Synthesis, structure, photophysical, electrochemical properties and antibacterial activity of brominated BODIPYs

Abstract

A series of mono- and di-brominated BODIPYs (1–5) was synthesized and characterized with a view to study the performance of dyes towards antibacterial activity. Regioselective bromination at the 2- and 2,6-positions of the BODIPY core was achieved with quantitative yield. The bromination of meso-(4-hydroxyphenyl) BODIPY (5) yielded an unexpected dibromo derivative, where the bromine groups were installed at the 3,5-positions of the phenyl ring rather than the 2,6-positions of the BODIPY core, which is confirmed and supported by UV-visible, fluorescence, and ¹H NMR spectroscopic analyses, electrochemical studies, and also by single crystal X-ray crystallography. We observed a red shift of ∼16 nm in the absorption and 20–29 nm in the emission spectra in CH₂Cl₂ for the installation of each bromine group at the BODIPY core. The small difference between the first reduction potentials of the parent and dibromo derivative (5 and 5b) reveal that dibromination does not occur on the pyrrolic moiety. The intermolecular interactions involving C⋯H, F⋯H, H⋯H, and Br⋯H are the key factors in stabilizing the molecular crystal packing. The antibacterial properties of these dyes were investigated and the brominated derivatives showed better antibacterial effects than their corresponding parent BODIPYs, particularly the unusual dibromo derivative, 5b.

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Introduction

The development of highly emissive dyes is of multifaceted interest for various applications ranging from materials chemistry to biology.¹ Out of the numerous organic dyes, such as fluorescein, cyanine, and rhodamine, BODIPYs (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) are intriguing because of their intense absorption profile, high fluorescent quantum yields, negligible triplet state formation, redox properties, decent solvatochromism, good solubility, and chemical robustness.^1,2 Architecting the BODIPYs with secondary units at the core/boron center/the meso-position is a thriving subject of interest and makes the system sensitive owing to their unusual spectroscopic behaviors.^2,3 Such renewed molecules could act as chemosensors, laser dyes, energy transfer cassettes, organic photovoltaics, biomolecular labels, supramolecular polymers, fluorescent pH probes, and photosensitizers for solar cells, and could also be used in photodynamic therapy.⁴

The synthetic difficulty in obtaining BODIPYs with an unsubstituted pyrrole is that none of the pyrrole-based carbons are blocked from electrophilic attack. However, effective synthetic strategies are available in the literature for diverse BODIPY derivatives; in the case of BODIPYs containing no substituents on the pyrrole ring,² modifications have to be allowed in the synthesis of dipyrromethane, to prevent the polymerization of pyrrole in the presence of catalytic amounts of TFA. Consequently, a ratio of 1 : 25 (aldehyde : pyrrole) for the dipyrromethane synthesis of BODIPYs with unsubstituted pyrrole is maintained, and pyrrole can act as both reactant as well as solvent. However, for the synthesis of BODIPYs with substituted pyrrole, the ratio is about 1 : 2 or 1 : 4 and the solvent employed is dichloromethane. Hence the chance of poly-pyrrole formation in the former is larger than that in the latter. The effect of halogen substitution at the pyrrole carbons of the boron dipyrrin core on structural and spectral properties is frequently studied.^3,5 The presence of heavy atoms, such as bromine, on the BODIPY nucleus enhances intersystem crossing, and these are potential sensitizers for PDT, as an alternative to porphyrin-based photosensitizers. As far as biological applications are concerned, fluorophores absorbing at longer wavelengths are particularly important, because at higher wavelength absorption by cells and water, light scattering and auto-fluorescence are reduced significantly, allowing for deeper light penetration, resulting in efficient therapy and diagnosis.² The regioselective electrophilic bromination of the 1, 2, 3, 5, 6, and 7-positions of BODIPY has recently been reported, using either liquid bromine or N-bromosuccinimide (NBS).⁵ It is also well documented in the literature that 2,6-halogenated BODIPYs could act as efficient singlet oxygen photosensitizers for photodynamic therapy (PDT), due to the heavy atom effect.⁶ Moreover, the introduction of a halogen atom onto the BODIPY core may facilitate the generation of longer-wavelength BODIPY dyes with further diverse derivatization through aromatic nucleophilic substitution or palladium-catalyzed coupling reactions, such as Suzuki, Stille, Heck, and Sonogashira reactions.^3,7

Reports on the antibacterial⁸ and antimicrobial⁹ activities of BODIPY dyes are very limited in the literature. Inspired by the report on singlet oxygen formation in brominated BODIPYs,¹⁰ we attempted studies on the antibacterial activity of synthesized brominated BODIPYs towards Gram-positive and Gram-negative bacteria under light and dark conditions. Furthermore, the halogenated BODIPYs had not been tested for any other biological activities so far. In order to delineate the heavy atom (bromine) effect on the BODIPYs towards antibacterial activity, we report here for the first time on mono-/dibromo derivatives of BODIPYs bearing 4-methyl, 4-t-butyl, 4-N,N′-dimethyl, and 4-hydroxyl groups at the meso-phenyl position (Schemes 1 and 2) and the present study is also directed towards a comparison on spectral, electrochemical, and photophysical properties.

Scheme 1 Synthesis of various BODIPYs and their mono- and di-brominated derivatives.

Scheme 2 Bromination reaction of 4-hydroxy BODIPY and its brominated derivatives.

Experimental

Materials and methods

All the chemicals used for the synthesis were reagent grade unless otherwise specified. Pyrrole and boron trifluoride etherate (BF₃·OEt₂), purchased from Spectrochem (India), was distilled over CaH₂ before use. Benzaldehyde, 4-hydroxybenzaldehyde, 4-methylbenzaldehyde, 4-tert-butylbenzaldehyde, 4-N,N-dimethylaminobenzaldehyde, and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) were obtained from Sigma Aldrich and were used as received. N-Bromosuccinimide (NBS), purchased from Merck (India), was recrystallized from hot water and dried at 80 °C for 6 h. Trifluoroacetic acid (TFA), purchased from Merck (India), was distilled over P₂O₅. Triethylamine (Et₃N), purchased from Qualigens, was used as received. Chloroform, dichloromethane, hexane, and methanol, purchased from AVRA Synthesis Pvt. Ltd, were purified by distilling over K₂CO₃. Toluene and acetonitrile were purchased from Fischer Scientific, and THF was obtained from SRL Chemicals Ltd, India. Hydrogen-1 NMR spectra were recorded with a Bruker 400 MHz FT-NMR spectrometer in CDCl₃/CD₃COCD₃, using tetramethylsilane as the internal reference. IR data was collected on a JASCO FT/IR-4700 with KBr pellets. Mass spectra of BODIPY complexes were performed either using liquid chromatography-mass spectrometry (LC-MS) (Shimadzu-LCMS-2010) or with a Waters Xevo G2 Quadrapole-Time-of-Flight (Q-TOF) high resolution mass spectrometer (HRMS). UV-visible spectra were recorded on a Shimadzu double-beam spectrometer 2450 instrument, using 1 cm matched quartz cuvettes at room temperature. Single crystal structure X-ray data collections were performed on a Bruker AXS Kappa Apex II CCD diffractometer with graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 298 K. Fluorescence spectra were recorded on a Perkin-Elmer LS-55 Luminescence spectrophotometer with a slit width of 10 at 470 nm excitation wavelength and emission from 490 to 750 nm. The fluorescence quantum yield (Φ_f) was computed using the equation below,

Φ_f = Φ^R_fF_SA_Rη_S²/F_RA_Sη_R²

where F_S and F_R are the integrated fluorescence intensities of the sample and reference, A_S and A_R are the absorbance of the sample and reference at the excitation wavelength, and η_S and η_R are the refractive indices of the solvents used for the sample and reference. Fluorescein in 0.1 M NaOH solution was used as the reference (Φ_f = 0.85, λ_ex = 470 nm). All Φ_f values were corrected for changes in refractive index. Both the BODIPYs and reference solutions were prepared with the same absorbance at the excitation wavelength (between 0.1 and 0.05 in a 1 cm quartz cell). Electrochemical measurements were performed on a CH-instruments Inc., USA (Model: CHI660E), equipped with a potentiostat/galvanostat with a Fourier Transform AC voltammeter. The electrochemical system utilized a three-electrode configuration, consisting of a glassy carbon (working), a platinum wire (auxiliary) and standard calomel reference (SCE) electrodes. The concentrations of the samples were maintained at 0.1 M, containing tetrabutylammonium hexafluorophosphate (NBu₄PF₆) as supporting electrolyte (0.1 M) in dichloromethane at 25 °C under N₂ atmosphere at a scan rate of 50 mV s⁻¹. The fluorescence imaging was performed using a Nikon ECLIPSE Ti-microscope attached to a Cool SNAP digital camera at 40× magnification. The images were processed using NIS Elements BR software.

Microorganism and growth conditions

Escherichia coli strain 2065 and Bacillus subtilis strain 168 (trpC2) were used for studying the antibacterial activity of BODIPY compounds. Both the organisms were grown aerobically at 37 °C in a standard Luria–Bertani (LB) broth containing 10 g L⁻¹ tryptone, 5 g L⁻¹ yeast extract, and 10 g L⁻¹ NaCl. To prepare LB agar Petri-plates, 12 g L⁻¹ agar powder was added to the LB broth before autoclaving.

Agar well diffusion assay for determining the growth inhibition of Escherichia coli and Bacillus subtilis under light and dark conditions

An agar well diffusion assay was performed as described earlier.^11a Briefly, Bacillus subtilis and Escherichia coli (EC) were grown overnight in 5 mL LB broth in a shaking incubator maintained at 37 °C, at 200 rotations per minute (RPM). LB agar-containing Petri plates were prepared by pouring the sterile LB agar into sterile Petri plates to a uniform depth of 4 mm, which is equivalent to approximately 25 mL in a 90 mm plate. The medium was allowed to solidify in the plates. The overnight culture of BS and EC was spread evenly on the solidified agar with the help of a sterile cotton swab. Wells were made in the agar plates for addition of the BODIPY solutions. All the BODIPYs were then added in equal volume and equal concentration (25 μg per well) into the wells. The Petri plates were distributed into two sets; one set was kept in the incubator with an inbuilt light source, while the other was kept in a dark incubator. Both incubators were programmed to maintain 37 °C, and organisms were allowed to grow overnight. In the incubator with the light source, irradiation was performed using a quartz line lamp (300 W). Light intensities were measured with a pyroelectric detector (RJP-735 from Laser Probe), which was connected to an energy ratio meter (RJ-7620 from Laser Probe). The study was conducted for an irradiation time of 12 h and the average light fluence rate was 95 μJ cm⁻². Tetracycline (10 μg per well) was used as a positive control for this experiment and DMSO was kept as the solvent control. After 12 h of incubation, the zone of inhibition was calculated using the formula given below:^11a

% of inhibition = (diameter of the inhibition zone × 100)/90(diameter of Petri plate)

Determination of half maximal inhibitory concentration (IC₅₀) and minimum inhibitory concentration (MIC) values for the BODIPYs using LB broth assay

The overnight culture of EC and BS cells was inoculated in 5 mL LB medium containing different concentrations of BODIPYs (5, 10, 20, 40, 60, and 80 μM). The growth inhibitory effect of the compounds was studied by monitoring the absorbance at 600 nm (A₆₀₀), at different time intervals (0, 4, 8, and 12 hours). The A₆₀₀ value of the control cells treated with the solvent control (DMSO) was subtracted from the A₆₀₀ value obtained immediately after adding the compounds in all cases, in order to eliminate the absorbance contributed by the compounds alone. The percentage of growth inhibition was determined for 12 h after the addition of BODIPYs to the bacterial cultures. The percentage inhibition of the cell growth was calculated using the equation:¹¹

% Inhibition = [1 − (XD₆₀₀/CD₆₀₀)] × 100

where XD₆₀₀ represents the A₆₀₀ value of X μM of the compound-treated culture at the different time points. CD₆₀₀ represents the A₆₀₀ value of the control culture of the same time. The IC₅₀ values of the compounds were determined by plotting the percentage inhibition of cell growth against various concentrations of BODIPYs. Furthermore, the concentration of the compound at which there was no visible growth of the organism was diluted and spread on to the LB agar plates, and incubated for an additional 12 h at 37 °C. The number of colony-forming units was calculated by counting the colonies on each plate. The concentration at which no visible growth was observed was considered as the MIC.¹¹

Intracellular localization of BODIPYs in EC and BS cells by fluorescence microscopy analysis

For studying the intracellular localization of the BODIPYs, EC and BS cells were grown in the liquid medium for 6 hours in the presence of compounds from the 5 series (5, 5a, and 5b), in a shaking incubator at 37 °C. The cells were then harvested and washed twice with 1× PBS in order to remove the un-internalized compounds. The cells were then incubated with Hoechst 33 342 (1.5 μg mL⁻¹) for 30 minutes to visualize the DNA.¹¹ This step was followed by washing the cells twice in 1× PBS. The cells were then mounted on clean glass slides and sealed for microscopic analysis. The compounds were excited using a green filter (490–510 nm) so as to excite the BODIPYs. The fluorescence imaging was performed using a Nikon ECLIPSE Ti-microscope attached with a Cool SNAP digital camera at 40× magnification. The images were processed using NIS Elements BR software.

Measurement of singlet oxygen species

DPBF (1,3-diphenylisobenzofuran) (4.9 × 10⁻⁵ M) solutions with and without BODIPYs (2.25 × 10⁻⁶ M) in DMSO, prepared in the dark, were irradiated at room temperature under gentle magnetic stirring. The singlet oxygen (¹O₂) acceptor, DPBF, gradually decolorized upon reaction with singlet oxygen species generated by BODIPYs. The breakdown of the DPBF molecules was monitored by measuring the decrease in absorbance at 410 nm at regular irradiation intervals. The relative rates of singlet oxygen production in selected BODIPYs (1, 1a, 1b, 2a, 3a, 4b, and 5b) were determined.

Synthesis

Synthesis of dipyrromethanes. To a solution of the corresponding aldehyde (1 equiv.) in freshly distilled pyrrole (25/50 equivalent), degassed with a stream of N₂, was added a catalytic amount of trifluoroacetic acid (TFA).¹⁰ The mixture was stirred at room temperature for 10–25 min. Upon completion of the reaction, dilute NaOH (0.1 M) was added to quench the reaction. The product was extracted with ethyl acetate, dried over anhydrous Na₂SO₄ and evaporated to dryness under vacuum. This crude product was then purified by recrystallization in an EtOH/water mixture or column chromatographed on silica gel using CHCl₃/hexane.
5-Phenyldipyrromethane. Prepared according to the general procedure.¹² Yield: 62%.
5-(4-Methylphenyl)dipyrromethane. Yield: 30%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 2.32 (s, 3H; methyl-H), 5.43 (s, 1H; meso-H), 5.91 (s, 2H; py), 6.15–6.13 (m, 2H; py), 6.68–6.66 (m, 2H; py), 7.13–7.08 (m, 4H; Ar-H), 7.89 (s br, 1H; py-NH).
5-(4-tert-Butylphenyl)-dipyrromethane. Yield: 64%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 1.30 (s, 9H; t-butyl-H), 5.45 (s, 1H; meso-H), 5.94–5.92 (m, 2H; py), 6.16–6.14 (m, 2H; py), 6.69–6.67 (m, 2H; py), 7.15–7.13 (m, 2H; Ar-H), 7.33–7.31 (m, 2H; Ar-H), 7.91 (s br, 1H; py-NH).
5-(4-N,N′-Dimethylaminophenyl)-dipyrromethane. Yield: 48%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 2.92 (s, 6H; dimethylamino-H), 5.38 (s, 1H; meso-H), 5.94–5.92 (m, 2H; py), 6.15–6.13 (m, 2H; py), 7.08–7.06 (m, 2H; py), 6.70–6.67 (d, 2H; Ar-H), 7.06–7.08 (d, 2H; Ar-H), 7.89 (s br, 1H; py-NH).
5-(4-Hydroxyphenyl)-dipyrromethane. Yield: 46%; ¹H NMR (400 MHz, acetone-d₆, δ in ppm): 5.33 (s, 1H; meso-H), 5.71 (d, 2H; py), 5.96–5.94 (q, 2H; py), 6.65 (d, 2H; py), 6.73 (d, 2H; Ar-H), 7.02 (d, 2H; Ar-H), 9.57 (s br, 2H; py-NH).

Synthesis of BODIPYs, 1–5. Into the corresponding dipyrromethane dissolved in dry CH₂Cl₂, nitrogen was purged for 15 min. DDQ (3-dichloro-5,6-dicyano-1,4-benzoquinone) was added and stirred vigorously for 0.5–1 h at room temperature until the complete consumption of dipyrromethane, as monitored by TLC. To this oxidized product, dipyrromethene, triethylamine was added and stirred further at room temperature for another 15 min, after which boron trifluoroetherate was added and stirring was continued. TLC analysis indicated the formation of the expected compound as a fluorescent orange-red spot. The reaction mixture was then washed with water and extracted with CH₂Cl₂, the organic layers were combined, dried over Na₂SO₄, filtered, and the solvent was removed under reduced pressure to give the crude product. This was further purified by silica gel column chromatography, using chloroform to afford the pure compound as an orange red/green fluorescent solid.
4,4-Difluoro-8-phenyl-4-bora-3a,4a-diaza-s-indacene (1). Compound 1 was obtained as an orange solid. Yield = 56%; UV/vis (CH₂Cl₂): λ_max (log ε) = 500 nm (4.66); fluorescence (CH₂Cl₂): λ_em = 527 nm (λ_ex = 470 nm). IR (KBr, cm⁻¹): 3063, 2923, 2852, 2345, 1638, 1577, 1542, 1413, 1389, 1352, 1321, 1260, 1226, 1157, 1116, 1076, 910, 780, 775, 751, 722, 665.
4,4-Difluoro-8-(4-methylphenyl)-4-bora-3a,4a-diaza-s-indacene (2). Compound 2 was afforded in pure form as a reddish-orange solid. Yield = 84%. ¹H NMR (400 MHz, CDCl₃, δ in ppm): 2.47 (s, 3H; –CH₃), 6.53–6.54 (d, 2H; py), 6.95–6.96 (d, 2H; py), 7.32–7.34 (d, 2H; Ar-H), 7.46–7.48 (d, 2H; Ar-H), 7.92 (s, 2H; py); UV/vis (CH₂Cl₂): λ_max (log ε) = 499 nm (4.22); fluorescence (CH₂Cl₂): λ_em = 514 nm (λ_ex = 470 nm). IR (KBr, cm⁻¹): 2923, 2958, 2853, 1730, 1574, 1545, 1476, 1413, 1387, 1262, 1223, 1186, 1156, 1121, 1098, 1081, 1050, 977, 911, 774, 757, 738.
4,4-Difluoro-8-(4-tert-butylphenyl)-4-bora-3a,4a-diaza-s-indacene (3). The crude solid was purified chromatographically to yield target compound 3 as a reddish orange solid in 57% yield; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 1.39 (s, 9H), 6.53–6.54 (d, 2H; py), 6.98–6.99 (d, 2H; py), 7.50–7.55 (m, 4H; ArH), 7.93 (s, 2H; py). UV/vis (CH₂Cl₂): λ_max (log ε) = 499 nm (4.75); fluorescence (CH₂Cl₂): λ_em = 515 nm (λ_ex = 470 nm). IR (KBr, cm⁻¹): 3114, 2923, 2956, 2852, 1537, 1603, 1735, 1475, 1353, 1077, 1117, 1260, 1223, 1198, 1157, 1312, 1384, 1411, 1475, 719, 775, 764, 803, 836, 851, 916, 649.
4,4-Difluoro-8-(4-N,N′-dimethylaminophenyl)-4-bora-3a,4a-s-diaza-s-indacene (4). A green solid was obtained and the yield was noted as 16%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 3.11 (s, 6H; dimethylamino-H), 6.53–6.54 (d, 2H; py), 6.78–6.81 (d, 2H; py), 7.04–7.05 (d, 2H; Ar-H), 7.55–7.57 (d, 2H; Ar-H), 7.87 (s, 2H; py). λ_max (log ε) = 489 nm (3.82); fluorescence (CH₂Cl₂): λ_em = 520 nm (λ_ex = 470 nm); IR (KBr, cm⁻¹): 2853, 2714, 1734, 1662, 1603, 1551, 1534, 1465, 1411, 1389, 1374, 1263, 1232, 1200, 1167, 1120, 1079, 941, 910, 824, 812, 742, 728, 775, 742, 728, 699, 596, 2923, 1465, 1475.
4,4-Difluoro-8-(4-hydroxyphenyl)-4-bora-3a,4a-diaza-s-indacene (5). An orange solid was obtained and the yield was 56%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 6.55 (d, 2H; py), 6.98 (d, 4H; Ar-H), 7.49 (d, 2H; py), 7.92 (s, 2H; py); UV/vis (CH₂Cl₂): λ_max (log ε) = 497 nm (4.77); fluorescence (CH₂Cl₂): λ_em = 512 nm (λ_ex = 470 nm). IR (KBr, cm⁻¹): 3576, 3439, 2918, 2801, 2682, 2324, 1757, 1708, 1604, 1510, 1474, 1446, 1413, 1587, 1539, 1510, 1474, 1446, 1413, 1353, 1299, 1278, 1259, 1227, 1178, 1119, 1077, 1040, 1009, 978, 963, 909, 844, 780, 765, 744, 710, 647.

Synthesis of brominated BODIPYs, 1–5(a–b). To a solution of BODIPY dissolved in dry CH₂Cl₂ was added dropwise three equivalents of N-bromosuccinimide (NBS) in dry CH₂Cl₂ over a period of 20 min. This mixture was left stirring at room temperature for an additional 15–17 h, washed with brine and extracted with CH₂Cl₂. The organic layers were combined, dried over Na₂SO₄ and evaporated to dryness under vacuum. The purification was performed by column chromatography on silica gel, using CHCl₃/hexane as eluent, which afforded two products, mono-brominated (1a–5a) as well as di-brominated (1b–5b) derivatives.
2-Bromo-4,4-difluoro-8-phenyl-4-bora-3a,4a-diaza-s-indacene (1a) and 2,6-dibromo-4,4-difluoro-8-phenyl-4-bora-3a,4a-diaza-s-indacene (1b)⁵. Purification was performed by column chromatography on silica gel, using CHCl₃/hexane (80/20) as eluent to provide compounds 1a and 1b. Compound 1a: yield = 20%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 6.59 (s, 1H; py), 6.88 (d, 1H; py), 6.99 (s, 1H; py-H), 7.53–7.59 (m, 5H; Ar-H), 7.79 (d, 1H; py), 8.00 (d, 1H; py); ¹³C NMR (400 MHz, CDCl₃): δ 147.1, 146.2, 142.1, 135.4, 134.3, 133.3–133.2, 131.16, 130.4, 130.3, 128.6, 119.61, 105.9. λ_max (log ε) = 516 nm (4.70); fluorescence (CH₂Cl₂): λ_em = 547 nm (λ_ex = 470 nm); IR (KBr, cm⁻¹): 3124, 2922, 2852, 2955, 1637, 1729, 1577, 1551, 1477, 1474, 1441, 1402, 1362, 1360, 1253, 1222, 1148, 1115, 1074, 1038, 985, 925, 903, 832, 772, 749, 723, 700, 665, 633, 614, 576. HRMS (ESI-TOF) m/z: 346.97 (calc.) 347.02 (found). Compound 1b: yield = 65%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 6.88 (s, 2H; py), 7.56 (m, 1H; Ar-H), 7.47 (m, 4H; Ar-H), 7.78 (s, 2H; py); ¹³C NMR (400 MHz, CDCl₃): δ 146.0, 143.2, 133.6, 131.8, 130.7, 130.5, 129.3, 127.8, 106.2. λ_max (log ε) = 538 nm (4.65); fluorescence (CH₂Cl₂): λ_em = 569 nm (λ_ex = 470 nm); IR (KBr, cm⁻¹): 3114, 2923, 2852, 2959, 1733, 1638, 1576, 1553, 1479, 1464, 1435, 1375, 1346, 1253, 1099, 1073, 992, 916, 848, 802, 748, 715, 633, 614, 573. LCMS m/z: 423.92 (calc.) 423 (found).
2-Bromo-4,4-difluoro-8-(4-methylphenyl)-4-bora-3a,4a-diaza-s-indacene (2a) and 2,6-dibromo-4,4-difluoro-8-(4-methylphenyl)-4-bora-3a,4a-diaza-s-indacene (2b). The reaction mixture was stirred at 45 °C for 72 h. The purification was performed by column chromatography on silica gel using CHCl₃/hexane (40/60) as eluent. Compound 2a: yield = 24%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 2.48 (s, 3H; methyl-H), 6.59 (d, 1H; py), 6.90 (s, 1H; py), 7.02 (d, 1H; py), 7.35 (d, 2H; Ar–H), 7.46 (d, 2H; Ar-H), 7.77 (s, 1H; py), 7.98 (s, 1H; py); ¹³C NMR (400 MHz, CDCl₃): δ 147.5, 145.8, 141.9, 141.8, 133.1, 135.3, 134.2, 130.5, 130.2, 129.4, 119.4, 105.7, 21.5. UV/vis (CH₂Cl₂): λ_max (log ε) = 515 nm (4.66); fluorescence (CH₂Cl₂): λ_em = 538 nm (λ_ex = 470 nm). IR (KBr, cm⁻¹): 3134, 2923, 1731, 1604, 1566, 1536, 1474, 1402, 1474, 1365, 1360, 1256, 1227, 1365, 1360, 1075, 1032, 1146, 1121, 1104, 1018, 905, 925, 984, 869, 772, 756, 740, 703, 646. HRMS (ESI-TOF) m/z: 360.99 (calc.) 361 (found). Compound 2b: yield = 67%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 2.41 (s, 3H; methyl-H), 6.90 (s, 2H; py), 7.29 (d, 2H; Ar-H), 7.38 (d, 2H; Ar-H), 7.76 (s, 2H; py); ¹³C NMR (400 MHz, CDCl₃): δ 147.4, 143.8, 142.4, 134.6, 131.7, 130.5–130.5, 130.1, 129.6–129.5, 107.0, 21.5. UV/vis (CH₂Cl₂): λ_max (log ε) = 535 nm (4.84); fluorescence (CH₂Cl₂): λ_em = 558 nm (λ_ex = 470 nm). IR (KBr, cm⁻¹): 3123, 2922, 2852, 2345, 2360, 1735, 1684, 1606, 1569, 1541, 1569, 1541, 1569, 1479, 1352, 1362, 1379, 1263, 1183, 1126, 1103, 1001, 982, 915, 835, 824, 754, 743, 705, 620, 644, 575. HRMS (ESI-TOF) m/z: 439.89 (calc.) 440.91 (found).
2-Bromo-4,4-difluoro-8-(4-tert-butylphenyl)-4-bora-3a,4a-diaza-s-indacene (3a) and 2,6-dibromo-4,4-difluoro-8-(4-tert-butylphenyl)-4-bora-3a,4a-diaza-s-indacene (3b). The brominated derivatives were obtained by column chromatography on silica gel using CHCl₃/hexane (80/20) as eluent. Compound 3a: yield = 5%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 1.35 (s, 9H), 6.53 (s, 1H; py), 6.87 (s, 1H; py), 6.99 (s, 1H; py), 7.44 (d, 2H; Ar-H), 7.49 (d, 2H; Ar-H), 7.71 (s, 1H; py), 7.92 (s, 1H; py); ¹³C NMR (400 MHz, CDCl₃): δ 154.0, 146.5, 144.7, 140.7, 134.3, 133.1, 132.2, 129.4–129.3, 124.6, 118.3, 104.7, 34.0, 30.1. λ_max (log ε) = 515 nm (4.57); fluorescence (CH₂Cl₂): λ_em = 537 nm (λ_ex = 470 nm); IR (KBr, cm⁻¹): 3119, 2923, 2959, 2852, 1732, 1539, 1364, 1642, 1478, 1463, 1402, 1032, 1069, 1088, 1128, 1260, 1256, 1146, 649, 906, 830, 719, 775, 760, 744, 714. LCMS m/z: 402.07 (calc.) 402 (found). Compound 3b: yield = 89%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 1.32 (s, 9H), 6.93 (s, 2H), 7.42 (m, 2H; Ar-H), 7.49 (d, 2H; Ar-H), 7.75 (s, 2H; py); ¹³C NMR (400 MHz, CDCl₃): δ 155.5, 144–143.7, 134.6, 131.8, 130.5–130.3, 129.5, 125.9–125.8, 114, 35.1, 31.1. λ_max (log ε) = 544 nm (4.59); fluorescence (CH₂Cl₂): λ_em = 564 nm (λ_ex = 470 nm); IR (KBr, cm⁻¹): 2923, 2959, 2852, 3116, 1556, 1640, 1746, 1352, 1464, 1075, 1101, 1227, 1243, 1352, 1397, 835, 755, 708, 1609, 1464, 1370, 1382, 741. HRMS (ESI-TOF) m/z: 481.97 (calc.) 481 (found).
2-Bromo-4,4-difluoro-8-(4-N,N′-dimethylaminophenyl)-4-bora-3a,4a-diaza-s-indacene (4a) and 2,6-dibromo-4,4-difluoro-8-(4-N,N′-dimethylaminophenyl)-4-bora-3a,4a-diaza-s-indacene (4b). Prepared according to the general procedure, 4,4-difluoro-8-(4-N,N′-dimethylaminophenyl)-4-bora-3a,4a-diaza-s-indacene (4) was treated with NBS for 22 h at room temperature. The crude reaction mixture after standard work up was purified on silica gel using CHCl₃/hexane (30/70) as eluent. Compound 4a: yield = 28%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 2.95 (s, 6H; dimethylamino-H), 6.54 (s, 1H; py), 6.86 (s, 1H; py), 6.98 (d, 1H; py), 7.47 (m, 2H; Ar-H), 7.64 (m, 2H; Ar-H), 7.71 (s, 1H; py), 7.91 (s, 1H; py); ¹³C NMR (400 MHz, CDCl₃): δ 154.6, 145.7, 141.7, 136.2, 135.0, 133.9, 132.7, 130.7, 129.9, 127.6, 119.7–119.5, 117.3, 105.8, 43.6. UV/vis (CH₂Cl₂): λ_max (log ε) = 518 nm (4.66); fluorescence (CH₂Cl₂): λ_em = 542 nm (λ_ex = 470 nm). IR (KBr, cm⁻¹): 3116, 2923, 2956, 2852, 1725, 1684, 1592, 1556, 1469, 1402, 1360, 1331, 1260, 1221, 1168, 1118, 1078, 1035, 1078, 988, 946, 933, 915, 825, 773, 756, 742, 720, 665, 642. ESI-MS m/z: 390.03 (calc.) [M + Na]⁺ = 413 (found). Compound 4b: yield = 42%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 2.98 (s, 6H; dimethylamino-H), 7.00–7.02 (d, 2H; Ar), 7.15–7.13 (d, 2H; py), 7.50–7.45 (m, 2H; py), 7.78–7.60 (d, 2H; Ar); ¹³C NMR (400 MHz, CDCl₃): δ 154.9, 148.4, 143.6, 142.4, 136.2, 134.8, 134.7, 131.3, 130.8, 119.8, 107.1, 43.6. UV/vis (CH₂Cl₂): λ_max (log ε) = 535 nm (4.60); fluorescence (CH₂Cl₂): λ_em = 548 nm (λ_ex = 470 nm). IR (KBr, cm⁻¹): 3107, 3125, 2851, 1688, 1590, 1553, 1499, 1474, 1375, 1349, 1263, 1189, 1166, 1120, 1090, 1035, 1375, 1349, 992, 946, 915, 886, 867, 821, 742, 720, 661, 643, 606, 566, 585. LCMS m/z: 468.93 (calc.) 468 (found).
2-Bromo-4,4-difluoro-8-(4-hydroxyphenyl)-4-bora-3a,4a-diaza-s-indacene (5a) and 2,6-dibromo-4,4-difluoro-8-(3,5-dibromo-4-hydroxyphenyl)-4-bora-3a,4a-diaza-s-indacene (5b). The reaction mixture was stirred for 15 h. Purification was performed by column chromatography on silica gel using CHCl₃/hexane (80/20) as eluent. Compound 5a: yield = 2%. ¹H NMR (400 MHz, CDCl₃, δ in ppm): 6.45 (s br, 1H; –OH), 6.59 (d, 1H; py), 6.95–6.68 (dd, 1H; py), 7.55–7.54 (m, 1H; py), 7.86–7.83 (m, 4H; Ar-H), 7.98 (s, 1H; py), 8.04 (s, 1H; py). UV/vis (CH₂Cl₂): λ_max (log ε) = 514 nm (4.39); fluorescence (CH₂Cl₂): λ_em = 541 nm (λ_ex = 470 nm). IR (KBr, cm⁻¹): 3449, 2923, 2853, 2957, 1719, 1640, 1560, 1534, 1465, 1401, 1362, 1260, 1222, 1184, 1080, 1021, 908, 861, 808, 728, 645. ESI-MS m/z: 362.96 (calc.) 363.25 (found). Compound 5b: yield = 90%; ¹H NMR (400 MHz, CDCl₃, δ in ppm): 6.23 (s, br, 1H; –OH), 6.51 (d, 2H; py), 6.87 (d, 2H; py-H), 7.88 (s, 2H; py), 7.62 (s, 2H; Ar-H); ¹³C NMR (400 MHz, CDCl₃): δ 151.7, 144.8, 143.2, 134.6, 133.7, 131.1, 128.2, 119, 110. UV/vis (CH₂Cl₂): λ_max (log ε) = 510 nm (4.58); fluorescence (CH₂Cl₂): λ_em = 529 nm (λ_ex = 470 nm). IR (KBr, cm⁻¹): 3423, 2925, 2853, 2963, 1640, 1560, 1536, 1467, 1409, 1399, 1261, 1098, 863, 801, 742, 727, 702, 663. HRMS (ESI-TOF) m/z: 441.86 (calc.) 440.90 (found).

Results and discussion

Synthesis and characterization

The required appropriate boron dipyrromethane precursors were synthesized by adopting the standard protocol.¹² In the first step, the corresponding dipyrromethanes were prepared by the condensation of substituted benzaldehyde, with a large excess of pyrrole (25/50 equiv.), in the presence of a catalytic amount of trifluoroacetic acid (TFA, 0.1 equiv.) for 10–20 min at room temperature. The crude compounds were purified by a column chromatography/recrystallization technique, affording meso-(4-phenyl substituted) dipyrromethanes in 30–64% yield. These dipyrromethanes were then subjected to a two-step one-pot reaction by oxidizing the dipyrromethanes in the first step with DDQ, and reacted in the second step with Et₃N, followed by BF₃·OEt₂ at room temperature. Column chromatography on silica gel yielded the meso-substituted BODIPY dyes, 1–3 and 5, in 56–84% and 4 in 16% yield. The yields of dipyrromethanes and unbrominated BODIPYs (1–3 and 5, except 4) are almost fair, and reasonably comparable to literature values.¹² The synthetic routes employed to prepare the meso-substituted BODIPYs and their mono- and dibromo derivatives are outlined in Schemes 1 and 2.

It has been reported that the regioselective bromination of unsubstituted BODIPY with 1.2 and 2.4 equivalents of NBS in dichloromethane at room temperature could yield mono- and di-brominated derivatives, respectively, in quantitative yield.⁷ In the present study, regioselective bromination of BODIPYs was achieved at 2- and 2,6-positions, except in the case of 4-HPhBODIPY, 5, with three equivalents of NBS in dichloromethane at room temperature. TLC analysis of the crude reaction mixture showed a major spot corresponding to the di-brominated BODIPYs (1b–4b), along with a minor amount of mono-brominated product (1a–4a) (Scheme 1). The crude products were then subjected to silica gel column chromatographic purification using CHCl₃/hexane. Here, all the dibromo derivatives were obtained in good yield; higher yields of the monobromo derivative could be obtained with 1–1.2 equivalents of NBS.^3,4 Hence, no further optimization of the protocol was performed. In addition, limiting the side-product formation and increasing the low separation yield has to be addressed to yield quantitative amount of products chromatographically.

The synthesized compounds were freely soluble in common organic solvents and were characterized by absorption, fluorescence, ¹H NMR spectroscopic methods, electrochemical studies, and XRD techniques. In contrast to other derivatives, bromination of 4-HPhBODIPY, 5, occurred at the 3,5-phenyl positions rather than the desired 2,6-pyrrole positions, as confirmed by spectroscopic and XRD analyses (Scheme 2).

Hydrogen-1 NMR spectra were taken for the dipyrromethanes and the integrated intensities of all the proton signals are very well in concordance with the expected structure, indicating their structural confirmation. Unlike other BODIPYs (1–4), the resonance structures of compound 5 bear the least positive charge at the beta pyrrole positions, hence dibromination occurred at the 3,5-phenyl positions, rather than the desired 2,6-pyrrole positions. The absence of proton signals corresponding to one and/or two β-pyrrole hydrogen(s) at 2- and/or 2,6-protons, respectively, in the spectra clearly indicated successful bromination at the beta pyrrole positions. In general, ¹H NMR spectra of BODIPYs indicate six pyrrole protons, whereas mono-brominated and di-brominated products indicate five and four proton signals, respectively. However, the number of pyrrole proton signals and their spin multiplicity vary with the symmetric/asymmetric nature of the compounds. Representative overlaid ¹H NMR spectra of compound 2 and their brominated derivatives (2a and 2b) are shown in Fig. 1. The β-pyrrole proton signals at 6.95 ppm in the parent BODIPY are deshielded to 7.01 ppm in mono-brominated product 2a, which in turn disappeared from the spectrum of di-brominated compound 2b. The aromatic phenyl protons with four-proton intensity in 5 at 6.98 ppm were deshielded to 7.62 ppm, with two-proton intensity in the corresponding brominated derivative (5b), which clearly indicated bromination on the meso-phenyl group, rather than the pyrrole ring of the BODIPY core. The ¹H NMR spectra of other dipyrromethanes, BODIPYs and their brominated derivatives are presented in the ESI.†

Fig. 1 Selected region of overlaid ¹H NMR spectra of compounds, 2, 2a, and 2b in CDCl₃ at 25 °C.

The FT-IR spectra of the synthesized compounds were taken with KBr pellets, and overlaid spectra of selected BODIPYs are shown in Fig. S27.† The IR spectra of all the BODIPYs showed three characteristic aromatic –C–H vibration peaks near 2923, 2959, and 2852 cm⁻¹. The sp² carbon (=CH) stretching frequencies were found between 3000 and 3019 cm⁻¹. The vibrations corresponding to B–F and –C=N of the BODIPY core were observed in the vicinity of 1537 and 1735 cm⁻¹, respectively.¹³ The β-H of the pyrrole ring¹³ was observed near 775 cm⁻¹ in unbrominated BODIPYs (1–5), but this peak was absent in the corresponding brominated products, 1b–4b (Fig. S27†). The B–N stretching frequency was observed in the proximity of 1411 cm⁻¹. The presence of para-disubstitution is shown by vibrations in the range 800 to 850 cm⁻¹. The absorption in the range 1032–1088 cm⁻¹ indicates the presence of aryl bromides. The presence of an alkyl C–H stretch at 2923 cm⁻¹ in compounds 2, 2a, and 2b indicates the presence of –CH₃. The –CH₃ deformations of the tert-butyl group in 3, 3a, and 3b arise at 1384, 1364, and 1352 cm⁻¹, respectively. The characteristic vibrations at 2852 cm⁻¹ indicate the presence of –N(CH₃)₂ in BODIPYs 4, 4b, and 4c, and C–N stretches are observed around 1260 cm⁻¹. In the case of hydroxyl BODIPYs, 5, 5a, and 5b, an OH stretching frequency of medium intensity around 3400 cm⁻¹ indicates the presence of H-bonding in these molecules.

Photophysical properties

The absorption and emission properties of the BODIPYs in dichloromethane, along with their molar absorption coefficients and quantum yield data are summarized in Table 1. The stock solutions (10⁻⁵ M) were freshly prepared in doubly distilled dichloromethane. The absorption spectra of the BODIPYs show a narrow, intense transition between 490–545 nm (S₀ → S₁), a shoulder at high energy centered around 480 ± 5 nm (0–1 vibrational transition), and a weak broad absorption band around ∼330–390 nm (S₀ → S₂ transition). Their molar absorption coefficient ranges from 38 000 to 46 000 M⁻¹ cm⁻¹ (Fig. 2).

Table 1 Photophysical properties of compounds 1–5

Code	λmax (nm)	log ε	λem (nm)	Φf^b	Max. brightness, Φ × ε/10^3 M^−1 cm^−1	Stokes shift, Δmax (cm^−1)
a Similar data reported for 1, 1a, and 1b in dichloromethane at room temperature by L. Jiao et al. taken from J. Org. Chem., 2011, 76, 9988–9996.b Fluorescence quantum yields (Φf) were calculated using the standard, rhodamine B, in anhydrous ethanol (Φf = 0.49) for 1b and fluorescein in aqueous NaOH (0.1) N (Φf = 0.90) for 1 and 1a.
1^a	500	4.66	527	0.03	1.37	1024
1a^a	516	4.70	547	0.08	4.02	1098
1b^a	538	4.65	569	0.08	3.63	1012
2	499	4.22	514	0.05	0.83	584
2a	515	4.66	538	0.06	2.77	830
2b	535	4.84	558	0.02	1.39	770
3	499	4.75	515	0.04	2.25	623
3a	515	4.57	537	0.05	1.87	796
3b	544	4.59	564	0.04	1.57	652
4	489	3.82	520	0.06	0.40	1219
4a	518	4.66	542	0.00056	0.03	854
4b	535	4.60	548	0.00014	0.006	443
5	497	4.77	512	0.06	3.53	589
5a	514	4.39	541	0.04	0.98	971
5b	510	4.58	529	0.05	1.91	704

---

Fig. 2 Normalized optical and fluorescence spectra of BODIPYs, 2, 2a, and 2b, in CH₂Cl₂.

It has been documented that the introduction of a substituent on a BODIPY affects the spectroscopic characteristics of the dye in particular; with an increasing number of bromine groups at the pyrrole carbons of the BODIPY core, a systematic increase in the magnitude of the bathochromic shift was observed and limited up to four bromine atoms.⁵ It is also reported that, in meso-(p-methoxyphenyl) BODIPY, each bromine substitution at the pyrrole carbon contributes an additional ∼10 nm red shift with respect to the absorption maxima.⁵ Consistent with the reported literature, we also observed a red shift of 10 nm with the installation of each bromine substituent at the pyrrole carbon of PhBODIPYs in toluene.¹⁴ However, in dichloromethane, a red shift of about 16 nm was observed in the absorption with each attached bromine group in compounds 1–3, with respect to their brominated derivatives (Tables 1 and S1†). Interestingly, this trend was not observed in 4 and its brominated derivatives, 4a and 4b, which may be described as an antagonistic inductive effect by bromines present at the pyrrole carbons of the BODIPY core.⁵ On the contrary, a red shift of ∼13 nm was observed for di-brominated product 5b with respect to 5, in which bromination occurs at the phenyl positions rather than at the β-pyrrole positions.

When excited into either the S₁ or S₂ states, a narrow, slightly Stokes-shifted emission band of mirror-image shape with λ_max positioned between 530–560 nm was also observed from the S₁ state. A remarkable red shift was also induced by bromine atoms in the emission spectra, which was observed to be higher in di-brominated than in mono-brominated compounds, which was in turn higher than that of the parent BODIPYs. For the standard compounds, 1, 1a, and 1b, in toluene, the extent of the red-shift in the emission maxima was the same as reported.¹⁴ The low emission yields (<0.1) in brominated derivatives is due to heavy atom quenching by bromine, in which intersystem crossing (S₁ to T₁) becomes prominent by spin orbit coupling and, consequently, these derivatives are likely to be sensitizers for PDT. Thus, the presence of bromine intensified the spin-forbidden process, either internal or external to the excited molecule.¹⁴ Yet another notable photophysical property of a fluorescent dye is its brightness; the products of the fluorescence quantum yields (Φ_f) and the molar extinction coefficients (ε) at given wavelengths, λ, are also compared in Table 1. The mono-brominated derivatives show higher quantum yields than their corresponding di-brominated analogues, and the brightness follows a similar trend.¹⁴

More often than not, photophysical processes in BODIPYs are closely associated with intermolecular interactions between the dye and its immediate environment, and this could be easily interpreted by solvatochromic studies, which in turn are associated with both the solvent polarity and viscosity. Both the absorption and emission spectra of all the compounds were acquired in five different solvents with varying polarity/refractive indices (dichloromethane, toluene, THF, acetonitrile, and methanol), and are summarized in Table S1.† Little change in the absorption or emission was observed as a result of changing the polarity of the solvent. For parent/unbrominated BODIPYs, the highest and lowest absorption and emission maxima were observed with toluene and methanol, respectively, and hence a Stokes shift was observed. The maximum red shifts were observed for compound 4 in toluene. Both the absorption and emission maxima show a bathochromic shift with decreasing solvent polarity, indicating the more polar character of the ground state.¹⁴ In general, each bromine substitution at the pyrrole carbon contributes an average bathochromic shift of 18 nm with respect to the parent/unbrominated BODIPY. The highest red shift was observed in toluene and lowest in methanol. Furthermore, the red shift could be immediately noticed and identified just after the purification of the products, as di-brominated derivatives have a pinkish appearance that is very different from the monobromo or parent derivatives, as shown in the photo-images of the compounds in visible and UV light (Fig. S29†).

Single crystal X-ray diffraction studies

The molecular structures of compounds 1, 2, 2a, 3, 5, and 5b were explored with the help of X-ray crystallography. Single crystals of compounds of a suitable size were obtained by evaporating a mixture of chloroform/hexane at ambient temperature over a period of one week. Compound 1 crystallized with three rotamers in the asymmetric unit, whereas 2, 2a, 3, 5, and 5b crystallized with monomers. The crystal structures of the compounds are presented in Fig. 3; the crystallographic data and selected geometrical parameters are included in Tables 2 and 3, respectively. In all the compounds, the BODIPY core displays characteristic bond lengths and angles (Table 3), with two pyrrole rings and a boron atom in one plane, and a central six-membered ring and both fluorine atoms in another plane.

Fig. 3 ORTEP representation of molecular structures of 1, 2, 2a, 3, 5, and 5b, shown with 40% probability ellipsoids (solvent molecules are omitted for clarity).

Table 2 Crystal data and structure refinement parameters for compounds 1, 2, 2a, 3, 5, and 5b

Parameters	1	2	2a	3	5	5b
a R1 = ∑||Fo| − |Fc||/∑|Fo|; Io > 2σ(Io).b wR2 = [∑w(Fo^2 − Fc^2)^2/∑w(Fo^2)^2]^1/2.
Molecular formula	C15H11BF2N2	C16H13BF2N2	C16H12BBrF2N2	C19H19BF2N2	C15H11BF2N2O·H2O	C15H9BBr2F2N2O
Formula weight	307.86	282.09	361.00	324.17	302.08	441.87
CCDC number	1434321	1434320	1434318	1434319	1041760	1434317
Temperature/K	293(2)	293(2)	293(2)	293(2)	293(2)	293(2)
Crystal system	Triclinic	Triclinic	Monoclinic	Orthorhombic	Orthorhombic	Monoclinic
Space group	P1̄	P1̄	C2/c	P212121	Pbca	P21/c
λ/Å	0.71073	0.71073	0.71073	0.71073	0.71073	0.71073
a/Å	8.1738(5)	8.0364(3)	20.8544(14)	9.1279(6)	18.456(2)	11.5754(8)
b/Å	10.1538(6)	8.0696(4)	7.3456(5)	11.6936(9)	7.9351(7)	15.3509(11)
c/Å	27.9370(17)	11.0822(5)	22.1366(17)	15.8335(12)	19.4327(19)	9.3750(5)
α (°)	80.058(3)	88.549(2)	90	90	90	90
β (°)	89.870(3)	84.664(2)	117.420(2)	90	90	112.989(2)
γ (°)	72.418(3)	71.997(2)	90	90	90	90
Volume/Å^3	2174.1(2)	680.53(5)	3010.1(4)	1690.0(2)	2846.0(5)	1533.57(17)
Z	6	2	8	4	8	4
μ/mm^−1	0.279	0.100	2.748	0.090	0.111	5.310
Densitycalcd/mg m^−3	1.411	1.377	1.593	1.274	1.410	1.914
F(000)	944	292	1440	680	1248	856
θ range (°)	2.373–24.997	2.654–25.997	2.200–25.994	2.165–25.996	2.207–24.369	2.327–25.000
e data/unique	57 302/7632	18 363/2671	24 867/2950	26 059/3333	34 754/2330	30 194/2693
Rint	0.0392	0.0227	0.0390	0.0393	0.0654	0.0459
Data/restraints/parameters	7632/183/615	2671/0/192	2950/0/200	3333/0/221	2330/3/208	2693/0/208
GOF on F^2	1.082	1.064	1.051	1.051	1.073	1.102
R1^a, wR2^b [I > 2σ(I)]	0.0548, 0.1220	0.0392, 0.0851	0.0444, 0.1117	0.0480, 0.1172	0.0390, 0.0907	0.0439, 0.1011
R1, wR2 (all data)	0.0901, 0.1462	0.0569, 0.1022	0.0712, 0.1273	0.0797, 0.1413	0.0831, 0.1176	0.0565, 0.1083

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Table 3 Selected bond lengths and angles for compounds 1, 2, 2a, 3, 5, and 5b

Parameter	PhBODIPY^a	1^b	2	2a	4-BPhBODIPY^a	3	5	5b
a Taken from J. Phys. Chem. B, 2005, 109, 20433–20443.b Average value obtained from three molecules in the asymmetric unit of 1.
CBod–CAryl	1.481 (11)	1.476 (4)	1.473 (2)	1.482 (5)	1.474 (3)	1.466 (5)	1.459 (3)	1.486 (6)
B–N1	1.547 (9)	1.529 (4)	1.534 (3)	1.536 (6)	1.546 (3)	1.530 (6)	1.517 (4)	1.529 (6)
B–N2	1.547 (9)	1.535 (4)	1.541 (2)	1.541 (6)	1.546 (3)	1.539 (6)	1.523 (4)	1.537 (7)
N–B–N	106.4 (8)	106.4 (3)	106.2 (14)	106.0 (3)	105.9 (15)	106.2 (3)	106.9 (2)	106.9 (4)
F–B–F	110.3 (12)	109.4 (3)	108.5 (15)	109.9 (3)	109.9 (16)	109.4 (4)	107.7 (2)	108.2 (4)
F1–B–C5	124.9	123.2	119.5	117.9	115.8	116.3	120.8	120.9
F2–B–C5	124.9	127.4	132.1	132.2	134.3	134.3	131.5	131.0
ΦAryl	—	51.4	54.6	53.6	—	52.9	47.7	50.2
ΦPyrrole	—	175.7	174.6	174.1	—	173.6	174.6	175.5

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The B–F distances are non-equivalent in all BODIPYs and present above and below the plane of the pyrrole moieties, and the F–B–F plane is almost perpendicular (86.7–88.5°) to the plane of the ring system. The boron atom in all the BODIPYs exhibits a distorted tetrahedral geometry with average angles of N–B–N, 106.4° and F–B–F, 108.9°. The average B–N distance present in all the BODIPYs is 1.53(5) Å, which is in-between a single bond (1.420 Å) and a coordination bond (1.630 Å) distance, indicating that the electron density on the N atom is delocalized through conjugation within the system.¹⁵

In all compounds, the two pyrrole units, along with a boron atom, form a rigid six-membered plane, and the mean plane deviations of the BODIPY core range from ±0.0405 to ±0.0723 Å. The BODIPY core is nearly planar in all compounds, because the dihedral angles between the two pyrrole rings are nearly 180° (Φ_Pyrrole values varying from 174.1–175.7°, Table 3).

The dihedral angles between the meso-aryl group and BODIPY core (Φ_Aryl values) are 51.4°, 54.6°, 53.6°, 52.9°, 47.7°, and 50.2° for 1, 2, 2a, 3, 5, and 5b, respectively, indicating that they are midway to orthogonal, and have parallel arrangements comparable to the reported BODIPYs.¹⁵ Such an arrangement of these two moieties restricts the possible resonance between the BODIPY core and the aryl substituent, and is confirmed by the presence of single-bond distances, C_Bod–C_Aryl, 1.476, 1.473, 1.482, 1.466, 1.459, and 1.486 Å in 1, 2, 2a, 3, 5, and 5b, respectively.

The molecular crystal packing of 1 is shown in Fig. 4a, and exhibits an anti-parallel 3D arrangement of molecules, with the solvent molecule (CHCl₃) acting as a bridging unit between the molecules through various intermolecular interactions, as listed in Table S2.† The crystal packing of 2 forms a herringbone-like structure (Fig. 4b), in which the BODIPY core is connected through B–F⋯H_(Ph), _(Pyr)H⋯C_(Ph), and _(Pyr)H⋯H_(Pyr) interactions. Compound 2a forms zig-zag 3D structural arrangements (Fig. 4c) that are connected through interactions such as C_(Pyr)⋯C_(Pyr), N–BH_(Pyr), B–F⋯H_(Ph), B–F⋯H_(Pyr), and _(Pyr)Br⋯C_(Ph). Interestingly, the packing of 3 forms a one-dimensional array (Fig. 4d) of BODIPY molecules connected exclusively via B–F⋯H_(Ph) interactions. The packing diagram of 5 is through the solvent water molecules that connect the BODIPY molecules, forming a trimer-like structure through various non-covalent interactions (Table S2†), which are further extended in all three directions to form an intricate 3D structural motif. In the packing of 5b, a dimer-like structure is formed through various interactions, resulting in a complex 3D structural arrangement.

Fig. 4 Molecular crystal packing of (a) 1, viewed down ‘a’ axis; (b) 2, viewed along ‘bc’ plane; (c) 2a, viewed along ‘bc’ plane; (d) 3, viewed along ‘ac’ plane; (e) 5, viewed down ac plane and (f) 5b, viewed along ‘bc’ plane.

We have quantitatively analyzed all the crystal structures with the help of Crystal Explorer 3.1 to determine the presence and importance of various intermolecular interactions; the data were presented in the form of Hirshfeld surfaces (HSs) and 2D fingerprint plots (FPs)¹⁶ (Fig. 5). In most cases, there is a red spot observed for intermolecular interactions involving boron and fluorine atoms (BF₂ moieties) with other atoms, such as carbon, hydrogen and oxygen, of different groups. Interestingly, in 5 and 5b, an intense red spot on the hydroxyl group indicates the presence of stronger H-bonding interactions. The observed FPs for each BODIPY are unique in nature with characteristic spikes of various lengths and thicknesses (Fig. 5b).

Fig. 5 (a) Hirshfeld surfaces with normalized contact distance ranging from −0.08 Å (red) to 1.67 Å (blue) and (b) 2D fingerprint plots of all the intermolecular contacts, with d_i and d_e ranging from 0.8 to 2.8 Å for 1, 2, 2a, 3, 5, and 5b.

Fig. 6 displays the percentage distribution of individual intermolecular interactions based on the results of HS analysis for all BODIPYs. It is observed that the relative contributions of C⋯H, F⋯H, and H⋯H contacts are comparable in all BODIPYs, except for 2a and 5b, where Br⋯H contacts also contribute an important role in the molecular crystal packing.

Fig. 6 Percentage contribution of non-covalent interactions in BODIPYs on the basis of HS analysis.

Electrochemical measurements

The electrochemical redox properties of BODIPYs were probed through cyclic voltammetric and differential pulse voltammetric studies, using tetrabutylammonium hexafluorophosphate (NBu₄PF₆) as supporting electrolyte (0.1 M) in dichloromethane at room temperature, with a scan rate of 50 mV s⁻¹. It has been reported that the BODIPY species usually exhibit one or two reductions, but oxidation is either irreversible or absent.¹⁷ In this study, BODIPY derivatives exhibited a reversible first reduction and a quasi-reversible second reduction in cyclic voltammetric studies; hence, differential pulse voltammetric studies were carried out in order to obtain the second reduction potentials, which are listed in Table 4.

Table 4 Electrochemical redox data of BODIPYs 1–5

Compound	Reduction potential (V)		Compound	Reduction potential (V)
	I	II		I	II
1	−0.82	−1.72	3b	−0.56	−1.46
1a	−0.71	−1.62	4	−0.93	−1.88
1b	−0.58	−1.52	4a	−0.70	−1.57
2	−0.85	−1.78	4b	−0.59	−1.47
2a	−0.73	−1.67	5	−0.88	−1.11
2b	−0.55	−1.50	5a	−0.69	−1.43
3	−0.86	−1.79	5b	−0.73	−1.55
3a	−0.71	−1.65

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Electrochemical redox data of BODIPYs 1–5 are presented in Table 4; overlaid cyclic voltammograms and differential pulse voltammograms of compounds 2, 2a, and 2b are depicted in Fig. 7 and S26.† The first reduction potentials of 2, 2a, and 2b are −0.85, −0.73, and −0.55 V, respectively; the more positive values of 2a and 2b compared to 2 indicate that they are more easily reducible, which also confirms the electron-deficient nature of the compounds. A similar trend is observed in all other cases, except in 5, 5a, and 5b (Table 4). The less positive reduction potential of 5b (−0.73 V) compared to 5 (−0.88 V) suggests that dibromination does not occur on the pyrrolic moiety, which is confirmed and further supported by various spectroscopic data (UV-visible, fluorescence and ¹H NMR), and also by single crystal X-ray crystallographic studies.

Fig. 7 Cyclic voltammograms of 2, 2a, and 2b in CH₂Cl₂ under N₂ at 25 °C.

The reduction potentials of unbrominated BODIPYs follow the trend, –N(Me)₂ > –OH > –t-Bu > –CH₃ > –H; and are found to increase with increasing electron donating nature of the substituent on the meso-aromatic ring, which may be due to the inductive stabilization effect of their anionic form.¹⁷ The presence of alkyl or tert-butyl (2 and 3, respectively) substituents in fact step up the reduction potentials compared to that of compound 1. Moreover, in unbrominated BODIPYs 1–5, the lack of substitutions at positions 2, 3, 5, or 6 tends to cause instability in the radical cation produced during oxidation, and the presence of meso-substitution usually results in less reactive radical formation during reduction.

Brominated BODIPYs 1a–5a and 1b–5b, with their electron-deficient nature, displayed one irreversible oxidation and one reversible reduction peak.¹⁷ Bromination actually lowers the oxidation potential,¹⁴ and this small redox potential value hints at the fact that these dyes could be easily reduced and oxidized. Their irreversible oxidation curve indicates that the radical cations produced on the anode could be quickly converted to other products.¹⁸ As reported earlier, a successive anodic shift in the reduction potential was observed with the introduction of bromine, compared to their respective parent BODIPY dyes, i.e., the ease of reduction follows the order: 1b > 1a > 1, 2b > 2a > 2, 3b > 3a > 3, 4b > 4a > 4, 5a > 5b > 5.⁵ The absence of any oxidation peaks between 0–2 V also throws light on the fact that the dyes are not electron-rich.

Antimicrobial activities

Agar well diffusion assay for determining the antimicrobial effect of BODIPYs on Gram-positive and Gram-negative bacteria. The diffusion method is a qualitative and quantitative screening technique to determine the antimicrobial activity of the molecules under study. The antimicrobial effect of BODIPYs was assessed against EC and BS cultures by zone-of-inhibition assay, using 25 μg of the compound per well. It was found that the BODIPYs showed more potency towards BS (Gram-positive bacteria) as compared to EC (Gram-negative bacteria). The experiment was divided into two sets, one set being illuminated and the other being left unilluminated. It was observed that the difference in the growth inhibitory effect of the compounds in the presence and absence of light was very minimal in the case of BS. For example, compound 5b produced 30% and 32.22% growth inhibition under unilluminated and illuminated conditions, respectively, with BS. Similarly, compound 1a showed 18.88% growth inhibition in the dark and 18.33% in the light, whereas compound 1b showed 15% growth inhibition in the dark and 15.55% in the light (Fig. 8). A few compounds, such as 4b, 5, and 2, exhibited a moderate increase in the growth inhibitory effect under illuminated conditions. For example, the inhibitory effect of compounds 4b, 5, and 2 under unilluminated conditions was found to be 15.55, 5.55, and 0%, respectively, while their activities increased to 21.11, 8.88, and 10%, respectively, upon illumination. The antibacterial activity of 5b, under illuminated (32.22%) and unilluminated conditions (30%) is comparable to that of the standard broad-spectrum anti-bacterial drug, tetracycline (10 μg per well), which exhibited a zone of inhibition of 22.22%. Compound 3 showed no activity against BS under both illuminated and unilluminated conditions.

Fig. 8 Comparison of the percentage of inhibition by BODIPYs, (a) under dark and (b) light conditions. All the BODIPYs were added in equal volume and equal concentrations (25 μg per well) into the wells. R refers to the standard anti-bacterial drug, tetracycline. Error bar represents standard deviation.

In the case of the Gram-negative bacteria, EC, 3, 3a, and 3b showed no activity, both in illuminated and unilluminated conditions. The maximum growth inhibition activity was exhibited by compound 4a, i.e., 25.55% under unilluminated conditions, which was slightly higher than the growth inhibitory activity of tetracycline. Compounds 5, 5a, and 2a showed higher growth inhibition activities of 16.66%, 14.44%, and 15%, respectively, under illuminated conditions, while in unilluminated conditions their activities were found to be 13.33%, 0%, and 11%, respectively.

The experiments were performed under dark conditions, assuming little or no activity as there will not be any reactive singlet oxygen production with BODIPYs in the absence of light. However, it was observed that most of the compounds had a significant activity towards Gram-positive/negative bacteria in the dark, which indicates that the singlet oxygen production is not the sole mechanistic pathway governing the antibacterial properties.⁹ Hence, we advanced the experiments under ambient light.

Determination of IC₅₀ and MIC values for the BODIPYs using LB broth assay. Inhibition of the growth of BS and EC cells in LB broth assay was studied only under ambient light conditions, as we could not see any significant difference in the anti-bacterial activity of the compound under illuminated or unilluminated conditions. In the case of BS, all the compounds, except compound 3, showed good antibacterial activity in a time and concentration-dependent manner (Fig. 9). Compound 5b showed the best anti-bacterial activity, with IC₅₀ values of 5 μM. Compound 2 showed the highest IC₅₀ value of 36 μM, which makes it the least potent compound amongst all the other studied test compounds against BS. In the case of EC, all compounds, except 1, 2, 3, 3a, and 3b, showed good anti-bacterial activity. Compound 5 showed the most potency with the lowest IC₅₀ value of 17 μM. Compound 4a showed the least potency with a high IC₅₀ value of 41 μM. Overall, these results are in compliance with the results obtained from the previous experiment, where all the compounds exhibited more potency against Gram-positive bacteria than against Gram-negative bacteria. The IC₅₀ values of all the BODIPYs are shown in Table S3.† The growth inhibitory effect of the most potent compound, 5b, against BS and EC is shown in Fig. 9.

Fig. 9 Effect of BODIPY 5b at different time points on the growth of (a) BS and (b) EC cultured in LB broth.

MIC values were also calculated for the active compounds against BS and EC. The lowest concentration of the compound at which no visible growth observed was considered as the MIC. In the case of Gram-positive bacteria, BS, compounds 4b and 5b showed the lowest MIC value of 60 μM, whereas the other compounds showed higher MIC values. A comparison of the MIC values of 5b against BS and EC is shown in Fig. 10. The dilution methods are considered as quantitative assays, as they determine the MIC values; the observed discrepancy between MIC and zone of inhibition values may be attributed to the extent of diffusion of molecules within solid and liquid media, which in turn is dependent on the structure and size of the molecule.

Fig. 10 Effect of different concentrations of BODIPYs 5, 5a, and 5b on the % growth inhibition of (a) BS and (b) EC cultured in LB broth.

The experiments with DPBF were done with the most bioactive compounds from each series. The results proved that the generation of singlet oxygen could be one of the possible mechanisms for the antibacterial properties of the dyes in the presence of light, and this was easily visualized in the presence of DPBF. It was observed that the gradual decolourisation of DPBF at 410 nm with increasing irradiation time was much quicker with 2 and 2,6-mono/dibromo derivatives than that with bromine substituted on the phenyl group (5b). The generation of singlet oxygen in 2- and 2,6-brominated derivatives has been well reported by Elezcano et al.⁶ A plot of absorbance vs. irradiation time reflects the singlet oxygen yield of the compounds, and the slope of the line with the relative rate of ¹O₂ generation was compared (Fig. 11). The higher the slope, the higher the ¹O₂ yield. From the plot, the order of the singlet oxygen generation rate was found to be 3a > 2a > 1a > 4b > 5b.

Fig. 11 Plot of change in absorbance of DPBF vs. irradiation time in the presence of selected BODIPYs.

The ¹O₂ production for series 1 was also performed and compared (Fig. S28†). The gradual decolourisation of DPBF occurs due to the quenching of singlet oxygen generated by the brominated derivatives. Fig. S28† is a representative to show that the dibromo-derivative shows quicker decolourisation of DPBF than that of mono-bromo, as well as the unbrominated derivative, which in turn reflects the fact that the singlet oxygen generation follows the order 1b > 1a > 1 if bromination occurred at the 2-/2-,6-positions of the pyrrole moiety. These results are in agreement with the antibacterial assays, except for 5b, where bromination happened at the meso-(3,5-phenyl) group of the BODIPY. Therefore, we strongly suggest that the formation of singlet oxygen is probably not the sole antibacterial mechanism associated with BODIPYs. Still, there are several other factors that contribute towards cell disruption.

Intracellular localization by fluorescent microscopic analysis. The amazing fluorescent properties of BODIPYs make them ideal fluorescent imaging agents. The comparison of images under control agents and BODIPY indicates effective internalization into cells. This permits specific evaluation of their cellular uptake. As expected, the untreated cells were non-fluorescent. The fluorescence of all the compounds was found to be uniformly distributed throughout the cells, indicating their cytoplasmic localization (Fig. 12). The fluorescent microscopic images confirm that the anti-microbial activity of BODIPYs in BS is due to their efficient uptake within the bacterial cells.

Fig. 12 Uptake of compounds 5, 5a, and 5b by BS bacterial cells. Cells were incubated with the compounds for 4 hours and processed for microscopic analysis. Fluorescence microscopic images of BS cells treated with solvent control (DMSO) and different BODIPYs (5, 5a, and 5b). (a) Untreated cells (b) bright field image, (c) Hoechst staining for control and BODIPYs: 5b, 5a, and 5 with respective Hoechst staining.

As a whole, it cannot be strongly proposed that the anti-bacterial activity of the tested BODIPYs is dependent on their photosensitization environment. The cytoplasmic localization of these compounds indicates that their possible mechanism of action may be through the generation of singlet oxygen within the bacterial cells or by targeting key cellular enzymes involved in cell-wall synthesis.^11,18 Compounds such as 4b and 5b showed an anti-bacterial activity comparable to that of tetracycline, which implicates the possible use of these compounds against multidrug-resistant strains of BS.¹⁸ Also, compound 5, which showed an anti-bacterial activity similar to that of tetracycline in EC, holds potential against EC strains exhibiting multi-drug resistant (MDR) phenotypes.¹⁸

Conclusions

We report here a series of brominated BODIPYs (mono- and di-) bearing electron-donating substituents (1–5), synthesized by the treatment of meso-aryl boron dipyrrin (BODIPY) with three equivalents of N-bromosuccinimide (NBS) in CH₂Cl₂ at room temperature. The 2- and 2,6-positions of the BODIPYs were regioselectively brominated with quantitative yield for all cases, except for meso-(4-hydroxyphenyl) BODIPY, 5. The unusual product obtained in the bromination of 5 yielded a dibromo derivative (5b); bromination occurred only at the 3,5-positions of the phenyl ring, which may possibly be ascribed to the more electrophilic nature of phenyl carbons. UV-visible absorption studies reveal a red shift of ∼16 nm in CH₂Cl₂ for the installation of each bromine group at the BODIPY core, which is consistent with the reported literature. Moreover, an unexpected red shift was observed for 5b compared to its parent compound 5, indicating the formation of an unusual di-brominated product. A similar trend was noticed in the emission spectra of 5 and 5b. Moreover, the difference in the first reduction potentials of parent to monobromo derivatives and parent to dibromo derivatives is ∼0.10 V and ∼0.26 V, respectively, for 1–3. However, in the case of 4 and 5, the values are 0.22 V and 0.34; 0.19 V and 0.15 V, respectively; the least difference in the reduction potentials of 5 compared to its dibromo derivative indicates that dibromination does not occur on the pyrrolic moiety, which is further confirmed and supported by conventional spectroscopic methods as well as by single crystal X-ray crystallographic studies. It is observed that the intermolecular interactions involving C⋯H, F⋯H, H⋯H, and Br⋯H play a key role in the molecular crystal packing. The antibacterial properties of these brominated dyes showed enhanced activity compared to the corresponding unbrominated BODIPYs. Among all, the unexpected product, 5b, showed the most potency with the lowest IC₅₀ value of 5 μM and an MIC value of 60 μM, with excellent activity, comparable to the standard antibacterial drug, tetracycline.

Acknowledgements

CA (SB/EMEQ-016/2013) thanks DST, New Delhi for the financial support. We are grateful to Dr Babu Varghese, SAIF, IIT Madras, Chennai, Tamilnadu for the single crystal data collection, structure solution, and refinement. We would also like to thank STIC, CUSAT, Kochi, Kerala for the NMR measurements.

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Footnote

† Electronic supplementary information (ESI) available: Crystallographic data for the porphyrins, 1, 2, 2a, 3, 5, and 5b. CCDC 1434321, 1434320, 1434318, 1434319, 1041760 and 1434317. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra12258b

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