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Novel 3-carboranyl thymidine analogues (3CTAs) were synthesized as potential boron delivery agents for boron neutron capture therapy (BNCT). This library includes six zwitterionic NH3⁺-nido-m-carborane-substituted thymidine analogues (Thds) and the corresponding neutral NH2-closo-m-carborane-substituted counterparts. All compounds of this library were good substrates for recombinant human thymidine kinase 1 (TK1) with phosphorylation rates up to 89% relative to that of Thd. One compound out of this library, 3-[3-(7-NH3⁺-nido-m-carboran-1-yl)propan-1-yl]thymidine (19b), showed selective retention in TK1-expressing murine L929 wild-type tumors versus L929 TK1 (−) tumors in biodistribution studies. The biological evaluation of the zwitterionic NH3⁺-nido-m-carborane-substituted Thds indicated improved aqueous solubility and similar or even superior potential as BNCT agents compared with different classes of 3CTAs (Cancer Res. 2004, 64, 6280−6286 and 6287−6295). To complete previous structure−activity relationship (SAR) studies, 3-[(closo-o-carboranyl)methyl]thymidine (4) was also synthesized and evaluated.

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 × 10⁵ receptor sites/cell with a Ka = 1.64 ± 0.32 × 10⁸ M^-1. F98 cells transfected with the gene encoding a mutant form of EGFR, designated the F98EGFRvIII glioma, expressed 1.07 ± 0.10 × 10⁵ receptor sites/cell with a Ka = 2.18 ± 0.54 × 10⁹ 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 ~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 μg B/g tumor was localized in F98EGFR gliomas versus 36.5 ± 18.8 μg B/g tumor in F98WT gliomas and 13.4 ± 6.1 μg in normal brain. In contrast, only 6.7 ± 3.6 μg 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.

Successful treatment of cancer by boron neutron capture therapy (BNCT) requires the selective delivery of ¹⁰B 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 ¹⁰B 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 PEG2000 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 ~15 decaborate clusters and ~1 PEG2000 unit with folic acid attached to the distal end, the other containing ~13 decaborate clusters, ~1 PEG2000 unit, and ~1 PEG800 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.

A small library consisting of two series of thymidine derivatives containing o-carboranylalkyl groups at the N-3 position was prepared. In both series, alkyl spacers of 2−7 methylene units were placed between the o-carborane cage and the thymidine scaffold. In one series, an additional dihydroxypropyl substituent was introduced at the second carbon atom of the carborane cage. In the series of N-3-substituted carboranyl thymidines without additional dihydroxypropyl substituent, three steps were required to obtain the target compounds in overall yields as high as 75%, while in the series of N-3-substituted carboranyl thymidines with additional dihydroxypropyl substituent, 9−10 steps were necessary with significantly lower overall yield. All target compounds were good substrates of human cytosolic thymidine kinase 1 while they were, if at all, poor substrates of the mitochondrial thymidine kinase 2. There was only a minor difference in phosphorylation rates between N-3-substituted carboranyl thymidines with additional dihydroxypropyl substituents with thymidine kinase 1 (range:  13−49% relative to thymidine) and their counterparts lacking this group (range:  11−57% relative to thymidine). Tether lengths of two and five methylene groups in both series gave the highest enzyme activities in the present study. A hypothesis for this result is presented.

Boron neutron capture therapy (BNCT) depends on the selective delivery of a sufficient number of ¹⁰B atoms to tumor cells to sustain a lethal ¹⁰B(n,α)⁷Li 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, Na2[B12H11SH] and Na3 (B20H17NH3), 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:  N⁵-(4-carboranylbutyl)spermidine·3HCl (SPD-5), N⁵-[4-(2-aminoethyl-o-carboranyl)butyl]spermidine·4HCl (ASPD-5). Three were the spermine derivatives:  N⁵-(4-carboranylbutyl)spermine·4HCl (SPM-5), N⁵-[4-(2-aminoethyl-o-carboranyl)butyl]spermine·5HCl (ASPM-5), and N⁵,N¹⁰-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 μg per 10⁹ cells) was observed with FR-targeted liposomes than with nontargeted control liposomes (up to 154 μg per 10⁹ 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.

Derivatives of thymidine containing o-carboranylalkyl groups at the N-3 position and derivatives of 2‘-deoxyuridine containing o-carboranylalkylmercapto groups at the C-5 position were synthesized. The alkyl spacers consist of 4−8 methylene units. The synthesis of the former compounds required 3−4 reaction steps in up to 75% overall yield and that of the latter 9−10 reaction steps with significantly lower overall yield. Derivatives of thymidine substituted with carboranylalkyl substituents at the N-3 position and short spacers were phosphorylated by both recombinant and purified cytosolic thymidine kinase (TK1) to a relatively high degree. None of the tested 2‘-deoxyuridine derivatives possessing carboranyl substituents at the C-5 position were phosphorylated by either recombinant or purified TK1. The amounts of phosphorylation product detected for some of the C-5-substituted nucleosides with recombinant mitochondrial thymidine kinase (TK2) were low but significant and decreased with increasing lengths of the alkyl spacer. The data obtained in this study do not seem to support the tether concept applied in the synthesis of the new C-5- and N-3-substituted carboranyl nucleosides intended to reduce possible steric interference in the binding of carboranyl nucleosides with deoxynucleoside kinases. Instead, it appeared that a closer proximity of the bulky carborane moiety to the nucleoside scaffold resulted in better substrate characteristics.

New boron-containing spermidine/spermine (SPD/SPM) analogues have been synthesized:  N⁵-[4-(2-aminoethyl-o-carboranyl)butyl] and N⁵-{4-[(2,3-dihydroxypropyl)-o-carboranyl]butyl} SPD/SPM derivatives (ASPD-5, ASPM-5, DHSPD-5, and DHSPM-5) as well as N⁵-{[4-(dihydroxyboryl)phenyl]methyl}spermidine (BBSPD-5). These boronated polyamines retain their ability to displace ethidium bromide from calf thymus DNA and are rapidly taken up in vitro by F98 rat glioma cells. The in vitro toxicities of ASPD-5, ASPM-5, DHSPD-5, and DHSPM-5 are lower than those previously reported for N⁵-[4-(o-carboranyl)butyl] SPD/SPM derivatives (SPD-5 and SPM-5) but similar to those of native SPD and SPM. Very low toxicity was also observed for BBSPD-5. In vivo studies of ASPD-5 and BBSPD-5 were performed in mice bearing intracerebral implants of the GL261 glioma and subcutaneous implants of the B16 melanoma. The biodistribution data found in both tumor models suggest that the polyamines synthesized to date do not appear to be suitable boron agents for BNCT.