Synthesis and Biological Evaluation of RGD Peptidomimetic–Paclitaxel Conjugates Bearing Lysosomally Cleavable Linkers

Abstract

Two small-molecule-drug conjugates (SMDCs, 6 and 7) featuring lysosomally cleavable linkers (namely the Val-Ala and Phe-Lys peptide sequences) were synthesized by conjugation of the αvβ3-integrin ligand cyclo[DKP-RGD]-CH2NH2 (2) to the anticancer drug paclitaxel (PTX). A third cyclo[DKP-RGD]-PTX conjugate with a nonpeptide "uncleavable" linker (8) was also synthesized to be tested as a negative control. These three SMDCs were able to inhibit biotinylated vitronectin binding to the purified αVβ3-integrin receptor at nanomolar concentrations and showed good stability at pH 7.4 and pH 5.5. Cleavage of the two peptide linkers was observed in the presence of lysosomal enzymes, whereas conjugate 8, which possesses a nonpeptide "uncleavable" linker, remained intact under these conditions. The antiproliferative activities of the conjugates were evaluated against two isogenic cell lines expressing the integrin receptor at different levels: the acute lymphoblastic leukemia cell line CCRF-CEM (αVβ3-) and its subclone CCRF-CEM αVβ3 (αVβ3+). Fairly effective integrin targeting was displayed by the cyclo[DKP-RGD]-Val-Ala-PTX conjugate (6), which was found to differentially inhibit proliferation in antigen-positive CCRF-CEM αVβ3 versus antigen-negative isogenic CCRF-CEM cells. The total lack of activity displayed by the "uncleavable" cyclo[DKP-RGD]-PTX conjugate (8) clearly demonstrates the importance of the peptide linker for achieving the selective release of the cytotoxic payload.

Multivalency Increases the Binding Strength of RGD Peptidomimetic‐Paclitaxel Conjugates to Integrin αVβ3

“…As a further step, to achieve selective release of PTX in the cancer cell environment, we synthesized conjugates of the cyclo [DKP‐RGD]‐CH 2 NH 2 ligand 2 with paclitaxel ( 3 ) via a 2′‐carbamate with a self‐immolative spacer and the lysosomally cleavable linkers (Val‐Ala and Phe‐Lys dipeptide sequences) . Notably, despite its remarkable size, the cyclo [DKP‐RGD]‐Val‐Ala‐PTX conjugate 4 (Figure ) retained a very good affinity for the α V β 3 integrin receptor (IC 50 =13.3±3.6 n m in competitive binding assays with biotinylated vitronectin) and displayed fairly effective integrin targeting …”

“…The synthesis of conjugates 5 – 9 was carried out according to a common synthetic strategy, shown in Scheme . The bis‐protected compound 15 , featuring the Val‐Ala linker connected to the para ‐aminobenzyl carbamate (PABC) ‐N , N′ ‐dimethylethylenediamine self‐immolative spacer, was prepared according to a methodology reported by our group . Compound 15 was Fmoc‐deprotected and the resulting crude free amine was coupled to scaffolds 10 – 14 , affording the corresponding amides 16 a – e in good yields (71–92 %).…”

Tumor Targeting with an isoDGR–Drug Conjugate

“…Conjugate 2 has been designed in a way similar to the corresponding RGD‐drug conjugate ( 4 , Figure ), which contains the RGD integrin ligand 3 . The iso DGR targeting moiety has been linked to the cytotoxic agent paclitaxel using the lysosomally cleavable dipeptide Val–Ala: this sequence showed high plasma stability, whereas it is rapidly cleaved by lysosomal cysteine proteases (such as cathepsins B and D) upon integrin‐mediated internalization by endocytosis …”

“…Mtr‐ (4‐methoxy‐2,3,6‐trimethylbenzenesulphonyl) and tert ‐butyl ester removal on macrolactam 16 afforded the desired iso DGR peptidomimetic 5 after HPLC purification and freeze‐drying. The benzylic amine of compound 5 was coupled with 18 , the N ‐hydroxysuccinimidyl ester of carboxylic acid 17 , to give compound 19 (Scheme ). Treatment of 19 with trifluoroacetic acid in dichloromethane afforded amine 20 which was reacted with carbonate 22 to obtain the final iso DGR‐PTX conjugate 2 .…”

Design and Synthesis of an RGD Peptidomimetic-Paclitaxel Conjugate Targeting αvβ3 Integrin for Tumour-Directed Drug Delivery

“…In order to reliably assess the selective cytotoxicity of conjugate 3 against αvβ3 positive cell lines, a "cell washout" cytotoxicity assay was performed. 16 Following this procedure -aimed at mimicking the in vivo conditions where the administered drug is rapidly cleared from the extracellular tumour environment -cells were incubated with the cytotoxic agents for a short period of time (6 h) and then the medium was washed away to remove the non-internalised conjugate 3. Using this method, the possible anti-proliferative activity arising from extracellular cleavage of the linker from 3 -and consequent undesired drug release -can be minimised.…”