In Vitro Anticancer Activity Screening of Novel Fused Thiophene Derivatives as VEGFR-2/AKT Dual Inhibitors and Apoptosis Inducers

Abstract

Protein kinases are seen as promising targets in controlling cell proliferation and survival in treating cancer where fused thiophene synthon was utilized in many kinase inhibitors approved by the FDA. Accordingly, this work focused on adopting fused thienopyrrole and pyrrolothienopyrimidine scaffolds in preparing new inhibitors, which were evaluated as antiproliferative agents in the HepG2 and PC-3 cell lines. The compounds 3b (IC₅₀ = 3.105 and 2.15 μM) and 4c (IC₅₀ = 3.023 and 3.12 μM) were the most promising candidates on both cells with good selective toxicity-sparing normal cells. A further mechanistic evaluation revealed promising kinase inhibitory activity, where 4c inhibited VEGFR-2 and AKT at IC₅₀ = 0.075 and 4.60 μM, respectively, while 3b showed IC₅₀ = 0.126 and 6.96 μM, respectively. Moreover, they resulted in S phase cell cycle arrest with subsequent caspase-3-induced apoptosis. Lastly, docking studies evaluated the binding patterns of these active derivatives and demonstrated a similar fitting pattern to the reference ligands inside the active sites of both VEGFR-2 and AKT (allosteric pocket) crystal structures. To conclude, these thiophene derivatives represent promising antiproliferative leads inhibiting both VEGFR-2 and AKT and inducing apoptosis in liver cell carcinoma.

Keywords: AKT; VEGFR-2; anticancer; apoptosis; thienopyrimidine; thiophene.

Figure 1

Thienopyrimidine derivatives with potential activities as anticancer agents; structures adopted from (I) [8,16], (II) [16,18], (III) [21], (IV) [8], (V) [23], and (VI) [25].

Figure 2

Development strategy of the novel thiophene candidates.

Scheme 1

Conditions and reagents: (a1) cyanoacetamide, ethanol, morpholine, and sulfur; (a2) ethyl cyanoacetate, ethanol, morpholine, and sulfur; (b) aldehyde derivatives, DMF, C. HCl, and reflux for 24 h; (c) isothiocyanates, ethanol, and reflux for 8 h; (d) substituted aniline derivatives 5a–d, potassium hydroxide, ethanol, and reflux for 18 h.

Figure 3

(a) Effect of treatment of HepG2 cell line with 3b and 4c compounds on VEGFR2 residual concentration using ELISA. (b) Kinase inhibitory activity assay (IC₅₀ in μM) of 3b and 4c on VEGFR-2 using ELISA and taking sorafenib as the reference compound. Values are given as mean ± S.D. for groups of 3. (****) significantly different from HepG2 group at p < 0.0001.

Figure 4

(a) Effect of treatment of HepG2 cell line with 3b and 4c compounds on AKT-1 residual concentration using ELISA. (b) Kinase inhibitory activity assay (IC₅₀ in μM) of 3b and 4c on AKT using ELISA and taking LY2780301 as the reference compound. Values are given as mean ± S.D. for groups of 3. (****) significantly different from HepG2 group at p < 0.0001.

Figure 5

Cell cycle analysis and DNA content in different cell cycle phases after treating HepG2 cells with 3b and 4c for 48 h. (a) Control HepG2 cells; (b) compound 3b; (c) compound 4c.

Figure 6

The proapoptotic effect of the novel compounds 3b and 4c on HepG2 cells after 48 h against control, untreated cells. Q1: viable cells; Q2: early apoptotic; Q3: late apoptotic; and Q4: necrotic cell content. (a) Control HepG2 cells; (b) compound 3b; (c) compound 4c.

Figure 7

The effect of treating HepG2 cells with 3b and 4c compounds on caspase-3 concentration using ELISA. Values are given as mean + S.D. for groups of 3. (****) significantly different from HepG2 group at p < 0.0001.

Figure 8

Binding interaction of 4c inside VEGFR-2 active site with CDOCKER score of −11.20 Kcal/mol. (a) The 2D interaction diagram; (b) the 3D interaction diagram; and (c) the binding interactions codes.

Figure 9

Binding interaction of 4c inside AKT-1 active site with CDOCKER score of −10.16 Kcal/mol. (a) the 2D interaction diagram; (b) the 3D interaction diagram; and (c) the binding interactions codes.