Design, Synthesis, and Biological Evaluation of Boron-Containing Macrocyclic Polyamines and Their Zinc(II) Complexes for Boron Neutron Capture Therapy

Abstract

Boron neutron capture therapy (BNCT) is a binary therapeutic method for cancer treatment based on the use of a combination of a cancer-specific drug containing boron-10 (¹⁰B) and thermal neutron irradiation. For successful BNCT, ¹⁰B-containing molecules need to accumulate specifically in cancer cells, because destructive effect of the generated heavy particles is limited basically to boron-containing cells. Herein, we report on the design and synthesis of boron compounds that are functionalized with 9-, 12-, and 15-membered macrocyclic polyamines and their Zn²⁺ complexes. Their cytotoxicity, intracellular uptake activity into cancer cells and normal cells, and BNCT effect are also reported. The experimental data suggest that mono- and/or diprotonated forms of metal-free [12]aneN₄- and [15]aneN₅-type ligands are uptaken into cancer cells, and their complexes with intracellular metals such as Zn²⁺ would induce cell death upon thermal neutron irradiation, possibly via interactions with DNA.

Scheme 1. Structures of Representative BNCT Agents

Scheme 2. Structures of Polyamines and Boron-Containing Spermidine Derivatives 5 and 6

Scheme 3. Hydrolytic Cleavage of C–B Bond of 7

Scheme 4. Structures of Macrocyclic Polyamine Derivatives and Their Zn²⁺ Complexes Synthesized in This Work

Scheme 5. Complexation of Zn²⁺–Cyclen 21 with the Deprotonated dT^– in Aqueous Solution at Neutral pH

Scheme 6. Synthesis of 12a,b, 15a,b, and 18a,b

Scheme 7. Synthesis of 13a,b, 16a,b, 7, and 19a,b

Scheme 8. Synthesis of 14a–c, 17a–c, and 20a–c

Figure 1

ORTEP drawing of 19a with a Zn²⁺—bound NO₃^–. Selected bond lengths: Zn(1)–N(1) 2.059 Å, Zn(1)–N(2) 2.167 Å, Zn(1)–N(3) 2.091 Å, Zn(1)–N(4) 2.962 Å, Zn(1)–O(3) 1.999 Å, C(13)–B(1) 1.561 Å, B(1)–O(1) 1.363 Å, and B(1)–O(2) 1.366 Å. One external nitrate anion, ethanol, and hydrogen atoms were omitted for clarity.

Figure 2

Comparison of intracellular boron atoms against HeLa S3 (open bars), A549 (shaded bars), and IMR-90 (closed bars) cells as determined by ICP–MS. All cells were treated with boron compounds 1, 2, 7, 12–17 (a), and 18–20 (b) (30 μM) in culture medium at 37 °C for 24 h. Data represent the mean ± standard deviation (SD) of at least three replicates.

Scheme 9. Typical Procedure Used for Measuring the Intracellular Uptake of Boron Compounds in Living Cells

Figure 3

Intracellular boron uptake-T/N selectivity profiles (a,c) and intracellular boron uptake-IC₅₀ value against normal cell profiles (b,d) of boron compounds 1, 2, 7, and 12–20. (a,c) Selectivity (T/N ratio) to HeLa S3 cells (a) and A549 cells (c) were calculated from the results for the intracellular uptake of the boron compounds into HeLa S3 and A549 cells in comparison to the uptake into IMR-90 cells, respectively. (b,d) IC₅₀ values (μM) of boron compounds 1, 2, 7, and 12–20 against IMR-90 cells and boron uptake (fmol/cell) into HeLa S3 cells (b) and A549 cells (d).

Scheme 10. Reported Deprotonation Constants (pK_a) of Macrocyclic Polyamines 9–11⁻ and Stability Constants (log K_ZnL) of Their Zn²⁺ Complexes 31–33 in Aqueous Solution at 25 °C

Figure 4

Effect of low temperature on the intracellular uptake of boron compounds 2 and 15a–17a (30 μM) into HeLa S3 (a) and A549 cells (b) at 37 °C (open bars) or 4 °C (closed bars) for 1 h. Data represent the mean ± SD of at least three replicates.

Figure 5

Relative uptake of 2 and 17a (30 μM) into HeLa S3 cells in the absence (open bars) and presence of inhibitors (closed bars), 1.5 mM of MβCD (a), 80 μM of dynasore (b), 2 mM of amiloride (c), and 2 mM of spermidine 3 (d). After pretreatment with the inhibitors for 1 h, the cells were incubated with 2 and 17a at 37 °C for 1 h in the presence of inhibitors. Data represent the mean ± SD of at least three replicates.

Scheme 11. Synthesis of ¹⁰B-Enriched 15b, 16b, and 17a (¹⁰B-15b, ¹⁰B-16b, and ¹⁰B-17a)

Figure 6

Anti-tumor effect of boron compounds 1, 2, 15b, ¹⁰B-15b, 16b, ¹⁰B-16b, 17a, ¹⁰B-17a, ¹⁰B-18b,¹⁰B-19b, and ¹⁰B-20a (30 μM) against A549 cells was examined by a colony formation assay: (a) control (in the absence of boron compound) (○), 1 (●), 2 (◇), 15b (◆), ¹⁰B-15b (□), and ¹⁰B-18b (■). (b) Control (○), 16b (●), ¹⁰B-16b (◇), 17a (◆), and ¹⁰B-17a (□), and ¹⁰B-19b (■), and ¹⁰B-20a (×). 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. Averaged thermal neutron flux was 1.4 × 10⁹ n/cm²·s for control (in the absence of boron compound), 1, 2, 15b, 16b, ¹⁰B-16b, 17a, and ¹⁰B-17a and 1.6 × 10⁹ n/cm²·s for ¹⁰B-15b, ¹⁰B-18b, ¹⁰B-19b, and ¹⁰B-20a, respectively. The survival fraction was determined by ImageJ-plugin Colony Area. Data represent the mean ± SD of at least three replicates.

Scheme 12. Evaluation of the Anti-tumor Effect of Boron Compounds in an In Vitro BNCT Study

Figure 7

Relationship between the intracellular uptake of boron compounds 1 (○), 2 (◇), ¹⁰B-15b (□), ¹⁰B-16b (■), ¹⁰B-17a (△) (30 μM) and control (in the absence of boron compound) (●) into A549 cells after incubation for 24 h and their BNCT effect (survival fractions after irradiation with thermal neutrons for 45 min; thermal neutron fluence: 4.1 ± 0.1 × 10¹² n/cm²).

Scheme 13. Proposed Scheme for the BNCT Effect of ¹⁰B-15b, ¹⁰B-16b, ¹⁰B-17a, and Their Zn²⁺ Complexes