**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

**Figure 13.**

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

**Figure 14.** *The synthetic route of SQAP derivatives developed by Aoki et al.*

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].

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.

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.

*Design, Synthesis, and Biological Applications of Boron-Containing Polyamine and Sugar… DOI: http://dx.doi.org/10.5772/intechopen.105998*

**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

#### **Figure 16.**

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

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**) [44–47]. 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**) [48–51]. 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 [52–54].

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 homoand 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

#### **Figure 17.** *Structures of B-containing macrocyclic polyamine monomers and their Zn2+ complexes.*

*Design, Synthesis, and Biological Applications of Boron-Containing Polyamine and Sugar… DOI: http://dx.doi.org/10.5772/intechopen.105998*

#### **Figure 18.**

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

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**.

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

#### **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.*

#### **Figure 20.**

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

**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**.

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

*Design, Synthesis, and Biological Applications of Boron-Containing Polyamine and Sugar… DOI: http://dx.doi.org/10.5772/intechopen.105998*

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.
