Boron-containing folate receptor-targeted liposomes as potential delivery agents for neutron capture therapy

Boron neutron capture therapy (BNCT) depends on the selective delivery of a sufficient number of (10)B atoms to tumor cells to sustain a lethal (10)B(n,alpha)(7)Li reaction. Expression of FR frequently is amplified among human tumors. The goal of the present study was to investigate folate receptor (FR)-targeted liposomes as potential carriers for a series of boron-containing agents. Two highly ionized boron compounds, Na(2)[B(12)H(11)SH] and Na(3) (B(20)H(17)NH(3)), were incorporated into liposomes by passive loading with encapsulation efficiencies of 6% and 15%, respectively. In addition, five weakly basic boronated polyamines were investigated. Two were the spermidine derivatives: N(5)-(4-carboranylbutyl)spermidine.3HCl (SPD-5), N(5)-[4-(2-aminoethyl-o-carboranyl)butyl]spermidine.4HCl (ASPD-5). Three were the spermine derivatives: N(5)-(4-carboranylbutyl)spermine.4HCl (SPM-5), N(5)-[4-(2-aminoethyl-o-carboranyl)butyl]spermine.5HCl (ASPM-5), and N(5),N(10)-bis(4-carboranylbutyl)spermine.4 HCl (SPM-5,10). These were incorporated into liposomes by a pH-gradient-driven remote-loading method with varying loading efficiencies, which were influenced by the specific trapping agent and the structure of the boron compound. Greater loading efficiencies were obtained with lower molecular weight boron derivatives, using ammonium sulfate as the trapping agent, compared to those obtained with sodium citrate. The in vitro uptake of folate-derivatized, boronated liposomes was investigated using human KB squamous epithelial cancer cells, which have amplified FR expression. Higher cellular boron uptake (up to 1584 microg per 10(9) cells) was observed with FR-targeted liposomes than with nontargeted control liposomes (up to 154 microg per 10(9) cells), irrespective of the chemical form of the boron and the method used for liposomal preparation. KB cell binding of the FR-targeted liposomes was saturable and could be blocked by 1 mM free folic acid. Our findings suggest that further evaluation of FR-targeted liposomes is warranted to assess their potential as boron carriers for neutron capture therapy.     

Synthesis and in vivo biodistribution of BPA-Gd-DTPA complex as a potential MRI contrast carrier for neutron capture therapy

p-boronophenylalanine (BPA) conjugated Gd-DTPA complex (3) was synthesized from the active methyne compound 6, the allylic carbonate 7, and BPA by the palladium-catalyzed allylation reaction followed by the DCC coupling reaction. The in vivo biodistribution of complex 3 was evaluated by prompt gamma-ray analysis and alpha-autoradiography using the tumor-bearing rats. High accumulation of gadolinium was observed in the kidney and the %ID values were 0.17 and 0.088 at 20 and 60 min after injection of 3, respectively. The accumulation was also observed in the tumor and the %ID values were 0.010 and 0.0025 at 20 and 60 min after injection, respectively. The visualization experiment of boron distribution in the tumor-bearing rat by alpha-autoradiography indicates that boron was accumulated in the tumor and the intestines at 20 min after injection.     

Boronated dipeptide borotrimethylglycylphenylalanine as a potential boron carrier in boron neutron capture therapy for malignant brain tumors.

Takagaki, M., Ono, K., Masunaga, S-I., Kinashi, Y., Oda, Y., Miyatake, S-I., Hashimoto, N., Powell, W., Sood, A. and Spielvogel, B. F. Boronated Dipeptide Borotrimethylglycylphenylalanine as a Potential Boron Carrier in Boron Neutron Capture Therapy for Malignant Brain Tumors. Radiat. Res. 156, 118-122 (2001).A boronated dipeptide, borotrimethylglycylphenylalanine (BGPA), was synthesized as a possible boron carrier for boron neutron capture therapy (BNCT) for malignant brain tumors. In vitro, at equal concentrations of (10)B in the extracellular medium, BGPA had the same effect in BNCT as p-boronophenylalanine (BPA). Boron analysis was carried out using prompt gamma-ray spectrometry and track-etch autoradiography. The tumor:blood and tumor:normal brain (10)B concentration ratios were 8.9 +/- 2.1 and 3.0 +/- 1.2, respectively, in rats bearing intracranial C6 gliosarcomas using alpha-particle track autoradiography. The IC(50), i.e. the dose capable of inhibiting the growth of C6 gliosarcoma cells by 50% after 3 days of incubation, was 5.9 x 10(-3) M BGPA, which is similar to that of 6.4 x 10(-3) M for BPA. The amide bond of BGPA is free from enzymatic attack, since it is protected from hydrolysis by the presence of a boron atom at the alpha-carbon position of glycine. These results suggest promise for the use of this agent for BNCT of malignant brain tumors. Further preclinical studies of BGPA are warranted, since BGPA has advantages over both BPA and BSH.     

Boron neutron capture synovectomy (BNCS) as a potential therapy for rheumatoid arthritis: boron biodistribution study in a model of antigen-induced arthritis in rabbits

Boron neutron capture synovectomy (BNCS) is explored for the treatment of rheumatoid arthritis (RA). The aim of the present study was to perform boron biodistribution studies in a model of antigen-induced arthritis (AIA) in female New Zealand rabbits, with the boron carriers boronophenylalanine (BPA) and sodium decahydrodecaborate (GB-10) to assess the potential feasibility of BNCS for RA. Rabbits in chronic phase of AIA were used for biodistribution studies employing the following protocols: intra-articular (ia) (a) BPA-f 0.14 M (0.7 mg ¹⁰B), (b) GB-10 (5 mg ¹⁰B), (c) GB-10 (50 mg ¹⁰B) and intravenous (iv), (d) BPA-f 0.14 M (15.5 mg ¹⁰B/kg), (e) GB-10 (50 mg ¹⁰B/kg), and (f) BPA-f (15.5 mg ¹⁰B/kg) + GB-10 (50 mg ¹⁰B/kg). At different post-administration times (13–85 min for ia and 3 h for iv), samples of blood, pathological synovium (target tissue), cartilage, tendon, muscle, and skin were taken for boron measurement by inductively coupled plasma mass spectrometry. The intra-articular administration protocols at <40 min post-administration both for BPA-f and GB-10, and intravenous administration protocols for GB-10 and [GB-10 + BPA-f] exhibited therapeutically useful boron concentrations (>20 ppm) in the pathological synovium. Dosimetric estimations suggest that BNCS would be able to achieve a therapeutically useful dose in pathological synovium without exceeding the radiotolerance of normal tissues in the treatment volume, employing boron carriers approved for use in humans. Radiobiological in vivo studies will be necessary to determine the actual therapeutic efficacy of BNCS to treat RA in an experimental model.     

Synthesis and biological evaluation of folate receptor-targeted boronated PAMAM dendrimers as potential agents for neutron capture therapy

Successful treatment of cancer by boron neutron capture therapy (BNCT) requires the selective delivery of (10)B to constituent cells within a tumor. The expression of the folate receptor is amplified in a variety of human tumors and potentially might serve as a molecular target for BNCT. In the present study we have investigated the possibility of targeting the folate receptor on cancer cells using folic acid conjugates of boronated poly(ethylene glycol) (PEG) containing 3rd generation polyamidoamine dendrimers to obtain (10)B concentrations necessary for BNCT by reducing the uptake of these conjugates by the reticuloendothelial system. First we covalently attached 12-15 decaborate clusters to 3rd generation polyamidoamine dendrimers. Varying quantities of PEG units with varying chain lengths were then linked to these boronated dendrimers to reduce hepatic uptake. Among all prepared combinations, boronated dendrimers with 1-1.5 PEG(2000) units exhibited the lowest hepatic uptake in C57BL/6 mice (7.2-7.7% injected dose (ID)/g liver). Thus, two folate receptor-targeted boronated 3rd generation polyamidoamine dendrimers were prepared, one containing approximately 15 decaborate clusters and approximately 1 PEG(2000) unit with folic acid attached to the distal end, the other containing approximately 13 decaborate clusters, approximately 1 PEG(2000) unit, and approximately 1 PEG(800) unit with folic acid attached to the distal end. In vitro studies using folate receptor (+) KB cells demonstrated receptor-dependent uptake of the latter conjugate. Biodistribution studies with this conjugate in C57BL/6 mice bearing folate receptor (+) murine 24JK-FBP sarcomas resulted in selective tumor uptake (6.0% ID/g tumor), but also high hepatic (38.8% ID/g) and renal (62.8% ID/g) uptake, indicating that attachment of a second PEG unit and/or folic acid may adversely affect the pharmacodynamics of this conjugate.     

Site-specific conjugation of boron-containing dendrimers to anti-EGF receptor monoclonal antibody cetuximab (IMC-C225) and its evaluation as a potential delivery agent for neutron capture therapy

The gene encoding EGFR often is amplified in human gliomas, and the receptor itself has been considered as a potential target for the specific delivery of therapeutic agents to brain tumors. The purpose of the present study was to investigate the use of the chimeric MoAb cetuximab (IMC-C225), which is directed against EGFR and EGFRvIII, as a boron delivery agent for neutron capture therapy (NCT) of brain tumors. As determined by 125I-cetuximab radioligand binding assays, F98 rat glioma cells, which had been transfected with the gene encoding EGFR (F98EGFR), expressed 1.60 +/- 0.13 x 10(5) receptor sites/cell with a Ka = 1.64 +/- 0.32 x 10(8) M-1). F98 cells transfected with the gene encoding a mutant form of EGFR, designated the F98EGFRvIII glioma, expressed 1.07 +/- 0.10 x 10(5) receptor sites/cell with a Ka = 2.18 +/- 0.54 x 10(9) M-1 compared to background levels expressed on F98 wild-type cells (F98WT). A heavily boronated, fifth generation polyamidoamine (PAMAM or "starburst") dendrimer, G5-B1100, was linked to oligosaccharide moieties, which were distant from antigen binding sites of cetuximab, by means of the heterobifunctional reagents N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and N-(k-maleimidoundecanoic acid) hydrazide (KMUH). The resulting bioconjugate, designated C225-G5-B1100, was separated from the unconjugated dendrimer using a Sephacryl S-300 column. On the basis of the relative concentration ratios of boron and protein, there were approximately 1100 boron atoms per molecule of cetuximab with only a slight reduction of Ka. The localization of C225-G5-B1100 or G5-B1100 in rats bearing intracerebral implants of either F98EGFR or F98WT gliomas was determined 24 h following direct intratumoral (i.t.) injection at which time 92.3 +/- 23.3 micrograms B/g tumor was localized in F98EGFR gliomas versus 36.5 +/- 18.8 micrograms B/g tumor in F98WT gliomas and 13.4 +/- 6.1 micrograms in normal brain. In contrast, only 6.7 +/- 3.6 micrograms B/g tumor of G5-B1100 was localized in F98EGFR gliomas following i.t. injection, thereby demonstrating specific molecular targeting of EGFR. Based on these data, BNCT studies will be initiated in F98EGFR glioma bearing rats to evaluate C225-G5-B1100 for the treatment of intracerebral brain tumors.     

Receptor-targeted liposomal delivery of boron-containing cholesterol mimics for boron neutron capture therapy (BNCT)

Liposomes have been a main focus of tumor-selective boron delivery strategies in boron neutron capture therapy (BNCT), a binary method for the treatment of cancer that is based on the nuclear reaction between boron atoms and low-energy thermal neutrons. Three novel carboranyl cholesterol derivatives were prepared as lipid bilayer components for the construction of nontargeted and receptor-targeted boronated liposomes for BNCT. A major structural feature of these novel boronated cholesterol mimics is the replacement of the B and the C ring of cholesterol with a carborane cluster. Computational analyses indicated that all three boronated compounds have structural features and physicochemical properties that are very similar to those of cholesterol. One of the synthesized boronated cholesterol mimics was stably incorporated into non-, folate receptor (FR)-, and vascular endothelial growth factor receptor-2 (VEGFR-2)-targeted liposomes. No major differences were found in appearance, size distribution, and lamellarity between conventional dipalmitoylphosphatidylcholine (DPPC)/cholesterol liposomes, nontargeted, and FR-targeted liposomal formulations of this carboranyl cholesterol derivative. FR-targeted boronated liposomes were taken up extensively in FR overexpressing KB cells in vitro, and the uptake was effectively blocked in the presence of free folate. In contrast, a boronated cholesterol mimic incorporated into nontargeted liposomes showed significantly lower cellular uptake. There was no apparent in vitro cytotoxicity in FR overexpressing KB cells and VEGFR-2 overexpressing 293/KDR cells when these were incubated with boronated FR- and (VEGFR-2)-targeted liposomes, respectively, although the former accumulated extensively in KB cells and the latter effectively interacted with VEGFR-2 by causing autophosphorylation and protecting 293/KDR cells from SLT (Shiga-like toxin)-VEGF cytotoxicity.     

Hyaluronan as carrier of carboranes for tumor targeting in boron neutron capture therapy

Boron neutron capture therapy (BNCT) represents a promising approach for tumor therapy. A critical requirement for BNCT is tumor targeting, a goal that is currently addressed with the development of low and high molecular weight agents capable of interacting with receptors expressed by cancer cells. Here, we describe a new bioconjugate (HApCB) composed by n-propyl carborane linked to hyaluronan (HA) via an ester linkage for a degree of substitution of approximately 30%, leading to a water-soluble derivative. The structure and main physicochemical characteristics of the new HA derivative were determined by means of Fourier transform infrared, fluorescence, and 1H, 13C, and 10B NMR analysis and are herein reported in detail. As HA is recognized by the CD44 antigen, densely populating the surface of many tumor cells, HApCB is expected to deliver boron atoms from the locally released carborane cages directly to target cells for antitumor application in BNCT. In vitro biological experiments showed that HApCB was not toxic for a variety of human tumor cells of different histotypes, specifically interacted with CD44 as the native unconjugated HA, and underwent uptake by tumor cells, leading to accumulation of amounts of boron atoms largely exceeding those required for a successful BNCT approach. Thus, HApCB may be regarded as a promising new BNCT agent for specific targeting of cancer cells overexpressing the CD44 receptor.     

Adjustable ‘cross-linking’ of neighboring DNA molecules in liquid-crystalline dispersions through (daunomycin-copper) polymeric chelate complexes

The interaction of daunomycin molecules with double-stranded DNA in the liquid-crystalline state was investigated. It was shown that at a certain extent of daunomycin binding a change of the mechanism of anthracycline orientation with reference to the DNA chain occurs. This is testified by the alteration of the sense of spatial packing of the DNA molecules in liquid-crystalline dispersions formed as a result of phase separation in poly(ethyleneglycol)-containing solutions, as well as by the onset of the reaction of daunomycin with divalent copper ions. Using this reaction, polymeric (daunomycin-copper) chelate cross-links between the DNA molecules fixed in the liquid-crystalline dispersions were formed. The length of such cross-links is adjusted by the distance between the DNA molecules, which, in turn, depends on the concentration of poly(ethyleneglycol) used for phase separation. The above molecular building mechanism may lead to new interesting applications.     

Boronated monoclonal antibody 225.28S for potential use in neutron capture therapy of malignant melanoma

The concept of conjugating boron cluster compounds to monoclonal antibodies has been examined by several groups of research workers in boron neutron capture therapy (BNCT). The procedures reported to date for boronation of monoclonal antibodies resulted in either an inadequate level of boron incorporation, the precipitation of the conjugates, or a loss of immunological activity. The present report describes the conjugation of dicesium-mercapto-undecahydrododecaborate (Cs2B12H11SH) to 225.28S monoclonal antibody directed against high molecular weight melanoma-associated antigens (HMW-MAA), using poly-L-ornithine as a "bridge" to increase the carrying capacity of the antibody and to minimize change in the conformational structure of antibody. The method produces a boron content of 1,300 to 1,700 B atoms per molecule 225.28S while retaining the immunoreactivity. Characterization in terms of the homogeneity of the conjugation of the boron-monoclonal antibody conjugates has been studied by gel electrophoresis and ion-exchange HPLC.     

A new approach for the synthesis of isonitrile carborane derivatives. Ligands for metal based boron neutron capture therapy (BNCT) and boron neutron capture synovectomy (BNCS) agents

A new approach for the synthesis of carborane isonitrile derivatives was developed. This approach involved the dehydration of both boron and carbon derived formamides using the Burgess reagent. The products, some of which were characterized by X-ray crystallography, can now be used as ligands for the synthesis of transition metal based boron neutron capture therapy and synovectomy agents and targeted radiopharmaceuticals.     

Folate receptor-mediated boron-10 containing carbon nanoparticles as potential delivery vehicles for boron neutron capture therapy of nonfunctional pituitary adenomas

Invasive nonfunctional pituitary adenomas (NFPAs) are difficult to completely resect and often develop tumor recurrence after initial surgery. Currently, no medications are clinically effective in the control of NFPA. Although radiation therapy and radiosurgery are useful to prevent tumor regrowth, they are frequently withheld because of severe complications. Boron neutron capture therapy (BNCT) is a binary radiotherapy that selectively and maximally damages tumor cells without harming the surrounding normal tissue. Folate receptor (FR)-targeted boron-10 containing carbon nanoparticles is a novel boron delivery agent that can be selectively taken up by FR-expressing cells via FR-mediated endocytosis. In this study, FR-targeted boron-10 containing carbon nanoparticles were selectively taken up by NFPAs cells expressing FR but not other types of non-FR expressing pituitary adenomas. After incubation with boron-10 containing carbon nanoparticles and following irradiation with thermal neutrons, the cell viability of NFPAs was significantly decreased, while apoptotic cells were simultaneously increased. However, cells administered the same dose of FR-targeted boron-10 containing carbon nanoparticles without neutron irradiation or received the same neutron irradiation alone did not show significant decrease in cell viability or increase in apoptotic cells. The expression of Bcl-2 was down-regulated and the expression of Bax was up-regulated in NFPAs after treatment with FR-mediated BNCT. In conclusion, FR-targeted boron-10 containing carbon nanoparticles may be an ideal delivery system of boron to NFPAs cells for BNCT. Furthermore, our study also provides a novel insight into therapeutic strategies for invasive NFPA refractory to conventional therapy, while exploring these new applications of BNCT for tumors, especially benign tumors.     

Dodecaborate lipid liposomes as new vehicles for boron delivery system of neutron capture therapy

closo-Dodecaborate lipid liposomes were developed as new vehicles for boron delivery system (BDS) of neutron capture therapy. The current approach is unique because the liposome shell itself possesses cytocidal potential in combination with neutron irradiation. The liposomes composed of closo-dodecaborate lipids DSBL and DPBL displayed high cytotoxicity with thermal neutron irradiation. The closo-dodecaborate lipid liposomes were taken up into the cytoplasm by endocytosis without degradation of the liposomes. Boron concentration of 22.7 ppm in tumor was achieved by injection with DSBL-25% PEG liposomes at 20 mg B/kg. Promising BNCT effects were observed in the mice injected with DSBL-25% PEG liposomes: the tumor growth was significantly suppressed after thermal neutron irradiation (1.8 × 10¹² neutrons/cm²).     

Boron-enriched streptavidin potentially useful as a component of boron carriers for neutron capture therapy of cancer.

A boron-enriched streptavidin has been prepared by chemical conjugation of a boron-rich compound, B(12)H(11)SH(2)(-) (BSH), to a genetically engineered streptavidin variant. The streptavidin variant used has 20 cysteine residues per molecule, derived from a C-terminal cysteine stretch consisting of five cysteine residues per subunit. Because natural streptavidin has no cysteine residues, the reactive sulfhydryl groups of the cysteine stretch serve as unique conjugation sites for sulfhydryl chemistry. BSH was conjugated irreversibly to the sulfhydryl groups of the streptavidin variant via a sulfhydryl-specific homobifunctional chemical cross-linker. Quantitative boron analysis indicates that the resulting streptavidin-BSH conjugate carries approximately 230 boron atoms/molecule. This indicates that the chemical conjugation of BSH to the streptavidin variant was highly specific and efficient because this method should allow the conjugation of a maximum of 240 boron atoms/streptavidin molecule. This boron-enriched streptavidin retained both full biotin-binding ability and tetrameric structure, suggesting that the conjugation of BSH has little, if any, effect on the fundamental properties of streptavidin. This boron-enriched streptavidin should be very useful as a component of targetable boron carriers for neutron capture therapy of cancer. For example, a monoclonal antibody against a tumor-associated antigen can be attached tightly to the boron-enriched streptavidin upon simple biotinylation, and the resulting conjugate could be used to target boron to tumor cells on which the tumor-associated antigen is overexpressed.     

A universal approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A universal approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with amino group in aglycone moiety can be conjugated with PBC carboxy derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a condensing agent. Both N-and O-glycosides differing in the conformation mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides isolated from natural sources can be transformed into N-glycosides with a functional group in aglycone. The approach was tested by conjugation of the carboxy derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand of lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

A uniform approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A uniform approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with an aglycone moiety amino group can be coupled with PBC carboxyl derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a coupling agent. Both N- and O-glycosides differing in the conformational mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides. For example, not only oligosaccharides obtained by chemical synthesis but also reducing oligosaccharides isolated from natural sources can be transformed into N-glycosides. The approach was tested by the example of conjugation of the carboxyl derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand for lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

Biodistribution of sodium borocaptate (BSH) for boron neutron capture therapy (BNCT) in an oral cancer model

Boron neutron capture therapy (BNCT) is based on selective accumulation of ¹⁰B carriers in tumor followed by neutron irradiation. We previously proved the therapeutic success of BNCT mediated by the boron compounds boronophenylalanine and sodium decahydrodecaborate (GB-10) in the hamster cheek pouch oral cancer model. Based on the clinical relevance of the boron carrier sodium borocaptate (BSH) and the knowledge that the most effective way to optimize BNCT is to improve tumor boron targeting, the specific aim of this study was to perform biodistribution studies of BSH in the hamster cheek pouch oral cancer model and evaluate the feasibility of BNCT mediated by BSH at nuclear reactor RA-3. The general aim of these studies is to contribute to the knowledge of BNCT radiobiology and optimize BNCT for head and neck cancer. Sodium borocaptate (50 mg ¹⁰B/kg) was administered to tumor-bearing hamsters. Groups of 3–5 animals were killed humanely at nine time-points, 3–12 h post-administration. Samples of blood, tumor, precancerous pouch tissue, normal pouch tissue and other clinically relevant normal tissues were processed for boron measurement by optic emission spectroscopy. Tumor boron concentration peaked to therapeutically useful boron concentration values of 24–35 ppm. The boron concentration ratio tumor/normal pouch tissue ranged from 1.1 to 1.8. Pharmacokinetic curves showed that the optimum interval between BSH administration and neutron irradiation was 7–11 h. It is concluded that BNCT mediated by BSH at nuclear reactor RA-3 would be feasible.     

Hydrophilically enhanced 3-carboranyl thymidine analogues (3CTAs) for boron neutron capture therapy (BNCT) of cancer

Five novel 3-carboranyl thymidine analogues (3CTAs) were designed and synthesized for boron neutron capture therapy (BNCT) of cancer. Phosphorylation of all five 3CTAs was catalyzed by recombinant human thymidine kinase (hTK1) using adenosine triphosphate (ATP) as the phosphate donor. The obtained phosphorylation rates ranged from 4% to 64.5% relative to that of thymidine. The compound with the most favorable hTK1 binding properties had a k(cat)/K(M) value of 57.4% relative to that of thymidine and an IC(50) of inhibition of thymidine phosphorylation by hTK1 of 92 microM. Among the five synthesized 3CTAs, this agent had also the overall most favorable physicochemical properties. Therefore, it may have the potential to replace N5-2OH, the current lead 3CTA, in preclinical studies. An in silico model for the binding of this compound to hTK1 was developed.