Halogenated Flavonoid Derivatives Display Antiangiogenic Activity

Abstract

Antiangiogenic agents attenuate tumours' growth and metastases and are therefore beneficial as an adjuvant or standalone cancer regimen. Drugs with dual antiproliferative and antiangiogenic activities can achieve anticancer efficacy and overcome acquired resistance. In this study, synthetic flavones (5a,b) with reported anticancer activity, and derivatives (4b and 6a), exhibited significant inhibition of endothelial cell tube formation (40-55%, 12 h) at 1 µM, which is comparable to sunitinib (50% inhibition at 1 µM, 48 h). Flavones (4b, 5a,b and 6a) also showed 25-37% reduction in HUVECs migration at 10 µM. In a Western blotting assay, 5a and 5b subdued VEGFR2 phosphorylation by 37% and 57%, respectively, suggesting that VEGFR2 may be their main antiangiogenic target. 5b displayed the best docking fit with VEGFR2 in an in silico study, followed by 5a, emphasizing the importance of the 7-hydroxyl group accompanied by a 4-C=S for activity. Conversely, derivatives with a 4-carbonyl moiety fitted poorly into the target's binding pocket, suggesting that their antiangiogenic activity depends on a different target. This study provides valuable insight into the Structure Activity Relationships (SAR) and modes of action of halogenated flavones with VEGFR2 and highlights their therapeutic potential as antiangiogenic/anticancer lead compounds.

Keywords: SAR; angiogenesis; antiangiogenic; cancer; flavones; flavonoids.

Scheme 1

Synthesis of oxo and thio-p-halo-phenyl flavone derivatives. (I) Halobenzoyl Chloride, DBU, pyridine, 75 °C, 2 h; (II) Pyridine, KOH, 50 °C, 2 h; (III) Glacial acetic acid, 1% H₂SO₄, 90–110 °C, 1 h; (IV) Dry toluene, Lawesson’s reagent, 110 °C, 4 h; (V, VI) Dry DCM, BBr₃, room temp, 4 h.

Figure 1

Cell viability of HUVECs at 40 µM of tested flavones. Data are expressed as mean ± standard error of the mean (SEM), n = 3.

Figure 2

Antiangiogenic activity of flavonoid derivatives on in vitro HUVEC tube formation after 12 h expressed as a ratio to the +ve control (10 ng/mL VEGF-enriched media). (A) Representative images of tube formation assay at 4× magnification. Images were analysed using Angiogenesis Analyzer macro in ImageJ software; (B) number of junctions, (C) number of meshes, (D) number and length of master segments and (E) number and length of segments. Data are expressed as mean ± standard error of the mean (SEM), n = 3. Statistical significance was estimated with respect to the +ve control by one-way ANOVA, followed by Dunnett’s multiple comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 2

Antiangiogenic activity of flavonoid derivatives on in vitro HUVEC tube formation after 12 h expressed as a ratio to the +ve control (10 ng/mL VEGF-enriched media). (A) Representative images of tube formation assay at 4× magnification. Images were analysed using Angiogenesis Analyzer macro in ImageJ software; (B) number of junctions, (C) number of meshes, (D) number and length of master segments and (E) number and length of segments. Data are expressed as mean ± standard error of the mean (SEM), n = 3. Statistical significance was estimated with respect to the +ve control by one-way ANOVA, followed by Dunnett’s multiple comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 3

In vitro HUVEC wound closure (migration) inhibition activity of flavonoid derivatives (4b, 5a,b and 6a) expressed as a ratio to the +ve control (10 ng/mL VEGF-enriched media). (A) Representative images of scratch assay at 0 h and 12 h at 10× magnification. Images were analysed using ImageJ software; (B) % wound closure after 12 h as a ratio to +ve control. Data are expressed as mean ± standard error of the mean (SEM), n = 3. Statistical significance was estimated with respect to the +ve control by one-way ANOVA, followed by Dunnett’s multiple comparison test (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Figure 4

VEGFR2 induced phosphorylation inhibition activity of flavonoid derivatives (4b, 5a,b and 6a) on HUVEC cell lysates. (A) Representative Western blot images of the HUVECs proteins; T-VEGFR2, P-VEGFR2 and β-Actin. Images were analysed using ImageJ software; (B) VEGFR2-induced phosphorylation inhibition expressed as a ratio to the +ve control (10 ng/mL VEGF-enriched media). Data are expressed as mean ± standard error of the mean (SEM), n = 3. Statistical significance was estimated with respect to the +ve control by one-way ANOVA, followed by Dunnett’s multiple comparison test (** p < 0.01, **** p < 0.0001).

Figure 5

Summary of SAR of the tested panel of flavones on VEGFR2 phosphorylation inhibition.

Figure 6

Comparison between tube formation (represented by nb junctions), scratch and Western blotting assay results of flavonoid derivatives (4b, 5a,b and 6a). (A) Chemical structures of 4a, 5a,b and 6a; (B) comparison of 10 µM concentration; (C) comparison of 1 µM concentration.

Figure 7

Docking of the original co-crystallized ligand (N-{4-[4-amino-6-(4-methoxyphenyl)furo[2,3-d]pyrimidin-5-yl]phenyl}-n’-[2-fluoro-5-(trifluoromethyl)phenyl]urea) with VEGFR2 (1YWN). (A) Ribbon representation showing ligand pose in chain A; (B) 2D interaction of ligand with binding pocket; (C) mesh representation of binding pocket surface interaction with ligand; (D) 3D interaction of ligand with binding pocket showing involved amino acids.

Figure 8

3D interaction of flavonoid derivatives (4b, 5a,b and 6a) (green) with the ATP-binding pocket of VEGFR2 and compared to co-crystallized ligand (pink). (A,B) Flavonoid 4b; (C,D) 5a; (E,F) 5b; (G,H) 6a.

Figure 8

3D interaction of flavonoid derivatives (4b, 5a,b and 6a) (green) with the ATP-binding pocket of VEGFR2 and compared to co-crystallized ligand (pink). (A,B) Flavonoid 4b; (C,D) 5a; (E,F) 5b; (G,H) 6a.

Figure 9

Mesh representation of flavonoid derivatives (4b, 5a,b and 6a) in the ATP-binding pocket of VEGFR2 compared to co-crystallized ligand (pink). (A) Less active horizontal orientation of flavonoids 4b (green) and 6a (blue); (B) more active vertical orientation of flavonoids 5a (green) and 5b (blue).

Figure 10

Cartoon representation showing the different orientations (horizontal or vertical) of flavonoid derivatives (4b, 5a,b and 6a) in ATP-binding pocket of VEGFR2 resulting from the highlighted structural differences (yellow shadow, C=S; red circle, OCH₃; red shadow, C=O). (A) 4b (green, horizontal orientation) vs. 5b (blue, vertical orientation); (B) 5a (blue, vertical orientation) vs. 6a (green, horizontal orientation).