Antitumor Activity of 3-Indolylmethanamines 31B and PS121912

The most simple and best known member of the 3-indolylmethanamine family is gramine, a compound made by many plants for defense due to its toxicity to animals (lethal dose 50% (LD50) ≈ 50 mg/kg in rodents) (1). In the laboratory, gramine is an important precursor for tryptophan, especially when converted into the quaternary ammonium salt (Figure 1A) (2). Importantly, thiols can react with the free gramine base (pKb = 3.4) at elevated temperature due to acidity of the thiol (pKa = 10.5) and the basicity of the amine. Thus, the amine is converted into a quaternary ammonium group making it a good leaving group and the sulfur anion a superior nucleophile (Figure 1B) (3). Figure 1 Mode of action of 3-indolylmethanamines. A: Synthesis of tryptophan based on nucleophilic substitution. B: Reaction of gramine with sulfur nucleophile. C: Developed 3-indolylmethanamines. In water, protonation of gramine is accelerated under acidic conditions inducing a self-condensation reaction leading to the formation of an insoluble polymer (4). In contrast, N-phenyl-substituted 3-indolylmethanamines are significantly more stable in water due to a higher nitrogen pKb value inducing selectivity towards protein targets. For instance, the 3-indolylmethanamine CCT036477 (Figure 1C) was identified as a selective inhibitor of wingless-type MMTV integration site family (WNT)-dependent transcription (5). Evan et al. reported that CCT036477 reduced the transcriptional activity of the T-cell factor/lymphoid enhancer factor transcription factor family at the β-catenin level. The compound was stable for 24 hours at pH 7 and pH 9 but decomposed at pH 4 within that time frame. In the presence of nucleophile glutathione at pH 7, CCT036477 formed the corresponding sulfide, which was detectable after one hour by liquid chromatography–mass spectrometry/mass spectrometry (LCMS/MS). Independently, we used high throughput screening (HTS) to identify the class of 3-indolylmethanamines as novel irreversible inhibitors of the interaction between the vitamin D receptor (VDR) and the co-regulator steroid receptor coactivator-2 using high-throughput screening (6). The initial hit compounds, similar to CCT036477, were pyridyl-substituted 3-indolylmethanamines. Structural analogs of the class of compounds bearing electron-donating groups in the para position alkylated VDR more readily than those with electron-withdrawing groups following a correlation with decreasing pKb values of the nitrogen. An additional strong free linear relationship using Hammett σ-values for different substituents confirmed the correlation between reaction rate and pKb, as well as the stabilization of the electrophilic transition state due to electron-donating phenyl substituents in conjugation with the protonated indole nitrogen. The selectivity of the 3-indolylmethanamine PS121912 for VDR over other nuclear receptors is remarkable (7). Like Ewan et al., we observed selective transcriptional inhibition of particular genes in the presence of these compounds (8). In addition, both groups observed the induction of antiproliferation in cancer cells mediated by the activation of caspases 3 and 7 (5, 8). Importantly, cytotoxic effects were predominantly observed in cancer cells, as well as certain types of cancer cell lines. Herein, we report the in vivo effects of 3-indolylmethanamines using xenografted mice to evaluate their antitumor activity. In addition, we demonstrated the effects of these compounds for antiproliferative pathways in cells.