Discovery of potent and selective spiroindolinone MDM2 inhibitor, RO8994, for cancer therapy

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Abstract

The field of small-molecule inhibitors of protein–protein interactions is rapidly advancing and the specific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published on this topic and multiple compounds are in various stages of clinical development. Building on the strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound, we have developed additional scaffolds that provide opportunities for future development. Here, we report the discovery and optimization of a highly potent and selective series of spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.

Introduction

Tumor suppressor p53 is a potent transcription factor that plays a central role in guarding the integrity of cell genome.1, 2, 3, 4 In non-stressed cells, p53 is tightly controlled by its master negative regulator MDM2.5 In about 50% of human cancers, p53 remains wild-type but its function is impaired by other mechanisms, such as overexpression of MDM2.6, 7, 8, 9 Thus, MDM2 has been deemed a valid target for cancer therapy.10 The early structure of a p53 peptide bound to MDM2 showed that the interactions were mediated by a limited set of amino acids and provided a reasonable expectation that small molecules could successfully interfere with the p53-MDM2 interaction.11 The discovery of Nutlins verified this approach.12 An advanced member of the Nutlin family of molecules, 1 (RG7112, Fig. 1), is in clinical evaluation.13 Internal efforts identified a novel pyrrolidine compound, 2 (RG7388, Fig. 1), which was found to display superior in vitro potency and in vivo efficacy and is progressing in clinical studies.14 Nevertheless, identification of candidates from distinctive chemical classes may also be desirable to enhance the chances for ultimate success in the clinic.

Since the discovery of Nutlins, several groups have identified additional compound classes and a few have even progressed into clinical development.15 The most notable one has been a spiroindolinone-3,3′-pyrrolidine MI-219 series reported by Ding et al.16 Just recently, this group published their latest findings in which the original stereochemistry was found to be unstable and converted over time to a more stable diastereomeric configuration and the most potent compound 3 (MI-888, Fig. 1) was found to achieve tumor regression in mice bearing SJSA-1 xenograft model at a relatively high dose.17, 18 Prior to their work, we also demonstrated that stereochemistry of the pyrrolidine core structure in which the two aryl rings (‘A’ and ‘B’) adopt a ‘Trans’ orientation as shown in 2 (RG7388, Fig. 1) was important for optimal binding to MDM2.14 Guided by X-ray co-crystal structures, we further explored the pyrrolidine analogues by combining the key structural elements of 2 (RG7388) and the spiroindolinone core structure of 3 (MI-888).18, 19 Here we report our work leading to the discovery of a potent and highly efficacious compound 4 (RO8994) with promising potential for clinical development (Fig. 1).19

Section snippets

Results and discussion

Compared to the identification of Nutlins by high throughput screenings,12 the discoveries of 2 (RG7388) and 4 (RO8994) were driven by the structure-based de novo design and information based approach. Our initial design started with the indolinone compound 5, which was confirmed to be a validated hit with an IC50 of 3.9 μM in a biochemical binding assay (Fig. 2).20 In the crystal structure, the 6-chlorooxindole (‘A’) fills the deep, narrow Trp23 pocket. Only the (S)-configuration of compound 5

Conclusion

In summary, our investigation of 5-membered spiroindolinones 8 led to the identification of a highly potent and selective p53-MDM2 inhibitor 4 (RO8994). The synthesis of compound 4 has been optimized for large scale and the compound has also demonstrated acceptable toxicity profiles in both rodent and non-rodent dose range finding studies (data not shown).29 Like 2 (RG7388), compound 4 (RO8994) represents a new generation of p53–MDM2 antagonists with marked improvement in both in vitro and in

General chemistry

All commercial reagents and anhydrous solvents were purchased and used without further purification, unless otherwise specified. Mass samples were analyzed on a MicroMass ZQ, ZMD, Quattro LC, or Quattro II mass spectrometer operated in a single MS mode with electrospray ionization. High resolution mass spectra (HRMS) were measured on a Bruker Daltonics APEXII 3 Tesla Fourier transform mass spectrometer. 1H NMR spectra (δ, ppm) were recorded using either a Bruker Avance 400 (400 MHz) or a Bruker

Acknowledgments

The authors would like to thank Theodore Lambros for SFC purification, Gino Sasso for detailed 1H and NOE NMR analysis of stereochemical assignments, Samir Serrano for high resolution mass spectrometry, Simon Yang, Roche China for optical rotation, and Francisco Talamas for proof reading of the manuscript.

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