Design, synthesis and application of fluorine-labeled taxoids as 19F NMR probes for the metabolic stability assessment of tumor-targeted drug delivery systems

Abstract

Novel tumor-targeting drug conjugates, BLT-F2 (1) and BLT-S-F6 (2), bearing a fluorotaxoid as the warhead, a mechanism-based self-immolative disulfide linker, and biotin as the tumor-targeting module, were designed and synthesized as 19F NMR probes. Fluorine atoms and CF3 groups were strategically incorporated into the conjugates to investigate the mechanism of linker cleavage and factors that influence their plasma and metabolic stability by real-time monitoring with 19F NMR. Time-resolved 19F NMR study on probe 1 disclosed a stepwise mechanism for release of a fluorotaxoid, which might not have been detected by other analytical methods. Probe 2 was designed to bear two CF3 groups in the taxoid moiety as “3-FAB” reporters for enhanced sensitivity and a polyethylene glycol oligomer insert to improve solubility. The clean analysis of the linker stability and reactivity of drug conjugates in blood plasma or cell culture media by HPLC and 1H NMR is troublesome, due to the overlap of key signals/peaks with background arising from highly complex ingredients in biological systems. Accordingly, the use of 19F NMR would provide a practical solution to this problem. In fact, our “3-FAB” probe 2 was proven to be highly useful to investigate the stability and reactivity of the self-immolative disulfide linker system in human blood plasma by 19F NMR. It has also been revealed that the use of polysorbate 80 as excipient for the formulation of probe 2 dramatically increases the stability of the disulfide linker system. This finding further indicates that the tumor-targeting drug conjugates with polysorbate 80/EtOH/saline formulation for in vivo studies would have high stability in blood plasma, while the drug release in cancer cells proceeds smoothly.

Design, Synthesis, and Biological Evaluation of Theranostic Vitamin–Linker–Taxoid Conjugates

“…3, 4, 11 Once a drug conjugate is internalized into tumor cells following target-specific binding and receptor-mediated endocytosis (RME), the “smart” linker is designed to release the drug warhead through thiol-disulfide exchange with endogenous thiols, e.g., glutathione (GSH) and thioredoxin, via facile benzothiolactonization. 4, 11, 13, 35 Since the GSH level in tumor tissues (2–8 mM) is more than 1,000 times higher than that in the blood stream (1–2 µM), GSH and other endogenous thiols serve as ideal tumor-specific triggers for drug release. 11, 13, 34, 36 …”

Quantitative NMR Studies of Multiple Compound Mixtures

“…Quantitative 1D 19 F NMR is widely applied in pharmaceutical, polymer, and organic industries [51][52][53]. Fluorine ( 19 F) nucleus is similar to hydrogen ( 1 H) with nuclear spin I ¼ 1/2, a large gyromagnetic ratio of 40.05 MHz/T, and natural abundance of 100% [54], which opens a way to selective, sensitive, and quantitative detection of fluorinated compounds in complex matrices.…”

Strategic incorporation of fluorine into taxoid anticancer agents for medicinal chemistry and chemical biology studies

“…We designed and synthesized two novel 19 F NMR probes, BLT-F 2 and BLT-S-F 6 in order to investigate the factors that influence the rate of disulfide cleavage and drug release in biologically relevant media such as human blood plasma [74]. This type of assessment by conventional HPLC or 1 H NMR analysis would be challenging, due to complex background peaks/signals.…”

“…For the construction of BLT-F 2 , fluorotaxoid SB-T-12145 was synthesized first as a close mimic of a highly efficacious next-generation taxoid, SB-T-1214 [19, 20, 30, 36], through the Ojima-Holton coupling of fluorobaccatin 6 with β-lactam 7 (Scheme 3) [74]. The linker construct 12 , incorporating another fluorine at the 4 position of the disulfanylphenylacetate moiety, i.e., para to the disulfide moiety was synthesized from 5-fluorobenzothiophene ( 8 ) via 5-fluoro-2-surfhydrylphenylacetic acid ( 10 ) (Scheme 4) [74].…”