Synthesis and fluorescent properties of boroisoquinolines, a new family of fluorophores

First representatives of a new family of isoquinolines, so called boroisoquinolines, were synthesized and characterized. The synthesis was based on the insertion of the difluoroboranyl group into the 1-methylidene-3,4-dihydroisoquinoline core. The optimization of the 2-difluoroboranyl-3,4-dihydroisoquinoline-1(2H)-ylidene core led to efficient fluorescence in a range of 400–600 nm with outstanding (>100 nm) Stokes shifts. The compounds might be suitable for reversible or irreversible labelling of proteins, particularly the cannabinoid receptor CB2.


Experimental General
All melting points were determined on a Jasco SRS OptiMelt apparatus and are uncorrected. 1 H and 13 C NMR spectra were recorded in CDCl 3 solution at room temperature, on a Varian Unity Inova 500 spectrometer (500 and 125 MHz for 1 H and APT NMR spectra, respectively), and on a Varian Unity Inova 300 spectrometer (282 MHz for 19 F NMR spectra), with the deuterium signal of the solvent as the lock and TMS as the internal standard.
Chemical shifts (δ) and coupling constants (J) are given in ppm and Hz, respectively. High resolution mass spectra were recorded on a Waters Q-TOF Premier mass spectrometer in positive ESI ionization mode. The microwave reactions were conducted in an Anton Paar Monowave 300 microwave reactor. The reactions were followed by analytical thin layer chromatography on silica gel 60 F 254 and HPLC-MS chromatography with a Shimadzu LCMS-2020 device using a Reprospher 100 C18 (5 µm; 100x3mm) column and positivenegative double ion source (DUIS) with a quadrupole MS analysator in a range of 50−1000 m/z. All reagents were purchased from commercial sources. Analytical samples of new compounds were obtained by trituration or recrystallization from the solvents or solvent mixtures given below in parentheses. Single crystal X-ray measurements were carried on a Rigaku R-Axis Rapid imaging plate area detector with graphite monochromated Cu-Kα radiation.

Photophysical measurements
The fluorescence and absorbance measurements were carried out on a Jasco FP8300 spectrofluorometer, in a standard cell quartz cuvette with 1 cm light path length. The widths of the excitation slit and the emission slit were both set to 2.5 nm with the scanning speed at 1000 nm/min. Pure solvents were used as blank correction. We used acetonitrile as solvent if not mentioned else, the concentration was 5 μM or 0.5 μM.

Method A
In a three-necked round bottom flask 5.5 g N- [2-(3,4-dimethoxyphenyl)ethyl]acetamide or 4.8 g of N- [2-(3-methoxyphenyl)ethyl]acetamide (24.7 mmol) was dissolved in 50 mL toluene and 2.3 mL phosphorus oxychloride (24.7 mmol, 1 equiv.) was added dropwise. The solution was stirred at reflux temperature for 1 h. The reaction mixture was cooled to room temperature, and the solvent was removed in vacuo. The brown crystallic hydrochloride salt was triturated with diethyl ether, and filtered. The remaining solid was suspended in 50 mL of dichloromethane, and 200 mL concd. NaHCO 3 solution was added. The layers were separated, and the aqueous phase was washed with 2 × 30 mL dichloromethane. The combined organic phases were dried (Na 2 SO 4 ), filtered and the solvent was removed in vacuo.

Method B
In a three-necked round bottom flask 4.6 g N- [2-(3-bromophenyl)ethyl]acetamide or 3.4 g N- [2-(3-fluorophenyl)ethyl]acetamide (18.9 mmol) was dissolved in 100 mL dichloromethane and 1.8 mL oxalyl chloride (2.6 g, 20.7 mmol, 1.1 equiv) was added dropwise under nitrogen atmosphere. The solution was stirred at room temperature for 30 minutes. Then the reaction mixture was cooled below -10 °C, and 3.7 g FeCl 3 (22.7 mmol, 1.2 equiv) was added. The mixture was stirred at room temperature overnight. Aqueous 2M HCl (10 mL, 20.0 mmol) was added to quench the reaction. The mixture was stirred at room temperature for 1 h, then the layers were separated. The organic layer was washed with brine, and dried over MgSO 4 ), filtered, and the solvent was removed in vacuo. The oxalyl adduct was dissolved in 180 mL methanol, 9.5 mL concentrated sulphuric acid was added, and stirred at reflux overnight. The next morning the reaction mixture was allowed to cool room temperature, and the solvent was removed in vacuo. The crude material was dissolved in ethyl acetate, and washed with water.
The aqueous layer was basified with 10 % sodium hydroxide solution, and extracted with ethyl acetate. The combined organic phases were dried (MgSO 4 ), filtered and the solvent was removed in vacuo.

Method D
In a sealed tube 4.4 g 6-fluoro-1-methyl-3,4-dihydroisoquinoline (16d) (27.0 mmol) and 10 mL pyrrolidine (11.5 g, 162 mmol, 6 equiv) were dissolved in 10 mL dioxane. The reaction mixture was heated at 80 °C for 3 days. The residue concentrated under reduced pressure, and purified with steam distillation. After all of the excess pyrrolidine was distilled, the leftover was dissolved in aqueous HCl, then extracted with dicholormethane (5x50 mL). For NMR purpose the crude product purified with reverse phase column chromatography using acetonitrile/water.