Exploring a Gemcitabine-Glucose Hybrid as a Glycoconjugate Prodrug

Abstract

Nucleoside analogues are established treatments for cancer and viral infection. Gemcitabine is a commonly employed nucleoside analogue displaying anticancer properties against a range of tumor types but is rapidly inactivated in vivo. Efforts to bolster its pharmaceutical profile include investigating prodrug forms. Herein, we explore the synthesis of a novel glucose-gemcitabine glycoconjugate, targeting uptake via glucose transport. We select a redox-reactive disulfide linker for conjugation of gemcitabine (through N4-cytosine) with glucose. Evaluation of this glycoconjugate reveals increased toxicity against androgen insensitive PC3 prostate cancer cells compared to LNCaP (which have lower levels of glucose transporter GLUT1). These preliminary results suggest that glycoconjugation of nucleosides may be an effective approach to targeting cells which display increased uptake and metabolism of glucose.

Figure 1

(a) Targeting nucleoside analogue transport into cells using a glucose-nucleoside analogue glycoconjugate. (b) Glycoconjugate design based upon a responsive cellular trigger to release both glucose and drug via a self-immolative linker.

Scheme 1. Initial Gemcitabine-Glucose Conjugate Design

Upon intracellular disulfide reduction, gemcitabine 4 and benign by-products 2 and 3 are released.

Scheme 2. Synthesis of Protected Glucose Ligand 10

Reagents and conditions: (i) I₂, PPh₃, imidazole, THF, 70 °C; (ii) Ac₂O, pyridine, 35 °C (76%, 2 steps); (iii) KSAc, acetone, reflux, 70%; (iv) NIS, I₂, MeCN, 77%; (v) H₂SO₄, AcOH, Ac₂O, 68%; (vi) DTT, MeCN; (vii) TCCA, THF, NaH, −20 °C to RT, 3-MPA (51%, 2 steps).

Scheme 3. Attempted Synthesis of Glycoconjugate 1

Reagents and conditions: (i) TBSCl, imidazole, DMF, 86%; (ii) EDC, DMAP, DCM, 10, 65%.

Figure 2

HSQC NMR (400 × 100 MHz, CDCl₃) data for glycoconjugate 12, highlighting anomeric shifts for each glucose anomer and the nucleoside.

Scheme 4. Toward Glycoconjugate 19via Thiol–Pyridyl Disulfide Exchange

Reagents and conditions: (a) previously reported synthesis of disulfide conjugate 14;(b) (i) EDC, DMAP, DCM, 80%; (ii) TBAF, THF, 0 °C, 66%; (c) (iii) AcOH, H₂SO₄, RT, 62%; (iv) Na, MeOH, RT, 85%; (d) unsuccessful pyridyl disulfide thiol exchange reaction.

Scheme 5. Synthesis of Glycoconjugate 23

Reagents and conditions: (i) 4-mercaptobutyric acid, MeCN, RT, 61%; (ii) EDC, DMAP, RT, 45%; (iii) TBAF, THF, 0 °C, 54%.

Figure 3

MTS cell viability assay measuring the formazan product formed at 490 nm in metabolically active cells following treatment of PC3 or LNCaP cells with varying concentrations of 23 for 72 h.