Abul K. M. Anisuzzaman

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.

Synthesis of a carboranyl nucleoside for potential use in neutron capture therapy of cancer

Abstract The reaction of 2′,3′-O-(dibutylstannylene)uridine (1) with 3-bromopropyne yielded 2′,(3′)-O-(3-propynyl)uridine (2 and 3). The mixture of 2 and 3 on acetylation followed by chromatographic purification yielded 3′,5′-di-O-acetyl-2′-O-(3-propynyl) uridine (4). Reaction of 4 with bis(acetonitrile)decaborane produced 3′,5′-di-O-acetyl-2′-O-(o-carboran-l-ylmethyl)uridine (5). Deacetylation of 5 gave 2′-O-(o-carboran-l-ylmethyl) uridine (6), a boronated nucleoside for potential use in neutron capture therapy of cancer.

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 1,3-Dl-O-Acetyl-5-O-Benzoyl-2-O -(O-Carboran-1-Ylmethyl)- D-Ribofuranose. A General Precursor for the Preparation of Carborane-Containing Nucleosides for Boron Neutron Capture Therapy

Abstract An eight-step synthesis of 1,3-di-O-acetyl-5-O-benzoyl-2-O-(o-carboran-1-ylmethyl)-D-ribofuranose 9 was carried out from 1,2:5,6-O-isopropylidene-α-D-allofuanose 1. Condensation of 9 with trimethylsilyl protected uracil in the presence of trimethylsilyl trifluoro-methanesulfonate, and subsequent deblocking of the resulting 1-[3-O-acetyl-5-O-benzoyl-2-O-(o-carboran-1-ylmethyl)-D-ribofuranosyl]uracil 10 (>95& β-configuration) by alkaline hydrolysis, yielded 1-[2-O-(o-carboran-1-ylmethyl)-β-D-ribofuranosyl]uracil 11.

Tumor Targeting Agents for Neutron Capture Therapy

The development of compounds for use as delivery agents in the treatment of cancer by neutron capture therapy has largely focused on the use of boron-10 even though there are other nuclides having higher capture cross section values for thermal neutrons. The major reasons are: (1) it is nonradioactive and comprises approximately 20% of naturally occurring boron; (2) boron-10 enriched materials are readily available commercially; (3) the products of the fission reaction are largely high LET recoiling 7Li and 4He (alpha) nuclei; (4) the pathlength of these particles is such that they are confined to a radius approximating the diameter of a single cell; and (5) the chemistry of boron is such that it may be incorporated into a multitude of different stable chemical structures.

Carboranyl Precursors of Nucleic Acids--Potential DNA Probes for BNCT

It has been determined that the biological effectiveness of BNCT will be maximized if the capture reaction were to occur in the cell nucleus in comparison with the cytoplasm, on cell membrane or in extracellular spaces.1–2 This has been the basis for the ongoing synthesis of boron compounds which are chemically similar to the building blocks of the nucleic acids. Initially, the approach concentrated on the preparation of purine and pyrimidine bases which contain boron.3–7 The rationale was that such structures might emulate the naturally-occurring purine and pyrimidine bases and become more selectively incorporated into tumor cell nuclei in comparison to normal cells due to the higher proliferative rates of the former. Of the different boron compounds which were initially synthesized, many were unstable, toxic or failed to become incorporated into nucleic acids. One structure which resembled more closely its natural counterpart was 5-dihydroxyboryluracil.8 Its synthesis encouraged Schinazi and Prusoff to prepare the first boron-containing pyrimidine nucleoside.9

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 of Carborane-Containing Nucleosides, Nucleotides, and Carbohydrates for Bnct

The radiobiological effectiveness of the neutron capture reaction is 2-5 times greater when it occurs in the cell nucleus rather than in the cytoplasm.1,2 Boronated nucleic acid precursors and components may be suitable agents to achieve high selective boron concentrations in the nucleus of tumor cells.

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.

Determination of Boron Concentration by Means of Direct Current Plasma - Atomic Emission Spectroscopy

The accurate measurement of total boron content in biological samples with a sensitivity in the ppm range is essential for evaluating the potential usefulness of various tumor localizing boron-containing compounds for Boron Neutron Capture Therapy (BNCT)1. Among the procedures that have been used are spectrophotometric analyses involving various complexing agents2–4. These methods are time consuming and require that the boron compounds can be oxidized to boric acid. Relatively low sensitivity and interference from various contaminants further limit their usefulness. Recently, inductively coupled plasma-atomic emission spectroscopy (ICP-AES) has been shown to be sensitive enough for the detection of microgram quantities of boron in biological samples5,6, although the sensitivity is adversely affected by high concentrations of inorganic salts in the samples7. Since alkaline fusion is not suitable for use with ICP8, samples were digested by exposure to either perchloric acid5,6 or nitric acid6. There may be a danger of explosion with the former, and with the latter, it may be necessary to decompose the tissues in teflon-lined digestion bombs. Both procedures, therefore, have their limitations. The objectives of the present study were to determine whether DCPAES could be used to quantify boron in a variety of chemical compounds including polyhedral boranes and carboranes, to improve the procedure for the digestion of tissue samples, to determine if this method could be used to quantify cellular uptake of boron, and finally to define the limits of boron detection in biologic samples by means of this method.