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Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

A heterocyclic, sp(3)-rich chemical scaffold was synthesised in just 6 steps via a highly regio- and diastereo-selective tandem nitrone formation/intramolecular nitrone-alkene [3+2] cycloaddition reaction. A library of 543 lead-like compounds based on the scaffold core has been produced.

In order to address the current downturn in the drug discovery pipeline, initiatives are being undertaken to synthesise screening libraries of sp3-rich, low molecular weight compounds. As part of the European Lead Factory initiative, the synthesis and derivatisation of a simple hexahydrooxazolo[5,4-c]pyridin-2(1H)-one bicyclic carbamate has been achieved. The synthetic route employed involved a telescoped hetero-Diels-Alder/[2,3]-sigmatropic rearrangement/cyclisation sequence to deliver the desired core scaffold containing two points for further diversification. When applied, this synthesis was found to be robust and scalable which allowed the production of a 155 compound library.

In response to cellular stress, the p53 tumor suppressor is activated to modulate cell cycle progression, DNA repair, and cell death. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 can therefore reactivate p53 and elicit an anti-cancer effect. Small molecule inhibitors of the MDM2-p53 interaction remains a promising strategy for cancer therapy and a number of these compounds are in clinical development. An isoindolinone series, identified by the Northern Institute for Cancer Research (NICR), has been used as a starting point for the development of potent MDM2-p53 inhibitors. Structure based drug design was applied during lead optimisation to gain potency whilst also focusing on stabilizing the main metabolically labile position and reducing lipophilicity. This approach led to potent compounds with EC50 Citation Format: Lynsey Fazal, Maria Ahn, Luke Bevan, Ildiko Buck, Juan Castro, Gianni Chessari, Ben Cons, Keisha Hearn, Steven Howard, Chris Johnson, Judith Reeks, Emiliano Tamanini, Neil Thompson, Hugh Walton, Pamela Williams, Ruth H. Bawn, Tim J. Blackburn, Celine Cano, Sarah J. Cully, Bernard Golding, Roger Griffin, Karen Haggerty, Ian Hardcastle, Herbie Newell, Martin Noble, Huw Thomas, Elaine Willmore, Yan Zhao, Steve Wedge. Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1652.

The application of a tandem condensation/cyclisation/[3+2]-cycloaddition/elimination reaction gives an sp(3)-rich tricyclic pyrazoline scaffold with two ethyl esters in a single step from a simple linear starting material. The successive hydrolysis and cyclisation (with Boc anhydride) of these 3-dimensional architectures, generates unprecedented 16-membered macrocyclic bisanhydrides (characterised by XRD). Selective amidations could then be achieved by ring opening with a primary amine followed by HATU-promoted amide coupling to yield an sp(3)-rich natural product-like library.

The potent and selective MDM2-p53 antagonist 4-chloro-3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one 1 displays promising activity in cellular tumour models; however, poor solubility precludes in vivo investigations.(Bioorg. Med. Chem. Letters 2011, 21, 5916-5919) A water-soluble succinate ester of 1 was designed as a pro-drug, based on an understanding of the interaction of the parent compound 1 with MDM2, and the cellular pharmacology and in vivo pharmacokinetics of the ester 2 were defined. Compound 2 was synthesised and evaluated as an antagonist of MDM2- and MDMX-p53 using ELISA assays. Cell growth inhibition induced by 2 and 1 were compared in paired p53 functional and p53 non-functional HCT116, A2780 and SJSA-1 cell lines. Conversion of 2 into 1 was studied in human and mouse plasma in vitro, as was uptake into tumour cells, by HPLC analysis. The pharmacokinetics of 2 was studied in vivo in tumour-bearing mice. Unexpectedly, the succinate ester 2 was 7-fold more potent than the parent compound 1 as an MDM2-p53 binding antagonist in cell-free assays (IC50 values 20 ± 13 nM versus 95 ± 96 nM, respectively) and maintained >100-fold selectivity for MDM2 over MDMX antagonism. In cell growth inhibition assays (GI50) 2 was 2.3-, 6.1- and 8.6-fold more potent in paired p53 functional versus non-functional HCT116, A2780 and SJSA-1 cell lines, respectively. As anticipated, the solubility of 2 (65 µM) was greatly improved over 1 (6.5 µM), and the succinate ester was converted to the parent compound in mouse plasma (>90% in 60 min), although interestingly not in human plasma. Following incubation of tumour cell lines with 2, both the pro-drug and parent compound could be detected. Administration of 2 to A2780 tumour-bearing mice i.v. at 10 mg/kg generated plasma concentrations approaching those required for cell growth inhibition (≥ 3 μM), and oral administration at 100 mg/kg resulted in 2 bio-availability of 7%, and an 1 AUC that was estimated to be 21% of the equivalent i.v. dose of 2. However, a major unidentified metabolite was observed which compromised the in vivo utility of the succinate ester 2. Protein-protein interaction inhibitors as exemplified by MDM2-p53 binding antagonists are a major new class of antitumour agent. However, targeting hydrophobic protein-protein interactions can result in molecules with poor physicochemical properties. A cleavable succinate ester approach can be applied to address this problem without compromising target interaction or cellular activity, as in the current study. Although additional metabolite pathways may limit the utility of this approach, the improved potency gained by addition of a solubilising group provides valuable information for further lead optimisation. (This work was supported by the CR-UK) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 919. doi:1538-7445.AM2012-919