Strategic Incorporation of Fluorine into Taxoid Anticancer Agents for Medicinal Chemistry and Chemical Biology Studies

Abstract

This account exemplifies our recent progress on the strategic incorporation of fluorine and organofluorine groups to taxoid anticancer agents and their tumor-targeted drug delivery systems (TTDDSs) for medicinal chemistry and chemical biology studies. Novel 3'-difluorovinyltaxoids were strategically designed to block the metabolism by cytochrome P-450, synthesized, and evaluated for their cytotoxicity against drug-sensitive and multidrug-resistant (MDR) human cancer cell lines. 3'-Difluorovinyltaxoids exhibited impressive activities against these cancer cell lines. More significantly, a representative 3'-difluorovinyltaxoid exhibited 230-33,000 times higher potency than conventional anticancer drugs against cancer stem cell-enriched HCT-116 cell line. Studies on the mechanism of action (MOA) of these fluorotaxoids were performed by tubulin polymerization assay, morphology analysis by electron microscopy (EM) and protein binding assays. Novel ¹⁹F NMR probes, BLT-F₂ and BLT-S-F₆, were designed by strategically incorporating fluorine, CF₃ and CF₃O groups into tumor-targeting drug conjugates. These ¹⁹F-probes were designed and synthesized to investigate the mechanism of linker cleavage and factors that influence their plasma and metabolic stability by real-time ¹⁹F NMR analysis. Time-resolved ¹⁹F NMR study on probe BLT-F₂ revealed a stepwise mechanism for the release of a fluorotaxoid, which might not be detected by other analytical methods. Probe BLT-S-F₆ were very useful to study the stability and reactivity of the drug delivery system in human blood plasma by ¹⁹F NMR. The clean analysis of the linker stability and reactivity of drug conjugates in blood plasma by HPLC and ¹H NMR is very challenging, but the use of ¹⁹F NMR and suitable ¹⁹F probes can provide a practical solution to this problem.

Keywords: 19F NMR; Anticancer agent; Fluorotaxoid; Plasma and metabolic stability; Structure-activity relationship; Taxoid; Tumor-Targeted Drug Delivery System.

Fig. 1

Primary sites of hydroxylation on the new-generation taxoids by the P450 family of enzymes. Adapted from Reference [32].

Fig. 2

(a) Proposed binding conformation of SB-T-12853 in tubulin; (b) overlay of SB-T-12853 (cyan) and SB-T-1213 (magenta) in tubulin. Adapted from Reference [32].

Fig. 3

Tubulin polymerization with SB-T-12851, SB-T-12852, SB-T-12854, paclitaxel and GTP: microtubule protein 1 mg/mL, 37 °C, GTP 1 mM, Drug 10 μM. Adapted from Reference [32].

Fig. 4

Electromicrographs of microtubules: (a) GTP; (b) paclitaxel; (c) SB-T-12851; (d) SB-T-12852; (e) SB-T-12854. Adapted from Reference [32].

Fig. 5

A model system for the mechanism-based drug release using cysteine as the trigger for thiolactonization. Adapted from Reference [15].

Fig. 6

Examples of vitamin-based SMDCs developed in our laboratory.

Fig. 7

BLT-F₂, BLT-S-F₆, SB-T-12145 and SB-T-12822-5.

Fig. 8

Time-resolved ¹⁹F NMR spectra for the disulfide linker cleavage and thiolactonization process of BLT-F₂ (2.5 mM) in 30% DMSO in D₂O beginning at 1 hour after the addition of 6 equivalents of GSH at 25 °C with 15 min intervals (128 scans/spectrum). Adapted from Reference [74].

Fig. 9

Time-resolved ¹⁹F NMR spectra for the drug release of the probe (BLT-S-F₆) (200 μM) in 86% blood plasma, 10% D₂O, 2% ethanol, and 2% polysorbate 80 at 37 °C without supplemental GSH at 0, 24, and 48 h (2048 scans/spectrum). The signals of 2-m-OCF₃ (left) and 3′-CF₃ (right) for the probe (P) and free taxoid (T, SB-T-12822-5) are shown, which indicates minimal drug release after 48 h. Adapted from Reference [74].

Fig. 10

Time-resolved ¹⁹F NMR spectra for the drug release of the probe (P, BLT-S-F₆) (200 μM) in 86% blood plasma, 2% ethanol, and 2% Tween 80 in D₂O at 30 min after the addition of 100 equivalents of GSH at 37 °C with 1 h intervals (1024 scans/spectrum) for 13 h. The signals of 2-m-OCF₃ (left) and the 3′-CF₃ (right) are shown, which indicate full drug (T, SB-T-12822-5) release after 13.5 h. Adapted from Reference [74].

Fig. 11

Normalized changes in integration of 3′-CF₃ peaks of the probe (BLT-S-F₆) with 100 equiv. of GSH in 86% blood plasma, 2% ethanol, 2% polysorbate 80, 10% D₂O and released free taxoid (SB-T-12822-5). Adapted from Reference [74].

Scheme 1

Synthesis of 3′-difluorovinyltaxoids 1 through the Ojima-Holton coupling.

Scheme 2

Preparation of (3R,4R)-1-t-Boc-3-TIPSO-4-difluorovinylazetidin-2-one (3).

Scheme 3

Synthesis of SB-T-12145.

Scheme 4

Synthesis of ¹⁹F-labeled linker construct 12.

Scheme 5

Synthesis of BLT-F₂.

Scheme 6

Synthesis of SB-T-12822-5.

Scheme 7

Synthesis of BLT-S-F₆