A novel hybrid drug between two potent anti-tubulin agents as a potential prolonged anticancer approach

We report the design, synthesis and biological characterisation of a novel hybrid drug by conjugation of two tubulin inhibitors, a hemiasterlin derivative A (H-Mpa-Tle-AHA-OH), obtained by condensation of three non-natural amino acids, and cis-3,4′,5-trimethoxy-3′aminostilbene (B). As we have previously demonstrated synergy between A and B, we used a monocarbonyl derivative of triethylene glycol as linker (L) to synthesise compounds A-L and A-L-B; via HPLC we analysed the release of its potential hydrolysis products A, A-L, B and B-L in physiological fluids: the hybrid A-L-B undergo hydrolysis in rat whole blood of the ester bond between A and L (half-life = 118.2 ± 9.5 min) but not the carbamate bond between B and L; the hydrolysis product B-L was further hydrolyzed, but with a slower rate (half-life = 288 ± 12 min). The compound A-L was the faster hydrolyzed conjugate (half-life = 25.4 ± 1.1 min). The inhibitory activity of the compounds against SKOV3 ovarian cancer cell growth was analysed. The IC50 values were 7.48 ± 1.27 nM for A, 40.3 ± 6.28 nM for B, 738 ± 38.5 nM for A-L and 37.9 ± 2.11 nM for A-L-B. The anticancer effect of A-L-B was evidenced to be obtained via microtubule dynamics suppression. Finally, we stated the expression of the active efflux transporters P-gp (ABCB1) and MRP1 (ABCC1) in the human normal colon epithelial NCM460 cell line by reverse-transcription PCR. Via permeation studies across NCM460 monolayers we demonstrate the poor aptitude of A to interact with active efflux transporters (AET): indeed, the ratio between its permeability coefficients for the basolateral (B) → apical (A) and B → A transport was 1.5 ± 0.1, near to the ratio of taltobulin (1.12 ± 0.06), an hemiasterlin derivative able to elude AETs, and significantly different form the ratio of celiprolol (3.4 ± 0.2), an AET substrate.