Metabolism-Guided Optimization of Tryptophanol-Derived Isoindolinone p53 Activators

Abstract

For the first time, the pharmacokinetic (PK) profile of tryptophanol-derived isoindolinones, previously reported as p53 activators, was investigated. From the metabolites' identification, performed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS), followed by their preparation and structural elucidation, it was possible to identify that the indole C2 and C3 are the main target of the cytochrome P450 (CYP)-promoted oxidative metabolism in the tryptophanol-derived isoindolinone scaffold. Based on these findings, to search for novel p53 activators a series of 16 enantiopure tryptophanol-derived isoindolinones substituted with a bromine in indole C2 was prepared, in yields of 62-89%, and their antiproliferative activity evaluated in human colon adenocarcinoma HCT116 cell lines with and without p53. Structural optimization led to the identification of two (S)-tryptophanol-derived isoindolinones 3.9-fold and 1.9-fold more active than hit SLMP53-1, respectively. Compounds' metabolic stability evaluation revealed that this substitution led to a metabolic switch, with the impact of Phase I oxidative metabolism being minimized. Through differential scanning fluorimetry (DSF) experiments, the most active compound of the series in cell assays led to an increase in the protein melting temperature (T_m) of 10.39 °C, suggesting an effective binding to wild-type p53 core domain.

Keywords: cancer; hit-to-lead optimization; metabolic stability; p53; tryptophanol-derived isoindolinones.

Figure 1

Chemical structure of p53-activating small molecules in clinical development for CRC.

Figure 2

Structural optimization of tryptophanol-derived isoindolinones as p53 activators.

Figure 3

LC-HRMS/MS-based metabolite profile of (S)-tryptophanol-derived isoindolinones SLMP53-1 (6) (in orange) and SLMP53-2 (7) (in pink). In grey boxes are highlighted the diagnostic ions that attested for the metabolic transformation at the indole ring.

Scheme 1

Direct biomimetic oxidation of SLMP53-1 (6). i. White–Chen catalyst (8) 10 mol%, H₂O₂ 0.45 eq. (35% v/v), AcOH 5.00 eq., ACN at 40 °C.

Figure 4

Possible SLMP53-1 (6) bioactivation pathways. In grey boxes are highlighted the diagnostic fragment ions that attested for the GSH addition at the indole ring.

Scheme 2

Synthesis of 2-bromo (S)-tryptophanol-derived isoindolinones 13a-i. Reagents and conditions: (i) 1. NaH, 0 °C DMF; 2. MeI, rt (7) or 2. PrBr, rt (13b); (ii) PyHBr₃, THF/DCM, 0 °C, instantaneous.

Scheme 3

Preparation of bromine-enriched isoindolinones 13j-o. Reagents and conditions: (i) 1. NaH, 0 °C DMF; 2. RI, rt (13j) or RCl, rt (13k, 13l, 13n and 13o) or R₂O, rt (13m).

Figure 5

SAR trends for halogen-enriched (S)-tryptophanol-derived isoindolinones 13a and 13c-o.

Figure 6

LC-HRMS-MS-based metabolite profile of compounds 13d (in blue) and 13k (in orange) and potential bioactivation pathways. In the grey box is the diagnostic fragment ion that attested for the metabolic transformation at the indole ring.