Direct C–H functionalization of difluoroboron dipyrromethenes (BODIPYs) at β-position by iodonium salts

A copper-catalyzed direct C–H arylation or vinylation of BODIPYs at the β-position by iodonium salts has been developed, which provides facile access to a variety of mono-substituted BODIPY dyes. Interestingly, β-styryl BODIPY compound 9b exhibits apparent cytotoxicity after laser irradiation, which has great potential for photodynamic therapy.


Introduction
Diuoroboron dipyrromethenes (BODIPYs) and their derivatives are most widely used as small molecule organic uorophores due to its excellent features, such as high uorescence quantum yield, narrow emission bandwidth with high peak intensity, good biocompatibility and high photophysical stability. 1 Modication of the BODIPY framework can tune for their uorescence prole and functionality for the specic application such as bio-imaging and photodynamic therapy (PDT). 2 Besides the meso-derivation for chemosensors, the extension of BODIPY core by the conjugated groups at the aor b-position of could lead the advantage of red-shied emission band and large Stokes shi. 1a,3 Introducing diverse functional groups based on the BODIPY directly is an efficient way to avoid the use of unstable intermediate and multiple-steps processes from pyrroles. Traditionally, the installation of functional conjugated groups has been proceeded by the coupling reaction from halogenated BODIPY or the condensation from active carbon (methyl group at a-position and formyl group at b-position). 3,4 Direct C-H functionalization of BODIPY chromophores has become an attractive alternative due to the economy and efficiency. 5 Several groups successfully developed the C-H aarylation of BODIPYs from the different aryl sources. 6 However, the conjugated b-derivation is rarely explored.
Burgess and coworkers reported a few examples of palladiumcatalyzed C-H Heck-type alkenylation at the b-position in 5 days convertion. 7 You and coworkers reported decarboxylative C-H arylation at the b-position from specic orthosubstituted ben-zoic acids in the harsh conditions to obtain the mono-and disubstituted products. 8 Herein, we report a direct C-H arylation or vinylation method for BODIPY postmodication by iodonium salts at the b-position for the mono-substitution as the major product, which could furtherly result the diversity at aand b-positions of the BODIPYs (Scheme 1). Scheme 1 Direct arylation or vinylation of BODIPY at b-position.

Results and discussion
Diaryliodonium salts as highly reactive species have been widely applied to versatile arylation reaction. 9 Direct a-selective arylation of BODIPYs by diaryliodonium salts could be smoothly proceeded through radical reaction pathway with metal free. 6c Since the b-position of the BODIPY is also potentially electron rich, 10 we hypothesized the direct b-arylation of BODIPYs using diaryliodonium salt could be proceed by blocking the active a-position (Scheme 2). We selected the readily available pentamethylsubstituted BODIPY 1 to investigate the b-arylation reaction since the methyl group could block the active a-position and be easily functionalized further. Under the metal-free conditions reported previously, 6c the reaction of BODIPY 1 with diaryliodonium triates 2a did not happen. Since diaryliodonium salt could behave as a highly activated aromatic electrophile by Cu catalyst, 11,12 the reaction was carried out in the presence of copper triuoromethanesulfonate (Cu(OTf) 2 , 0.1 equiv.) and 2,6-di-tertbutylpyridine (DTBP, 1.2 equiv.) as base in DCM at 70 C for 6 hours. As expected, the desired mono-arylated product 3a was obtained in 48% yield with an excellent selectivity. Among the different solvents (Table 1, entries 1-4), the DCM is best. By the detailed analysis of the reaction, we found that de-boron diuoride byproduct was mainly formed in DCM and the starting material 1 could be easily recovered in toluene. Since the acidic conditions might cause the deboronation of the BODIPY, 13 we tried to the mixture solvents of DCM and toluene. The cosolvents (DCM : toluene ¼ 3 : 1) gave the best yield of 3a with high selective ratio ($10 : 1) with the recovered starting material 1. Without base or with inorganic base, the reaction didn't give the satised results (Table 1, entries 8, 9). In addition, counter anions of diaryliodonium such as PF 6 or Cl were tested (Table 2, entries 10, 11). The results uncovered the observation that electron-withdrawing counter anion PF 6 afforded the similar yield and selectivity as OTf. By increasing the equivalents of iodonium triate salt to 2.4, mono-arylated 3a and diarylated 4a were obtained in 21% and 50%, respectively.
To explore the phenyl substituent scope for this novel Cu(II)catalyzed C-H arylation process, various asymmetrical diaryliodonium triates 2 were evaluated to react with 1 under the optimized conditions (Scheme 3). Gerenally, the results showed that both electron-withdrawing and electron-donating substituted diaryliodonium triates 2 were suitable to this reaction, and desired products 3 were obtained in modest yields with good selectivity by using the 0.8 equiv. of 2 (condition A). Unfortunately, methyl group at ortho-position didn't form the desired product due to the probable steric hindrance of diaryliodonium salt (Scheme 3, compound 3d). Signicantly, the bromo substituted diaryliodonium triates performed well under this reaction conditions, producing compounds 3j and 3k, which could be further modied by coupling reactions. Increasing the amount of diaryliodonium triates 2 to 2.4 equivalent (condition B), the overall yields of 3 and 4 could be improved with less selectivity. Since the resultant monosubstituted BODIPYs could be arylated by the different diaryliodonium triates to generate the diversity, we applied compound 3e (Scheme 4A) as the substrate with para-methoxycarbonyl group as electron-donor to react with substituted diaryliodonium triate 2g with electron-acceptor. As expected, Scheme 2 The possible reaction mechanism for b-arylation of BODIPY. the asymmetrical diarylated BODIPY 5 containing D-p-A (donor-p-acceptor) structure was obtained in 52% yield. In addition, meso-phenyl BODIPY 6 could react with diaryliodonium 2a (Scheme 4B) under the condition A to achieve product 7 in 46% yield. The photophysical properties of 3a-3k, 5 and 7 were evaluated in different solvents system (Table 2, Fig. S1-5 †). As shown in Table 2, 2-aryl BODIPYs 3a-3k exhibited the absorption maxima and excitation maxima among 501-504 nm no matter electron-withdrawing or electron-donating substituents. All 2aryl BODIPYs showed high absorption coefficients and relatively high uorescence quantum yields. Methoxyl substituent 3e showed a lower uorescence quantum yield ($24%) correspondingly. On the other hand, 3e exhibited the longest emission maxima at 551 nm and the largest stokes shi (47 nm) among 2-aryl BODIPYs. These data indicate that slight modication of BODIPY could change their photophysical property signicantly. The asymmetrical diarylated BODIPY 5 containing D-p-A has the desired properties with red uorescence, high quantum yield and large Stokes shi.
Next, we explored the reaction of BODIPY 1 with the variety of styryliodonium triates 8 to generate the different conjugated  systems. Due to the higher reactivity of vinyliodonium than aryliodonium salts, 12b the reaction temperature at 50 C gave the similar efficiency at 70 C. To our delight, the different 2-styryl BODIPYs 9a-9e were delivered (Scheme 5). Additionally, alkyl vinyliodonium triates can also be successfully applied in this reaction, maintaining 51% yield of 2-vinyl BODIPY 9f. Photophysical properties of 9a-9f were investigated as shown in Table 3. The 2-vinyl BODIPYs 9 exhibited emission maxima up to 598-620 nm with large stokes shis. Probably due to the geometry relaxation of uorophores upon photoexcitation, the quantum yields of those dyes were as low as 0.78%. Dye with photoproperties such as high extinction coefficient, low quantum yield and red emission could be used as photosensitizer for photodynamic therapy. 2a,2d The BODIPY 9b was thus chosen to evaluate photocytotoxic activity in vitro. The half maximal inhibitory concentration (IC 50 ) of compound 9b with or without light irradiation was assessed in human breast cancer cell line MDA-MB-231 and epidermoid carcinoma cell line A-431. Nine different concentrations (ranging from 0.195 to 50 mM) of 9b were applied to cells and 12 h exposure to mercury lamp decreased the cell viability signicantly in presence of 9b (P < 0.0001) in both cells (Fig. 1). For example, light irradiation reduced the IC 50 value from 43.9 mM to 4.0 mM ($88% decline) in MDA-MB-231 cells. Photophysical property and laser-induced cytotoxicity of compound 9b were also characterized and visualized by a Leica confocal microscopy. As shown in Fig. 2 and S7, † high uorescence signals were detected in cytoplasm within 5 min aer 9b (2.5 mM) feeding. Then the photothermal treatment was performed by continuous irradiation of the cells using a 552 nm laser for 10 min, leading to a $200 mm diameter irradiation spot. Cell blebbing, a characteristic feature of injured cells, was only observed in compound 9b-treated cells but not in the vehicle-treated cells. Similar photothermal effects were exhibited in 9a-or 9f-treated cells (Fig. S7 †). These results suggest that 2-vinyl BODIPYs could be excellent photosensitizers and have potential for photodynamic therapy of cancer.
Scheme 5 Scope of the direct C-H vinylation of BODIPY 1 with different vinyliodonium triflates. a Isolated yield. Table 3 Photophysical properties of BODIPY dyes in CH 3 CN at room temperature   This journal is © The Royal Society of Chemistry 2018

Conclusions
In conclusion, we have developed the Cu-catalyzed direct C-H arylation and vinylation of BODIPYs at the b-position. This methodology provides a convenient synthetic procedure to prepare diversied BODIPY derivatives. These reactions exhibited good selectivity for mono-substituted product to allow the different modication of BODIPY at 2,6-position. Through the evaluation of photophysical properties of those dyes, 2-styryl BODIPYs showed intrinsic large Stokes shi up to 101 nm and low quantum yield, suggesting the potential for photodynamic therapy of cancer. In this way, the novel functionality of uorophores with asymmetrical substitution can be synthesized economically and efficiently.

General
All reactions were carried out under an atmosphere of argon in oven-dried glassware with magnetic stirring. All commercially available reagents were used as received. Chromatographic puri-cations were performed by ash chromatography with silica gel (40-63 mm) packed in glass columns. The eluting solvent for the purication of each compound was determined by thin-layer chromatography (TLC) on glass plates coated with silica gel 60 F254 and visualized by UV light (254 nm or 365 nM). 1 H NMR spectra and proton-decoupled 13 C NMR spectra were obtained on a 400 MHz or 500 MHz Bruker NMR spectrometer. 1 H chemical shis (d) are reported in parts per million (ppm) relative to TMS (s, d 0). 13 C NMR chemical shis are reported relative to CDCl 3 (t, d 77.4). High-resolution mass data were obtained on an Agilent 6224 accurate-mass TOF LC/MS (ESI). Absorption spectra were acquired using a Varian Cary 300 spectrophotometer. Fluorescence measurements were carried out on a Horiba FluoroMax-4 spectrometer. Quantum yields were determined in reference to either uorescein or rhodamine 6G and corrected for solvent refractive index. The extinction coefficients were determined through Beer's law plots. All data were measured at room temperature. Human breast cancer cell line MDA-MB-231 and human epidermoid carcinoma cell line A-431 were obtained from the American Type Culture Collection (Manassas, VA, USA) and were cultured in DMEM (high glucose) and RPMI 1640 medium respectively, supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). Cells were incubated at 37 C in 5% CO 2 in air.
General procedure for the Cu(II)-catalyzed diarylation of BODIPYs (reaction condition B) Copper(II) triuoromethanesulfonate (14.5 mg, 0.04 mmol) was adequately suspended in 3 mL mixture solution in 10 mL sealed tube, then 1 (52.4 mg, 0.20 mmol), appropriate diaryliodonium salt (0.48 mmol) and 2,6-di-tert-butylpyridine (109 mL, 0.48 mmol) were sequentially added to the solution. Seal up the tube with argon atmosphere. The purication procedures were the same with reaction condition A to afford 4a-4k. The yield data was calculated by BODIPY 1 stoichiometric equivalence.

Cell viability assay for photocytotoxic effect
Cells were seeded in 96-well plates at a density of 1600-3000 cells per well in triplicate. Twenty-four hours later, the cells were incubated with fresh medium with compounds at different concentrations (ranging from 0.195 to 50 mM). The cells were exposed under the mercury lamp (HXP R120W/45C; Osram, Germany) for 12 h, and then were kept under normal culture condition for another 72 h. At the endpoint, the cells were xed with 10% pre-cooled trichloroacetic acid (Sigma, St Louis, MO, USA) over 4 h followed by staining with 4 mg mL À1 sulforhodamine B (SRB; Sigma) in 1% acetic acid for 15 min. SRB in the cells was dissolved in 10 mmol L À1 , Tris-HCl and measured at 560 nm using a microplate reader (SpectraMAX190; Molecular Devices, Sunnyvale, CA, USA). The half maximal inhibitory concentration (IC 50 ) was decided using (log(inhibitor) vs. responsevariable slope (four parameters)) method in the GraphPad Prism Soware. The data were presented as the mean AE SD. Differences were considered statistically signicant at P < 0.0001 by 2 way ANOVA test.

Visualization of laser-induced cytotoxicity
Cells were grown on the 8-well chambered coverglass (Thermo Fisher Scientic, Waltham, MA, USA) for 36 h. Immediately aer adding the compound with indicated concentrations to cells, a small region of interest (ROI) was randomly chosen and irradiated using the laser line at 552 nm from a confocal microscopy (Leica TCS SP8 STED, Germany) over 10 min. During that period, the uorescent and differential interference contrast (DIC) images were acquired at 0 min, 5 min and 10 min, respectively. The power of the laser was kept at 80% output to ensure the consistent irradiation between experiments.

Conflicts of interest
There are no conicts to declare.