MATERIALS AND METHODS
IR spectra were measured on a Shimadzu FT/IR-8100 spectrometer. 1H- and 13C-NMR spectra were obtained on a JEOL JNM ECZ600R at 25°C. Chemical shifts are expressed in δ ppm relative to the solvent peaks for 1H-NMR [(DMSO-d6) (2.50 ppm)] and 13C-NMR [DMSO-d6 (39.52 ppm)]. The signal assignments were confirmed by 1H–1H two-dimensional (2D) correlation spectroscopy (COSY), 1H–13C heteronuclear multiple-quantum coherence (HMQC), and 1H–13C heteronuclear multiple-bond connectivity (HMBC) spectra. High-resolution FAB-MS spectra [HR-MS (FAB)] were obtained by a JEOL JMS-700T mass spectrometer. Microanalyses were performed with a Yanaco MT-6 CHN corder.
Preparation of C2-Symmetrical DerivativesCompounds (1a, b and 1d–g) and C3-type compound 1i were prepared according to the reported procedure. The physical and spectroscopic data for these compounds shown in Fig. 1 have already been reported.7,12,13) Thioamide analogue 1e and new mode C2-symmetrical compound 1h were prepared by a procedure described below in good yields.
Preparation of N1,N8-Bis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl) Octanebis(thioamide) (1c)To a solution of N1,N8-bis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)octanediamide (576.3 mg, 1.00 mmol) in tetrahydrofuran (THF) (5 mL) was added Lawesson’s reagent (445.0 mg, 1.10 mmol). The resulting mixture was stirred at 70°C for 3 h. After cooling to room temperature, the solvents that were used were evaporated under reduced pressure. The obtained crude material was washed with CH2Cl2 to give the desired product (1c) (337.4 mg, 55% yield) as a white solid. Mp 230–233°C. 1H-NMR (DMSO-d6) δ: 1.30 (24H, s, CH3), 1.34–1.44 [4H, m, C(=S)-CH2-CH2-CH2-CH2-CH2-CH2-C(=S)], 1.72–1.84 [4H, m, C(=S)-CH2-CH2-CH2-CH2-CH2-CH2-C(=S)], 2.74 [4H, t, J = 7.5 Hz, C(=S)-CH2-CH2-CH2-CH2-CH2-CH2-C(=S)], 7.40 (2H, dd, J = 7.2, 8.4 Hz, Ar H-5), 7.51 (2H, d, J = 7.2 Hz, Ar H-4), 7.93 (2H, s, Ar H-2), 8.08 (2H, d, J = 8.4 Hz, Ar H-6), 11.48 (2H, s, NH); 13C-NMR (DMSO-d6)16) δ: 24.7 (CH3), 28.1 [C(=S)-CH2-CH2-CH2], 29.3 [C(=S)-CH2-CH2-CH2], 46.9 [C(=S)-CH2-CH2-CH2], 83.8 (B-O-C-C-O-B), 126.2 (Ar C-6), 128.0 (Ar C-5), 129.3 (Ar C-2), 131.7 (Ar C-4), 139.2 (Ar C-1), 204.1 (C = O). IR (neat) 3152, 2978, 2928, 1532, 1353 cm−1. MS m/z: 609 [(M + H)+]. HR-MS (FAB) m/z: 609.3156 (Calcd for C32H47B2N2O4S2+: 609.3158). Anal. Calcd for C32H46B2N2O4S2·0.9H2O: C, 61.53; H, 7.71; N, 4.48. Found: C, 61.65; H, 7.43; N, 4.20.
Preparation of 5,12-Dihydro-5,12-diborabenzo[c]isochromeno[3,4-g]chromene-5,12-diol (1h)To 2,5-diphenylhydroquinone (262.3 mg, 1.00 mmol) were added BCl3 in n-hexane (3.000 mL, 3.00 mmol) and AlCl3 (26.66 mg, 0.2000 mmol). The resulting mixture was stirred at 75°C for 6 h. After cooling to room temperature, ice was added slowly and diethyl ether was added to the solution. After stirring for 20 min, the product was extracted with diethyl ether (× 3) and washed with brine, and the combined organic extract containing a white solid material was filtered. The isolated material was washed with diethyl ether to afford the desired product (1h) (108.4 mg, 35% yield) as a white solid. Mp>250°C. 1H-NMR (DMSO-d6) δ: 7.52 (2H, dd, J = 7.2 Hz, J = 7.5 Hz, H-3, 10), 7.72–7.79 (2H, m, H-2, 9), 8.06 (2H, s, H-7, 14), 8.12 (2H, d, J = 7.5 Hz, H-4, 11), 8.34 (2H, d, J = 8.4 Hz, H-1, 8), 9.43 (2H, s, OH); 13C-NMR (DMSO-d6)16) δ: 113.3 (C-7, 14), 122.4 (C-1, 8), 123.2 (C-7a, 14a), 127.6 (C-3, 10), 132.5 (C-2, 9), 133.5 (C-4, 11), 139.2 (C-7b, 14b), 146.5 (C-6a, 13a). IR (neat) 3397 cm−1. MS m/z: 313 [(M−H)−]. HR-MS(FAB) m/z: 313.1044 (Calcd for C18H11B2O4−: 313.0849).
Cell CultureTwo human cancer cell lines (U251, KB3-1) were used. The cells were incubated at 37°C in an atmosphere of 5% CO2 and 95% air.
Anticancer AssayAnticancer activities of the synthesized compounds against the two cancer cell lines (U251 and KB3-1) were evaluated by the reported procedure.18) Cell proliferation in vitro was estimated (measured) by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation in vitro was assessed by the MTT colorimetric assay in 96-well plates. Cells (5 × 103) were inoculated into each well. After overnight incubation (37°C in 5% CO2), synthesized compounds (1a–h) were added to the culture and incubated for 5 d. Thereafter, 50 µL of MTT (1 mg/mL) was added to each well and the plates were incubated for an additional 4 h. After aspiration of the culture medium, the resulting formazan was dissolved with 100 µL of dimethylsulfoxide (DMSO). The plates were read at 570 nm using a micro-plate reader. The 50% tumor growth inhibitory ratios (IC50 values) were estimated and used for structure–activity relationship (SAR) discussion of the compounds. The determined IC50 values are summarized in Table 1.
Table 1. Anticancer Activities (IC
50) of Target Molecules against Human Brain Glioma Cells (U251 Cells) and Human Carcinoma Cells (KB3-1 Cells), Cytotoxicities (IC
50) against Vero Cells and Anti-HSV-1 Activities (EC
50)
| Compound | IC50 (µM)a) | EC50 (µM)b) |
|---|
| Vero cellsb) | U251 | KB3-1 | Anti-HSV-1 |
|---|
| 1a | 25.2 | >100 | >100 | 8.0 |
| 1b | 28.8 | 44.4 | >100 | >100 |
| 1c | 14.8 | >100 | >100 | 4.0 |
| 1d | 76.2 | >100 | >100 | >100 |
| 1e | >200 | >100 | 33.9 | >100 |
| 1f | 5.43 | 39.6 | 32.5 | >100 |
| 1g | >200 | 19.0 | 3.78 | >100 |
| 1h | 11.5 | 48.6 | 64.9 | 5.5 |
| 1i | >200 | >100 | 26.3 | 65.2 |
| Cisplatin | — | 3.06 | 6.90 | — |
a) IC50: concentration that produces 50% inhibition of proliferation after 5 d of incubation. b) Data were taken from refs. 7, 12, and 13.
The minimum inhibitory concentration (MIC) values of the synthesized symmetrical boronic acid derivatives against standard strains (Gram-positive and Gram-negative bacteria) were determined by the authentic microdilution method to monitor bacterial growth turbidity in Muller–Hinton broth according to the Japanese Society of Chemotherapy.19,20) The anti-herpes simplex virus (HSV)-1 activities (EC50) of the synthesized boronic acid derivatives were determined by using a plaque reduction assay21) and their cytotoxicity against Vero cells (IC50) was also determined by our previously described method.7,12,13) The cytotoxic activities against Vero cells (IC50) are shown in Table 1.
RESULTS AND DISCUSSION
The structures of the tested phenylboronic acid-related compounds are shown in Fig. 1. The cytotoxicities of the compounds (1a–i) (IC50 values) against Vero cells, their anti-HSV-1 activities and their anti-proliferative activities (IC50 values) against a human brain glioma cell line (U251) and a human carcinoma cell line (KB3-1) are also summarized in Table 1. As shown in Table 1, many of the tested compounds showed significant anti-proliferative activities against U251 and KB3-1 cells. Three compounds, 1a, 1c and 1d, showed no anti-proliferative activity against either of the cancer cell lines. Among compounds that are structurally free phenylboronic acid derivatives and have a slightly different methylene linker length (1d–g) (see Table 1), three compounds (1e–g) showed significant anti-proliferative activities against U251 or KB3-1 cells. Among the tested compounds, C2-symmetrical bivalent phenylboronic acid derivative 1g showed the highest activities against both cancer cell lines (IC50 = 19.0 and 3.78 µM, respectively), and the activity tended to increase as the linker methylene chain became longer. It is notable that the anti-proliferative activity of compound 1g against KB3-1 cells was higher than that of the control drug cisplatin (IC50 = 6.90 µM). The phenylboronic acid ester 1a, thioamide analogue 1c and compound 1d, having the shortest linker length, showed no anti-proliferative activity against either cancer cell line (IC50 = >100 µM).
Although compound 1g in the series of compounds 1d–g showed the highest level of anti-proliferative activity, it is noteworthy that the bivalent C2-symmetrical phenylboronic acid derivative 1a in the ester series corresponded to compounds 1d–g showed the highest levels of antibacterial activity and anti-HSV-1 activity (MIC = 27.1 µM and 8.0 µM, respectively).7) A possible reason for compound 1g having the highest anti-proliferative activity against both U251 and KB3-1 cells with no anti-HSV-1 activity (EC50 = >100 µM) is that the length of methylene linkers in these bivalent C2-symmetrical molecules contributes to the potential of anti-proliferative activity.
Symmetrical no linker mode new compound 1h, which showed a high level of cytotoxic activity against Vero cells (IC50 = 11.5 µM), also showed moderate anti-proliferative activities against both U251 and KB3-1 cells (IC50 = 48.6 and 64.9 µM, respectively), but notable characteristics were not observed. In contrast, it is noteworthy that the C3-symmetrical phenylboronic acid pinacol ester derivative 1i12,22) showed about 5-times higher anti-proliferative activity (IC50 = 26.3 µM) against human carcinoma cells (KB3-1 cells) than that against U251 cancer cells (IC50=>100 µM).
Regarding cytotoxic activities (IC50 values) for the listed compounds (1a–i) against Vero cells, there were few distinct correlations between IC50 values and anticancer activities against U251 and KB3-1 cancer cells (IC50 values) as shown in Table 1.
Based on the above-described interesting discovery, we consider that some molecules may serve as a guide for the development of more interesting anticancer leads. Thus, we are further developing molecular modifications of derivatives that are structurally similar to the bivalent phenylboronic acid 1g, and we will perform biological evaluation of the obtained boronic acid derivatives using other human cancer cell lines in addition to the above cancer cell lines. The finding of selectivity of C3-type phenylboronic acid ester 1i against species of cancer cells also seems to be interesting for a lead molecule, and we are considering addressing this issue in the future as well. The results of bioassays of newly targeted molecules will be reported separately in detail.
