Synthesis and Antineoplastic Activity of a Dimer, Spiroindolinone Pyrrolidinecarboxamide

Abstract

The mutation or function loss of tumour suppressor p53 plays an important role in abnormal cell proliferation and cancer generation. Murine Double Minute 2 (MDM2) is one of the key negative regulators of p53. p53 reactivation by inhibiting MDM2-p53 interaction represents a promising therapeutic option in cancer treatment. Here, to develop more effective MDM2 inhibitors with lower off-target toxicities, we synthesized a dimer, spiroindolinone pyrrolidinecarboxamide XR-4, with potent MDM2-p53 inhibition activity. Western blotting and qRT-PCR were performed to detect the impact of XR-4 on MDM2 and p53 protein levels and p53 downstream target gene levels in different cancers. Cancer cell proliferation inhibition and clonogenic activity were also investigated via the CCK8 assay and colony formation assay. A subcutaneous 22Rv1-derived xenografts mice model was used to investigate the in vivo anti-tumour activity of XR-4. The results reveal that XR-4 can induce wild-type p53 accumulation in cancer cells, upregulate the levels of the p53 target genes p21 and PUMA levels, and then inhibit cancer cell proliferation and induce cell apoptosis. XR-4 can also act as a homo-PROTAC that induces MDM2 protein degradation. Meanwhile, the in vivo study results show that XR-4 possesses potent antitumour efficacy and a favourable safety property. In summary, XR-4 is an interesting spiroindolinone pyrrolidinecarboxamide-derivative dimer with effective p53 activation activity and a cancer inhibition ability.

Keywords: MDM2 inhibitor; cancer treatment; dimer spiroindolinone pyrrolidinecarboxamide; p53 activation.

Scheme 1

The mechanism of action of XR-4.

Figure 1

The design strategy of dimer spiroindolinone pyrrolidinecarboxamide: (A) the chemical structures of compound 2 and XR-2; (B) the binding model of compound 2 and XR-2 with MDM2 (PDB: 5TRF); (C) the chemical structure of XR-4.

Scheme 2

Reagents and conditions: (a) 2,2′-((Oxybis(ethane-2,1-diyl)) bis (oxy)) diethanol; (b) 1-chloroethyl chloroformate; (c) linker intermediate Bis(1-chloroethyl) (((oxybis(ethane-2,1-diyl)) bis(oxy)) bis(ethane-2,1-diyl)) bis(carbonate); (d) compound 2; (e) XR-4; (f) acetone and Cs₂CO₃.

Figure 2

XR-4 effectively promotes p53 protein accumulation in different cancer cells in both dose-dependent and time-dependent manners: (A) 22Rv1 cells were treated with XR-4 at the different concentrations for 24 h, and the protein levels of p53 and β-actin were detected via Western blotting; (B) LNCaP cells were treated with the gradually rising concentrations of XR-4 for 24 h, and the protein levels of p53 and GAPDH were detected via Western blotting; (C) Western blotting was used to detect the levels of p53 and β-actin in HepG2 cells treated with XR-4 at the indicated concentrations for 24 h; (D) Western blotting was performed at different time points after 22Rv1 cells were treated with 5 μM XR-4 and the protein levels of p53 and GAPDH were determined; (E) LNCaP cells treatment with XR-4 at the indicated concentrations for 24 h; qRT-PCR was performed to measure p53 mRNA levels and normalised to GAPDH; (F) HepG2 cell treatment with XR-4 at the indicated concentrations for 24 h and qRT-PCR was performed to measure p53 mRNA levels and normalised to GAPDH. Experiments were performed in triplicates.

Figure 3

XR-4 activates p53 downstream target genes: (A,B) LNCaP cell treatment with XR-4 at the indicated concentrations for 24 h and qRT-PCR were performed to measure p21 and PUMA mRNA levels and normalised to GAPDH; (C,D) HepG2 cell treatment with XR-4 at the indicated concentrations for 24 h and qRT-PCR were performed to measure p21 and PUMA mRNA levels and normalised to GAPDH. Experiments were performed in triplicates. All results are shown as mean ± SD. ** p < 0.01; *** p < 0.001 vs. the control group.

Figure 4

XR-4 downregulates MDM2 protein levels: (A) 22Rv1 cells were treated with XR-4 at the indicated concentrations for 24 h, and the protein levels of MDM2 and GAPDH were determined via Western blotting; (B) ImageJ was used to analyse the relative protein levels. Experiments were performed in triplicates. All results are represented as mean ± SD. *** p < 0.001 vs. the control group.

Figure 5

XR-4 inhibits cellular proliferation and promotes the apoptosis of wild-type p53 cancer cells in vitro. (A) After 14 days of treatment with different concentrations of XR-4, 22Rv1 cells were stained with crystal violet. (B) The protein levels of cleaved PARP and GAPDH were measured via Western blot after LNCaP cells were treated with XR-4 at indicated concentrations for 24 h. (C) LNCaP cells were treated with different concentrations of XR-4, flow cytometry analysis were performed to detect the cell apoptosis levels.

Figure 6

XR-4 suppressed wild-type p53 tumour progression in vivo: (A) Tumour volume of 22Rv1 xenografts treated with 50 mg/kg XR-4 or vehicle control for 15 days (n = 8); (B) mice weight of 22Rv1 xenografts treated with 50 mg/kg XR-4 or vehicle control for 15 days (n = 8).