Human P-glycoprotein (Pgp) is as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. The aim of this study is to evaluate whether Pgp modulators can be engineered to exhibit high-affinity binding using polyvalency. Five bivalent homodimeric polyenes based on stipiamide linked with polyethylene glycol ethers in the range of 3-50 A were synthesized and quantitatively characterized for their effect on Pgp function. The stipiamide homodimers displaced [(125)I]iodoarylazidoprazoin (IAAP), an analogue of the Pgp substrate prazosin. A minimal spacer of 11 A is necessary for inhibition of IAAP labeling, beyond which there is an inverse correlation between the length of the spacer and the IC(50) for the displacement of IAAP. ATP hydrolysis by Pgp on the other hand is stimulated by the dimers with spacers of up to 22 A, whereas dimers with longer spacers inhibit ATP hydrolysis. Finally, the homodimers reverse Pgp-mediated drug efflux in intact cells overexpressing Pgp, and 11 A is a threshold beyond which the effectiveness of the homodimers increases exponentially and levels off at 33 A. We demonstrate that dimerization and identification of an optimal spacer length increase by 11-fold the affinity of stipiamide, and this is reflected in the efficacy with which Pgp-mediated drug efflux is reversed. These results suggest that polyvalency could be a useful strategy for the development of more potent Pgp modulators.
The synthesis of (−)-stipiamide (1) is reported
together with the designed enynes 2
(6,7-dehydrostipiamide)
and 3 that are now shown to reverse the multidrug resistance
(MDR) of human breast cancer cells (MCF-7adrR).
Stipiamide was assembled using a Stille coupling with
(E)-vinyl iodide 17 and (Z)-stannyl
amide 16 in 78% yield.
(E)-Vinyl iodide 17 was made using a Takai
reaction and a selective dihydroxylation of the terminal olefin
of
nonconjugated diene 7 using the Sharpless AD-mix reagent.
The precursor to 16, (E,Z)-stannyl diene
ester 13, was
assembled with high selectivity in a single operation using a tandem
syn-addition of tributyltin cuprate to
acetylene
followed by conjugate addition to ethyl propiolate. Structural
variants 2 and 3 were assembled using
palladium-catalyzed Sonogashira couplings with vinyl iodides 17 and
35 and acetylenes 22 and 26 in high
yield at near 1:1
stoichiometry. Compound 2 was found to be far less
toxic than stipiamide and performed much better as an MDR
reversal agent. Compound 3 was better still due to even
lower toxicity.
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