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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.

One category of boron neutron capture therapy (BNCT) agents that has received extensive attention during recent years is 3-carboranyl thymidine analogues (3CTAs). These molecules are phosphorylated to the corresponding 5'-monophosphates by human thymidine kinase 1 (TK1), an enzyme that is up-regulated in dividing malignant cells. Thus, these phosphorylated molecules are selectively entrapped in tumor cells due to the acquired negative charge. This review will analyze design strategies applied for the synthesis of boron-containing nucleosides in general and in particular reference to 3CTAs. Results of biological studies with these molecules will be discussed.

Concise synthetic methods for synthesizing 3-carboranyl thymidine analogues (3CTAs) modified with cyclic and acyclic alcohols have been developed. The synthesis of these potential boron neutron capture therapy (BNCT) agents and their preliminary biological evaluation is described.

Novel 3-carboranyl thymidine analogues (3CTAs) were synthesized as potential boron delivery agents for boron neutron capture therapy (BNCT). This library includes six zwitterionic NH(3)(+)-nido-m-carborane-substituted thymidine analogues (Thds) and the corresponding neutral NH(2)-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-NH(3)(+)-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 NH(3)(+)-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.

Several thymidine analogues substituted with closo- and nido-carborane at the N-3 position were synthesized. The nido-carboranyl thymidine analogues were designed to be effective substrates for human thymidine kinase 1 in combination with an increased water solubility sufficient for clinical application in boron neutron capture therapy. This was done because N-3 substituted closo-carboranyl thymidine analogues previously synthesized in our laboratories were good TK1 substrates but were poorly water-soluble. Newly synthesized zwitterionic amino nido- and the corresponding neutral closo-m-carboranyl thymidine analogues exhibited excellent TK1 phosphorylation rates up to 75% relative to thymidine, indicating that these compounds were good substrates for thymidine kinase 1. Thin layer chromatographic studies were indicative of increased hydrophilicity of the synthesized nido-carboranyl thymidine analogues compared with their closo-carboranyl counterparts and previously reported closo-carboranyl thymidine analogues.

Eleven 3-carboranyl thymidine analogues (3CTAs) containing highly hydrophilic and flexible ethyleneoxide moieties were synthesized as potential agents for boron neutron capture therapy (BNCT) and their biochemical and physicochemical properties were evaluated. Based on specific structural features, this library of 3CTAs was divided into three subgroups. The first group contained 3CTAs with 1-4 ethyleneoxide units between the thymidine (Thd) scaffold and a carborane cluster. The second group of 3CTAs contained a pentylene spacer between Thd and the carborane and 2-4 ethyleneoxide units additionally attached to the carborane cluster. The third group contained three 3CTAs all with pentylene spacers and four ethylene units but with different carborane cages. The ethyleneoxide modified 3CTAs were good substrates of thymidine kinase 1 (TK1) and poor substrates of human mitochondrial thymidine kinase 2 (TK2) as determined in phosphoryl transfer assays. In the first group of 3CTAs, all the compounds were efficiently phosphorylated regardless of varying spacer lengths (37-42% of the activity of Thd). The second group of 3CTAs was less effectively phosphorylated (17-26% of the activity of Thd) probably due to a less favorable sterical orientation of Thd within the active site of TK1 and/or an increased lipophilicity compared with the first group. In the third group of structural isomers, no significant differences in phosphorylation rates were observed (17-25%). A structure-function hypothesis explaining these results is presented.

The purpose of the present study was to evaluate both in vitro and in vivo a series of boron-containing nucleosides that potentially could be used as delivery agents for neutron capture therapy. The rationale for their synthesis was based on the fact that proliferating neoplastic cells have increased requirements for nucleic acid precursors, and, therefore, they should preferentially localize in the tumor. A series of 3-carboranlyalkyl thymidine analogs has been synthesized and a subset, designated N4, N5, and N7, and the corresponding 3-dihydroxypropyl derivatives, designated N4-2OH, N5-2OH, and N7-2OH, have been selected for evaluation. Using these compounds as substrates for recombinant human thymidine kinase-1 and the mitochondrial isoenzyme thymidine kinase-2, the highest phosphorylation levels relative to thymidine were seen with N5 and the corresponding dihydroxypropyl analog N5-2OH. In contrast, N4, N4-OH, N7, and N7-OH had substantially lower phosphorylation levels. To compare compounds with high and low thymidine kinase-1 substrate activity, N5 and N7 and the corresponding dihydroxypropyl derivatives were selected for evaluation of their cellular toxicity, uptake and retention by the F98 rat glioma, human MRA melanoma, and murine L929 cell lines, all of which are thymidine kinase-1(+), and a mutant L929 cell line that is thymidine kinase-1(-). N5-2OH was the least toxic (IC50, 43-70 microm), and N7 and N7-2OH were the most toxic (IC50, 18-49 microm). The highest boron uptake was seen with N7-2OH by the MRA 27 melanoma and L929 wild-type (wt) cell lines. The highest retention was seen with L929 (wt) cells, and this ranged from 29% for N5-2OH to 46% for N7. Based on the in vitro toxicity and uptake data, N5-2OH was selected for in vivo biodistribution studies either in rats bearing intracerebral implants of the F98 glioma or in mice bearing either s.c. or intracerebral implants of L929 (wt) tumors. At 2.5 hours after convection-enhanced delivery, the boron values for the F98 glioma and normal brain were 16.2 +/- 2.3 and 2.2 microg/g, respectively, and the tumor to brain ratio was 8.5. Boron values at 4 hours after convection-enhanced delivery of N5-2OH to mice bearing intracerebral implants of L929 (wt) or L929 thymidine kinase-1(-) tumors were 39.8 +/- 10.8 and 12.4 +/- 1.6 microg/g, respectively, and the corresponding normal brain values were 4.4 and 1.6 microg/g, thereby indicating that there was selective retention by the thymidine kinase-1(+) tumors. Based on these favorable in vitro and in vivo data, neutron capture therapy studies will be initiated using N5-2OH in combination with two non-cell cycle dependent boron delivery agents, boronophenylalanine and sodium borocaptate.

Thymidine analogs containing o-carboranylalkyl groups at the 3-position were screened as potential substrates for human thymidine kinase 1 (TK1), an enzyme that is selectively expressed in a variety of rapidly proliferating cells, including tumor cells. On the basis of previous studies, 12 of these were identified as potential delivery agents for boron neutron capture therapy, a therapeutic method used for the treatment of high-grade brain tumors. Compound 4 with a pentylene spacer between the o-carborane cage and the thymidine scaffold and compound 10, which has an additional dihydroxypropyl substituent at the o-carborane cage, were the best substrates for TK1 with kcat/Km values of 27% and 36% relative to that of thymidine, respectively. These compounds showed partial competitive inhibition for thymidine phosphorylation by TK1. Neither compound was a substrate of recombinant human thymidine phosphorylase nor were their respective 5'-monophosphates substrates of 5'-deoxynucleotidase 1, thereby indicating potential in vivo stability. The octanol/water partition coefficient for compound 10 was 2.09, suggesting that it has excellent physiochemical properties for crossing the blood brain barrier and penetrating brain tissue. The in vitro cytotoxic effect of the 12 analogs was moderate to low in mammalian cell cultures with IC50 values between 10 and 160 micromol/L. Compounds 4 and 10 were taken up selectively and retained by the murine fibroblast L929 cell line, in contrast to its TK1-deficient variant. These findings suggest that compound 10 is a promising candidate for selective delivery of boron-10 to malignant cells, and additional in vivo studies are planned to evaluate it for boron neutron capture therapy of brain tumors.

The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1-(2-deoxy-beta-D-ribofuranosyl)-isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were <1% relative to Thd, where as the phosphorylation rates for EN1 were 1.4% and 1.1% with dCK and dGK relative to dCyd and dGuo, respectively. The analogue 1-(2-deoxy-beta-D-ribofuranosyl)-7-iodoisocarbostyril (EN2) showed poor relative-phosphorylation efficiencies (kcat/Km) with both TK1 and dGK, but not with TK2. The kcat/Km value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluorotoluene (JW1) and 1-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluoro-5-iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with kcat/Km values of 45% relative to that for Thd. 2,5-Difluoro-4-[1-(2-deoxy-beta-L-ribofuranosyl)]-aniline (JW5), a L-nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril- and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated.

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) is a chemoradio-therapeutic method for the treatment of cancer. It depends on the selective targeting of tumor cells by boron-containing compounds. One category of BNCT agents with potential to selectively target tumor cells may be thymidine derivatives substituted at the 3'-position with appropriate boron moieties. Thus, several thymidine analogues were synthesized with a carborane cluster bound to the 3'-position either through an ether or a carbon linkage. The latter are the first reported carborane-containing nucleosides in which the carboranyl entity is directly linked to the carbohydrate portion of the nucleoside by a carbon-carbon bond. Low but significant phosphorylation rates in the range of 0.18% that of thymidine were observed for the carbon-linked 3'-carboranyl thymidine analogues in phosphoryl transfer assays using recombinant preparations of thymidine kinases 1 (TK1) and thymidine kinases 2 (TK2). Some of the ether-linked 3'-carboranyl thymidine analogues appeared to be slightly unstable under acidic as well as phosphoryl transfer assay conditions and were, if at all, poor substrates for TK1.

Abstract: Thymidine kinase 1 (TK1) is a key target for antiviral and anticancer chemotherapy. Three-dimensional quantitative structure–activity relationship (3D-QSAR) using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques was applied to analyze the phosphorylation capacity of a series of 31 TK1 substrates. The optimal predictive CoMFA model with 26 molecules provided the following values: cross-validated r2 (q2)=0.651, non-cross-validated r2=0.980, standard error of estimate (s)=0.207, F=129.3. For the optimal CoMSIA model the following values were found: q2=0.619, r2=0.994, s=0.104, F=372.2. The CoMSIA model includes steric, electrostatic, and hydrogen bond donor fields. The predictive capacity of both models was successfully validated by calculating known phosphorylation rates of five TK1 substrates that were not included in the training set. Contour maps obtained from CoMFA and CoMSIA models correlated with the experimentally developed SAR. [Copyright &y& Elsevier]