Structure-Based Design and Evaluation of Reversible KRAS G13D Inhibitors.

Oncogenic KRAS mutations were identified decades ago, yet the selective inhibition of specific KRAS mutant proteins represents an ongoing challenge. Recent progress has been made in targeting certain P-loop mutant proteins, in particular KRAS G12C, for which the covalent inhibition of the GDP state via the Switch II pocket is now a clinically validated strategy. Inhibition of other KRAS mutant proteins such as KRAS G13D, on the other hand, still requires clinical validation. The remoteness of the D13 residue relative to the Switch II pocket in combination with the solvent exposure and conformational flexibility of the D13 side chain, as well as the difficulties of targeting carboxylate residues covalently, renders this specific protein particularly challenging to target selectively. In this report, we describe the design and evaluation of potent and KRAS G13D-selective reversible inhibitors. Subnanomolar binding to the GDP state Switch II pocket and biochemical selectivity over WT KRAS are achieved by leveraging a salt bridge with D13.

1-Aminomethyl SAR in a novel series of flavagline-inspired eIF4A inhibitors: Effects of amine substitution on cell potency and in vitro PK properties.

Flavaglines such as silvestrol (1) and rocaglamide (2) constitute an interesting class of natural products with promising anticancer activities. Their mode of action is based on inhibition of eukaryotic initiation factor 4A (eIF4A) dependent translation through formation of a stable ternary complex with eIF4A and mRNA, thus blocking ribosome scanning. Herein we describe initial SAR studies in a novel series of 1-aminomethyl substituted flavagline-inspired eIF4A inhibitors. We discovered that a variety of N-substitutions at the 1-aminomethyl group are tolerated, making this position pertinent for property and ADME profile tuning. The findings presented herein are relevant to future drug design efforts towards novel eIF4A inhibitors with drug-like properties.

Targeting Oncogene mRNA Translation in B-Cell Malignancies with eFT226, a Potent and Selective Inhibitor of eIF4A.

The PI3K/AKT/mTOR pathway is often activated in lymphoma through alterations in PI3K, PTEN, and B-cell receptor signaling, leading to dysregulation of eIF4A (through its regulators, eIF4B, eIF4G, and PDCD4) and the eIF4F complex. Activation of eIF4F has a direct role in tumorigenesis due to increased synthesis of oncogenes that are dependent on enhanced eIF4A RNA helicase activity for translation. eFT226, which inhibits translation of specific mRNAs by promoting eIF4A1 binding to 5'-untranslated regions (UTR) containing polypurine and/or G-quadruplex recognition motifs, shows potent antiproliferative activity and significant in vivo efficacy against a panel of diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma models with ≤1 mg/kg/week intravenous administration. Evaluation of predictive markers of sensitivity or resistance has shown that activation of eIF4A, mediated by mTOR signaling, correlated with eFT226 sensitivity in in vivo xenograft models. Mutation of PTEN is associated with reduced apoptosis in vitro and diminished efficacy in vivo in response to eFT226. In models evaluated with PTEN loss, AKT was stimulated without a corresponding increase in mTOR activation. AKT activation leads to the degradation of PDCD4, which can alter eIF4F complex formation. The association of eFT226 activity with PTEN/PI3K/mTOR pathway regulation of mRNA translation provides a means to identify patient subsets during clinical development.

Strategic Diastereoselective C1 Functionalization in the Aza-Rocaglamide Scaffold toward Natural Product-Inspired eIF4A Inhibitors.

Rocaglates, rocaglamides, and related flavagline natural products exert their remarkable anticancer activity through inhibition of eukaryotic initiation factor 4A (eIF4A) but generally display suboptimal drug-like properties. In our efforts to identify potent drug-like eIF4A inhibitors, we developed synthetic strategies for diastereoselectively functionalizing the C1 position of aza-rocaglamide scaffolds (cf. 14 and 18), which proceed via retention or inversion of configuration at C1 depending on the C2 substituent (cf. 15 and 21) and ultimately enabled the discovery of novel and potent eIF4A inhibitors such as 25.

Design of Development Candidate eFT226, a First in Class Inhibitor of Eukaryotic Initiation Factor 4A RNA Helicase.

Dysregulation of protein translation is a key driver for the pathogenesis of many cancers. Eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase, is a critical component of the eIF4F complex, which regulates cap-dependent protein synthesis. The flavagline class of natural products (i.e., rocaglamide A) has been shown to inhibit protein synthesis by stabilizing a translation-incompetent complex for select messenger RNAs (mRNAs) with eIF4A. Despite showing promising anticancer phenotypes, the development of flavagline derivatives as therapeutic agents has been hampered because of poor drug-like properties as well as synthetic complexity. A focused effort was undertaken utilizing a ligand-based design strategy to identify a chemotype with optimized physicochemical properties. Also, detailed mechanistic studies were undertaken to further elucidate mRNA sequence selectivity, key regulated target genes, and the associated antitumor phenotype. This work led to the design of eFT226 (Zotatifin), a compound with excellent physicochemical properties and significant antitumor activity that supports clinical development.

Bioisosteric Exchange of Csp3 -Chloro and Methyl Substituents: Synthesis and Initial Biological Studies of Atpenin†A5 Analogues.

Asymmetric synthesis and initial biological studies of two analogues of a naturally occurring chlorinated antifungal agent, atpenin A5, are described. These analogues were selected on the basis of Cl→CH3 or H3 C→Cl exchanges in the side-chain of atpenin A5. The interchange of chloro and methyl substituents led to complex II inhibitors with equal IC50 values. This suggests that Cl↔Me bioisosteric exchange can be realized in aliphatic settings.

Synthesis and Biological Evaluation of Chlorinated Analogs of Leukotoxin Diol.

This study documents that chlorinated analogs of leukotoxin diol 1, in which the vic-diol has been replaced with vic-chlorides (2), induce caspase 3 activity and apoptosis on HepG2 cells in a dose-dependent manner in analogy to the parent diol. This suggests that chlorides may substitute for hydroxyls in certain lipids as bioisosteres in defined biological settings.

Synthesis and biological evaluation of dimeric furanoid macroheterocycles: discovery of new anticancer agents.

A recently developed dimerization/macrocyclization was employed to synthesize a series of macroheterocycles which were biologically evaluated, leading to the discovery of a number of potent cytotoxic agents (e.g., 27: GI50 = 51 nM against leukemia CCRF-CEM cell line; 29: GI50 = 99 nM against melanoma MDA-MB-435 cell line). Further biological studies support an apoptosis mechanism of action for these compounds involving deregulation of the tricarboxylic acid cycle activity and suppression of mitochondrial function in cancer cells.

Total synthesis of viridicatumtoxin B and analogues thereof: strategy evolution, structural revision, and biological evaluation.

The details of the total synthesis of viridicatumtoxin B (1) are described. Initial synthetic strategies toward this intriguing tetracycline antibiotic resulted in the development of key alkylation and Lewis acid-mediated spirocyclization reactions to form the hindered EF spirojunction, as well as Michael-Dieckmann reactions to set the A and C rings. The use of an aromatic A-ring substrate, however, was found to be unsuitable for the introduction of the requisite hydroxyl groups at carbons 4a and 12a. Applying these previous tactics, we developed stepwise approaches to oxidize carbons 12a and 4a based on enol- and enolate-based oxidations, respectively, the latter of which was accomplished after systematic investigations that revealed critical reactivity patterns. The herein described synthetic strategy resulted in the total synthesis of viridicatumtoxin B (1), which, in turn, formed the basis for the revision of its originally assigned structure. The developed chemistry facilitated the synthesis of a series of viridicatumtoxin analogues, which were evaluated against Gram-positive and Gram-negative bacterial strains, including drug-resistant pathogens, revealing the first structure-activity relationships within this structural type.

Directed ortho metalation strategies. Effective regioselective routes to 1,2-, 2,3-, and 1,2,3-substituted naphthalenes.

The regioselective synthesis of 2,3-di- and 1,2,3-trisubstituted naphthalenes via Directed ortho Metalation (DoM) strategies of N,N-diethyl-O-naphthyl-2-carbamate (1) is presented. Sequential LiTMP metalation-electrophile quench and s-BuLi/TMEDA (or t-BuLi)-electrophile quench of naphthyl-2-carbamate 1 provides a general route to contiguously substituted naphthalenes (6) with full regioselectivity. Further derivatization via ipso-halodesilylation and Suzuki-Miyaura cross-coupling leads ultimately to substituted halonaphthalenes and benzonaphthopyranones (9).

Constructing molecular complexity and diversity: total synthesis of natural products of biological and medicinal importance.

The advent of organic synthesis and the understanding of the molecule as they occurred in the nineteenth century and were refined in the twentieth century constitute two of the most profound scientific developments of all time. These discoveries set in motion a revolution that shaped the landscape of the molecular sciences and changed the world. Organic synthesis played a major role in this revolution through its ability to construct the molecules of the living world and others like them whose primary element is carbon. Although the early beginnings of organic synthesis came about serendipitously, organic chemists quickly recognized its potential and moved decisively to advance and exploit it in myriad ways for the benefit of mankind. Indeed, from the early days of the synthesis of urea and the construction of the first carbon-carbon bond, the art of organic synthesis improved to impressively high levels of sophistication. Through its practice, today chemists can synthesize organic molecules--natural and designed--of all types of structural motifs and for all intents and purposes. The endeavor of constructing natural products--the organic molecules of nature--is justly called both a creative art and an exact science. Often called simply total synthesis, the replication of nature's molecules in the laboratory reflects and symbolizes the state of the art of synthesis in general. In the last few decades a surge in total synthesis endeavors around the world led to a remarkable collection of achievements that covers a wide ranging landscape of molecular complexity and diversity. In this article, we present highlights of some of our contributions in the field of total synthesis of natural products of biological and medicinal importance. For perspective, we also provide a listing of selected examples of additional natural products synthesized in other laboratories around the world over the last few years.

A total synthesis trilogy: calicheamicin γ1(I), Taxol®, and brevetoxin A.

Detailed behind-the-scenes accounts of the total syntheses of calicheamicin γ(1)(I), Taxol(®), and brevetoxin A are discussed with particular emphasis placed on strategies and tactics employed in these campaigns.

Conformational and configurational analysis in the study and synthesis of chlorinated natural products.

The first detailed study of the J-based configuration analysis method in chlorinated hydrocarbons and chlorohydrins is presented along with the development of a spectroscopic database that facilitates configurational assignment of these structures. The data are generated through the investigation of model structures in solution by NMR spectroscopic methods and in the solid state by X-ray crystallography. Consequently, complete conformational analysis of trichlorinated hexane-1,2- and -1,3-diols is presented. The investigations in chlorinated systems for the first time attest to the relevance, reliability, and accuracy of the spectroscopic approach in configurational assignment, which had been otherwise developed for polyketides. During the synthesis of the various molecules that constitute the database and exemplify the various possible stereochemical patterns, a number of observations were made that underscore the unique features of these chlorinated systems. Thus, certain diastereomeric subclasses of 4,5-dichloro-2,3-epoxyhexane-1-ols display a propensity to undergo ring-opening reactions at C-3 with concomitant inversion of configuration at the neighboring C-Cl at C4, implicating the intermediacy of chloronium ions. The observations of positional and stereochemical scrambling in polychlorinated hydrocarbons underscore the necessity of a spectroscopic database that enables rapid, reliable configurational assignment of chlorinated natural products and intermediates en route to these.

Total synthesis of a chlorosulpholipid cytotoxin associated with seafood poisoning.

Each year, there are many cases of seafood poisoning in humans worldwide. Among the various toxins isolated that contribute to these poisonings, the chlorosulpholipids are particularly intriguing because of their structural and stereochemical complexity. The mechanism of biological activity remains unknown and, although chlorosulpholipids are associated with membranes in the organisms from which they are isolated, little is understood about their role within biological membranes. The lack of availability of the natural products has impaired more in-depth biochemical studies. So far, none of the chlorosulpholipids have been obtained from total synthesis, and efficient routes to their synthesis would be desirable for the preparation of material for pharmacological characterization and proper evaluation of the risk to human health. Despite the notable advances in the science of organic synthesis, reliable methods for stereoselective construction of polychlorinated acyclic substrates are lacking, although some preliminary investigations have appeared. Here we report the synthesis of a chlorosulpholipid cytotoxin, leading to confirmation of the proposed structure and the discovery of unanticipated reactivity of polychlorinated hydrocarbons. The concise synthetic approach should enable the preparation of material in sufficient quantities to facilitate biological studies.