Synthesis, biological evaluation and network pharmacology based studies of 1,3,4-oxadiazole bearing azaphenols as anticancer agents

Guifen Chen; Minjie Zhang; Yafang Chen; Yan Zhang; Guoyong Luo; Yi Long; Wude Yang; Xiang Yu

doi:10.1016/j.arabjc.2023.105386

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01 2023

:17;

105386

doi:

10.1016/j.arabjc.2023.105386

Synthesis, biological evaluation and network pharmacology based studies of 1,3,4-oxadiazole bearing azaphenols as anticancer agents

Guifen Chen^{^a,1}, Minjie Zhang^{^a,1}, Yafang Chen^{^a}, Yan Zhang^{^a,^b}, Guoyong Luo^{^a,^b}, Yi Long^{^a,^b}, Wude Yang^{^a,^b,⁎}, Xiang Yu^{^a,^b,⁎}

a College of Pharmacy, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China

b Guizhou Joint Laboratory for International Cooperation in Ethnic Medicine (Ministry of Education), Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China

⁎Corresponding authors at: College of Pharmacy, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China. yangwude476@gzy.edu.cn (Wude Yang), yuxiang@gzy.edu.cn (Xiang Yu)

Received: 2023-8-29, Accepted: 2023-10-22,

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

1 These authors contribute equally to this work.

Abstract

To discover novel and effective potential anticancer agents, a series of azophenol derivatives containing 1,3,4-oxadiazoles moiety was synthesized and investigated for their anticancer activities against several human cancer cell lines by MTT method. Their structures were characterized by ¹H NMR, ¹³C NMR, IR and HRMS spectral analyses. Among the prepared compounds, 5df displayed significant anti-proliferative activity against HCT116 cancer cells with an IC₅₀ value of 4.09 ± 0.04 µM. Moreover, this compound had low cytotoxicity against normal cells. Flow cytometric analysis indicated that compound 5df arrested the cell cycle at S phase and induced apoptosis in a dose-dependent manner. Additionally, network pharmacology analysis calculated that 5df might target several key proteins, including AKT serine/threonine kinase 1 (AKT1), SRC proto-oncogene, non-receptor tyrosine kinase (SRC). Furthermore, molecular docking study indicated that 5df exhibited potentially high binding affinity to these target proteins with binding energies lower than −8 kcal/mol. These findings provide valuable insights for the development of azophenol derivatives as potential anticancer agents.

Keywords

Azophenol

1,3,4-Oxadiazole

Anticancer activity

Network pharmacology

Molecular docking

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1 Introduction

Cancer is one of the leading causes of death in the twenty-first century and a critical challenge that needs to be addressed to increase human life expectancy (Bray et al., 2018). Recent statistics revealed a staggering 19.3 million new cancer cases and nearly ten million cancer-related deaths in 2020 (Sung et al., 2011). Colorectal cancer ranks as the third most prevalent cancer globally and the second deadliest in terms of mortality rate (Sawicki et al., 2021). The emergence and progression of colorectal cancer result from a complex interplay of factors, including age, family history, gender, geographical location, and personal medical history (Peng et al., 2018; Shen et al., 2018). Conventional treatment approaches for colorectal cancer encompass surgical intervention and chemotherapy. Chemotherapeutic agents induce DNA damage or activate diverse signaling pathways to prompt cancer cell demise, including cell cycle arrest, inhibition of global translation, blockage of DNA repair, and other mechanisms (Woods and Turchi, 2013). Hence, the discovery and advancement of novel chemical entities that are more efficacious and less toxic would signify a groundbreaking stride in cancer research.

Azo derivatives, characterized by their nitrogen-nitrogen double bond (—N⚌N—), rank among the most crucial chromophores and find diverse applications in the realms of science, industry, and pharmaceuticals (Benkhaya et al., 2020; Tahir et al., 2021). Presently, the synthesis of azo derivatives has attracted significant attention due to their varied bioactivities, such as antibacterial (Atay et al., 2017), antioxidant (Mohammadi et al., 2015), antifungal (Matada et al., 2020), anti-inflammatory (Manjunatha and Bodke, 2021), and antitubercular (Manjunatha et al., 2021). Furthermore, azo derivatives also displayed substantial anticancer potentials. For example, Keshavayya et al. reported a robust anticancer heterocyclic azo derivative, dimethyl-[4-(5-methyl-thiazol-2-ylazo)-phenyl]-amine (I, Fig. 1), which manifested potent anticancer effects against A-549 and K-562 cell lines (Ravi et al., 2020). Another novel azo derivative, II (Fig. 1), exhibited notable activity against the human colon cell line (HCT116) by inhibiting the growth of the cancerous cells (Maliyappa et al., 2020).

Design of the azophenol derivatives containing 1,3,4-oxadiazoles moiety.

On the other hand, 1,3,4-oxadiazole stands as a widely studied pharmacophore that has garnered significant research attention in recent years, owing to its metabolic characteristics and capacity to establish hydrogen bonds with receptor sites. These compounds serve as excellent bioisosteres of amides and esters, as the incorporation of the oxadiazole core with the azole (—N⚌C—O—) motif enhances lipophilicity. This property plays a pivotal role in facilitating the transmembrane diffusion of drugs, enabling them to effectively reach their intended targets (Bajaj et al., 2015; Andreani et al., 2001). These molecules exhibit diverse biological activities, including anti-inflammatory (Abd-Ellah et al., 2017), antidiabetic (Shingalapur et al., 2010), antianxiety (Harfenist et al., 1996), antifungal (Wang et al., 2021), antibacterial (Guo et al., 2019) and antitubercular etc (Ahsan et al., 2011). Notably, numerous distinct 1,3,4-oxadiazoles also displayed substantial anticancer properties against various cancer cell lines. For instance, compounds III and IV (Fig. 1) have demonstrated potent anticancer activities (Zhang et al., 2011; Abou-Seri, 2010).

The development of small molecules through molecular hybridization from known structural motifs is one of the current trends in drug discovery. It is anticipated that improved cytotoxicity may be achieved through the structural conjugation of two potent pharmacophoric units. However, there are few literature reports on the combination of azo group and 1,3,4-oxadiazole for the development of anti-tumor drug molecules. Drawing inspiration from the aforementioned facts and our prior research (Yu et al., 2021, 2023), we devised and synthesized a novel series of azophenol derivatives containing the 1,3,4-oxadiazoles moiety (Fig. 1) as potential anticancer agents. The in vitro anticancer activities of all target derivatives were evaluated via the MTT method against several human cancer cell lines, including lung cancer cells (A549), cervical carcinoma cells (HeLa), colon cancer cells (HCT116), hepatocellular carcinoma cells (HePG₂), breast cancer cells (MCF-7), HT1080 fibrosarcoma cells (HT1080). The mechanism of antiproliferative effects of this class of compounds was studied via cell cycle arrest and cell apoptosis assay. Additionally, network pharmacology and molecular docking study were conducted to identify potential targets of these derivatives. This study may provide lead molecules for the discovery and development of anticancer candidates.

2 Experimental

2.1

2.1 Chemistry

All reagents and solvents were of reagent grade or purified according to standard methods before use. Analytical thin-layer chromatography (TLC) was performed with silica gel plates using silica gel 60 GF₂₅₄ (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Melting points were determined on an X-5A micro melting point tester (Gongyi Kerui instrument Co., Ltd.) and were uncorrected. Infrared absorption spectra (IR) were recorded by IRTracer-100 (Shimadzu, Wikipedia, Japan). ¹H/¹³C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance neo 400 or 600 MHz instrument (Bruker, Bremerhaven, Germany) in CDCl₃ or DMSO‑d₆ using TMS (tetramethylsilane) as the internal standard. High-resolution mass spectrometry (HRMS) was determined by a Xevo G2-SQTOF instrument (Waters, Milford, MA, USA).

2.1.1

2.1.1 General procedure for the synthesis of (E)-N'-(2-nitrobenzylidene)arylhydrazides (2aa ∼ eg)

To a solution of 2-nitrobenzaldehydes (1a ∼ e, 1 mmol) and aryl hydrazides (1 mmol) in ethanol (EtOH, 10 mL), two drops of acetic acid (HOAc) was added, and the mixture was reflux for 1–5 h. When the reaction was complete, the mixture was cooled to room temperature until no more precipitate was observed. Then the solvent was removed under reduced pressure to give the intermediates (2aa ∼ eg). The compounds were not purified and went straight to the next step.

2.1.2

2.1.2 General procedure for the synthesis of 2-(2-nitrophenyl)-5-aryl-1,3,4-oxadiazoles (3aa ∼ eg)

The intermediates 3aa ∼ eg (1.1 mmol), K₂CO₃ (3.0 mmol, 414.6 mg) and iodine (1.1 mmol, 279.4 mg) were dissolved in dimethyl sulfoxide (DMSO, 10 mL), then the reaction mixture was stirred at 100 °C. When the reaction was complete according to TLC analysis, the mixture was treated with saturated Na₂S₂O₃ (20 mL) and extracted with ethyl acetate (EtOAc, 3 × 20 mL). The combined organic layer was washed with brine (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered and evaporated. The given residues were purified by flash chromatography on silica gel to get the compounds 3aa ∼ eg in 61–98 % yields. Their spectral data were provided in Supplementary Material.

2.1.3

2.1.3 General procedure for the synthesis of 2-(5-aryl-1,3,4-oxadiazol-2-yl)anilines (4aa ∼ eg)

A mixture of compounds 3aa ∼ eg (1 mmol), hydrated tin(II) chloride (5 mmol, 1128.5 mg) in EtOAc (10 mL) was reflux for 4-12 h. While the reaction was complete, the mixture was cooled to room temperature and adjusted to pH 8–9 with saturated NaHCO₃. Then the mixture was filtered and extracted with EtOAc (2 × 50 mL). The combined organic phase was washed with saturated brine (100 mL), dried over anhydrous Na₂SO₄, concentrated, and purified by flash chromatography on silica gel to obtain compounds 4aa ∼ eg in 48–93 % yields. Their spectral data were provided in Supplementary Material.

2.1.4

2.1.4 General procedure for the synthesis of (E)-4-((2-(5-aryl-1,3,4-oxadiazol-2-yl)phenyl)diazenyl) phenol derivatives (5aa ∼ eg)

To a mixture of compounds 4aa ∼ eg (1 mmol), water (6 mL) and concentrated HCl (12 mol/L, 0.5 mL) at 0 °C, a solution of sodium nitrite (NaNO₂, 1.2 mmol) in water (6 mL) was added dropwise while maintaining the temperature below 5 °C. After stirring for 0.5–1 h, a solution of diazonium chloride was prepared. Subsequently, The solution of diazonium chloride was added gradually to a mixture of phenol (1.1 mmol), sodium hydroxide (NaOH, 2 mmol), EtOH (10 mL) and water (10 mL) at 0–5 °C. After the addition of the above diazonium solution, the mixture was continued to stir for 4–6 h until a lot of precipitate was produced. The solid was collected, washed with water (3 × 10 mL), dried and purified by flash chromatography on silica gel to afford the target products 5aa ∼ eg in 38–90 % yields. The spectroscopic and analytical data of these compounds are as follows:

2.1.4.1

2.1.4.1 (E)-4-((2-(5-phenyl-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5aa)

Yield: 48 %, Red solid, M.p.: 281–283 ℃; IR cm⁻¹ (KBr): 1606, 1510, 1369, 1246, 1141, 837, 704; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.50 (s, 1H), 8.20 (d, J = 7.7 Hz, 1H), 8.03 (d, J = 6.8 Hz, 2H), 7.85 – 7.78 (m, 3H), 7.74 – 7.70 (m, 2H), 7.68 – 7.59 (m, 3H), 6.95 (d, J = 8.8 Hz, 2H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.3, 163.0, 161.2, 149.9, 145.2, 132.4, 131.7, 129.9 × 2, 129.1 × 2, 126.2 × 2, 125.1 × 2, 122.9, 120.4, 116.6, 115.6 × 2; HRMS (ESI) calcd for C₂₀H₁₄N₄O₂Na ([M + Na]⁺) 365.1008, found 365.1002.

2.1.4.2

2.1.4.2 (E)-4-((2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ab)

Yield: 89 %, Red solid, M.p.: 273–275 ℃; IR cm⁻¹ (KBr): 1589, 1498, 1384, 1284, 1143, 1095, 837, 731; ¹H NMR (600 MHz, DMSO‑d₆) δ 10.45 (s, 1H), 8.19 (dd, J = 7.6, 1.4 Hz, 1H), 8.09 – 8.07 (m, 2H), 7.82 (d, J = 8.8 Hz, 2H), 7.80 – 7.77 (m, 1H), 7.73 – 7.70 (m, 2H), 7.47 (t, J = 8.8 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.4, 163.9, 163.3, 162.7, 161.6, 150.3, 145.6, 132.7, 130.3, 130.2, 129.3, 125.5 × 2, 120.8, 120.0, 117.0, 116.8, 116.6, 116.0 × 2; HRMS (ESI) calcd for C₂₀H₁₃FN₄O₂Na ([M + Na]⁺) 383.0914, found 383.0909.

2.1.4.3

2.1.4.3 (E)-4-((2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ac)

Yield: 73 %, Red solid, M.p.: 238–239 ℃; IR cm⁻¹ (KBr): 1591, 1463, 1382, 1286, 1143, 835,754, 731; ¹H NMR (600 MHz, DMSO‑d₆) δ 10.47 (s, 1H), 8.19 (dd, J = 7.8, 1.4 Hz, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.82 (d, J = 8.8 Hz, 2H), 7.78 – 7.77 (m, 1H), 7.73 – 7.67 (m, 4H), 6.96 (d, J = 8.8 Hz, 2H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 163.5, 163.1, 161.2, 149.9, 145.2, 136.4, 132.4, 129.9, 129.8, 129.2 × 2, 127.9 × 2, 125.1 × 2, 121.8, 120.3, 116.6, 115.6 × 2; HRMS (ESI) calcd for C₂₀H₁₃ClN₄O₂Na ([M + Na]⁺) 399.0619, found 399.0613.

2.1.4.4

2.1.4.4 (E)-4-((2-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ad)

Yield: 38 %, Red solid, M.p.: 214–216 ℃; IR cm⁻¹ (KBr): 1587, 1508, 1313, 1282, 1143, 842, 725; ¹H NMR (600 MHz, DMSO‑d₆) δ 10.47 (s, 1H), 8.22 (dd, J = 8.0, 1.5 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 7.5 Hz, 1H), 7.79 – 7.77 (m, 2H), 7.72 – 7.69 (m, 2H), 7.49 – 7.44 (m, 2H), 6.96 (d, J = 8.8 Hz, 2H), 2.37 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.8, 163.3, 161.6, 150.3, 145.6, 138.9, 132.8, 132.7, 130.2, 130.1, 129.3, 126.8, 125.5 × 2, 123.7, 123.2, 120.6, 117.0, 116.0 × 2, 20.8; HRMS (ESI) calcd for C₂₁H₁₆N₄O₂Na ([M + Na]⁺) 379.1165, found 379.1159.

2.1.4.5

2.1.4.5 (E)-4-((2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ae)

Yield: 89 %, Red solid, M.p.: 253–255 ℃; IR cm⁻¹ (KBr): 1598, 1508, 1327, 1278, 1143, 854, 731; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.50 (s, 1H), 8.21 (d, J = 8.9 Hz, 1H), 7.84 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.9 Hz, 1H), 7.75 – 7.68 (m, 2H), 7.61 (d, J = 7.7 Hz, 1H), 7.55 – 7.46 (m, 2H), 7.22 (dd, J = 8.3, 2.7 Hz, 1H), 6.95 (d, J = 8.9 Hz, 2H), 3.80 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.6, 163.4, 161.6, 159.7, 150.3, 145.6, 132.8, 130.7, 130.3, 130.2, 125.5 × 2, 124.5, 120.7, 118.8, 118.1, 117.0, 116.0 × 2, 111.2, 55.33; HRMS (ESI) calcd for C₂₁H₁₆N₄O₃Na ([M + Na]⁺) 395.1114, found 395.1105.

2.1.4.6

2.1.4.6 (E)-4-((2-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5af)

Yield: 69 %, Red solid, M.p.: 289–291 ℃; IR cm⁻¹ (KBr): 1591, 1504, 1386, 1286, 1143, 837, 721; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.45 (s, 1H), 8.18 (d, J = 7.4 Hz, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.77 (t, J = 7.6 Hz, 1H), 7.71 – 7.68 (m, 2H), 7.57 (d, J = 8.1 Hz, 1H), 7.48 (s, 1H), 7.13 (d, J = 8.1 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 6.18 (s, 2H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.5, 162.9, 161.6, 150.5, 150.2, 148.2, 145.6, 132.6, 130.2, 130.1, 125.5 × 2, 121.8, 120.8, 116.9, 116.0 × 2, 115.9, 109.1, 106.2, 102.1; HRMS (ESI) calcd for C₂₁H₁₄N₄O₄Na ([M + Na]⁺) 409.0907, found 409.0901.

2.1.4.7

2.1.4.7 (E)-4-((2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ag)

Yield: 59 %, Red solid, M.p.: 250–251 ℃; IR cm⁻¹ (KBr): 1589, 1504, 1328, 1280, 1141, 840, 723; ¹H NMR (400 MHz, DMSO‑d₆) δ 9.18 (d, J = 2.3 Hz, 1H), 8.82 (d, J = 4.9 Hz, 1H), 8.39 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.84 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 7.3 Hz, 1H), 7.73 – 7.64 (m, 3H), 6.94 (d, J = 8.5 Hz, 2H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 163.9, 163.2, 161.9, 152.7, 150.6, 147.4, 145.8, 134.3, 133.1, 130.5, 130.4, 125.7 × 2, 124.6, 120.7, 120.1, 117.2, 116.2 × 2; HRMS (ESI) calcd for C₁₉H₁₃N₅O₂Na ([M + Na]⁺) 366.0961, found 366.0955.

2.1.4.8

2.1.4.8 (E)-4-((4-methoxy-2-(5-phenyl-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ba)

Yield: 55 %, Green solid, M.p.: 256–257 ℃; IR cm⁻¹ (KBr): 1598, 1504, 1332, 1278, 1141,831, 725; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.33 (s, 1H), 8.06 (d, J = 6.6 Hz, 2H), 7.82 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.70 – 7.61 (m, 4H), 7.34 (dd, J = 9.0, 2.9 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 3.95 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.0, 163.6, 161.2, 160.8, 145.7, 144.2, 132.3, 129.7 × 2, 126.8 × 2, 125.2 × 2, 123.5, 123.4, 119.1, 118.5, 116.1 × 2, 114.4, 56.2; HRMS (ESI) calcd for C₂₁H₁₆N₄O₃Na ([M + Na]⁺) 395.1114, found 395.1107.

2.1.4.9

2.1.4.9 (E)-4-((2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-4-methoxyphenyl)diazenyl)phenol (5bb)

Yield: 73 %, Brown solid, M.p.: 253–255 ℃; IR cm⁻¹ (KBr): 1597, 1496, 1328, 1278, 1141, 1091, 833, 734; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.32 (s, 1H), 8.13 – 8.09 (m, 2H), 7.81 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 2.8 Hz, 1H), 7.48 (t, J = 8.8 Hz, 2H), 7.34 (dd, J = 9.0, 2.9 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 3.95 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.6, 164.3, 163.5, 163.1, 161.2, 160.8, 145.7, 144.2, 129.5, 125.2 × 2, 123.3, 120.2, 119.0, 118.5, 117.1, 116.8, 116.1 × 2, 114.4, 56.2; HRMS (ESI) calcd for C₂₁H₁₅FN₄O₃Na ([M + Na]⁺) 413.1020, found 413.1008.

2.1.4.10

2.1.4.10 (E)-4-((2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-methoxyphenyl)diazenyl)phenol (5bc)

Yield: 46 %, Red solid, M.p.: 233–235 ℃; IR cm⁻¹ (KBr): 1600, 1504, 1325, 1280, 1141, 833, 750, 729; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.33 (s, 1H), 8.06 (d, J = 7.7 Hz, 2H), 7.85 – 7.63 (m, 6H), 7.34 (d, J = 11.7 Hz, 1H), 6.91 (d, J = 8.4 Hz, 2H), 3.95 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.3, 163.7, 161.2, 160.8, 145.7, 144.2, 137.1, 129.9 × 2, 128.6 × 2, 125.2 × 2, 123.3, 122.4, 119.1, 118.5, 116.1 × 2, 114.5, 56.2; HRMS (ESI) calcd for C₂₁H₁₅ClN₄O₃Na ([M + Na]⁺) 429.0724, found 429.0718.

2.1.4.11

2.1.4.11 (E)-4-((4-methoxy-2-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5bd)

Yield: 60 %, Brown solid, M.p.: 201–202 ℃; IR cm⁻¹ (KBr): 1598, 1506, 1328, 1280, 1141, 835, 725; ¹H NMR (400 MHz, DMSO‑d₆) δ 7.91 – 7.75 (m, 5H), 7.67 (s, 1H), 7.49 (d, J = 9.0 Hz, 2H), 7.34 (d, J = 9.2 Hz, 1H), 6.91 (d, J = 8.1 Hz, 2H), 3.95 (s, 3H), 2.39 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.0, 163.4, 161.1, 160.6, 145.5, 144.0, 139.0, 132.7, 129.4, 126.9, 125.0 × 2, 123.8, 123.2, 123.0, 118.9, 118.3, 115.9 × 2, 114.1, 56.0, 20.8; HRMS (ESI) calcd for C₂₂H₁₈N₄O₃Na ([M + Na]⁺) 409.1271, found 409.1264.

2.1.4.12

2.1.4.12 (E)-4-((4-methoxy-2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5be)

Yield: 90 %, Green solid, M.p.: 202–203 ℃; IR cm⁻¹ (KBr): 1589, 1506, 1330, 1290, 1141, 839, 729; ¹H NMR (400 MHz, DMSO‑d₆) δ 7.81 (d, J = 9.0 Hz, 1H), 7.76 (d, J = 8.9 Hz, 2H), 7.66 (d, J = 2.8 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.57 – 7.51 (m, 2H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 7.23 (dd, J = 8.3, 2.7 Hz, 1H), 6.93 (d, J = 8.9 Hz, 2H), 3.95 (s, 3H), 3.82 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.8, 163.5, 161.5, 160.5, 159.7, 145.4, 144.1, 130.8, 125.0 × 2, 124.5, 123.0, 119.1, 118.9, 118.4, 118.2, 116.1 × 2, 114.2, 111.4, 56.0, 55.4; HRMS (ESI) calcd for C₂₂H₁₈N₄O₄Na ([M + Na]⁺) 425.1220, found 425.1209.

2.1.4.13

2.1.4.13 (E)-4-((2-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)-4-methoxyphenyl)diazenyl)phenol (5bf)

Yield: 61 %, Brown solid, M.p.: 271–272 ℃; IR cm⁻¹ (KBr): 1589, 1502, 1325, 1288, 1141, 837, 731; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.42 (s, 1H), 7.80 – 7.74 (m, 3H), 7.64 – 7.57 (m, 2H), 7.52 (d, J = 1.8 Hz, 1H), 7.33 (dd, J = 9.0, 2.9 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 6.93 (d, J = 8.9 Hz, 2H), 6.19 (s, 2H), 3.95 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.6, 162.9, 161.1, 160.6, 150.5, 148.2, 145.5, 143.9, 125.0 × 2, 123.2, 121.9, 118.8, 118.3, 116.9, 115.9 × 2, 114.1, 109.2, 106.3, 102.2, 56.0; HRMS (ESI) calcd for C₂₂H₁₆N₄O₅Na ([M + Na]⁺) 439.1012, found 439.1010.

2.1.4.14

2.1.4.14 (E)-4-((4-methoxy-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5bg)

Yield: 45 %, Red solid, M.p.: 288–289 ℃; IR cm⁻¹ (KBr): 1597, 1504, 1336, 1280, 1141, 839, 725; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.43 (s, 1H), 9.22 (s, 1H), 8.84 (s, 1H), 8.42 (d, J = 7.5 Hz, 1H), 7.82 – 7.75 (m, 3H), 7.68 (s, 2H), 7.35 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 3.96 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 163.7, 163.1, 161.1, 160.5, 152.6, 147.3, 145.5, 144.0, 134.2, 125.0 × 2, 124.4, 122.8, 119.9, 119.0, 118.4, 115.9 × 2, 114.2, 56.0; HRMS (ESI) calcd for C₂₀H₁₅N₅O₃Na ([M + Na]⁺) 396.1067, found 396.1056.

2.1.4.15

2.1.4.15 (E)-4-((4-ethoxy-2-(5-phenyl-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ca)

Yield: 76 %, Green solid, M.p.: 242–244 ℃; IR cm⁻¹ (KBr): 1597, 1477, 1332, 1240, 1141, 833, 725; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.36 (s, 1H), 8.06 (d, J = 6.8 Hz, 2H), 7.78 – 7.75 (m, 3H), 7.72 – 7.60 (m, 4H), 7.32 (dd, J = 9.0, 2.8 Hz, 1H), 6.91 (d, J = 8.4 Hz, 2H), 4.22 (q, J = 7.0 Hz, 2H), 1.41 (t, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.8, 163.4, 161.0, 159.9, 145.6, 143.9, 132.2, 129.5 × 2, 126.6 × 2, 125.0 × 2, 123.3, 123.2, 119.2, 118.4, 115.9 × 2, 114.7, 64.1, 14.5; HRMS (ESI) calcd for C₂₂H₁₈N₄O₃Na ([M + Na]⁺) 409.1271, found 409.1263.

2.1.4.16

2.1.4.16 (E)-4-((4-ethoxy-2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5cb)

Yield: 74 %, Red solid, M.p.: 281–283 ℃; IR cm⁻¹ (KBr): 1595 1496, 1328, 1278, 1141, 1093, 833, 734; ¹H NMR (400 MHz, DMSO‑d₆) δ 8.13 – 8.10 (m, 2H), 7.81 (d, J = 9.0 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 2.8 Hz, 1H), 7.48 (t, J = 8.9 Hz, 2H), 7.33 (dd, J = 9.0, 2.8 Hz, 1H), 6.89 (d, J = 8.8 Hz, 2H), 4.23 (q, J = 6.9 Hz, 2H), 1.41 (t, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.4, 164.0, 163.3, 162.9, 161.4, 159.8, 145.3, 143.9, 129.3, 125.0 × 2, 123.1, 120.0, 119.1, 118.3, 116.9, 116.6, 116.0 × 2, 114.7, 64.1, 14.5; HRMS (ESI) calcd for C₂₂H₁₇FN₄O₃Na ([M + Na]⁺) 427.1176, found 427.1165.

2.1.4.17

2.1.4.17 (E)-4-((2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-ethoxyphenyl)diazenyl)phenol (5 cc)

Yield: 81 %, Brown solid, M.p.: 260–262 ℃; IR cm⁻¹ (KBr): 1600, 1483, 1327, 1278, 1141, 833, 750, 729; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.31 (s, 1H), 8.06 (d, J = 8.6 Hz, 2H), 7.80 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 2.9 Hz, 1H), 7.32 (dd, J = 9.1, 2.8 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 4.22 (q, J = 7.0 Hz, 2H), 1.40 (t, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.0, 163.4, 161.0, 159.9, 145.5, 143.8, 136.8, 129.6 × 2, 128.3 × 2, 125.0 × 2, 123.0, 122.2, 119.2, 118.3, 115.9 × 2, 114.7, 64.1, 14.5. HRMS (ESI) calcd for C₂₂H₁₇ClN₄O₃Na ([M + Na]⁺) 443.0881, found 443.0873.

2.1.4.18

2.1.4.18 (E)-4-((4-ethoxy-2-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5 cd)

Yield: 63 %, Green solid, M.p.: 220–222 ℃; IR cm⁻¹ (KBr): 1597, 1506, 1325, 1278, 1143, 833, 684; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.33 (s, 1H), 7.87 – 7.82 (m, 2H), 7.79 – 7.76 (m, 3H), 7.64 (d, J = 2.8 Hz, 1H), 7.53 – 7.45 (m, 2H), 7.31 (dd, J = 9.1, 2.8 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 4.22 (q, J = 7.0 Hz, 2H), 2.39 (s, 3H), 1.41 (t, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.9, 163.3, 161.0, 159.8, 145.5, 143.8, 138.9, 132.7, 129.3, 126.9, 125.0 × 2, 123.7, 123.2, 123.0, 119.1, 118.3, 115.9 × 2, 114.5, 64.0, 20.8, 14.5; HRMS (ESI) calcd for C₂₃H₂₀N₄O₃Na ([M + Na]⁺) 423.1427, found 423.1417.

2.1.4.19

2.1.4.19 (E)-4-((4-ethoxy-2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ce)

Yield: 82 %, Red solid, M.p.: 230–232 ℃; IR cm⁻¹ (KBr): 1598, 1467, 1325, 1278, 1141, 833, 729; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.33 (s, 1H), 7.78 (t, J = 8.4 Hz, 3H), 7.68 – 7.60 (m, 2H), 7.55 – 7.49 (m, 2H), 7.31 (dd, J = 9.1, 2.8 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 6.91 (d, J = 8.5 Hz, 2H), 4.22 (q, J = 6.9 Hz, 2H), 3.82 (s, 3H), 1.40 (t, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.7, 163.4, 161.0, 159.9, 159.7, 145.5, 143.9, 130.7, 125.0 × 2, 124.5, 123.1, 119.1, 118.9, 118.3, 118.1, 115.9 × 2, 114.6, 111.3, 64.1, 55.3, 14.5. HRMS (ESI) calcd for C₂₃H₂₀N₄O₄Na ([M + Na]⁺) 439.1376, found 439.1364.

2.1.4.20

2.1.4.20 (E)-4-((2-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)-4-ethoxyphenyl)diazenyl)phenol (5cf)

Yield: 71 %, Red solid, M.p.: 251–253 ℃; IR cm⁻¹ (KBr): 1597 1502, 1323, 1278, 1141, 833, 731; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.32 (s, 1H), 7.77 (t, J = 8.6 Hz, 3H), 7.64 – 7.57 (m, 2H), 7.52 (s, 1H), 7.30 (dd, J = 9.0, 2.8 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 6.18 (s, 2H), 4.21 (q, J = 7.0 Hz, 2H), 1.40 (t, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.5, 162.9, 160.9, 159.9, 150.5, 148.1, 145.5, 143.8, 125.0 × 2, 123.2, 121.8, 119.0, 118.2, 116.9, 115.9 × 2, 114.9, 109.1, 106.2, 102.1, 64.0, 14.5. HRMS (ESI) calcd for C₂₃H₁₈N₄O₅Na ([M + Na] ⁺) 453.1169, found 453.1163.

2.1.4.21

2.1.4.21 (E)-4-((4-ethoxy-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5cg)

Yield: 74 %, Red solid, M.p.: 269–271 ℃; IR cm⁻¹ (KBr): 1598 1504, 1338, 1280, 1143, 833, 727; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.32 (s, 1H), 9.23 (s, 1H), 8.83 (d, J = 3.7 Hz, 1H), 8.42 (d, J = 8.1 Hz, 1H), 7.78 – 7.75 (m, 3H), 7.68 – 7.61 (m, 2H), 7.33 (dd, J = 9.0, 2.9 Hz, 1H), 6.91 (d, J = 8.9 Hz, 2H), 4.23 (q, J = 6.9 Hz, 2H), 1.41 (t, J = 6.9 Hz, 3H); ¹³C NMkR (101 MHz, DMSO‑d₆) δ 163.6, 163.0, 161.0, 159.8, 152.6, 147.3, 145.5, 143.9, 134.2, 125.0 × 2, 124.4, 122.8, 119.9, 119.2, 118.3, 115.9 × 2, 114.7, 64.1, 14.4; HRMS (ESI) calcd for C₂₁H₁₇N₅O₃Na ([M + Na]⁺) 410.1223, found 410.1213.

2.1.4.22

2.1.4.22 (E)-4-((2-(5-phenyl-1,3,4-oxadiazol-2-yl)-4-propoxyphenyl)diazenyl)phenol (5da)

Yield: 82 %, Red solid, M.p.: 247–248 ℃; IR cm⁻¹ (KBr): 1598, 1467, 1330, 1236, 1141, 844, 721; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.34 (s, 1H), 8.06 (d, J = 7.3 Hz, 2H), 7.81 – 7.75 (m, 3H), 7.69 – 7.59 (m, 4H), 7.32 (d, J = 6.2 Hz, 1H), 6.92 (d, J = 8.4 Hz, 2H), 4.11 (t, J = 6.6 Hz, 2H), 1.80 (h, J = 7.1 Hz, 2H), 1.03 (t, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.6, 163.1, 160.8, 159.2, 145.3, 143.6, 131.9, 129.2 × 2, 126.4 × 2, 124.8 × 2, 123.1, 123.0, 118.9, 118.0, 115.7, 115.6, 114.5, 69.6, 21.7, 10.1; HRMS (ESI) calcd for C₂₃H₂₀N₄O₃Na ([M + Na]⁺) 423.1427, found 423.1419.

2.1.4.23

2.1.4.23 (E)-4-((2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-4-propoxyphenyl)diazenyl)phenol (5db)

Yield: 74 %, Green solid, M.p.: 235–236 ℃; IR cm⁻¹ (KBr): 1597 1496, 1328, 1278, 1141, 1093, 839, 734; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.33 (s, 1H), 8.11 (s, 2H), 7.80 – 7.773 (m, 3H), 7.62 (s, 1H), 7.48 (t, J = 8.6 Hz, 2H), 7.31 (d, J = 9.2 Hz, 1H), 6.92 (d, J = 8.5 Hz, 2H), 4.10 (t, J = 6.4 Hz, 2H), 1.81 (p, J = 6.8 Hz, 2H), 1.03 (t, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.6, 164.2, 163.5, 163.1, 161.1, 160.2, 145.7, 144.0, 129.5, 125.1 × 2, 123.3, 120.2, 119.2, 118.4, 117.0, 116.7, 116.1 × 2, 114.9, 70.0, 22.0, 10.4. HRMS (ESI) calcd for C₂₃H₁₉FN₄O₃Na ([M + Na]⁺) 441.1333, found 441.1327.

2.1.4.24

2.1.4.24 (E)-4-((2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-propoxyphenyl)diazenyl)phenol (5dc)

Yield: 64 %, Red solid, M.p.: 209–210 ℃; IR cm⁻¹ (KBr): 1600, 1483, 1328, 1284, 1141, 833, 752, 731; ¹H NMR (400 MHz, DMSO‑d₆) δ 8.05 (d, J = 8.6 Hz, 2H), 7.79 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 8.9 Hz, 2H), 7.69 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 2.8 Hz, 1H), 7.31 (dd, J = 9.0, 2.8 Hz, 1H), 6.93 (d, J = 8.8 Hz, 2H), 4.10 (t, J = 6.5 Hz, 2H), 1.80 (q, J = 7.0 Hz, 2H), 1.03 (t, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.0, 163.5, 161.0, 160.0, 145.5, 143.9, 136.8, 129.6 × 2, 128.3 × 2, 124.9 × 2, 123.0, 122.2, 119.1, 118.3, 115.9 × 2, 114.7, 69.8, 21.9, 10.2. HRMS (ESI) calcd for C₂₃H₁₉ClN₄O₃Na ([M + Na]⁺) 457.1037, found 457.1030.

2.1.4.25

2.1.4.25 (E)-4-((4-propoxy-2-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5dd)

Yield: 59 %, Red solid, M.p.: 214–216 ℃; IR cm⁻¹ (KBr): 1598, 1506, 1328, 1280, 1141, 842, 727; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.34 (s, 1H), 7.89 – 7.82 (m, 2H), 7.78 (d, J = 8.8 Hz, 3H), 7.65 (d, J = 2.8 Hz, 1H), 7.53 – 7.43 (m, 2H), 7.32 (dd, J = 9.0, 2.8 Hz, 1H), 6.92 (d, J = 8.5 Hz, 2H), 4.11 (t, J = 6.6 Hz, 2H), 2.39 (s, 3H), 1.80 (q, J = 7.0 Hz, 2H), 1.03 (t, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.9, 163.3, 160.9, 160.0, 145.5, 143.8, 138.9, 132.6, 129.3, 126.8, 124.9 × 2, 123.7, 123.2, 123.0, 119.1, 118.3, 115.8 × 2, 114.5, 69.8, 21.8, 20.7, 10.2. HRMS (ESI) calcd for C₂₄H₂₂N₄O₃Na ([M + Na]⁺) 437.1584, found 437.1575.

2.1.4.26

2.1.4.26 (E)-4-((2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-4-propoxyphenyl)diazenyl)phenol (5de)

Yield: 73 %, Red solid, M.p.: 198–199 ℃; IR cm⁻¹ (KBr): 1595, 1465, 1330, 1273, 1145, 842, 731; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.35 (s, 1H), 7.79 – 7.74 (m, 3H), 7.64 – 7.61 (m, 2H), 7.55 – 7.50 (m, 2H), 7.31 (dd, J = 9.1, 2.8 Hz, 1H), 7.23 (dd, J = 8.3, 1.6 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 4.11 (t, J = 6.5 Hz, 2H), 3.82 (s, 3H), 1.80 (q, J = 7.0 Hz, 2H), 1.03 (t, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.1, 163.8, 161.4, 160.4, 160.1, 145.9, 144.3, 131.1, 125.3 × 2, 124.9, 123.4, 119.5, 119.2, 118.7, 118.4, 116.3 × 2, 115.1, 111.7, 70.2, 55.7, 22.3, 10.6. HRMS (ESI) calcd for C₂₄H₂₂N₄O₄Na ([M + Na]⁺) 453.1533, found 453.1524.

2.1.4.27

2.1.4.27 (E)-4-((2-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)-4-propoxyphenyl)diazenyl)phenol (5df)

Yield: 77 %, Green solid, M.p.: 251–252 ℃; IR cm⁻¹ (KBr): 1598 1502, 1384, 1280, 1141, 842, 731; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.37 (s, 1H), 7.78 – 7.74 (m, 3H), 7.63 – 7.56 (m, 2H), 7.53 (s, 1H), 7.32 (dd, J = 9.0, 2.9 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 6.91 (d, J = 8.9 Hz, 2H), 6.18 (s, 2H), 4.12 (t, J = 6.5 Hz, 2H), 1.80 (h, J = 6.9 Hz, 2H), 1.03 (t, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.1, 163.5, 161.6, 160.6, 151.1, 148.8, 146.1, 144.4, 125.5 × 2, 123.8, 122.4, 119.6, 118.9, 117.6, 116.5 × 2, 115.2, 109.7, 106.8, 102.7, 70.4, 22.5, 10.8. HRMS (ESI) calcd for C₂₄H₂₀N₄O₅Na ([M + Na]⁺) 467.1325, found 467.1320.

2.1.4.28

2.1.4.28 (E)-4-((4-propoxy-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5dg)

Yield: 52 %, Red solid, M.p.: 237–238 ℃; IR cm⁻¹ (KBr): 1597 1504, 1384, 1282, 1139, 840, 725; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.48 (s, 1H), 9.25 (d, J = 2.2 Hz, 1H), 8.86 (d, J = 4.9 Hz, 1H), 8.48 (d, J = 8.1 Hz, 1H), 7.80 – 7.71 (m, 4H), 7.66 (d, J = 2.8 Hz, 1H), 7.33 (dd, J = 9.0, 2.8 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H), 4.12 (t, J = 6.5 Hz, 2H), 1.81 (p, J = 7.1 Hz, 2H), 1.03 (t, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 163.5, 162.6, 160.9, 159.8, 151.7, 146.5, 145.3, 143.7, 134.6, 124.7 × 2, 124.4, 122.5, 119.9, 119.0, 118.1, 115.7 × 2, 114.6, 69.6, 21.6, 10.0. HRMS (ESI) calcd for C₂₂H₁₉N₅O₃Na ([M + Na]⁺) 424.1380, found 424.1371.

2.1.4.29

2.1.4.29 (E)-4-((4-(allyloxy)-2-(5-phenyl-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ea)

Yield: 65 %, Green solid, M.p.: 238–239 ℃; IR cm⁻¹ (KBr): 1597, 1504, 1330, 1276, 1240, 1141, 835, 727; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.33 (s, 1H), 8.07 (d, J = 6.9 Hz, 2H), 7.81 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.70 – 7.61 (m, 4H), 7.36 (d, J = 9.1 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 6.16 – 6.06 (m, 1H), 5.48 (d, J = 17.3 Hz, 1H), 5.34 (d, J = 10.5 Hz, 1H), 4.78 (d, J = 5.1 Hz, 2H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.3, 163.8, 161.5, 159.9, 146.0, 144.5, 133.5, 132.6, 129.9 × 2, 127.0 × 2, 125.5 × 2, 123.8, 123.6, 119.8, 118.8, 118.5, 116.4 × 2, 115.5, 69.4; HRMS (ESI) calcd for C₂₃H₁₈N₄O₃Na ([M + Na]⁺) 421.1271, found 421.1263.

2.1.4.30

2.1.4.30 (E)-4-((4-(allyloxy)-2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5eb)

Yield: 87 %, Green solid, M.p.: 236–237 ℃; IR cm⁻¹ (KBr): 1595, 1496, 1328, 1278, 1141, 1093, 837, 734; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.34 (s, 1H), 8.13 – 8.10 (m, 2H), 7.80 (d, J = 9.0 Hz, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 2.6 Hz, 1H), 7.48 (t, J = 8.8 Hz, 2H), 7.36 (d, J = 9.0 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.16 – 6.06 (m, 1H), 5.49 (d, J = 17.3 Hz, 1H), 5.34 (d, J = 10.5 Hz, 1H), 4.78 (d, J = 5.2 Hz, 2H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.9, 164.5, 163.7, 161.5, 159.9, 146.0, 144.5, 133.4, 129.8, 129.7, 125.5 × 2, 123.5, 120.4, 119.8, 118.7, 118.5, 117.3, 117.1, 116.4 × 2, 115.5, 69.4; HRMS (ESI) calcd for C₂₃H₁₇FN₄O₃Na ([M + Na]⁺) 439.1176, found 439.1171.

2.1.4.31

2.1.4.31 (E)-4-((4-(allyloxy)-2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ec)

Yield: 50 %, Brown solid, M.p.: 241–243 ℃; IR cm⁻¹ (KBr): 1589, 1506, 1328, 1282, 1141, 833, 750, 729; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.53 (s, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 2.2 Hz, 1H), 7.36 (d, J = 9.0 Hz, 1H), 6.96 (d, J = 8.7 Hz, 2H), 6.18 – 6.04 (m, 1H), 5.49 (d, J = 17.4 Hz, 1H), 5.34 (d, J = 10.5 Hz, 1H), 4.78 (d, J = 4.6 Hz, 2H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.5, 163.9, 161.7, 159.9, 145.9, 144.5, 137.3, 133.4, 130.1 × 2, 128.8 × 2, 125.4 × 2, 123.4, 122.6, 119.9, 118.8, 118.5, 116.4 × 2, 115.5, 69.41; HRMS (ESI) calcd for C₂₃H₁₇ClN₄O₃Na ([M + Na]⁺) 455.0881,found 455.0874.

2.1.4.32

2.1.4.32 (E)-4-((4-(allyloxy)-2-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ed)

Yield: 86 %, Red solid, M.p.: 213–214 ℃; IR cm⁻¹ (KBr): 1597, 1506, 1327, 1278, 1141, 842, 727; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.36 (s, 1H), 7.88 – 7.76 (m, 5H), 7.69 (d, J = 2.7 Hz, 1H), 7.53 – 7.46 (m, 2H), 7.35 (dd, J = 9.0, 2.8 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.19 – 6.05 (m, 1H), 5.49 (dd, J = 17.3, 1.7 Hz, 1H), 5.34 (dd, J = 10.6, 1.8 Hz, 1H), 4.78 (d, J = 4.9 Hz, 2H), 2.39 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.9, 163.3, 161.1, 159.5, 145.6, 144.0, 138.9, 133.0, 132.7, 129.3, 126.9, 125.0 × 2, 123.8, 123.2, 123.0, 119.4, 118.3, 118.0, 115.9 × 2, 114.9, 68.9, 20.8. HRMS (ESI) calcd for C₂₄H₂₀N₄O₃N ([M + Na]⁺) 435.1427, found 435.1420.

2.1.4.33

2.1.4.33 (E)-4-((4-(allyloxy)-2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ee)

Yield: 54 %, Red solid, M.p.: 186–187 ℃; IR cm⁻¹ (KBr): 1598, 1506, 1325, 1278, 1141, 837, 729; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.34 (s, 1H), 7.79 (t, J = 9.3 Hz, 3H), 7.70 – 7.62 (m, 2H), 7.57 – 7.50 (m, 2H), 7.36 (dd, J = 9.0, 2.9 Hz, 1H), 7.23 (dd, J = 8.2, 2.7 Hz, 1H), 6.91 (d, J = 8.4 Hz, 2H), 6.19 – 6.05 (m, 1H), 5.49 (d, J = 17.3 Hz, 1H), 5.34 (d, J = 10.6 Hz, 1H), 4.78 (d, J = 5.2 Hz, 2H), 3.82 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 165.3, 164.0, 161.5, 160.3, 160.1, 146.2, 144.7, 133.6, 131.4, 125.6 × 2, 125.1, 123.6, 120.0, 119.5, 119.0, 118.8, 118.7, 116.5 × 2, 115.6, 112.0, 69.5, 55.9. HRMS (ESI) calcd for C₂₄H₂₀N₄O₄Na ([M + Na]⁺) 451.1376, found 451.1369.

2.1.4.34

2.1.4.34 (E)-4-((4-(allyloxy)-2-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ef)

Yield: 68 %, Brown solid, M.p.: 245–247 ℃; IR cm⁻¹ (KBr): 1597, 1502, 1458, 1323, 1280, 1141, 835, 731; ¹H NMR (400 MHz, DMSO‑d₆) δ 10.33 (s, 1H), 7.78 (t, J = 8.8 Hz, 3H), 7.67 (d, J = 2.9 Hz, 1H), 7.60 (dd, J = 8.1, 1.8 Hz, 1H), 7.52 (d, J = 1.7 Hz, 1H), 7.34 (dd, J = 9.0, 2.9 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 6.92 (dd, J = 8.9, 2.4 Hz, 2H), 6.18 (s, 2H), 6.14 – 6.06 (m, 1H), 5.48 (dd, J = 17.3, 1.7 Hz, 1H), 5.34 (dd, J = 10.5, 1.6 Hz, 1H), 4.77 (d, J = 5.2 Hz, 2H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 164.6, 162.8, 161.0, 159.5, 150.5, 148.2, 145.6, 144.0, 133.0, 125.0 × 2, 123.1, 121.9, 119.3, 118.3, 118.0, 116.9, 115.9, 115.8, 114.9, 109.2, 106.3, 102.1, 68.9. HRMS (ESI) calcd for C₂₄H₁₈N₄O₅Na ([M + Na]⁺) 465.1169, found 465.1166.

2.1.4.35

2.1.4.35 (E)-4-((4-(allyloxy)-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5eg)

Yield: 70 %, Red solid, M.p.: 230–232 ℃; IR cm⁻¹ (KBr): 1597, 1506, 1321, 1278, 1141, 835, 725; ¹H NMR (400 MHz, DMSO‑d₆) δ 9.22 (s, 1H), 8.83 (d, J = 3.2 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.71 – 7.63 (m, 2H), 7.36 (dd, J = 9.0, 2.8 Hz, 1H), 6.88 (d, J = 8.4 Hz, 2H), 6.17 – 6.05 (m, 1H), 5.49 (d, J = 19.3 Hz, 1H), 5.34 (d, J = 9.4 Hz, 1H), 4.78 (d, J = 5.2 Hz, 2H); ¹³C NMR (101 MHz, DMSO‑d₆) δ 163.9, 163.2, 162.3, 159.5, 152.8, 147.5, 145.2, 144.4, 134.4, 133.2, 125.4 × 2, 124.6, 122.8, 120.1, 119.7, 118.6, 118.3, 116.4 × 2, 115.2, 69.1; HRMS (ESI) calcd for C₂₂H₁₇N₅O₃Na ([M + Na]⁺) 422.1223, found 422.1215.

2.2

2.2 Biology assays

2.2.1

2.2.1 Cell line and culture conditions

The human lung cancer cells (A549), human cervical carcinoma cells (HeLa), human colon cancer cells (HCT116), human hepatocellular carcinoma cell (HePG₂), human breast cancer cells (MCF-7), HT1080 human fibrosarcoma cells (HT1080), human lung epithelial cells (BEAS-2B) were obtained from Cell Bank/Stem Cell Bank, Chinese Academy of Sciences. We used RPMI-1640 and DMEM medium supplemented with 10 % fetal bovine serum and 1 % penicillin for cell growth under the condition of 37 °C in 5 % CO₂. 5- Fluorouracil (5-FU) was employed as positive control drug.

2.2.2

2.2.2 Cytotoxic assays

The MTT assay was used to evaluate the cytotoxicity of novel synthesized derivatives (5aa ∼ eg) on cell viability. When cells were in exponentially growing phase, seeded at a concentration of 10,000 cells/well on 96 tissue culture plate overnight. After the culture medium replaced with a fresh one, a solution of synthesized compounds in DMSO was added to reach the concentration of 200 μmol/L. After 72 h, the drug solutions discarded and cells were washed with PBS. Then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL, in PBS) was added into each well and cultured for another 4 h. Subsequently, the supernatant replaced with DMSO to soften the formed formazan. In parallel, we used negative control, blank and positive control (5-fluorouracil, 5-FU) in the same conditions. At the final step, absorbance at the wavelength of 490 nm was read by a microplate reader. the IC₅₀ values for some compounds were also calculated. All the experiments were repeated three times.

2.2.3

2.2.3 Cell cycle arrest assay

HCT116 cells (1 × 10⁶/well) were spread in 6-well plates overnight; the cells were treated with different concentration of compound 5df for 48 h. The cells were then washed twice with PBS and digested with trypsin. The supernatant was removed via centrifugation and fixed overnight in 70 % ethanol. The cells were washed twice with PBS and resuspended in a 190 µL staining buffer containing 4 µL of 1 mg/mL PI (propidium iodide), 4 µL of 10 mg/mL RNaseA (ribonuclease), and 0.2 µL Tritonx-100. After staining for 20 min in the dark, the cell cycle distribution was detected using flow cytometry (American BD,Canto II plus).

2.2.4

2.2.4 Cell apoptosis assay

HCT116 cells (2.2 × 10⁶/well) were grown in 6-well plates and treated with different concentrations of compound 5df for 48 h. After incubation, the cells were trypsinised and washed with PBS. The obtained cell pellet was resuspended in 1x Annexin binding buffer. 5 μL of annexin V/FITC and 10 μL of PI were added to the resuspended cells and incubated for 15 min at room temperature. Thereafter, the stained cells were analyzed by flow cytometry.

2.3

2.3 Analysis of network pharmacology

2.3.1

2.3.1 Prediction of disease targets of compound 5df

Compound 5df was draw in ChemBioDraw and saved as “sdf” file format. This file was then imported into PharmMapper database (https://lilab-ecust.cn/pharmmapper/submitfile.html) (Wang et al., 2016), Swiss target prediction database (https://www.swisstargetprediction.ch) (Gfeller et al., 2013), inputting the protein names with the species limited to “Homo sapiens”, and we could receive their official symbol. After these operations, proteins information of compound targets and known targets was obtained.

2.3.2

2.3.2 Gene screening of HCT116 related targets

Using “HCT116” as the search keywords, we searched Gene Cards database (https://www.genecards.org/) (Stelzer et al., 2016) to excavate potential targets associated with HCT116. Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/) software was used to identify the targets of compound 5df against HCT116 (Sun et al., 2019).

2.3.3

2.3.3 Construction of protein–protein interaction (PPI) network

To further identify the core regulatory targets, PPI analysis was performed by submitting overlapping targets of active compounds in HCT116 to the STRING 11.5 database (https://string-db.org) (Szklarczyk et al., 2019). The species type was set to “Homo sapiens”, the minimum interaction threshold was set to “highest confidence” >0.4, and the rest was set as the default. Finally, the PPI result was imported into Cytoscape 3.8.0 software to construct a PPI network. Finally, the core targets of the top 20 were also exhibited.

2.3.4

2.3.4 Go and KEGG enrichment analysis

To further understand the functions of core target genes and the main action pathways of the active substances of 5df, the disease-related targets obtained from the above screening were entered into the DAVID database (https://david.ncifcrf.gov/home.jsp) (Sherman et al., 2022). The list of genes of 5df targets against HCT116 was uploaded to the DAVID searching tool. Here, the species section was selected as “homo sapiens”, and all target gene names were corrected to their official names (official gene symbol), and the threshold of p < 0.05 was set for GO and KEGG enrichment before running the tool.

2.4

2.4 Molecular docking

Molecular docking was performed among the top three potential target proteins of HCT116 with 5df. The 2D structure of compound 5df was drawn by ChemDraw2019, and saved as cdx file. Then, MM2 force field was used to optimize the 3D structure of the ligand. The 3D structures of the target proteins were downloaded from the PDB database (https://www.rcsb.org) (AKT1, PDB: 3MV5; SRC, PDB: 1Y57; EGFR, PDB: 1 M17. The water molecules and the original ligands were removed from the target protein through Discovery studio. Later, the target proteins were imported into AutoDock Tools 1.5.6 for hydrogenation, charge calculation, and non-polar hydrogen combination, and then the result was stored in PDBQT format. The 3D coordinates of active sites (x, y, z) were as follows: AKT1 (1.931, −0.696, 25.291), SRC (9.48, 38.365, 38.325), EGFR (30.465, 9.823, 59.384). Finally, run AutoDock Tools 1.5.6 for molecular docking, and use Discovery studio 2021 to visualize the results (Trott & Olson, 2010).

2.5

2.5 Data analyses

The cell growth inhibition rate of compounds against six human cancer cell lines were calculated by the following formula: Inhibition rate (%) = (ODnegative control – ODexperiment)/(ODnegative control – ODblank) × 100 %. The relative cell viability was calculated with the formula: Cell viability rate (%) = (ODexperiment – ODblank)/(ODnegative control – ODblank) × 100 %. All data are expressed as mean ± SD, and each group was performed three times. Statistical analysis was processed by the SPSS 21.0 software (SPSS Inc., Chicago, USA).

3 Results and discussion

3.1

3.1 Chemistry

As illustrated in Scheme 1, the synthesis of the azophenol derivatives containing 1,3,4-oxadiazoles moiety was performed starting from the available chemicals. Firstly, according to the method we reported, a nucleophilic addition and elimination reaction between o-nitrobenzaldehyde and substituted hydrazides yielded acylhydrazone intermediates (2aa ∼ eg) in the presence of acetic acid. Subsequently, these intermediates underwent a ring-closing reaction facilitated by iodine to form 1,3,4-oxadiazoles (3aa ∼ eg) (Yu et al., 2021). In the subsequent step, the corresponding oxadiazoles were reacted with hydrated tin(II) chloride to yield intermediates 4aa ∼ eg, with yields ranging from 48 % to 93 %. Finally, compounds 4aa ∼ eg reacted with sodium nitrite to produce diazonium salts, which were further reacted with phenol to give azophenol derivatives containing the 1,3,4-oxadiazoles moiety (5aa ∼ eg) in moderate to good yields (Table 1).

General synthetic procedure of azophenol derivatives containing 1,3,4-oxadiazoles moiety (5aa ∼ eg).

Table 1 The structure, yield, and melting point of all target compounds.

Compound	R¹	R²	Yield (%)	M.p. (^oC)
5aa	H	Ph	48	281–283
5ab	H	(4-F)Ph	89	273–275
5ac	H	(4-Cl)Ph	73	238–239
5ad	H	(3-CH₃)Ph	38	214–216
5ae	H	(3-OCH₃)Ph	89	253–255
5af	H	(3,4-OCH₂O)Ph	69	289–291
5ag	H	4-Py	59	250–251
5ba	OCH₃	Ph	55	256–257
5bb	OCH₃	(4-F)Ph	73	253–255
5bc	OCH₃	(4-Cl)Ph	46	233–235
5bd	OCH₃	(3-CH₃)Ph	60	201–202
5be	OCH₃	(3-OCH₃)Ph	90	202–203
5bf	OCH₃	(3,4-OCH₂O)Ph	61	271–272
5bg	OCH₃	4-Py	45	288–289
5ca	OCH₂CH₃	Ph	76	242–244
5cb	OCH₂CH₃	(4-F)Ph	74	281–283
5cc	OCH₂CH₃	(4-Cl)Ph	81	260–262
5cd	OCH₂CH₃	(3-CH₃)Ph	63	220–222
5ce	OCH₂CH₃	(3-OCH₃)Ph	82	230–232
5cf	OCH₂CH₃	(3,4-OCH₂O)Ph	71	251–253
5cg	OCH₂CH₃	4-Py	74	269–271
5da	OCH₂CH₂CH₃	Ph	82	247–248
5db	OCH₂CH₂CH₃	(4-F)Ph	74	235–236
5dc	OCH₂CH₂CH₃	(4-Cl)Ph	64	209–210
5dd	OCH₂CH₂CH₃	(3-CH₃)Ph	59	214–216
5de	OCH₂CH₂CH₃	(3-OCH₃)Ph	73	198–199
5df	OCH₂CH₂CH₃	(3,4-OCH₂O)Ph	77	251–252
5dg	OCH₂CH₂CH₃	4-Py	52	237–238
5ea	OCH₂CH = CH₂	Ph	65	238–239
5eb	OCH₂CH = CH₂	(4-F)Ph	87	236–237
5ec	OCH₂CH = CH₂	(4-Cl)Ph	50	241–243
5ed	OCH₂CH = CH₂	(3-CH₃)Ph	86	213–214
5ee	OCH₂CH = CH₂	(3-OCH₃)Ph	54	186–187
5ef	OCH₂CH = CH₂	(3,4-OCH₂O)Ph	68	245–247
5eg	OCH₂CH = CH₂	4-Py	70	230–232

The structural characterization of all synthesized compounds was carried out using ¹H NMR, ¹³C NMR and HRMS (see Supplementary Materials). For example, the ¹H NMR spectrum of 5af showed a single peak at 10.45 ppm which could be assigned to –OH. two double peaks at 7.83 ppm (d, J = 8.7 Hz, 2H) and 6.95 ppm (d, J = 8.8 Hz, 2H) were attributed to the protons of phenol. The other a single peak at 7.45 ppm and two double peaks at 7.57 (d, J = 8.1 Hz, 1H) and 7.13 (d, J = 8.1 Hz, 1H) ppm belonged to the protons of benzo[d][1,3]dioxole. The single peaks of OCH₂O protons of benzo[d][1,3]dioxole moiety appeared at 6.18 ppm. In the ¹³C NMR spectrum of 5af, the signals at 164.5 and 162.9 ppm were assigned to the carbons in 1,3,4-oxadiazole. The carbon linked to hydroxyl groups appeared at 161.6 ppm. The signals at 125.5 and 116.0 ppm were assigned to the carbons of phenol. The single peaks at 102.1 ppm belonged to OCH₂O. The remaining multiple single peaks observed at 150.5–106.2 ppm were attributed to aromatic rings. The HRMS spectrum of 5af showed a molecular ion peak at m/z 409.0907 as [M + Na]⁺.

3.2

3.2 Biology assays

3.2.1

3.2.1 In vitro anticancer activity

The synthesized azophenol derivatives (5aa ∼ eg) were assessed for their in vitro anticancer activities at 200 µmol/L against six human cancer cell lines, which included lung cancer cells (A549), cervical carcinoma cells (HeLa), colon cancer cells (HCT116), hepatocellular carcinoma cells (HePG2), breast cancer cells (MCF-7), and fibrosarcoma cells (HT1080). The MTT assay was used for this evaluation, and 5-fluorouracil (5-Fu) served as the positive control. As indicated in Table 2, most of the target compounds displayed significant anticancer activity, particularly against HCT116 and HePG2. For instance, compounds 5be, 5cb, 5db, 5dc, 5df, 5ec and 5eg exhibited potent cytotoxicity against HCT116 and HePG2, with growth inhibition rates exceeding 60 %. Besides, 5db, 5dc, 5df and 5ec also demonstrated noteworthy cytotoxicity against HeLa, with growth inhibition rates of 85.81 %, 88.68 %, 78.87 %, and 80.13 %, respectively. Of particular note, compound 5df exhibited substantial antineoplastic activity across all six tumor cell lines, with growth inhibition rates surpassing 50 %. These results underscored the potent anticancer effects of azophenol derivatives containing the 1,3,4-oxadiazoles moiety. Furthermore, preliminary structure–activity relationship (SAR) studies were also conducted. For example, to HCT116 and HePG2 cells, introduction of ethoxy or propoxy groups on R² led to more potent compounds (5ca, 5da vs 5aa). When R¹ was a ethoxy group, the compound 5be with R² = (4-F)Ph exhibited superior anticancer activity compared to other 4-substituted phenyl variations. Similarly, when R¹ was a propoxy group, compounds 5cb and 5df, with the introduction of (4-F)Ph or benzo[d][1,3]dioxole groups on R² displayed more potent antitumor activity. On the contrary, introducing pyridine heterocycles in R² did not improve the activity of the compounds, such as 5ag, 5bg, 5cg. This outcome indicated that the rational incorporation of propoxy group on R¹ and benzo[d][1,3]dioxole group on R² can distinct influence the anticancer effect.

Table 2 The anticancer activities of target compounds 5aa ∼ eg against six human cancer cells at 200 µmol/L.

Compound	Growth Inhibition Rate (%)^a
Compound	A549	HeLa	HCT116	HePG2	MCF-7	HT1080
5aa	NA^b	NA	NA	42.95 ± 2.14	NA	NA
5ab	NA	48.68 ± 5.23	13.58 ± 4.46	70.65 ± 1.38	33.92 ± 6.35	NA
5ac	NA	NA	52.93 ± 7.12	40.93 ± 3.09	NA	NA
5ad	14.74 ± 4.77	31.15 ± 4.6	33 ± 5.13	79.03 ± 3.57	NA	NA
5ae	NA	43.51 ± 8.65	51.48 ± 2.66	67.95 ± 3.58	NA	NA
5af	NA	NA	28.99 ± 2.96	54.34 ± 3.1	NA	NA
5ag	16.51 ± 6.66	3.66 ± 30.35	14.78 ± 3.12	55.55 ± 3.99	NA	NA
5ba	NA	NA	35.7 ± 3.03	41.06 ± 2.88	NA	24.62 ± 1.62
5bb	NA	NA	17.31 ± 4.67	17.5 ± 6.29	13.51 ± 4.94	38.74 ± 3.47
5bc	19.15 ± 4.28	32.4 ± 5.1	75.9 ± 3.42	35.54 ± 5.84	NA	NA
5bd	–	NA	NA	40 ± 4.3	NA	44.17 ± 7.21
5be	13.41 ± 2.33	39.82 ± 4.11	79.71 ± 0.37	73.11 ± 3.36	NA	NA
5bf	NA	18.38 ± 2.55	NA	59.67 ± 5.65	NA	34.15 ± 0.14
5bg	NA	NA	NA	55.72 ± 5.02	47.86 ± 2.99	18.74 ± 2.87
5ca	51.96 ± 2.67	NA	35.5 ± 5.92	60.69 ± 3.29	NA	NA
5cb	NA	NA	65.22 ± 1.04	69.22 ± 4.16	NA	NA
5cc	45.69 ± 0.37	83.7 ± 2.34	62.66 ± 3.94	42 ± 5.84	58.52 ± 4.6	18.69 ± 3.16
5cd	NA	NA	22.29 ± 3.14	39.95 ± 5.51	9.04 ± 4.29	NA
5ce	NA	NA	23.38 ± 6.7	84.57 ± 3.19	28.25 ± 3.77	NA
5cf	NA	NA	7.24 ± 4.65	42.05 ± 4.48	NA	23.93 ± 6.05
5cg	12.42 ± 2.64	67.37 ± 5.11	12.58 ± 1.59	23.11 ± 2.47	10.13 ± 2.64	6.81 ± 1.76
5da	33.56 ± 5.71	27.72 ± 5.08	48 ± 2.28	83.85 ± 5.15	18.49 ± 5.43	NA
5db	NA	85.81 ± 1.26	85.41 ± 2.35	78.24 ± 2.01	49.24 ± 2.78	6.81 ± 1.76
5dc	NA	88.68 ± 1.39	62.39 ± 5.71	88.24 ± 2.01	NA	NA
5dd	NA	14.05 ± 4.84	46.51 ± 2.11	87.08 ± 2.39	44.5 ± 3	NA
5de	NA	59.43 ± 6.2	53.91 ± 2.04	53.55 ± 5.42	NA	45.35 ± 5.2
5df	62.09 ± 4.68	78.87 ± 3.78	92.47 ± 3.5	73.66 ± 5.79	64.22 ± 0.94	54.23 ± 1.94
5dg	NA	27.97 ± 8.79	26.95 ± 3.78	78.72 ± 3.32	NA	18.14 ± 2.83
5ea	NA	6.07 ± 15.84	7.63 ± 5.3	44.62 ± 5.6	NA	7.91 ± 1.11
5eb	NA	NA	36.93 ± 2.22	43.53 ± 4.97	26.18 ± 4.18	38.09 ± 5.78
5ec	40.9 ± 4.76	80.13 ± 1.58	65.03 ± 3.51	81.13 ± 7.22	28.74 ± 3.64	24.6 ± 7.42
5ed	NA	20.96 ± 5.82	36.75 ± 4.74	55.64 ± 5.34	21.08 ± 8.97	35.06 ± 6.57
5ee	NA	NA	30.62 ± 2.63	62.64 ± 4.96	10.03 ± 7.33	NA
5ef	NA	NA	41.56 ± 0.64	56.18 ± 5.23	31.93 ± 5.4	NA
5eg	53.9 ± 1.02	50.16 ± 2.47	70.52 ± 1.31	87.69 ± 0.11	38.81 ± 4.15	21.06 ± 2.16
5-FU	81.16 ± 2.95	83.74 ± 2.93	92.36 ± 3.64	88.02 ± 3.21	88.84 ± 1.26	69.01 ± 3.90

a The values given are means of three experiments.

b NA means no action.

Additionally, the IC₅₀ values (50 % inhibitory concentrations) of the tested compounds, where growth inhibition rates were over 70 %, were evaluated and consolidated in Table 3. Among these derivatives, the IC₅₀ values of 5db and 5df against HCT116 were measured at 9.96 and 4.09 μM, respectively. For HePG2 cells, the IC₅₀ value of 5dc was determined to be 9.39 μM. These values are in close proximity to or even surpass the IC₅₀ of 5-FU, the utilized positive control. In order to evaluate the safety of these new synthetic compounds, we selected the most active compound 5df and tested tis toxicity to human normal lung epithelial cells (BEAS-2B). The result showed that 5df displayed low cytotoxicity against BEAS-2B with the cell viabilities over 80 % even at concentration of 160 μM, which indicated that compound 5df showed higher selectivity to tumor cells than normal cells (Fig. 2).

Table 3 IC₅₀ values of target compounds against selected human cancer cells.

Compound	IC₅₀ (μM)^a
Compound	HeLa	HCT116	HePG2
5ab	ND^b	ND	>100
5ad	ND	ND	>100
5bc	ND	>100	ND
5be	ND	40.56 ± 0.45	29.6 ± 0.73
5cc	>100	ND	ND
5ce	ND	ND	>100
5da	ND	ND	13.7 ± 0.72
5db	70.77 ± 0.73	9.96 ± 0.08	44.64 ± 0.59
5dc	24.97 ± 0.16	ND	9.39 ± 0.45
5dd	ND	ND	38.43 ± 0.69
5dg	ND	ND	>100
5df	>100	4.09 ± 0.04	>100
5ec	21.79 ± 0.35	ND	36.77 ± 0.51
5eg	ND	61.69 ± 0.77	>100
5-FU	51.65 ± 0.78	2.20 ± 0.13	32.56 ± 0.64

a The values given are means of three experiments.

b ND means no detection.

Relative cell viabilities of BEAS-2B treated with compound 5df.

3.2.2

3.2.2 Cell cycle analysis

Motivated by the initial in vitro antiproliferative screening outcomes, the underlying anticancer mechanism of this series compounds was further explored. Among the compounds, the most potent candidate, 5df, was chosen to delve into its impact on the cell cycle progression of HCT116 cancer cells, employing flow cytometry analysis. For this investigation, HCT116 cancer cells were subjected to treatment with either DMSO (control group) or varying concentrations of 5df (10, 20, 40 μM) for a duration of 48 h. As depicted in Fig. 3, the percentage of cells in the S phase in presence of 5df exhibited values of 50.3 %, 52.8 %, and 59.0 %, respectively, while 26.7 % of the S phase was detected for the control group. These finding demonstrated that compound 5df elicited a noticeable concentration-dependent S-phase arrest.

Effect of compound 5df on cell cycle in HCT116 cells. Flow cytometry of HCT116 cells treated with 5df for 48 h (** p < 0.01; *** p < 0.001).

3.2.3

3.2.3 Cell apoptosis analysis

Moreover, cell apoptosis analysis was conducted on HCT116 cells exposed to escalating concentrations of 5df (0, 10, 20, 40 μM), utilizing the annexin V-FITC-PI assay. As illustrated in Fig. 4, following a 48-hour incubation period with 5df, the collective percentages of late (Q2) and early (Q3) apoptotic cells exhibited increases to 17.5 %, 18.1 %, and 26.0 %, respectively. In contrast, the control group displayed a mere 3.0 % of apoptotic cells. Consequently, these results demonstrated that compound 5df had antiproliferative activity through dose-dependently inducing cellular apoptosis.

Effect of compound 5df on cell apoptosis in HCT116 cells. Flow cytometric analysis of apoptotic cells after treatment of HCT116 cells with 5df for 48 h (*** p < 0.001).

3.3

3.3 Analysis of network pharmacology

Network pharmacology, an emerging discipline centered around the interplay of disease-gene-drug targets, has evolved into an efficacious research methodology for exploring active compounds and comprehending the mechanisms underlying the pharmacodynamic actions of these compounds (Hopkins, 2008; Zhu et al., 2022). In the context of this study, the network pharmacology approach was employed to uncover the mechanism of action and potential targets associated with compound 5df in its application for HCT116 treatment.

As illustrated in Fig. 5, by searching the public databases (Swiss target prediction, PharmMapper, Gene Cards), confining the result to “Homo sapiens”, 378 targets related with compound 5df and 5412 targets with HCT116 were collected, respectively. The utilization of Venny 2.1.0 facilitated the calculation of the intersection between 5df targets and HCT116 targets, resulting in 86 intersection targets (Fig. 5, A). Subsequently, high-confidence protein interaction data were extracted by employing target proteins and their corresponding components from the STRING database. This process eliminated uninvolved proteins, thereby yielding 86 shared proteins between 5df and HCT116 (Fig. 5, B). The obtained protein–protein interaction (PPI) outcomes were imported into Cytoscape 3.8.0 software, enabling the construction of a PPI network (Fig. 5, C). The visualization also highlighted the twenty core targets. Notably, the top three targets, AKT serine/threonine kinase 1 (AKT1), SRC proto-oncogene, non-receptor tyrosine kinase (SRC), and epidermal growth factor receptor (EGFR), emerged as the most plausible targets of action for compound 5df (Table 4). AKT1 belongs to a family of kinases AKT1, AKT2, AKT3, can include the coexpression of multiple family members in normal tissues and tumors. AKT is part of the PI3K/AKT pathway that encompasses multiple receptor tyrosine kinases (PI3K and mTOR) and integrates extracellular signals with cell growth, proliferation, and survival. It is often activated in malignant cells, and is a promising point of therapeutic intervention for many cancers (Nicholson and Anderson, 2002; Cheng et al. 2005). SRC kinase is a group of proteins with tyrosine protein kinase activity that are overexpressed and highly activated in various human tumor cells, it can activate the MAPK and PI3K/Akt pathways related to tumor growth (Boggon and Eck, 2004; Peiró et al., 2014). EGFR is a member of the epidermal growth factor receptor (HER) family, widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, and other cells. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation, and differentiation. When EGFR undergoes mutations, it leads to sustained activation of the EGFR signaling pathway, leading to abnormal cell proliferation (Sabbah et al., 2020). The above three proteins were closely related to the development of tumor cells and might be the main targets of compound 5df.

Analysis of network pharmacology of 5df-HCT116. (A) The Venn diagram of compound 5df and HCT116 targets; (B) Protein-protein interaction (PPI) network; (C) Topological network schematic of proteins targeted by 5df and associated with HCT116; (D) Analysis of potential targets of 5df for anti-HCT116 based on GO enrichment; (E) Analysis of potential targets of 5df for anti-HCT116 based on KEGG enrichment; (F) Constituent-target-pathway network of top 20 pathways.

Table 4 The twenty core targets of compound 5df against HCT116.

Target ID	Gene name	Degree	Betweenness	Closeness
AKT1	AKT Serine/Threonine Kinase 1	57	807.0940761	0.008695652
SRC	SRC Proto-Oncogene, Non-Receptor Tyrosine Kinase	50	470.9976093	0.008130081
EGFR	Epidermal Growth Factor Receptor	48	569.0928943	0.008064516
CASP3	Caspase 3	44	378.868961	0.007692308
ESR1	Estrogen Receptor 1	44	497.4969143	0.007633588
TNF	Tumor Necrosis Factor	43	470.5896637	0.007692308
MAPK1	Mitogen-Activated Protein Kinase 1	35	168.7924717	0.007092199
PIK3CA	Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha	35	150.0517497	0.006944444
SIRT1	Sirtuin 1	34	221.1860693	0.007142857
MDM2	MDM2 Proto-Oncogene	34	265.0791359	0.006993007
CDC42	Cell Division Cycle 42	33	250.7977111	0.006849315
MMP9	Matrix Metallopeptidase 9	31	233.3490098	0.006944444
HDAC1	Histone Deacetylase 1	29	265.6142845	0.006578947
RPS6KB1	Ribosomal Protein S6 Kinase B1	28	100.9209659	0.006756757
CDK2	Cyclin Dependent Kinase 2	26	130.9764011	0.006578947
MMP2	Matrix Metallopeptidase 2	25	127.7503376	0.006578947
RAC1	Rac Family Small GTPase 1	24	107.5536357	0.00617284
PPARA	Peroxisome Proliferator Activated Receptor Alpha	22	366.0219331	0.006329114
PTPN1	Protein Tyrosine Phosphatase Non-Receptor Type 1	20	200.0227838	0.00617284
HMOX1	Heme Oxygenase 1	18	224.9469155	0.006134969

GO enrichment analysis and KEGG pathway enrichment analysis were also executed to shed light on the functions and enriched pathways attributed to the potential HCT116 genes targeted by compound 5df. The analysis of biological processes (BP) outcomes indicated that compound 5df's targets involved in anti-HCT116 were prominently enriched in processes such as protein phosphorylation, protein autophosphorylation, negative regulation of the apoptotic process, and other biological processes. Cellular components (CC) analysis underscored the significance of cellular elements like cytosol, cytoplasm, nucleoplasm, and other cellular components. The molecular functions (MF) analysis results underscored that protein serine/threonine/tyrosine kinase activity, ATP binding, protein serine/threonine kinase activity and other molecular functions played an important role (Fig. 5, D). The KEGG pathway enrichment analysis yielded a notable result, 136 signaling pathways relevant to the interaction between 5df and HCT116, were statistically significant. These included pathways associated with pathways in cancer, proteoglycans in cancer, prostate cancer, ErbB signaling pathway, and more. A visual representation in the form of a bubble diagram depicted the top 20 pathways which were significant enrichment potential, along with the highest count of associated genes (Fig. 5, E). Furthermore, a network was constructed to depict the relationships between ingredients, targets, and the top 20 pathways (Fig. 5, F). Among the pathways with the highest counts, such as pathways in cancer, proteoglycans in cancer, and prostate cancer, may contribute to the anti-HCT116 effect exhibited by 5df.

3.4

3.4 Molecular docking

Molecular docking is a cutting-edge scientific method that can match small molecules to the binding sites of proteins and evaluate their binding strength to predict the binding mode and binding free energy of a given protein and small molecule ligand, and then study their function and mechanism of action (Kitchen et al., 2004; Saikia and Bordoloi, 2019). In recent years, molecular docking has played an important role in the validation of drug targets, and provided effective tools for the development of novel anticancer drugs (Premkumar et al., 2016; Shaheen et al., 2020; Eze et al., 2022 ). In this work, molecular docking studies were conducted between the compound 5df and the three key targets, AKT1 (PDB: 3MV5), SRC (PDB: 1Y57), and EGFR (PDB: 1 M17) which were high-degree nodes in the interaction network to verify the results of network pharmacology analysis. The docking results showed that compound 5df could be bound into the docking pockets with binding energies lower than −8 kcal/mol, and had good docking activities between the target proteins, suggesting they played a critical role in the response to compound 5df in HCT116. For example, compound 5df bound to AKT1 by forming hydrogen bonds with GLY159, ALA230, TYR437 (Fig. 6, A), which had the same binding sites and similar binding patterns as the AKT1 inhibitors that Freeman-Cook reported (Freeman-Cook et al., 2010). As shown in Fig. 6, B, Cowan-Jacob et al. demonstrated that the binding site of RSC inhibitors is composed of amino acid residues GLY274, LEU273, MET341, ALA293, LEU393, VAL281, GLN275 etc, by cultivating protein and drug complexes (Cowan-Jacob et al., 2005). There were also four hydrogen bonds, LYS295, TYR340, MET341 and ASP404 formed between 5df and SRC, indicates that the compound could bind to the active site of RSC. Similarly, compound 5df was predicted to dock into the binding pocket of EGFR via hydrogen bonds LYS721, MET769, GLY772 and ASP831 (Fig. 6, C), which was the active site of EGFR (Stamos et al., 2002). The above data suggested that compound 5df could potentially influence their function by inhibiting the binding of the docking pockets to the target receptors and played an crucial role in anti-HCT116.

Molecular models of 5df binding to AKT1 (A), SRC (B), and EGFR (C).

4 Conclusion

In summary, this study encompassed the synthesis of thirty-five azophenol derivatives containing the 1,3,4-oxadiazoles moiety, whose structures were elucidated through ¹H NMR, ¹³C NMR and HRMS analyses. The anticancer activity of all synthesized compounds was evaluated in vitro against six different human cancer cell lines via the MTT assay. Remarkably, compound 5df exhibited a notable inhibition of HCT116 cell proliferation with an IC₅₀ value of 4.09 ± 0.04 µM, and low toxicity against normal human cells. Further mechanism studies showed that 5df significantly arrested cell cycle at S phase, induced apoptosis via flow cytometric analysis. In addition, network pharmacology computations pointed towards AKT1, SRC, and EGFR as plausible target proteins for 5df against HCT116 cells. Molecular docking studies further confirmed the good binding interactions of 5df with these three target proteins. Collectively, these results demonstrated that compound 5df could be a potent anticancer candidate reagent with low toxicity, and worthy for further investigation.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (82260833), Guizhou Provincial Natural Science Foundation [QKHJ-ZK(2022)YB503], Science and Technology Planning Project of Guizhou Province [QKHF-ZK(2023)G426].

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Appendix A

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2023.105386.

Appendix A

Supplementary material

The following are the Supplementary data to this article:

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Introduction

Experimental

Chemistry
Biology assays
Analysis of network pharmacology
Molecular docking
Data analyses

Results and discussion

Chemistry
Biology assays
Analysis of network pharmacology
Molecular docking

Conclusion

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[1] Abd-Ellah H.S., Abdel-Aziz M., Shoman M.E., Beshr E.A.M., Kaoud T., Ahmed A.F.F., . New 1,3,4-oxadiazole/oxime hybrids: design, synthesis, anti-inflammatory, COX inhibitory activities and ulcerogenic liability. Bioorg. Chem.. 2017;74:15-29.
[CrossRef] [Google Scholar]

[2] Abou-Seri S.M., . Synthesis and biological evaluation of novel 2,4'-bis substituted diphenylamines as anticancer agents and potential epidermal growth factor receptor tyrosine kinase inhibitors. Eur. J. Med. Chem.. 2010;45(9):4113-4121.
[CrossRef] [Google Scholar]

[3] Ahsan M.J., Samy J.G., Khalilullah H., Nomani M.S., Saraswat P., Gaur R., Singh A., . Molecular properties prediction and synthesis of novel 1,3,4-oxadiazole analogues as potent antimicrobial and antitubercular agents. Bioorg. Med. Chem. Lett.. 2011;21(24):7246-7250.
[CrossRef] [Google Scholar]

[4] Andreani A., Granaiola M., Leoni A., Morigi R., Ramballdi M., . Synthesis and antitubercular activity of imidazo [2,1-b]thiazoles. Eur. J. Med. Chem.. 2001;36:743-746.
[CrossRef] [Google Scholar]

[5] Atay Ç.K., Gökalp M., Tuncer B.Ö., Tilki T., . Antimicrobial activities and absorption properties of disazo dyes containing imidazole and pyrazole moieties. J. Macromol. Sci., Part A Pure Appl. Chem.. 2017;54(4):236-242.
[CrossRef] [Google Scholar]

[6] Bajaj S., Asati V., Singh J., Roy P.P., . 1,3,4-Oxadiazoles: an emerging scaffold to target growth factors, enzymes and kinases as anticancer agents. Eur. J. Med. Chem.. 2015;97:124-141.
[CrossRef] [Google Scholar]

[7] Benkhaya S., M'rabet S., El Harfi A., . Classifications, properties, recent synthesis and applications of azo dyes. Heliyon. 2020;6(1):e03271.
[Google Scholar]

[8] Boggon T.J., Eck M.J., . Structure and regulation of Src family kinases. Oncogene. 2004;23(48):7918-7927.
[CrossRef] [Google Scholar]

[9] Bray F., Ferlay J., Soerjomataram I., Siegel R.L., Torre L.A., Jemal A., . Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin.. 2018;68(6):394-424.
[CrossRef] [Google Scholar]

[10] Cheng J.Q., Lindsley C.W., Cheng G.Z., Yang H., Nicosia S.V., . The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene. 2005;24(50):7482-7492.
[CrossRef] [Google Scholar]

[11] Cowan-Jacob S.W., Fendrich G., Manley P.W., Jahnke W., Fabbro D., Liebetanz J., Meyer T., . The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation. Structure. 2005;13(6):861-871.
[CrossRef] [Google Scholar]

[12] Eze C.C., Ezeokonkwo A.M., Ugwu I.D., Eze U.F., Onyeyilim E.L., Attah I.S., Okonkwo I.V., . Azole-pyrimidine hybrid anticancer agents: a review of molecular structure, structure activity relationship, and molecular docking. Anticancer Agents Med Chem.. 2022;22(16):2822-2851.
[CrossRef] [Google Scholar]

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[CrossRef] [Google Scholar]

[14] Gfeller D., Michielin O., Zoete V., . Shaping the interaction landscape of bioactive molecules. Bioinformatics. 2013;29(23):3073-3079.
[CrossRef] [Google Scholar]

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Synthesis, biological evaluation and network pharmacology based studies of 1,3,4-oxadiazole bearing azaphenols as anticancer agents

Abstract

Keywords

Azophenol

1,3,4-Oxadiazole

Anticancer activity

Network pharmacology

Molecular docking

1 Introduction

2 Experimental

2.1 Chemistry

2.1.1 General procedure for the synthesis of (E)-N'-(2-nitrobenzylidene)arylhydrazides (2aa ∼ eg)

2.1.2 General procedure for the synthesis of 2-(2-nitrophenyl)-5-aryl-1,3,4-oxadiazoles (3aa ∼ eg)

2.1.3 General procedure for the synthesis of 2-(5-aryl-1,3,4-oxadiazol-2-yl)anilines (4aa ∼ eg)

2.1.4 General procedure for the synthesis of (E)-4-((2-(5-aryl-1,3,4-oxadiazol-2-yl)phenyl)diazenyl) phenol derivatives (5aa ∼ eg)

2.1.4.1 (E)-4-((2-(5-phenyl-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5aa)

2.1.4.2 (E)-4-((2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ab)

2.1.4.3 (E)-4-((2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ac)

2.1.4.4 (E)-4-((2-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ad)

2.1.4.5 (E)-4-((2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ae)

2.1.4.6 (E)-4-((2-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5af)

2.1.4.7 (E)-4-((2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ag)

2.1.4.8 (E)-4-((4-methoxy-2-(5-phenyl-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ba)

2.1.4.9 (E)-4-((2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-4-methoxyphenyl)diazenyl)phenol (5bb)

2.1.4.10 (E)-4-((2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-methoxyphenyl)diazenyl)phenol (5bc)

2.1.4.11 (E)-4-((4-methoxy-2-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5bd)

2.1.4.12 (E)-4-((4-methoxy-2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5be)

2.1.4.13 (E)-4-((2-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)-4-methoxyphenyl)diazenyl)phenol (5bf)

2.1.4.14 (E)-4-((4-methoxy-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5bg)

2.1.4.15 (E)-4-((4-ethoxy-2-(5-phenyl-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ca)

2.1.4.16 (E)-4-((4-ethoxy-2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5cb)

2.1.4.17 (E)-4-((2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-ethoxyphenyl)diazenyl)phenol (5 cc)

2.1.4.18 (E)-4-((4-ethoxy-2-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5 cd)

2.1.4.19 (E)-4-((4-ethoxy-2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ce)

2.1.4.20 (E)-4-((2-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)-4-ethoxyphenyl)diazenyl)phenol (5cf)

2.1.4.21 (E)-4-((4-ethoxy-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5cg)

2.1.4.22 (E)-4-((2-(5-phenyl-1,3,4-oxadiazol-2-yl)-4-propoxyphenyl)diazenyl)phenol (5da)

2.1.4.23 (E)-4-((2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-4-propoxyphenyl)diazenyl)phenol (5db)

2.1.4.24 (E)-4-((2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-propoxyphenyl)diazenyl)phenol (5dc)

2.1.4.25 (E)-4-((4-propoxy-2-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5dd)

2.1.4.26 (E)-4-((2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-4-propoxyphenyl)diazenyl)phenol (5de)

2.1.4.27 (E)-4-((2-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)-4-propoxyphenyl)diazenyl)phenol (5df)

2.1.4.28 (E)-4-((4-propoxy-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5dg)

2.1.4.29 (E)-4-((4-(allyloxy)-2-(5-phenyl-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ea)

2.1.4.30 (E)-4-((4-(allyloxy)-2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5eb)

2.1.4.31 (E)-4-((4-(allyloxy)-2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ec)

2.1.4.32 (E)-4-((4-(allyloxy)-2-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ed)

2.1.4.33 (E)-4-((4-(allyloxy)-2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ee)

2.1.4.34 (E)-4-((4-(allyloxy)-2-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5ef)

2.1.4.35 (E)-4-((4-(allyloxy)-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)diazenyl)phenol (5eg)

2.2 Biology assays

2.2.1 Cell line and culture conditions

2.2.2 Cytotoxic assays

2.2.3 Cell cycle arrest assay

2.2.4 Cell apoptosis assay

2.3 Analysis of network pharmacology

2.3.1 Prediction of disease targets of compound 5df

2.3.2 Gene screening of HCT116 related targets

2.3.3 Construction of protein–protein interaction (PPI) network

2.3.4 Go and KEGG enrichment analysis

2.4 Molecular docking

2.5 Data analyses

3 Results and discussion

3.1 Chemistry

3.2 Biology assays

3.2.1 In vitro anticancer activity

3.2.2 Cell cycle analysis

3.2.3 Cell apoptosis analysis

3.3 Analysis of network pharmacology

3.4 Molecular docking

4 Conclusion

Acknowledgements

Declaration of Competing Interest

References

Appendix A

Supplementary material

Appendix A

Supplementary material

Supplementary Data 1

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