We recently reported the discovery of AM-8553 (1), a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Continued research investigation of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface, led to the discovery of a one-carbon tethered sulfone which gave rise to substantial improvements in biochemical and cellular potency. Further investigation produced AMG 232 (2), which is currently being evaluated in human clinical trials for the treatment of cancer. Compound 2 is an extremely potent MDM2 inhibitor (SPR KD = 0.045 nM, SJSA-1 EdU IC50 = 9.1 nM), with remarkable pharmacokinetic properties and in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft model (ED50 = 9.1 mg/kg).

Structure-based rational design led to the discovery of novel inhibitors of the MDM2-p53 protein-protein interaction. The affinity of these compounds for MDM2 was improved through conformational control of both the piperidinone ring and the appended N-alkyl substituent. Optimization afforded 29 (AM-8553), a potent and selective MDM2 inhibitor with excellent pharmacokinetic properties and in vivo efficacy.

Restoration of p53 function through the disruption of the MDM2-p53 protein complex is a promising strategy for the treatment of various types of cancer. Here, we present kinetic, thermodynamic, and structural rationale for the remarkable potency of a new class of MDM2 inhibitors, the piperidinones. While these compounds bind to the same site as previously reported for small molecule inhibitors, such as the Nutlins, data presented here demonstrate that the piperidinones also engage the N-terminal region (residues 10-16) of human MDM2, in particular, Val14 and Thr16. This portion of MDM2 is unstructured in both the apo form of the protein and in MDM2 complexes with p53 or Nutlin, but adopts a novel β-strand structure when complexed with the piperidinones. The ordering of the N-terminus upon binding of the piperidinones extends the current model of MDM2-p53 interaction and provides a new route to rational design of superior inhibitors.

Contents 1. Introduction 449 2. Ramoplanin Basics 452 2.1. Structural Overview 452 2.2. Antimicrobial Activity 453 2.3. Clinical Status 454 3. Mechanism of Action of RamoplaninsEarly Work 454 3.1. Cellular Targets of Antibiotics 454 3.2. Peptidoglycan Structure and Biosynthesis 454 3.3. MurG Is Proposed as the Target of Ramoplanin 455 3.4. Ramoplanin Is Shown To Bind to an Intermediate in Peptidoglycan Biosynthesis 456 3.5. Ramoplanin Is Proposed To Block the Transglycosylation Step of Peptidoglycan Biosynthesis 456 4. Mechanism of Action of RamoplaninsRecent Work 457 4.1. Technical Advances in the Study of Peptidoglycan-Synthesizing Enzymes 457 4.2. Expected Inhibition Kinetics for Substrate Binders 457 4.3. Inhibition Kinetics of Ramoplanin 458 4.3.1. Transglycosylase Inhibition 458 4.3.2. MurG Inhibition 459 4.4. Evaluating the Proposed Cellular Targets of Ramoplanin 459 5. Molecular Recognition by Ramoplanin 460 5.1. Problem of Fibril Formation 460 5.2. Comparison of Substrate-Binding Affinities 461 5.3. Structural Studies on Ramoplanin and Ramoplanin Complexes 462 6. Total Synthesis of Ramoplanin and Key Analogues 463 6.1. Preparation of Key Amino Acids 463 6.1.1. aThr (allo-Threonines) 463 6.1.2 HAsn (L-threo-β-Hydroxyasparagine) 463 6.2. Total Synthesis of the Ramoplanin A2 and Ramoplanose Aglycon 464 6.3. Total Synthesis and Structure of the Ramoplanin A1 and A3 Aglycons 467 6.4. [N-Acetyl-Asn 1 ]ramoplanin Aglycon 467 6.5. Total Synthesis of Ramoplanin Amide Analogues 468 6.6. Solid-Phase Synthesis of a Simplified Analogue 469 7. Degradation and Semisynthetic Studies 470 7.1. Ramoplanin Aglycons 470 7.2. Depsipeptide Hydrolysis 470 7.3. Lipid Side-Chain Reduction 470 7.4. Orn 4 and Orn 10 Derivatization 470 7.5. Lipid Side-Chain Replacement 471 7.6. Summary 471 8. Structure−Activity Studies on Ramoplanin and Its Synthetic and Semisynthetic Analogues 471 8.1. Antimicrobial Activity of Key Derivatives and Analogues 471 8.2. Mechanistic Analysis of Ramoplanin AnaloguessThe Path Forward 472 9. Conclusion 473 10. Abbreviations 473 11. References 474