Novel VEGFR2 inhibitors with thiazoloquinoxaline scaffold targeting hepatocellular carcinoma with lower cardiotoxic impact

Abstract

Hepatocellular carcinoma (HCC) is a fatal tumor which is usually diagnosed at advanced stage. Molecular targeted drugs were used recently to treat HCC, however, due to serious side effects, mainly cardiotoxicity and emergence of resistance, there is demanding to explore new chemotherapeutics. 10 novel thiazoloquinoxaline derivatives coupled with different sulfonamide moieties 4(a-j) were designed and synthesized fulfilling pharmacophoric features of VEGFR-2 inhibition. Structures of all new compounds were verified via spectral and microanalytical data. After carrying in-vitro VEGFR-2 assay for compounds 4(a-j); sulfapyridine and sulfamethoxazole derivatives 4d and 4f showed potential inhibitory effect [61.04 and 83.35 nM], respectively, comparable to standard sorafenib [51.41 nM]. Both were then further evaluated for their cytocidal activity against HepG2 cell-line and against myocardium cells using H9C2 cell-line. As a result, only sulfapyridine derivative 4d exhibited a significant inhibition of HepG2 cells viability [IC₅₀ = 4.31 μM]. Furthermore, it showed relatively lower cytotoxic impact against normal H9C2 myocardium cells [IC₅₀, 33.47 μM] compared to that of sorafenib [IC₅₀, 98.07 μM]. In-vivo study was carried out to determine myocardium safety of compound 4d on irradiated mice (8 Gy). In-vivo results of sulfapyridine derivative 4d showed normal cardiac enzyme function (CK) and serum catalase activity with significant reductions in LDH, cardiac TNF-α and caspase-9 levels, alongside with its efficacy in suppressing the expression of hepatic VEGF. In conclusion, sulfapyridine derivative 4d could be considered a promising candidate as VEGFR-2 inhibitor with less myocardium side effect.

Figure 1

Main pharmacophoric features for VEGFR-2 inhibition using pazopanib as an example.

Scheme 1

Synthetic route of compounds 4 (a–j).

Figure 2

Effect of sorafenib and compound 4d [0.01, 0.1, 1, 10 and 100 μM] on H9C2 cells viability and IC₅₀.

Figure 3

Effect of daily treatment (3 days) of sorafenib or compound 4d [50 mg/ kg, p.o] on heart creatine kinase activity (CK, ng/mg) in 8 Gy irradiated mice. All values are plotted as mean ± S.E. * represented significance from Normal at P ≤ 0.05, # represented significance from IRR at P ≤ 0.05, s represented significance from IRR + sorafenib at P ≤ 0.05.

Figure 4

Effect of daily treatment (3 days) of sorafenib or compound 4d [50 mg/ kg, p.o] on serum lactate dehydrogenase (LDH, IU/ml) and Catalase activity (IU/ml) in 8 Gy irradiated mice. All values are plotted as mean ± S.E. * represented significance from Normal at P ≤ 0.05, # represented significance from IRR at P ≤ 0.05, s represented significance from IRR + sorafenib at P ≤ 0.05.

Figure 5

Effect of daily treatment (3 days) of sorafenib or compound 4d [50 mg/ kg, p.o] on heart levels of caspase-9 (ng/mg Tp) and TNF-α (pg/ mg Tp) in 8 Gy irradiated mice. All values are plotted as mean ± S.E. * represented significance from Normal at P ≤ 0.05, # represented significance from IRR at P ≤ 0.05, s represented significance from IRR + sorafenib at P ≤ 0.05.

Figure 6

In-vivo Immunohistochemistry expression of VEGF in liver tissue of irradiated (8 Gy) mice after daily treatment of sorafenib or compound 4d (50 mg/kg, 3 days, p.o). (a) Normal group, (b) IRR group, (c) IRR + sorafenib group, (d) IRR + compound 4d group (VEGF X400).

Figure 7

3D and 2D binding interactions of compound 4d into VEGFR-2 (PDB: 4ASD).