1,2-Diphenyl-o-carborane and Its Chromium Derivatives: Synthesis, Characterization, X-ray Structural Studies, and Biological Evaluations

The objective of this study is to design and synthesize substituted η6-chromium(0) tricarbonyl metal complexes carrying o-carborane units as potential boron neutron capture therapy (BNCT) agents. In this study, 1,2-diphenyl-o-carborane (1) units were used as starting materials to generate biologically active species. We investigated how the structural changes of 1 substituted with chromium(0) tricarbonyl affect the biological properties, and 1-(Phenyl-η6-chromium(0) tricarbonyl)-2-phenyl-o-carborane (2) and 1,2-bis(phenyl-η6-chromium(0) tricarbonyl)-o-carborane (3) species were produced in moderate yields. The molecular structures of compounds 1–3 were identified and established by infrared (IR); 1H, 11B, and 13C nuclear magnetic resonance (NMR) and X-ray crystallography analyses. Crystal structures of 1,2-diphenyl-o-carborane and the corresponding chromium complexes 1, 2, and 3 were obtained. In an in vitro study using B16 and CT26 cancer cells containing the triphenyl-o-carboranyl chromium(0) complexes Ph3C2BCr2 and Ph3C2BCr3, which we reported previously, compounds 2 and 3 accumulated at higher levels than compounds Ph3C2BCr2 and Ph3C2BCr3. However, the phenylated o-carboranyl chromium complexes have been found to be more cytotoxic than p-boronophenylalanine (BPA).


Introduction
Boron neutron capture therapy (BNCT) is a promising treatment for a variety of central nervous system (CNS) disorders, especially brain tumors. For example, BNCT is currently used as an adjunct to surgery for the treatment of glioblastoma multiforme [1]. The effect of BNCT is to suppress brain micrometastases that cannot be treated surgically or for which other treatment methods are not available [2]. BNCT destroys cancer cells with energy from targeted radiation bursts by reacting boron isotopes delivered to malignant cells with neutrons. Upon uptake into the cells, 10 B is externally irradiated with neutrons and becomes unstable [ 11 B]*, decaying to lithium ( 7 Li 3+ ) and releasing high-energy particles ( 4 He 2+ ). Another advantage of boron is that many 10 B atoms form a polyhedral cluster, such as B 10 H 10 2− and B 12 H 12 2− , enabling a high concentration of boron to be present in the molecule so that a satisfactory therapeutic effect can be expected. The high selectivity of neutron capture by the boron atoms and the effectiveness of treatment using it makes it an acceptable alternative to other chemotherapy methods that destroy cancer cells via high toxicity levels.
Compound o-carborane, namely 1,2-dicarba-closo-dodecaborane, C 2 B 10 H 12 , is a boronrich compound with an icosahedral structure and a diameter similar to that of a rotating benzene ring, nearly 1 nm, with high symmetry and remarkable stability under various conditions [3]. This cluster contains ten boron and two carbon atoms, making it well-suited as a BNCT agent [4,5], and also has potential in other fields of drug discovery, molecular To the best of our knowledge, this is the first example of the application of η 6 -arene chromium(0) tricarbonyl-substituted o-carborane compounds in BNCT. Until now, few η 6arene chromium(0) tricarbonyl-substituted o-carborane complexes [16][17][18] have been structurally characterized. Moreover, many studies have reported the introduction of various aryl groups to carbons and/or borons in o-carborane. However, no biological characteristics have been reported yet for them. This study is significant because it demonstrates the potential of phenylated o-carboranes with η 6 -chromium(0) tricarbonyl groups as potential BNCT agents.

Synthesis of 1,2-Diphenyl-o-carborane and Corresponding Chromium Metal Complexes
We previously reported the detailed synthesis of the compounds Ph3C2B, Ph3C2BCr2, and Ph3C2BCr3 [8,9]. Compound 1 was synthesized by a procedure modified according to previous results [19,20], as shown in Scheme 1. Compound 1,2-Diphenylo-carborane (1) was prepared from decaborane (B10H14) and diphenylacetylene in moderate yield in toluene solvent using N,N-dimethylaniline as a base. As a result, we first synthesized icosahedral o-carborane with phenyl substituents on the two carbons (Scheme 1). For comparison with previous results, we tried to confirm the electron-withdrawing ability of o-carborane by reacting chromium(0) hexacarbonyl [Cr(CO)6] with two phenyl groups introduced into o-carborane [21]. The reaction of 1 with Cr(CO)6 produced η 6 -phenyl-coordinated mono-and bis-chromium complexes (2 and 3) in moderate yields To the best of our knowledge, this is the first example of the application of η 6 -arene chromium(0) tricarbonyl-substituted o-carborane compounds in BNCT. Until now, few η 6 -arene chromium(0) tricarbonyl-substituted o-carborane complexes [16][17][18] have been structurally characterized. Moreover, many studies have reported the introduction of various aryl groups to carbons and/or borons in o-carborane. However, no biological characteristics have been reported yet for them. This study is significant because it demonstrates the potential of phenylated o-carboranes with η 6 -chromium(0) tricarbonyl groups as potential BNCT agents.

Synthesis of 1,2-Diphenyl-o-carborane and Corresponding Chromium Metal Complexes
We previously reported the detailed synthesis of the compounds Ph3C2B, Ph3C2BCr2, and Ph3C2BCr3 [8,9]. Compound 1 was synthesized by a procedure modified according to previous results [19,20], as shown in Scheme 1. Compound 1,2-Diphenyl-o-carborane (1) was prepared from decaborane (B 10 H 14 ) and diphenylacetylene in moderate yield in toluene solvent using N,N-dimethylaniline as a base. As a result, we first synthesized icosahedral o-carborane with phenyl substituents on the two carbons (Scheme 1). plexes, η 6 -arene chromium(0) tricarbonyl complexes have been the development due to their significant applications in organic synthesi the use of metal carbonyls in bioorganometallic chemistry is being inc to enhance the potential of chromium complexes [12][13][14][15]. To the best of our knowledge, this is the first example of the ap chromium(0) tricarbonyl-substituted o-carborane compounds in BNC arene chromium(0) tricarbonyl-substituted o-carborane complexes structurally characterized. Moreover, many studies have reported the ious aryl groups to carbons and/or borons in o-carborane. However, n teristics have been reported yet for them. This study is significant beca the potential of phenylated o-carboranes with η 6 -chromium(0) tricar tential BNCT agents.

Synthesis of 1,2-Diphenyl-o-carborane and Corresponding Chromium M
We previously reported the detailed synthesis of the co Ph3C2BCr2, and Ph3C2BCr3 [8,9]. Compound 1 was synthesized by fied according to previous results [19,20], as shown in Scheme 1. Comp o-carborane (1) was prepared from decaborane (B10H14) and diphenyl ate yield in toluene solvent using N,N-dimethylaniline as a base. As a thesized icosahedral o-carborane with phenyl substituents on the two For comparison with previous results, we tried to confirm the el ability of o-carborane by reacting chromium(0) hexacarbonyl [Cr(CO groups introduced into o-carborane [21]. The reaction of 1 with Cr(CO nyl-coordinated mono-and bis-chromium complexes (2 and 3) For comparison with previous results, we tried to confirm the electron-withdrawing ability of o-carborane by reacting chromium(0) hexacarbonyl [Cr(CO) 6 ] with two phenyl groups introduced into o-carborane [21]. The reaction of 1 with Cr(CO) 6 produced η 6phenyl-coordinated mono-and bis-chromium complexes (2 and 3) in moderate yields

IR and NMR Spectroscopy
The structure of 1,2-diphenyl-o-carborane (1) was proposed based on the assig of the 1 H, 11 B, and 13 C NMR resonances before confirmation by X-ray diffraction The 1 H NMR spectrum of compound 1 showed resonances at around δ 7.45~7.1 two phenyl rings. The 11 B NMR spectrum showed resonances at around δ −11.3 The 13 C NMR spectrum showed resonances at around δ 130.55~85. 16. The infra spectra of compounds 2 and 3 showed characteristic absorption bands of C≡O 1965, 1890 (2), 1965, and 1892 cm −1 (3). These values are lower than those of (C6H6) [22][23][24][25][26]. The IR and NMR spectra clearly indicate Cr coordination with the pheny of 2 and 3, with chemical shifts to the downfield region of δ 7.715-7.250 and 137.5 in the 1 H and 13 C NMR spectra, respectively (see Figures S1-S9 in Supplementary als).

X-ray Structural Studies of 1,2-Diphenyl-o-carborane and Corresponding Chromium Complexes
The selected crystallographic data and a summary of the intensity data collec rameters for 1, 2, and 3 are presented in Tables 1 and 2, respectively. Detailed info on the structural determinations and structural features of compounds 1, 2, and 3 vided in the Supplementary Materials and Appendix A. Additionally, the molecul tures and structural characteristics of Ph3C2B, Ph3C2BCr2, and Ph3C2BCr3 are cluded (Figures S1-S3 and Tables S10 and S11). Single-crystal X-ray structure de tion revealed the structural authenticity of each compound and suggested altera the electronic structure based on the changes in the C-C distance of o-carborane c The C1-C2 bond distance of 1 was 1.726(2) Å, which is significantly longer than C2 bond distance of the unsubstituted o-carborane [1.629(6) and 1.630(6) Å]. M our comparison of the C-C distances in compounds 1 and Ph3C2B (Table S11) that the bonds in Ph3C2B were slightly longer than those in compound 1. Com Ph3C2B contained further phenyl decorations at the boron atom while maintai active compound 1 platform [28,29].

IR and NMR Spectroscopy
The structure of 1,2-diphenyl-o-carborane (1) was proposed based on the assignments of the 1 H, 11 B, and 13 C NMR resonances before confirmation by X-ray diffraction (XRD). . These values are lower than those of (C 6 H 6 )Cr(CO) 3 [22][23][24][25][26]. The IR and NMR spectra clearly indicate Cr coordination with the phenyl groups of 2 and 3, with chemical shifts to the downfield region of δ 7.715-7.250 and 137.55-130.06 in the 1 H and 13 C NMR spectra, respectively (see Figures S1-S9 in Supplementary Materials).

X-ray Structural Studies of 1,2-Diphenyl-o-carborane and Corresponding Chromium Complexes
The selected crystallographic data and a summary of the intensity data collection parameters for 1, 2, and 3 are presented in Tables 1 and 2, respectively. Detailed information on the structural determinations and structural features of compounds 1, 2, and 3 are provided in the Supplementary Materials and Appendix A. Additionally, the molecular structures and structural characteristics of Ph3C2B, Ph3C2BCr2, and Ph3C2BCr3 are also included (Figures S1-S3 and Tables S10 and S11). Single-crystal X-ray structure determination revealed the structural authenticity of each compound and suggested alterations to the electronic structure based on the changes in the C-C distance of o-carborane cage [27]. The C1-C2 bond distance of 1 was 1.726(2) Å, which is significantly longer than the C1-C2 bond distance of the unsubstituted o-carborane [1.629(6) and 1.630(6) Å]. Moreover, our comparison of the C-C distances in compounds 1 and Ph3C2B (Table S11) showed that the bonds in Ph3C2B were slightly longer than those in compound 1. Compound Ph3C2B contained further phenyl decorations at the boron atom while maintaining the active compound 1 platform [28,29].
Single crystals of 1 and its chromium(0) tricarbonyl complexes, 2 and 3, suitable for X-ray crystallography, were obtained from dichloromethane solutions by slow evaporation. The general structural characteristics of compound 1 and its chromium complex are shown in Table 2 [30]. By comparing the data, it can be seen that the bond lengths, angles, and dihedral angles were nearly identical ( Figure 2).             Single crystals of 1 and its chromium(0) tricarbonyl complexes, 2 and 3, suitable for X-ray crystallography, were obtained from dichloromethane solutions by slow evaporation. The general structural characteristics of compound 1 and its chromium complex are shown in Table 2 [30]. By comparing the data, it can be seen that the bond lengths, angles, and dihedral angles were nearly identical ( Figure 2). As shown in Figure 3, the chromium atom of compound 2 was coordinated to the phenyl ring of the carbon atom of o-carborane via a π-bond, similar to the previous results [8,9]. The chromium metal adopted the typical three-legged "piano stool" geometry, with the expected geometrical parameters. The chromium metals were centered approximately  The Cr-CPh and Cr-CO bonds had average values of 2.210 and 1.856 Å, respectively, which were within the normal range [31]. The average C-O bond length in the chromium-coordinated carbonyl ligands was 1.143 Å, slightly shorter than that of the reported η 6 -arene chromium(0) tricarbonyl complexes [32][33][34][35][36]. As expected, the C1-C2 bond distance was 1.740(2) Å, slightly greater than compound 1, and was essentially in the range of nonbonding character [37][38][39][40]. Furthermore, as shown in Tables 2 and S6, the torsion angles of the two phenyl rings of 1 and 2 changed upon coordination with the chromium atom. The X-ray crystal structure of 3 ( Figure 4) reveals that the dichromium atoms adopted an η 6 -coordination with the two phenyl rings. The single crystal X-ray diffraction study of 3 revealed crystallization in the triclinic space group P-1. Interestingly, even though it was a triclinic space group, the APEX3 program identified the Z value as 8, which made it difficult to solve. For this reason, we attempted to obtain better results by changing the crystal system and the space group, but we were unable to obtain satisfactory results. In the end, this was considered to be recognized by four molecules as one molecule in the unit cell, and the structure was interpreted by adjusting the Z value to 2. The geometrical parameters of complex 3 were within the expected ranges ( Table 2). The average C-C bond length in the chromium-coordinated phenyl rings was 1.406 Å, 0.03 Å longer than the average bond length of 1.376 Å for the C-C bonds in 1. The Cr-CPh and Cr-CO bond lengths were within the normal range [31], with average values of 2.212 and 1.851 Å, respectively. The average C-O bond length in the chromium-coordinated carbonyl ligands was 1.138 Å, significantly shorter than the lengths of reported η 6 -arene chromium(0) tricarbonyl complexes [32][33][34][35][36]. As shown in Table 2, the chromium metals were centered approximately over the phenyl rings, giving rise to Cr1/Cr2-C6H5 face (centroid) distances of 1.687 and 1.705 Å, respectively. Interestingly, the C1-C2 bond length of 3 was shorter than that of 1 and 2. It was shown that the contraction of this bond was due to a favorable phenyl ring π*, and carboranyl σ* orbital interactions did not occur in 3, as has been observed in previous results [8,9]. As shown in Tables 2 and S9, the torsion angles of the phenyl rings of 1 and 3 or 2 and 3 changed upon coordination with the chromium atoms.
We next examined the level of intracellular accumulation of compounds 2, 3, Ph3C2BCr2, and Ph3C2BCr3 by determining their boron concentrations using ICP-OES. The intracellular boron uptake of compounds 2, 3, Ph3C2BCr2, and Ph3C2BCr3 in B16 and CT26 cells was higher than that of BPA (Table 3). Boron uptake from both bis-and tris-chromium(0) tricarbonyl-substituted compounds, which included the 1,2,3-triphenylo-carboranyl chromium tricarbonyl complexes (i.e., Ph3C2BCr2 and Ph3C2BCr3), was lower. These results show that the introduction of phenyl groups or chromium metals into the carborane backbone increases cytotoxicity rather than increasing boron accumulation in cancer cells. As shown in Figure 3, the chromium atom of compound 2 was coordinated to the phenyl ring of the carbon atom of o-carborane via a π-bond, similar to the previous results [8,9]. The chromium metal adopted the typical three-legged "piano stool" geometry, with the expected geometrical parameters. The chromium metals were centered approximately over the phenyl rings, giving rise to Cr1-C 6 H 5 face (centroid) distances of 1.702 Å. The average C-C bond length in the coordinated phenyl ring was 1.410 Å, 0.028 Å longer than the average bond length of 1.382 Å for C-C bonds within the non-coordinated phenyl ring. The Cr-C Ph and Cr-CO bonds had average values of 2.210 and 1.856 Å, respectively, which were within the normal range [31]. The average C-O bond length in the chromium-coordinated carbonyl ligands was 1.143 Å, slightly shorter than that of the reported η 6 -arene chromium(0) tricarbonyl complexes [32][33][34][35][36]. As expected, the C1-C2 bond distance was 1.740(2) Å, slightly greater than compound 1, and was essentially in the range of non-bonding character [37][38][39][40]. Furthermore, as shown in Tables 2 and S6, the torsion angles of the two phenyl rings of 1 and 2 changed upon coordination with the chromium atom.
The X-ray crystal structure of 3 ( Figure 4) reveals that the dichromium atoms adopted an η 6 -coordination with the two phenyl rings. The single crystal X-ray diffraction study of 3 revealed crystallization in the triclinic space group P-1. Interestingly, even though it was a triclinic space group, the APEX3 program identified the Z value as 8, which made it difficult to solve. For this reason, we attempted to obtain better results by changing the crystal system and the space group, but we were unable to obtain satisfactory results. In the end, this was considered to be recognized by four molecules as one molecule in the unit cell, and the structure was interpreted by adjusting the Z value to 2. The geometrical parameters of complex 3 were within the expected ranges ( Table 2). The average C-C bond length in the chromium-coordinated phenyl rings was 1.406 Å, 0.03 Å longer than the average bond length of 1.376 Å for the C-C bonds in 1. The Cr-C Ph and Cr-CO bond lengths were within the normal range [31], with average values of 2.212 and 1.851 Å, respectively. The average C-O bond length in the chromium-coordinated carbonyl ligands was 1.138 Å, significantly shorter than the lengths of reported η 6 -arene chromium(0) tricarbonyl complexes [32][33][34][35][36]. As shown in Table 2, the chromium metals were centered approximately over the phenyl rings, giving rise to Cr1/Cr2-C 6 H 5 face (centroid) distances of 1.687 and 1.705 Å, respectively. Interestingly, the C1-C2 bond length of 3 was shorter than that of 1 and 2. It was shown that the contraction of this bond was due to a favorable phenyl ring π*, and carboranyl σ* orbital interactions did not occur in 3, as has been observed in previous results [8,9]. As shown in Tables 2 and S9, the torsion angles of the phenyl rings of 1 and 3 or 2 and 3 changed upon coordination with the chromium atoms.

Determination of IC 50 and Incorporation of Boron into B16 and CT26 Cells
B16 mouse melanoma and CT26 colon carcinoma cells were treated with compounds 2, 3, Ph3C2BCr2, and Ph3C2BCr3 for 72 h, after which the cell viability was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Compounds 2, 3, Ph3C2BCr2, and Ph3C2BCr3 showed higher cytotoxicity than BPA (Table 3), with IC 50 (the half maximal inhibitory concentration) values in the range of 0.091-0.736 µM. Interestingly, the 1,2,3-triphenyl-o-carboranyl chromium(0) tricarbonyl complexes (Ph3C2BCr2 and Ph3C2BCr3) showed higher cytotoxicity to the B16 and CT26 cells than complexes 2 and 3. The higher cytotoxicity of compounds Ph3C2BCr2 and Ph3C2BCr3 to B16 cells may be a result of the difference in the mechanism by which the phenyl groups and chromium(0) tricarbonyl moieties induce cell toxicity. Compounds 2, 3, Ph3C2BCr2, and Ph3C2BCr3 exhibited similar activities in the CT26 and B16 cells, with IC 50 values in the range of 0.089-0.833 µM. Table 3. Cytotoxicity (IC 50 ) and boron accumulation of B16 melanoma and CT26 colon carcinoma cells. We next examined the level of intracellular accumulation of compounds 2, 3, Ph3C2BCr2, and Ph3C2BCr3 by determining their boron concentrations using ICP-OES. The intracellular boron uptake of compounds 2, 3, Ph3C2BCr2, and Ph3C2BCr3 in B16 and CT26 cells was higher than that of BPA (Table 3). Boron uptake from both bis-and tris-chromium(0) tricarbonyl-substituted compounds, which included the 1,2,3-triphenyl-o-carboranyl chromium tricarbonyl complexes (i.e., Ph3C2BCr2 and Ph3C2BCr3), was lower. These results show that the introduction of phenyl groups or chromium metals into the carborane backbone increases cytotoxicity rather than increasing boron accumulation in cancer cells.

General Procedure
All manipulations were performed under either a dry nitrogen or argon atmosphere using standard Schlenk techniques. Tetrahydrofuran (THF) was distilled from sodium and benzophenone under a nitrogen atmosphere. Elemental analyses were performed using a Carlo Erba Instruments CHNS-O EA 1108 analyzer. High-resolution tandem mass spectrometry (JMS-HX 110/110A, Jeol Ltd.) data were acquired at the Korean Basic Science Institute. 1 H, 11 B, and 13 C NMR spectra were recorded using a Bruker 600 spectrometer operating at 600.1, 150.9, and 192.6 MHz, respectively. All 11 B chemical shifts were referenced to BF 3 ·O(C 2 H 5 ) 2 (0.0 ppm), with a negative sign indicating an upfield shift. All proton and carbon chemical shifts were measured relative to the internal residual CHCl 3 in the lock solvent (99.9% CDCl 3 ). Decaborane was purchased from Katchem. N,N-dimethylaniline, 1,2-diphenylacetylene, n-BuLi (2.5 M in hexane), and chromium hexacarbonyl [Cr(CO) 6 ] were purchased from Aldrich Chemicals.

Crystal Structure Determination
Crystals of 1, 2, and 3 were obtained from toluene or CH 2 Cl 2 , sealed in glass capillaries under argon atmosphere, and mounted on a diffractometer. 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 (40 kV × 50 mA), using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å). The preliminary unit cell constants were determined using a set of forty-five narrow-frame (0.3 • in ω) scans. Doublepass 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 was used for data collection and SAINT was used for frame integration [41]. The final cell constants were determined by global refinement of the xyz centroids of the reflections harvested from the entire dataset. Structure solution and refinement were performed using the SHELXTL-PLUS software package [42].

Cell Viability Assay (MTT Assay)
B16 and CT26 cells were obtained from the Bioevaluation Center of the Korea Research Institute of Bioscience and Biotechnology, Korea. B16 and CT26 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Welgene, Gyeongsan-si, Republic of Korea) containing 10% fetal bovine serum (FBS; Welgene). The cells were cultured at 37 • C in an incubator with 5% CO 2 . The boron compounds were dissolved in dimethylsulfoxide (DMSO), and the resulting solution was diluted with Dulbecco's modified Eagle's medium (DMEM) (10% FBS), or p-boronophenylalanine (BPA) was directly dissolved in the same medium. In a 96-well culture plate (Falcon 3072), B16 mouse melanoma and CT26 colon carcinoma cells (5 × 10 3 cells/well) were cultured in five wells with a medium containing the boron compounds at various concentrations. This was followed by incubation for 72 h at 37 • C in a CO 2 incubator. DMSO is non-toxic at concentrations less than 0.5%, and control experiments confirmed the non-toxicity 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 to count the cells on a microplate reader. The concentration that resulted in a cell culture with 50% of the number of cells in the corresponding untreated group (IC 50 ) is summarized in Table 3.

In Vitro Boron Incorporation into B16 and CT26 Cancer Cells
B16 and CT26 cancer cells were cultured in Falcon 3025 dishes (150 mm in diameter). When the cell population increased to fill the dish (5 × 10 5 cells/dish), the boron compounds and BPA (10 µM) were added to the dishes. The cells were then incubated for 3 h at 37 • C in DMEM (20 mL of 10% FBS). The cells were washed three times with Ca/Mg-free PBS (-), collected with a rubber policeman, digested with a mixture of 60% HClO 4 -30% H 2 O 2 (1:2) solution (2 mL), and finally, decomposed for 1 h at 75 • C. After filtration through a membrane filter (Millipore, 0.22 mm), the boron concentration was determined using an inductively coupled plasma optical emission spectroscopy (ICP-OES) instrument (Avio 220 Max, PerkinElmer, Waltham, MA, USA). Each experiment was performed in triplicate.

Conclusions
In conclusion, this study describes the synthesis, X-ray structures, and biological activities of a series of 1,2-diphenyl-and 1,2,3-triphenyl-o-carborane compounds and their corresponding chromium(0) tricarbonyl transition metal complexes, which can be easily stoichiometrically substituted with transition metals to produce highly active biological molecules for BNCT. We presented a general and versatile method for the sequential introduction of phenyl groups into o-carborane and stoichiometrically-coordinated transition metal complexes using chromium(0) hexacarbonyl. Interestingly, it was confirmed that the C1-C2 bond length of compound 1 was the longest in compound 2 and the shortest in compound 3. We found that this affected the interaction of the π* orbital of the phenyl group with the σ* orbital of the carborane carbon by functioning as a strong π-receptor to form stable new types of organometallic complexes (2 and 3) through metal(Cr)-to-ligand(Ph) back-bonding interactions. The results clearly show the electron withdrawal properties of the o-carborane when the two phenyl groups are placed face-to-face with each other. Diphenyl-or triphenyl-o-carborane and the corresponding chromium complexes showed higher cytotoxicity than p-boronophenylalanine in B16 and CT26 cancer cell lines; however, boron accumulation was higher than that of p-boronophenylalanine. As a result, it was confirmed that as more chromium metal atoms and/or phenyl groups were substituted in the o-carborane backbone, cytotoxicity increased, but boron accumulation decreased.