Synthesis and in vitro evaluation of 4-substituted furano[3,2-c] tetrahydroquinolines as potential anti-cancer agents

Abstract

A convenient and mild method for the synthesis of substituted furano [3,2-c]tetrahydroquinoline derivatives was developed, using the multi-component Povarov reaction. Of the synthesized tetrahydroquinoline derivatives, compound 10a displayed the greatest cellular proliferation inhibitory activities with IC₅₀ values of 2.5–16.7 μmol/l. In addition, 10a induced murine C6 glioma cell apoptosis in a dose-dependent manner by up-regulating the expression of Bax, caspase-3, and caspase-9, and by down-regulating Bcl-2. Our findings suggest that these novel compounds have potential as therapeutic agents via inducing mitochondrial apoptosis.

• Antitumor activity
• apoptosis
• diastereoselectivity
• tetrahydroquinoline derivatives

Introduction

Tetrahydroquinolines are common structures and a very important class of bioactive heterocycles1, which show neuroleptic2, antipsychotic3, antagonistic4^,5, antimalarial6^,7, antitumor8^,9, anti-HIV10, antibacterial11, anti-allergic12, and anti-inflammatory activities13. In addition, 4-heteroaromatic substituted anilines are another class of bioactive heterocycles with antitumor activity. Meanwhile we plan to incorporate these two moieties to explore, it is a remarkable fact that the substituted furano[3,2-c] tetrahydroquinoline moiety, as an important scaffold that has potential antitumor biological activities (Figure 1). Due to their structural novelty and important biological activities, the synthesis of complex chiral molecules around a biologically relevant framework has played an important role in the discovery of drugs14. Furthermore, some 2-phenylbenzofuran analogues have showed potent anticancer activities15–17. The [4 + 2] cycloaddition between N-arylimines and electron-rich alkenes, the multicomponent Povarov reaction has been catalyzed by various lewis acid catalysts, such as I₂, InCl₃, TFA and p-TsOH18–22.

Figure 1. Chemical structure of bioactive substituted tricyclic tetrahydroquinoline derivatives.

Although numerous methods for the synthesis of furano[3,2-c] tetrahydroquinoline derivatives have been reported in recent years, to our knowledge, the synthesis and potential antitumor activity of heteroaromatic substituted furano[3,2-c] tetrahydroquinoline derivatives have not been fully understand until now. As a part of our ongoing research on the synthesis of diverse drug candidates, the development of efficient synthetic methods to access bioactive substituted furano[3,2-c] tetrahydroquinoline derivatives would be of great utility for subsequent drug discovery.

Herein, we report a mild and efficient method for the synthesis of eight novel heteroaromatic substituted furano[3,2-c] tetrahydroquinoline derivatives and the in vitro biological evaluation of their antitumor activity, selective cytotoxicity and apoptosis-inducing effects.

Experimental

Chemistry

General

¹H NMR spectra were recorded with a Bruker Avance 400 MHz spectrometer (Billerica, MA) at 298 K with TMS as an internal standard. MS spectra were recorded on a Mariner Mass Spectrum (ESI). All compounds were routinely checked by TLC and ¹H NMR. TLCs and preparative thin-layer chromatography were performed on silica gel GF254, and the chromatograms were conducted on silica gel (300–400 mesh, Merck, Kenilworth, IL) and visualized under UV light at 254 and 365 nm. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33258 were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against Bax, Bcl-2, caspase-3, caspase-8, caspase-9, β-actin, and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All solvents were of reagent grade and, when necessary, were purified and dried by standards methods.

Abbreviation: PE = Petroleum ether, EA = Ethyl acetate, InCl₃ = Indium Chloride

General procedure for the preparation of compounds 2–8

4-(Pyridin-4-yl)benzenamine (170 mg; 1.00 mmol) and InCl₃ (43.8 mg; 20 mol%) were dissolved into CH₃CN (5 mL),then the aldehyde (1.20 mmol) was added to the mixture. After 5 min stirring at room temperature, 2,3-dihydro-2H-furan (97 μl; 1.20 mmol) was added. This mixture was stirred for 30 min at room temperature, then the solvent was removed under reduced pressure. The residue was diluted with ethyl acetate and filtered. The organic layer was washed with water and brine, and dried over Na₂SO₄. The mixture was filtered, evaporated to dryness, and the residue was purified by column chromatography (PE:EA = 10:1) to afford the products, yield: 59–71%.

General procedure for the preparation of compounds 9–11

Substituted anilines (1.00 mmol) and 2,3-dihydro-2H-furan (2.00 mmol) were dissolved in MeCN (10 ml), then InCl₃ (0.2 mmol) was added to the mixture. This mixture was stirred for 30 min at room temperature. After complete conversion, the solvent was removed under reduced pressure. The residue was diluted with ethyl acetate and filtered. The organic layer was washed with water and brine, and dried over Na₂SO₄. The mixture was filtered, evaporated to dryness, and the residue was purified by column chromatography (PE:AC = 5:1) to afford the products, yield: 65–73%.

2-Phenyl-6-(pyridin-4-yl)-furano[3,2-c]-1,2,3,4-tetrahydroquinoline (compound 2): cis: ¹H NMR (400 MHz, CDCl₃) δ 8.55 (2H, d, J = 6.0 Hz, H₂, H₃), 7.94 (s, 1H, H₈), 7.57 (2H, dd, J = 6.4 Hz, J = 1.8 Hz, H₄, H₅), 7.46 (m, 3H, H₁₅, H₁₆ and H₁₇), 7.40 (m, 2H, H₁₄ and H₁₈), 7.20 (m, 2H, H₆ and H₇), 4.68 (d, J = 5.6 Hz, 1H, H₁₃), 4.08 (m, 1H, H₁₂), 3.82 (t, J = 6.2 Hz, 1/2 2H, H₉), 3.11 (t, J = 6.3 Hz, 1/2 H, H₉), 2.51 (m, 1H, H₁₁), 2.07 (m, 1/2 2H, H₁₀), 1.76 (m, 1/2 2H, H₁₀). ¹³C NMR (101 MHz, CDCl₃) 157.39, 152.96, 148.20, 145.44, 144.96, 137.65, 129.65, 129.05, 127.49, 120.67, 120.05, 115.88, 115.10, 114.73, 98.37, 72.67, 67.67, 55.89, 39.25, 29.67, 20.13. HRMS (EI): [M + H]⁺ 329.1642 measured, [C₂₂H₂₁NO₂]⁺ Calcd 329.1648.

2-(2′,4′-Dichlorophenyl)-6-(pyridin-4-yl)-furano[3,2-c]-1,2,3,4-tetrahydroquinoline (compound 3): cis: ¹H NMR (400 MHz, CDCl₃) δ 8.57 (2H, d, J = 6.0 Hz, H₂, H₃), 7.92 (s, 1H, H₈), 7.59 (2H, dd, J = 6.4 Hz, J = 1.8 Hz, H₄, H₅) 7.47 (m, J = 7.5 Hz, 2H, H₁₄ and H₁₅), 7.31 (s, 1H, H₁₆), 7.20 (m, 2H, H₆ and H₇), 4.69 (d, J = 5.7 Hz, 1H, H₁₃), 4.10 (m, 1H, H₁₂), 3.78 (dd, J = 15.7, 8.3 Hz, 1/2 2H H₉), 2.50 (s, 1H, 1/2 2H, H₉), 2.10 (m, 1/2 2H, H₁₀), 1.47 (m, 1/2 2H, H₁₀). ¹³C NMR (101 MHz, CDCl₃) 158.20, 155.63, 148.40, 146.79, 135.55, 130.96, 129.87, 127.53, 126.22, 123.96, 120.87, 116.98, 115.32, 98.92, 74.47, 67.05, 55.23, 39.12, 29.32, 21.09. HRMS (EI): [M + H]⁺ 397.0863 measured, [C₂₂H₁₉Cl₂NO₂]⁺ Calcd 397.0869.

2-(3′-Hydroxyphenyl)-6-(pyridin-4-yl)-furano[3,2-c]-1,2,3,4-tetrahydroquinoline (compound 4): cis: ¹H NMR (400 MHz, CDCl₃) δ 8.56 (2H, d, J = 6.0 Hz, H₂, H₃), 7.90 (s, 1H, H₈), 7.60 (2H, dd, J = 6.4 Hz, J = 1.8 Hz, H₄, H₅), 7.20 (m, 3H, H₆, H₇ and H₁₄), 6.96 (m, 2H, H₁₇ and H₁₆), 6.79 (d, J = 8.0 Hz, 1H, H₄), 4.66 (d, J = 4.8 Hz, 1H, H₁₂), 4.02 (d, J = 10.9 Hz, 1H, H₁₃), 3.85 (d, J = 12.3 Hz, 1/2 2H, H₉), 3.75 (d, J = 8.3 Hz, 1/2 2H, H₉), 2.47 (s, 1H, H₁₁), 2.04 (m, 2H, H₁₀). ¹³C NMR (101 MHz, CDCl₃) δ 158.99, 149.51, 146.52, 134.33, 131.29, 127.59, 126.88, 125.06, 121.76,118.49, 115.45, 102.85, 71.35, 61.80, 50.06, 36.56, 34.31, 29.59, 27.35, 18.98. HRMS (EI): [M + H]⁺ 345.1592 measured, [C₂₂H₂₁NO₃]⁺ Calcd 345.1598.

2-(4′-Acetylamidophenyl)-6-(pyridin-4-yl)-furano[3,2-c]-1,2,3,4-tetrahydroquinoline (compound 5): cis: ¹H NMR (400 MHz, CDCl₃) δ 8.56 (2H, d, J = 6.0 Hz, H₂, H₃),7.95 (s, 1H, H₈), 7.60 (2H, dd, J = 6.4 Hz, J = 1.8 Hz, H₄, H₅), 7.43 (t, J = 7.5 Hz, 2H, H₁₅ and H₁₆), 7.25–7.17 (m, 4H, H_6,H₇, H₁₄ and H₁₇), 4.68 (d, J = 5.7 Hz, 1H, H₁₃), 4.09 (m, 1H, H₁₂), 3.76 (dt, J = 18.6, 7.2 Hz, 2H, H₉), 2.49 (m, 1H, H₁₁), 2.22 (s, 3H,–CH₃), 2.07 (m, 2H, H₁₀). ¹³C NMR (101 MHz, CDCl₃) 164.56, 156.46, 148.39, 144.36, 146.82 137.89, 129.25, 129.02, 127.69, 126.36, 123.89, 120.85, 116.87, 115.69, 96.42, 74.87, 67.36, 55.28, 38.97, 29.34, 23.59, 21.52. HRMS (EI): [M + H]⁺ 386.1879 measured, [C₂₄H₂₄N₂O₃]⁺ Calcd 386.1863.

2-(3′-Methoxyphenyl)-6-(pyridin-4-yl)-furano[3,2-c]-1,2,3,4-tetrahydroquinoline (compound 6): cis: ¹H NMR (400 MHz, CDCl₃) δ 8.58 (2H, d, J = 6.0 Hz, H₂, H₃), 7.93 (s, 1H, H₈), 7.62 (2H, dd, J = 6.4 Hz, J = 1.8 Hz, H₄, H₅), 7.31(t, J = 7.9 Hz, 1H, H₁₆), 7.19 (m, 2H, H₆ and H₇,), 7.07–7.00 (m, 3H, H₁₇, H₁₅ and H₁₄), 4.67 (d, J = 4.9 Hz, 1H, H₁₃), 4.06 (m, 1H, H₁₂), 3.88 (m, 2H, H₉), 3.84 (s, 3H, –CH₃), 2.49 (m, 1H, H₁₁), 2.06 (m, 1/2 2H, H₁₀), 1.78 (m, 1/2 2H, H₁₀). ¹³C NMR (101 MHz, CDCl₃) 161.17, 156.58, 155.20, 149.11, 145.34, 137.74, 129.99, 127.57, 126.35, 123.64, 120.20, 116.88, 115.02, 111.75, 98.96, 75.37, 68.18, 54.56, 39.87, 29.47, 20.12. HRMS (EI): [M + H]⁺ 359.1750 measured, [C₂₃H₂₃NO₃]⁺ Calcd 359.1754.

2-Phenyl-6-(pyrimidin-4-yl)-furano[3,2-c]-1,2,3,4-tetrahydroquinoline (compound 7): cis: ¹H NMR (400 MHz, CDCl₃) δ 9.16 (1H, s, H₂), 8.69 (1H, dd, J = 5.2 Hz, J = 1.2 Hz, H₃), 7.92 (s, 1H, H₈), 7.73 (1H, s, H₄), 7.45 (m, 3H, H₁₅, H₁₆ and H₁₇), 7.41 (m, 2H, H₁₄ and H₁₈), 7.19 (m, 2H, H₆ and H₇), 4.69 (d, J = 5.6 Hz, 1H, H₁₃), 4.10 (m, 1H, H₁₂), 3.80 (t, J = 6.2 Hz, 1/2 2H, H₉), 3.10 (t, J = 6.3 Hz, 1/2 H, H₉), 2.53 (m, 1H, H₁₁), 2.05 (m, 1/2 2H, H₁₀), 1.75 (m, 1/2 2H, H₁₀). ¹³C NMR (101 MHz, CDCl₃) 154.54, 149.01, 145.38, 129.74, 127.38, 126.22, 124.05, 120.87, 114.65, 94.28, 74.38, 68.74, 54.87, 39.18, 30.45, 29.15, 22.10.HRMS (EI): [M + H]⁺ 329.1605 measured, [C₂₁H₂₀N₃O]⁺ Calcd 330.1601.

2-Phenyl-6-(pyrimidin-2-yl)-furano[3,2-c]-1,2,3,4-tetrahydroquinoline (compound 8): cis: ¹H NMR (400 MHz, CDCl₃) δ 8.79 (2H, d, J = 5.2 Hz, H₂, H₃), 7.93 (s, 1H, H₈), 7.44 (m, 3H, H₁₅, H₁₆ and H₁₇), 7.40 (m, 2H, H₁₄ and H₁₈), 7.21 (m, 2H, H₆ and H₇), 4.69 (d, J = 5.6 Hz, 1H, H₁₃), 4.12 (m, 1H, H₁₂), 3.81 (t, J = 6.2 Hz, 1/2 2H, H₉), 3.12 (t, J = 6.3 Hz, 1/2 H, H₉), 2.52 (m, 1H, H₁₁), 2.07 (m, 1/2 2H, H₁₀), 1.76 (m, 1/2 2H, H₁₀). HRMS (EI): [M + H]⁺ 329.1607 measured, [C₂₁H₂₀N₃O]⁺ Calcd 330.1601.

2-(3′-Hydroxypropyl)-6-(pyridin-4-yl)-furano[3,2-c]-1,2,3,4-tetrahydroquinoline (compound 9): cis: ¹H NMR (400 MHz, DMSO) δ 8.55 (2H, d, J = 6.0 Hz, H₂, H₃), 7.94 (s, 1H, H₈), 7.65 (2H, dd, J = 6.4 Hz, J = 1.8 Hz, H₄, H₅), 7.20 (m, 2H, H₆ and H₇), 5.05 (d, J = 5.7 Hz, 1H, H₁₃), 4.48 (m, 1H, H₁₂), 3.44 (dd, J = 12.9, 7.3 Hz, 4H, H₉ and H₁₆), 2.72 (d, J = 8.5 Hz, 1H, H₁₁), 1.88–1.75 (m, 4H, H₁₄ and H₁₅), 1.57 (m, 2H, H₁₀). ¹³C NMR (101 MHz, CDCl₃) 154.54, 149.01, 145.38, 129.74, 127.38, 126.22, 124.05, 120.87, 114.65, 94.28, 74.38, 68.74, 54.87, 39.18, 30.45, 29.15, 22.10. HRMS (EI): [M + H]⁺ 311.1752 measured, [C₁₉H₂₃N₂O₂]⁺ Calcd 311.1754.

2-(3′-Hydroxypropyl)-6-(pyrimidin-4-yl)-furano[3,2-c]-1,2,3,4-tetrahydroquinoline (compound 10): cis: ¹H NMR (400 MHz, DMSO) δ 9.18 (1H, s, H₂), 8.71 (1H, dd, J = 5.2 Hz, J = 1.2 Hz, H₃), 7.96 (s, 1H, H₈), 7.74 (1H, s, H₄), 7.22 (m, 2H, H₆ and H₇), 5.08 (d, J = 5.7 Hz, 1H, H₁₃), 4.49 (m, 1H, H₁₂), 3.48 (dd, J = 12.9, 7.3 Hz, 4H, H₉ and H₁₆), 2.69 (d, J = 8.5 Hz, 1H, H₁₁), 1.85–1.76 (m, 4H, H₁₄ and H₁₅), 1.55 (m, 2H, H₁₀). ¹³C NMR (101 MHz, CDCl₃) 169.52, 163.64, 158.54, 144.88, 129.64, 126.58, 126.22, 124.55, 117.49, 112.25, 92.38, 75.18, 68.14, 52.17, 38.25, 31.15, 28.65, 20.55. HRMS (EI): [M + H]⁺ 312.1701 measured, [C₁₈H₂₂N₃O₂]⁺ Calcd 312.1707.

2-(3′-Hydroxypropyl)-6-(pyrimidin-4-yl)-furano[3,2-c]-1,2,3,4-tetrahydroquinoline (compound 11): cis: ¹H NMR (400 MHz, DMSO) δ 8.78 (2H, d, J = 5.2 Hz, H₂, H₃), 7.95 (s, 1H, H₈), 7.20 (m, 2H, H₆ and H₇), 7.15 (1H, t, J = 4.8 Hz, H₁), 5.11 (d, J = 5.7 Hz, 1H, H₁₃), 4.50 (m, 1H, H₁₂), 3.51 (dd, J = 12.9, 7.3 Hz, 4H, H₉ and H₁₆), 2.71 (d, J = 8.5 Hz, 1H, H₁₁), 1.84–1.75 (m, 4H, H₁₄ and H₁₅), 1.56 (m, 2H, H₁₀). ¹³C NMR (101 MHz, CDCl₃) 168.32, 162.24, 157.54, 145.38, 129.34, 127.78, 126.52, 124.15, 116.89, 93.68, 75.33, 68.54, 53.27, 39.38, 31.25, 30.45, 21.38. HRMS (EI): [M + H]⁺ 312.1709 measured, [C₁₈H₂₂N₃O₂]⁺ Calcd 312.1707.

In vitro antitumor evaluation

MTT assay

The cellular proliferation inhibitory effects of test compounds on human lung cancer cells (A549), human hepatocellular carcinoma cells (HepG2), human breast cancer cells (MCF-7), murine glioma cells (C6), and murine melanoma cells (B16) were measured by MTT assay. The cells were suspended at a density of 4 × 10⁴/mL and added to 96-well flat bottom micro titer plates 100 μl per well. After incubation for 24 h, cells were treated with different concentrations of the drug. After incubation for indicated time, cells growth was measured by MTT assay. The percentage of inhibitory ratio was calculated as follows:

Hoechst 33258 staining

After incubation with test compounds for 24 h, the C6 cells were stained with Hoechst 33258 at 37 °C for 30 min, and then the morphology was observed by a fluorescence microscopy (TE2000-T, Nikon, Tokyo, Japan).

Flow cytometry assay of cellular apoptosis

The annexin V/PI dual staining assay was employed to determine the involvement of apoptotic cell death, using Annexin-V-FLUOS Staining Kit (Roche, Basel, Switzerland) as the instructions of the manufacturer. C6 glioma cells were cultured overnight and incubated with tested compounds or control for 24 h. The percentage of apoptotic cells was determined by flow cytometry (Beckman Coulter, Brea, CA) analysis.

Western blot analysis

The C6 glioma cells were harvested, washed twice with cold PBS and then lysed in whole cell lysis buffer, supplemented with the protease inhibitors 100 μg/mL at 4 °C for 1 h. After 12 000 g centrifugation at 4 °C for 10 min, the protein concentration was determined by a BCA Protein Assay Kit (CWBIO, Beijing, China). Equal amounts of total proteins were separated by 12% SDS-PAGE, and transferred onto Immobilon-P Transfer Membrane (Millipore Corporation, Billerica, MA). The membranes were blocked with 5% skimmed milk at room temperature for 1 h, incubated with indicated primary antibodies at 4 °C overnight and horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 h, then visualized by using ECL reagents.

Results and discussion

In this study, we report an imino Diels–Alder reaction to synthetize heteroaromatic substituted furano[3,2-c]-tetrahydroquinoline derivatives. This reaction is a LUMO-diene-controlled Diels–Alder reaction between an electron-deficient azadiene and electron-rich dienophiles, such as cyclic enol ethers. First, we optimized the reaction conditions (see Supporting information). After optimization of the conditions, different aromatic aldehydes were then treated with 1 and 2,3-dihydro-2H-furan to obtain a series of furano[3,2-c]tetrahydroquinoline derivatives. In addition, to synthesize more furano[3,2-c]tetrahydroquinoline derivatives for activity screening, we used 4-(nitrogen containing heteroaromatic substituted)benzenamine, 2,3-dihydro-2H-furan, and InCl₃ catalyst providing compounds 9–11 in good yields (Table 1).

Table 1. Imino Diels–Alder reactions catalyzed by InCl3 for the synthesis of furano[3,2-c]quinolines 2a–11a and 2b–11b..

Entry	X	Y	Z	R′	Catalyst %	Time (min)	Product	Yield (%)	cis/trans*
1	N	CH	CH	Phenyl	20 mol%	30	2a + 2b	71	67:33
2	N	CH	CH	2,4-Dichlorophenyl	20 mol%	30	3a + 3b	59	80:20
3	N	CH	CH	3-Hydroxyphenyl	20 mol%	30	4a + 4b	68	75:25
4	N	CH	CH	4-Acetylamidophenyl	20 mol%	30	5a + 5b	62	72:28
5	N	CH	CH	3-Methoxyphenyl	20 mol%	30	6a + 6b	65	87:13
6	N	N	CH	Phenyl	20 mol%	30	7a + 7b	63	68:32
7	CH	N	N	Phenyl	20 mol%	30	8a + 8b	64	70:30
8	N	CH	CH	/	20 mol%	30	9a + 9b	73	73:27
9	N	N	CH	/	20 mol%	30	10a + 10b	65	79:21
10	CH	N	N	/	20 mol%	30	11a + 11b	68	78:22

*Diastereomeric ratios (cis/trans) were determined by means of ¹H NMR analysis.

The anti-proliferation activities of compounds 2a–11a

Having synthesized a variety of substituted furano[3,2-c] tetrahydroquinoline derivatives, we set out to evaluate these compounds for their biological activities. The cis products in Table 1 were tested for selective antitumor activity against the A549 human lung cancer cells, HepG2 human hepatocellular carcinoma cells, MCF-7 human breast cancer cells, C6 murine glioma cells, and B16 murine melanoma cells using a proliferation-based assay. Selected results of the broad compound screening for 2a–11a are shown in Table 2. Compound 10a caused a remarkable anti-proliferative effect on C6 cell growth in a time- and dose-dependent manner, and the treatment with 2.5 μM (7.8 μg/mL) 10a for 24 h resulted in almost 50% inhibition.

Table 2. The IC₅₀ values of 2a–11a against five tumor cell lines.

	IC50 (µmol/L)
Compd.	A549	HepG2	MCF-7	C6	B16
2a	>50	13.1	25.9	19.5	>50
3a	>50	33.5	>50	26.7	18.3
4a	23.7	11.5	26.4	9.9	17.2
5a	36.9	30.6	16.9	7.4	15.9
6a	43.6	41.4	>50	10.7	>50
7a	>50	19.8	38.6	27.7	>50
8a	>50	>50	47.2	20.2	>50
9a	18.7	23.6	9.4	5.1	8.3
10a	16.7	7.3	9.7	2.5	9.6
11a	>50	8.1	7.3	10.2	14.8

Compound 10a inhibits cell growth and induces apoptosis in C6 glioma cells

To characterize the 10a-induced C6 cell growth inhibition, we observed the morphologic changes in the cells. When the cells were cultured with 2.5 μM 10a for 24 h, marked apoptotic morphologic alterations were observed by inverted microscopy. These changes were further confirmed by hoechst 33258 staining. The cells in the control group showed uniform dispersion of low-density fluorescence, but 10a-treated cells showed condensed, bright fluorescence, and nuclear fragmentation (Figure 2A). The flow cytometry apoptosis assay was further investigated employing C6 cells which were incubated with blank control alone, with compound 10a treated with pan-caspase inhibitor Z-VAD-FMS, and without Z-VAD-FMS. The percentages of apoptotic cells were determined by staining with FITC-Annexin V/PI staining. The C6 cell apoptosis was rarely seen in the control group and the percentages of apoptosis significantly increased from 3.5% to 37.4% by treated with compound 10a. Moreover, the total apoptotic cell percentage was reduced from 37.4% to 16.6% when co-treated with 10 μM Z-VAD-FMS. These results suggest that 10a-induced cellular apoptosis involves caspase activation.

Figure 2. Compound 10a induced C6 murine glioma cell apoptosis in vitro. (A) The cells were treated with 10a (2.5 μM) for 24 h then stained with hoechst 33258 and observed by fluorescence microscope (×200 magnification). (B) The cells were treated with 10a (2.5 μM) for 24 h without or with co-incubation of pan-caspase inhibitor Z-VAD-FMK (10 μM), then revealed by Annexin-V/PI double staining using flow cytometry analysis. (C) The C6 glioma cells were treated with 10a (2.5 μM) for 0, 6, 12, and 24 h, followed by western blot analysis for the detection of Bax, Bcl-2, caspase-3, caspase-8, and caspase-9 levels. β-Actin was used as an equal loading control.

The activation of caspases suggested that compound 10a-induced apoptosis may involve the mitochondrial-mediated intrinsic apoptosis pathway. In intrinsic apoptosis, Bcl-2 (an anti-apoptotic protein) is down-regulated and Bax (a pro-apoptotic protein) is up-regulated, which disrupts the mitochondrial membrane potential (MMP). In addition, intrinsic apoptosis can be either caspase dependent or independent. In order to probe the specific mechanism of apoptosis, i.e. whether Bcl-2 family proteins (Bax and Bcl-2) and specific caspases (-3, -8, and -9), are induced by 10a treatment, C6 gliomia cells were treated with 2.5 μM 10a for 24 h and analyzed by western blot. Results demonstrated that the expression level of Bax slightly increased, Bcl-2 decreased, caspase-3 and -9 increased greatly, and caspase-8 was not changed (Figure 2C). These results are consistent with caspase-dependent intrinsic apoptotis. Thus, compound 10a-induced apoptosis is mainly through intrinsic mitochondria pathway.

Conclusion

We reported a convenient and mild method for the synthesis of substituted furano [3,2-c]tetrahydroquinoline derivatives, using the multi-component Povarov reaction. We examined different catalysts and reaction conditions, and found the best reaction conditions to be with InCl₃ as a catalyst at room temperature. These tetrahydroquinoline derivatives were obtained in moderate to good yields. Our results suggest that compound 10a may be a potential therapeutic agent for the treatment of glioma. Our results also demonstrate that compound 10a can induce mitochondrial apoptosis in a time-dependent manner by the up-regulation of the expression of Bax, caspase-9, and caspase-3 and down-regulation of Bcl-2.

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Supporting information

Declaration of interest

The authors gratefully acknowledge the support by the National Natural Science Foundation of China (Nos. 30901837 and 81402500); the Fund of the Health Department of Sichuan Province (No. 120486); the program for young scholar scientific and technological innovative research team in Sichuan province (No. 2014TD0021), and the program for provincial universities innovative research team in Sichuan province (No. 14TD0023).