Synthesis, Characterization and in vitro Studies of a Cathepsin B-Cleavable Prodrug of the VEGFR Inhibitor Sunitinib

Abstract

Since several decades, the prodrug concept has raised considerable interest in cancer research due to its potential to overcome common problems associated with chemotherapy. However, for small-molecule tyrosine kinase inhibitors, which also cause severe side effects, hardly any strategies to generate prodrugs for therapeutic improvement have been reported so far. Here, we present the synthesis and biological investigation of a cathepsin B-cleavable prodrug of the VEGFR inhibitor sunitinib. Cell viability assays and Western blot analyses revealed, that, in contrast to the non-cathepsin B-cleavable reference compound, the prodrug shows activity comparable to the original drug sunitinib in the highly cathepsin B-expressing cell lines Caki-1 and RU-MH. Moreover, a cathepsin B cleavage assay confirmed the desired enzymatic activation of the prodrug. Together, the obtained data show that the concept of cathepsin B-cleavable prodrugs can be transferred to the class of targeted therapeutics, allowing the development of optimized tyrosine kinase inhibitors for the treatment of cancer.

Keywords: cathepsin B; inhibitors; prodrugs; sunitinib; tyrosine kinase inhibitor; vascular endothelial growth factor receptor (VEGFR).

Figure 1

Schematic overview of the cathepsin B‐cleavable prodrug concept for sunitinib. Hydrogen bonds are indicated by dashed lines.

Scheme 1

A) Synthetic route of prodrug 8. Reagents and conditions: a) NHS, DCC, THF abs., 0 °C to room temperature; b) H‐Lys(alloc)‐OH, NaHCO₃, DME, H₂O; c) PABOH, EEDQ, THF; d) HCl conc., EtOH; e) morpholin‐4‐yl‐acetic acid, EEDQ, Et₃N, THF/EtOH; f) bis(4‐nitrophenyl) carbonate, DIPEA, DMF abs.; g) sunitinib, 4‐DMAP, DMF abs.; h) Pd(PPh₃)₄, NDMBA, DMF abs. B) Chemical structure of the Gly‐Gly reference compound 14.

Figure 2

Inhibitory effects of prodrug 8 in comparison to sunitinib on RTK signaling. Caki‐1 cells were grown in medium with FCS and treated with the indicated drug for 4 h. Then, they were harvested and lysed, and the impact on ERK phosphorylation as readout for RTK signaling was analyzed by Western blotting.

Figure 3

Cathepsin B assay. A), B): Time curves of (A) compound 8 and (B) compound 14 after incubation with and without (‘control’) human liver cathepsin B at pH 5 and 37 °C. C), D): Chromatograms of the incubation study of compound 8 (C) with human liver cathepsin B, and (D) without cathepsin B.

Scheme 2

Postulated mechanism of the rearrangement of the enol form of 8 and 14 from carbamate to direct linkage in aqueous solution.