Synthesis and Biological Evaluation of Novel Dispiro-Indolinones with Anticancer Activity

Abstract

Novel variously substituted thiohydantoin-based dispiro-indolinones were prepared using a regio- and diastereoselective synthetic route from 5-arylidene-2-thiohydantoins, isatines, and sarcosine. The obtained molecules were subsequently evaluated in vitro against the cancer cell lines LNCaP, PC3, HCT^wt, and HCT^(-/-). Several compounds demonstrated a relatively high cytotoxic activity vs. LNCaP cells (IC₅₀ = 1.2-3.5 µM) and a reasonable selectivity index (SI = 3-10). Confocal microscopy revealed that the conjugate of propargyl-substituted dispiro-indolinone with the fluorescent dye Sulfo-Cy5-azide was mainly localized in the cytoplasm of HEK293 cells. P388-inoculated mice and HCT116-xenograft BALB/c nude mice were used to evaluate the anticancer activity of compound 29 in vivo. Particularly, the TGRI value for the P388 model was 93% at the final control timepoint. No mortality was registered among the population up to day 31 of the study. In the HCT116 xenograft model, the compound (170 mg/kg, i.p., o.d., 10 days) provided a T/C ratio close to 60% on day 8 after the treatment was completed. The therapeutic index-estimated as LD₅₀/ED₅₀-for compound 29 in mice was ≥2.5. Molecular docking studies were carried out to predict the possible binding modes of the examined molecules towards MDM2 as the feasible biological target. However, such a mechanism was not confirmed by Western blot data and, apparently, the synthesized compounds have a different mechanism of cytotoxic action.

Keywords: MDM2; PPI; anticancer activity; cytotoxicity; dispiro-indolinones; in vivo trials; molecular docking; p53.

Figure 1

Small-molecule MDM2 inhibitors investigated in different clinical trials.

Figure 2

Representative examples of spiro-indolinones with anticancer activity.

Figure 3

Examples of physiologically active spiro-thiohydantoins.

Scheme 1

Synthesis of dispiro-indolinones 4–69.

Figure 4

Molecular structures of dipiro-indolinones 29 (left) and 69 (right); atoms are displayed as thermal ellipsoids at a 30% probability level.

Figure 5

(a) Structures of the template molecules XX and XXI used for modeling; (b,c) the overlapping of compound XX (yellow) with the hotspots in the domain recognition element of the p53 peptide (orange); three key hydrophobic binding points and an α-helix mimetic core.

Figure 6

Compound XX (a), compound XXI (b), and compound 29 (c) docked in the MDM2 pocket. Yellow: X-ray data for compounds XX (a) (PDB code: 4JVR, resolution: 1.70 Å) and XXI (b) (PDB code: 4QOC, resolution: 1.70 Å); grey—docking results. For (a): E^score = −8.49 kcal/mol; for (b): E^score = −7.62 kcal/mol; for (c): E^score = −5.31 (compound 26); low E^score values correspond to more favorable binding.

Scheme 2

Click reaction between the fluorescent dye Sulfo-Cy5-azide and dispiro-indolinones 69 and 35.

Figure 7

The localization of the compound 35–Sulfo-Cy5-azide conjugate in 22RV1 cells ((left) stained by click reaction product; (right) Sulfo-Cy5-azide only).

Figure 8

Western blot data showing the effects of compound 29 on the expression of the p53 and PUMA proteins, as well as caspase-3 and caspase-8—caspases involved in p53-dependent apoptosis in HCTwt, HCT^(−/−),and LNCaP cells. Nutlin-3a (20 µM) was used as a positive control.

Figure 9

Changes in P388 volume upon the treatment with compound 29 (1176 mg/kg, o.d., 10 days, i.p.).

Figure 10

Lifespan observed among the P388-inoculated mice after the treatment (o.d., 10 days i.p.) with compound 29 at doses of 295 mg/kg (g1), 882 mg/kg (g2), and 1176 mg/kg (g3).

Figure 11

Changes in P388 nude volume upon the treatment with compound 29 (1000 mg/kg, o.d., 10 days, i.p.) as compared to the control group.

Figure 12

Changes in HCT116 tumor xenografts’ volume upon treatment with compound 29 (1000 mg/kg, o.d., 10 days, i.p.).