David Chow

Structure-based design of novel inhibitors of the MDM2-p53 interaction.

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.

Discovery of AMG 232, a Potent, Selective, and Orally Bioavailable MDM2–p53 Inhibitor in Clinical Development

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).

Rational Design and Binding Mode Duality of MDM2–p53 Inhibitors

Structural analysis of both the MDM2-p53 protein-protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone 10, an MDM2 inhibitor with a biochemical IC50 of 1.0 μM. The cocrystal structure of 10 with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones 16 and 27 possessing distinct binding modes. Both analogues were potent MDM2 inhibitors in biochemical and cellular assays, and morpholinone 27 (IC50 = 0.10 μM) also displayed suitable PK profile for in vivo animal experiments. A pharmacodynamic (PD) experiment in mice implanted with human SJSA-1 tumors showed p21(WAF1) mRNA induction (2.7-fold over vehicle) upon oral dosing of 27 at 300 mg/kg.

Discovery and Optimization of Potent GPR40 Full Agonists Containing Tricyclic Spirocycles.

GPR40 (FFAR1 or FFA1) is a target of high interest being pursued to treat type II diabetes due to its unique mechanism leading to little risk of hypoglycemia. We recently reported the discovery of AM-1638 (2), a potent full agonist of GPR40. In this report, we present the discovery of GPR40 full agonists containing conformationally constrained tricyclic spirocycles and their structure-activity relationships leading to more potent agonists such as AM-5262 (26) with improved rat PK profile and general selectivity profile. AM-5262 enhanced glucose stimulated insulin secretion (mouse and human islets) and improved glucose homeostasis in vivo (OGTT in HF/STZ mice) when compared to AM-1638.

Selective and potent morpholinone inhibitors of the MDM2-p53 protein-protein interaction.

We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.

Novel Inhibitors of the MDM2-p53 Interaction Featuring Hydrogen Bond Acceptors as Carboxylic Acid Isosteres.

We previously reported the discovery of potent and selective morpholinone and piperidinone inhibitors of the MDM2-p53 interaction. These inhibitors have in common a carboxylic acid moiety that engages in an electrostatic interaction with MDM2-His96. Our continued search for potent and diverse inhibitors led to the discovery of novel replacements for these acids uncovering new interactions with the MDM2 protein. In particular, using pyridine or thiazole as isosteres of the carboxylic acid moiety resulted in very potent analogues. From these, AM-6761 (4) emerged as a potent inhibitor with remarkable biochemical (HTRF IC50 = 0.1 nM) and cellular potency (SJSA-1 EdU IC50 = 16 nM), as well as favorable pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 11 mg/kg. Optimization efforts toward the discovery of these inhibitors as well as the new interactions observed with the MDM2 protein are described herein.

Discovery of Potent and Simplified Piperidinone-Based Inhibitors of the MDM2-p53 Interaction.

Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone-pyridine inhibitors 6, 7, 14, and 15 with improved pharmacokinetic properties in rats. Reducing structure complexity of the N-alkyl substituent led to the discovery of 23, a potent and simplified inhibitor of MDM2. Compound 23 exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model.

Synthesis and Evaluation of the Metabolites of AMG 221, a Clinical Candidate for the Treatment of Type 2 Diabetes.

All eight of the major active metabolites of (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221, compound 1), an inhibitor of 11β-hydroxysteroid dehydrogenase type 1 that has entered the clinic for the treatment of type 2 diabetes, were synthetically prepared and confirmed by comparison with samples generated in liver microsomes. After further profiling, we determined that metabolite 2 was equipotent to 1 on human 11β-HSD1 and had lower in vivo clearance and higher bioavailability in rat and mouse. Compound 2 was advanced into a pharmacodynamic model in mouse where it inhibited adipose 11β-HSD1 activity.

The Intermolecular Disulfide Bridge of Human Glial Cell Line-Derived Neurotrophic Factor: Its Selective Reduction and Biological Activity of the Modified Protein

Recombinant human glial cell line-derived neurotrophic factor has been implicated to have therapeutic potential in the treatment of neurodegenerative diseases. The mature protein is a single polypeptide of 134 amino acid residues and functions as a disulfide-linked dimer. Reduction of the protein with dithiothreitol at pH 7.0 and in the absence of denaturant showed that the single intermolecular cystine bridge was reduced preferentially. Direct alkylation of the generated free sulfhydryl group using iodoacetamide or iodoacetate without denaturant was incomplete. Unfolding the protein in 6 M guanidine hydrochloride prior to the modification showed rapid disulfide scrambling. However, the sulfhydryl-modifying reagent N-ethylmaleimide was able to label quantitatively the free cysteinyl residue in the absence of any added chaotropic agent. By a combination of peptide mapping, Edman degradation, and mass spectrometric analysis, the labeled residue was identified to be Cys101, hence verifying the location of the intermolecular disulfide bond. The modified protein behaved as a noncovalent dimer when chromatographed through a Superdex 75 column under nondenaturing conditions and was comparable in biological activity to an unmodified control sample. The results therefore indicate that the intermolecular disulfide bridge of the protein is not essential for its biological function.

C5-Alkyl-2-methylurea-Substituted Pyridines as a New Class of Glucokinase Activators

Glucokinase (GK) activators represent a class of type 2 diabetes therapeutics actively pursued due to the central role that GK plays in regulating glucose homeostasis. Herein we report a novel C5-alkyl-2-methylurea-substituted pyridine series of GK activators derived from our previously reported thiazolylamino pyridine series. Our efforts in optimizing potency, enzyme kinetic properties, and metabolic stability led to the identification of compound 26 (AM-9514). This analogue showed a favorable combination of in vitro potency, enzyme kinetic properties, acceptable pharmacokinetic profiles in preclinical species, and robust efficacy in a rodent PD model.