Development of Small Boron-containing Molecules as BNCT Agents

2. Development of Small Boron-containing Molecules as BNCT Agents

Author: Martin Kellert and Evamarie Hey-Hawkins

Various boron-containing low molecular-weight compounds were designed to interact with receptors or transporters that are overexpressed on the surface of tumor cells. Such overexpressed receptors or transporters are highly attractive targets for selective BNCT, as the boronated agents are designed to specifically interact with these receptors or transporters to undergo a selective accumulation after transportation in the malignant cells. Main task in the synthesis of such boron compounds is the generation of bioavailable molecules that possess a sufficient water solubility, result in high accumulation of boron in the cells, show predictable behavior in living matter and low cytotoxicity. Boronic acid , carboranes , and various boron clusters have been chosen as ¹⁰B moieties. Promising boron-containing molecules designed and synthesized in the last decade are divided into six classes: amino acid derivatives, nucleic acid derivatives, porphyrins and related derivatives, carbohydrates, peptides and DNA- or mitochondria-targeting molecules.

         2.1. Amino Acid Derivatives
         2.2. Nucleic Acid Derivatives
         2.3. Boronated Porphyrins and Their Analogs
         2.4. Carbohydrates
         2.5. Peptides
         2.6. DNA- or Mitochondria-targeting Molecules

2.1. Amino Acid Derivatives

As L-¹⁰BPA was found to be actively accumulated in malignant melanoma cells , much attention has been focused on the development of boron-containing amino acids and related peptides , e.g. carborane-functionalized glutamine derivatives (1), benzoxaborole-containing phenylalanine analogs (2), various boron-containing cyclic amino acids (3) or novel carborane-based amino acids (4 and 5). Biodistribution studies in mice bearing melanoma tumors and rats using the F98 rat glioma model indicated that selected boronated amino acids (6 and 7) were taken up by the tumors selectively. Furthermore, fluorinated (¹⁸F) BPA derivatives were synthesized to put an effort to devise practical tools for both magnetic resonance imaging (MRI) and BNCT agents. More recently, research focused on the synthesis of cobalt bis(dicarbollide)-containing amino acids (8) which contain 18 boron atoms to enhance the boron loading.

Fig. 2.1 Structures of boron-containing amino acids

2.2. Nucleic Acid Derivatives

Initial approaches focused on incorporation of boron into nucleobases , such as purines (1) and pyrimidines. Another approach was the direct conjugation of a boron moiety to nucleic acids; for example, 2’-deoxycytidine was modified with methyl boronic acid (2). Various boronic acid- and carborane-conjugated nucleic acids were developed in the 1990s. Recent advances in boron-containing nucleoside conjugates include carborane-containing thymidine analogs (3 and 4) and metallacarborane derivatives as thymidine kinase 1 (TK1) substrate, which were also tested with the RG2 rat glioma model and the F98 rat glioma model . There are also derivatives known bearing amino, amido or more complex nitrogen-containing groups in the linking unit or no linker between the nucleoside and the carborane. Metallacarborane derivatives of all four of the canonical nucleosides, thymidine (T), 2′-O-deoxycytidine (dC), 2′-O-deoxyadenosine (dA), and 2′-O-deoxyguanosine (dG) (5) were prepared. The availability of this methodology has made studies of a broad spectrum of nucleoside conjugates bearing metals and the incorporation of these metal centers into DNA oligomers at designated locations possible. Novel uridine derivatives bearing ortho-carborane linked via an ethynyl and/or triazole ring were also developed (6). These compounds show only low to medium cytotoxicity and moderate phosphorylation rates, which is an indicator of the possible DNA incorporation. Additionally, the phosphorylation rate of these derivatives by TK1 and TK2 were investigated. It was found that nucleosides containing the cobalt bis(dicarbollide) moiety or the dicarba-closo-dodecaboranyl moiety tend to aggregate, whereas nucleosides bearing a nido-carborate form a stable aqueous solution. More exotic examples are the synthesis of azanonaborane derivatives of purine, adenine, guanine and 2,6-diaminopurine (7). Here, the exo-NH₂R group of the azanonaborane can be exchanged by one nitrogen atom of the pyrimidine ring, and except for guanine, also by an N atom of the imidazole ring.

Fig. 2.2 Structures of boron-containing nucleosides

2.3. Boronated Porphyrins and Their Analogs

Porphyrins and their analogs such as chlorins , phthalocyanines and porphyrazines are known to accumulate selectively in a wide variety of tumors, and these compounds are used in photodynamic therapy (PDT) for cancer treatment as photosensitizers .
Boronated porphyrins are used in both BNCT and PDT, and as theranostic drugs because their distribution in cells and tissues can be visualized easily by fluorescence imaging .
The boronated porphyrin BOPP (1) was selectively taken up by tumor cells in xenograft models of glioma and localized predominantly in the mitochondria . A phase I drug evaluation of BOPP was subsequently carried out. However, BOPP could not be used clinically because thrombocytopenia was observed in patients due to its toxic effect and/or that of its metabolites on the platelets.
Various other boronated porphyrins and their analogs have been synthesized and evaluated as boron carriers for BNCT to overcome these side effects. Most of the boronated porphyrins containing one or more boronic acid groups or carboranes in the porphyrin backbone have poor water solubility. Therefore, porphyrins with ionic boron clusters such as nido-carborate, dodecaborate, and cobalt bis(dicarbollide) were developed to increase the water solubility. A dodecaborate containing protoporphyrin (2) was developed, which showed high water solubility and low cytotoxicity, and exhibited higher intercellular boron concentrations in various tumor cells than BOPP. A chlorin derivative with two BPA moieties (3) as substituents showed good water solubility (100 mg/mL) and tumor selectivity.
Carborane-containing porphyrins were conjugated with linear and branched polyamine and opioid peptide (Tyr-D-Arg-Phe-β-Ala; YRFA) (4) to increase the bloodbrain barrier (BBB) permeability. These conjugates showed low cytotoxicity (> 400 μM) and a high BBB permeability coefficient in an in vitro model .

Fig. 2.3 Boronated Porphyrins and Chlorin

2.4. Carbohydrates

An increased rate of glycolysis is often observed in many cancer cells, and therefore, boron-containing carbohydrates have recently sparked much interest, like boronic acid- and boronic acid ester-containing monosaccharides prepared from D-glucal (1). The sugar moiety is expected to not only confer water solubility to the otherwise hydrophobic boron cluster but also accumulate selectively in tumor tissues through the glucose transport system . Various water-soluble ortho-carborane-containing glycosides , carboranyl-C-deoxyriboses (2 to 5), functionalized glycosylated carboranes (6), carborane-bearing 5-thio-D-glucopyranose derivatives (7), mono-, bis- and tris-glucuronylated carboranes (8), ribofuranosylaminobutylazanonaborates (9), boron-rich bis-carborane-bridged bis-glycophosphonates (10) and multivalent dumbbell-shaped galactosyl carboranes have been synthesized and their biological properties as boron carriers investigated. It was shown that o-carboranylglucose significantly accumulated in F98 glioma cells . A different approach follows the synthesis of boronic acid derivatives mimicking monosaccharides (11). Furthermore, glycosylated carboranylquinazolines (12) were synthesized and in vitro toxicity studies with B16 melanoma cells were performed. It was shown that derivatives containing the ortho-carborane exhibit cell toxicity with an LD₅₀ value of >200 µg boron per mL and derivatives containing the respective nido-species with an higher water solubility exhibit no cell toxicity up to a boron concentration about 3000 µg boron per mL.

Fig. 2.4 Carbohydrate-containing boron compounds

2.5. Peptides

The groups of Hey-Hawkins and Beck-Sickinger have focused on different peptides which show selectivity towards G protein-coupled receptors which are overexpressed in different cancer types. Recent research included the synthesis of closo-carborane-modified neuropeptide Y analogs or metallacarborane derivatives (1); a second approach was the incorporation of meta-carboranes in novel ghrelin receptor agonists. More interesting is the improvement of these derivatives by introduction of water-soluble side chains at the carborane moieties (2), whereas the change from D-galactosyl to L-galactosyl groups increased the selectivity of these derivatives due to a lower unspecific uptake of bioconjugates into liver tissue.

Fig. 2.5a Metallacarborane conjugate 1 with tumor-selective neuropeptide Y

Fig. 2.5b Bis-D-galactosylated meta-carborane 2 as a water-soluble building block for tumor-selective peptides

Another attractive target is the integrin α_νβ₃, which has a specific expression in proliferating endothelial and tumor cells of various origins. In vitro cell adhesion assays of cyclic Arg-Gly-Asp (RGD) peptides conjugates with icosahedral boron clusters (BSH or ortho-carborane) (3) demonstrated the high binding affinity of the conjugates to integrin α_νβ₃. Biodistribution experiments showed comparable tumor uptake and a significantly longer retention of the carborane conjugates in tumors compared with BSH.

Fig. 2.5c cRGD-peptide conjugate with ortho-carborane

2.6. DNA- or Mitochondria-targeting Molecules

One of the important characteristics of boron carriers is the localization in the cytosol and/or nucleus of cancer cells. If boron compounds can be specifically accumulated in the nucleus, the DNA of cancer cells will be destroyed more effectively by BNCT. Based on this strategy, the accumulation of boron-containing compounds such as polyamine, DNA binding agents, and DNA intercalators have been reported. Within this approach, a carborane-containing-polyamine, ASPD-5 (1), was prepared and showed a high binding affinity for DNA and high tumor selectivity. Furthermore, in some cases it might be possible to combine BNCT properties with chemotherapeutic aspects to increase the overall therapeutic effect, for example, with boronated cisplatin -containing closo-carborane (2) or nido-carborate or carborane-containing benzo[b]acridones (DNA intercalator ) (3).
Another specific intracellular target are mitochondria . One strategy to address mitochondria as BNCT target is the combination of carboranes or dodecaborates with selected amino acid sequences which are known to be selective towards mitochondria; one example is the conjugation of BSH with an RLA (arginine-leucine-alanine) peptide (4).

Fig. 2.6 Boron compounds for targeting DNA or mitochondria

1. Basic Requirements of BNCT – BSH and BPA

2. Development of Small Boron-containing Molecules as BNCT Agents
         2.1. Amino Acid Derivatives
         2.2. Nucleic Acid Derivatives
         2.3. Boronated Porphyrins and Their Analogs
         2.4. Carbohydrates
         2.5. Peptides
         2.6. DNA- or Mitochondria-targeting Molecules

3. Boron Compounds Conjugated with Biomolecules
         3.1. Boron-conjugated Growth Factors
         3.2. Boron-Conjugated Antibodies
         3.3. Boron-conjugated Proteins

4. Boronated Polymers, Liposomes, and Other Nanoparticles as Boron-delivery Systems
         4.1. Boronated Polymers and Micelles
         4.2. Boronated Biopolymers
         4.3. Emulsions
         4.4. Liposomes
         4.5. Boronated Nanoparticles
         4.6. Boronated Nanotubes

5. Strategy for MRI-guided BNCT