Studies directed toward the asialoglycoprotein receptor mediated delivery of 5-fluoro-2′-deoxyuridine for hepatocellular carcinoma

Article abstract of DOI:10.1016/j.bmcl.2018.06.002

The anticancer nucleoside 5-fluoro 2′-deoxyuridine-5′-phosphate (5-FdU-P) was attached via an amide chain linker to a triantennary GalNAc cluster as a means to deliver the drug to hepatic cells that recognize the amino sugar units.

Full text of DOI:10.1016/j.bmcl.2018.06.002

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Studies directed toward the asialoglycoprotein receptor mediated delivery
of 5-fluoro-2′-deoxyuridine for hepatocellular carcinoma
Lorena Ricoa, Michael E. Østergaardb, Melanie Bellc, Punit P. Sethb, Stephen Hanessiana,⁎
a
Department of Chemistry, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada
Department of Medicinal Chemistry, Ionis Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA
Department of Antisense Drug Discovery, Ionis Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
The anticancer nucleoside 5-fluoro 2′-deoxyuridine-5′-phosphate (5-FdU-P) was attached via an amide chain
linker to a triantennary GalNAc cluster as a means to deliver the drug to hepatic cells that recognize the amino
sugar units.
Liver cancer
Drug delivery
Nucleoside
GalNAc cluster
Phosphoramidite
Hepatocellular carcinoma (HCC) is responsible for almost a third of
cancer-related deaths globally.1 Current treatment options include liver
resection, transplantation, radiofrequency ablation and sorafenib, a
multikinase inhibitor.2 However, treatment benefits are limited and
newer more effective modalities are needed. 5-Fluoro-2′-deoxyuridine
(5-FdU) is a nucleoside anticancer agent but its efficacy is limited by
inefficient phosphorylation by kinases.3 Peng and coworkers recently
employed phosphoramidate pro-drugs of 5-FdU to enhance potency and
hepatic delivery for the treatment of HCC.4 The phosphoramidate
prodrugs are metabolized to liberate the 5′-phosphorylated nucleotides
within cancer cells and exhibit cytotoxicity by inhibition of thymidylate
synthetase.5
D-galactosamine (GalNAc) sugar residues.11 The receptor is comprised
of two subunits (ASGR1 and ASGR2) which form a hetero-oligomeric
receptor of varying ratios (2–5:1).12 The receptor binds D-galactose and
N-acetyl-D-galactosamine terminated carbohydrate residues with high
affinity.13 The structure-activity relationships of ligand-receptor inter-
actions have been elucidated.14 The ASGR is internalized by clathrin
mediated endocytosis and delivers the cargo to endocytic compart-
ments inside the cell while the receptor is recycled back to the cell-
surface.15
Recently, we reported on a strategy to deliver a prodrug to the liver
by attaching a triantennary GalNAc conjugate to the 5′-end of an oli-
gonucleotide via a phosphodiester linkage.16,17 In the present work, we
relied on the same premise of drug delivery whereby the triantennary
GalNAc 5′-phosphorylated 5dFU nucleoside-linked conjugate is di-
rected to hepatocytes via ASGR-receptor mediated internalization, fol-
lowed by release of 5-FdU upon metabolism.
We hypothesized that the activity of cytotoxic nucleoside drugs like
5-FdU could be enhanced by exploring a ligand based receptor re-
cognition for targeted delivery to the liver (Fig. 1).
Targeted delivery of chemotherapeutic agents presents the oppor-
tunity to enhance potency and therapeutic index by enhancing selective
delivery to cancer cells versus healthy tissues.6 Devarajan and cow-
orkers recently showed that asialoglycoprotein receptor (ASGR)-medi-
ated delivery of doxorubicin nanoparticles showed enhanced efficacy in
HCC models.7 The ASGR has also been recently investigated for the
delivery of nucleic acid based therapeutic agents to liver hepatocytes.8,9
Recently Mascitti and coworkers10 have reported on the efficient tar-
geting of the asialoglycoprotein receptor with a 1,6-anhydro N-acetyl D-
galactosamine ligand in a polyvalent construct. The ASGR receptor is
expressed primarily on hepatocytes and regulates levels of plasma
glycoproteins terminating with sialic acid α2,6-D-galactose and N-acetyl
2′-Deoxy-5-fluoro-uridine 1 was converted to the 3′-benzoate 2.18
Treatment with 2-cyanoethyl diisopropylchlorophosphoramidite and
diisopropylamine19 led to the key coupling partner 5′-phosphoramidite
5-FdU 3 as a 1:1 mixture of P-diastereoisomers as evidenced by 31P
NMR. (δ150.39, 149.98. 1:1 d.r.) (Scheme 1).
Amide formation by treatment of the PFP ester 416 with 6-amino-
hexanol led to 5 as a white solid. Coupling of fragments 5 and 3 using
1H-tetrazole20 led to the corresponding phosphoramidite 6 which was
21
oxidized in situ with I2 to give the corresponding protected 5′-trian-
tennary GalNAc-5-FdU conjugate in 81% yield as a 1:1 mixture of P-
diasteroisomers. Treatment with NH4OH effected global deprotection of
⁎
Corresponding author.
E-mail address: Stephen.hanessian@umontreal.ca (S. Hanessian).
https://doi.org/10.1016/j.bmcl.2018.06.002
Received 26 April 2018; Accepted 1 June 2018
0960-894X/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Rico,L.,Bioorganic&MedicinalChemistryLetters(2018),https://doi.org/10.1016/j.bmcl.2018.06.002

L. Rico et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 1. Conception of a 5′-FdU triantennary GalNAc cluster as a prodrug.
Scheme 1. Synthesis of phosphoramidite 5-FdU 3. Reagents and conditions: a.
(i) TBDPSCl, imidazole, DMF, r.t., 80%, (ii) Bz2O, DMAP, pyridine, r.t, 81% (iii)
TBAF, THF, r.t., 88%, b. [iPr]2NP(Cl)OEtCN, DIPEA, CH2Cl2, r.t., 86%.
acetate esters to yield the intended 5′- triantennary GalNAc-5-FdU
phosphate conjugate 7 (Scheme 2).
Scheme 2. Synthesis of 5′-triantennary GalNAc-5FdU phosphate 7. Reagents
and conditions: a. 6-Amino1-hexanol, CH2Cl2, r.t, 93%, b. 3, 1H-tetrazole,
MeCN, r.t., then, I2/Py/THF, H2O, r.t., 81%, c. NH4OH, r.t.,85%.
Biological assays
HepG2 cells were treated with 7 to evaluate cytotoxicity. 5-FdU
(Fig. 1) was used as a positive control as it has previously been shown to
be cytotoxic in HepG2 cells.22 Cells were treated with varying con-
centrations of 7 or 5-FdU from 1.5 to 200 μM and incubated at 37 °C for
72 h. Cytotoxicity was measured using WST-1, a commercially available
cell viability reagent.
The unconjugated 5-FdU was found to be slightly more potent than
GalNAc conjugated 7 (Fig. 2). The result was in contrast to the data
with GalNAc-ASOs which showed improved potency in cells and in
mice relative to unconjugated ASOs.16,17 The difference in behavior
between the GalNAc-conjugated antisense oligonucleotide versus the
nucleoside in cell culture could be attributed to the differences in cel-
lular uptake pathways between a small molecule versus an anionic
macromolecular cargo. A small molecule such as 5-FdU is capable of
entering cells by passive diffusion versus an oligonucleotide which is
taken up by endocytic pathways. It is conceivable that the delivery of a
small molecule membrane permeable drug to cells in culture is not in
enhanced by receptor-mediated delivery. In contrast, receptor-medi-
ated delivery in an animal could enhance activity by delivering a
greater fraction of the administered dose to the targeted cell-type. This
is often the case with antibody-drug conjugates which typically do not
enhance activity versus the unconjugated drug in cell culture but im-
prove therapeutic index in vivo by delivering the cytotoxic drug to
cancer cells versus healthy tissue.23
Fig. 2. Cytotoxicity of 5-fluoro-2′-deoxyuridine and Tris-GalNac 5-FdU phos-
phate conjugate 7 in HepG2 Cells via WST-1.
Acknowledgments
The University of Montreal group thanks Ionis Pharmaceuticals for
financial support. Support is also acknowledged from NSERC.
A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2018.06.002.
In conclusion, we report the efficient synthesis and biological eva-
luation of 5-FdU phosphate conjugated to triantennary GalNAc for
ASGR-mediated delivery to hepatocellular carcinoma cells. Further
evaluation in hepatic cancer mouse models will determine the validity
of this approach for the enhancing activity of 5-FdU and related ther-
apeutic agents for treatment of liver cancer.
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