Approaches to improving the biological properties of natural products typically strive to modify their structures to identify the essential pharmacophore, or make functional group changes to improve biological target affinity or functional activity, change physical properties, enhance stability, or introduce conformational constraints. Aside from accessible semisynthetic modifications of existing functional groups, rarely does one consider using chemical synthesis to add molecular complexity to the natural product. In part, this may be attributed to the added challenge intrinsic in the synthesis of an even more complex compound. Herein, we report synthetically derived, structurally more complex vinblastines inaccessible from the natural product itself that are a stunning 100-fold more active (IC50 values, 50–75 pM vs. 7 nM; HCT116), and that are now accessible because of advances in the total synthesis of the natural product. The newly discovered ultrapotent vinblastines, which may look highly unusual upon first inspection, bind tubulin with much higher affinity and likely further disrupt the tubulin head-to-tail α/β dimer–dimer interaction by virtue of the strategic placement of an added conformationally well-defined, rigid, and extended C20′ urea along the adjacent continuing protein–protein interface. In this case, the added molecular complexity was used to markedly enhance target binding and functional biological activity (100-fold), and likely represents a general approach to improving the properties of other natural products targeting a protein–protein interaction.

Many natural products, including vinblastine, have not been easily subjected to simplifications in their structures by synthetic means or modifications by late-stage semi-synthetic derivatization in ways that enhance their biological potency. Herein, we detail a synthetic vinblastine that incorporates added benign complexity (ABC), which improves activity 10-fold, and is now accessible as a result of advances in the total synthesis of the natural product. The compound incorporates designed added molecular complexity but no new functional groups, and maintains all existing structural and conformational features of the natural product. It constitutes a member of an analogue class presently inaccessible by semi-synthetic derivatization of the natural product, by its late-stage functionalization, or by biosynthetic means. Rather, it was accessed by synthetic means, using an appropriately modified powerful penultimate single-step vindoline-catharanthine coupling strategy that proceeds with a higher diastereoselectivity than found for the natural product itself.

A series of 180 vinblastine 20’ amides were prepared in three steps from commercially available starting materials, systematically exploring a typically inaccessible site in the molecule enlisting a powerful functionalization strategy. Clear structure–activity relationships and a structural model were developed in the studies which provided many such 20’ amides that exhibit substantial and some even remarkable enhancements in potency, many that exhibit further improvements in activity against a Pgp overexpressing resistant cancer cell line, and an important subset of the vinblastine analogs that display little or no differential in activity against a matched pair of vinblastine sensitive and resistant (Pgp overexpressing) cell lines. The improvements in potency directly correlated with target tubulin binding affinity and the reduction in differential functional activity against the sensitive and Pgp overexpressing resistant cell lines was found to correlate directly with an impact on Pgp-derived efflux.

The total synthesis and evaluation of a key systematic series of vinblastines that incorporate the first deep-seated changes to the substituent at C4 are detailed. The synthetic approach features an expanded and redefined scope of a 1,3,4-oxadiazole [4 + 2]/[3 + 2] cycloaddition cascade in which electronically mismatched electron-deficient trisubstituted alkenes and unactivated trisubstituted alkenes were found to productively initiate the cycloaddition cascade with tethered electron-deficient 1,3,4-oxadiazoles. Such cycloaddition cascades were used to directly introduce altered C4 substituents, providing the basis for concise total syntheses of a series of C4 modified vindolines and their subsequent single-step incorporation into the corresponding synthetic vinblastines in routes as short as 8–12 steps. Evaluation of the synthetic vinblastines revealed a surprisingly large impact and role of the C4 substituent on activity even though it was previously not thought to intimately interact with the biological target tubulin. Only the introduction of a C4 methyl ester, a constitutional isomer of vinblastine in which the carbonyl carbon and ester oxygen of the C4 acetate are transposed, provided a synthetic vinblastine that matched the potency of the natural product. In contrast, even introduction of a C4 acetamide or N-methyl carboxamide, which incorporate single heavy atom exchanges (amide NH for ester oxygen) in vinblastine or the C4 methyl ester, provided compounds that were ≥10-fold less active than vinblastine. Other C4 acetate replacements, including a C4 amine, carboxylic acid, hydroxymethyl or acetoxymethyl group, led to even greater reductions in potency. Even replacement of the C4 acetoxy group or its equally active C4 methyl ester with an ethyl or isopropyl ester led to 10-fold or more reductions in activity. These remarkable trends in activity, which correlate with relative tubulin binding affinities, retrospectively may be ascribed to the role the substituent serves as a H-bond acceptor for α-tubulin Lys336 and Asn329 side chains at a site less tolerant of a H-bond donor, placing the methyl group of the C4 acetate or C4 methyl ester in a spatially restricted and well-defined hydrophobic half pocket created by a surrounding well-ordered loop. This remarkable impact of the C4 substituent, its stringency, and even the magnitude of its effect are extraordinary, and indicate that its presence was selected in Nature to enhance the effects of vinblastine and related natural products.