8-Triazolylpurines: Towards Fluorescent Inhibitors of the MDM2/p53 Interaction

Small molecule nonpeptidic mimics of alpha-helices are widely recognised as protein-protein interaction (PPIs) inhibitors. Protein-protein interactions mediate virtually all important regulatory pathways in a cell, and the ability to control and modulate PPIs is therefore of great significance to basic biology, where controlled disruption of protein networks is key to understanding network connectivity and function. We have designed and synthesised two series of 2,6,9-substituted 8-triazolylpurines as alpha-helix mimetics. The first series was designed based on low energy conformations but did not display any biological activity in a biochemical fluorescence polarisation assay targeting MDM2/p53. Although solution NMR conformation studies demonstrated that such molecules could mimic the topography of an alpha-helix, docking studies indicated that the same compounds were not optimal as inhibitors for the MDM2/p53 interaction. A new series of 8-triazolylpurines was designed based on a combination of docking studies and analysis of recently published inhibitors. The best compound displayed low micromolar inhibitory activity towards MDM2/p53 in a biochemical fluorescence polarisation assay. In order to evaluate the applicability of these compounds as biologically active and intrinsically fluorescent probes, their absorption/emission properties were measured. The compounds display fluorescent properties with quantum yields up to 50%

Inhibition of the MDM2/p53 Interaction Design, Synthesis and Evaluation of MDM2 Inhibitors

Numerous essential cellular processes are regulated by protein-protein interactions (PPIs) and PPIs have therefore been recognised as potential new drug targets. The transcription factor p53 is often referred to as the guardian of the genome due to its involvement in DNA repair, induction of cell cycle arrest and cellular apoptosis. The amount of p53 in a cell is mainly controlled by the negative regulator MDM2, which upon complex formation with p53 leads to an overall reduction of the p53 level. Consequently, inhibition of the MDM2/p53 interaction has emerged as a promising new therapeutic strategy for the treatment of cancers retaining wild-type p53. This thesis describes the design, synthesis and evaluation of β-hairpins, 8-(triazolyl)purines and 2,5-diketopiperazines as MDM2/p53 interaction inhibitors. β-Hairpin derivatives were synthesised using automated solid phase peptide synthesis followed by a head to tail cyclisation in solution. Evaluation of the MDM2 inhibitory activity of the β-hairpin derivatives together with solution conformational analysis using NAMFIS calculations revealed that molecular flexibility is important to gain highly potent MDM2 inhibitors. Two series of 8-(triazolyl)purines and 2,5- diketopiperazines (2,5-DKPs) were evaluated as MDM2 inhibitors. The first series were designed to directly mimic an α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions. Conformational analyses indicated that both 8- (triazolyl)purines and 2,5-DKP derivatives were able to place substituents in the same spatial orientation as an α-helical template. The second series were designed primarily based on structure-based docking studies. The most potent inhibitors identified were from the latter series and displayed micromolar IC50-values in a biochemical fluorescence polarization assay. Binding to MDM2 was confirmed by WaterLOGSY experiments. Efficient synthetic protocols for the synthesis of both tetrasubstituted 8- (triazolyl)purines and tetrasubstituted 2,5-DKPs have been developed. Furthermore, an efficient bromination protocol for 8-bromination of electron rich purines utilising pyridiniumtribromide was developed. The fluorescent properties of the 8- (triazolyl)purines were determined and it was found that the regioisomerism of the triazole has an important impact on the quantum yield

Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction

<div><p>The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2) <i>via</i> a direct protein—protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the <i>i</i>, <i>i+4</i> and <i>i+7</i> positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC₅₀-values in a biochemical fluorescence polarisation assay.</p></div

Stereochemical assignment of non-spiro-DKPs.

<p>NOE correlations are shown by a double headed arrow.</p

Synthesis of spiro-2-DKPs 17–21.

<p>Synthesis of spiro-2-DKPs 17–21.</p

General structures of spiro-2-DKPs and non-spiro-DKPs.

<p>Hydrophobic substituents are indicated by light grey, while hydrophilic substituents are shown in dark grey.</p

Reduction of 17S.

<p>Reagents and reaction conditions: i) NaBH₄ (3.0 eq.), EtOH, r.t., 5 days.</p

<i>N</i>4-Alkylation of non-spiro-DKPs afforded 40–45.

<p><i>N</i>4-Alkylation of non-spiro-DKPs afforded 40–45.</p

Synthesis of spiro-DKPs 7–9.

<p>Reagents and reaction conditions: i) PhCHO (1.2 eq.), Et₃N (1.2 eq.), NaCNBH₃ (1.0 eq.), MeOH, r.t. ii) (CH₃)₃SiCHN₂ (6.4 eq.), MeOH/toluene (1:3), r.t. iii) <b>4</b> or <b>5</b>: R₁CHO (1.2–1.5 eq.), Et₃N (1.2 eq.), NaCNBH₃ (1.0 eq.), MeOH, r.t. iv) Phe-OMe (2.0 eq.), HATU (2.0 eq.), DIPEA (12 eq.), DMF, 60°C, 30 min. <b>6</b>: iii) Boc₂O, 3M NaOH and 1,4-dioxane (1:2, pH~12), r.t. iv) Phe-OMe (2.0 eq.), HATU (2.0 eq.), DIPEA (6.0 eq.), DMF, 60°C, 30 min. v) <b>4</b>: water, MW, 160°C, 30 min; <b>5</b>: HCl (1M, aq.)/acetone (1:1), 55°C, 72 h <b>6</b>: water, MW, 160°C, 90 min.</p

<i>N</i>4-alkylation of non-spiro-DKPs 35–39.

<p><i>N</i>4-alkylation of non-spiro-DKPs 35–39.</p