Design, synthesis and evaluation of PD176252 analogues for ameliorating cisplatin-induced nephrotoxicity

Abstract

Cisplatin is a clinical chemotherapy drug for cancers; however, its remarkably high kidney toxicity and other toxicities pose a danger to patients. As the small molecule inhibitor of GRPR, PD176252 can inhibit the growth and proliferation of various cancer cells, but the characteristics of high toxicity and poor water solubility has limited its use as a drug. When we studied PD176252 for the reduction of toxicity of cisplatin, we modified its structure to synthesize 16 analogues. Surprisingly, the analogues showed reduced cisplatin-induced renal toxicity, and unlike PD176252, the analogues 5d and 5m were almost non-toxic to the normal HK2 cells. Furthermore, the analogue 5d and PD176252 were subjected to cisplatin-induced inflammatory response in vitro. The results showed that 5d was able to better prevent this condition by effectively inhibiting its inflammatory response. Thus, this study will help in clinically reducing the side effects of cisplatin.

Fig. 1. The general design strategy.

Scheme 1. Synthesis of compounds 5a–p. Reagents and conditions: (i) EDCI, HoBt, TEA, DCM, rt., 24 h; (ii) Na₂CO₃/CH₃OH, 3–5 h; HCl/H₂O; (iii) EDCI, HoBt, TEA, DCM, rt., 24 h.

Fig. 2. Effect of compounds on cell viability with cisplatin treatment. Compounds and PD176252 (4 μM) restored cell viability in cisplatin-treated HK2 cells (ATCC, USA). HK2 cells were pre-cultured for 24 h, the cells were then treated with the indicated concentrations of compounds for 6 h, and then exposed to 20 μM cisplatin for 24 h. The results are shown as means ± SD (n = 3) for at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the cisplatin-stimulated group. CIS, cisplatin; compounds, 5a–p; PD, PD 176252.

Fig. 3. Effect of different concentrations of 5d or 5m on cell viability with cisplatin treatment. 5d and 5m restored cell viability in cisplatin-treated HK2 cells (ATCC, USA). HK2 cells were pre-cultured for 24 h, the cells were then treated with the indicated concentrations of compounds for 6 h, and then exposed to 20 μM cisplatin for 24 h. The results are shown as means ± SD (n = 3) of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with cisplatin-stimulated group. CIS, cisplatin.

Fig. 4. Cytotoxicity of different concentrations of 5d, 5m or PD 176252 on HK2 cells. 5d and 5m reduce the viability of HK2 cells (ATCC, USA). HK2 cells were pre-cultured for 24 h, the cells were then treated with the indicated concentrations of compounds for 24 h. The results are shown as means ± SD (n = 3) of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group.

Fig. 5. Effect of 5d or 5m for inflammatory response in HK2 cells with cisplatin treatment. Real-time PCR in HK2 cells (ATCC, USA). The results demonstrate that the treatment of compounds reduced cisplatin-induced mRNA levels of TNF-α, IL-6, and MCP-1. Data represent the mean ± SEM for 3–4 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared to the cisplatin-treated group. CIS, cisplatin; PD, PD176252.

Fig. 6. Effect of 5d or 5m for inflammatory response in HK2 cells with cisplatin treatment. ELISA in HK2 cells (ATCC, USA). The results demonstrate that the treatment of compounds reduced cisplatin-induced mRNA levels of TNF-α, IL-6, and MCP-1. Data represent the mean ± SEM for 3–4 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared to cisplatin-treated group. CIS, cisplatin; PD, PD 176252.

Fig. 7. Effect of 5d and PD 176252 on cisplatin-induced phosphorylation of NF-κB p65. Results of western blot and quantitative data indicated that 5d and PD176252 had an effect on the phosphorylation of p65 for cisplatin-treated HK2 cells (ATCC, USA). Data represent the mean ± SEM for 3–4 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared to the cisplatin-treated group. CIS, cisplatin; PD, PD 176252.