Synthesis of quinoxalines and assessment of their inhibitory effects against human non-small-cell lung cancer cells

Abstract

Twenty-six quinoxalin derivatives were synthesized to assess their biological activities against human non-small-cell lung cancer cells (A549 cells). Compound 4b (IC₅₀ = 11.98 ± 2.59 μM) and compound 4m (IC₅₀ = 9.32 ± 1.56 μM) possess anticancer activity comparable to 5-fluorouracil (clinical anticancer drug) (IC₅₀ = 4.89 ± 0.20 μM). Western blot tests further confirmed that compound 4m effectively induced apoptosis of A549 cells through mitochondrial- and caspase-3-dependent pathways. The introduction of bromo groups instead of nitro groups into the quinoxaline skeleton has been shown to provide better inhibition against lung cancer cells in this article. This modification in the molecular structure could enhance the biological activity and effectiveness of quinoxaline derivatives in the design and synthesis of anticancer drugs, making bromo-substituted quinoxalines a promising avenue for further research and development in anticancer therapeutics.

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1 Introduction

Quinoxaline, known as benzopyrazine, is naphthalene with some carbon atoms replaced with nitrogen atoms¹ (Fig. 1a). The nitrogen atoms of quinoxalines contribute to the stabilization of radical ion species. Naturally occurring quinoxalines are scarce,^2,3 and their derivatives are mostly derived from synthetic products. Quinoxaline's skeleton is in frame with some natural antibiotics, such as echinomycin,^4,5 quinornycin A,⁶ triostins⁷ and actinoleutin which show activities against Gram-positive bacteria⁸ and tumors.⁹ Furthermore, the synthetic quinoxaline derivatives possess antimicrobial,^1,10,11 anti-inflammatory,^12,13 antiviral,^1,14 antioxidant,^13,15 antitumor,^1,9 receptor antagonist,¹⁶ antitubercular,^1,17,18 antifungal,^{1,11,15,19–22} antiparasitic²³ and antidiabetic^1,24–28 activities. Besides the pharmacological interest in quinoxalines, they are also employed in material sciences, such as in OLEDs²⁹ and dye-sensitized solar cells.³⁰

Fig. 1 The skeletons of quinoxaline (a) and C6-substituted alkenyl quinoxalines (b) are reported in this article, and a quinoxaline derivative (c) is reported in the literature with potent biological activity.

Quinoxaline urea derivatives were evaluated in the treatment of pancreatic therapy.³¹ Therefore, we have reported the synthesis of cinnamils and 2,3-dialkenyl-substituted quinoxalines to evaluate their activities against pancreatic cancer cells.³² Among them, one of cinnamils exhibited a more potent anticancer effect than that of quinoxalines. However, that series of quinoxalines presented a moderate inhibitory effect. Compared with other quinoxalines, synthesizing 2,3-dialkenyl-substituted quinoxalines is relatively rare.^33,34 They exhibited photophysical properties³⁴ or as a detector,³⁵ however, their biological activity is not well studied. A C6-fluoro substituted quinoxaline was reported as a potent inhibitor of JNK stimulatory phosphatase-1 (JSP-1) in an in vitro biological assay (Fig. 1c).³⁶ Due to quinoxalines for their pharmacological interests, it prompted us to introduce an electron-withdrawing group to the C6-position of 2,3-dialkenyl-substituted quinoxalines. Therefore, the bromo or nitro groups were selected to assess their activities against non-small-cell lung cancer cells (Fig. 1b, X = Br, or NO₂) and pursue hit compounds. According to reports, the NO₂ functional group may be genotoxic, so the genotoxicity of 5a–5m needs further discussion. However, this article aims to investigate the apoptotic mechanisms of the most effective compound 4m.

2 Results and discussion

2.1 Chemistry

There are at least three methods available for the synthesis of quinoxalines. First, the condensation of cinnamils with 1,2-phenylenediamine derivatives under either the catalytic amount of I₂ in DMSO³⁷ or reflux acetic acid,³⁸ which condition is used for the synthesis of target molecules in this article (Path a). Secondly, oxalic acid is condensed with 1,2-phenylenediamine in an acidic condition and then treated with POCl₃ followed by reacting with nucleophiles (Path b).¹⁰ Finally, the key intermediate, iminoethanone, was prepared from aldehyde and imidoyl chloride under the NHC catalyst (path c). The intermediate, iminoethanone, was condensed with 1,2-phenylenediamine catalyzed by hypervalent iodine³⁹ to obtain quinoxaline. It is noticed that Path b and Path c are more readily available for the synthesis of unsymmetric quinoxalines (Scheme 1).

Scheme 1 The reported strategies for the synthesis of quinoxalines.

The previous synthesis of 2,3-dialkenyl-substituted quinoxalines employed a Heck method (Fig. 2).³³ We applied the condensation of 1 or 2 with the corresponding 3a–m in acetic acid under reflux conditions.³² The resulting solid was filtered by suction and washed with distilled water to obtain 4a–m and 5a–m in moderate to high yields without column chromatography (Scheme 2, see experimental section).

Fig. 2 The synthesis of 2,3-dialkenyl-substituted quinoxalines via the Heck method.

Scheme 2 Synthesis of quinoxalines 4a–m and 5a–m.

2.2 Biological evaluation

The cytotoxic effects of all twenty-six synthesized compounds were evaluated by their activity to suppress human non-small cell lung cancer cells (A549 cells). As shown in Table 1, compound 4m (IC₅₀ = 9.32 ± 1.56 μM) exhibited the most potent inhibitory activity against A549 cells. Although compound 4m showed slightly weaker anticancer activity than 5-fluorouracil (5-FU) against A549 cells. We further compared the cytotoxicity of 4m and the clinical anticancer drug 5-FU on normal human cells. The cytotoxicity of 4m (IC₅₀ = 9.29 ± 1.25 μM) to human foreskin fibroblast (Hs68) cells is approximately 3 times lower than that of 5-FU (IC₅₀ = 3.08 ± 1.40 μM) (Table 2). Therefore, it shows that 4m possesses research and development value. In addition, compounds 4a, 4b, 4e, 4g, 4l, and 5g also exhibited cytotoxic activities against A549 cells.

Table 1 Inhibitory effects of compounds against human non-small-cell lung cancer cells (A549)^a

Compounds	IC50^b (μM)
a Results are presented as averages ±SD (n = 3).b Concentration necessary for 50% inhibition (IC50).c 5-Fluorouracil (5-FU) was used as a positive control.
4a	54.94 ± 2.13**
4b	11.98 ± 2.59***
4c	>100
4d	>100
4e	35.23 ± 9.29*
4f	>100
4g	42.39 ± 1.40**
4h	>100
4i	>100
4j	>100
4k	>100
4l	38.17 ± 0.48*
4m	9.32 ± 1.56**
5a	>100
5b	>100
5c	>100
5d	>100
5c	>100
5d	>100
5e	>100
5f	>100
5g	75.02 ± 4.50**
5h	>100
5i	>100
5j	>100
5k	>100
5l	>100
5m	>100
5-FU^c	4.89 ± 0.20**

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Table 2 In vitro cytotoxic effects of compound 4m and 5-fluorouracil against Hs68 (human foreskin fibroblast) cells^a

Compounds	IC50^b (μM)
a Results are presented as averages ±SD (n = 3).b Concentration necessary for 50% inhibition (IC50).c 5-Fluorouracil (5-FU) was used as a positive control.
4m	9.29 ± 1.25
5-FU^c	3.08 ± 0.40

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The effect of treating A549 cells with compound 4m on the expression of apoptosis-related proteins was investigated. Fig. 3a shows that the expression of the pro-apoptotic protein Bax treated with 20 μM of compound 4m was higher than that treated with 5 or 10 μM of 4m. In contrast, cells treated with 20 μM of compound 4m showed lower Bcl-2 (anti-apoptotic protein) expression than those treated with 5 or 10 μM (Fig. 3b). The results show that compound 4m suppressed the expression of Bcl-2 and increased the expression of Bax.

Fig. 3 Western blot analysis for Bcl-2 (a), Bax (b), pro-caspase 3 (c), and cleaved caspase 3 (d) in each group on A549 cells. 4m means compound 4m. 5-Fluorouracil (5-FU) was used as a positive control. Asterisks indicate significant differences (*p < 0.05) compared with the control group.

Besides, caspase 3 activation is a hallmark of apoptosis. We further investigated whether compound 4m could influence the enzymatic activities of caspase-3. The results show that compound 4m suppressed pro-caspase 3 and increased the cleaved caspase-3 (Fig. 3c and d). Furthermore, compound 4m markedly induced apoptosis of A549 cells through caspase-3-dependent pathways.

3 Conclusions

The development of quinoxalines as anticancer agents has gained attention in recent years.^40,41 Synthesis of quinoxalines 4a–m and 5a–m was facile. Their purifications were simple filtration, then washed with water to obtain the pure target molecules. This series of compounds showed less potency on A549 cells than 5-FU. However, it provides important information that the C6-bromo rather than C6-nitro group is a suitable quinoxaline substitute for designing potential drug candidates. Furthermore, we found compounds 4f, 4k, 5f, and 5k, which are with two nitro groups, reduced the solubility. Compound 4m was found to have the highest cytotoxic activity against A549 cells out of all synthesized molecules. The activity of compound 4m was comparable to that of 5-FU (clinical anticancer drug). Furthermore, compound 4m markedly induced apoptosis of A549 cells through the mitochondrial- and caspase-3-dependent pathways (Fig. 4). This suggests that the halo substituted at C6-position of 2,3-dialkenyl quinoxalines may be necessary. Thus, compound 4m is worth further investigation and might be developed as a candidate for treating or preventing human non-small-cell lung cancer.

Fig. 4 Schematic diagram for cancer cell apoptosis mechanism of compound 4m in A549 cells.

4 Experimental

All chemicals were purchased from either Acros or Alfa from Uniworld in Taiwan and used from received without further purification except otherwise mentioned. The structure's determinations were based on ¹H and ¹³C-NMR data recorded on a 600 MHz Bruker Ultrashield instrument. The chemical shifts were reported in part per million (ppm) for the residual solvent (CDCl₃: 7.26 ppm for ¹H; 77.0 ppm for ¹³C). The splitting constant (J) was represented in hertz (Hz), and the splitting patterns were designated as s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublets), t (triplet), and m (multiplet). The purity of compounds was performed by high-resolution mass spectrometry (HRMS) using a Bruker UltraFlex II instrument (from Bruker Taiwan Co. Ltd) for electrospray ionization (ESI). The reaction processes were monitored by thin layer chromatography (Analtech Silica gel HLF UV254) and visualized by UV (256 or 365 nm) or stained with KMnO₄ or p-anisaldehyde solution. The melting points were determined using an MP-2D apparatus and were uncorrected.

4.1 General procedure for the synthesis of compounds 4a–m and 5a–m

Compound 1 (4-bromo-1,2-diaminobenzene, 1.0 equivalent) and the corresponding cinnamil 3a–m (1.2 equivalent) were dissolved in acetic acid (0.2 M). This resulting mixture was heated at 75 °C for 6 h and then cooled in an ice bath until a precipitate formed. The precipitate was filtrated by suction and washed with H₂O to obtain the target molecules 4a–m, respectively. Synthesis of compounds 5a–m followed the method mentioned above.

4.2 Spectra data

4.2.1 6-Bromo-2,3-bis[(E)-2-(furan-2-yl)vinyl]quinoxaline (4a). Use compound 1 (0.093 g, 0.495 mmol) and compound 3a (0.10 g, 0.413 mmol) to afford compound 4a (0.144 g, 0.366 mmol). Yield: 89%. Mp 213–214 °C. A brown solid. ¹H NMR (600 MHz, CDCl₃) δ 8.15 (d, J = 1.7 Hz, 1H), 7.83 (d, J = 13.4 Hz, 2H), 7.82 (d, J = 9.6 Hz, 1H), 7.70 (dd, J = 8.9, 1.6 Hz, 1H), 7.55 (d, J = 3.2 Hz, 1H), 7.54 (d, J = 15.2 Hz, 2H), 7.53 (s, 1H), 6.62 (t, J = 4.8 Hz, 2H), 6.51 (d, J = 1.4 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 152.9, 152.8, 149.4, 148.9, 143.8, 143.7, 142.1, 140.3, 132.7, 131.0, 130.0, 125.1, 124.8, 123.1, 119.8, 119.7, 113.1, 112.9, 112.3. HRMS (ESI) calculated for C₂₀H₁₄BrN₂O₂ [M + H]⁺ 393.0239. Found: 393.0225.

4.2.2 6-Bromo-2,3-bis[(E)-2-(thiophen-2-yl)vinyl]quinoxaline (4b). Use compound 1 (0.082 g, 0.437 mmol) and compound 3b (0.10 g, 0.364 mmol) to afford compound 4b (0.118 g, 0.276 mmol). Yield: 76%. Mp 210–211 °C. A yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 8.17 (d, J = 2.1 Hz, 1H), 8.15 (d, J = 3.5 Hz, 1H), 8.12 (d, J = 3.5 Hz, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 8.8, 2.1 Hz, 1H), 7.38 (d, J = 15.3 Hz, 2H), 7.37 (d, J = 15.3 Hz, 2H), 7.32 (t, J = 3.4 Hz, 2H), 7.11–7.08 (m, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 149.2, 148.7, 142.1, 141.9, 141.8, 140.3, 132.8, 131.2, 131.0, 130.9, 129.4, 129.3, 128.1, 126.9, 126.8, 123.2, 121.1, 121.0. HRMS (ESI) calculated for C₂₀H₁₄BrN₂S₂ [M + H]⁺ 424.9782. Found: 424.9798.

4.2.3 6-Bromo-2,3-di[(E)-styryl]quinoxaline (4c). Use compound 1 (0.257 g, 1.373 mmol) and compound 3c (0.30 g, 1.144 mmol) to afford compound 4c (0.448 g, 1.084 mmol). Yield: 95%. Mp 190–191 °C. A yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 8.20 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 15.5 Hz, 1H), 7.99 (d, J = 15.5 Hz, 1H), 7.87 (d, J = 8.9 Hz, 1H), 7.72 (d, J = 8.9, 2.0 Hz, 1H), 7.67 (d, J = 7.7 Hz, 4H), 7.60 (d, J = 15.5 Hz, 1H), 7.59 (d, J = 15.5 Hz, 1H), 7.44 (d, J = 7.4 Hz, 1H), 7.43 (d, J = 7.4 Hz, 2H), 7.38 (t, J = 7.3 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 149.7, 149.2, 142.0, 140.3, 138.6, 138.3, 136.3, 136.2, 132.9, 131.1, 130.1, 129.3, 129.2, 128.9, 127.7, 127.6, 123.3, 122.2, 122.1. HRMS (ESI) calculated for C₂₄H₁₈BrN₂ [M + H]⁺ 413.0653. Found: 413.0640.

4.2.4 6-Bromo-2,3-bis[(E)-2-(naphthalen-2-yl)vinyl]quinoxaline (4d). Use Compound 1 (0.031 g, 0.165 mmol) and compound 3d (0.050 g, 0.138 mmol) to afford compound 4d (0.065 g, 0.126 mmol). Yield: 91%. Mp 234–235 °C. A yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 8.25 (d, J = 1.5 Hz, 1H), 8.20 (d, J = 15.5 Hz, 2H), 8.07 (s, 2H), 7.93–7.85 (m, 9H), 7.78–7.75 (m, 3H), 7.55–7.51 (br m, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 149.8, 149.4, 142.2, 140.4, 138.8, 138.5, 133.9, 133.8, 133.6, 132.9, 131.2, 130.2, 129.1, 129.0, 128.6, 128.5, 128.4, 127.8, 126.8, 126.7, 126.6, 123.7, 123.3, 122.4, 122.3. HRMS (ESI) calculated for C₃₂H₂₂BrN₂ [M + H]⁺ 513.0966. Found: 513.0971.

4.2.5 6-Bromo-2,3-bis[(E)-2-methoxystyryl]quinoxaline (4e). Use compound 1 (0.139 g, 0.744 mmol) and compound 3e (0.20 g, 0.620 mmol) to afford compound 4e (0.284 g, 0.601 mmol). Yield: 97%. Mp 182–183 °C. A yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 8.28 (d, J = 15.7 Hz, 1H), 8.27 (d, J = 15.7 Hz, 1H), 8.24 (d, J = 2.1 Hz, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.75 (d, J = 15.7 Hz, 1H), 7.73 (d, J = 15.7 Hz, 1H), 7.72 (dd, J = 8.7, 2.2 Hz, 1H), 7.70 (d, J = 7.6 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.02 (t, J = 7.5 Hz, 2H), 6.97 (d, J = 8.3 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) d 158.0, 150.5, 150.1, 142.2, 140.3, 133.9, 133.6, 132.5, 131.2, 130.3, 130.2, 130.1, 128.2, 125.6, 125.5, 123.5, 123.3, 122.9, 120.8, 111.1, 55.5. HRMS (ESI) calculated for C₂₆H₂₂BrN₂O₂ [M + H]⁺ 473.0865. Found: 473.0858.

4.2.6 6-Bromo-2,3-bis[(E)-3-nitrostyryl]quinoxaline (4f). Use compound 1 (0.032 g, 0.170 mmol) and compound 3f (0.050 g, 0.14 mmol) to afford compound 4f (0.061 g, 0.121 mmol). Yield: 85%. Mp 290–291 °C. A yellow solid. ¹H and ¹³C-NMR spectra were not obtained in perfect forms because of insolubility in CDCl₃, DMSO-d₆, CD₃OD, and acetone-d₆. HRMS (ESI) calculated for C₂₄H₁₆BrN₄O₄ [M + H]⁺ 503.0355. Found: 503.0335.

4.2.7 6-Bromo-2,3-bis[(E)-3-chlorostyryl]quinoxaline (4g). Use compound 1 (0.068 g, 0.362 mmol) and compound 3g (0.10 g, 0.302 mmol) to afford compound 4g (0.123 g, 0.254 mmol). Yield: 85%. Mp 94–95 °C. A yellow-brown solid.¹H NMR (600 MHz, CDCl₃) δ 8.21 (d, J = 1.9 Hz, 1H), 7.94 (d, J = 15.5 Hz, 2H), 7.88 (d, J = 8.9 Hz, 1H), 7.76 (dd, J = 8.9, 2.0 Hz, 1H), 7.66 (s, 2H), 7.57 (d, J = 3.6 Hz, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.54 (s, 1H), 7.39–7.34 (m, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 149.1, 148.7, 142.2, 140.4, 138.1, 138.0, 137.3, 137.0, 134.9, 133.3, 131.2, 130.2, 130.1, 129.2, 129.1, 127.4, 125.9, 125.8, 123.7, 123.2, 123.1. HRMS (ESI) calculated for C₂₄H₁₆BrCl₂N₂ [M + H]⁺ 480.9874. Found: 480.9870.

4.2.8 6-Bromo-2,3-bis[(E)-4-fluorostyryl]quinoxaline (4h). Use compound 1 (0.113 g, 0.603 mmol) and compound 3h (0.150 g, 0.503 mmol) to afford compound 4h (0.179 g, 0.399 mmol). Yield: 80%. Mp 212–213 °C. A yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 8.21 (s, 1H), 7.97 (d, J = 15.5 Hz, 1H), 7.96 (d, J = 15.5 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.75 (dd, J = 8.9, 1.2 Hz, 1H), 7.66 (d, J = 7.9 Hz, 2H), 7.65 (d, J = 7.9 Hz, 2H), 7.52 (d, J = 15.5 Hz, 1H), 7.50 (d, J = 15.5 Hz, 1H), 7.13 (t, J = 8.3 Hz, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 164.1, 163.3 (¹J_C–F = 249.0 Hz), 149.5, 149.0, 142.1, 140.3, 137.4, 137.1, 133.0, 132.6, 132.5, 131.1, 130.1, 129.4 (³J_C–F = 7.5 Hz), 129.3, 123.4, 121.8, 121.7, 116.0 (²J_C–F = 22.5 Hz). HRMS (ESI) calculated for C₂₄H₁₆BrF₂N₂ [M + H]⁺ 449.0465. Found: 449.0455.

4.2.9 6-Bromo-2,3-bis[(E)-4-chlorostyryl]quinoxaline (4i). Use compound 1 (0.068 g, 0.362 mmol) and compound 3i (0.10 g, 0.302 mmol) to afford compound 4i (0.081 g, 0.168 mmol). Yield: 56%. Mp 219–220 °C. A brown solid. ¹H NMR (600 MHz, CDCl₃) δ 8.22 (s, 1H), 7.96 (d, J = 15.5 Hz, 2H), 7.89 (d, J = 8.8 Hz, 1H), 7.76 (dd, J = 8.8, 1.7 Hz, 1H), 7.61 (d, J = 8.3 Hz, 4H), 7.57 (d, J = 15.5 Hz, 1H), 7.56 (d, J = 15.5 Hz, 1H), 7.41 (d, J = 8.1 Hz, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 149.3, 148.9, 142.2, 140.4, 137.4, 137.1, 135.1, 134.8, 134.7, 133.2, 131.1, 130.1, 129.2, 128.8, 128.7, 123.6, 122.5, 122.4. HRMS (ESI) calculated for C₂₄H₁₆BrCl₂N₂ [M + H]⁺ 480.9880. Found: 480.9874.

4.2.10 6-Bromo-2,3-bis[(E)-4-bromostyryl]quinoxaline (4j). Use compound 1 (0.053 g, 0.286 mmol) and compound 3j (0.10 g, 0.238 mmol) to afford compound 4j (0.116 g, 0.203 mmol). Yield: 85%. Mp 241–242 °C. A yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 8.22 (d, J = 2.1 Hz, 1H), 7.95 (d, J = 15.5 Hz, 1H), 7.94 (d, J = 15.5 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.76 (dd, J = 8.9, 2.1 Hz, 1H), 7.59–7.52 (m, 10H). ¹³C NMR (150 MHz, CDCl₃) δ 149.3, 148.9, 142.2, 140.4, 137.5, 137.2, 135.2, 135.1, 133.2, 132.1, 131.9, 131.5, 131.2, 130.4, 130.2, 129.1, 129.0, 123.6, 123.4, 123.3, 122.6, 122.5. HRMS (ESI) calculated for C₂₄H₁₆Br₃N₂ [M + H]⁺ 568.8864. Found: 568.8877.

4.2.11 6-Bromo-2,3-bis[(E)-4-nitrostyryl]quinoxaline (4k). Use compound 1 (0.032 g, 0.170 mmol) and compound 3k (0.050 g, 0.142 mmol) to afford compound 4k (0.061 g, 0.121 mmol). Yield: 85%. Mp 307–308 °C. A yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 8.32–8.28 (m, 5H), 8.11 (d, J = 15.2 Hz, 2H), 7.95 (d, J = 8.6 Hz, 1H), 7.84 (d, J = 8.0 Hz, 5H), 7.75 (d, J = 15.2 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 148.0, 147.9, 147.8, 142.3, 142.2, 140.5, 136.2, 135.9, 133.9, 131.2, 130.2, 128.1, 128.0, 125.7, 125.6, 124.4, 124.2. HRMS (ESI) calculated for C₂₄H₁₆BrN₄O₂ [M + H]⁺ 503.0355. Found: 503.0356.

4.2.12 6-Bromo-2,3-bis[(E)-4-methoxystyryl]quinoxaline (4l). Use compound 1 (0.069 g, 0.372 mmol) and compound 3l (0.10 g, 0.310 mmol) to afford compound 4l (0.092 g, 0.195 mmol). Yield: 63%. Mp 190–191 °C. A yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 8.20 (d, J = 2.0 Hz, 1H), 7.97 (d, J = 15.5 Hz, 1H), 7.96 (d, J = 15.5 Hz, 1H), 7.87 (d, J = 8.9 Hz, 1H), 7.71 (dd, J = 8.9, 2.0 Hz, 1H), 7.64 (d, J = 8.3 Hz, 4H), 7.50 (d, J = 15.5 Hz, 1H), 7.49 (d, J = 15.5 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 3.87 (s, 6H). ¹³C NMR (150 MHz, CDCl₃) δ 160.6, 160.5, 150.0, 149.6, 142.0, 140.2, 138.1, 137.8, 132.5, 131.0, 130.0, 129.2, 129.1, 122.8, 120.0, 119.9, 114.3, 55.4. HRMS (ESI) calculated for C₂₆H₂₂BrN₂O₂ [M + H]⁺ 473.0865. Found: 473.0864.

4.2.13 N,N′-[{(1E,1′E)-(6-bromoquinoxaline-2,3-diyl)bis(ethene-2,1-diyl)}bis(4,1-phenylene)] diacetamide (4m). Use compound 1 (0.029 g, 0.159 mmol) and compound 3m (0.050 g, 0.133 mmol) to afford compound 4m (0.057 g, 0.108 mmol). Yield: 81%. Mp 239 °C (decomposed). A yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 10.12 (s, 1H), 10.1 (s, 1H), 8.18 (d, J = 1.6 Hz, 1H), 7.95–7.82 (m, 10H), 7.68 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 8.0 Hz, 2H), 2.07 (s, 6H). ¹³C NMR (150 MHz, CDCl₃) δ 168.6, 149.7, 149.3, 141.5, 140.4, 140.3, 139.7, 137.6, 132.6, 130.8, 130.7, 130.4, 130.3, 128.9, 128.7, 122.3, 120.3, 120.3, 119.0. HRMS (ESI) calculated for C₂₈H₂₄BrN₄O₂ [M + H]⁺ 527.1083. Found: 527.1087.

4.2.14 2,3-Bis[(E)-2-(furan-2-yl)vinyl]-6-nitroquinoxaline (5a). Use compound 2 (0.076 g, 0.495 mmol) and compound 3a (0.10 g, 0.413 mmol) to afford compound 5a (0.124 g, 0.345 mmol). Yield: 84%. Mp 218–219 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.83 (d, J = 2.2 Hz, 1H), 8.37 (dd, J = 9.1, 2.2 Hz, 1H), 8.03 (d, J = 9.1 Hz, 1H), 7.92 (d, J = 15.2 Hz, 1H), 7.89 (d, J = 15.2 Hz, 1H), 7.56 (s, 1H), 7.55 (s, 1H), 7.52 (d, J = 15.2 Hz, 1H), 7.51 (d, J = 15.2 Hz, 1H), 6.68 (d, J = 3.1 Hz, 1H), 6.67 (d, J = 3.1 Hz, 1H), 6.53 (d, J = 1.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 152.6, 151.4, 150.8, 147.3, 144.3, 144.1, 144.0, 140.2, 130.0, 126.6, 126.0, 125.1, 122.5, 119.0, 118.8, 114.2, 113.9, 112.5, 112.4. HRMS (ESI) calculated for C₂₀H₁₄N₃O₄ [M + H]⁺ 360.0984. Found: 360.0995.

4.2.15 6-Nitro-2,3-bis[(E)-2-(thiophen-2-yl)vinyl]quinoxaline (5b). Use compound 2 (0.067 g, 0.437 mmol) and compound 3b (0.10 g, 0.364 mmol) to afford compound 5b (0.109 g, 0.279 mmol). Yield: 91%. Mp 223–224 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.86 (d, J = 2.4 Hz, 1H), 8.26 (d, J = 15.2 Hz, 1H), 8.23 (d, J = 15.2 Hz, 1H), 8.06 (d, J = 9.1 Hz, 1H), 7.41 (d, J = 5.1 Hz, 1H), 7.40 (d, J = 5.1 Hz, 1H), 7.38–7.34 (m, 4H), 7.13–7.10 (m, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 151.1, 150.6, 147.3, 144.0, 141.6, 140.2, 133.0, 132.4, 130.3, 130.1, 130.0, 128.3, 128.2, 127.8, 127.5, 125.1, 122.6, 120.3, 120.1. HRMS (ESI) calculated for C₂₀H₁₄N₃O₂S₂ [M + H]⁺ 392.0527. Found: 392.0540.

4.2.16 6-Nitro-2,3-di[(E)-styryl]quinoxaline (5c). Use compound 2 (0.070 g, 0.457 mmol) and compound 3c (0.10 g, 0.381 mmol) to afford compound 5c (0.109 g, 0.286 mmol). Yield: 75%. Mp 195–196 °C. A brown solid. ¹H NMR (600 MHz, CDCl₃) δ 8.91 (d, J = 2.5 Hz, 1H), 8.41 (dd, J = 9.1, 2.5 Hz, 1H), 8.13 (d, J = 15.5 Hz, 1H), 8.11 (d, J = 9.1 Hz, 1H), 8.09 (d, J = 15.5 Hz, 1H), 7.70 (d, J = 7.4 Hz, 1H), 7.62 (d, J = 15.5 Hz, 1H), 7.61 (d, J = 15.5 Hz, 1H), 7.46 (t, J = 7.4 Hz, 4H), 7.42–7.39 (m, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 151.6, 151.1, 147.4, 144.0, 140.5, 140.3, 140.0, 136.0, 135.9, 130.2, 129.9, 129.7, 129.0, 127.9, 127.8, 125.2, 122.7, 121.4, 121.1. HRMS (ESI) calculated for C₂₄H₁₈N₃O₂ [M + H]⁺ 380.1399. Found: 380.1405.

4.2.17 2,3-Bis[(E)-2-(naphthalen-2-yl)vinyl]-6-nitroquinoxaline (5d). Use compound 2 (0.051 g, 0.331 mmol) and compound 3d (0.10 g, 0.276 mmol) to afford compound 5d (0.094 g, 0.196 mmol). Yield: 70%. Mp 228–229 °C. An orange solid. ¹H NMR (600 MHz, CDCl₃) δ 8.93 (d, J = 2.3 Hz, 1H), 8.42 (dd, J = 9.1, 2.8 Hz, 1H), 8.31 (d, J = 15.4 Hz, 1H), 8.28 (d, J = 15.4 Hz, 1H), 8.14 (d, J = 9.1 Hz, 1H), 8.08 (s, 2H), 7.92–7.86 (m, 9H), 7.78 (d, J = 15.4 Hz, 1H), 7.77 (d, J = 15.4 Hz, 1H), 7.55–7.53 (m, 5H). ¹³C NMR (150 MHz, CDCl₃) δ 151.7, 151.2, 147.4, 144.1, 140.7, 140.3, 140.0, 134.1, 134.0, 133.5, 133.4, 130.2, 130.0, 129.5, 128.8, 128.7, 128.5, 127.8, 127.1, 127.0, 126.8, 126.7, 125.2, 123.6, 122.7, 121.6, 121.4. HRMS (ESI) calculated for C₃₂H₂₂N₃O₂ [M + H]⁺ 480.1712. Found: 480.1719.

4.2.18 2,3-Bis[(E)-2-methoxystyryl]-6-nitroquinoxaline (5e). Use compound 2 (0.057 g, 0.372 mmol) and compound 3e (0.10 g, 0.310 mmol) to afford compound 5e (0.122 g, 0.278 mmol). Yield: 90%. Mp 219–220 °C. An orange solid. ¹H NMR (600 MHz, CDCl₃) δ 8.95 (s, 1H), 8.40 (dd, J = 9.4, 2.5 Hz, 1H), 8.39 (d, J = 15.7 Hz, 1H), 8.38 (d, J = 15.7 Hz, 1H), 8.14 (d, J = 9.1 Hz, 1H), 7.78 (d, J = 15.7 Hz, 1H), 7.77 (d, J = 15.7 Hz, 1H), 7.71 (d, J = 7.5 Hz, 2H), 7.40–7.35 (m, 2H), 7.04 (t, J = 7.4 Hz, 2H), 6.99 (d, J = 8.3 Hz, 2H), 3.97 (s, 3H), 3.96 (s, 3H). ¹³C NMR (150 MHz, CDCl₃) δ 158.3, 158.2, 152.5, 152.0, 147.2, 135.8, 135.1, 130.9, 130.7, 130.2, 128.6, 128.4, 125.3, 125.2, 125.1, 122.7, 122.5, 122.4, 120.9, 120.8, 119.2, 55.6. HRMS (ESI) calculated for C₂₆H₂₂N₃O₄ [M + H]⁺ 440.1610. Found: 440.1594.

4.2.19 6-Nitro-2,3-bis[(E)-3-nitrostyryl]quinoxaline (5f). Use compound 2 (0.052 g, 0.341 mmol) and compound 3f (0.10 g, 0.284 mmol) to afford compound 5f (0.069 g, 0.146 mmol). Yield: 86%. Mp 197–198 °C. A yellow solid. ¹HNMR (600 MHz, CDCl₃) δ 8.99 (d, J = 2.4 Hz, 1H), 8.57 (d, J = 2.1 Hz, 2H), 8.51 (dd, J = 9.3, 2.7 Hz, 1H), 8.28–8.24 (m, 3H), 8.21 (dd, J = 9.3, 1.8 Hz, 2H), 8.06–8.01 (m, 2H), 7.76 (d, J = 15.6, 3.0 Hz, 2H), 7.67 (t, J = 7.8 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 150.6, 150.1, 148.9, 148.0, 144.1, 140.6, 138.2, 137.6, 137.5, 133.7, 133.5, 130.6, 130.1, 125.4, 124.2, 124.1, 124.0, 123.8, 123.5, 122.4, 122.2. HRMS (ESI) calculated for C₂₄H₁₆N₅O₆ [M + H]⁺ 470.1101. Found: 470.1094.

4.2.20 2,3-Bis[(E)-3-chlorostyryl]-6-nitroquinoxaline (5g). Use compound 2 (0.044 g, 0.289 mmol) and compound 3g (0.080 g, 0.242 mmol) to afford compound 5g (0.092 g, 0.205 mmol). Yield: 85%. Mp 200–201 °C. A brown solid. ¹H NMR (600 MHz, CDCl₃) δ 8.93 (s, 1H), 8.45 (d, J = 8.9 Hz, 1H), 8.15 (d, J = 8.9 Hz, 1H), 8.08 (d, J = 15.6 Hz, 1H), 8.05 (d, J = 15.6 Hz, 1H), 7.69 (s, 2H), 7.62–7.57 (br m, 4H), 7.45–7.38 (br m, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 151.1, 150.6, 147.7, 144.1, 140.4, 139.2, 138.6, 137.7, 135.0, 130.4, 129.7, 129.6, 127.7, 127.5, 126.2, 125.3, 123.0, 122.6, 122.4. HRMS (ESI) calculated for C₂₄H₁₆Cl₂N₃O₂ [M + H]⁺ 448.0620. Found: 448.0521.

4.2.21 2,3-Bis[(E)-4-fluorostyryl]-6-nitroquinoxaline (5h). Use compound 2 (0.062 g, 0.402 mmol) and compound 3h (0.10 g, 0.335 mmol) to afford compound 5h (0.080 g, 0.195 mmol). Yield: 83%. Mp 258–259 °C. An orange-yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 8.95 (s, 1H), 8.45 (d, J = 9.5 Hz, 1H), 8.15 (d, J = 9.5 Hz, 1H), 8.13 (d, J = 15.5 Hz, 1H), 8.09 (d, J = 15.5 Hz, 1H), 7.70 (br s, 4H), 7.56 (d, J = 15.5 Hz, 1H), 7.55 (d, J = 15.5 Hz, 1H), 7.16 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.3 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 167.0, 163.7 (¹J_C–F = 249.0 Hz), 163.6 (¹J_C–F = 249.0 Hz), 151.5, 151.0, 147.5, 144.0, 140.3, 139.4, 138.7, 132.2, 130.2, 130.0, 129.7 (²J_C–F = 24.0 Hz), 129.6 (³J_C–F = 7.5 Hz), 125.2, 122.8, 121.1 (²J_C–F = 28.5 Hz), 116.2 (⁴J_C–F = 4.5 Hz), 116.0 (⁴J_C–F = 4.5 Hz). HRMS (ESI) calculated for C₂₄H₁₆F₂N₃O₂ [M + H]⁺ 416.1222. Found: 416.1215.

4.2.22 2,3-Bis[(E)-4-chlorostyryl]-6-nitroquinoxaline (5i). Use compound 2 (0.031 g, 0.199 mmol) and compound 3h (0.055 g, 0.166 mmol) to afford compound 5h (0.062 g, 0.138 mmol). Yield: 89%. Mp 221–222 °C. A yellow-brown solid. ¹H NMR (600 MHz, CDCl₃) δ 8.93 (s, 1H), 8.44 (d, J = 7.4 Hz, 1H), 8.14 (d, J = 8.8 Hz, 1H), 8.11 (d, J = 15.8 Hz, 1H), 8.06 (d, J = 15.8 Hz, 1H), 7.64 (d, J = 6.8 Hz, 1H), 7.59 (d, J = 15.8 Hz, 1H), 7.58 (d, J = 15.8 Hz, 1H), 7.43 (d, J = 7.4 Hz, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 151.3, 150.8, 147.6, 144.1, 140.4, 139.3, 138.6, 135.7, 135.6, 134.4, 130.3, 129.3, 129.1, 129.0, 125.3, 122.9, 121.9, 121.7. HRMS (ESI) calculated for C₂₄H₁₆Cl₂N₃O₂ [M + H]⁺ 448.0620. Found: 448.0617.

4.2.23 2,3-Bis[(E)-4-bromostyryl]-6-nitroquinoxaline (5j). Use compound 2 (0.022 g, 0.143 mmol) and compound 3j (0.050 g, 0.119 mmol) to afford compound 5h (0.059 g, 0.111 mmol). Yield: 93%. Mp 247–248 °C. A yellow-brown solid. ¹H NMR (600 MHz, CDCl₃) δ 8.93 (d, J = 2.3 Hz, 1H), 8.44 (dt, J = 9.2, 1.2 Hz), 8.14 (d, J = 9.2 Hz, 1H), 8.08 (d, J = 15.4 Hz, 1H), 8.04 (d, J = 15.4 Hz, 1H), 7.62–7.55 (m, 11H). ¹³C NMR (150 MHz, CDCl₃) δ 150.9, 150.4, 147.2, 143.7, 140.0, 138.9, 138.3, 134.5, 134.4, 131.9, 131.8, 129.9, 128.9, 128.8, 124.9, 123.7, 123.5, 122.6, 121.5, 121.3. HRMS (ESI) calculated for C₂₄H₁₆Br₂N₃O₂ [M + H]⁺ 535.9609. Found: 535.9607.

4.2.24 6-Nitro-2,3-bis[(E)-4-nitrostyryl]quinoxaline (5k). Use compound 2 (0.052 g, 0.341 mmol) and compound 3k (0.10 g, 0.284 mmol) to afford compound 5k (0.108 g, 0.231 mmol). Yield: 81%. Mp 297–298 °C. A yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 9.00 (s, 1H), 8.53 (d, J = 9.1 Hz, 1H), 8.34 (d, J = 8.5 Hz, 4H), 8.25–8.20 (m, 3H), 7.87 (d, J = 8.2 Hz, 4H), 7.78 (d, J = 16.2 Hz, 1H), 7.76 (d, J = 16.2 Hz, 1H). No satisfaction of the ¹³C NMR spectrum was obtained due to solubility. HRMS (FAB) calculated for C₂₄H₁₆N₅O₆ [M + H]⁺ 470.1101. Found: 470.1101.

4.2.25 2,3-Bis[(E)-4-methoxystyryl]-6-nitroquinoxaline (5l). Use compound 2 (0.057 g, 0.372 mmol) and compound 3l (0.10 g, 0.335 mmol) to afford compound 5l (0.069 g, 0.184 mmol). Yield: 89%. Mp 144–145 °C. A dark-orange solid. ¹H NMR (600 MHz, CDCl₃) δ 8.85 (d, J = 2.4 Hz, 1H), 8.36 (dd, J = 9.1, 2.4 Hz, 1H), 8.05 (d, J = 15.2 Hz, 1H), 8.04 (d, J = 9.1 Hz, 1H), 8.02 (d, J = 15.2 Hz, 1H), 7.63 (d, J = 8.5 Hz, 4H), 7.45 (d, J = 15.4 Hz, 1H), 7.44 (d, J = 15.4 Hz, 1H), 6.97 (d, J = 8.5 Hz, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 161.0, 160.9, 151.9, 151.4, 147.1, 144.0, 140.1, 140.0, 139.3, 129.9, 129.5, 129.4, 128.9, 128.8, 125.0, 122.3, 119.1, 118.9, 114.4, 114.3, 55.4. HRMS (ESI) calculated for C₂₆H₂₂N₃O₄ [M + H]⁺ 440.1610. Found: 440.1613.

4.2.26 N,N′-[(1E,1′E)-(6-nitroquinoxaline-2,3-diyl)bis(ethene-2,1-diyl)bis(4,1-phenylene)]diacetamide (5m). Use compound 2 (0.024 g, 0.159 mmol) and compound 3m (0.050 g, 0.133 mmol) to afford compound 5m (0.060 g, 0.121 mmol). Yield: 91%. Mp 325–326 °C. An orange solid. ¹H NMR (600 MHz, CDCl₃) δ 10.15 (s, 1H), 10.14 (s, 1H), 8.66 (d, J = 2.1 Hz, 1H), 8.34 (d, J = 9.1 Hz, 1H), 8.09 (d, J = 9.1 Hz, 1H), 7.99 (d, J = 15.6 Hz, 1H), 7.95 (d, J = 15.6 Hz, 1H), 7.85–7.81 (m, 6H), 7.67 (d, J = 8.0 Hz, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 168.7, 151.7, 151.1, 146.8, 143.6, 140.9, 140.7, 139.6, 139.5, 138.9, 130.6, 130.5, 130.1, 129.3, 129.2, 124.4, 122.6, 119.9, 119.7, 119.0, 24.2. HRMS (FAB) calculated for C₂₈H₂₄N₅O₄ [M + H]⁺ 494.1828. Found: 494.1824.

4.3 Cell culture

Prof. Y. Su of National Yang-Ming Chiao Tung University, Taipei, Taiwan kindly provided human non-small lung cancer cells (A549 cells). The cells were stored in liquid nitrogen at −196 °C. After the cells were thawed, the cells were incubated at 37 °C in 5% CO₂ and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U per mL penicillin, 100 μg per mL streptomycin, 2 μM L-glutamine, and 1 mM sodium pyruvate.^42,43

4.4 In vitro cytotoxicity assay

The cell viability was evaluated with MTT assay, as previously reported to further assess the cytotoxicity.^32,43 In brief, the cells were plated in 96-well culture plates at a concentration of 3 × 10³ cells in 200 μl per well. After 24 hours, cells were treated with different concentrations (6.25, 12.5, 25, 50, and 100 μM) of all twenty-six synthesized compounds, and 5-fluorouracil (5-FU) was used as a positive control. After 72 hours, the attached cells were added with MTT reagent at 0.5 mg mL⁻¹ in 100 μL per well and incubated at 37 °C for 3 h. Then, the MTT reagent was removed, 100 μL of DMSO was added per well to dissolve the formazan metabolite, and the amount of formazan was measured by the absorbance at 570 nm, using an ELISA plate reader, TECAN Spark (Tecan Group Ltd) (μ Quant). The optical density recorded previously in the control group (untreated cells) was considered to be 100% cell viability.

4.5 Western blot

Western blot analysis was performed according to the method previously described.^32,43 Briefly, the cells were seeded to 6-well culture plates. After reaching 85–90% confluence, cells were treated with compound 4m (5, 10, and 20 μM) and 5-FU (5 μM). The cells were incubated for 48 h after treatment. Afterward, the cells were collected and lysed using radioimmunoprecipitation assay (RIPA) buffer. Lysates of total protein were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Then, the membranes were blocked with 2% bovine serum albumin (BSA) solution and incubated with anti-Bax, anti-Bcl-2 (Cell Signaling Inc., Danvers, MA, USA), anti-caspase-3, and anti-β-actin (GeneTex Inc., Irvine, CA, USA) primary antibodies at 4 °C overnight. Each membrane was washed with tris-buffered saline containing 0.1% Tween 20 (TBST) three times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. Finally, the membranes were developed with an enhanced chemiluminescence (ECL) detection kit and visualized by ImageQuant LAS 4000 Mini bio-molecular imager (GE Healthcare, MA, USA). ImageJ software quantified the band densities (BioTechniques, NY, USA).

Data availability

The data supporting this article have been included as part of the ESI.† No code was produced as part of this review.

Author contributions

Jia-Hua Liang performed the bioassay and analyzed the data and manuscript writing. Shu-Tse Cho conducted the isolation and structure elucidation of the constituents. Jih-Jung Chen and Tzenge-Lien Shih planned, designed, and organized this study's research and the manuscript's preparation. All authors read and approved the final version of the manuscript.

Conflicts of interest

The authors declare no conflicts of financial interest.

Acknowledgements

This research was supported by grants from the National Science and Technology Council (NSTC) and the Ministry of Science and Technology (MOST) of Taiwan (No. NSTC 112-2320-B-A49-028-MY3 and MOST 111-2113-M-032-004), awarded to J.-J. C. and T.-L. S., respectively.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Spectroscopic data of ¹H NMR and ¹³C NMR for new compounds. See DOI: https://doi.org/10.1039/d4ra04453c

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