Design, Synthesis, and Biological Applications of Boron-Containing Polyamine and Sugar Derivatives

Abstract

Boron (B), an element that is present in ultratrace amounts in animal cells and tissues, is expected to be useful in many scientific fields. We have found the hydrolysis of C–B bond in phenylboronic acid-pendant cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) and the full decomposition of ortho-carborane attached with cyclen and ethylenediamines in aqueous solution at neutral pH upon complexation with intracellular metals. The change in the chemical shift of the 11B signals in 11B-NMR spectra of these boron-containing metal chelators can be applied to the magnetic resonance imaging (MRI) of metal ions in solutions and in living cells.

1. Introduction

Boron (B) is an element that is found in ultratrace amounts in mammalian cells and consists of two stable isotopes, boron-10 (¹⁰B) and boron-11 (¹¹B), with a natural abundance ratio (¹⁰B/¹¹B = 19.9/80.1). The most important properties of boron compounds with respect to biological and medical sciences would be: (1) ¹¹B atoms have a higher NMR sensitivity (16.5% for ¹¹B and 2.0% for ¹⁰B relative to ¹H NMR), thus permitting the detection of B-containing drugs themselves and analytes that react with B-containing probes in living systems [1]; and (2) the ¹⁰B nucleus possesses a high reactivity with thermal neutrons resulting in the generation of two radioactive species (⁴He and ⁷Li particles), which induce the excitation and ionization of molecules within short path lengths [2]. For the above reasons, boron compounds can be useful in biological applications for the treatment and diagnosis of cancer and other diseases [3].

In 1936, Locher proposed the concept of boron neutron capture therapy (BNCT) based on the aforementioned nuclear reaction between ¹⁰B and thermal neutrons [4]. Because the destructive effect of the two heavy particles (⁴He and ⁷Li particles) that are generated by the decomposition of ¹⁰B lies within 5–9 μm, which is close to the size of living cells, single-cell treatment would be possible by the achievement of cancer-specific delivery of ¹⁰B and irradiation with a sufficient intensity of thermal neutrons [5, 6, 7].

BNCT systems have been installed in clinical facilities as a method for the noninvasive treatment of certain types of cancers such as recurrent head and neck cancer and malignant gliomas [8]. The selective and efficient accumulation of boron into tumor tissues is one of the important clues for successful BNCT and, as described below, two boron compounds have been approved for use as BNCT drugs. In addition, monitoring the distribution of boron in patients is required for planning treatment protocols to determine the irradiation doses and positions of the patient [9].

In this review, we introduce the applications of boron compounds to ¹¹B NMR (nuclear magnetic resonance)/MRI (magnetic resonance imaging) probes for the sensing of intracellular metal ions and BNCT agents for use in the treatment of cancer. The d-block metal ion probes take advantage of changes in the chemical shift in ¹¹B NMR spectra due to the cleavage of the carbon-boron bond in phenylboronic acid-pendant cyclen (1,4,7,10-tetraazacyclododecane) and the decomposition of the ortho-carborane moieties of carborane-metal chelator hybrids upon complexation with metal ions in aqueous solution at neutral pH. In the second half of this review, the development of novel BNCT agents bearing sugar and macrocyclic polyamine scaffolds is described.

2. ¹¹B NMR and MRI probes for metal ions in solutions and in living cells based on carbon-boron bond cleavage and the decomposition of ortho-carboranes upon metal complexation of chelator units

2.1 General

Biologically essential d-block metal ions such as zinc (Zn²⁺), copper (Cu²⁺), manganese (Mn²⁺), and iron (Fe²⁺) are involved in a variety of physiological processes in living systems as cofactors for various enzymes, intracellular second messengers, and related processes [10]. It was reported that a metal imbalance in cells and tissues causes a number of disorders such as Alzheimer’s disease, Parkinson’s disease, Willson’s disease, etc. [10]. Therefore, the development of fluorescence-based probes for the detection of these intracellular metal ions has contributed to our understanding of their functions and metabolism in living cells, while some limitations to detecting their emission from tissues remain due to their impermeability [10, 11, 12].

It is well known that MRI is one of the useful noninvasive methods for in vivo visualization and that it permits three-dimensional images of organisms and drug distributions to be obtained [13]. Although MRI is powerful method, there are only a few examples of MRI probes such as Gd³⁺-based contrast agents [14, 15].

2.2 Development of d-block metal ions probes based on the cleavage of C–B bonds in B-containing probes

It is well established that macrocyclic polyamine ligands such as 1,4,7-triazacyclononane ([9]aneN₃) 1, 1,4,7,10-tetraazacyclododecane ([12]aneN₄, cyclen) 2, and 1,4,7,10,13-pentaazacyclopentadecane ([15]aneN₅) 3 are able to form more stable complexes 4–6 with metal ions such as Cu²⁺, Ni²⁺, and Zn²⁺ in aqueous solution (Figure 1) than metal complexes of linear polyamine types [16, 17]. In addition, metal ions in these complexes, especially the Zn²⁺ ion in Zn²⁺-cyclen complex (5), possess strong Lewis acidity and the deprotonated Zn²⁺-bound H₂O (HO⁻) functions as a nucleophile and a base in aqueous solution at neutral pH [18, 19, 20, 21, 22, 23].

Figure 1.

The structures of 9-, 12-, and 15-membered macrocyclic polyamines 1–3 and their metal complexes 4–6.

Bendel and coworkers reported that ¹¹B NMR/MRI would be a potential technique for the imaging of boron agents in the body [24, 25]. However, a functional system for achieving this has not been established yet. In this context, we hypothesized that the sp² boron in 7 and 8 would be changed to the sp³ boron due to the formation of metal complexes 9a and 10a and the following interaction of metal-bound H₂O (OH⁻) with boron at neutral pH, resulting in change in the ¹¹B NMR signals (Figure 2) [26]. However, the products obtained after the addition of Zn²⁺ to 7 (L¹) (Figure 3a) were 11a (ZnL³) and boric acid (B(OH)₃), as confirmed by an X-ray structure analysis (Figure 3b). The findings strongly indicated that the Zn²⁺-bound H₂O (9a and 10a) is efficiently deprotonated due to the double activation by Zn²⁺ and B to produce the Zn²⁺-bound HO⁻ (9b and 10b), which hydrolyzes the C–B bond. The hydrolytic cleavage of the C–B bond of 7 (L¹) was also observed by the measurement of ¹¹B NMR upon the addition of Zn²⁺, in which the ¹¹B NMR signal of 7 (L¹) at 31.1 ppm was shifted to 19.4 ppm that corresponds to B(OH)₃.

Figure 2.

The C–B bond hydrolysis of phenylboronic acid-pendant 12-membered tetraamine (cyclen) to produce inorganic boric acid.

Figure 3.

X-ray crystal structures of (a) 7 (L¹) and (b) 11a (ZnL³) with B(OH)₃.

The ¹¹B NMR spectral change of 7 (L¹) was promoted by Cu²⁺, Fe²⁺, Co²⁺, and Ni²⁺ but not by Ca²⁺ and Mg²⁺ (Table 1). Hydrolysis of the C–B bond of 7 (L¹) with Cd²⁺ was faster than that with Zn²⁺, possibly due to the strong nucleophilicity of the Cd²⁺-bound HO^– [27]. Meanwhile, the C–B bond cleavage of 7 (L¹) by Mn²⁺ and Fe³⁺ was slow.

	δ (ppm)	Δδ (ppm)b	time (h)c		δ (ppm)	Δδ (ppm)b	time (h)c
7 (L^1) alone	31.1	–	–	Mn^2+	20.6	–10.5	48
Zn^2+	19.4	–11.7	0.5	Ni^2+	19.8	–11.3	2
Cu^2+	19.5	–11.6	1.5	Cd^2+	19.2	–11.9	0.1
Fe^2+	19.7	–11.6	0.5	Ca^2+	31.7	0.6	–
Fe^3+	30.8	–0.3	–	Mg^2+	31.9	0.8	–
Co^2+	19.6	–11.5	1

Table 1.

¹¹B NMR spectral change of 7 (L¹) (20 mM) upon the addition of d-block metal ions (20 mM) in 1 M HEPES buffer at pD 7.4 and 25 °C [26].a

^a

All data are referenced to external BF₃·Et₂O in CDCl₃ (δ = 0 ppm).

^b

Δδ = δ (7 (L¹) with metal ions) – δ (7 (L¹)).

^c

Approximate reaction time for the completion of C–B bond cleavage.

The intracellular uptake of boron in 7 and 8 into Jurkat T cells was determined by ICP-AES, and the results indicated that the uptake of 8 was higher than that of 7, possibly due to the hydrophobicity of the boronic ester group. The Zn²⁺-induced C–B bond cleavage of 8 (L²) by intracellular Zn²⁺ was observed in living cells. The Jurkat T cells were sequentially treated with 8 (L²) and Zn²⁺ complex of pyrithione (Zn²⁺ ionophore to transfer Zn²⁺ into cells) for 20 min and 1 h, respectively. The cells were washed with CS-RPMI and PBS and then transferred to a quartz NMR tube, whose ¹¹B NMR spectra were measured in D₂O containing PBS. As shown in Figure 4, the ¹¹B signal for B(OH)₃ (ca. 19 ppm) in Jurkat T cells was observed with a positive correlation to the concentrations of Zn²⁺-pyrithione complex, indicating the successful detection of the intracellular Zn²⁺ ions. It should be noted that the ¹¹B signal for 8 (ca. 31 ppm) in the absence of Zn²⁺ was observed as a broad signal.

Figure 4.

In-cell ¹¹B NMR spectra of 8 (L²) in the absence of Zn²⁺–pyrithione (ionophore) and in the presence of Zn²⁺–pyrithione (BF₃·Et₂O was used as an external references). The Jurkat T cells (4 × 10⁸ cells) were incubated with 33 μM 8 (L²) in culture medium at 37 °C for 1 h, and then (a) DMSO (as negative control), (b) 2.5 μM Zn²⁺–pyrithione, and (c) 10 μM Zn²⁺–pyrithione at 37 °C for 20 min.

2.3 Development of Cu²⁺ ion probes based on decomposition reaction of ortho-carborane–metal chelator hybrids

It is known that the reaction of the o-carborane 12 with Brønsted or Lewis bases affords the corresponding nido-form 13 and B(OH)₃ and that the further degradation of 13 proceeds slowly under harsh conditions such as in acidic solutions and/or at high temperatures (Figure 5) [28]. On the other hand, we found that o-carborane derivatives such as 12, 14, and 15a–c generate 4–9 equiv. of B(OH)₃ upon the reaction with Cu²⁺ and Mn²⁺ via the corresponding nido-forms 12’, 14’, and 15a’–c’ under physiological conditions (Figure 6a) [29]. Our studies also indicated that the modification of nido-o-carborane (16 (L⁵)) with N,N,N’-trimethylethylenediamine (TriMEDA) as a chelator unit facilitates the Cu-promoted decomposition of the molecule (Figure 6b) via the Cu²⁺-complex 17 (CuL⁵) to produce 9 B(OH)₃ in aqueous solution [30].

Figure 5.

Decomposition of o-carborane 12 in the presence of a Brønsted or Lewis base.

Figure 6.

Decomposition of o-carborane-pendant chelators (a) the ¹¹B NMR/MRI detection of Cu²⁺ ion based on decomposition reaction of o-carborane derivatives and (b).

Changes in the ¹¹B NMR spectra of 16 (L⁵) in the presence of various d-block metal ions are shown in Figure 7. A strong ¹¹B signal at ca. 20 ppm corresponding to B(OH)₃ was observed in the presence of Cu²⁺, while, in the presence of other metal ions, the change was negligible. These results showed good agreement with the results of an azomethine-H assay, which also indicate the Cu²⁺ selectivity.

Figure 7.

Decomposition of 16 (L⁵) (1.4 mM) in the presence of Cu²⁺, Cu⁺, Cu⁺+NaAsc, Mg²⁺, Ca²⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺ and Pb²⁺ (2 mM) in DMSO/0.5 M HEPES buffer (pH 7)/D₂O (5:4:1, 0.5 mL in total) at 37 °C after incubation for 4 h measured by ¹¹B{¹H} NMR. For ¹¹B{¹H} NMR experiments, 2.5% BF₃·Et₂O in CDCl₃ was used for an external reference.

As shown in Figure 8, the oxidation potentials of 12′, 14′, and 16 are +0.57, +0.51, and +0.38 V (vs Ag/AgCl), respectively (determined by cyclic voltammetry), which are less positive than +0.7 V (vs Ag/AgCl) for [Cu(TMEDA)]⁺/[Cu(TMEDA)]²⁺ . These data may explain the reasons why 12′, 14′, and 16 are oxidized by Cu(TMEDA)]²⁺ complex. More efficient oxidation of 16 by Cu²⁺ than that of 12′ and 14′ is possibly due to the order of oxidation potentials (+0.38 V for 16 vs +0.57 and +0.51 V for 12′ and 14′) and the close contact between the o-carborane unit and stable Cu²⁺-TMEDA complex part in 17 and 18 (Figure 6).

Figure 8.

Summary of the oxidation potentials of 12′, 14′, and 16 (nido-form) with redox potentials of Cu, Fe, Pb, and Zn.

In addition, the chemical yields of B(OH)₃ from 16 (L⁵) with Cu⁺ were decreased when antioxidants (sodium ascorbate, NaAsc) were added to the reaction mixture. According to these results and DFT calculations, a proposed mechanism for the decomposition of o-carborane moieties by Cu²⁺ is shown in Figure 9. Initially, the nido-form 20 is generated from the closo-form 19 by reaction with a nucleophile such as HO⁻. Following the oxidation of the electronegative B10 (B at the 10 position) of 20 by Cu²⁺, the closo-form 21 is produced by a ring-closure reaction. The unstable intermediate 21 would react with H₂O at the B9 position and is then completely decomposed to 9 equiv. of B(OH)₃ and other products via the transition state 22.

Figure 9.

Proposed mechanism for the decomposition reaction (arrows indicate positively charged boron atoms, which are susceptible to attack by H₂O or HO⁻).

¹¹B MRI experiments were conducted by using an aqueous solution of B(OH)₃ (10 mM) and Cu(bpy) (1 mM) in a larger vial (S_out) and a o-carborane analogue 14 (Figure 6) (1 mM) in a smaller vial (S_in) that was nested in the larger vial (Figure 10). To detect these boron compounds separately, BF1 (the basic transmitter frequency) values for B(OH)₃ and 14 are set ca. 128.392 and 128.387 MHz, respectively, because they have different chemical shifts (a-i and b-i in Figure 10). Besides, ¹¹B NMR images are obtained by using a two-dimensional ultra-short echo time sequence (UTE2D) with TE (echo time) of 199 μsec and TR (repetition time) of 30 msec. The ¹¹B signals for both B(OH)₃ and the o-carborane derivatives 14 were clearly observed, as shown in Figure 10 (a-ii and b-ii).

Figure 10.

¹¹B MRI images differentiating B(OH)₃ and 14. Curves (a-i) and (b-i) show typical ¹¹B NMR spectra of solutions in two vials (inside vial contains 1 mM 14 and outside contains 10 mM B(OH)₃). Images (a-ii) and (b-ii) show ¹¹B MRI of the inside vial (S_in) containing 1 mM 14 and the outside vial (S_out) including 10 mM B(OH)₃ + 1 mM Cu(bpy). Both ¹¹B NMR images were acquired by a two dimensional ultra-short echo time sequence (UTE2D) with TE = 199 μsec and TR = 30 msec.

The detection of Cu²⁺ by a ¹¹B NMR probe 16 (L⁵) (2 mM) was carried out by the measurement of ¹¹B MRI and NMR at the increasing concentrations of Cu²⁺ (0, 0.02, 0.1, 0.2, 1.0, and 2.0 mM) in aqueous solution at neutral pH. The ¹¹B MRI/NMR signals of B(OH)₃ were successfully observed, and the signal intensities were increased in a dose-dependent manner due to the Cu²⁺-promoted decomposition of 16 (L⁵), as shown in Figure 11.

Figure 11.

¹¹B MRI and ¹¹B{¹H} NMR (128 MHz) spectra of 16 (L⁵) (2 mM) in DMSO/0.5 M HEPES buffer (pH 7)/D₂O (5:4:1, 0.5 mL in total) after incubation with various concentrations (0 (a), 0.02 (b), 0.1 (c), 0.2 (d), 1 (e), 2 mM (f)) of Cu²⁺ at 37 °C for 8 h (A 2.5% solution of BF₃·Et₂O in CDCl₃ was used as the external reference). ¹¹B NMR images were acquired by a two dimensional ultra-short echo time sequence (UTE2D) with BF1 values ≈ 128.392 MHz, TE = 199 μsec and TR = 30 msec.

3. Design and synthesis of boron-containing agents for boron neutron capture therapy (BNCT)

3.1 General

As described in the Introduction, BNCT is one of the powerful cancer treatment methods utilizing two heavy particles, ⁴He and ⁷Li, which are produced from ¹⁰B by a neutron capture reaction [¹⁰B (n, α)⁷Li] and induce the damage of biomolecules such as DNA, RNA, and so on within a short range of 5–9 μm [4, 5, 6, 7, 8]. For this BNCT to be achieved, the development of cancer-specific ¹⁰B carriers is urgently needed. To date, only two boron compounds, namely disodium mercaptoundecahydrododecaborate (BSH) 23 and L-4-boronophenylalanine (BPA) 24 (used as a complex with D-fructose), have been approved for use as BNCT agents in clinical settings (Figure 12) [31, 32], but they are not sufficiently effective for the treatment of various tumor types. Because more selective and more efficient BNCT agents are required, the design and synthesis of new boron carriers based on sugar and macrocyclic polyamine scaffolds were conducted.

Figure 12.

Structures of representative BNCT agents.

3.2 Design and synthesis of boron-containing sugars for BNCT

Sulfoquinovosyl acylglycerol (SQAG) 25 was isolated from sea algae and characterized by Sakaguchi et al., and 25 and its derivative sulfoquinovosyl acylpropanediol (SQAP) 26 were reported to be accumulated in cancer cells and exhibit weak toxicity against normal cells (Figure 13a) [33]. Because the modification of the long alkyl chain of SQAG has negligible effect on its biological activity, the design and synthesis of SQAP derivatives 27 and 28 containing a boron cluster unit and iodine atoms as BNCT agents and imaging agents for X-ray computed tomography (CT) were conducted [34, 35].

Figure 13.

Structures of (a) SQAG and SQAP derivatives and (b) 2-boryl-1,2-dideoxy-D-glucose derivatives.

The synthesis route for preparing SQAG analogues 27 and 28 is presented in Figure 14. The intermediate 32 was obtained by the α selective glycosylation of 30 with 31 in CH₂Cl₂/tert-butyl methyl ether (1/3), followed by the oxidation of thioacetate and the deprotection of p-methoxybenzyl (PMB) group. The condensation of 32 with a long chain fatty acid unit and subsequent deprotection of the benzyl groups could give the desired product 35, which would be ideal for the synthesis of SQAP analogues containing base-sensitive functional groups such as carborane. Furthermore, the conversion of a nucleophile (-OH) of 32 to a leaving group (-OMs) enables the introduction of various acyl moieties by S_N2 reaction to give 35, which corresponds to 27 and 28. This novel synthesis route, as presented in Figure 14, would be useful for preparing a wide variety of SQAP derivatives.

Figure 14.

The synthetic route of SQAP derivatives developed by Aoki et al.

The design and synthesis of 2-boryl-1,2-dideoxy-D-glucose derivatives 29a–e were also carried out (Figure 13b) [36]. It is well known that cancer cells exhibit high glucose consumption and upregulation of glucose transporters (GLUTs) for rapid growth and proliferation, a process that is known as the Warburg effect [37]. It was also reported that hydrogen bonding interactions between the hydroxy groups of D-glucose and amino acid residues of GLUT trigger the intracellular uptake of glucose, and that the modification of D-glucose with bulky moieties at the C2 and C6 positions is tolerated [38]. In clinical applications, for instance, the D-glucose analogue, 2-deoxy-2-[¹⁸F]fluoro-D-glucose, has been used for the diagnosis of cancer by means of positron emission tomography (PET) based on the aforementioned issues [39].

We therefore performed the regio- and stereoselective hydroboration of D-glucal 36 at the C1-C2 double bond, esterification with a diol, and deprotection of the hydroxy groups to provide 29a–e via the intermediate 37 (Figure 15). Although hydroboration is one of traditional methods for the conversion of alkenes into alcohols such as 38 after the treatment of a boryl intermediate such as 37 with H₂O₂/NaOH, 37 was directly converted into 29. Further investigations of their biological activity indicated that these sugar derivatives exhibit the moderate intracellular uptake against cancer cell lines through GLUT1, while their BNCT activity was not satisfying.

Figure 15.

Synthesis of 2-boryl-1,2-dideoxy-D-glucose derivatives 29a–e via the hydroboration of the protected D-glucal 36.

3.3 Design and synthesis of boron-containing macrocyclic polyamines for BNCT

It is known that natural polyamines play multiple roles in cellular functions, including gene expression and the stabilization of chromatin structure, and that the activated polyamine transport system and biosynthesis in cancer cells are related to the increase in polyamine concentrations and proliferation activity [40, 41]. Therefore, it is expected that polyamines would be desirable scaffolds for cancer selective and DNA-targeting boron delivery agents [42, 43].

Kimura and coworkers reported that Zn²⁺–cyclen complexes 39 selectively recognize thymidine (dT) units in DNA to form a stable complex 40 in aqueous solution at neutral pH by coordination bonding between the deprotonated imide part of dT (dT^–) and Zn²⁺ and by hydrogen bonding between the NH of cyclen and the imide oxygens of dT^– (Figure 16a) [44, 45, 46, 47]. In addition, the bis(Zn²⁺–cyclen) complexes 41 strongly bind two adjacent thymidine (thymidyl(3´–5´)thymidine, d(TpT)) 42, yielding a very stable 1:1 complex 43 (Figure 16b) [48, 49, 50, 51]. The dissociation constants (K_d) were reported to be 0.3 mM for 40 (1:1 complex of dT^– with 39) and 0.6 μM for 43 (1:1 complex of d(T^–pT^–) with 41), respectively, at physiological pH in aqueous solution [52, 53, 54].

Figure 16.

Complexation of (a) Zn²⁺–cyclen 39 with the deprotonated form of thymidine (dT^–) and (b) bis(Zn²⁺–cyclen) 41 with d(T⁻pT⁻) 42 in aqueous solution at neutral pH.

In this context, we designed and synthesized some novel DNA-targeting BNCT agents containing macrocyclic polyamine scaffolds such as [9]aneN₃, [12]aneN₄, and [15]aneN₅ and their Zn²⁺ complexes, which contain phenylboronic acid units, as shown in Figures 17 and 18 [55, 56]. It was assumed that these boron-containing macrocyclic polyamine monomers 44–49 (L⁶–L¹²) and their Zn²⁺ complexes 50–52 (ZnL⁶–ZnL¹²) would be efficiently transferred into cancer cells and that thermal neutron irradiation would induce effective DNA damage in cancer cells due the ¹⁰B atoms being located in close proximity to DNA molecules (Figure 17). We also expected that the interaction of homo- and heterodimer of macrocyclic polyamines 53–62 (L¹³–L²²) and their corresponding monozinc(II) complexes 63–68 (ZnL¹³–ZnL²¹) and dizinc(II) complexes 69–78 (Zn₂L¹³–Zn₂L²²) with DNA would be stronger than that of monomeric polyamines, resulting in efficient DNA damage upon thermal neutron irradiation (Figure 18). These mono- and dimeric macrocyclic polyamines were first prepared with boron in a natural abundance ratio (¹⁰B/¹¹B = 19.9/80.1) to evaluate their cytotoxicity and intracellular uptake in several cancer cell lines, and some of the promising compounds were synthesized in the corresponding ¹⁰B-enriched forms for the BNCT experiments. It should also be noted that these compounds possess macrocyclic polyamine units at the m- or p-position, but not at the o-position, of the C–B bonds to avoid the C–B hydrolysis upon metal complexation, as described in Figures 2 and 3.

Figure 17.

Structures of B-containing macrocyclic polyamine monomers and their Zn²⁺ complexes.

Figure 18.

Structures of B-containing macrocyclic polyamine dimers 53–62 (L¹³–L²²) and their Zn²⁺ complexes 63–78 (ZnL¹³–ZnL²¹ and Zn₂L¹³–Zn₂L²²).

The results of biological studies suggested that the boron-containing macrocyclic polyamine monomers 47b (L⁷), 48b (L⁹), and 49a (L¹⁰) have a weak cytotoxicity against normal cells and are efficiently transferred into cancer cells such as A549 and HeLa S3 cells, possibly via a polyamine transport system. In addition, it was found that ditopic macrocyclic polyamines possess much less cytotoxicity than that of the monomers and moderate uptake activity into cancer cells. Therefore, some of the more promising compounds were selected and their ¹⁰B-enriched forms (>99% of ¹⁰B) were prepared for BNCT experiments.

In vitro neutron irradiation experiments using A549 cells in the presence of the ¹⁰B-enriched ¹⁰B-47b (L⁷), ¹⁰B-48b (L⁹), and ¹⁰B-49a (L¹⁰) were performed at the Institute for Integrated Radiation and Nuclear Science, Kyoto University, and the BNCT effect of these drugs was evaluated by colony formation assays. It was found that ¹⁰B-47b (L⁷), ¹⁰B-48b (L⁹), and ¹⁰B-49a (L¹⁰) showed higher cytotoxic effects than ¹⁰B-BSH 23 and ¹⁰B-BPA 24 and that the BNCT effect of ¹⁰B-enriched dimers is nearly the same as ¹⁰B-BPA (Figure 19). The BNCT effect of ¹⁰B-47b (L⁷) and ¹⁰B-50b (ZnL⁷) is almost identical and that of ¹⁰B-50b (ZnL⁷) is even better, although the intracellular uptake of the Zn²⁺ complexes is generally lower than that of the corresponding Zn²⁺-free ligands. It is possibly due to weak complexation of the 9-membered ring of ¹⁰B-47b (L⁷) with Zn²⁺. In addition, 12- and 15-membered macrocycles ¹⁰B-48b and ¹⁰B-49a effectively inhibited the proliferation of cancer cells upon irradiation with thermal neutrons, while their intracellular uptake was lower than that of the [9]aneN₃-type 47b.

Figure 19.

BNCT effect of macrocyclic polyamine monomers 23, 24, 47b, ¹⁰B-47b, 48b, ¹⁰B-48b, 49a, ¹⁰B-49a, ¹⁰B-50b,¹⁰B-51b, and ¹⁰B-52a (30 μM) against A549 cells was examined by a colony formation assay: (a) control (in the absence of a boron compound) (○), 23 (•), 24 (◇), 47b (◆), ¹⁰B-47b (□), and ¹⁰B-50b (■). (b) Control (○), 48b (•), ¹⁰B-48b (◇), 49a (◆), and ¹⁰B-49a (□), and ¹⁰B-51b (■), and ¹⁰B-52a (×). After treatment with the boron compound for 24 h, the cells were irradiated with thermal neutrons for 0, 15, 30, and 45 min and then incubated without neutron irradiation for 7 days.

According to the results of biological evaluations and DNA interaction studies using double-stranded calf-thymus DNA, it was concluded that metal-free monomers would be efficiently taken up by cancer cells and then form complexes with intracellular Zn²⁺. Both the cationic metal-free macrocycles and their Zn²⁺ complexes would bind to DNA via electrostatic interactions between cationic macrocyclic polyamine moieties and anionic double-stranded DNA (79 in Figure 20), or via the selective recognition of Zn²⁺-complexes such as ¹⁰B-51b with dT⁻ units in DNA as depicted in Figure 16 (and 80 in Figure 20), resulting in effective DNA damage upon thermal neutron irradiation (Figure 20). These findings suggest that ¹⁰B delivery agents equipped with monomeric [12]aneN₄- and [15]aneN₅-type macrocycles are preferable for use in BNCT.

Figure 20.

Proposed scheme for BNCT effect of ¹⁰B-47b, ¹⁰B-48b, ¹⁰B-49a and their Zn²⁺ complexes.

4. Conclusion

In this review, we summarize the current state of knowledge regarding the design and synthesis of ¹⁰B and/or ¹¹B containing agents for biomedical applications such as ¹¹B NMR probes and BNCT agents. We developed the d-block metal ion probes based on changes in ¹¹B NMR signals due to the hydrolysis of C–B bond in 7 (L¹) and 8 (L²) and the decomposition of o-carborane moieties in derivatives such as 14 and 16 (L⁵) upon complexation with metal ions in aqueous solution at physiological pH. Some novel BNCT agents based on sugar and macrocyclic polyamine scaffolds were also designed and synthesized. The findings indicate that ¹⁰B-enriched monomeric macrocyclic polyamines ¹⁰B-48b (L⁹) and ¹⁰B-49a (L¹⁰) exhibit potent BNCT activity upon thermal neutron irradiation, possibly due to interaction with DNA, resulting in the efficient damage of DNA molecules that are in close proximity to the boron compounds.

We believe that this review provides useful information for the future design and synthesis of novel boron-containing compounds and their applications for the treatment and diagnosis of cancer and other diseases, as well as in related research fields.

Acknowledgments

We wish to thank our collaborators and coworkers for their contributions to work described in this review. We appreciate Dr. Motoo Shiro (Rigaku Co. Ltd.), Prof. Reiko Kuroda (Chubu University), and Dr. Yasuyuki Yamada (Nagoya University) for their great assistance and helpful discussion. Financial supports from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, the Uehara Memorial Foundation, the Tokyo Ohka Foundation for the Promotion of Science and Technology, Kanagawa, Japan, the Tokyo Biochemical Research Foundation, Tokyo, Japan, Japan Society for the Promotion of Science (JSPS), and Tokyo University of Science are gratefully acknowledged.

Conflicts of interest

The authors declare no conflict of interest.