o-Carboranylalkoxy-1,3,5-Triazine Derivatives: Synthesis, Characterization, X-ray Structural Studies, and Biological Activity

Morpholine- and bis(2-methoxyethyl)amine-substituted 1,3,5-triazine derivatives containing an alkoxy-o-carborane in the 6-position of the triazine ring were successfully synthesized. The molecular structures of the methoxy- and ethoxy-o-carboranyl-1,3,5-triazines were established by X-ray crystallography. In vitro studies showed that the methylene bridged morpholine- and bis(2-methoxyethyl)amine-substituted o-carboranyl-1,3,5-triazines accumulated to high levels in B16 melanoma cells and exhibited higher cytotoxicity than p-boronophenylalanine.


Introduction
Boron neutron capture therapy (BNCT) is a binary treatment modality for cancer that involves the selective accumulation of chemical agents containing a 10 B isotope in cancer cells and subsequent irradiation with thermal neutrons.Capture of a thermal neutron by the 10 B nucleus initiates a nuclear reaction in which the decay of the excited 11 B nucleus produces a high linear energy transfer α-particle and a lithium nucleus.Because of the short trajectories of these heavy particles (5-9 µm; approximately one cell diameter), radiation damage is limited to those cells that containing 10 B. Thus, side effects typically associated with ionizing radiation can be prevented if 10 B agents can be selectively targeted to tumor cells [1][2][3][4][5][6].
For BNCT to be successful in the treatment of cancer, the following criteria must be completely addressed: (i) preferential or selective uptake of 10 B-containing agent(s) by tumor tissue relative to normal tissue at concentrations high enough to deliver a therapeutic dose of 10 B atoms (20-30 µg 10 B per gram of tumor tissue or 10 9 atoms of 10 B per cell); (ii) a tumor/normal tissue differential greater than 1 and preferably in the range of 3-5; and (iii) sufficiently low cytotoxicity and rapid clearance of all 10 B delivery agents from blood and normal tissue [3][4][5][6][7][8][9][10][11][12].The only two BNCT delivery agents currently used in clinical trials are sodium mercaptoundecahydro-closo-dodecaborate (Na 2 B 12 H 11 SH), commonly known as sodium borocaptate (BSH), and the boron-containing amino acid (L)-4-dihydroxy-borylphenylalanine, known as boronophenylalanine or BPA [10].Neither of these agents adequately fulfills the aforementioned criteria, and for this reason, third-generation agents incorporating one or more polyhedral borane anions or carboranes have been investigated.With the development of new synthetic techniques and increased awareness of the biochemical requirements needed for effective boron-containing agents and their modes of delivery, several new boron agents have emerged.
o-Carborane is a stable, lipophilic molecule that resembles benzene in terms of reactivity and bulkiness [13,14].Its remarkable thermal and chemical stabilities make it a unique candidate molecule for use in several specialized applications in the fields of materials science, coordination compounds, and radiopharmaceuticals.The medicinal chemistry of o-carborane, which contains ten boron atoms, gives it a clear advantage for use in BNCT [15].We previously synthesized 1,2,3,4-tetrahydroisoquinolines [16], 1,3,5-triazines [17][18][19], and piperidines [20,21] containing the o-carborane unit as potential BNCT agents.However, since carborane cages consist only of C-H and B-H units, they have a lipophilic character [22,23].This lipophilicity necessitates the introduction of a second functional group into the o-carboranyl triazine that endows the molecule with water solubility.To meet the requirements for BNCT agents, we designed and synthesized many candidate molecules, increasing their water solubility while maintaining their high boron uptake and low toxicity [20,21].Among the numerous candidates explored, the 1,3,5-triazine derivatives of the o-carboranyl system [19,24,25] appeared promising in that they showed high boron uptake in cancer cells.Moreover, the water solubility of these molecules was found to improve via the introduction of a second functional group such as an alkylamine moiety [19].
It has been suggested that the incorporation of alkylamine or morpholine functionalities into molecules will increase their water solubilities in biological systems.Recently, we reported morpholineand alkylamine-substituted o-carboranyl-1,3,5-triazine derivatives 1-16 [26].However, we have confirmed that the purity of compounds 1-16 was not satisfactory when they were prepared in dimethylformamide (DMF) solvent.For this reason, we had difficulty performing spectroscopic and structural analyses and conducting meaningful biological experiments.Thus, we developed a modified procedure using tetrahydrofuran (THF) solvent that significantly improved the purity.In the present study, we report the improved synthesis of mono-or bis(triazinyl)-substituted o-carborane derivatives containing dimorpholine or di(methoxyethyl)amine side groups on nitrogen atoms of the triazine ring.The compounds were characterized by 1 H and 13 C nuclear magnetic resonance (NMR), and X-ray crystallographic studies, and the cytotoxicity and accumulation of selected molecules were tested in vitro.
Treatment of alkynyloxy-1,3,5-triazines with decaborane (B 10 H 14 ) and N,N-dimethylaniline as the base in toluene gave the target compounds 5-8 in moderate yields (5 51%, 6 49%, 7 40%, 8 40%).Compounds 5-8 showed the characteristic vibrational absorption bands of the B-H unit in their infrared (IR) spectra at 2588 and 2596 cm −1 .Diagnostic signals for compounds 5-7 were observed at δ 4.45 and 3.89 in the 1 H NMR spectra and at δ 73.0 and 72.3 in the 13 C NMR spectra of the cage C-H unit of the alkynyl group (see Figures S1-S4 for 1 H NMR and Figures S5-S8 for 13 C NMR, Supplementary Materials).To validate the NMR-based assignments of the final compounds, X-ray structural study of 5 and 6 were conducted to confirm their basic structures (Figures 1 and 2, respectively).Crystals suitable for X-ray crystallography were obtained from dichloromethane solutions of 5 and 6 by slow evaporation at ambient temperature; subsequent X-ray analysis provided definitive proof of their structures.

X-ray Structural Studies on 5 and 6
The X-ray structures of compounds 5 and 6 were consistent with those proposed on the basis of the NMR assignments.Selected crystallographic data and selected bond lengths and angles of 5 and 6 are summarized in Tables 1 and 2, respectively.Detailed information on the structural determinations and structural features of compounds 5 and 6 are provided in the Supplementary Materials and Appendix A. The ORTEP diagram in Figure 1   The single-crystal X-ray diffraction study of 6 revealed that it crystallized in the triclinic space group P-1 (Figure 2).The C-N bond lengths in the 1,3,5-triazine ring varied from 1.311( 2

Determination of IC 50 and Incorporation of Boron into B16 Cells
B16 mouse melanoma and HeLa human cervical carcinoma cells were treated with compounds 5-8 and 13-16 for 3 days, after which the cell viability was determined by the MTT [3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.As can be seen from Table 3, compounds 5-8 and 13-16 showed higher cytotoxicity than BPA, with IC 50 (the half maximal inhibitory concentration) values in the range of 13.1-28.6µM.Interestingly, methyleneand ethylene-bridged compounds (5, 6, 13, and 14) showed slightly higher cytotoxicity than the propylene-and 1,2-disubstituted ortho-carboranes (7, 8, 15, and 16) in B16 and HeLa cells.The higher cytotoxicity of compounds 5-8 in B16 cells may be a result of the difference between the natures of the morpholine-substituted compounds 5-8 and the bis(2-methoxyethyl)amine-substituted compounds 13-16.In HeLa cervical carcinoma cells, compounds 5-8 and 13-16 exhibited similar activities, with IC 50 values in the range of 15.9-21.5 µM.We next examined the level of intracellular accumulation of the compounds 5-8 and 13-16 by determining their boron concentrations via inductively coupled plasma atomic emission spectroscopy (ICP-AES).As shown in Figure 3, the intracellular boron uptake of compounds 5-8 and 13-16 was higher than that of BPA in B16 cells.Among the compounds synthesized, methylene-bridged compounds 5 and 13 showed more than six times higher boron accumulation than BPA.The boron uptake from both morpholine-and bis(2-methoxyethyl)amine-substituted compounds having a higher number of bridge carbon atoms, which included ethylene-and propylene-bridged compounds (i.e., 6, 7, 14, and 15), was lower.However, it should be noted that the accumulated boron concentrations of 1,2-bis[(4,6-disubstituted-1,3,5-triazin-2-yloxy)methyl]-o-carboranes (8 and 16) were lower than those of compounds 5-7 and 13-15 despite the similar boron concentrations of all these compounds; this result is attributed to the differences in the molecular size of these compounds.In addition, the intracellular boron uptake into B16 cells appears to correlate with the cytotoxicity of compounds.The compounds with greater cellular boron uptake had lower IC 50 values for cytotoxicity in B16 cells.

General Considerations
All manipulations were performed under either a dry nitrogen atmosphere using standard Schlenk techniques or a vacuum atmosphere in a KK-011AS glove box.THF and toluene were purchased from Samchun Pure Chemical Company, Ltd. (Seoul, Korea), and dried over sodium/benzophenone before use.Glassware, syringes, magnetic stirring bars, and needles were dried overnight in a convection oven.Decaborane was purchased from Katchem (Kralupy nad Vltavou, Czech Republic) and used after sublimation.Bis(2-methoxyethyl)amine, morpholine, cyanuric chloride, 2-butyn-1,4-diol, t-BuOK, triethylamine, prop-2-yn-1-ol, but-3-yn-1-ol, pent-4-yn-1-ol, and N,N-dimethylaniline were purchased from Sigma-Aldrich Chemicals (Merck KGaA, Darmstadt, Germany).IR spectra of the samples were recorded on an Agilent Cary 600 Series Fourier transform (FT)-IR spectrometer (Victoria, Australia) using KBr disks. 1 H and 13 C NMR spectra were recorded on a JEOL-JNM-AL300 spectrometer at 300.1 and 75.4 MHz, respectively. 11B NMR spectra were recorded on a Bruker Ascend 400 spectrometer (Billerica, MA, USA) (operating at 128.4 MHz) at the Korea Basic Science Institute (KBSI) Ochang Center.All 11 B chemical shifts were referenced to BF 3 •O(C 2 H 5 ) 2 (0.0 ppm), where a negative sign indicated an upfield shift.All 1 H and 13 C chemical shifts were measured relative to internal residual peaks arising from the lock solvent (99.5% CDCl 3 ) and then referenced to Me 4 Si (0.00 ppm).All melting points were uncorrected.

Crystal Structure Determination
Crystals of 5 and 6 were obtained from their CH 2 Cl 2 solutions, sealed in glass capillaries under argon, and mounted on the diffractometer.The preliminary examination and data collection were performed using a Bruker SMART CCD detector system single-crystal X-ray diffractometer equipped with a sealed-tube X-ray source (50 kV × 30 mA) using graphite monochromated Mo Kα radiation (λ = 0.71073 Å).The preliminary unit cell constants were determined using a set of 45 narrow-frame (0.3 • in ω) scans.The double pass method of scanning was used to exclude noise.The collected frames were integrated using an orientation matrix determined from the narrow-frame scans.The SMART software package (version 5.0, Madison, WI, USA) was used for data collection and SAINT (version 6.0, Madison, WI, USA) was used for frame integration [34].The final cell constants were determined through global refinement of the xyz centroids of the reflections harvested from the entire dataset.Structure solution and refinement were carried out using the SHELXTL-PLUS software package (version 4.1, Madison, WI, USA) [35].

Cell Viability Assay (MTT Assay)
The boron compounds were dissolved in DMSO, and the resulting solution was diluted with Dulbecco's modified Eagle's medium (DMEM) (10% FCS), or BPA was directly dissolved in the same medium.In a 96-well culture plate (Falcon 3072), B16 melanoma and HeLa cervical carcinoma cancer cells (1 × 10 3 cells/well) were cultured in five wells with the medium containing boron compounds at various concentrations, and then incubated for 72 h at 37 • C in a CO 2 incubator.DMSO is nontoxic at concentrations less than 0.5% and control experiments confirmed the nontoxicity of DMSO at the concentrations used in the present experiments.After incubation, the medium was removed, the cells were washed three times with phosphate-buffered saline [PBS (-)], and the CellTiter 96 ® AQueous Non-Radioactive Cell Proliferation Assay (MTT) was used for counting cells on a microplate reader.The results are presented in Table 3 as the agent concentration that resulted in a cell culture with 50% of the number of cells of the corresponding untreated group (IC 50 ).

Figure 1 .
Figure 1.Molecular structure of 5 with thermal ellipsoids drawn at the 30% level.Hydrogen atoms are omitted for clarity.

Figure 2 .
Figure 2. Molecular structure of 6 with thermal ellipsoids drawn at the 30% level.Hydrogen atoms are omitted for clarity.

Figure 3 .
Figure 3. Intracellular boron uptake of compounds 5-8 and 13-16.B16 melanoma cells were incubated for 3 h in the presence of each boron compounds or BPA (10 µM).Then the cells were digested and their boron concentrations were determined by ICP-AES.

Table 1 .
Crystal data and structure refinement of 5 and 6.
a B16 melanoma and HeLa cervical cancer cells were incubated for 72 h in the presence of compounds 5-8 and 13-16, and then the percentages of viable cells were determined by MTT assay.The drug concentrations required to inhibit cell viability by 50% (IC 50 ) were determined from semi-logarithmic concentration-response plots, and the results represent the means ± s.d. of triplicate samples.b n.d., not determined.