Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2016 Supplementary Information Novel fluorescent lapachone-based BODIPY: Synthesis, computational and electrochemical aspects, and subcellular localisation of a potent antitumour hybrid quinone Talita B. Gontijo,a Rossimiriam P. de Freitas,a Guilherme F. de Lima,a Lucas C. D. de Rezende,b Leandro F. Pedrosa,c Thaissa L. Silva,d Marilia O. F. Goulart,d Bruno C. Cavalcanti,e Claudia Pessoa,e,f Marina P. Bruno,g José R. Correia,g Flavio S. Emeryb* and Eufrânio N. da Silva Júniora* aInstitute of Exact Sciences, Department of Chemistry, Federal University of Minas Gerais, CEP 31270-901, Belo Horizonte, MG, Brazil; bFaculty of Pharmaceutical Sciences, University of São Paulo, CEP 14040-903, Ribeirão Preto, SP, Brazil; cInstitute of Exact Sciences, Department of Chemistry, Fluminense Federal University, CEP 27213-145, Volta Redonda, RJ, Brazil; dInstitute of Chemistry and Biotechnology, Federal University of Alagoas, CEP 57072-970, Maceió, AL, Brazil; eDepartment of Physiology and Pharmacology, Federal University of Ceará, CEP 60180-900, Fortaleza, CE, Brazil; fFiocruz-Ceará, CEP 60180-900, Fortaleza, CE, Brazil; gInstitute of Chemistry, University of Brasília, CEP 70904970, Brasília, DF, Brazil. E-mail: [email protected], [email protected] Contents Chemistry S2 NMR spectra of compounds S6 HRMS spectra S14 Photophysical Parameters S15 Cell Staining Procedure S18 Cytotoxicity against cancer cell lines – MTT assay S27 Analysis of reduced glutathione content and TBARS assay S29 Electrochemical studies S31 Computational details S35 S1 Chemistry Materials and methods Melting points were obtained on Thomas Hoover and are uncorrected. Analytical grade solvents were used. Column chromatography was performed on silica gel (SilicaFlash G60 UltraPure 60-200 µm, 60 Å). 1H and 13C NMR spectra were recorded at 300 K using a Bruker AVANCE DRX400 spectrometer. All samples for NMR were prepared in CDCl3 containing TMS as internal reference. Chemical shifts (δ) are given in ppm and coupling constants (J) in Hertz. High resolution mass spectra (electrospray ionization) were obtained using a MicroTOF Ic – Bruker Daltonics instrument. Synthesis of the Quinone and BODIPY derivatives 3-Azido-nor-- Lapachol Na CO Nor-lapachol lapachone 2 3 Br O O 2 O 1. H2O2 1. CH Cl OH OH 2 2 O THF, 60 ºC 0 ºC 2. THF N3 NaN3 O NaOH/Na2CO3 O 2. O CH2Cl2 CuSO4 (70%) (100%) Scheme S1. Synthesis of the 3-azido-nor-β-lapachone (4). Lapachol was initially extracted from the heartwood of Tabebuia sp. (Tecoma) and purified by a series of recrystallizations. Initially, nor-lapachol was synthesized by Hooker oxidation methodology1 and data are consistent with those reported in the literature.2 Nor-lapachol was obtained as an orange solid (160 mg, 0.7 mmol, 70% yield); 33 1 m.p. 121-122 °C. H NMR (400 MHz, CDCl3, 303 K) δ: 8.13 (ddd, J = 7.5, 1.5 and 0.5 Hz, 1H), 8.10 (ddd, J = 7.5, 1.5 and 0.5 Hz, 1H), 7.76 (td, J = 7.5, 7.5 and 1.5 Hz, 1H), 7.69 (td, J = 7.5, 7.5 and 1.5 Hz, 1H), 6.03-5.99 (m, 1H), 2.0 (d, J = 1.5 Hz, 3H), 1.68 (d, 13 J = 1.2 Hz, 3H). C NMR (100 MHz, CDCl3, 303 K) δ: 184.7, 181.5, 151.1, 143.6, 134.9, 133.0, 132.9, 129.5, 126.9, 126.0, 120.9, 113.6, 26.5, 21.7. Synthesis of 3-azido-2,2-dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5- dione, 3-azido-nor-β-lapachone (4): To a solution of nor-lapachol (228 mg, 1.0 mmol) in 25 mL of chloroform, 2 mL of bromine was added. The bromo intermediate precipitated immediately as an orange solid. After removal of bromine, by adding S2 dichloromethane and then removing the organic solvent with dissolved bromine by rotary evaporator, an excess of sodium azide (2 mmol) was added in CH2Cl2 and the mixture was stirred overnight. The crude reaction mixture was poured into 50 mL of water. The organic phase was extracted with organic solvent, dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The azido product 4 was obtained after recrystallization as an orange solid (263 mg, 0.98 mmol, 98% yield); m.p. 200-202 °C. 1 H NMR (400 MHz, CDCl3, 303 K) δ: 8.14 (ddd, J = 6.9, 2.1 and 0.9 Hz, 1H), 7.72-7.65 13 (3H, m), 4.77 (1H, s), 1.67 (3H, s), 1.55 (3H, s). C NMR (100 MHz, CDCl3, 303 K) δ: 180.3, 175.2, 170.2, 134.5, 132.7, 131.1, 113.5, 129.5, 125.1, 126.7, 95.5, 67.3, 27.1, 21.9. Data are consistent with those reported in the literature.3 BODIPYs 1, 2 and 3 were synthesized according to the literature.4,5,6 For the synthesis of BODIPY 2, we have followed the procedure previously described by Jiao 5 . and coworkers. A mixture of BODIPY 1 (230 mg, 0.684 mmol) and CuCl2 2H2O (176 mg, 1.03 mmol) in CH3CN (15 mL) was stirred at reflux for 2 h. After cooling down to room temperature, the reaction mixture was poured in diethyl ether (50 mL), washed with water (3 × 50 mL), dried over anhydrous Na2SO4, filtered and evaporated under vacuum. The residue was purified through column chromatography on silica, using C6H14/DCM (4:1 – 1:1) as eluent, from which the desired product 2 was obtained in 56% yield (142 5 1 mg). Data are consistent with those reported in the literature. H NMR (400 MHz, CDCl3, 303 K) δ: 7.97 (s, 1H), 7.52-7.42 (m, 3H), 6.70 (d, J = 4.3 Hz, 1H), 6.66 (d, J = 4.3 Hz, 13 1H), 6.55 (d, J = 4.4 Hz, 1H), 6.42 (d, J = 4.3 Hz, 1H). C NMR (75 MHz, CDCl3, 303 K) δ: 145.9, 145.7, 135.4, 133.8, 131.7, 131.6, 130.7, 130.3, 130.2, 130.0, 128.4, 119.7, 119.2. For the synthesis of BODIPY 3: To a solution of compound 2 (90 mg, 0.242 mmol) in acetonitrile (10 mL) under stirring at room temperature, propargylamine (39 µL, 33 mg, 0.608 mmol, 2.5 eq) was added. After 1 hour under stirring at room temperature TLC control showed full conversion of the starting material. Solvent was evaporated under reduced pressure and, after purification via silica column chromatography (C6H14/DCM, 3:2 – 1:3), the desired product 3 (61 mg, 0,156 mmol, 65% 1 yield) was obtained. H NMR (400 MHz, CDCl3, 303 K) δ: 7.46-7.42 (m, 3H), 7.38-7.32 (m, 1H), 6.73 (d, J = 4.9 Hz, 1H), 6.55 (br, 1H), 6.31 (dd, J = 3.6, 2.3 Hz, 1H), 6.28 (d, J = 4.9 Hz, 1H), 6.20 (d, J = 3.6 Hz, 1H), 4.22-4.17 (m, 2H), 2.40 (s, 1H). 13C NMR (101 MHz, CDCl3, 303 K) δ: 160.6, 135.0, 133.6, 132.6, 131.5, 131.0, 130.4, 129.5, 127.2, S3 127.1, 118.4, 113.0, 109.8, 76.6, 72.9, 33.0. ESI/HRMS (m/z) [M+Na]+: 412.0310 Cald. + for [C32H23BCl2F2N6O3Na] : 412.0367 Synthesis of the lapachone-based BODIPY 5 In a round bottom flask, 3-azido-nor-β-lapachone (4) (52 mg, 0.19 mmol), alkyne 3 (50 mg, 0.128 mmol), and 10 mL of CH3CN were added. The reaction mixture was stirred until complete solubilization of the reagents after which, CuI (10% per mole) was added. The system was kept under inert atmosphere (Ar) until complete consumption of BODIPY (3) and monitored by TLC until its completion (20 hours). The solvent from the crude was evaporated under reduced pressure and it was purified by column chromatography on silica-gel, using eluents with an increasing polarity gradient mixture of hexane and ethyl acetate. Lapachone-based BODIPY (5) was obtained as a red solid 1 (67 mg, 0.101 mmol, 79% yield); m.p. 179-183 ºC. H NMR (400 MHz, CDCl3, 303 K) δ: 1.15 (s, 3H), 1.75 (s, 3H), 4.68-4.78 (m, 2H), 5.98 (sl, 1H), 6.14 (d, J = 4.0 Hz, 1H), 6.27-6.27 (m, 1H), 6.31 (d, J = 4.0 Hz, 1H), 6.65 (d, J = 4.0 Hz, 1H), 6.93 (sl, 1H), 7.31 (sl, 1H), 7.31-7.33 (m, 1H), 7.41-7.43 (m, 2H), 7.66-7.74 (m, 3H), 7.78-7.80 (m, 1H), 13 8.09 (d, J = 8 Hz, 1H). C NMR (100 MHz, CDCl3, 303 K) δ: 21.1, 27.7, 40.2, 67.2, 95.9, 111.2, 112.0, 113.7, 118.5, 125.7, 126.7, 127.0, 128.1, 129.9, 130.5, 131.3, 131.5, 131.6, 132.0, 133.3, 133.9, 134.7, 134.8, 136.0, 136.1, 162.3, 171.5, 174.8, 180.2. + + EI/HRMS (m/z) [M+Na] : 681.1141 Cald. for [C32H23BCl2F2N6O3Na] : 681.1168. Synthesis of nor-β-lapachone and β-lapachone General procedure for the synthesis of nor-β-lapachone and β-lapachone: Sulfuric acid was slowly added to lapachol (1 mmol, 242 mg) or nor-lapachol (1 mmol, 228 mg) until complete dissolution of the quinone. Then, the solution was poured into ice and the precipitate formed was filtered and washed with water.