Vinblastine 20' Amides: Synthetic Analogues That Maintain or Improve Potency and Simultaneously Overcome Pgp-Derived Efflux and Resistance

Abstract

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 analogues 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.

Figure 1

Natural product structures and 20’ amide analogs of vinblastine.

Figure 2

Vinblastine aliphatic 20’ amides.

Figure 3

Vinblastine 20’ benzoyl amides.

Figure 4

Plots of Hammett σ_p constant versus –log IC₅₀ (nM) for an initial series of 4-substituted benzamides that were examined. A. HCT116. B. L1210.

Figure 5

Tubulin binding assay measuring the % displacement of tubulin-bound BODIPY-vinblastine under conditions where vinblastine results in 50% displacement. Compounds 64 (88 ± 5%), 24 (72 ± 4%), and 54 (56 ± 5%) are the 20’ benzamide derivatives containing a 4-NMe₂, 4-H, and 4-CN substituent, respectively. IC₅₀ (HCT116) = 0.18, 0.8, 3.1 and 6.8 nM for 64, 24, 54 and vinblastine, respectively.

Figure 6

Vinblastine 20’ acrylamides.

Figure 7

Polycyclic benzoyl-like 20’ amides.

Figure 8

Monocyclic heterocyclic 20’ amides.

Figure 9

Polycyclic heterocyclic 20’ amides.

Figure 10

Plot of 20’ amide cLogP vs differential activity (ratio) for 28, 121, 173 and closely related compounds that progressively exchange in heteroatoms.

Figure 11

Vinblastine 20’ sulfonamides.

Figure 12

10’-Fluorovinblastine 20’ amides.

Figure 13

Tubulin binding assay measuring the % displacement of tubulin-bound BODIPY-vinblastine, comparing compounds 28, 121 and 173 with vinblastine.

Figure 14

Caco-2 bidirectional permeability and stimulated Pgp ATPase activity in membranes for vinblastine and compounds 173, 121 and 28.

Figure 15

Evaluation of 28, 121, and 173 against additional representative human cancer cell lines and an independent assessment HCT116 versus HCT116/VM46 activity.

Figure 16

Models of 28 (panels C), 121 (panels A) and 173 (panels B) bound to tubulin generated from the X-ray structure of a vinblastine-bound complex (pdb 5J2T). Left: Stick figures highlighting the key H-bonds of the 20’ amide: the amide N–H with the backbone carbonyl of Pro222 (2.0 Å) and the amide carbonyl with the backbone amide N–H of Tyr224 (3.0 Å). Right: Space filling models of tubulin highlighting the extension of the 20’ amides into a narrow channel formed along the continuing protein–protein interaction at the tubulin α/β dimer–dimer interface.

Scheme 1