Nutlin-3a chiral

Exploration of liquid and supercritical fluid chromatographic chiral separation and purification of Nutlin-3--a small molecule antagonist of MDM2

Inhibition of the MDM2-p53 interaction can stabilize the p53 protein and offer a novel strategy for cancer therapy. The imidazoline compound (Nutlin-3) is a promising small molecule antagonist of the MDM2-p53 interaction. This compound was synthesized as a racemic mixture, and one enantiomer is 100-200-fold more active than the other enantiomer. In this study, various enantiomeric separation approaches were explored to resolve the Nutlin-3 enantiomers using chiral supercritical fluid chromatography (SFC) as well as chiral liquid chromatography (LC) under normal phase mode, reversed phase mode and polar organic phase mode. The chiral SFC method based on Chiralcel OD column showed superior separation in terms of selectivity and efficiency. Optimization of the chiral separation method enabled high throughput preparative scale purification. Ultimately, 5 g of racemic mixture were purified on Prep-SFC in 75 min with the recovery rate above 92%.

Preparation of (-)-Nutlin-3 using enantioselective organocatalysis at decagram scale

Chiral nonracemic cis-4,5-bis(aryl)imidazolines have emerged as a powerful platform for the development of cancer chemotherapeutics, stimulated by the Hoffmann-La Roche discovery that Nutlin-3 can restore apoptosis in cells with wild-type p53. The lack of efficient methods for the enantioselective synthesis of cis-imidazolines, however, has limited their more general use. Our disclosure of the first enantioselective synthesis of (-)-Nutlin-3 provided a basis to prepare larger amounts of this tool used widely in cancer biology. Key to the decagram-scale synthesis described here was the discovery of a novel bis(amidine) organocatalyst that provides high enantioselectivity at warmer reaction temperature (-20 °C) and low catalyst loadings. Further refinements to the procedure led to the synthesis of (-)-Nutlin-3 in a 17 g batch and elimination of all but three chromatographic purifications.

An α-quaternary chiral latam derivative, YH-304 as a novel broad-spectrum anticancer agent

Previously, we reported that α-quaternary chiral lactam derivatives have broad spectrum anticancer activity. However, the underlying molecular mechanisms and its relevance are largely unknown. In the present study, we report progress on α-quaternary chiral lactam analogues that address this, focusing on the novel analogue YH-304 as a candidate to broadly target human cancer cells. The effect of YH-304 on cell transformation was assessed by clonogenic assay in non-small cell lung cancer cells (NSCLCs) A549 and 226B. Proapoptotic activity of YH-304 was determined by TUNEL assay and cleaved PARP, cleaved caspase-9, and Bax as markers for apoptosis. The p53-dependency and therapeutic spectrum of YH-304 was assessed by western blot analysis, real-time PCR, and cell viability assays in cells expressing endogenous wild or mutant p53. The effect of YH-304 on angiogenesis in vivo was examined by bFGF-mediated angiogenesis assay in zebrafish. Finally, the effect of YH-304 on AKT and ERK activation (phosphorylation) as a putative mechanism underlying the effect of YH-304 on bFGF-mediated angiogenesis was assessed using western blotting. We found that YH-304 significantly decreases the colony-forming activities of both A549 and 226B cells, inducing cellular apoptosis. Unlike nutlin-3 (p53 pathway activator), YH-304 did not affect the expression levels of p53 and its target gene such as p21 and thus showed p53-independent anticancer activity with broad spectrum. In addition, YH-304 inhibited bFGF-induced angiogenesis in vivo through mediating AKT and ERK signaling pathway, which plays an important role in bFGF activation and angiogenesis. Taken together, our data indicate that YH-304 may represent a novel therapeutic option for the treatment of cancer in a p53-independent manner.

Understanding the interplay of weak forces in [3,3]-sigmatropic rearrangement for stereospecific synthesis of diamines

Chiral diamines are important building blocks for constructing stereoselective catalysts, including transition metal based catalysts and organocatalysts that facilitate oxidation, reduction, hydrolysis, and C-C bond forming reactions. These molecules are also critical components in the synthesis of drugs, including antiviral agents such as Tamiflu and Relenza and anticancer agents such as oxaliplatin and nutlin-3. The diaza-Cope rearrangement reaction provides one of the most versatile methods for rapidly generating a wide variety of chiral diamines stereospecifically and under mild conditions. Weak forces such as hydrogen bonding, electronic, steric, oxyanionic, and conjugation effects can drive this equilibrium process to completion. In this Account, we examine the effect of these individual weak forces on the value of the equilibrium constant for the diaza-Cope rearrangement reaction using both computational and experimental methods. The availability of a wide variety of aldehydes and diamines allows for the facile synthesis of the diimines needed to study the weak forces. Furthermore, because the reaction generally takes place cleanly at ambient temperature, we can easily measure equilibrium constants for rearrangement of the diimines. We use the Hammett equation to further examine the electronic and oxyanionic effects. In addition, computations and experiments provide us with new insights into the origin and extent of stereospecificity for this rearrangement reaction. The diaza-Cope rearrangement, with its unusual interplay between weak forces and the equilibrium constant of the reaction, provides a rare opportunity to study the effects of the fundamental weak forces on a chemical reaction. Among these many weak forces that affect the diaza-Cope rearrangement, the anion effect is the strongest (10.9 kcal/mol) followed by the resonance-assisted hydrogen-bond effect (7.1 kcal/mol), the steric effect (5.7 kcal/mol), the conjugation effect (5.5 kcal/mol), and the electronic effect (3.2 kcal/mol). Based on both computation and experimental data, the effects of these weak forces are additive. Understanding the interplay of the weak forces in the [3,3]-sigmatropic reaction is interesting in its own right and also provides valuable insights for the synthesis of chiral diamine based drugs and catalysts in excellent yield and enantiopurity.

Organocatalytic, diastereo- and enantioselective synthesis of nonsymmetric cis-stilbene diamines: a platform for the preparation of single-enantiomer cis-imidazolines for protein-protein inhibition

The finding by scientists at Hoffmann-La Roche that cis-imidazolines could disrupt the protein-protein interaction between p53 and MDM2, thereby inducing apoptosis in cancer cells, raised considerable interest in this scaffold over the past decade. Initial routes to these small molecules (i.e., Nutlin-3) provided only the racemic form, with enantiomers being enriched by chromatographic separation using high-pressure liquid chromatography (HPLC) and a chiral stationary phase. Reported here is the first application of an enantioselective aza-Henry approach to nonsymmetric cis-stilbene diamines and cis-imidazolines. Two novel mono(amidine) organocatalysts (MAM) were discovered to provide high levels of enantioselection (>95% ee) across a broad range of substrate combinations. Furthermore, the versatility of the aza-Henry strategy for preparing nonsymmetric cis-imidazolines is illustrated by a comparison of the roles of aryl nitromethane and aryl aldimine in the key step, which revealed unique substrate electronic effects providing direction for aza-Henry substrate-catalyst matching. This method was used to prepare highly substituted cis-4,5-diaryl imidazolines that project unique aromatic rings, and these were evaluated for MDM2-p53 inhibition in a fluorescence polarization assay. The diversification of access to cis-stilbene diamine-derived imidazolines provided by this platform should streamline their further development as chemical tools for disrupting protein-protein interactions.