Feng-Guang Rong

Strategies for the design and synthesis of boronated nucleic acid and protein components as potential delivery agents for neutron capture therapy

PURPOSE Strategies for the design and synthesis of boronated nucleosides, amino acids, and peptides as potential delivery agents for boron neutron capture therapy (BNCT) are described. METHODS AND MATERIALS For BNCT to be a useful treatment modality, there is a need to design and synthesize nontoxic boron compounds that selectively target tumor cells, accumulate in sufficient amounts (20-30 micrograms 10B/g of tumor) and persist at therapeutic levels for a sufficient time prior to neutron irradiation. Boronated nucleosides, amino acids and peptides are such promising target compounds. Such structures may be selectively used by proliferating neoplastic cells compared with mitotically less active normal cells and therefore achieve the tissue differentials necessary for BNCT. RESULTS The rationale for synthesis of boronated nucleic acid and protein components is discussed. Results of biological and clinical studies of some boronated nucleosides, nucleotides, amino acids and peptides are presented. CONCLUSION Boronated nucleosides, amino acids and peptides can be considered as potential targeting agents for BNCT.

Identification, development, synthesis and evaluation of boron-containing nucleosides for neutron capture therapy

Abstract The development of boron compounds with the capacity for selectively targeting tumor cells would offer the potential for specifically destroying such cells using the capture reaction of the nonradioactive 10 B nuclide and thermal neutrons. The key problem is the development of compounds with the ability to discriminate between tumor cells and contiguous normal cells and to concentrate in the former at suitable concentration levels. One category of agents that has been explored is boron-containing nucleosides. Such recent structures have been biochemically converted in vitro to their corresponding nucleotides by the action of human thymidine kinase. These studies have attempted to correlate a compound’s physiochemical properties with its biochemical attributes. Since only a fraction of cells are undergoing replication at any one time, requiring the need for nucleic acid precursors, such boron compounds must be only one component of a cocktail of agents that are targeting malignant cells. This presentation is selective, focusing on those boron-containing nucleosides that have been designed for studies with kinases.

Synthesis of 5-Tethered Carborane-Containing Pyrimidine Nucleosides as Potential Agents for DNA Incorporation

Abstract Several 5-substituted-2′-deoxyuridines have been prepared in which the carborane moiety is attached at the terminus of a flexible hydrocarbon chain containing an ester linkage. These boron moieties as the B-10 enriched compounds have potentiality for use in the treatment of cancer by means of boron neutron capture therapy. A convenient synthetic route, in high yield, has been developed for the preparation of these 5-tethered carborane-containing pyrimidine nucleosides.

Synthesis and Biochemical Activity of 5-Tethered Carborane-Containing Pyrimidine Nucleosides as Potential Agents for DNA Incorporation

Abstract Synthesis and biochemical evaluation of a series of 5-tethered carborane-containing pyrimidine nucleosides have been undertaken. The biochemical studies with human TK demonstrated that these compounds can be phosphorylated to the corresponding 5′-monophosphate and that the most significant conversion occurred when seven atoms were inserted between the carborane cage and the pyrimidine base, and of these the best was the one with saturated ester.

Synthesis and Biochemical Activity of Hydrophilic Carborane-Containing Pyrimidine Nucleosides as Potential Agents for DNA Incorporation and BNCT

Abstract A novel type of hydrophilic 5-tethered carborane-containing 2′-deoxyuridine derivative, SUB-7-DIOL, has been developed. This compound is a potential agent for DNA incorporation and BNCT, because it showed satisfactory aqueous solubility and demonstrated an excellent rate of phosphorylation by human thymidine kinase. This study also demonstrated the importance of tethering a flexible hydrocarbon chain between the carborane cage and the 5-position of the nucleoside, and the effectiveness of a water-solubilizing moiety attached to the carbon atom of the lipophilic carborane cage.

Evaluation of Carboranyl 2′-Deoxyuridine Derivatives as Substrates for Human Thymidine Kinases 1 and 2

Cellular nucleoside kinases play a pivotal role in the use of nucleosides for cancer and antiviral therapy.1 For BNCT, the cytosolic thymidine kinase (TK1) may be a particularly important target enzyme. TK1 activity is present in proliferating cells but is virtually absent from all quiescent cells.2 Boron-containing thymidine derivatives, that are good substrates for TK1, may be entrapped in proliferating neoplastic cells after conversion to the monophosphate. Therefore, such agents may be excellent vehicles for the selective delivery of boron-10 to those compartments of a tumor consisting of viable cells. A substantial number of nucleosides modified with various boron moieties at different positions have been synthesized and evaluated biologically for use in BNCT.3 Some thymidine derivatives were found to be phosphorylated in vitro 4 and in phos-phoryl transfer assays with purified human thymidine kinases.5 In these experiments, the observed rates of phosphorylation were generally low compared to natural nucleosides and did not distinguish between phosphorylation by TK1 and mitochondr-ial thymidine kinase (TK2) which appears to be equally active in proliferating and non-proliferating cells, thus providing no basis for selective uptake of boronated nucleosides in tumor cells.6

Synthesis and Biochemical Evaluation of 5-Tethered Boron-Containing Pyrimidine Nucleosides for BNCT

In order for BNCT to be successful, a major need is the development of boron compounds, which penetrate the blood-brain barrier (BBB), target tumor cells selectively, and maintain a relatively high concentration of boron-10 in the tumor, ca. 30 ug/g of tumor tissue, vis-a-vis surrounding normal tissues. Boron-containing nucleosides that use a carrier-mediated transport process for penetrating the CNS and have the potential for becoming incorporated into proliferating cells may be useful agents for BNCT.1 This fact led to the attempts to synthesize carborane-containing nucleoside derivatives. Yamamoto2 and Schinazi3 have attached the carborane moiety to the 5-position on the pyrimidine nucleosides, either directly or through a bulky substituent. Unfortunately, these compounds are very poor substrates for nucleoside kinases, and are unlikely to demontrate the requisite biological activity. This may be due to the fact that having a bulky substituent, like the carborane moiety, attached directly on the 5-position may inhibit enzymatic conversion of the 5-carboranyl-2′-deoxyuridine to their corresponding nucleotides. The formation of the latter is a necessary precondition for the incorporation of such nucleotides into DNA.

Synthesis of 5-S-Alkylcarboranyl-2′-Deoxyuridines

Since glioma cells are more mitotically active than normal brain cells, they have an increased demand for nucleic acids to synthesize DNA. Several boronated nucleosides have been synthesized to take advantage of this differential to selectively target boron to brain tumors for treatment by BNCT.1 5-Methylmercapto-2′-deoxyuridine has been reported to be phosphorylated2 and incorporated into DNA3 at comparable, if not greater, levels than thymidine. On this basis, we have designed the 5-S-alkyl carboranyl deoxyuridines 1–5 (Figure 1). Our objective was to not only synthesize boron-containing nucleosides but compounds that would use the enzyme systems that act on the natural nucleoside, thymidine. If the nucleoside portion of these compounds undergoes normal cellular metabolism, a triphosphate will be produced that can ultimately become incorporated into the cell’s DNA. Such subcellular localization of boron within the cell’s nucleus would increase the RBE of the neutron capture reaction by at least two fold.4 The nucleosides 1–5 were designed with a long, flexible hydrocarbon tether between the carborane and nucleoside portions unlike other boronated nucleosides which have positioned the carborane immediately next to nucleoside component.1 The positioning of such bulky groups immediately attached to the nucleoside may interfere with proper binding to kinases, the enzymes that phosphorylate the nucleoside. Addition of the tether, however, projects the bulky boron moiety away from the nucleoside component thereby decreasing steric interference and allowing better binding. The concept of using a tether has proven useful in the application of affinity chromatography in which the binding of enzymes to substrates covalently linked to a solid support was increased when an appropriate tether length between the ligand and support matrix was used.5 The synthesis of 1–3 and a preliminary in vitro evaluation of 1 is presented in this paper. It is our objective to use the in vitro phosphorylation assay presented to direct our synthetic efforts. The assay also serves as a screening method to select the best nucleoside candidate for future cell culture studies and in vivo assays.