Article pubs.acs.org/joc
Synthesis and Spectroscopic and Cellular Properties of Near-IR [a]Phenanthrene-Fused 4,4-Difluoro-4-bora-3a,4a-diaza‑s‑indacenes Ning Zhao, Sunting Xuan, Zehua Zhou, Frank R. Fronczek, Kevin M. Smith, and M. Graça H. Vicente* Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States S Supporting Information *
ABSTRACT: A new synthetic method to build aryl-fused 4,4difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPYs) is reported. The intramolecular cyclization step was completed in a short time (1−2 h) and in high yields (>90%), due to the intrinsic rigid structural conformation of the precursor BODIPY and the high reactivity of its 1,7-bromo groups. The [a]phenanthrene-fused BODIPYs 4a−c were characterized by NMR spectroscopy, HRMS, DFT calculations, and, in the case of 4a, by X-ray crystallography. Spectroscopic studies show that 4a−c strongly absorb and emit in the NIR spectral region, in the range 642−701 nm. In addition, BODIPYs 4b and 4c exhibit no toxicity in the light or dark in HEp2 cells and accumulate intracellularly in a time-dependent manner, mainly in the cell endoplasmic reticulum. These results suggest the potential use of [a]phenanthrene-fused BODIPYs as NIR bioimaging probes.
B
of aryl-fused pyrrole precursors, followed by boron complexation under basic conditions, and (i and iii) via the preparation of BODIPY precursors from cyclohexane-fused pyrroles7c,e or bicyclo[2.2.2]octadiene-fused pyrroles,7a,12 followed by DDQ oxidation or retro-Diels−Alder reactions, respectively. However, several disadvantages remain for the above-reported methods: (1) noncommercially available pyrrole precursors require multistep synthesis; (2) the preparation of benzo[a]fused BODIPYs usually suffers from low yields (200 °C),7a,12 which can easily cause the polymerization or decomposition of BODIPYs. Therefore, new, low-cost, convenient, and easy synthetic methods are required to synthesize [a]aryl-fused BODIPYs. In this work, we report an efficient synthetic methodology for the synthesis of [a]phenanthrene-fused BODIPYs from 2,6dichloro-BODIPYs, which in turn can be easily prepared from inexpensive commercially available pyrroles. The extended πsystem is built via the introduction of two biphenyl groups at positions 2 and 6, followed by bromination at the 1,7-positions and then Pd(0)-catalyzed intramolecular cyclization in high overall yields. There are several advantages to our strategy: (i) the 1,7-bromo groups are highly reactive under the Pd(0)catalyzed reaction conditions;14 (ii) due to steric hindrance caused by the 3,5-aryl groups, the 2,6-biphenyl groups are restricted to the same side as the 1,7-bromo groups, enhancing the intramolecular cyclization reactivity; and (iii) all of the starting materials are commercially available or readily obtained. Furthermore, “push−pull” BODIPY 4c was prepared using this
ODIPYs (4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes or boron dipyrromethenes) have found a wide range of applications as metal ion sensors, photodynamic therapy (PDT) sensitizers, and biological probes, due to their excellent spectroscopic properties. These include large extinction coefficients and fluorescence quantum yields, narrow emission bandwidths, high photophysical and chemical stabilities, and high structural tunability.1 BODIPYs that absorb and emit in the near-infrared (NIR) region (650−1000 nm) are particularly attractive, especially in the field of bioimaging1c,2 due to the enhanced tissue penetration and minimum background interference of the NIR light in the so-called biological window.3 Additionally, NIR-absorbing and -emitting BODIPY dyes, especially those bearing electron-donating and electronwithdrawing groups, have emerged as potential sensitizers for dye-sensitized solar cells (DSSC).4 In the last 2 decades considerable effort has been dedicated to the development of NIR BODIPY dyes.1c,2 A particularly promising strategy relies on the extension of the π-conjugated system of BODIPYs, which typically causes increased rigidity of structures and enhanced fluorescence quantum yields, as well as strong intermolecular π−π stacking effects in the solid state.5 Thus, B,O-chelated,6 benzo[a]-7 or -[b]-fused,5,8 and meso-arylfused9 BODIPYs have been reported. Among these, the benzo[a]-fused BODIPYs are particularly interesting, due to the availability of the α-(3,5)- and meso-(8)-positions, bearing larger MO coefficients of the HOMO and LUMO respectively,1c for further functionalization. However, unlike the synthesis of benzo[b]-fused BODIPYs,5,8,10 introduction of π-extended conjugation systems at the β,β′-positions is challenging, due to the low reactivity of the unsubstituted 1,7-positions of BODIPYs.5,11 Currently, there are three major synthetic strategies to build [a]aryl-fused BODIPYs, as shown in Scheme 1: (ii) by direct condensation © 2017 American Chemical Society
Received: August 1, 2017 Published: August 28, 2017 9744
DOI: 10.1021/acs.joc.7b01940 J. Org. Chem. 2017, 82, 9744−9750
Article
The Journal of Organic Chemistry Scheme 1. Approaches to Benzo[a]-Fused BODIPYs
Scheme 2. Synthesis of [a]Phenanthrene-Fused BODIPYs 4a−c
donating methoxy groups at the 3,5-p-phenyl positions and an electron-withdrawing benzyloxycarbonyl group at the 8-pphenyl position of the [a]phenanthrene-fused BODIPY 4c. In addition, debenzylation of 4c can provide a free carboxylic acid for further conjugation with biomolecules or other synthetic molecules or platforms. In contrast, other reported Pd(0)-mediated intramolecular cyclization reactions on BODIPYs usually require longer refluxing times (>12 h),10,19,20 which is probably due to the free rotation of the phenoxyl or aniline groups on the precursors. It is worth noting that in this work the cyclization reactions were completed much faster (1−2 h for BODIPY 4a− c), in nearly quantitative yields (>90%). These surprisingly high yields and high reactivity can be explained by the following factors: (i) the highly reactive 1,7-bromo groups, as previously reported;14 (ii) the steric hindrance caused by the 3,5-diphenyl groups, which forces the 2,6-biphenyl groups to be on the same side as the 1,7-bromos, as shown in Figures S8−S13 of the Supporting Information (SI); and (iii) BODIPYs 3a−c with bulkier bromo groups at the 1,7-positions, compared with 2a− c, further restricted the free rotation of the 2,6-biphenyl groups, as indicated by the increased dihedral angles between the 2,6biphenyl groups and the BODIPY cores of 3a−c. Suitable crystals of the bis-chloroform solvate of 4a were obtained for X-ray analysis by slow evaporation from chloroform, and the results are illustrated in Figure 1. The presence of the phenyl substituent at the meso-8-position imparts a pronounced twist of the molecule about its local 2fold axis, as shown in Figure 1b. The central C3N2B ring is twisted by 12.9° out of coplanarity. The best planes of the two phenanthrene subunits form dihedral angles of 22.9° and 31.0° with the best plane of the C3N2B ring. The phenyl ring at the
method to further tune the properties of this type of BODIPY. To investigate their potential use as bioimaging agents, the cytotoxicity, cellular uptake, and fluorescence microscopy studies were performed in human HEp2 cells on BODIPYs 4b and 4c. Recently, our group has been investigating the synthesis and reactivity of polyhalogenated BODIPY platforms. The results showed the following order of reactivity of chloro groups at different positions, under the conditions of Pd(0)-catalyzed15 and substitution reactions: 8-Cl > 3,5-Cl > 2,6-Cl.15b,16 On the basis of these studies, 3,5,8-triaryl-BODIPYs 1a−c were prepared from 2,3,5,6,8-pentachloro-BODIPY via regioselective Suzuki coupling reactions, as previously reported by our group.15b,17 As shown in Scheme 2, treatment of BODIPYs 1a− c with 20 equiv of 2-biphenylboronic acid in the presence of 5% Pd(PCy3)G2 and 1 M Na2CO3 (aq) in refluxing toluene for 5 h produced the corresponding BODIPYs 2a−c as the major products, in high yields (71−85%). Bromination reactions on BODIPYs 2a−c were performed using 10 equiv of Br2 and 10 equiv of NaHCO3 in CH2Cl218 to provide the 1,7-dibromoBODIPYs 3a−c in 91−94% yields. In this way, the unreactive 1,7-H atoms were converted to highly reactive 1,7-bromo groups for further intramolecular cyclization catalyzed by Pd(0), as recently reported.19 By treating BODIPYs 3a−c with 50 equiv of K2CO3 and Pd(PPh3)4 in refluxing toluene, [a]phenanthrene-fused BODIPYs 4a−c were obtained within 2 h, in high yields (91−92%). The functionalized BODIPY 4c was prepared to illustrate the versatility of this synthetic strategy to approach push−pull BODIPYs for applications as potential bioimaging agents or as DSSC sensitizers.17a Due to the large MO coefficients of the HOMO at the 3,5-positions and of the LUMO at the 8-position,1c we introduced electron9745
DOI: 10.1021/acs.joc.7b01940 J. Org. Chem. 2017, 82, 9744−9750
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The Journal of Organic Chemistry
could be attributed to the increased dihedral angles (12°−13°) between the 2,6-biphenyl groups and BODIPY cores with substitution at the 1,7-positions and to the heavy atom effect, respectively. DFT calculations, as shown in Figures S8−S13 (SI), suggest that the 1,7-bromo groups lead to less-conjugated BODIPY systems. Intramolecular cyclization of BODIPYs 3a−c afforded the corresponding [a]phenanthrene-fused BODIPYs 4a−c, characterized by large red-shifts in both the absorption (55−75 nm) and emission (52−68 nm) spectra, due to the extension of the π-conjugated systems. Among the BODIPYs, the push−pull BODIPYs 1c−4c exhibited the most red-shifted absorption and emission bands as a result of the decreased HOMO−LUMO gap, along with larger Stokes shifts, presumably due to the large difference of dipole moment between the ground and excited states.21 On the other hand, the push−pull BODIPYs also showed lower fluorescence quantum yields, probably induced by internal charge transfer (ICT), as previously reported.17a For example, as shown in Figure 2, BODIPY 4c, bearing both electron-donating groups at the 3,5-positions and an electronwithdrawing group at the 8-position, exhibited 16 and 30 nm bathochromic shifts in the absorption and emission spectra, respectively, and a 2-fold decrease in fluorescence quantum yield, compared with 4a. DFT calculations of all the BODIPYs were conducted to gain information on their molecular orbital characteristics, and the results are summarized in Table S2, Figure 3, and Figures S14− S16 (SI). For example, the installation of methoxy and benzyloxycarbonyl groups at the 3,5- and 8-p-phenyl positions, respectively, as in the case of BODIPY 4c, shifted the MO coefficients to the 8-positions for the LUMO and LUMO+1 and to the 3- and 5-positions for the HOMO−1 and HOMO. Overall, the HOMO−LUMO gaps decrease in the order of BODIPYs 3a−c > 2a−c ≫ 4a−c and a > b > c, as shown in Figure 3. Such a trend is in agreement with the results obtained from the spectroscopic studies (Table 1). [a]Phenanthrene-fused BODIPYs 4b and 4c were chosen for biological evaluation in human HEp2 cells, including their cellular uptake, cytotoxicity, and subcellular distribution, by fluorescence microscopy, and the results are summarized in Figures 4 and S4−S7 (SI). None of the BODIPYs were toxic to cells, both in the dark (IC50 ≥ 200 μM) and upon irradiation with 1.5 J/cm2 light dose (IC50 ≥ 100 μM). The BODIPYs were readily taken up by the human HEp2 cells, rapidly in the first 1 h (for 4b) or 4 h (for 4c) after which a slower accumulation was observed, as shown in Figure 4. Compared with 4b, 4c accumulated to a significantly higher extent (2−4fold) at all times investigated, up to 24 h; this could be due to the enhanced amphiphilic character of 4c. ChemBio3D log P calculations (Table S1, SI) suggest that 4c (log P = 15.4) is slightly more hydrophobic than 4b (log P = 13.6), thus favoring its cellular uptake. Furthermore, the main subcellular localization sites of the BODIPYs were investigated in HEp2 cells using fluorescence microscopy, and the results are shown in Figures 4 and S6 and S7 (SI). Both compounds preferentially localized in the cell endoplasmic reticulum (ER) and to a lower extent in the lysosomes, in agreement with previously reported studies on this type of compound.7c,16,17 In summary, a convenient and efficient method for the synthesis of [a]phenanthrene-fused BODIPYs was developed. The intramolecular cyclization step was completed in less than 2 h, in high yields (>90%). Spectroscopic studies of all the BODIPYs were conducted in CH2Cl2, and the results are
Figure 1. X-ray structure of 4a: top view (a) and side view (b) with 50% ellipsoids.
meso-8-position forms a dihedral angle of 45.6° with the central ring, and for the phenyl groups at the 3,5-positions, analogous dihedral angles are 64.8° and 67.4°. The spectroscopic properties of BODIPYs 1a−4a, 1b−4b, and 1c−4c in CH2Cl2 were evaluated, and the results are shown in Table 1 and Figures 2 and S1−S3 (SI). All the BODIPYs Table 1. Spectroscopic Properties of BODIPYs in CH2Cl2 at Room Temperature BODIPY
λabs (nm)
log ε
λem (nm)
Φfa
ssb (nm)
1a 2a 3a 4a 1b 2b 3b 4b 1c 2c 3c 4c
566 584 567 642 581 595 587 642 594 607 593 658
4.78 4.83 4.87 4.94 4.68 4.79 4.78 4.76 4.71 4.58 4.53 4.81
597 617 603 671 621 629 626 678 638 649 633 701
0.23 0.61 0.19 0.37 0.29 0.62 0.43 0.30 0.08 0.21 0.35 0.18
31 33 36 29 30 34 39 36 44 42 40 43
a
Cresyl violet (Φ = 0.55 in methanol) was used as the standard for BODIPYs 1a−3a, 1b−3b, and 1c−3c; methylene blue (Φ = 0.03 in methanol) was used as the standard for BODIPYs 4a−c. bStokes shift.
strongly absorb in the red and NIR spectral region (λmax = 566−658 nm, log ε > 4.5). The introduction of biphenyl groups at the 2,6-positions of BODIPYs 2a−c caused bathochromic shifts both in the absorption (13−18 nm) and emission (8−30 nm) spectra and significantly increased the fluorescence quantum yields. However, moderate blue-shifts (8−17 nm) and fluorescence quenching for BODIPYs 3a−c were observed after introducing the bromo groups at the 1,7-positions. This 9746
DOI: 10.1021/acs.joc.7b01940 J. Org. Chem. 2017, 82, 9744−9750
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Figure 2. Normalized absorption (a) and emission (b) spectra of BODIPYs 3c (black), 4a (red), and 4c (blue).
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EXPERIMENTAL SECTION
General. All the commercially available chemical reagents were used without further purification. All the reactions were monitored by TLC using precoated silica gel plates with 254 nm indicator (0.2 mm, 60 Å, polyester backed) and a UV lamp. Liquid chromatography (performed on either preparative TLC plates or silica gel column (230−400 mesh, 60 Å) was used for all the purifications. NMR spectra were obtained on 400 or 500 MHz spectrometers at 300 K. Chemical shifts (δ) are given in ppm in acetone-d6 (2.50 and 205.87 ppm for 1H NMR and 13C NMR, respectively), CD2Cl2 (5.30 and 53.4 ppm for 1H NMR and 13C NMR, respectively), or CDCl3 (7.27 and 77.0 ppm for 1 H NMR and 13C NMR, respectively). Coupling constants (J) are given in hertz. High-resolution mass spectra (HRMS) were obtained by ESI-TOF with negative mode. Spectroscopy Methods. Absorption spectra were collected on a Varian Cary spectrophotometer; fluorescence emission spectra were obtained on a PerkinElmer LS55 spectrophotometer, at room temperature. Quartz cuvettes (1 cm) and HPLC-grade solvents were used for all the spectroscopic studies. The plots of integrated absorbance (A = 0.1−1) vs concentration were used to determine the molar extinction coefficients. The relative quantum yields were measured by using several different dilute solutions (A = 0.03−0.08) at the particular excitation wavelength. Cresyl violet (Φ = 0.55 in methanol) and methylene blue (Φ = 0.03 in methanol) were used as the external standards for BODIPYs 1a−3a, 1b−3b, 1c−3c, and 4a− c.22 The relative fluorescence quantum yield (Φf) was calculated based on the following equation:23
Figure 3. DFT-calculated frontier MOs (top) of BODIPYs 4a and 4c, and the MO energies (bottom) of BODIPYs 4a−c. Small black squares represent the occupied MOs. Black and gray lines refer to LUMO/HOMO and LUMO+1/HOMO−1 MOs, respectively. The HOMO−LUMO gaps are distinguished by a secondary axis and are represented by red triangles.
Φx = Φstd
Grad x n x 2 Grad std nstd 2
where the subscripts std and x represent the standards and tested samples, respectively; Φ stands for fluorescence quantum yields; Grad stands for the gradient of integrated fluorescence intensity vs absorbance; and n stands for the refractive indices. X-ray Methods. The crystal structure of the chloroform disolvate of 4a was determined using data collected at low temperature with Mo Kα radiation on a Bruker Kappa Apex-II diffractometer equipped with a Triumph curved monochromator. Refinement was by SHELX2014. Hydrogen atoms were visible in difference maps, and were placed in idealized positions and treated as riding. Crystal data: CCDC 1523512; C51H31BF2N2·2CHCl3, MW = 959.32, monoclinic, space group I2/a; a = 28.2486(5) Å, b = 9.3345(2) Å, c = 35.1839(9) Å, β = 103.7927(9)°, V = 9010.0(3) Å3, Z = 8, d = 1.414 g cm−3, T = 100 K. The total number of reflections measured to θmax = 35.6° was 78 841, yielding 20 739 independent data, of which 12 940 were observed with
consistent with computational studies. Push−pull BODIPY 4c was prepared to verify the versatility of this method and also to further tune the properties of BODIPYs. In addition, the [a]phenanthrene-fused BODIPYs 4a−c strongly absorb and emit in the NIR region (λmax = 642−701 nm). The representative BODIPYs 4b and 4c showed no cytotoxicity toward HEp2 cells and mainly localized in the cell ER and to a lower extent in the lysosomes. These results indicate that the synthetic method developed can be used to approach NIR dyes for potential use as bioimaging agents. BODIPY 4c with push− pull moieties may also be a good sensitizer for dye-sensitized solar cells. 9747
DOI: 10.1021/acs.joc.7b01940 J. Org. Chem. 2017, 82, 9744−9750
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The Journal of Organic Chemistry
Figure 4. Time-dependent cellular uptake (left) of BODIPYs 4b (black) and 4c (red) and subcellular localization (right) of 4b (a−c) and 4c (d−f) in HEp2 cells at 10 μM for 6 h: (a) 4b fluorescence, (d) 4c fluorescence, (b, e) ER Tracker Blue, and (c, f) overlay of ER Tracker Blue with 4b and 4c fluorescence, respectively. F2 > 2σ(F2), Rint = 0.052, R = 0.055, wR(F2) = 0.144 for all data and 577 refined parameters. Computational Methods. The ground-state geometries were optimized by the DFT method with the B3LYP/6-31G(d) basis sets by using Gaussian 09 software package.24 The partition coefficients (log P) were calculated using ChemBio3D Ultra 12.0. Cell Culture. All the cell culture media and reagents were purchased from Invitrogen and used as received. All the cell studies were conducted by adapting reported procedures.16 The human HEp2 cells were purchased from ATCC and maintained in a 50:50 mixture of DMEM:AMEM containing 5% FBS and 1% penicillin/streptomycin antibiotic. The cells were subcultured twice weekly to maintain subconfluent stocks. Cell toxicity was evaluated using the CellTiterBlue (Promega) assay. Dark Cytotoxicity. The HEp2 cells were plated at 7500 cells per well in a Costar 96-well plate (BD Biosciences) and allowed to grow for 48 h. BODIPY solutions in 100% DMSO (32 mM) were diluted to different working concentrations (0, 6.25, 12.5, 25, 50, 100, and 200 μM) with Eagle’s minimum essential medium (EMEM). The above BODIPY working solutions up to 200 μM were added to cells and incubated for 24 h (37 °C, 95% humidity, 5% CO2), after which the working solutions were removed and the cells were washed with 1× PBS buffer three times. The cells were exposed to the medium containing 20% CellTiter-Blue and incubated for 4 h (37 °C, 95% humidity, 5% CO2). The viability of the cells is measured by reading the fluorescence of the medium at an excitation wavelength of 570 nm and an emission wavelength of 615 nm using a BMG FLUOstar Optima microplate reader. The fluorescence signal of untreated cells in media only was normalized to 100%. Photocytotoxicity. The human HEp2 cells were prepared as described above and exposed to BODIPY working solutions in EMEM at different concentrations (100, 50, 25, 12.5, 6.25, 3.125, and 0 μM) for 24 h. The BODIPY working solutions were removed and the cells were washed with 1× PBS buffer three times followed by adding 100 μL of EMEM containing 10% FBS and 1% penicillin to each well. The cells were irradiated with a light dose of approximately 1.5 J/cm2 provided by a 600 W halogen lamp light source filtered with a water filter (transmits radiation 250−950 nm) and a beam-turning mirror with 200 nm to 30 μm spectral range (Newport), for 20 min. After light exposure, the cells were incubated for 24 h (37 °C, 95% humidity, 5% CO2), and the cell viability was evaluated using CellTiter-Blue as described above. Time-Dependent Cellular Uptake. Human HEp2 cells were prepared as described above. Each BODIPY solution (10 μM) was added to the cells and incubated for 0, 1, 2, 4, 8, and 24 h. At the end of each incubation period, the BODIPY solution was removed, and the cells were washed with 1× PBS buffer three times followed by solubilizing the cells with 0.25% Triton X-100 in 1× PBS buffer. The standard curve of each BODIPY was prepared by using BODIPY solutions with concentrations of 10, 5, 2.5, 1.25, 0.625, and 0.3125 μM in 1× PBS buffer containing 0.25% X-100. The standard curve of cell number was prepared using 104, 2 × 104, 4 × 104, 6 × 104, 8 × 104,
and 105 cells per well and the cell number was quantified using a CyQuant Cell Proliferation Assay (Life Technologies). The BODIPY concentration in cells at the end of each incubation period was determined using a BMG FLUOstar Optima microplate reader at the excitation wavelength and emission wavelength of each BODIPY. Microscopy. Human HEp2 cells were plated in a 6-well plate (MatTeck) and allowed to grow for 24 h (37 °C, 95% humidity, 5% CO2). Each BODIPY solution in medium (1−10 μM) was added to the above cells and incubated for another 6 h. Organelle tracers (50 nM LysoSensor Green, 250 nM MitoTracker Green, 100 nM ER Tracker Blue/White, and 50 nM BODIPY FL C5 Ceramide) were added subsequently and incubated for 30 min. The above solution in each well was removed, and the cells were washed with 1× PBS buffer three times before imaging. The images were obtained using a Leica DMRXA2 upright microscopy with a water immersion objective and DAPI, GFP, and Texas Red filter cubes (Chroma Technologies). Synthesis and Characterization of BODIPYs. BODIPYs 1a−c were synthesized by adapting the published procedures.15b,17a General Procedure for the Synthesis of BODIPYs 2a−c. Into a flask equipped with a stir bar were added the starting BODIPY 1a−c (0.04 mmol), 2-biphenylboronic acid (0.8 mmol), and Pd(PCy3)G2 (10% mol). The flask was evacuated and refilled with N2 five times. Anhydrous toluene (4 mL) and 1 M Na2CO3 (1 mL) were injected into the flask. The mixture was stirred and refluxed for 5 h and the reaction was monitored by TLC. The reaction was stopped after all the starting material was consumed, according to TLC. The mixture was poured into water (30 mL), and CH2Cl2 (10 mL × 3) was used to extract the organic components. The layers were combined and washed with brine, followed by drying with anhydrous Na2SO4. The organic solvents were removed under reduced pressure. The residue was subjected to column chromatography (CH2Cl2/hexanes as eluents) to afford the desired products 2a−c. BODIPY 2a. Yield 20.6 mg (as a blue solid), 71%; mp 149−151 °C; 1 H NMR (400 MHz, CD2Cl2) δ 7.52−7.56 (m, 1H), 7.43−7.46 (m, 2H), 7.35−7.37 (m, 2H), 7.24−7.28 (m, 2H), 7.13−7.28 (m, 14H), 7.05−7.09 (t, J = 7.7 Hz, 4H), 6.94−6.96 (d, J = 7.5 Hz, 4H), 6.85− 6.87 (dd, J = 7.9, 1.5 Hz, 4H), 6.73 (s, 2H); 13C NMR (126 MHz, CD2Cl2) δ 156.6, 143.6, 141.29, 141.27, 134.32, 134.23, 134.0, 132.4, 131.6, 131.2, 131.0, 130.8, 130.2, 130.1, 129.7, 129.1, 128.4, 128.2, 128.0, 127.7, 127.2, 127.0, 126.3; 11B NMR (128 MHz, CD2Cl2) δ 0.63 (t, J = 30.9 Hz); HRMS (ESI-TOF) m/z [M]− 723.2919, calcd for C51H35BF2N2 723.2903. BODIPY 2b. Yield 27.7 mg (as a blue solid), 85%; mp 288−290 °C; 1 H NMR (400 MHz, CDCl3) δ 7.26−7.30 (m, 4H), 7.15−7.24 (m, 12H), 6.69−7.01 (d, J = 8.8 Hz, 4H), 6.94−6.96 (d, J = 8.7 Hz, 2H), 6.88−6.90 (m, 4H), 6.74 (s, 2H), 6.58−6.61 (d, J = 8.8 Hz, 4H), 3.92 (s, 3H), 3.76 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 161.2, 159.6, 155.9, 141.9, 141.4, 141.1, 134.3, 133.8, 132.9, 132.4, 131.32, 131.30, 131.0, 130.8, 130.2, 129.0, 127.9, 127.5, 127.1, 126.8, 126.1, 124.1, 113.6, 113.0, 55.5, 55.1; 11B NMR (128 MHz, CDCl3) δ 0.84 (t, J = 9748
DOI: 10.1021/acs.joc.7b01940 J. Org. Chem. 2017, 82, 9744−9750
Article
The Journal of Organic Chemistry 31.4 Hz); HRMS (ESI-TOF) m/z 813.3197 [M]−, calcd for C54H41BF2N2O3 813.3220. BODIPY 2c. Yield 29.8 mg (as a blue solid), 81%; mp 220−222 °C; 1 H NMR (400 MHz, CDCl3) δ 8.11−8.14 (d, J = 8.3 Hz, 2H), 7.50− 7.52 (m, 2H), 7.38−7.52 (m, 5H), 7.26−7.28 (m, 2H), 7.15−7.23 (m, 10H), 7.10−7.13 (m, 2H), 7.01−7.03 (d, J = 8.8 Hz, 4H), 6.86−6.89 (m, 4H), 6.60−6.62 (m, 6H), 5.46 (s, 2H), 3.76 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 165.7, 159.8, 157.0, 141.3, 141.2, 140.0, 138.8, 135.9, 134.3, 134.1, 132.5, 131.3, 130.9, 130.7, 130.5, 130.2, 129.4, 129.0, 128.7, 128.4, 128.2, 128.0, 127.7, 127.1, 126.2, 123.9, 113.1, 67.0, 55.1; 11B NMR (128 MHz, CDCl3) δ 0.84 (t, J = 31.1 Hz); HRMS (ESI-TOF) m/z 917.3457 [M]−, calcd for C61H45BF2N2O4 917.3482. General Procedure for BODIPYs 3a−c. The BODIPY (2a−c, 0.02 mmol) was dissolved in CH2Cl2 (4 mL) followed by the addition of NaHCO3 (16.8 mg, 0.2 mmol). Br2 (0.2 mmol) was then added into the mixture dropwise. The mixture was stirred at room temperature for another 30 min. The reactions were monitored by TLC and stopped when the bromination was complete. The mixture was poured into aq saturated sodium thiosulfate solution (20 mL), and CH2Cl2 (10 mL × 3) was used to extract the organic components. The layers were combined and washed with brine followed by drying over anhydrous Na2SO4. The organic solvents were removed under reduced pressure. The residue was subjected to column chromatography (CH2Cl2/hexanes as eluents) to afford the desired products 3a−c. BODIPY 3a. Yield 16.6 mg (as a purple solid), 94%; mp >160 °C (dec); 1H NMR (400 MHz, acetone-d6) δ 7.54−7.65 (m, 5H), 7.36− 7.45 (m, 6H), 7.11−7.26 (m, 10H), 6.94−6.99 (m, 4H), 6.76−6.78 (m, 3H), 6.67−6.72 (m, 5H); 13C NMR (126 MHz, acetone-d6) δ 157.3, 156.8, 144.95, 144.81, 142.7, 142.5, 141.2, 141.1, 137.2, 137.1, 133.06, 133.04, 132.33, 132.30, 131.3, 131.1, 130.6, 130.5, 130.41, 130.39, 130.33, 130.23, 130.21, 130.16, 130.13, 130.11, 129.8, 129.7, 129.5, 129.28, 129.25, 129.20, 129.13, 129.11, 128.44, 128.37, 127.6, 127.4, 127.3, 127.23, 127.15, 122.75, 122.57; 11B NMR (128 MHz, acetone-d6) δ 0.07−0.57 (m); HRMS (ESI-TOF) m/z 879.1122 [M]−, calcd for C51H33BBr2F2N2 879.1113. BODIPY 3b. Yield 17.7 mg (as a dark blue solid), 91%; mp >145 °C (dec); 1H NMR (400 MHz, acetone-d6) δ 7.33−7.45 (m, 8H), 7.14− 7.25 (m, 10H), 6.65−6.76 (m, 8H), 6.51−6.55 (m, 1H), 3.91 (m, 3H), 3.71 (s, 6H); 13C NMR (126 MHz, acetone-d6) δ 162.0, 161.9, 160.9, 160.8, 157.0, 156.5, 143.97, 143.83, 142.8, 142.6, 141.4, 141.2, 136.93, 136.78, 133.2, 133.1, 132.1, 131.87, 131.84, 131.81, 131.79, 131.72, 130.97, 130.86, 130.4, 130.2, 129.2, 129.1, 128.45, 128.37, 127.7, 127.2, 127.1, 124.53, 124.48, 123.8, 123.6, 122.36, 122.18, 115.3, 115.1, 114.9, 113.0, 112.9, 55.5, 55.2; 11B NMR (128 MHz, acetoned6) δ 0.11−0.65 (m); HRMS (ESI-TOF) m/z 969.1402 [M]−, calcd for C54H39BBr2F2N2O3 969.1430. BODIPY 3c. Yield 19.6 mg (as a blue solid), 91%; mp >100 °C (dec); 1H NMR (400 MHz, CDCl3) δ 8.29 (m, 2H), 7.52−7.74 (m, 4H), 7.39−7.46 (m, 3H), 7.32−7.37 (m, 4H), 7.25−7.31 (m, 2H), 7.07−7.19 (m, 8H), 6.69−6.72 (m, 4H), 6.61−6.64 (m, 4H), 6.46− 6.50 (m, 4H), 5.44 (s, 2H), 3.71 (m, 6H); 13C NMR (126 MHz, CDCl3) δ 166.2, 160.03, 160.01, 156.6, 156.1, 142.2, 142.0, 140.7, 140.53, 140.47, 137.1, 136.6, 136.3, 135.8, 132.4, 132.3, 131.5, 131.17, 131.11, 131.05, 131.02, 130.87, 130.82, 130.6, 130.4, 130.28, 130.24, 130.1, 130.0, 129.9, 129.74, 129.65, 128.7, 128.56, 128.46, 128.4, 127.82, 127.75, 126.9, 126.51, 126.46, 123.1, 122.8, 121.6, 121.4, 112.74, 112.69, 67.1, 55.1; 11B NMR (128 MHz, CDCl3) δ 0.21−0.71 (m); HRMS (ESI-TOF) m/z 1073.1651 [M] − , calcd for C61H43BBr2F2N2O4 1073.1693. General Procedure for BODIPYs 4a−c. Into a flask equipped with a stir bar were added the BODIPY (3a−c, 0.01 mmol), K2CO3 (70 mg, 0.5 mmol), and Pd(PPh3)4 (10% mol). The flask was evacuated and refilled with N2 five times. Anhydrous toluene (3 mL) was injected into the flask. The mixture was stirred and refluxed for 1− 2 h. The reaction was stopped when the starting material was totally consumed, according to TLC. Catalyst and K2CO3 were removed by filtration through a Celite cake using CH2Cl2 as the eluent. The resulting residue was subjected to preparative TLC (CH2Cl2/hexanes 2:1 as eluent) to afford the desired pure products 4a−c.
BODIPY 4a. Yield 6.63 mg (as a teal solid), 92%; mp 255−257 °C; H NMR (400 MHz, CD2Cl2) δ 8.46−8.49 (d, J = 8.3 Hz, 2H), 8.38− 8.40 (d, J = 8.2 Hz, 2H), 7.53−7.61 (m, 12H), 7.43−7.46 (m, 2H), 7.36−7.40 (m, 1H), 7.27−7.31 (m, 2H), 7.15−7.19 (m, 6H), 6.95− 6.96 (d, J = 8.3 Hz, 2H), 6.70−6.73 (m, 2H); 13C NMR (126 MHz, CD2Cl2) δ 153.8, 143.5, 136.4, 136.2, 133.7, 133.4, 132.0, 131.1, 130.6, 130.1, 129.9, 129.64, 129.56, 129.2, 128.3, 127.6, 127.1, 126.8, 126.6, 126.1, 125.0, 123.6, 123.2, 123.1; 11B NMR (128 MHz, CD2Cl2) δ 1.16 (t, J = 30.6 Hz); HRMS (ESI-TOF) m/z 719.2604 [M]−, calcd for C51H31BF2N2 719.2590. BODIPY 4b. Yield 7.38 mg (as a green solid), 91%; mp 284−285 °C; 1H NMR (400 MHz, CDCl3) δ 8.47−8.49 (d, J = 8.2 Hz, 2H), 8.38−8.40 (d, J = 8.2 Hz, 2H), 7.59−7.61 (m, 4H), 7.41−7.48 (m, 4H), 7.22−7.35 (m, 6H), 7.01−7.06 (m, 6H), 6.76−6.80 (m, 2H), 6.67−6.69 (m, 2H), 3.92 (s, 6H), 3.79 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 162.3, 160.4, 153.8, 142.5, 136.2, 135.3, 132.0, 131.7, 130.7, 129.48, 129.43, 128.9, 128.0, 127.1, 126.8, 126.6, 126.0, 125.8, 125.4, 124.8, 123.8, 123.2, 123.1, 115.5, 114.0, 55.6, 55.3; 11B NMR (128 MHz, CDCl3) δ 1.25 (t, J = 30.3 Hz); HRMS (ESI-TOF) m/z 809.2902 [M]−, calcd for C54H37BF2N2O3 809.2907. BODIPY 4c. Yield 8.32 mg (as a green solid), 92%; mp 267−269 °C; 1H NMR (400 MHz, CDCl3) δ 8.47−8.50 (d, J = 8.2 Hz, 2H), 8.38−8.40 (d, J = 8.1 Hz, 2H), 7.83−7.85 (d, J = 8.3 Hz, 2H), 7.60− 7.62 (m, 5H), 7.40−7.50 (m, 8H), 7.33−7.35 (m, 2H), 7.23−7.29 (m, 4H), 7.05−7.07 (d, J = 8.8 Hz, 4H), 6.88−6.89 (d, J = 7.6 Hz, 2H), 6.64−6.67 (t, J = 7.3 Hz, 2H), 5.41 (s, 2H), 3.93 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 165.7, 160.5, 154.8, 141.0, 140.7, 136.0, 135.9, 133.9, 132.1, 131.8, 131.6, 130.75, 130.69, 129.5, 129.2, 128.6, 128.3, 127.9, 127.2, 127.0, 126.33, 126.29, 126.1, 125.1, 124.8, 123.8, 123.3, 114.0, 66.9, 55.3; 11B NMR (128 MHz, CDCl3) δ 1.21 (t, J = 30.1 Hz); HRMS (ESI-TOF) m/z 913.3154 [M] − , calcd for C61H41BF2N2O4 913.3169. 1
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b01940. X-ray data for BODIPY 4a in CIF format (CIF) Experimental procedures, additional UV−vis and emission spectra and tables, cytotoxicity graphs, microscopy images, DFT calculation results, and 1H, 13C, and 11B NMR spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Ning Zhao: 0000-0003-3699-3369 M. Graça H. Vicente: 0000-0002-4429-7868 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by grants from the NIH (R01 CA179902) and the NSF (CHE 1362641). REFERENCES
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DOI: 10.1021/acs.joc.7b01940 J. Org. Chem. 2017, 82, 9744−9750
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