Synthesis and Evaluation of Folate-Conjugated Phenanthraquinones for Tumor-Targeted Oxidative Chemotherapy

Abstract

Almost all cells are easily killed by exposure to potent oxidants. Indeed, major pathogen defense mechanisms in both animal and plant kingdoms involve production of an oxidative burst, where host defense cells show an invading pathogen with reactive oxygen species (ROS). Although cancer cells can be similarly killed by ROS, development of oxidant-producing chemotherapies has been limited by their inherent nonspecificity and potential toxicity to healthy cells. In this paper, we describe the targeting of an ROS-generating molecule selectively to tumor cells using folate as the tumor-targeting ligand. For this purpose, we exploit the ability of 9,10-phenanthraquinone (PHQ) to enhance the continuous generation of H₂O₂ in the presence of ascorbic acid to establish a constitutive source of ROS within the tumor mass. We report here that incubation of folate receptor-expressing KB cells in culture with folate-PHQ plus ascorbate results in the death of the cancer cells with an IC₅₀ of ~10 nM (folate-PHQ). We also demonstrate that a cleavable spacer linking folate to PHQ is significantly inferior to a noncleavable spacer, in contrast to most other folate-targeted therapeutic agents. Unfortunately, no evidence for folate-PHQ mediated tumor regression in murine tumor models is obtained, suggesting that unanticipated impediments to generation of cytotoxic quantities of ROS in vivo are encountered. Possible mechanisms and potential solutions to these unanticipated results are offered.

Keywords: Cancer; Folate Receptor; Reactive Oxygen Species.

Figure 1

Oxygen consumption traces in air-saturated solutions containing 10 μM quinone and 1.00 mM ascorbate in 20 mM phosphate buffer at pH 7.4 and 37°C. The arrow indicates the time when ascorbate was injected. The numbers labeling the curves identify the quinone used to promote redox cycling.

Figure 2

Effect of different combinations of ascorbate +/− various quinones on growth of FR+ tumor xenografts in athymic nude mice. (a) KB tumor implantation: 1 million KB cells were injected subcutaneously in 0.1 ml FD medium/mouse. Treatment began after the 20th day of tumor implantationwhen the average tumor size was 100 - 350 mm³. The controls are untreated mice. 3 was i.v. injected at a dose of 2.0 μmol/kg, 3 days/week for two weeks. Sodium ascorbate was injected intraperitoneally at a dose of 4 g/kg or 80 mg per mouse 30 min after i.v. injection of 3 or 11.8 mg per mouse every 30 min for 90-min, 30 min after i.v. injection of 3; (b) FR+ MDA-MB-231 tumor implantation: 1 million FR+ MDA-MB-231 cells were injected subcutaneously in 0.1 ml FD medium/mouse. Treatment began after the 12th day of tumor implantation when the average tumor size was 75 - 100 mm³. The controls are untreated mice. 3 was i.v. injected at a dose of 4.0 μmol/kg 3 days/week for two weeks. Sodium ascorbate was injected i.p. (4 g/kg or 80 mg/mouse) 30 min after 3 was injected and also on days when mice were not injected with 3. Tumor growth % values are averages ± standard errors of the mean at least four determinations.

Scheme 1

Synthesis of 3-carboxyllic phenanthrenequinone.

Scheme 2

Synthesis of 3: reagents and conditions: (a) 3-carboxy-9,10-phenanthraquinone, PyBOP, DIPEA, DMF; (b) (i) 20% piperidine, DMF, (ii) Fmoc-Glu(OtBu)-OH, PyBOP, DIPEA, DMF; (c) (i) 20% piperidine, DMF, (ii) N(10)-TFA-pteroic acid, PyBOP, DIPEA, DMF; d) (i) TFA:TIPS:water (95:2.5:2.5:), (ii) sat.Na₂CO₃, pH = 10 - 11, 30 min.

Scheme 3

Synthesis of 2. Reagents and conditions: (a) (i) HOBt, DCC, dioxane, (ii) BocNH(CH₂)₂NH₂, Et₃N, CH₂Cl₂; (b) HCl, CH₃COOH.

Scheme 4

Synthesis of 5.

Scheme 5

Synthesis of 6: reagents and conditions: (a) (i) 20% piperidine/DMF, (ii) Fmoc-Glu(O^tBu)-OH, PyBOP, DIPEA/DMF; (b) (i) 20% piperidine/DMF, (ii) α-protected Fmoc-Glu(O^tBu)-OH, PyBOP, DIPEA/DMF; (c) (i) 20% piperidine/DMF, (ii) N¹⁰TFA-Pteroic acid, PyBOP, DIPEA/DMF; (d) (i) 2% NH₂NH₂/DMF, (ii) TFA:TIPS:Water:EDT (92.5:2.5:2.5:2.5).

Scheme 6

Synthesis of 4: (a) 5, THF:H₂O (1:1), pH = 7.0, argon bubbling, rt, 45 min.