Synthesis and anti-tumor activity of new benzofuran-based chalcone derivatives as potent VEGFR-2 inhibitors

Abstract

Cancer is one of the most significant public health problems worldwide, and the discovery and development of efficient VEGFR-2 inhibitors has been a research hotspot in cancer treatment. In the present work, a series of novel benzofuran-based chalcone derivatives have been prepared, and in vitro anti-tumor activities of them have been evaluated. The results indicated that the compounds displayed potent anticancer activity against HCC1806, HeLa and A549 cell lines. The preliminary mechanism study showed that 4g could effectively induce the apoptosis of HCC1806 cells, and showed inhibitory effect on VEFGR-2. The molecular docking study indicated that 4g had an obvious binding site with the target VEGFR-2 (PDB ID: 4BSK). Therefore, the benzofuran-based chalcone derivatives could be considered as potent VEGFR-2 inhibitors.

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Introduction

Cancer, a genetic disease caused by genetic defects or mutations that cause uncontrolled cell growth, is one of the most significant public health problems worldwide, which has become a serious threat to the health of residents, economic and social development.¹ The International Agency for Research on Cancer (IARC) has reported the latest global cancer statistics for 2022, with nearly 20 million new cancer cases and 9.7 million deaths from cancer.^2,3 Over the past decades, various new technologies and drugs for the treatment of cancers have been developed with the concerted efforts of the scientific community. However, traditional methods such as surgery, chemotherapy and radiotherapy can no longer meet the requirements of individualized and precise treatment of cancers in clinical practice. As the incidence of cancer continues to rise and the problem of drug resistance becomes more prominent, drug treatment of cancer still faces great challenges. Therefore, there is an urgent need for exploring highly effective anti-tumor drugs.

Recently, small molecule targeted anti-tumor drugs have been widely studied by researchers and gradually applied in clinics. The latest studies have shown that overexpression of tyrosine kinases is closely related to cancer proliferation and/or metastasis.^4,5 Among them, the vascular endothelial growth factor (VEGF) is a functional glycoprotein with high biological activity, and is the most important angiogenesis stimulator.^6,7 VEGF inhibitors can inhibit cancer angiogenesis so as to achieve the effect of treating cancers. There are more than 15 anti-tumor drugs targeting VEGF or its receptor in the world.⁸ VEGFR-2, a member of the protein tyrosine kinase receptor superfamily, could inhibit the formation of new blood vessels for cancer treatment by blocking the VEGF/VEGFR-2 signaling pathway. Therefore, the discovery and development of efficient VEGFR-2 inhibitors has become a hotspot.

Natural active ingredients come from a wide range of sources, and they are a shortcut to drug exploration in modifying the structure and eventually developing them into drugs with better efficacy and pharmacokinetic properties.⁹ Benzofurans are one of the typical oxygen-containing heterocyclic compounds, which are widely present in plants and animals, such as Dorsmerunin A–C and Moracin A–Z (Scheme 1).¹⁰ A large number of studies have indicated that benzofurans showed good pharmacological effects, including anti-tumor, anti-HIV, antibacterial, anti-inflammatory, antioxidant and anti-cardiovascular aging properties.^11–13 To date, more than 30 benzofuran drugs have been used in clinical applications. For example, fruquintinib, amiodarone and benzbromarone are used for colorectal cancer, cardiac arrhythmias and hyperuricemia, respectively (Scheme 1). In former works, we have carried out a series of studies on the structure–activity relationship of benzofuran derivatives, and found that benzofuran compounds bearing heterocyclic fragments showing the characteristics of high anticancer activity and low toxicity.^14–16

Scheme 1 Natural benzofuran products and drugs.

In recent years, there have been a lot of studies on benzofurans worldwide, including natural products and derivatives. Among them, benzofuran-chalcone hybrids are a class of compounds with a special structure, containing both benzofuran and chalcone structural units, which have been proved to show pharmacological activities, such as anti-tumor, antibacterial, anti-inflammatory and anti-tubulin activities.^17–19 In the present work, we have designed new benzofuran-chalcone hybrids based on fragment-based drug design (FBDD) by recombination of active benzofuran and chalcone fragments (Scheme 2), and the active unit of the anticancer drugs fruquintinib or gefitinib was introduced into the molecule. Moreover, the in vitro anti-tumor activity, mechanism and docking study of hybrids have been carried out. The results indicated that the benzofuran-based chalcone derivatives could be considered as potent VEGFR inhibitors.

Scheme 2 Design of new benzofuran-based chalcone derivatives.

Results and discussion

Chemistry

The new benzofuran-based chalcone derivatives (4a–4r) have been prepared by a general procedure as shown in Scheme 3. Firstly, benzofuran 1 was formed by substitution and condensation of 2-hydroxy-5-nitrobenzaldehyde with chloroacetone in CH₃OH. Then, reduction of the nitro group with Fe to an amino group gave compound 2 in reflux saturated ammonium chloride solution in high yield. Subsequently, compound 2 was substituted with 4-chloro-6,7-dimethoxy-quinazoline to obtain the key intermediate 3 in reflux isopropanol. Finally, a series of benzofuran-chalcone derivatives were synthesized by the aldol reaction with aldehydes in 22–95% yields (Table 1). However, during the preparation of the title compounds, we found that the bases (KOH, piperidine and t-BuOK) affected the aldol reaction greatly. The effect of piperidine and t-BuOK catalyzed reaction was poor; even if the reaction time was prolonged or the reaction temperature was increased, the yield of the product did not enhance obviously. When piperidine is used as the catalyst, the yield of the reaction is the best compared to other bases. In addition, in order to investigate the effects of different chalcone fragments on the anti-tumor activity of the title hybrids, we introduced different types of aldehydes (Ar–CHO or Het–CHO) into the molecules. Under the optimized reaction conditions, 18 new benzofuran-chalcone hybrids (4a–4r) have been obtained (Table 1). All prepared new benzofuran-based chalcone derivatives were characterized by ¹H NMR and ¹³C NMR, and some compounds were detected by HRMS analysis (ESI†).

Scheme 3 The synthetic route of benzofuran-chalcone derivatives.

Table 1 The structure and yields of derivatives 4a–4r

Compound	R	Yield^a (%)
a Isolated yields.
4a		59
4b		88
4c		39
4d		39
4e		78
4f		30
4g		47
4h		22
4i		78
4j		75
4k		80
4l		90
4m		33
4n		48
4o		65
4p		58
4q		43
4r		95

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Biological assay

In vitro anti-tumor activity. The thiazole blue (MTT) method is one of the main methods for screening the cytotoxic activity of anti-tumor drugs in vitro. It can detect cell survival and growth, and can perform high-throughput screening. It has the characteristics of simple operation, high sensitivity and accuracy, good repeatability, wide application range and economy, so it is widely used in the determination of cell activity.²⁰ In this work, the in vitro anticancer activity of benzofuran-chalcone hybrids (4a–4r) was tested by the MTT method. We used cisplatin (DDP) as the positive control drug, and HeLa (human cervical cancer cells), A549 (human lung cancer cells), and HCC1806 (human mammary squamous cancer cells) as the tested cancer cell lines, and the results are shown in Table 2. The results indicated that the benzofuran-chalcone derivatives showed good cytotoxic activity against HCC1806 and HeLa cell lines, but had little inhibitory activity against A549 cell lines. Among all compounds, eight derivatives showed selective inhibitory activity against tumor cell lines. Compound 4d only showed inhibitory activity against HCC1806, with IC₅₀ of 17.13 μmol L⁻¹. Compound 4g showed good cytotoxic activity against HeLa and HCC1806 with IC₅₀ of 5.61 μmol L⁻¹ and 5.93 μmol L⁻¹, respectively. The compound 4j showed inhibitory activity against both HCC1806 and HeLa, with IC₅₀ values less than 25 μmol L⁻¹. Compound 4o showed cytotoxic activity against both HCC1806 and HeLa with IC₅₀ values of 6.40 μmol L⁻¹ and 6.36 μmol L⁻¹, respectively. Compound 4p also had inhibitory activity against HCC1806 only, with IC₅₀ value less than 20 μmol L⁻¹. Compound 4q also showed cytotoxic activity against HCC1806 and HeLa with IC₅₀ values of 9.52 μmol L⁻¹ and 4.95 μmol L⁻¹, respectively.

Table 2 The in vitro anti-tumor activity of 4a–4r

Compound	IC50/(μmol L^−1)
	HCC1806	HeLa	A549
4a	>25	>25	>25
4b	>25	>25	>25
4c	>25	>25	>25
4d	17.13	>25	>25
4e	>25	>25	>25
4f	>25	>25	>25
4g	5.93	5.61	>25
4h	>25	>25	>25
4i	>25	>25	>25
4j	18.45	24.79	>25
4k	>25	>25	>25
4l	6.60	6.19	6.27
4m	>25	>25	>25
4n	7.03	3.18	21.63
4o	6.40	6.36	>25
4p	16.58	>25	>25
4q	9.52	4.95	>25
4r	>25	>25	>25
DDP	2.65	7.10	2.59

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In addition, compound 4l exhibited the highest cytotoxicity against HeLa (IC₅₀ = 6.19 μmol L⁻¹), A549 (IC₅₀ = 6.27 μmol L⁻¹) and HCC1806 (IC₅₀ = 6.60 μmol L⁻¹). Compound 4n also showed good cytotoxic activity against three tumor cell lines, and its inhibitory activity was HeLa (IC₅₀ = 3.18 μmol L⁻¹), HCC1806 (IC₅₀ = 7.03 μmol L⁻¹), and A549 (IC₅₀ = 21.63 μmol L⁻¹), respectively. Among all the derivatives, 5 compounds showed better cytotoxic activity against HeLa, and the inhibitory activity was better than that of DDP (IC₅₀ = 7.10 μmol L⁻¹). The compounds with their IC₅₀ values were 4g (IC₅₀ = 5.61 μmol L⁻¹), 4l (IC₅₀ = 6.19 μmol L⁻¹), 4n (IC₅₀ = 3.18 μmol L⁻¹), 4o (IC₅₀ = 6.36 μmol L⁻¹), and 4q (IC₅₀ = 4.95 μmol L⁻¹), respectively.

The preliminary structure–activity relationship is shown in Scheme 4. The structures of the chalcones have an obvious effect on the anti-tumor activity of the derivatives. In general, aryl chalcones displayed better cytotoxic activity than heterocyclic chalcones. In addition, when there were EDGs on the benzene ring, the compounds showed good anticancer activity against cancer cell lines, such as CH₃O and OH. Among heterocyclic derivatives, pyridine was more conducive to the activity than indole. The SAR results provided a basis for further structural optimization and mechanism research of benzofuran-chalcone compounds.

Scheme 4 Preliminary SAR analysis of derivatives.

Apoptosis test. From the preliminary screening activity results, it could be seen that most benzofuran-chalcone derivatives showed good inhibitory activity against HeLa and HCC1806. To further investigate the anti-tumor mechanism of this class of compounds, we carried out induced apoptosis experiments. Annexin V-FITC/PI double staining was used to analyze the apoptosis induced by compound 4g in the tumor cell line HCC1806, which provided a basis for further research on the anti-tumor mechanism of benzofuran chalone derivatives. 4g was prepared as a mixture of two concentrations (3 μmol L⁻¹ and 6 μmol L⁻¹), and then incubated with HCC1806 cells for 24 hours, and finally analyzed and detected by flow cytometry, as shown in the following figure (Fig. 1). We found that compared with the blank control group, when the concentration of derivative 4g was 3 μmol L⁻¹, the proportion of apoptotic cells was 9.05%, and when the concentration was 6 μmol L⁻¹, the proportion of apoptotic cells was increased to 29.4%, and the proportion of apoptotic cells was 5.29% in the blank control group. Flow cytometry showed that compound 4g could effectively trigger the apoptosis of HCC1806 cells. When the concentration increased from 3 μmol L⁻¹ to 6 μmol L⁻¹, the induced apoptosis rate of HCC1806 was significantly increased, and showed a significant dose-dependent relationship.

Fig. 1 Compound 4g induced apoptosis of HCC1806.

In vitro VEGFR-2 kinase assay. We found that compound 4g not only showed the best anticancer activity, but also induced the apoptosis rate of HCC1806. In order to investigate the inhibitory activity of 4g on VEGFR-2 kinase, we have performed the in vitro VEGFR-2 kinase study and pazopanib was used as a positive control. As shown in Fig. 2, the inhibitory rate of compound 4g on VEGFR-2 kinase was 38% at 100 μM, 16% at 50 μM and 10% at 25 μM, respectively. The result indicated that 4g showed the inhibitory effect on VEGFR-2.

Fig. 2 Inhibitory rate of 4g on VEGFR-2 kinase.

Molecular docking. Fruquintinib is a selective vascular endothelial growth factor receptor tyrosine kinase inhibitor (VEGFR-TKI), which can limit cancer angiogenesis and prevent cancer cell growth by inhibiting the phosphorylation of VEGFR. Because the structure of the prepared benzofuran-based chalcone hybrids is similar to that of fruquintinib, they may have similar targets. Based on this, we selected the 4BSK fragment as the intended target protein to conduct molecular docking experiments with compound 4g, and obtained protein data from PDB data for molecular docking. The results showed that compound 4g had an obvious binding site with the target protein 4BSK (Fig. 3). The hydrophobic amino acids of compound 4g interacting with the target protein are LEU176, LEU197 and ALA201, the amino acid residue interacting with the hydrogen bond in the compound is ASN167, and the final docking score is 105.469, indicating that the interaction between compound 4g and the target protein 4BSK is strong, which could be considered as a potent VEGFR-2 inhibitor.

Fig. 3 Molecular docking of compound 4g with the target protein 4BSK.

Conclusions

In the present work, a series of novel benzofuran-based chalcone derivatives have been prepared, which were screened for their in vitro anti-tumor activities. The results indicated that most compounds showed good cytotoxic activity against HCC1806, HeLa and A549 cell lines. In particular, compound 4g showed the best anti-tumor activity against HCC1806 and HeLa with IC₅₀ of 5.93 μmol L⁻¹ and 5.61 μmol L⁻¹, respectively. The preliminary mechanism assessment showed that compound 4g could effectively trigger the apoptosis of HCC1806 cells in a dose dependent manner. Moreover, the molecular docking study showed that compound 4g had an obvious binding site with the target VEGFR-2 (PDB ID: 4BSK). Therefore, the benzofuran-chalcone derivatives may be considered as new potent VEGFR-2 inhibitors.

Experimental

General information

All chemical reagents and solvents were commercially available. NMR data was recorded on a Bruker AV 400 spectrometer (Bruker), with TMS as the standard. HRMS data was obtained on a Triple TOF 5600+ spectrometer (AB Sciex). Human cervical cancer cells (HeLa), human lung cancer cells (A549), and human mammary squamous cancer cells (HCC1806) were purchased from Guangzhou Cellcook Biotech Co., Ltd. Biological data was obtained using a BSM-120.4 electronic balance, carbon dioxide incubator and flow cytometer. Thin-layer chromatography (TLC) analysis was performed on silica gel plates GF₂₅₄.

General preparation of compound (1)

To a stirred solution of 5-nitrosalicylaldehyde (1.67 g, 10 mmol) in 50 mL methanol, potassium hydroxide (0.56 g, 10 mmol) was added and left to react at room temperature for half an hour, and the reaction system was stirred in an ice water bath for 10 min. Then chloracetone (12 mmol) was slowly added to the reaction mixture and heated to 75 °C in an oil bath for 5–6 h. When the reaction was completed (detected by TLC), the mixture was cooled, and poured into a separating funnel. Then the mixture was extracted with methylene chloride (100 mL) and washed with water for three times, and the organic phase was dried by anhydrous sodium sulfate and concentrated by vacuum. The residue was subjected to silica gel column chromatography to give a yellow solid (1.30 g, 63% yield).

Synthesis of compound (2)

To a stirred solution of iron powder (1.41 g, 25 mmol) and 30 mL of saturated ammonium chloride aqueous, the solution of compound 1 (1.30 g, 6.3 mmol) in 8 mL of anhydrous ethanol was slowly added and left to react at 100 °C for 5 h. After the reaction was completed by TLC, the reaction mixture was cooled and then extracted with ethyl acetate (50 mL) for three times. The organic phase was combined and dried with Na₂SO₄, concentrated in vacuo and purified by column chromatography to afford an orange solid (0.91 g, 82% yield).

Synthesis of key intermediate (3)

To a stirred solution of compound 2 (0.91 g. 5.19 mmol) and 4-chloro-6,7-dimethoxyquinazoline (1.17 g, 5.19 mmol) in isopropyl alcohol (25 mL), the mixture was heated in reflux overnight. After the reaction was completed, the mixture was filtered and washed with isopropyl alcohol (15 mL) for 3–4 times to afford a pale yellow solid after drying.

General synthetic procedure for title derivatives (4a–4r)

To a stirred solution of compound 3 (145.3 mg, 0.4 mmol) and various aldehydes (0.48 mmol) in ethanol (5 mL), piperidine (2 d) was added and the mixture was refluxed for 5 h. After completion of the reaction as indicated by TLC, the mixture was concentrated in vacuo, and the residue was purified by column chromatography to give the corresponding products.

Compound 4a. ¹H NMR(400 Hz, DMSO-d₆) δ: 8.53(s, 1H), 7.96–7.99(m, 2H), 7.79(d, J = 15.6 Hz, 1H), 7.57–7.60(m, 1H), 7.42–7.47(m, 4H), 7.30(d, J = 15.4 Hz, 1H), 7.17(d, J = 11.8 Hz, 2H), 6.80(d, J = 8.9 Hz, 2H), 3.93(s, 3H), 3.90(s, 3H), 3.24–3.26(m, 4H), 2.48(t, J = 17.0 Hz, 4H), 2.26(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 179.90, 157.04, 155.01, 154.86, 153.71, 153.09, 153.00, 149.63, 147.51, 145.39, 134.99, 130.74, 127.98, 124.88, 124.41, 117.22, 116.74, 114.75, 112.75, 112.64, 109.28, 107.82, 100.28, 56.66, 56.38, 54.89, 47.64, 46.25, 29.83.
Compound 4b. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.76(s, 1H), 8.49(s, 1H), 8.27(d, J = 2.0 Hz, 1H), 8.22(s, 1H), 7.85–7.91(m, 1H), 7.51–7.79(m, 4H), 7.68(d, J = 15.6 Hz, 1H), 7.21(s, 1H), 7.03(d, J = 8.9 Hz, 2H), 3.98(s, 3H), 3.95(s, 3H), 3.76(t, J = 4.6 Hz, 4H), 3.27–3.30(m, 4H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 178.72, 157.17, 154.84, 154.78, 153.29, 153.12, 153.53, 149.45, 144.49, 135.95, 131.16, 127.63, 125.21, 124.81, 118.00, 117.09, 114.56, 112.41, 109.17, 107.22, 102.54, 66.36, 56.74, 56.32, 47.54; HRMS-ESI calcd for C₃₁H₂₉N₄O₅ (M + H)⁺ 537.2133, found 537.2129.
Compound 4c. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.80(s, 1H), 8.64(d, J = 2.5 Hz, 1H), 8.50(s, 1H), 8.35(d, J = 0.5 Hz, 1H), 8.30(d, J = 2.0 Hz, 1H), 8.07(d, J = 8.8 Hz, 2H), 7.99(d, J = 8.8 Hz, 2H), 7.90–7.94(m, 3H), 7.78–7.89(m, 2H), 7.20(s, 1H), 6.60–6.61(m, 1H), 3.98(s, 3H), 3.94(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 178.76, 157.18, 154.86, 154.39, 153.04, 152.73, 149.47, 142.99, 142.11, 141.59, 136.04, 132.58, 130.90, 128.53, 127.53, 125.65, 122.07, 118.87, 117.24, 115.79, 112.51, 109.15, 108.90, 107.09, 102.54, 56.75, 56.33.
Compound 4d. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.66(s, 1H), 8.48(s, 1H), 8.29(d, J = 1.7 Hz, 2H), 7.54–8.25(m, 7H), 7.49(t, J = 8.1 Hz, 1H), 7.34(t, J = 7.6 Hz, 2H), 7.20(s, 1H), 3.98(s, 3H), 3.94(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 183.09, 161.77, 160.33, 159.48, 158.85, 158.12, 158.01, 157.81, 157.39, 154.14, 152.05, 141.02, 135.39, 133.45, 132.45, 132.35, 130.36, 128.93, 127.57, 126.68, 121.73, 120.38, 119.06, 117.24, 116.64, 114.01, 112.35, 107.15, 61.45, 61.03, 60.12.
Compound 4e. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.64(s, 1H), 8.47(s, 1H), 8.29(d, J = 2.0 Hz, 1H), 8.23(d, J = 0.6 Hz, 1H), 7.93–8.03(m, 1H), 7.83–7.89(m, 3H), 7.49(d, J = 9.0 Hz, 1H), 7.73(d, J = 3.3 Hz, 1H), 7.53(d, J = 15.4 Hz, 1H), 7.20–7.23(m, 2H), 3.98(s, 3H), 3.94(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 183.09, 161.77, 160.33, 159.48, 158.85, 158.12, 158.01, 157.81, 157.39, 154.14, 152.05, 141.02, 135.39, 133.45, 132.45, 132.35, 130.36, 128.93, 127.57, 126.68, 121.73, 120.38, 119.06, 117.24, 116.64, 114.01, 112.35, 107.15, 61.45, 61.03, 60.12.
Compound 4f. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.63(s, 1H), 8.46(s, 1H), 8.25(d, J = 2.0 Hz, 1H), 8.10(d, J = 0.7 Hz, 1H), 7.87–7.90(m, 2H), 7.78(d, J = 9.0 Hz, 1H), 7.61(d, J = 15.4 Hz, 1H), 7.39(d, J = 15.4 Hz, 1H), 7.19(s, 1H), 7.04(d, J = 3.3 Hz, 1H), 6.36–6.37(m, 1H), 3.97(s, 3H), 3.93(s, 3H), 2.41(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 183.26, 161.76, 159.45, 159.13, 157.19, 154.93, 154.11, 152.06, 140.91, 135.13, 132.38, 129.98, 125.15, 121.93, 121.62, 119.29, 117.14, 115.32, 114.01, 112.35, 107.14, 61.43, 61.00.
Compound 4g. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.88(s, 1H), 8.50(s, 1H), 8.38(d, J = 0.6 Hz, 1H), 8.33(d, J = 2.0 Hz, 1H), 7.95(s, 1H), 7.85–7.91(m, 2H), 7.73–7.79(m, 2H), 7.21(s, 1H), 7.10(d, J = 2.2 Hz, 2H), 6.62(t, J = 4.6 Hz, 1H), 3.99(s, 3H), 3.94(s, 3H), 3.83(s, 6H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 183.56, 166.00, 161.97, 159.67, 159.03, 157.64, 157.52, 154.25, 148.78, 141.51, 140.77, 132.23, 130.44, 127.59, 122.00, 120.84, 117.26, 113.89, 112.07, 111.67, 108.30, 107.41, 61.56, 61.09, 60.70, 57.25.
Compound 4h. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.69(s, 1H), 8.47(s, 1H), 8.30–8.32(m, 2H), 7.85–7.89(m, 2H), 7.77–7.79(m, 3H), 7.55(d, J = 2.0 Hz, 1H), 7.41–7.44(m, 1H), 7.20(s, 1H), 7.07(d, J = 8.4 Hz, 1H), 3.98(s, 3H), 3.94(s, 3H), 3.89(s, 3H), 3.84(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 183.54, 161.84, 159.51, 159.29, 157.98, 157.33, 156.79, 154.29, 154.15, 151.90, 149.17, 140.88, 132.42, 132.29, 132.14, 130.10, 129.42, 124.67, 121.72, 120.01, 117.21, 116.87, 116.00, 114.00, 112.26, 107.18, 61.47, 61.05, 60.98, 60.87; HRMS-ESI calcd for C₂₉H₂₆N₃O₆ (M + H)⁺ 512.1816, found 512.1816.
Compound 4i. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.97(s, 1H), 8.52(s, 1H), 8.34(d, J = 0.7 Hz, 1H), 8.29(d, J = 2.0 Hz, 1H), 7.91–7.97(m, 3H), 7.83–7.90(m, 1H), 7.78(d, J = 9.3 Hz, 2H), 7.56(d, J = 8.5 Hz, 2H), 7.20(s, 1H), 3.99(s, 3H), 3.94(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 183.45, 162.03, 159.75, 159.00, 157.57, 154.30, 147.20, 140.65, 140.59, 138.58, 135.86, 134.26, 132.22, 130.55, 127.82, 122.18, 120.77, 117.24, 113.79, 111.28, 107.53, 61.59, 61.12.
Compound 4j. ¹H NMR(400 Hz, DMSO-d₆) δ: 10.06(s, 1H), 8.54(s, 1H), 8.35(d, J = 0.6 Hz, 1H), 8.28(d, J = 2.0 Hz, 1H), 7.99(s, 1H), 7.86–7.94(m, 4H), 7.78–7.82(m, 2H), 7.71(d, J = 8.5 Hz, 2H), 7.22(s, 1H), 3.99(s, 3H), 3.95(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 183.48, 162.15, 159.87, 159.03, 157.66, 154.38, 147.34, 140.51, 138.91, 137.20, 136.07, 132.24, 130.68, 129.52, 127.87, 122.37, 120.79, 117.30, 113.71, 110.93, 107.61, 61.62, 61.16.
Compound 4k. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.69(s, 1H), 8.73(d, J = 4.6 Hz, 1H), 8.48(s, 1H), 8.27–8.29(m, 2H), 8.15(d, J = 15.4 Hz, 1H), 7.80–7.94(m, 6H), 7.46–7.49(m, 1H), 7.20(s, 1H), 3.98(s, 3H), 3.94(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 183.80, 161.81, 159.52, 158.83, 157.65, 157.45, 154.16, 147.79, 142.54, 140.96, 132.31, 130.70, 130.48, 130.31, 130.00, 121.85, 117.29, 113.98, 112.26, 107.14, 61.45, 61.05.
Compound 4l. ¹H NMR(400 Hz, DMSO-d₆) δ: 10.21(s, 1H), 9.70(s, 1H), 8.47(s, 1H), 8.29(d, J = 2.0 Hz, 2H), 8.24(s, 2H), 7.87–7.90(m, 4H), 7.65–7.79(m, 1H), 7.20(s, 1H), 6.89(d, J = 8.6 Hz, 2H), 3.98(s, 3H), 3.94(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 178.83, 160.92, 157.06, 154.70, 154.59, 153.32, 152.49, 149.35, 147.24, 144.43, 136.12, 131.64, 127.57, 125.96, 125.27, 124.00, 122.50, 118.77, 116.97, 116.40, 114.91, 112.40, 109.25, 107.55, 102.43, 56.71, 56.27; HRMS-ESI calcd for C₂₇H₂₁N₃O₅ (M + H)⁺ 468.1554, found 468.1545.
Compound 4n. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.72(s, 1H), 8.48(s, 1H), 8.30–8.33(m, 2H), 7.85–7.92(m, 6H), 7.79(d, J = 9.0 Hz, 1H), 7.48–7.49(m, 3H), 7.19(s, 1H), 3.98(s, 3H), 3.93(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 178.83, 157.06, 154.74, 154.27, 153.21, 152.64, 149.38, 147.00, 143.93, 136.16, 134.84, 131.36, 129.46, 129.43, 127.49, 125.56, 122.32, 117.06, 115.86, 112.46, 109.21, 107.39, 102.43, 56.71, 56.27.
Compound 4o. ¹H NMR(400 Hz, DMSO-d₆) δ: 10.73(s, 1H), 8.72(d, J = 6.0 Hz, 2H), 8.66(s, 1H), 8.44(d, J = 0.5 Hz, 1H), 8.26(d, J = 2.0 Hz, 1H), 8.16(d, J = 12.8 Hz, 1H), 7.89–8.09(m, 1H), 7.87–7.89(m, 3H), 7.77–7.84(m, 2H), 7.28(s, 1H), 4.01(s, 3H), 3.97(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 183.32, 162.76, 160.56, 158.87, 158.22, 155.63, 154.82, 146.82, 145.83, 139.70, 132.20, 131.36, 127.81, 117.52, 113.18, 108.46, 108.30, 61.86, 61.38.
Compound 4p. ¹H NMR(400 Hz, DMSO-d₆) δ: 9.75(s, 1H), 8.49(s, 1H), 8.37(d, J = 24.8 Hz, 1H), 8.16(s, 1H), 8.14(t, J = 10.0 Hz, 1H), 7.90(s, 3H), 7.78–7.82(m, 3H), 7.61(q, J = 8.9 Hz, 1H), 7.21(s, 1H), 3.99(s, 3H), 3.95(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 178.79, 154.81, 154.19, 153.15, 152.76, 149.43, 141.75, 136.32, 127.44, 117.20, 116.28, 112.53, 109.17, 102.43, 56.72, 56.32;
Compound 4q. ¹H NMR(400 Hz, DMSO-d₆) δ: 12.06(s, 1H), 10.15(s, 1H), 8.56(s, 1H), 8.30(d, J = 1.9 Hz, 1H), 8.23(s, 1H), 8.13–8.17(m, 3H), 8.04(s, 1H), 7.80–7.87(m, 2H), 7.65(d, J = 15.6 Hz, 1H), 7.52–7.54(m, 1H), 7.27–7.29(m, 2H), 7.24(s, 1H), 4.00(s, 3H), 3.96(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 178.87, 157.47, 155.15, 155.07, 152.63, 152.41, 149.65, 139.21, 138.11, 135.44, 134.65, 127.81, 125.50, 125.13, 123.36, 121.80, 121.05, 117.50, 115.46, 113.72, 113.21, 113.05, 112.46, 108.93, 102.97, 56.90, 56.42; HRMS-ESI calcd for C₂₉H₂₃N₄O₄ (M + H)⁺ 491.1714, found 491.1710.
Compound 4r. ¹H NMR(400 Hz, DMSO-d₆) δ: 12.17(s, 1H), 9.66(s, 1H), 8.48(s, 1H), 8.23–8.36(m, 4H), 8.13(d, J = 15.6 Hz, 1H), 7.79–7.90(m, 3H), 7.62(d, J = 15.6 Hz, 1H), 7.50(d, J = 8.6 Hz, 1H), 7.41(d, J = 8.4 Hz, 1H), 7.21(s, 1H), 3.99(s, 3H), 3.95(s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 178.95, 157.06, 154.84, 154.69, 153.40, 152.37, 149.35, 147.40, 138.28, 136.73, 136.03, 135.08, 127.74, 127.32, 125.96, 124.85, 122.97, 116.90, 116.32, 114.94, 114.54, 114.21, 112.77, 112.39, 109.28, 107.68, 102.39, 56.70, 56.28; HRMS-ESI calcd for C₂₉H₂₂BrN₄O₄ (M + H)⁺ 569.0819, found 569.0815.

In vitro anti-tumor activity

The anticancer activity of the synthetic derivatives (4a–4r) was tested by the MTT method, with cisplatin as the positive control drug, and HeLa (human cervical cancer cell), A549 (human lung cancer cell) and HCC1806 (human mammary squamous cancer cell) were selected as the tumor strains to be detected. At first, a constant temperature incubator (37 °C, 5% CO₂) was used to provide a culture environment. When cells were found at the bottom of the petri dish, passage culture was started for later experiments. On a 96-well plate, tumor cells with a logarithmic growth stage (5 × 10⁴ cells per mL) were inoculated, cultured overnight, and then compounds were added to them, and five concentration gradients of 100 μmol L⁻¹, 50 μmol L⁻¹, 25 μmol L⁻¹, 12.5 μmol L⁻¹ and 6.25 μmol L⁻¹ were set. Each concentration has 3 compound pores. After 48 hours of reaction, 20 μL and 5 mg ml⁻¹ of MTT were added to each well and cultured continuously for 4 h. Finally, it was centrifuged at 3000 rpm for 10 min, the culture solution was absorbed and discharged, 150 μL DMSO was added to finish the reaction, and the reaction was shaken in the shaker for 10 min. The absorption was measured at 570 nm wavelength by an enzymograph device, the absorbance was determined, and the IC₅₀ value was calculated.

Apoptosis test

The logarithmic growth phase HCC1806 (human breast squamous carcinoma cell) cells were digested for cell suspension and the cell density was diluted to 4 × 10⁵ cells per mL. 1 mL of cell suspension was added to each six-well plate, and then the six-well plate was cultured at 37 °C in an incubator with 5% CO₂ for 24 h. One day later (24 h), 1 mL of the sample solution with a certain concentration gradient (3 μmol L⁻¹, 6 μmol L⁻¹) was added to the six-well plate, and the culture was continued for 24 h at 37 °C. A day later (24 h), the collected cells were immersed and washed twice with cold PBS, and finally centrifuged at 2000 rpm for 5 min, then the supernatant was absorbed and discarded, and precipitated in an ice bath at 0 °C. 500 μL Annexin binding buffer was gently suspended into each well. Then the 5 μL red fluorescent dye (PI) and 5 μL green fluorescent dye (Annexin V) were added into the mixture, mixed slowly and evenly, and placed in an ice water bath for 10 minutes away from light. The results were detected by flow cytometry.

VEGFR-2 test

The inhibitory effect of compound 4g on VEGFR-2 kinase was determined using a VEGFR-2 (KDR) kinase assay kit. The compound was prepared at 100-fold the highest desired concentration in 100% DMSO and then diluted 10-fold in 1× kinase buffer 1 to prepare the highest concentration of the 10-fold intermediate dilutions. For positive and negative controls, 10% DMSO was prepared in 1× kinase buffer 1 (v/v) so that all wells contained the same amount of DMSO (diluent solution). Then 25 μL of the master mix and 5 μL of the compound solution were added to each well. 20 μL of 1× kinase buffer 1 and 5 μL of diluent solution were added to the “blank” and “positive control” wells as well. Subsequently, the reaction was initiated by adding 20 μL of diluted VEGFR-2 kinase to the wells designated “positive control” and “test inhibitor.” Then the mixture was incubated at 30 °C for 45 min and reacted for 45 min, and 50 μL of Kinase-Glo™ MAX reagent was added. The plate was covered with aluminum foil and incubated at room temperature for 15 min, and were immediately read using a luminometer or microplate reader capable of reading luminescence.

Molecular docking experiment

The 2D planar structure of the desired compound was drawn in ChemDraw 2D and saved as a mol format file. The three-dimensional crystal structure of VEGFR-1 in PDB format was obtained from the PDB database (https://www.rcsb.org/), and the 4BSK fragment was selected as the intended target protein of the synthesized drug. The obtained protein was pre-treated with water removal and hydrogen replenishment in Discovery Studio and saved as a PBD format file. Docking between small molecules and target proteins was carried out in Discovery Studio, the scoring results were obtained, the docking results were visualized, and interaction diagrams of the docking sites were obtained.

Data availability

Data for this article, including experimental details and spectra data, are available as part of the ESI.†

Conflicts of interest

There is no conflict of interest to declare.

Acknowledgements

This work was financially supported by the Key Project of Applied Basic Research of Yunnan Province (202401AS070007), and the Yunnan Provincial Science and Technology Department-Applied Basic Research Joint Special Funds of Yunnan University of Chinese Medicine (202001AZ070001-007).

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Footnotes

† Electronic supplementary information (ESI) available: NMR and HRMS spectra. See DOI: https://doi.org/10.1039/d4md00621f

‡ These authors contributed equally to this work.

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