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

Synthesis of spiro-DKPs 9–16.

<p>Reagents and reaction conditions: i) AA-OMe (1.5–1.7 eq.). HATU (2.0 eq.), DIPEA (6.0 eq.), DMF, 60°C, 30 min. ii) water, MW, 160°C, 70 min. iii) R₃-NH₂ (1.7–2.0 eq.), PEMB (1.0 eq.), glacial acetic acid (2.3–2.6 eq.), MeOH, r.t. o.n.</p

2,5-DKPs as inhibitors of the MDM2-p53 interaction.

<p>2,5-DKPs as inhibitors of the MDM2-p53 interaction.</p

Boc-deprotection of 55–56 and 58S.

<p>Reagents and reaction conditions: i) TFA:DCM (1:1 v/v), 1h, r.t.</p

Conformational analysis of 57RS.

<p>(<b>A</b>) Model of two low energy conformations of <b>57RS</b>; (<b>B</b>) Chemical structure of <b>57RS</b> with atom numbers; (<b>C</b>) ¹H NMR signals from H7 and H6 of <b>57RS</b> at 25°C and 55°C.</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

Examples of type II and III inhibitors previously reported.

<p>Examples of type II and III inhibitors previously reported.</p

Ester hydrolysis of 30S and 31S.

<p>Reagents and reaction conditions: i) Conc. HCl (aq.) for R₁ = Et, 70°C o.n. For R₁ = <i>t</i>Bu, r.t. o.n.</p

Amidation of 2,5-DKPs afforded 55–58.

<p>Amidation of 2,5-DKPs afforded 55–58.</p