Simultaneous structure-activity studies and arming of natural products by C-H amination reveal cellular targets of eupalmerin acetate

Abstract

Natural products have a venerable history of, and enduring potential for the discovery of useful biological activity. To fully exploit this, the development of chemical methodology that can functionalize unique sites within these complex structures is highly desirable. Here, we describe the use of rhodium(II)-catalysed C-H amination reactions developed by Du Bois to carry out simultaneous structure-activity relationship studies and arming (alkynylation) of natural products at 'unfunctionalized' positions. Allylic and benzylic C-H bonds in the natural products undergo amination while olefins undergo aziridination, and tertiary amine-containing natural products are converted to amidines by a C-H amination-oxidation sequence or to hydrazine sulfamate zwitterions by an unusual N-amination. The alkynylated derivatives are ready for conversion into cellular probes that can be used for mechanism-of-action studies. Chemo- and site-selectivity was studied with a diverse library of natural products. For one of these-the marine-derived anticancer diterpene, eupalmerin acetate-quantitative proteome profiling led to the identification of several protein targets in HL-60 cells, suggesting a polypharmacological mode of action.

Figure 1

A two-step C–H Amination or Aziridination-Conjugation Sequence for Simultaneous Arming and SAR Studies of Natural Products.

Figure 2. Synthesis of alkynyl sulfamate 9 and exploratory C–H amination with carvone

a, Preparation of the nitrene precursor, alkynyl sulfamate 9. b, Catalyst screening for C–H amination versus aziridination with L-(−)-carvone 10.

Figure 3

Natural Product Remodeling: (a) Cyclization of the carvone C–H amination product, (b) Rearrangement of gibberellic acid methyl ester derived aziridine and (c) Deprotection of betaines.

Figure 4. Cytoxicity and Proteomic Profiling of EPA and EPAyne

(A) Inhibition of HL-60 cell proliferation by EPA and EPAyne as measured using a WST-1 assay. (B) In situ treatment of HL-60 cells with EPAyne alone or in competition with EPA followed by conjugation to an azide-rhodamine reporter tag, SDS-PAGE, and fluorescent scanning. Red arrows highlight competed signals. (C) Schematic of competitive ABPP-SILAC. Cells are enriched with heavy or light isotopic tags and treated with DMSO or EPA, respectively, and followed by addition of EPAyne. EPA labeled proteomes are mixed, conjugated to biotin using click chemistry, enriched, and analyzed by MudPIT. (D) List of putative EPA targets in HL-60 cells. Average ratios are from duplicate runs and standard error results are reported. In the control experiment, heavy and light cells are treated with EPAyne alone (E) Validation of three high affinity targets DERL1, CYB5B, and TBXAS1 by labeling of transiently transfected 293T cells with EPAyne (5 μM) ± EPA (15 μM). Red arrows indicate the target’s expected molecular weight.