A covalent p97/VCP ATPase inhibitor can overcome resistance to CB-5083 and NMS-873 in colorectal cancer cells.

Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins involved in tumorigenesis and virus infections. Multiple p97 inhibitors identified from previous high-throughput screening studies are thiol-reactive compounds targeting Cys522 in the D2 ATP-binding domain. Thus, these findings suggest a potential strategy to develop covalent p97 inhibitors. We first used purified p97 to assay several known covalent kinase inhibitors to determine if they can inhibit ATPase activity. We evaluated their selectivity using our dual reporter cells that can distinguish p97 dependent and independent degradation. We selected a ß-nitrostyrene scaffold to further study the structure-activity relationship. In addition, we used p97 structures to design and synthesize analogues of pyrazolo[3,4-d]pyrimidine (PP). We incorporated electrophiles into a PP-like compound 17 (4-amino-1-tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18 (N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent selectivity in an in vitro ATPase activity assay: IC50 of 0.6 µM, 300 µM, and 100 µM for wild type p97, yeast Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified and shown to increase IC50 values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with Cysteine decreased the IC50 by 10-fold. The molecular modeling suggested the hydrogen bonds and hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA. Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was inhibited by PPA with IC50 of 2.7 µM, 6.1 µM, and 3.4 µM, respectively. In addition, PPA is able to inhibit proliferation of two HCT116 cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future exploration to improve the potency of p97 inhibitors with different mechanisms of action.

Oxidative Fragmentations and Skeletal Rearrangements of Oxindole Derivatives.

An oxidative sequence for the conversion of oxindoles to structurally distinct heterocyclic scaffolds and aniline derivatives is disclosed by the combination of a copper-catalyzed C-H peroxidation and subsequent base-mediated fragmentation reaction. In contrast to classic enzymatic (i.e., kynurenine pathway) and biomimetic methods (i.e., Witkop-Winterfeldt oxidation) for oxidative indole cleavage, this protocol allows for the incorporation of external nucleophiles. The new transformation displays broad functional group tolerance and is applicable to tryptophan derivatives, opening potential new avenues for postsynthetic modification of polypeptides, bioconjugation, and unnatural amino acid synthesis.

Highly functionalized donor-acceptor cyclopropanes applied toward the synthesis of the Melodinus alkaloids.

A series of highly substituted vinylcyclopropanes were prepared and examined as reaction partners in a palladium-catalyzed (3 + 2) cycloaddition with nitrostyrenes. Described herein are our efforts to synthesize an elusive 1,1-divinylcyclopropane by several distinct approaches, and to apply surrogates of this fragment toward the synthesis of the Melodinus alkaloids.

Cobalt-catalyzed hydrogenation of esters to alcohols: unexpected reactivity trend indicates ester enolate intermediacy.

The atom-efficient and environmentally benign catalytic hydrogenation of carboxylic acid esters to alcohols has been accomplished in recent years mainly with precious-metal-based catalysts, with few exceptions. Presented here is the first cobalt-catalyzed hydrogenation of esters to the corresponding alcohols. Unexpectedly, the evidence indicates the unprecedented involvement of ester enolate intermediates.

Stereoselective Lewis acid mediated (3+2) cycloadditions of N-H- and N-sulfonylaziridines with heterocumulenes.

Alkyl and aryl isothiocyanates and carbodiimides are effective substrates in (3+2) cycloadditions with N-sulfonyl-2-substituted aziridines and 2-phenylaziridine for the synthesis of iminothiazolidines and iminoimidazolidines. Additionally, the stereoselective (3+2) cycloaddition of N-H- and N-sulfonylaziridines with isothiocyanates can be accomplished, allowing for the synthesis of highly enantioenriched iminothiazolidines. Evidence for an intimate ion-pair mechanism is presented herein in the context of these chemo-, regio-, and diastereoselective transformations. The demonstrated ability to remove the sulfonyl group from the heterocyclic products displays the utility of these compounds for further derivatization and application.

Lewis acid mediated (3 + 2) cycloadditions of donor-acceptor cyclopropanes with heterocumulenes.

Isocyanates, isothiocyanates, and carbodiimides are effective substrates in (3 + 2) cycloadditions with donor-acceptor cyclopropanes for the synthesis of five-membered heterocycles. These reactions exhibit a broad substrate scope, high yields, and well-defined chemoselectivity. Discussed herein are the implications of Lewis acid choice on the stereochemical outcome and the reaction mechanism.

A palladium-catalyzed vinylcyclopropane (3+2) cycloaddition approach to the Melodinus alkaloids.

A palladium-catalyzed (3+2) cycloaddition of a vinylcyclopropane and a ß-nitrostyrene is employed to rapidly assemble the cyclopentane core of the Melodinus alkaloids. The ABCD ring system of the natural product family is prepared in six steps from commercially available materials.