Syntheses of L-Rhamnose-Linked Amino Glycerolipids and Their Cytotoxic Activities against Human Cancer Cells

Abstract

A major impediment to successful cancer treatment is the inability of clinically available drugs to kill drug-resistant cancer cells. We recently identified metabolically stable L-glucosamine-based glycosylated antitumor ether lipids (GAELs) that were cytotoxic to chemotherapy-resistant cancer cells. In the absence of commercially available L-glucosamine, many steps were needed to synthesize the compound and the overall yield was poor. To overcome this limitation, a facile synthetic procedure using commercially available L-sugars including L-rhamnose and L-glucose were developed and the L-GAELs tested for anticancer activity. The most potent analog synthesized, 3-amino-1-O-hexadecyloxy-2R-(O-α-L-rhamnopyranosyl)-sn- glycerol 3, demonstrated a potent antitumor effect against human cancer cell lines derived from breast, prostate, and pancreas. The activity observed was superior to that observed with clinical anticancer agents including cisplatin and chlorambucil. Moreover, like other GAELs, 3 induced cell death by a non-membranolytic caspase-independent pathway.

Keywords: L-rhamnose-based glycolipids; caspase independent; chemotherapy resistant; glycosylated antitumor ether lipids.

Figure 1

Reference and newly synthesized compounds used in this study. Compound 1 is reference l-GAEL, Compounds 2–6 are newly synthesized l-sugar-derived GAELs, and compound 7 is reference d-GAEL.

Scheme 1

Synthesis of compounds 2–4. Reagents and conditions: (a) Ac₂O, DMAP, Pyridine, 18 h, rt; (b) PhSH, BF₃·Et₂O, DCM,18 h, rt; (c) AgOTf, NIS, DCM, 3 h, rt; (d) MeONa, MeOH, 1 h; (e) TsCl, Pyridine, DMAP, 0 °C to rt, 18 h; (f) DMF, NaN₃, 70 °C; (g) P(CH₃)₃, THF, H₂O, 2 h, rt; (h) TBTU, DMF, 5 h. Abbreviations: 4-dimethyl amino pyridine (DMAP), dichloromethane (DCM), N,N-dimethylformamide (DMF), room temperature (rt), N-iodosuccinimide (NIS).

Scheme 2

Synthesis of compound 5. Reagents and conditions: (a) Ac₂O, DMAP, Pyridine, 18 h, rt; (b) PhSH, BF₃·Et₂O, DCM,18, h, rt; (c) AgOTf, NIS, DCM, 3 h, rt; (d) MeONa, MeOH, 1 h; (e) P(CH₃)₃, THF, H₂O, 2 h, rt; (h) TBTU, DMF, argon, 5 h. Abbreviations: 4-dimethyl amino pyridine (DMAP), dichloromethane (DCM), N,N-dimethylformamide (DMF), room temperature (rt), N-iodosuccinimide (NIS).

Scheme 3

Synthesis of compound 6. Reagents and conditions: (a) Ac₂O, DMAP, Pyridine, 18 h, rt; (b) PhSH, BF₃·Et₂O, DCM,18, h, rt; (c) MeONa, MeOH, 1 h; (d) TsCl, Pyridine, DMAP, 0 °C to rt, 18 h; (e) DMF, NaN₃, 70 °C; (f) AgOTf, NIS, DCM, 3 h, rt; (g) P(CH₃)₃, THF, H₂O, 2 h, rt. Abbreviations: 4-dimethyl amino pyridine (DMAP), dichloromethane (DCM), N,N-dimethylformamide (DMF), room temperature (rt), N-iodosuccinimide (NIS).

Figure 2

Effects of compounds 2–6 on the viability of MDA-MB-231, JIMT-1, DU-145, and MiaPaCa2 cells lines. Twenty four hours after plating, cells were incubated with compounds 2–6 (0–30 µM) for 48 h. Cell viability relative to vehicle control was measured by MTS assay. The results represent the mean ± standard deviation of 6 independent determinations.

Figure 3

Dose-dependent alteration of cell viability. Human breast adenocarcinoma cell lines were treated with increasing concentrations of 3. Twenty four hours after plating, cells were incubated with 0–15 µM 3 for 48 h. Cell viability relative to vehicle control was measured by MTS assay. The results represent the mean ± standard deviation of 6 independent determinations.

Figure 4

Demonstration of caspase/apoptosis independent cell death. DU-145 or JIMT-1 cell lines were treated with increasing concentrations of 3 or Adriamycin in the presence or absence of 40 µM QVD. Four hours after plating, cells were treated with QVD. After 20 h, the cells were incubated with 0–15 µM 3 or 0–4 µM Adriamycin for 48 h. Cell viability relative to vehicle control was measured by MTS assay. The results represent the mean ± standard deviation of 6 independent determinations.

Figure 5

Evaluation of effect of compound 3 on cell membrane of DU-145 and JIMT-1 cell lines using cell impermeant ethidium homodimer-1 (EthD-1) dye that emits red fluorescence upon binding to DNA.

Figure 6

Hemolytic properties of GAEL 3 using freshly isolated ovine erythrocytes. 1% ammonium hydroxide was used for 100% hemolysis, the control. The values were determined as % of control.