Discovery of Bivalent GalNAc-Conjugated Betulin as a Potent ASGPR-Directed Agent against Hepatocellular Carcinoma

Abstract

Herein, we describe the design, synthesis, and biological evaluation of novel betulin and N-acetyl-d-galactosamine (GalNAc) glycoconjugates and suggest them as targeted agents against hepatocellular carcinoma. We prepared six conjugates derived via the C-3 and C-28 positions of betulin with one or two saccharide ligands. These molecules demonstrate high affinity to the asialoglycoprotein receptor (ASGPR) of hepatocytes assessed by in silico modeling and surface plasmon resonance tests. Cytotoxicity studies in vitro revealed a bivalent conjugate with moderate activity, selectivity of action, and cytostatic properties against hepatocellular carcinoma cells HepG2. An additional investigation confirmed the specific engagement with HepG2 cells by the enhanced generation of reactive oxygen species. Stability tests demonstrated its lability to acidic media and to intracellular enzymes. Therefore, the selected bivalent conjugate represents a new potential agent targeted against hepatocellular carcinoma. Further extensive studies of the cellular uptake in vitro and the real-time microdistribution in the murine liver in vivo for fluorescent dye-labeled analogue showed its selective internalization into hepatocytes due to the presence of GalNAc ligand in comparison with reference compounds. The betulin and GalNAc glycoconjugates can therefore be considered as a new strategy for developing therapeutic agents based on natural triterpenoids.

Development of Asialoglycoprotein-Mediated Hepatocyte-Targeting Antitumor Prodrugs Triggered by Glutathione

“…In other words, the hepatocyte-targeting capacity followed the W-1-5 (monoga-lactose) < W-2-9 (digalactose) < W-3-8 (trigalactose) order, obeying the rules of the galactose-recognition cluster effect toward ASGPR. Interestingly, it was reported that the galactose-modified bis-fluorophore conjugate, 24 the galactosamine-modified betulin conjugate, 25 and the trivalent galactosamine-modified Cy5 dye 26 showed a similar hepatocytetargeted tendency, also confirming the reliability of our results.…”

Design and synthesis of atorvastatin derivatives with enhanced water solubility, hepatoselectivity and stability

“…The receptor was immobilised on the surface of the CM5 sensor chip, as was reported previously. 18,19 Results are presented as equilibrium dissociation constants ( K D , Table 1). The obtained conjugates demonstrated greater affinity to the ASGPR compared to unmodified atorvastatin.…”

Semisynthetic Derivatives of Pentacyclic Triterpenes Bearing Heterocyclic Moieties with Therapeutic Potential

“…Betulin glycoconjugates obtained as a result of attaching N-acetyl-D-galactosamine fragments to the C-3 or/and C-28 positions in betulin’s molecule also using triazole as a linker were tested in vitro against HepG2 and Huh7 hepatocellular carcinoma cells, PC3 prostate cancer cells, and A549 lung adenocarcinoma cells. The bivalent derivatives, in particular, compound Bet 25, bearing two modified galactosamine fragments at C-3 and C-28, respectively, exerted stronger cytostatic effects against HepG2 cells compared with monovalent glycoconjugates by inducing G0/G1 cell cycle arrest [ 39 ]. In opposition to these findings, Grymel et al, who synthesized a series of betulin glycoconjugates by attaching D-glucose or D-galactose through triazole linkers to the C-3 and/or C-28 positions, reported that the glycoconjugates containing only one sugar unit exhibited equal or lower biological activity compared with the parent compound.…”