Abstract
Diaryl-substituted carboranes, as a new class of HIF-1α inhibitors, were synthesized from the corresponding diaryl-substituted alkynes by decaborane coupling. The microwave-irradiated conditions with a combination of N,N-dimethylaniline and chlorobenzene were effective to obtain the diaryl-substituted carboranes in good to high yields. Among the compounds synthesized, compounds 1a and 1d showed significant inhibition of HIF-1 mediated transcriptional activity under hypoxia. Both compounds similarly suppressed hypoxia-induced HIF-1α accumulation in a concentration-dependent manner without affecting HIF-1α mRNA expression.
Introduction
Hypoxic regions in solid tumors are often associated with tumor angiogenesis, proliferation, invasion, and metastasis [1]. Hypoxia-inducible factor 1 (HIF-1), is a transcription factor principally by which cancer cells adapt to the hypoxic microenvironment caused by rapid proliferations [2, 3]. It regulates the expression of hundreds of genes in response to hypoxia. HIF-1 is a heterodimeric transcription factor consisting of two subunits, HIF-1α and HIF-1β/ARNT, functions to stimulate the physiological expression of those angiogenesis factors [3]. HIF-1α undergoes hydroxylation of proline residues by prolyl hydroxylase (PHD) under normoxia [4, 5]. This modification is recognized by the von Hippel–Lindau (VHL) tumor suppressor protein that ubiquitinates HIF-1α to lead proteasomal degradation [6]. On the other hand, the hydroxylation by PHD is inactivated under hypoxia and the unmodified HIF-1α is sufficiently stabilized to form a heterodimeric complex with HIF-1
Combretastatin A-4 (CA-4) is a mitotic agent isolated from the bark of the South African bush willow Combretum caffrum. CA-4 and its phosphate prodrug (CA4P) also possesse vascular disruption that selectively damages tumor neovasculature with induction of extensive blood flow shutdown in metastatic tumor compared to normal tissues [8]. Dachs et al. found that CA4P reduced HIF-1 accumulation in endothelial cells under hypoxia and adversely affected members of the HIF-1 signal transduction pathway [9].
Carboranes (icosahedral dicarba-closo-dodecaboranes; C2B10H12) possess a stable B–B–B three-center two-electron bond and show remarkable thermal and catabolic stability [10]. Because of their icosahedral geometry and exceptional hydrophobicity, carboranes have been used as an alternative hydrophobic pharmacophore [11–15] in pharmaceutical drug design [16–20]. We recently synthesized diaryl-substituted ortho- and meta-carboranes as mimics of manassantin A and found their significant inhibitory activities toward HIF-1α protein accumulation, resulting in suppression of HIF-1 transcriptional activity in human cervical cancer cells, HeLa cells, under hypoxia [21]. According to the structure-activity relationship study, a diaryl substituted carborane framework is essential for inhibition of HIF-1α protein accumulation. In this paper, we summarized the design and synthesis of diaryl-substituted ortho-carborane analogs 1 based on the cis geometry of the stilbene framework in CA-4 (Fig. 1) and the target identification [22].
Design of diaryl-substituted ortho-carborane analogs 1.
Results and discussion
Chemistry
Synthesis of diaryl-substituted ortho-carborane analogs 1 is shown in Scheme 1. The terminal alkynes 2 derived from the corresponding aldehydes in two steps [23] underwent the Sonogashira coupling with aromatic iodides (ArI) in the presence of Pd(PPh3)4 catalyst (10 mol%) in THF to give corresponding diaryl-substituted alkynes 3a–d in 42–90 % yields. The decaborane coupling of alkyne 3a with decaborane (B10H14) proceeded in the presence of acetonitrile as the Lewis base under toluene reflux conditions [24]. The yields are shown in the parenthesis of Table 1. However, 1a was obtained only in 20 % yield. Furthermore, in the cases of 3b–d, corresponding diaryl-substituted ortho-carboranes 1b–d were obtained in poor yields (5–12 %) under similar conditions. Therefore, we optimized the reaction condition for decaborane coupling using 1,2-diphenylethyne as a model compound. The results are summarized in Table 2. The reaction of 4 with decaborane proceeded in the presence of acetonitrile for 8 h under toluene-refluxed conditions to give 1,2-diphenyl-ortho-carborane 5 in 30 % yield (entry 1). Prolonging the reaction time did not increase the yield (entry 2). Next, the reaction was carried out by microwave irradiation in a sealed tube. Although the reaction time was reduced by the microwave irradiation, the condition did not increase the yields of 5 (entries 3 and 4). A higher reaction temperature did not affect the reaction yield (entry 5). We also employed propionitrile, which was efficient in our previous experiments, as a Lewis base, however it was not effective for the coupling (entry 6). Recently, Nagasawa et al. reported that the combination of N,N-dimethylaniline and chlorobenzene was effective for the decaborane coupling under microwave irradiated conditions. Indeed, the coupling of decaborane with 1,2-diphenylethyne proceeded smoothly in the presence of three equivalents of N,N-dimethylaniline as the Lewis base in chlorobenzene by microwave irradiation for 15 min, giving 1,2-diphenyl-ortho-carborane in 75 % yield (entry 7). Therefore, we synthesized ortho-carborane analogs 1 again by coupling decaborane with 3 under the optimum conditions. As shown in Table 1, the diaryl-substituted ortho-carborane analogs 1a–g were obtained in 50–78 % yields by microwave irradiation in the presence of N,N-dimethylaniline in chlorobenzene.
Synthesis of diaryl-substituted ortho-carborane analogs 1.
Chemical yieldsa and inhibition of HIF-1 transcriptional activity in cell-based HRE reporter assay.b
| Compound | Yield of 1 (%) |
HRE reporter assay IC50 (μM) | |
|---|---|---|---|
| (a) Toluene-reflux | (b) Microwave | ||
| 1a | 20 | 61 | 0.6 |
| 1b | 6 | 50 | 1.2 |
| 1c | 12 | 71 | 5.1 |
| 1d | 9 | 53 | 0.8 |
| 1e | 20 | 78 | 6.4 |
| 1f | 14 | 53 | 5.5 |
| 1g | 22 | 63 | 4.6 |
| YC-1 | – | – | 1.5 |
aReactions were carried out in the presence of three equivalent of N,N-dimethylanline as a Lewis base in chlorobenzene under microwave-irradiated conditions in a sealed tube for 15 min. bHeLa cells expressing HRE-dependent firefly luciferase reporter construct (HRE–Luc) and constitutively expressing CMV-driven Renilla luciferase reporter with SureFECT Transfection Reagent were incubated for 12 h with or without drugs under normoxic or hypoxic conditions. After removal of the supernatant, luciferase assay was performed using a Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The fluorescence intensity of HRE–Luc was normalized to that of constitutively expressing CMV-driven Renilla luciferase control. The drug concentration required to inhibit relative light units by 50 % (IC50) was determined from semi-logarithmic dose–response plots.
Optimization of reaction conditions for the decaborane coupling.
 |
|||||
|---|---|---|---|---|---|
| Entry | Base | Solvent | Temp/°C | Time | Yield/% |
| 1 | CH3CN | Toluene | 110a | 8 h | 30 |
| 2 | CH3CN | Toluene | 110a | 22 h | 30 |
| 3 | CH3CN | Toluene | 110b | 30 min | 7 |
| 4 | CH3CN | Toluene | 110b | 1 h | 35 |
| 5 | CH3CN | Toluene | 150b | 1 h | 31 |
| 6 | CH3CH2CN | Toluene | 110b | 1 h | 30 |
| 7 | N,N-dimethylaniline | Chlorobenzene | 150b | 15 min | 75 |
aReactions were carried out under toluene reflux conditions. bReactions were carried out under microwave-irradiated conditions in a sealed tube.
Biology
The synthesized diaryl-substituted ortho-carborane analogues 1a–g were evaluated for their ability to inhibit transcriptional activity of HIF-1 by a cell-based reporter gene assay under hypoxic condition. We conduced HRE-dependent firefly luciferase reporter (HRE-Luc) and constitutively expressing CMV-driven Renilla luciferase reporter expressing HeLa cells [25]. The concentrations of compounds required to inhibit relative light units by 50 % (IC50) are summarized in Table 1 and YC-1 (3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole) was used as the positive control [26, 27]. All the synthesized ortho-carborane analogs 1a–g significantly inhibited the transcriptional activity of HIF-1 under hypoxic condition with IC50 values between 0.6 to 6.4 μM. It is notable that compounds 1a, 1b, and 1d exhibited higher inhibitory activity than YC-1, and the IC50 values being 0.6, 1.2, and 0.8 μM, respectively.
Because significant inhibition of the hypoxia-induced HIF-1 transcriptional activity by ortho-carborane analogs 1a and 1d was observed in HeLa cells, we next examined the effects of those two compounds on HIF-1α protein accumulation and HIF-1α mRNA expression under hypoxic condition. The results are shown in Fig. 2. According to the western blotting analysis, both compounds 1a and 1d similarly suppressed accumulation of HIF-1α in a concentration-dependent manner without affecting the expression of tubulin protein under hypoxic condition (Fig. 2a). RT-PCR analysis revealed that both compounds 1a and 1d inhibited VEGF mRNA expression, but did not suppress the HIF-1α mRNA expression (Fig. 2b). These results indicate that the inhibition of HIF-1 transcriptional activation by compounds 1a and 1d is mediated by the suppression of HIF-1α protein accumulation without affecting HIF-1α mRNA level in HeLa cells under hypoxic conditions.
Effect of compounds 1a and 1d on HIF-1α protein and mRNA expression under hypoxic condition. (a) HeLa cells were incubated for 4 h with compounds 1a and 1d at the indicated concentrations under hypoxic condition. HIF-1β was used as the loading control and ‘–’ is DMSO treated control. The levels of each protein were detected by immunoblot analysis with HIF-1α or HIF-1β specific antibodies. (b) After incubation with compounds 1a and 1d for 4 h, mRNA level of HIF-1α and VEGF was detected by RT-PCR analysis. GAPDH was used as the loading control and ‘–’ is DMSO treated control.
Chemical biology
To further clarify the mechanism of compounds 1a and 1d against the inhibition of HIF-1 transcriptional activity, we designed and synthesized multifunctional molecular probe 10 based on conventional chemical biology techniques, including photo-affinity labelling and click conjugation, which are highly useful tools for the identification of target proteins of biologically active molecules having undefined mode of action [28–30]. As shown in Scheme 2, we synthesized a multifunctional molecular probe of 1a by substituting a benzophenone moiety that would form covalent bonds with target proteins by UV irradiation (photo-affinity labelling), and an acetylene moiety that would conjugate with fluorescent azide or azide-linked biotin by the click reaction. Aniline derivative 5, the starting material, was converted into iodoketone 6. The Sonogashira coupling of ethynylbenzene 2a with iodoketone 6 gave the diaryl-substituted alkyne 7, which underwent the coupling with decaborane to afford diaryl-substituted ortho-carborane 8. Hydrogenesis of 8 was carried out in the presence of Pd/C and the resulting phenoxy group of 9 was propargylated under basic conditions to give multifunctional probe molecule 10.
Synthesis of a multifunctional probe molecule.
To confirm whether the synthesized probe 10 exhibits HIF inhibitory activity, we examined the effects of probe 10 on activation of HIF in HeLa cells under hypoxic condition. As shown in Fig. 3, probe 7 inhibited the hypoxia-induced HIF-1α protein accumulation similar to compounds 1a and 1d.
Effect of compounds 1a, 1d, and 10 on HIF-1α protein accumulation induced by hypoxia. HeLa cells were incubated with compounds at 3 and 30 μM under hypoxic condition for 4 h. Tubulin was used as the loading control and ‘–’ is DMSO treated control. The levels of each protein were detected by immunoblot analysis with HIF-1α or tubulin specific antibodies.
We next performed photo-affinity labeling in HeLa cell lysate and click conjugation with Alexa Fluor 488-azide to visualize the target protein. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), proteins bound to the probe-Alexa Fluor 488 were visualized by direct in-gel fluorescence detection. As shown in Fig. 4a, a major fluorescent band around the 55 kDa molecular weight was detected by the probe 10 in a concentration-dependent manner. We also performed photo-affinity labeling of the probe 10 with tubulin in vitro and analyzed by SDS-PAGE. The band conjugated to tubulin is similar molecular weight to the band indicated by arrow. To confirm the specificity of probe binding, we performed competition assays with 1d. As shown in Fig. 4b, the fluorescent band around the 55 kDa molecular weight was diminished by addition of 1d. These results raise the possibility that tubulin may be one of the major target proteins of 1d and its probe 10.
Fluorescence imaging of photo-affinity labeling followed by click conjugation using the probe 10 in HeLa cell lysate. (a) HeLa cell lysate was irradiated for 30 min at 360 nm with various concentrations (10–1000 μM) of the probe 10. The conjugation of probe and Alexa Flour 488-azide was performed by click reaction. (b) HeLa cell lysates was photoaffinity-labeled with 10 (2–200 μM) in the presence of 1 (300 μM).
We next examined effects of compounds 1a, 1d, and 10 on levels of acetylated tubulin, an indication of polymerized tubulin. As shown in Fig. 5, compound 1a strongly inhibited acetylation of tubulin. Compounds 1d also inhibited the acetylation of tubulin at 30 μM, but compound 10 did not show inhibitory effect. These results indicate that compounds 1a and 1d exhibit the inhibitory activity against tubulin polymerization.
Effects of compounds 1a, 1d, and 10 on level of acetylated tubulin. HeLa cells were incubated with compounds at 3 and 30 μM for 4 h. Tubulin was used as the loading control and ‘–’ is DMSO treated control. The levels of each protein were detected by immunoblot analysis with acetylated (Ac) tubulin or tubulin specific antibodies.
Conclusion
We succeeded in the synthesis of diaryl-substituted ortho-carboranes 1a–g as analogs of CA-4 by coupling decaborane with alkynes 3a–g in good yields. Microwave irradiation was effective for the coupling in the presence of N,N-dimethylaniline as the Lewis base in chlorobenzene. Significant inhibition of the hypoxia-induced HIF-1 transcriptional activity was observed in all the synthesized diaryl-substituted ortho-carborane analogs 1a–g with IC50 values between 0.6 to 6.4 μM. In particular, compounds 1a and 1d had high potency: both compounds similarly suppressed the hypoxia-induced HIF-1α accumulation in a concentration-dependent manner without affecting HIF-1α mRNA expression. The photo-affinity labeling revealed that tubulin is one of the target proteins of diaryl-substituted ortho-carborane analogs 1, indicating that the suppression mechanism of 1 toward the hypoxia-induced HIF-1α accumulation is similar to that of CA-4.
Experimental
General Information
Analytical thin layer chromatography (TLC) was performed on a glass plates of silica gel 60 GF254 (Merck), which were visualized by the quenching of UV fluorescence (254 nm), and/or by an aqueous alkaline KMnO4 solution followed by heating. Column chromatography was conducted on silica gel (Merck Kieselgel 70–230 mesh). Most commercially supplied chemicals were used without further purification. 1H and 13C NMR spectra were recorded on a JEOL JNM-AL 300 (300 MHz) or a VARIAN UNITY-INOVA 400 (400 MHz) spectrometer. The chemical shifts are reported in δ units relative to internal tetramethylsilane. IR spectra were recorded on a Shimadzu FTIR-8200A spectrometer. Liquid chromatogram mass spectrometer was recorded on Shimadzu LCMS-2010EV. High-resolution mass spectra (ESI) were recorded on a Bruker Daltonics micro TOF-15 focus. Decaborane was purchased from Katchem spol. s r. o. (Praha, Czech Republic). Most commercially supplied chemicals were used without further purification. Compound 3a–g were synthesized according to the literature procedure [31].
Typical procedure for synthesis of 1-(3-Hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl) -ortho-carborane (1a)
A mixture of decaborane (17.5 mg, 0.14 mmol), 3a (30 mg, 0.095 mmol), N,N-dimethylaniline (22.2 μL, 0.28 mmol) was dissolved in chlorobenzene (1 mL) and microwave irradiation was carried out at 120 °C for 15 min under nitrogen. The reaction mixture was extracted with EtOAc three times and combined organic layers were washed with water, 0.5 N HCl aq., and brine, dried over MgSO4 and concentrated. The residue was purified by column chromatography on silica gel (Hexane/EtOAc = 10:1 to 1:1) to give 1a as a white solid (25.5 mg, 61 %). M.p. 152–153 °C; 1H NMR (400 MHz; CDCl3) δ 7.08 (m, 1H), 6.94 (m, 1H), 6.63 (s, 2H), 6.60 (s, 1H), 6.58 (d, J = 8.0 Hz, 1H), 5.50 (s, 1H), 3.83 (s, 3H), 3.78 (s, 3H), 3.73 (s, 3H); 13C NMR (75 MHz; CDCl3) δ 152.2, 148.0, 144.9, 125.9, 124.0, 123.1, 117.3, 109.7, 108.5, 85.8, 85.5, 77.4, 77.2, 77.0, 76.6, 60.8, 56.2, 55.9; 11B NMR (96.3 MHz; CDCl3) δ –15.42, –7.71; IR (KBr) 2548, 1589, 1508, 1337, 1130, 835 cm–1; HRMS (ESI-TOF) m/z calcd for C18H27B10O5 [M–] 431.2871; found 431.2876.
1-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-ortho-carborane (1b)
White solid; m.p. 100–102 °C; 1H NMR (400 MHz; CDCl3) δ 7.36 (d, J = 9.2 Hz, 2H), 6.67 (d, J = 9.2 Hz, 2H), 6.60 (s, 2H), 3.77 (s, 3H), 3.74 (s, 3H), 3.71 (s, 6H); 13C NMR (100 MHz; CDCl3) δ 161.0, 152.3, 139.6, 132.1, 126.0, 123.1, 113.6, 108.5, 85.9, 85.4, 60.8, 56.2, 55.3; 11B NMR (96.3 MHz; CDCl3) δ –14.74, –7.22; IR (KBr) 2548, 1589, 1508, 1337, 1130, 835 cm–1; HRMS (ESI-TOF) m/z calcd for C18H28B10O4Na [M + Na+] 439.2893; found 439.2892.
1-(3-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-ortho-carborane (1c)
White solid; m.p. 128–129 °C; 1H NMR (400 MHz; CDCl3) δ 7.04–7.12 (m, 2H), 6.98 (s, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.61 (s, 2H), 3.77 (s, 3H), 3.70 (s, 9H); 13C NMR (75 MHz; CDCl3) δ 161.0, 152.3, 139.6, 132.1, 126.0, 123.1, 113.6, 108.5, 85.9, 85.4, 60.8, 56.2, 55.3; 11B NMR (96.3 MHz; CDCl3) δ –15.24, –7.22; IR (KBr) 2592, 1586, 1509, 1455, 1415, 1335, 1249, 1130, 1000 cm–1; HRMS (ESI-TOF) m/z calcd for C18H28B10O4Na [M + Na+] 439.2887; found 439.2892.
1-(2-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-ortho-carborane (1d)
White solid; m.p. 137–140 °C; 1H NMR (400 MHz; CDCl3) δ 7.53 (d, J = 9.6 Hz, 1H), 7.23–7.28 (m, 1H), 6.74–6.81 (m, 2H), 6.65 (s, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.69 (s, 6H); 13C NMR (100 MHz; CDCl3) δ 159.0, 152.2, 139.4, 134.6, 132.1, 126.7, 120.6, 118.6, 112.6, 108.1, 86.6, 83.8, 60.8, 56.1, 55.4; 11B NMR (96.3 MHz; CDCl3) δ –15.04, –8.40, –6.57; IR (KBr) 2563, 1585, 1508, 1128, 754, 628 cm–1; HRMS (ESI-TOF) m/z calcd for C18H28B10O4Na [M + Na+] 439.2888; found 439.2892.
1-(4-Methoxyphenyl)-2-(3,4-dimethoxyphenyl)-ortho-carborane (1e)
White solid; m.p. 119–120 °C; 1H NMR (400 MHz; CDCl3) δ 7.35 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.65–6.60 (m, 3H), 3.80 (s, 3H), 3.73 (s, 6H); 13C NMR (100 MHz; CDCl3) δ 160.9, 150.4, 148.0, 132.1, 124.0, 123.3, 123.1, 113.6, 113.5, 110.2, 85.9, 55.8, 55.3; 11B NMR (96.3 MHz; CDCl3) δ –15.44, –13.86, –7.51; IR (KBr) 2960, 2636, 1518, 1332, 1022, 862, 615 cm–1; HRMS (ESI-TOF) m/z calcd for C17H26B10O3Na [M + Na+] 409.2783; found 409.2784.
1-(3-Methoxyphenyl)-2-(3,4-dimethoxyphenyl)-ortho-carborane (1f)
White solid; m.p. 95–98 °C; 1H NMR (400 MHz; CDCl3) δ 7.02–7.09 (m, 3H), 6.98 (s, 1H), 6.86 (s, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.61 (d, J = 8.8 Hz, 1H), 3.80 (s, 3H), 3.72 (s, 3H), 3.70 (s, 3H); 13C NMR (100 MHz; CDCl3) δ 159.0, 150.5, 148.0, 132.1, 129.2, 124.0, 123.1, 117.2, 115.1, 113.5, 110.2, 85.6, 85.0, 55.9, 55.3; 11B NMR (96.3 MHz; CDCl3) δ –15.21, –13.67, –7.22; IR (KBr) 2567, 1601, 1520, 1414, 1271, 1153, 1024, 854 cm–1; HRMS (ESI-TOF) m/z calcd for C17H26B10O3Na [M + Na+] 409.2783; found 409.2784.
1-(2-Methoxyphenyl)-2-(3,4-dimethoxyphenyl)-ortho-carborane (1g)
White solid; m.p. 132–134 °C; 1H NMR (400 MHz; CDCl3) δ 7.52 (d, J = 8.4 Hz, 1H), 7.23 (t, J = 8.4 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H), 6.90 (s, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.73 (t, J = 8.0, 1H), 6.60 (d, J = 8.8, 1H), 3.79 (s, 6H), 3.68 (s, 3H); 13C NMR (100 MHz; CDCl3) δ 159.0, 150.2, 147.8, 134.5, 132.0, 124.0, 123.7, 120.5, 113.3, 112.6, 110.1, 87.1, 83.8, 55.8, 55.4; 11B NMR (96.3 MHz; CDCl3) δ –15.21, –8.59, –6.54; IR (KBr) 2554, 1599, 1518, 1267, 1155, 1024, 758 cm–1; HRMS (ESI-TOF) m/z calcd for C17H26B10O3Na [M + Na+] 409.2783; found 409.2785.
(4-Benzyloxy)phenyl- (4-Iodophenyl)methanone (6)
To a solution of 5 (80 mg, 0.3 mmol) in acetonitrile (1.3 mL) were added HCl (1N, 1.2 mL) and NaNO2 (25 mg, 0.36 mmol) dissolved in water (0.5 mL) at 0 °C, and the mixture was stirred for 30 min. An aqueous solution of KI (125 mg, 0.75 mmol, 0.5 mL) was added and the mixture was stirred for 5 h at 0 °C. The reaction mixture was extracted with CH2Cl2 three times and combined organic layers were washed with water followed by brine, dried over MgSO4 and concentrated. The residue was purified by column chromatography on silica gel (Hexane/EtOAc = 10:1) to give 6 as a white solid (53 mg, 76 %): 1H NMR (400 MHz; CDCl3) δ 7.81 (dd, J = 9.6, 8.8 Hz, 4H), 7.33–7.49 (m, 7H), 7.04 (d, J = 12.0 Hz, 2H), 5.15 (s, 2H); 13C NMR (100 MHz; CDCl3) δ 193.4, 163.1, 149.5, 143.7, 135.9, 132.6, 130.3, 129.1, 128.7, 128.3, 127.4, 123.5, 114.8, 70.3; IR (KBr) 1641, 1605, 1506, 1252, 1175, 1007, 930, 853, 756, 692 cm–1; MS (ESI, negative) [M + Na+] m/z = 437.0.
(4-Benzyloxyphenyl)-5-(2-(3,4,5-trimethoxyphenyl)ethynyl)methanone (7)
A mixture of 2a (23 mg, 0.12 mmol), 6 (50 mg, 0.12 mmol), CuI (1.0 mg, 0.012 mmol), Pd(PPh3)4 (7.0 mg, 0.006 mmol), and triethylamine (50 μL, 0.36 mmol) was dissolved in THF and the mixture was refluxed for 4 h under nitrogen. The reaction mixture was then concentrated and the residue was purified by column chromatography on silica gel (Hexane/EtOAc = 10:1) to give 7 as a white solid (51 mg, 89 %): 1H NMR (400 MHz; CDCl3) δ 7.81 (d, J = 2.4 Hz, 2H), 7.76 (d, J = 6.4 Hz, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.36–7.47 (m, 5H), 7.05 (d, J = 8.8, 2H), 6.80 (s, 2H), 5.17 (s, 2H), 3.89 (d, J = 4.4 Hz, 9H); 13C NMR (100 MHz; CDCl3) δ 194.7, 162.5, 153.2, 139.2, 137.4, 136.2, 132.5, 131.3, 129.8, 128.7, 128.3, 127.5, 114.5, 108.9, 92.1, 88.6, 70.2, 56.2; IR (KBr) 1601, 1410, 1246, 1126, 843 cm–1; MS (ESI, negative) [M + Na+] m/z = 501.1.
1-(4-(Benzyloxybenzoyl)phenyl)-2-(3,4,5-trimethoxyphenyl)-ortho-carborane (8)
A mixture of decaborane (38 mg, 0.31 mmol), 7 (100 mg, 0.21 mmol), N,N-dimethylaniline (80 μL, 0.63 mmol) was dissolved in chlorobenzene (1 mL) and microwave irradiation was carried out at 120 °C for 10 min under nitrogen. The reaction mixture was extracted with EtOAc three times and combined organic layers were washed with water, 0.5 N HCl aq., and brine, dried over MgSO4 and concentrated. The residue was purified by column chromatography on silica gel (Hexane/EtOAc = 2:1) to give 8 as a white solid (79 mg, 63 %): m.p. 142–144 °C; 1H NMR (400 MHz; CDCl3) δ 7.68 (d, J = 8.8 Hz, 2H), 7.58–7.54(m, 4H), 7.40–7.42 (m, 5H), 7.05 (d, J = 9.2 Hz, 2H), 6.62 (s, 2H), 5.14 (s, 2H), 3.77 (s, 3H), 3.71 (s, 6H); 13C NMR (100 MHz; CDCl3) δ 194.0, 162.8, 152.4, 140.0, 136.0, 134.0, 132.5, 130.6, 129.4, 128.7, 128.3, 127.5, 125.5, 114.6, 108.5, 85.5, 84.2, 70.2, 60.8, 56.2; 11B NMR (96.3 MHz; CDCl3) δ –14.74, –7.22; IR (KBr) 2571, 1655, 1585, 1501, 1252, 1128, 1005, 841 cm–1; HRMS (ESI-TOF) m/z calcd for C31H36B10O5Na [M + Na+] 620.3443; found 620.3445.
1-(4-(Propargyloxybenzoyl)phenyl)-2-(3,4,5-trimethoxyphenyl)-ortho-carborane (10)
To mixture of 8 (34.1 mg, 0.057 mmol) in MeOH/THF (1:1 v/v, 1.5 mL) was added Pd/C (12 mg) and the mixture was stirred under H2 gas for 1 h at room temperature. The reaction mixture was filtered with a celite pad and the solvent was removed under reduced pressure. The residue involved with compound 9 was dissolved in acetone (1.2 mL) and propargyl bromide (5.2 μL, 0.068 mmol) and K2CO3 (78.7 mg, 0.57 mmol) were added with stirring. The reaction was quenched with water and the mixture was extracted with EtOAc three times. The combined organic layers washed with water, 0.5 N HCl aq., and brine, dried over MgSO4 and concentrated. The residue was purified by column chromatography on silica gel (hexane:EtOAc = 20:1 to 3:2) to give 7 as a white solid (14.6 mg, 2 steps 47 %). M.p. 158–159 °C; 1H NMR (400 MHz; CDCl3) δ 7.70 (d, J = 8.8 Hz, 2H), 7.58–7.56 (m, 4H), 7.03 (d, J = 8.8 Hz, 2H), 6.23 (s, 2H), 4.77 (s, 2H), 3.77 (s, 3H), 3.71 (s, 6H), 2.56 (s, 1H); 13C NMR (75 MHz; CDCl3) δ 194.0, 161.4, 152.4, 140.0, 139.6, 134.1, 132.3, 130.6, 130.1, 129.4, 125.5, 114.6, 108.5, 85.5, 84.1, 77.6, 77.2, 76.3, 60.8, 56.2, 55.9; 11B NMR (96.3 MHz; CDCl3) δ –14.85, –7.19; IR (KBr) 3296, 2552, 1599, 1252, 847, 633 cm–1; HRMS (ESI-TOF) m/z calcd for C27H32B10O5Na [M + Na+] 567.3158; found 567.3161.
Cell culture
The human cervical carcinoma cell line HeLa cells were obtained the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). The cells were cultured under 5 % CO2 at 37 °C in RPMI 1640 medium (Wako Pure Chemical, Osaka, Japan) supplemented with 10 % fetal bovine serum (FBS, HyClone, Logan, UT), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA). For subsequent experiments, the cells were seeded at a density of 2.5 x 105 cells/mL/well in a 12-well TC plate (Greiner Japan, Tokyo, Japan), and incubated at 37 °C for 12 h. Hypoxic condition was achieved by replacing cells to 1 % O2, 95 % N2 and 5 % CO2 in a multigas incubator (Astec, Fukuoka, Japan).
Reporter gene assay
HeLa cells expressing HRE-dependent firefly luciferase reporter construct (HRE-Luc) and constitutively expressing CMV-driven Renilla luciferase reporter with SureFECT Transfection Reagent were established with Cignal™ Lenti Reporter (SABiosciences, Frederick, MD, USA) according to the manufacturer’s instructions. The consensus sequence of HRE was 5′-TACGTGCT-3′ from the erythropoietin gene. Cells stably expressing the HRE-reporter gene were selected with puromycin. The cells were incubated for 12 h with or without compounds under the normoxic or hypoxic condition. After removal of the supernatant, the luciferase assay was performed using a Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The drug concentration required to inhibit the relative light units by 50 % (IC50) was determined from semi-logarithmic dose–response plots.
Western blotting
HeLa cells were treated with compounds and incubated for 4 h under hypoxic condition. The cells were washed with PBS (Ca/Mg-free) three times, dipped in 100 μL of ice-cold lysis buffer (20 mM HEPES, pH = 7.4, 1 % triton X-100, 10 % glycerol, 1 mM EDTA, 5 mM sodium fluoride, 2.5 mM p-nitrophenylene phosphate, 10 μg/mL phenylmethylsulfonylfluoride, 1 mM sodium vanadate, and 10 μg/mL leupeptin) for 15 min, and disrupted with a Handy Sonic Disrupter, and the lysate was boiled for 5 min in a sample buffer (50 mM Tris, pH 7.4, 4 % SDS, 10 % glycerol, 4 % 2-thioethanol, and 50 μg/mL bromophenol blue) at a ratio of 4:1. The cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride (PVDF) membrane (GE Healthcare Buckinghamshire, UK), and immunoblotted with anti-HIF-1α antibody (BD Transduction Laboratories, Lexington, KY, USA) and anti-Tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After further incubation with horseradish peroxidase (HRP)-conjugated secondary antibody, the blot was treated with ECL kit (GE Healthcare) and protein expression was visualized with a Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA).
Reverse transcription polymerase chain reaction (RT-PCR)
HeLa cells were treated with compounds and incubated for 4 h under hypoxic condition. Total RNA was extracted with an ISOGEN II (Wako Pure Chemicals, Osaka, Japan) according to the manufacturer’s instructions. The extracted RNA (1 μg) was reverse transcribed at 40 °C for 50 min by adding 5 μM random hexamer oligonucleotides (Promega, Madison, WI), and 2.5 mM dNTP (Bioline, London, UK). The PCR primers used were 5′-CTC AAA GTC GGA CAG CCT CA-3′ (sense) and 5′-CCC TGC AGT AGG TTT CTG CT-3′ (antisense) for HIF-1α and 5′-ACC ACA GTC CAT GCC ATC AC-3′ (sense) and 5′- TCC CCA CCC TGT TGC TGT A -3′ (antisense) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR was carried out with 10 μL of template DNA and 40 μL of PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl2) containing each primer (0.2 μM), dNTP (0.2 mM), and BIOTAQTM DNA polymerase (1.25 units) (Bioline). The PCR conditions for the primer sets were as follows: initial denaturation at 94 °C for 5 min, 25 cycles of amplification consisting of a denaturation step at 94 °C for 0.5 min, an annealing step at 58 °C for 0.5 min, an extension step at 72 °C for 1 min, and a final extension at 72 °C for 7 min using PCR thermal cycler (Astec, Fukuoka, Japan). After the PCR, 10 μL of the reaction mixture was subjected to electrophoresis on 2 % agarose gel. The PCR products were stained with ethidium bromide and HIF-1α and GAPDH mRNA expression was visualized with the Molecular Imager ChemiDoc XRS System.
Photoaffinity labeling, click chemistry, and fluorescent gel imaging
Soluble extract of HeLa cells was prepared using glass beads disruption in lysis buffer by centrifugation at 13,200 rpm for 20 min. The cell lysate (200 μg) was incubated for 10 min at 4 °C with various concentrations of the probe 10 in the presence or absence of 1d, and then irradiated with 360 nm UV light (longwave ultraviolet lamp model B-100A, UVP, Upland, CA) on ice for 30 min. Click reactions of the probe 10 and Alexa Fluor 488 azide (Alexa Fluor 488 5-carboxamide-(6-azidohexanyl), bis(triethylammonium salt), Invitrogen) were established with Click-iT Protein Reaction Buffer Kit (Invitrogen) according to the manufacturer’s instructions. After the click reaction, proteins were precipitated with methanol/chloroform/water (60/15/40, v/v) and denatured by boiling for 5 min in a sample buffer. Proteins were separated by SDS-PAGE, and fluorescence of Alexa Fluor 488 was visualized in-gel using a Molecular Imager ChemiDoc XRS System.
Article note
A collection of invited papers based on presentations at the 15th International Meeting on Boron Chemistry (IMEBORON-XV), Prague, Czech Republic, 24–28 August 2014.
Acknowledgments
We thank Dr. Hyun Seung Ban (Korea Research Institute of Bioscience and Biotechnology) for technical suggestions regarding hypoxia-induced HIF-1α protein and mRNA analyses. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemical Biology of Natural Products” from The Ministry of Education, Culture, Sports, Science and Technology, Japan.
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