281, found 224.1292.

3-(Piperidin-1-ylmethyl)-5-(propan-2-ylidene)furan-2(5H)-one (8b): Light yellow solid, yield 50%. ¹H NMR (400 MHz, CDCl₃) δ: 7.54 (s, 1H), 3.32 (s, 2H), 2.48 (brs, 4H), 2.02 (s, 3H), 1.96 (s, 3H), 1.63 (brs, 4H), 1.50~1.42 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.8, 144.9, 136.1, 128.6, 121.9, 54.5, 53.1, 29.7, 25.8, 24.1, 18.6. HRMS (ESI) calcd for C₁₃H₂₀NO₂ [M+H]⁺ 222.1489, found 222.1483.

3-((4-Methylpiperazin-1-yl)methyl)-5-(propan-2-ylidene)furan-2(5H)-one (8c): Light yellow solid, yield 63%. ¹H NMR (400 MHz, CDCl₃) δ: 7.48 (s, 1H), 3.35 (s, 2H), 2.57 (brs, 8H), 2.32 (s, 3H), 2.01 (s, 3H), 1.95 (s, 3H). HRMS (ESI) calcd for C₁₃H₂₁N₂O₂ [M+H]⁺ 237.1598, found 237.1595.

5-(Propan-2-ylidene)-3-((p-tolylamino)methyl)furan-2(5H)-one (8d): White solid, yield 50%. ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (s, 1H), 6.81 (d, J＝8.9 Hz, 2H), 6.62 (d, J＝8.9 Hz, 2H), 4.11 (s, 2H), 2.43 (s, 3H), 2.01 (s, 3H), 1.91 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.1, 152.7, 144.7, 141.2, 134.6, 129.9, 122.5, 114.9, 114.7, 55.7, 40.5, 29.7, 18.7. HRMS (ESI) calcd for C₁₅H₁₈NO₂ [M+H]⁺ 244.1332, found 244.1314.

3-(((4-Methoxyphenyl)amino)methyl)-5-(propan-2-ylidene)furan-2(5H)-one (8e): Light yellow solid, yield 55%. ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (s, 1H), 7.09 (d, J＝8.9 Hz, 2H), 6.72 (d, J＝8.9 Hz, 2H), 4.11 (s, 2H), 3.77 (s, 3H), 2.01 (s, 3H), 1.91 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.5, 144.8, 136.4, 129.6, 127.7, 122.8, 122.7, 114.9, 114.7, 66.7, 52.7, 29.4, 18.7. HRMS (ESI) calcd for C₁₅H₁₈NO₃ [M+H]⁺ 260.1281, found 260.1273.

3-(((4-Methylbenzyl)amino)methyl)-5-(propan-2-ylidene)furan-2(5H)-one (8f): White solid, yield 70%. ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (s, 1H), 7.02(d, J＝8.1 Hz, 2H), 6.57 (d, J＝8.4 Hz, 2H), 4.13 (s, 2H), 3.79 (s, 2H), 2.26 (s, 3H), 2.01 (s, 3H), 1.90 (s, 3H). HRMS (ESI) calcd for for C₁₆H₂₀NO₂ [M+H]⁺ 258.1489, found 258.1476.

3-(((4-Methoxybenzyl)amino)methyl)-5-(propan-2-ylidene)furan-2(5H)-one (8g): Yellow solid, yield 75%. ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (s, 1H), 6.81 (d, J＝8.9 Hz, 2H), 6.62 (d, J＝8.9 Hz, 2H), 4.28 (s, 2H), 4.11 (s, 2H), 3.76 (s, 3H), 2.01 (s, 3H), 1.91 (s, 3H). HRMS (ESI) calcd for C₁₆H₂₀NO₃ [M+H]⁺ 274.1438, found 274.1467.

4.1.6 General procedure for the synthesis of compounds 9a~9p

In a round-bottomed flask with a magneton, compound 7a (183 mg, 1 mmol), substituted aldehyde or ketone (1 mmol), ethanediamine (6 mg, 0.1 mmol) and methanol (0.4 mL) were added. The reaction system was stirred at room temperature for 36 h. The progress was detected by thin-layer chromatography (TLC). Upon completion, a lot of solid were filtered, dried and crystallized from methanol.

(Z)-3-(Morpholinomethyl)-5-(thiophen-2-ylmethylene)-furan-2(5H)-one (9a1): Yellow soild, yield 11%. ¹H NMR (400 MHz, CDCl₃) δ: 7.49 (d, J＝5.1 Hz, 1H), 7.37 (d, J＝3.7 Hz, 1H), 7.33 (s, 1H), 7.07 (dd, J＝5.0, 3.8 Hz, 1H), 6.28 (s, 1H), 3.77~3.69 (m, 4H), 3.36 (s, 2H), 2.56~2.48 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 169.6, 145.6, 140.0, 136.2, 131.0, 130.4, 128.7, 127.8, 106.8, 66.9, 53.6, 52.9. HRMS (ESI) calcd for C₁₄H₁₆NO₃S [M+H]⁺ 278.0845, found 278.0856.

(Z)-3-(Morpholinomethyl)-5-(thiophen-3-ylmethylene)-furan-2(5H)-one (9a2): Yellow soild. ¹H NMR (400 MHz, CDCl₃) δ: 7.72 (t, J＝3.9 Hz, 1H), 7.52 (dd, J＝11.2, 7.0 Hz, 1H), 7.34 (dd, J＝4.3, 2.1 Hz, 1H), 7.33 (s, 1H), 6.09 (s, 1H), 3.77~3.70 (overlap, 4H), 3.36 (s, 2H), 2.52 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.0, 146.4, 140.8, 134.4, 128.8, 128.5, 128.2, 126.2, 107.4, 66.9, 53.6, 52.8. HRMS (ESI) calcd for C₁₄H₁₆NO₃S [M+H]⁺ 278.0845, found 278.0857.

(Z)-5-((5-Methylfuran-2-yl)methylene)-3-(morpholinomethyl)furan-2(5H)-one (9a3): Brown solid, yield 75%. m.p. 114~115 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.05 (s, 1H), 6.46 (d, J＝3.2 Hz, 1H), 6.34 (s, 1H), 6.11 (d, J＝3.1 Hz, 1H), 3.79~3.76 (m, 4H), 3.43 (s, 2H), 2.61~2.56 (m, 4H), 2.42 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ: 169.6, 155.3, 147.9, 146.1, 138.4, 116.6, 108.9, 102.6, 66.9, 53.6, 52.9, 14.8. HRMS (ESI) calcd for C₁₅H₁₈NO₄ [M+H]⁺ 276.1230, found 276.1238.

(Z)-5-Benzylidene-3-(morpholinomethyl)furan-2(5H)-one (9b): White solid, yield 30%. m.p. 145~146 ℃; ¹H NMR (400 MHz, CDCl₃) 7.79 (s, 1H), 7.35~7.38 (overlap, 5H), 6.02 (s, 1H), 3.80~3.73 (m, 4H), 3.40 (s, 2H), 2.61~2.52 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.2, 147.3, 141.4, 133.0, 130.4, 129.1, 128.9, 128.9, 128.7, 113.5, 66.9, 53.6, 52.8. HRMS (ESI) calcd for C₁₆H₁₈NO₃ [M+H]⁺ 272.1281, found 272.1273.

(Z)-5-(2-Chlorobenzylidene)-3-(morpholinomethyl)-furan-2(5H)-one (9c): White solid, yield 35%. m.p. 147~148 ℃; ¹H NMR (400 MHz, CDCl3) δ: 7.34~7.36 (overlap, 5H), 6.51 (s, 1H), 3.87~3.68 (m, 4H), 3.40 (s, 2H), 2.66~2.45 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 169.9, 148.4, 141.5, 134.3, 132.0, 130.9, 130.0, 129.6, 127.3, 108.5, 66.9, 53.6, 52.8. HRMS (ESI) calcd for C₁₆H₁₇ClNO₃ [M+H]⁺ 306.0891/308.0862, found 306.0886/308.0851.

(Z)-5-(3-Chlorobenzylidene)-3-(morpholinomethyl)-furan-2(5H)-one (9d): White solid, yield 78%. m.p. 114~115 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.76 (s, 1H), 7.67 (dd, J＝7.0, 1.6 Hz, 1H), 7.40~7.31 (m, 3H), 5.95 (s, 1H), 3.81~3.73 (m, 4H), 3.40 (s, 2H), 2.61~2.53 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 169.8, 148.1, 141.1, 134.7, 130.0, 129.6, 129.0, 128.5, 111.7, 66.9, 53.6, 52.8. HRMS (ESI) calcd for C₁₆H₁₇ClNO₃ [M+H]⁺ 306.0891/308.0862, found 306.0886/ 308.0851.

(Z)-5-(4-Chlorobenzylidene)-3-(morpholinomethyl)-furan-2(5H)-one (9e): White solid, yield 60%. m.p. 113~114 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.73 (d, J＝8.6 Hz, 2H), 7.38 (d, J＝8.6 Hz, 2H), 7.29 (s, 1H), 5.98 (s, 1H), 3.82~3.65 (m, 4H), 3.40 (s, 2H), 2.56 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 169.9, 147.6, 141.2, 135.1, 131.6, 129.1, 112.0, 66.9, 61.0, 56.3, 54.0, 52.8. HRMS (ESI) calcd for C₁₆H₁₇ClNO₃ [M+H]⁺ 306.0891/308.0862, found 306.0886/308.0851.

(Z)-5-(2-Methoxybenzylidene)-3-(morpholinomethyl)-furan-2(5H)-one (9f): Light yellow solid, yield 40%. m.p. 98~99 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.73 (d, J＝8.6 Hz, 2H), 7.38 (overlap, 3H), 5.97 (s, 1H), 3.83 (s, 3H), 3.81~3.73 (m, 4H), 3.40 (s, 2H), 2.64~2.51 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.1, 159.8, 147.5, 141.3, 134.2, 129.8, 128.8, 123.3, 115.3, 113.3, 66.9, 55.4, 53.6, 52.8. HRMS (ESI) calcd for C₁₇H₂₀NO₄ [M+H]⁺ 302.1387, found 302.1373.

(Z)-5-(3-Methoxybenzylidene)-3-(morpholinomethyl)-furan-2(5H)-one (9g): Yellow solid, yield 67%. m.p. 96~97 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.32~7.36 (overlap, 4H), 6.91 (d, J＝8.1 Hz, 1H), 5.99 (s, 1H), 3.87 (s, 3H), 3.81~3.71 (m, 4H), 3.39 (s, 2H), 2.61~2.52 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.1, 159.8, 147.5, 141.3, 134.2, 129.8, 128.8, 123.3, 115.3, 113.3, 66.9, 55.4, 53.6, 52.8. HRMS (ESI) calcd for C₁₇H₂₀NO₄ [M+H]⁺ 302.1387, found 302.1373.

(Z)-5-(4-methoxybenzylidene)-3-(morpholinomethyl)-furan-2(5H)-one (9h): Light yellow solid, yield 60%. m.p. 95~96 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.76(d, J＝8.6 Hz, 2H), 7.36 (d, J＝8.6 Hz, 2H), 6.93 (s, 1H), 5.97 (s, 1H), 3.83 (s, 3H), 3.82~3.65 (m, 4H), 3.40 (s, 2H), 2.56 (brs, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 170.6, 160.4, 145.4, 141.6, 132.3, 127.1, 125.4, 113.6, 66.9, 61.0, 56.3, 53.6, 52.8. HRMS (ESI) calcd for C₁₇H₂₀NO₄ [M+H]⁺ 302.1387, found 302.1373.

(Z)-3-(morpholinomethyl)-5-(3, 4, 5-trimethoxybenzylidene)furan-2(5H)-one (9i): Yellow solid, yield 50%. m.p. 120~121 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.32 (s, 1H), 7.02 (s, 2H), 5.91 (s, 1H), 3.90 (d, J＝6.7 Hz, 9H), 3.71~3.74 (overlap, 4H), 3.37 (s, 2H), 2.57~2.48 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.1, 153.3, 146.8, 141.3, 139.4, 128.5, 128.1, 113.5, 107.9, 66.9, 61.0, 56.3, 53.6, 52.8. HRMS (ESI) calcd for C₁₉H₂₄NO₆ [M+H]⁺ 362.1598, found 362.1587.

(Z)-5-(2, 3-dimethoxybenzylidene)-3-(morpholinomethyl)furan-2(5H)-one (9j): White solid, yield 46%. m.p. 108~109 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.84 (d, J＝8.1 Hz, 1H), 7.42 (s, 1H), 7.12 (t, J＝8.1 Hz, 1H), 6.93 (d, J＝7.0 Hz, 1H), 6.52 (s, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.79~3.74 (m, 4H), 3.40 (s, 2H), 2.60~2.53 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.3, 152.6, 147.9, 141.8, 128.5, 127.2, 123.1, 113.3, 107.2, 61.4, 55.8, 53.6, 52.8, 29.7. HRMS (ESI) calcd for C₁₈H₂₂NO₅ [M+H]⁺ 332.1492, found 332.1490.

(Z)-5-(4-fluorobenzylidene)-3-(morpholinomethyl)-furan-2(5H)-one (9k): Light yellow solid, yield 77%. m.p. 116~117 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.79 (m, 2H), 7.37 (s, 1H), 7.11 (m, 2H), 5.98 (s, 1H), 3.81~3.70 (m, 4H), 3.39 (s, 2H), 2.61~2.50 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.1, 164.2, 161.7, 147.0, 141.2, 132.5, 129.3, 128.6, 116.1, 115.9, 112.1, 66.9, 53.6, 52.8. HRMS (ESI) calcd for C₁₆H₁₇FNO₃ [M+H]⁺ 290.1187, found 290.1171.

(Z)-5-(4-(tert-Butyl)benzylidene)-3-(morpholinomethyl)-furan-2(5H)-one (9l): White solid, yield 55%. m.p. 145~146 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.81~7.67 (d, J＝8.5 Hz, 2H), 7.44 (d, J＝8.5 Hz, 2H), 7.29 (s, 1H), 6.09 (s, 1H), 3.92~3.78 (m, 4H), 3.52 (s, 2H), 2.72 (brs, 4H), 1.35 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.2, 150.5, 147.2, 137.5, 130.7, 129.9, 128.0, 126.3, 66.9, 53.6, 52.8, 34.1, 29.7, 23.8. HRMS (ESI) calcd for C₂₀H₂₆NO₃ [M+H]⁺ 328.1907, found 328.1918.

(Z)-5-(4-Isopropylbenzylidene)-3-(morpholinomethyl)-furan-2(5H)-one (9m): Light yellow solid, yield 47%. m.p. 98~99 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.73 (d, J＝8.3 Hz, 2H), 7.37 (s, 1H), 7.28 (d, J＝8.4 Hz, 2H), 6.01 (s, 1H), 3.75 (overlap, 4H), 3.39 (s, 2H), 3.03~2.85 (m, 1H), 2.54 (overlap, 4H), 1.26 (d, J＝8.3 Hz, 6H); .¹³C NMR (101 MHz, CDCl₃) δ: 170.4, 150.5, 146.8, 141.5, 130.7, 128.0, 127.0, 113.7, 53.6, 52.8, 34.1, 29.7, 23.8. HRMS (ESI) calcd for C₁₉H₂₄NO₃ [M+H]⁺ 314.1751, found 314.1755.

(Z)-5-(4-(4-methylpiperazin-1-yl)benzylidene)-3-(morpholinomethyl)furan-2(5H)-one (9n): Yellow soild, yield 16%. ¹H NMR (400 MHz, CDCl₃) δ: 7.73~7.67 (d, J＝9.0 Hz, 2H), 7.31 (s, 1H), 6.88 (d, J＝9.0 Hz, 2H), 5.93 (s, 1H), 3.77~3.70 (overlap, J＝4.1 Hz, 4H), 3.36 (s, 2H), 3.31~3.34 (overlap, J＝11.8, 6.7 Hz, 4H), 2.57 (brd, 6.7 Hz, 4H), 2.52 (brd, 4H), 2.47 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ: 151.3, 141.5, 132.2, 130.4, 126.1, 123.7, 115.4, 114.9, 66.9, 54.8, 53.6, 53.6, 48.0, 47..7, 46.1. HRMS (ESI) calcd for C₁₉H₂₄NO₃ [M+H]⁺ 370.2125, found 370.2134.

(Z)-5-(3-Fluoro-4-(4-methylpiperazin-1-yl)benzylidene)-3-(morpholinomethyl)furan-2(5H)-one (9o): Yellow soild, yield 19%. ¹H NMR (400 MHz, CDCl₃) δ: 7.57 (dd, J＝14.4, 1.9 Hz, 1H), 7.39 (dd, J＝8.5, 1.7 Hz, 1H), 7.31 (s, 1H), 6.92 (t, J＝20.5, 8.9 Hz, 1H), 5.90 (s, 1H), 3.76~3.70 (overlap, 4H), 3.37 (s, 2H), 3.24~3.15 (overlap, 4H), 2.64~2.57 (m, 4H), 2.55~2.49 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 170.2, 146.5, 141.2, 136.2, 133.4, 127.4, 118.4, 112.5, 66.9, 55.0, 53.7, 53.6, 52.8, 49.9, 46.1. HRMS (ESI) calcd for C₂₁H₂₇FN₃O₃ [M+H]⁺ 388.2031, found 388.2035.

5-Cyclohexylidene-3-(morpholinomethyl)furan-2(5H)-one (9p): Light yellow solid, yield 42%. m.p. 109~110 ℃; ¹H NMR (400 MHz, DMSO) δ: 7.96 (s, 1H), 3.61~3.55 (overlap, 4H), 3.22 (s, 2H), 2.41 (overlap, 8H), 1.59 (s, 6H); ¹³C NMR (101 MHz, DMSO) δ: 170.3, 142.3, 137.8, 130.4, 127.5, 66.6, 53.5, 52.5, 28.7, 28.1, 27.3, 26.1. HRMS (ESI) calcd for C₁₅H₂₂NO₃ [M+H]⁺ 264.1594, found 264.1591.

4.1.7 Synthesis of (Z)-3-(4-(tert-butyl)benzylidene)- indolin-2-one (11)

To a solution of indolin-2-one (200 mg, 1.5 mmol) in CH₃OH (1 mL) was added 4-(tert-butyl)benzaldehyde (365.6 mg, 2.25 mmol), and the mixture was stirred with ethylenediamine(10 mg) for 10 h at room temperature. Then, the system was concentrated to remove CH₃OH and purified by silica gel column chromatography (ethyl acetate/petroleum ether, V:V＝1:7) to give 3-(4-(tert-butyl)benzylidene)indolin-2-one (11), 68 mg, yield 16% (yellow soild). ¹H NMR (400 MHz, CDCl₃) δ: 10.59 (s, 1H), 7.66 (overlap, 3H), 7.61 (s, 1H), 7.55 (d, J＝8.3 Hz, 2H), 7.24 (d, J＝7.8 Hz, 1H), 6.88 (t, J＝7.9 Hz, 2H), 1.33 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ: 174.0, 158.0, 148.1, 141.0, 136.8, 135.2, 134.6, 132.1, 130.8, 127.6, 126.3, 126.2, 115.3, 39.9, 36. HRMS (ESI) calcd for C₁₉H₂₀NO [M+H]⁺ 278.1539, found 278.1544.

4.1.8 Synthesis of (Z)-3-(4-(tert-butyl)benzylidene)-1- ((tetra-hydro-2H-pyran-4-yl)methyl)indolin-2-one (12)

Under ice bath conditions, to a solution of 3-(4-(tert- butyl)benzylidene)indolin-2-one (25 mg, 0.09 mmol) in DMF (1 mL) was added NaH (7.22 mg, 0.3 mmol), and the mixture was stirred for 30 min. Then, 4-(bromomethyl)-tetrahydro-2H-pyran (24 μL, 0.16 mmol) was added to the system. After reacting for 9 h, the mixture was diluted into water (10 mL) and extracted with ethyl acetate (10 mL×4). The combined organic layers were dried over MgSO₄, concentrated, and purified by silica gel column chromatography [V(acetone):V(petroleum ether)＝1:5] to give 3-(4-(tert-butyl)-benzylidene)-1-(tetrahydro-2H-pyran-4-yl)-indolin-2-one (12), 14 mg, yield 44% (yellow oil). ¹H NMR (400 MHz, CDCl₃) δ: 7.43 (d, J＝7.9 Hz, 2H), 7.38 (d, J＝8.2 Hz, 2H), 7.25 (t, J＝7.2 Hz, 1H), 6.88 (d, J＝7.8 Hz, 1H), 6.78 (t, J＝7.8 Hz, 1H), 6.55 (d, J＝7.5 Hz, 1H), 4.80 (s, 1H), 3.99 (d, J＝10.2 Hz, 2H), 3.67 (d, J＝7.2 Hz, 2H), 3.35 (t, J＝11.5 Hz, 2H), 2.12~2.08 (m, 1H), 1.63 (d, J＝12.8 Hz, 2H), 1.53~1.44 (m, 2H), 1.33 (s, 9H). HRMS (ESI) calcd for C₂₅H₃₀NO₂ [M+H]⁺ 376.2271, found 376.2278.

4.2 Biological evalution

4.2.1 Material

Fetal bovine serum (FBS), Roswell Park Memorial Institute 1640 (RPMI-1640) medium, MEM, DMEM, and penicillin-streptomycin were purchased from HyClone (Victoria, Australia). MTT, NAC, and Z-VAD-fmk were purchased from Selleck Chemicals (Houston, TX). The FITC/Annexin V Apoptosis Detection Kit was purchased from BestBio (Shanghai, China). DCFH-DA and diaminobenzidine (DAB) were purchased from Beyotime Biotechnology (Shanghai, China). Antibodies specific for actin, caspase-3, and PARP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for caspase-9was purchased from Cell Signaling Technology (Danvers, MA). The antibody specific for survivin, cleaved caspase-3, and cleaved caspase-9 were purchased from Abcam (Cambridge, MA). Peroxidase labeled anti-rabbit, anti-mouse, and anti-goat polyclonal immunoglobulins were purchased from Bioss (Shanghai, China). The enhanced chemiluminescence (ECL) kit was purchased from Thermo Fisher Scientific (Waltham, MA).

All the tested compounds were dissolved in dimethyl sulfoxide (DMSO) to 10 mmol/L stock solutions and stored at －20 ℃. For each experiment, the stock solutions were diluted in the culture medium to obtain the desired concentration. The final DMSO content in cell culture was ≤0.5% (volume fraction), which was found to be nontoxic to cells.

4.2.2 Cell lines and cultures

Human gastric cancer cell lines (MGC803, HGC27, and SGC7901) and human gastric epithelial cells (GES1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured at 37 ℃ in an atmosphere containing 5% CO₂, with 10% heat-inactivated fetal bovine serum supplemented with RPMI1640 medium, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. All cell lines were from ATCC and were cultured under standard conditions as recommended by the supplier.

4.2.3 Analysis of cell proliferation

For MGC803 cells, cell survival rates were determined by MTT assay. The cells were seeded in a 96-well plate (4000 well) for 24 h, followed by adding solution of compounds (200 μmol/L) to the respective wells in the indicated final concentrations and incubated for 24 h. Then, 20 μL of 5 mg/mL MTT was added to the medium, and the cells were incubated for 4 h at 37 ℃ and 5% CO₂. After removal of the culture medium, 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance was read by an enzyme labeling instrument with a 490-nm wavelength resolution. The viability of untreated cells was set as 1008%, and the viability of the other groups was calculated by comparing the optical density reading of the control cells. The IC₅₀ values were calculated by nonlinear regression analysis.

4.2.4 Apoptosis analysis

The cells were treated with different concentrations of compound 9l for 24 h. Cells were collected and washed with PBS twice, and incubated with fluorescein isothiocyanate (FITC) conjugated annexin V by use of anannexin V/FITC apoptosis kit following the step-by-step protocol provided by the manufacturer. Annexin V+/PI− cells were considered as early apoptotic and Annexin V+/PI+ cells as late apoptotic/necrotic.

4.2.5 Western blot analysis

MGC803 cells were seeded in a 100-mm tissue culture plate, cultivated for 24 h, and then treated with different concentrations of compound 9l for 24 h. After treatment, cells were collected and lysed with ice-cold lysis buffer (Beyotime, Shanghai, China). Protein concentrations of the lysates were determined by a micro-BCA protein assay kit. After centrifugation at 12000 r/min for 30 min, the total cellular protein extracts were boiled with 5× loading buffer, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blocking with 5% skimmed milk in PBST for 2 h, membranes were incubated with appropriate antibodies overnight at 4 ℃, followed by horseradish peroxidase (HRP) conjugated anti-mouse, anti-goat, or anti-rabbit secondary antibodies. The detection of specific proteins was carried out with an enhanced chemiluminescence (ECL) Western blot kit according to the recommended procedure.

Images were captured on a Canon image scanner (LIDE110, Japan). The relative protein levels were calculated by use of β-actin protein as an endogenous control and quantified by Image J software.

4.2.6 Statistical analysis

The data are expressed as means±SD. Significant differences between the groups were determined by the unpaired Student's t-test. *P < 0.05 and **P < 0.01 were accepted as indications of statistical significance.

Supporting Information The NMR spectra for synthetic compounds. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn.

[1]
Manchoju, A.; Annadate, R. A.; Desquien, L.; Pansare, S. V. Org. Biomol. Chem. 2018, 16, 6224. doi: 10.1039/C8OB01511B
[2]
Fáková, H.; Pour, M.; Kuneš, J.; Šenel, P. Tetrahedron Lett. 2005, 46, 8137. doi: 10.1016/j.tetlet.2005.09.128
[3]
David, S.; Peter, B.; Andreas, R.; Wilhelm, S. Toxicol. Lett. 2018, 295, 369. doi: 10.1016/j.toxlet.2018.07.017
[4]
Dasmahapatra, G.; Lembersky, D.; Son, M. P.; Patel, H.; Peterson, D.; Attkisson, E.; Fisher, R. I.; Friedberg, J. W.; Dent, P.; Grant, S. Mol. Cancer Ther. 2019, 18, 1180. doi: 10.1158/1535-7163.MCT-19-0470
[5]
Germain, A. D. S.; Clavé, G.; Badetdenisot, M. A.; Pillot, J. P.; Cornu, D. Nat. Chem. Biol. 2016, 12, 1552.
[6]
Xu, H. W.; Zhao, L. J.; Liu, H. F.; Zhao, D.; Luo, J.; Xie, X. P.; Liu, W. S.; Zheng, J. X.; Dai, G. F.; Liu, H. M.; Liu, L. H.; Liang, Y. B. Bioorg. Med. Chem. Lett. 2014, 24, 2388. doi: 10.1016/j.bmcl.2014.03.012
[7]
徐海伟, 贾世龙, 谢晓平, 罗姣, 王树, 有机化学, 2017, 37, 902. doi: 10.6023/cjoc201612007Xu, H.W.; Jia, S. L.; Xie, X. P.; Luo, J.; Wang, S. H. Chin. J. Org. Chem. 2017, 37, 902(in Chinese). doi: 10.6023/cjoc201612007
[8]
Necula, L.; Matei, L.; Dragu, D.; Neagu, A. I.; Mambet, C.; Nedeianu, S.; Bleotu, C.; Diaconu, C. C.; Chivu-Economescu, M. World J. Gastroenterol. 2019, 17, 2029.
[9]
Xu, H. W.; Liu, G. Z.; Zhu, S. L.; Hong, G. F.; Liu, H. M.; Wu, Q. Steroids 2010, 75, 419. doi: 10.1016/j.steroids.2010.02.007
[10]
Xu, H. W.; Wang, J. F.; Liu, G. Z.; Hong, G. F.; Liu, H. M. Org. Biomol. Chem. 2007, 5, 1247. doi: 10.1039/B701051F
[11]
Caroppi, P.; Sinibaldi, F.; Fiorucci, L.; Santucci, R. Curr. Med. Chem. 2009, 16, 4058. doi: 10.2174/092986709789378206
[12]
Fulda, S.; Debatin, K. M. Oncogene 2006, 25, 4798. doi: 10.1038/sj.onc.1209608
[13]
Danial, N. N.; Korsmeyer, S. J. Cell 2004, 116, 205. doi: 10.1016/S0092-8674(04)00046-7
[14]
Fulda, S.; Debatin, K. M. Eur. J. Cancer 2005, 41, 786. doi: 10.1016/j.ejca.2004.12.020
[15]
崔庆, 王娇, 杨海申, 谢龙观, 徐效华, 有机化学, 2010, 30, 1705.Cui, Q; Wang, J. A.; Yang, H. S.; Xie, L. G. Chin. J. Org. Chem. 2010, 30, 1705(in Chinese).

Figure 1 Structures of BH3I-1, obaloclax and selected natural butenolides

下载: 全尺寸图片幻灯片

Scheme 1 Synthesis of compound 6

Reagents and conditions: (a) Me₃PhNBr₃, dioxane, r.t., 10 h; (b) LiBr/Li₂CO₃, TBAB, CH₃CN, r.t., 20 h; (c) ethyl 4-aminobutanoate hydrochloride, Et₃N, dry THF, 0 ℃~r.t., 26 h; (d) acetone, CH₃OH, r.t., 72 h

下载: 全尺寸图片幻灯片

Scheme 2 Synthesis of compound 8

Reagents and conditions: (a) various substituted amine, K₂CO₃/ Et₃N, THF, r.t., 2 h; (b) Acetone, CH₃OH, reflux, 1 h

下载: 全尺寸图片幻灯片

Scheme 3 Synthesis of compound 9

Reagents and conditions: (a) 5-methylfuran-2-carbaldehyde, ethylenediamine, CH₃OH, reflux; (b) cyclohexanone, ethylenediamine, CH₃OH, reflux; (c) substitued benzaldehyde, ethylenediamine, CH₃OH, reflux

下载: 全尺寸图片幻灯片

Scheme 4 Synthesis of compounds 11 and 12

Reagents and conditions: (a) 4-butylbenzaldehyde, ethylenediamine, CH₃OH, reflux; (b) 4-bromomethyl-tetrahy-dro-2H-pyran, DMF, NaH, 0 ℃~r.t.

下载: 全尺寸图片幻灯片

Figure 2 Apoptosis induced by compound 9l in MGC803 cells

(A) The cytotoxicity of compound 9l by MTT assay. Cells were treated with indicated concentrations for 24 h. **P < 0.01 vs. untreated group. (B) MGC803 cells were incubated with compound 9l (20 μmol/L) for 24 h, and then stained with DAPI. The stained nuclei were observed under a fluorescent microscope (magnification, ×200). (C) A dose-dependent induction of apoptosis by compound 9l was demonstrated in MGC803 cells by flow cytometric analysis of annexin V/PI stain assay. (D) The effects of compound 9l-induced (20 μmol/L) protein changes were detected by Western blot analysis for 24 h in MGC803 cells. (E) The effects of caspase inhibitor Z-VAD-fmk (100 μmol/L) on compound 9l-induced (20 μmol/L) apoptosis of MGC803 cells were shown through DAPI solution (magnification, ×200). (F) Annexin V/PI stained cells showed the effects of caspase inhibitor Z-VAD-fmk (100 μmol/L) on compound 9l-induced (20 μmol/Lol/L) apoptosis of MGC803 cells

下载: 全尺寸图片幻灯片

Table 1. Antiproliferative activity of synthetic compounds on MGC803

Compd.	IC₅₀^a/(μmol•L^－1)
8a	42.7±3.1
8b	> 128
8c	> 128
8d	> 128
8e	> 128
8f	> 128
8g	119.8±3.5
5-Fu^b	7.1±1.3
9a1	> 50
9a2	44.4±1.6
9a3	> 128
9b	23.1±2.9
9c	18.6±2.5
9d	10.9±1.7
9e	23.2±2.9
Uncinine	> 128
9f	28.1±3.1
9g	14.7±2.1
9h	13.3±2.1
9i	26.3±2.7
9j	19.3±2.2
9k	20.4±1.2
9l	2.9±0.1
9m	5.6±0.8
9n	17.1±1.2
9o	26.7±1.4
9p	23.0±1.4
11	7.5±0.9
12	12.4±1.1
^a MTT assay: All data are mean values from triplicate experiments. ^b Positive control: fluorouracil.

下载: 导出CSV

新型丁烯内酯衍生物的设计合成及诱导胃癌细胞凋亡研究

通讯作者: 王振亚, barton118@163.com 金成允, cyjin@zzu.edu.cn

English