Affinity guided isolation, structure elucidation and total synthesis of laetirobin a and its analogue synthesis for therapeutic development 1 4

Appendix 11 Appendix Experimental Section Appendix 174 Experimental Section General Remarks Chemicals and working techniques Unless otherwise stated, all reagents were obtained from Acros, Aldrich, Alfa Aesar, Fluka, Merck or TCI America and used without further purification. Commercial anhydrous solvents were used throughout and transferred under an argon atmosphere. Additionally, DCM was dried by distillation over CaH2, and THF was dried by distillation over sodium benzophenone ketyl. Absolute triethylamine and diisoproylamine were distilled over CaH2 prior to use. All reactions were performed under argon atmosphere and stirred magnetically in oven-dried glassware fitted with rubber septa. Inorganic salts and acids were used in aqueous solution and are reported in % w/v. NMR spectroscopy All spectra were measured on a Bruker Avance ACF 300 or Bruker Avance AMX 500 spectrometer. The Bruker Advance 300 spectrometer operated at 300 MHz for the 1H and 75 MHz for 13 C nuclei, respectively. The Bruker Advance 500 spectrometer operated at 500 MHz for the 1H and 125 MHz for 13C nuclei, respectively. Spectra were recorded at 295 K in CDCl3 unless noted otherwise. Chemical shifts are calibrated to the residual proton and carbon resonances of the solvents: CDCl3 (δH = 7.26 ppm, δC = 77.0 ppm). Data are reported as follow: chemical shift (multiplicity: s = singlet, d = doublet, t = triplet, dd = doublet of doublet, m = multiplet, b = broadened, J = coupling constant (Hz), integration). Experimental Section 175 Mass spectrometry Low resolution mass spectral analyses were recorded on Finnigan LCQ (ESI ionisation source). High resolution mass spectral analyses were recorded on Finnigan MAT95XL. The used mass spectrometric ionisation sources were electron impact (EI) and electrospray ionisation (ESI). High resolution mass (HRMS) analyses were referenced against perfluorokerosene. Some of the mass spectra were measured on a Shimadzu ESI-TOF. Low resolution mass is reported as follow: ionisation sourceionisation mode : found mass (percent of adduct). High resolution mass (HRMS) is reported as follow: (ionisation source) found mass [calcd. [related species] of calculated mass for formula of related species]. Infrared spectroscopy FT-IR spectra were recorded on a Fourier Transform infrared spectrometer model IR Prestige21 (Shimadzu). Solid or crystalline samples were pulverized with potassium bromide (KBr) and percent transmittance (T%) was measured. The percent transmittance (T%) of liquid samples or oils were measured in film between sodium chloride (NaCl) discs. Absorption band frequencies are reported in cm-1. Chromatographic methods Analytical thin layer chromatography (TLC) was performed on pre-coated with silica gel 60 F254 glass plates (Merck). The compounds were visualised by UV254 light. Non-UV active compounds were visualized by staining the developed glass plates with an aqueous solution of molybdophosphorous acid or an aqueous solution of potassium permanganate (heating with a hot gun). Staining solutions were prepared as follow: Ceric ammonium molybdate: 24 g ammonium molybdate [(NH4)6Mo7O24•4H2O] and 0.5 g Ce(NH4)2(NO3)6 were dissolved in 400 mL of aqueous 10% H2SO4. Potassium permanganate: 2.5 g KMnO4 and 12.5 g Na2CO3 in 250 mL H2O. 176 Appendix Flash chromatography and dry column vacuum chromatography (DCVC) were performed using EMD or Silicycle® silica gel 40–63 µm particle size, 40 or 60 Å pore size and 25–40 µm particle size, 60 Å pore size, respectively. Analytical HPLC-MS was performed using Shimadzu Prominence series connected with ESI/TOF; column: XTerraTM MS C18, 2.5µm, 2.1·30mm; gradient: 0-0.5 min, 10% B; 0.5-3.5 min, 100% B; 3.5-9.0 min, 100% B; 9.0-9.5 min, 10% B; 9.5-15.0 min, 10% B; flow: 0.4 mL•min-1. Preparative HPLC was performed using a Gilson machine (liquid handler GX-271, UV detector UV/VIS 151 and pump model 321); column: XTerraTM prep MS C18 OBDTM, 5µm, 19·50mm; gradient: optimized for each sample; flow: 10.0 mL•min-1. X-ray crystallography Crystals were mounted on glass fibres. X-ray data were collected with a Bruker AXS SMART APEX diffractometer, using Mo-Kα radiation at 223K or at 100K, with the SMART suite of programs (SMART, version 5.628, 2001. Bruker AXS Inc., Madison, Wisconsin, USA). Data were processed and corrected for Lorentz and polarization effects with SAINT (SAINT+, version 6.22a, 2001. Bruker AXS Inc., Madison, Wisconsin, USA), and for absorption effect with SADABS (SADABS, version 2.10, 2001. G. W. Sheldrick, University of Goettingen, Germany). Structural solution and refinement were carried out with the SHELXTL, suite of programs (SHELXTL, version 6.14, 2000. Bruker AXS Inc., Madison, Wisconsin, USA). All structures were solved by direct methods to locate the heavy atoms, followed by difference maps for the light, non-hydrogen atoms. All non-hydrogen atoms were generally given anisotropic displacement parameters in the final mode. All H-atoms were put at calculated positions. Experimental Section 177 Note: Copies of the coordinates for selected structures have been uploaded to the Cambridge Crystallographic Data Centre (http://www.ccdc.cam.ac.uk/). Experimental procedures All experimental procedures are arranged in the ascending order of numbers of the compounds. 2,4-dihyroxy-5-iodoacetophenone (3-11)1, 2. Iodine monochloride (10.65 g, O HO 65.6 mmol) in 30 mL of acetic acid was added over 30 to a solution of I 2,4-dihydroxyacetophenone (2-5) (9.5 g, 62.5 mmol) in 77 mL of glacial OH 3-11 acetic acid. After h, the reaction was quenched by addition of a saturated solution of sodium thiosulfate. The aqueous layer was extracted times and the combined organic layers were washed four times with water, dried over Na2SO4 and evaporated under vacuum. The residue was purified by silica gel column chromatography (hexane/EtOAc, 8/2) to afford compound 3-11 (8.688 g, 50%) as a clear oil. IR (KBr): νmax = 3291, 2917, 2735, 1636, 1615, 1270 cm-1; 1H NMR (300 MHz, CDCl3) δ 12.48 (s, H), 8.02 (s, 1H), 6.59 (s, H), 2.56 (s, 3H); 13 C NMR (75 MHz, CDCl3) δ 201.69, 165.21, 160.87, 140.76, 116.82, 103.56, 73.44, 26.25; EIMS+: m/z (%): 278 (80) [M]+, 263 (100), 136 (25), 108 (12), 41 (37); HRMS (EI) m/z: 277.9440 [calcd. [M]+ of 277.9440 for C8H7O3I]. 2,4-bis(tosyloxy)-5-iodoacetophenone (3-2)2. A mixture of 2,4-dihydroxy- O TsO 5-iodoacetophenone (3-11) (2.7 g, 9.71 mmol), p-toluenesulfonyl chloride I OTs 3-2 (5.7 g, 29.90 mmol) and anhydrous K2CO3 (11.5 g, 83.21 mmol) in 150 mL of anhydrous acetone was refluxed for 45 min. The reaction mixture was cooled, filtered through Celite, washed with acetone and the filtrate was concentrated by rotary evaporation. The residue was dissolved in water and extracted three times with 200 mL of DCM. The Appendix 178 combined organic layers were washed with 70 mL of 1M HCl, water, and brine. The organic layer was dried over Na2SO4 and concentrated via rotary evaporation. The resulting crude product was purified by flash column chromatography (5:1 hexane/EtOAc) to afford compound 3-2 in 94% yield (5.3 g, 8.66 mmol) as white crystals. IR (KBr): νmax = 2960, 2927, 1695, 1596, 1585, 1466, 1355 cm-1; 1H-NMR (500 MHz, CDCl3) δ 8.03 (s, 1H), 7.75 (dd, J = 1.4, 6.8 Hz, 2H), 7.71 (dd, J = 1.4, 7.0 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.34 (dd, J = 8.2 Hz, 2H), 7.12 (s, 1H), 2.52 (s, 3H), 2.48 (s, 3H), 2.47 (s, 3H); 13 C-NMR (125 MHz, CDCl3) δ 195.31, 152.38, 147.83, 146.64, 146.40, 140.92, 132.86, 132.17, 131.36, 130.31, 130.05, 128.83, 128.56, 117.58, 88.12, 30.43, 21.80; EIMS+: m/z (%): 586 (10) [M]+, 417 (32), 154 (89), 138 (15), 91 (100); HRMS (EI) m/z: 585.9623 [calcd. [M]+ of 585.9617 for C22H19O7IS2]. ((3-methylpenta-1,4-diyn-3-yloxy)methyl)benzene (3-3). A solution of 4OBn 3-3 trimethylsilyl-3-butynl-2-one (3-1) (1.4 g, 10.0 mmol) in 10 mL of anhydrous THF was added to a 0.5 M solution of ethynylmagnesium bromide in THF (60 mL, 30.0 mmol) at °C. After the addition, the reaction mixture was slowly warmed up to 23 °C and then refluxed for h. After cooling to 23 °C, the resulting brown solution was diluted with 100 mL of Et2O and treated slowly with 30 mL of saturated NH4Cl. The organic layer was collected and the aqueous layer was washed twice with 100 mL of Et2O. The combined organic layers were extracted sequentially with 30 mL of saturated NH4Cl, water and brine, dried over Na2SO4 and the solvent was removed on a rotary evaporator at < 23 °C (note: the product is highly volatile). The resulting brown oil was dissolved in anhydrous DMF. NaH (0.6 g. 15 mmol) was added to this solution in portions. After 15 min, n-Bu4NI (0.369 g, mmol) and benzyl bromide (1.7 g, 10.0 mmol) were added sequentially. After 18 h, the solvent was removed on a rotary evaporator at 40 °C. The crude product was dissolved in water and extracted three times with 70 mL of Et2O. The combined organic layers were Experimental Section 179 washed with 50 mL of 1M HCl, water and brine, dried over Na2SO4 and concentrated via rotary evaporation. The crude product was purified by flash chromatography (98:2 hexane/EtOAc) to afford intermediate 3-3 as a viscous colourless oil, which crystallised after storage for 24 h at -20 °C giving white crystals in 27% yield (0.501 g, 2.72 mmol). IR (KBr): νmax = 3281, 3246, 3007, 2861, 2116, 1378 cm-1; 1H-NMR (500 MHz, CDCl3) δ 7.41-7.28 (m, 5H), 4.78 (s, 2H), 2.60 (s, 2H), 1.85 (s, 3H); 13 C-NMR (125 MHz, CDCl3) δ 137.82, 128.33, 128.09, 127.68, 82.42, 72.56, 68.37, 30.74; EIMS+: m/z (%): 153 (65), 141 (20), 91 (100), 77 (63); HRMS (EI) m/z: 183.0808 [calcd. [M–H]ˉ of 184.0888 for C13H12O]. For synthetic intermediate 3-3, a small colourless block, 0.8 x 0.64 x 0.16 mm, was used for X-ray crystallographic data collection at 223(2) K using Mo (Kα) radiation. 7372 reflections were collected and 2458 were unique (Rint = 0.0271). No symmetry higher than monoclinic was observed and the centrosymmetric alternative, P21/c, was chosen based on the results of refinement. Direct methods were used to solve the structure and all non-hydrogen atoms were refined anisotropically. All H atoms were placed in idealized locations. For C13H12O, monoclinic, P21/c, a = 12.5415(10), b = 6.7365(5), c = 13.0122(11) Å, α = 90, β = 102.770(2), γ = 90 °, V = 1072.15(15) Å3, Z = 4, Dx = 1.141 Mg/m3, R1 = 0.0468, wR2 = 0.1108 based on 2σ(I) data. A copy of the coordinates for structure 3-3 has been uploaded to the Cambridge Crystallographic Data Centre (http://www.ccdc.cam.ac.uk/). 1,1’-(5,5’-(3-(benzyloxy)-3-methylpenta-1,4-diyne-1,5diyl)-bis(2,4-tosyl-5,1-phenylene))-diethanone (3-24). A solution of acetophenone 3-2 (10.98 g, 18.71 mmol), acetylene derivative 3-3 (1.724 g, 9.36 mmol), PPh3 (589 mg, 2.24 mmol), palladium dichloride (199.0 mg, 1.12 mmol), cuprous iodide (213.0 mg, 1.12 mmol) in a mixture of degassed DMF/triethylamine (23 mL/70 mL) was heated to 80˚C for h. After addition of 1M HCl, the mixture is extracted three times with 100 mL EtOAc. Appendix 180 The combined organic layers were washed successively with 70 mL of 1M HCl, 70 mL of water and brine, dried over Na2SO4 and evaporated under vacuum. The residue was purified by silica gel chromatography (hexane/EtOAc, 65/35 to hexane/EtOAc, 50/50) to afford compound 3-24 (8.30 g, 81%) as a yellow wax. IR (NaCl): νmax = 3064, 2925, 1700, 1593, 1480, 1384, cm-1; 1H-NMR (500 MHz, CDCl3) δ 7.77 (s, 2H), 7.71 (d, J = 8.3 Hz, 4H), 7.69 (d, J = 8.4 Hz, 4H), 7.34 (d, J = 8.3 Hz, 4H), 7.23 (d, J = 8.3 Hz, 4H), 7.14 (s, 2H), 4.77 (s, 2H), 2.50 (s, 6H), 2.47 (s, 6H), 2.36 (s, 6H), 1.85 (s, 3H); 13 C-NMR (75 MHz, CDCl3) δ 195.61, 151.85, 147.18, 146.56, 146.15, 137.66, 135.10, 134.64, 134.55, 134.46, 131.81, 131.24, 130.19, 129.97, 129.68, 128.45, 128.5, 128.25, 128.01, 127.62, 117.45, 116.02, 94.01, 77.63, 68.67, 66.55, 30.33, 30.28, 21.70, 21.57; ESIMS+: m/z (%): 1123 (100) [M+Na]+, 861 (17), 707 (31); HRMS (ESI) m/z: 1123.1775 [calcd. [M+Na]+ of 1123.1768 for C57H48O15Na S4]. 1,1’-(5,5’-(3-(benzyloxy)-3-methylpenta-1,4-diyne-1,5-diyl)bis(2,4-hydroxy-5,1-phenylene))diethanone (3-4). For reliability, we activated our magnesium turnings by stirring over 1M HCl for mins, filtering, washing with acetone and Et2O, and drying under high vacuum for hours. A solution of compound 3-24 (8.00 g, 7.26 mmol) and freshly activated magnesium turnings (7.06 g, 290.42 mmol) in 150 mL of MeOH was stirred for 20 h. After addition of 300 mL of 10% citric acid, the mixture was extracted three times with 200 mL of EtOAc. The combined organic layers were washed twice with 200 mL of water and 200 mL of brine, dried over Na2SO4 and evaporated under vacuum. The resulting crude product was purified by flash column chromatography (3:2 hexane/EtOAc) to afford compound 3-4 in 61% yield (2.14 g, 4.41 mmol) as a clear oil. IR (NaCl): νmax = 2922, 1640, 1490, 1411, 1368, 1293 cm-1; 1H-NMR (500 MHz, CDCl3) δ 12.74 (s, OH, 2H), 7.80 (s, 2H), 7.40 (d, J = 7.5 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.30 (d, J = 7.4 Hz, 1H), 6.50 (s, Experimental Section 181 2H), 4.91 (s, 2H), 2.58 (s, 6H), 2.07 (s, 3H); 13 C-NMR (75 MHz, CDCl3) δ 202.6, 166.0, 163.0, 136.0, 128.7, 128.2, 128.0, 127.1, 114.8, 104.1, 103.6, 101.1, 94.1, 78.4, 68.9, 67.3, 31.5, 26.5; ESIMSˉ: m/z (%): 967 (74) [2M–H]ˉ,637 (12), 505 (28), 483 (100) [M–H]ˉ, 375 (14) [M–OBn]ˉ; HRMS (ESI) m/z: 483.1455 [calcd. [M–H]ˉ of 483.1449 for C29H23O7]. 1,1'-(2,2'-(1-(benzyloxy)ethane-1,1-diyl)bis(6-hydroxylbenzofuran-5,2-diyl))diethanone (3-5). A solution of dialkyne 3-4 (2.0 g, 4.13 mmol) , cuprous iodide (200.0 mg, 1.05 mmol) and triethylamine (4.2 g, 41.5 mmol) in 30 mL of DMF was heated at 60˚C for hours. The reaction mixture was poured into 100 mL of 1M HCl and extracted three times with 30 mL of Et2O. The combined organic layers were washed with 50 mL of 1M HCl, saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4 and the solvent was removed on a rotary evaporator. The resulting crude product was purified by flash column chromatography (3:1 hexane/EtOAc) to afford 3-5 in 75% yield (1.51 g, 3.11 mmol) as a light yellow oil. IR (NaCl): νmax = 3435, 2927, 1645, 1448, 1371, cm-1; 1H-NMR (500 MHz, CDCl3) δ 12.45 (s, 2H), 7.97 (s, 2H), 7.34-7.27 (m, 5H), 7.01 (s, 2H), 6.80 (s, 1H), 6.80 (s, 1H), 4.52 (s, 2H), 2.69 (s, 6H), 2.11 (s, 3H); 13 C-NMR (75 MHz, CDCl3) δ 203.96, 161.33, 159.58, 157.88, 128.56, 128.35, 127.62, 127.45, 126.97, 124.02, 120.63, 117.17, 105.18, 99.95, 99.67, 74.88, 66.66, 65.40, 26.83, 23.35; ESIMSˉ: m/z (%): 483 (28) [M–H]ˉ, 375 (100) [M–OBn]ˉ; HRMS (ESI) m/z: 483.1416 [calcd. [M–H]ˉ of 483.1438 for C29H23O7]. Iso-laetirobin A (3-26). A 1.0M solution of BCl3 in hexane (154 µL, 0.154 mmol) was added to 3-5 (50 mg, 0.103 mmol) in mL of anhydrous DCM at -78 °C. The reaction mixture was stirred at this temperature for 15 min. The 182 Appendix reaction was terminated by the addition of mL of water and slowly warmed to 23 °C over 1.5 h. The reaction mixture was extracted three times with 10 mL of DCM. The combined organic layers were washed with 10 mL of 5% NaHCO3, water and brine. The organic layer was dried over Na2SO4 and the solvent was removed on a rotary evaporator. The resulting crude product was purified by preparative HPLC on a Waters SunFire C18 OBD 19x50 mm, µm with a gradient: 10 to 100% CH3CN in 30 to afford iso-laetirobin A (3-26) in 24% yield, as a yellow-brown oil (9.5 mg, 0.013 mmol). IR (NaCl): νmax = 3434, 1638, 1452, 1355, 1241 cm-1; 1H-NMR (500 MHz, CDCl3) δ 12.86 (s, 1H), 12.57 (s, 1H), 12.55 (s, 1H), 12.39 (s, 1H), 7.92 (s, 1H), 7.89 (s, 1H), 7.85 (s, 1H), 7.27 (s, 1H), 7.01 (s, 1H), 6.77 (s, 1H), 6.57 (s, 1H), 6.30 (s, 1H), 6.28 (s, 1H), 6.19 (s, 1H), 4.80 (s, 1H), 4.18 (d, J = 18.0 Hz, 1H), 3.68 (d, J = 18.0 Hz, 1H), 2.73 (s, 3H), 2.67 (s, 3H), 2.63 (s, 3H), 2.01 (d, J = 2.0 Hz, 3H) 1.63 (s, 3H); 13C-NMR (125 MHz, CDCl3) δ 204.19, 204.13, 204.04, 202.66, 165.92, 163.83, 161.45, 158.59, 155.64, 153.47, 150.83, 127.83, 124.30, 123.70, 120.16, 116.86, 115.07, 110.14, 105.28, 105.20, 100.32, 99.97, 99.80, 98.82, 47.53, 27.20, 27.15, 27.05, 25.27, 11.69; ESIMSˉ: m/z (%): 751 (100) [M–H]ˉ; HRMS (EI) m/z: 752.1892 [calcd. [M]+ of 752.1894 for C44H32O12]. Crystals were obtained upon recrystallisation from a dichloromethane/methanol mixture. For synthetic intermediate 3-26, a small block, 0.40 x 0.20 x 0.18 mm, was used for X-ray crystallographic data collection at 223(2) K using Mo (Kα) radiation. 12405 reflections were collected and 8291 were unique (Rint = 0.0276). No symmetry higher than triclinic was observed and the centrosymmetric alternative, Pī, was chosen based on the results of refinement. Direct methods were used to solve the structure and all non-hydrogen atoms were refined anisotropically. All H atoms were placed in idealized locations. For C44H32O12, triclinic, Pī, a = 11.9232(17), b = 12.0133(16), c = 13.613(2) Å, α = 97.200(3), β = 109.150(3), γ = 90.874(3) °, V = 1824.1(4) Å3, Z = 2, Dx = 1.400 Mg/m3, R1 = 0.0759, wR2 = 13 C-NMR Data O 5-39 C12H14O2 OH 299 O 5-41 C12H12O2 O 300 Appendix HO O O 5-28 C14H16O3 13 C-NMR Data 301 HO O 5-33 C14H16O3 OH 302 Appendix AcO O O 5-32 C16H18O4 13 C-NMR Data 303 AcO O OAc 5-35 C18H20O5 304 Appendix AcO O 5-38 C18H20O6 OAc O 13 C-NMR Data 305 HO O O 5-40 C14H16O4 OH 306 Appendix Appendix 2D-NMR Data HO O HO O O O 2-1 C44H32O12 O O HO O O OH 308 Appendix 2D-NMR Data 309 310 Appendix HO O HO O O O 2-1 C44H32O12 O O HO O O OH 2D-NMR Data 311 312 Appendix HO O HO O O O 2-1 C44H32O12 O O HO O O OH 2D-NMR Data 313 314 Appendix [...]... 15 5.32, 15 3 .44 , 15 3 .18 , 15 3 . 14 , 15 3 .11 , 14 4. 91, 13 9 .17 , 13 9 .12 , 13 9 .10 , 13 5. 01, 13 4. 96, 13 4. 92, 13 4. 66, 13 4. 30, 13 9 .12 , 13 3.39, 13 1.98, 13 1.79, 13 1.60, 12 9.93, 12 9. 74, 12 8.27, 12 7.99, 12 7.86, 12 7.83, 12 7.78, 12 4. 31, 11 6. 41 , 26.02, 21. 69; 31P NMR (203 MHz, CDCl3) δ 23.39 Appendix 1 192 Crystals were obtained upon recrystallisation from a hexane/ethylacetate mixture For the side product 5 - 14 , a small colourless... Hz, 1H), 5. 01 (s, 1H), 4. 25 (d, J = 18 Hz, 1H), 3.76 (dd, J = 1. 6 Hz, 18 Hz, 1H), 2.06 (d, J = 2 Hz, 3H) ; 13 C-NMR (12 5 MHz, CDCl3) δ 15 8.97, 15 7.98, 15 5.88, 15 4. 77, 15 4. 56, 15 3.86, 15 3.55, 15 0.66, 13 0.96, 12 8.29, 12 7 .16 , 12 5. 54, 12 2.82, 12 2.68, 12 2.03, 12 1.57, 12 1 .11 , 11 8.32, 11 1.66, 11 1.39, 11 0.90, 11 0 . 14 , 10 9.55, 10 6. 74, 10 4. 95, 10 3.72, 52. 64, 47 .66, 34. 66, 29.93, 11 .77, 11 .76; ESIMS+: Experimental... 12 1.29, 12 0.66, 11 1 .49 , 11 1.23, 11 1. 01, 11 0.55, 10 6 .10 , 10 4. 92, 10 3.99, 99.99, 49 . 34, 45 .07, 33.33, 22. 61; ESIMS+: m/z (%): 559 (15 ) [M+K]+, 543 (60) [M+Na]+, 5 21 (10 0) [M+H]+, 41 9 (11 ), 2 61 (13 ) [M/2+H]+; HRMS (ESI) m/z: 543 .15 69 [calcd [M+Na]+ of 543 .15 67 for C36H24O4Na] Crystals were obtained upon recrystallisation from a dichloromethane/methanol mixture For the [4+ 2]-cycloadduct 5 -4, a small colourless... 2H), 7 .19 (d, J = 1. 0 Hz, 1H), 6.97 (d, J = 1. 0 Hz, 1H), 2.53 (s, 3H), 2 .44 (s, 3H); 13 C-NMR (12 5 MHz, CDCl3) δ 19 7 .40 , 15 8.55, 14 6.08, 14 4. 33, 13 1. 61, 13 0 .11 , 12 9.99, 12 8.52, 12 7.93, 12 1.08, 11 7 .48 , 10 6 . 14 , 98.82, 30 .49 , 21. 71 1 ,1- Bis (4, 4,5,5-tetramethyl -1, 3,2-dioxaborolane-2-yl)ethane (3-38)6, 7 A solution of bis(pinacolato)diboron (2. 54 g, 10 .0 mmol) and vinyl bromide (1. 93 g, 3 .19 M in THF, 18 .0... 11 9.5, 11 7 .4, 11 7.2, 11 7.2, 11 5.9, 10 6.0, 10 5.6, 10 4. 4, 10 0 .4, 10 0 .1, 10 0 .1, 10 0.0, 43 .5, 34. 7, 31. 3, 27 .1, 27 .1, 27 .1, 26.9, 25.5; EIMSˉ: m/z (%): 7 51 (33) [M–H]ˉ; HRMS (EI) m/z: 752 .18 82 [calcd [M]+ of 752 .18 94 for C44H32O12] Crystals were obtained upon recrystallisation from a dichloromethane/methanol mixture For synthetic laetirobin A (2 -1) , a small orange block, 0 .16 x 0 .10 x 0.08 mm, was used for. .. 2855, 17 32, 15 97, 14 62, 12 61, 11 34, 10 34, 10 15, 999, 799, 752 cm -1; 1H NMR (500 MHz, CDCl3) δ 7 .16 (m, 1H), 7 .11 (m, 1H), 6.92 (m, 2H), 4. 93 (d, J = 9 .45 Hz, 1H), 4. 08 (m, 1H), 2.52 (m, 1H), 2 .43 (m, 1H), 2 .16 (m, 1H), 1. 96 (m, 2H), 1. 80 (m, 1H); 13 C NMR (12 5 MHz, CDCl3) δ 208.09, 15 9. 54, 12 8.67, 12 8. 41 , 12 3.29, 12 1 .42 , 11 0.22, 85 .13 , 44 .73, 38.62, 26.53, 21. 24 Crystals were obtained upon storage of. .. 14 5.85, 13 7.97, 13 4. 32, 13 2 . 14 , 13 1.78, 13 0. 04, 12 9.89, 12 8.60, 12 8 .48 , 12 8.32, 12 8 .11 , 12 7.63, 12 0.99, 11 7 .16 , 11 6 .12 , 93.59, 78.28, 68.65, 66. 74, 30.50, 21. 75, 21. 66; ESIMS+: m/z (%): 10 39 (36) [M+Na]+, 10 34 (10 0) [M+NH4]+, 500 (63), 352 (38); HRMS (ESI) m/z 10 34. 20 34 [calcd [M+NH4]+ of 10 34. 2008 for C53H48O13NS4] Bis(6-methoxybenzo[b]furan-2-yl)methanone (3 -40 ) A neat mixture of 2-hydroxy -4- methoxy-benzaldehyde... (m, 1H), 3.29 (m, 1H), 3. 21 (m, 1H), 3 .13 (m, 1H), 2. 34 (s, 3H), 2 .18 (m, 1H), 1. 79 (m, 2H), 1. 44 (m, 2H), 1. 26 (m, 1H); 13 C NMR (12 5 MHz, CDCl3) δ 16 9. 64, 16 9.52, 14 8 .43 , 13 5.75, 12 9 .17 , 12 9.03, 12 7.57, 12 7 .47 , 12 6.55, 12 6.26, 12 2. 84, 12 2.09, 55.90, 55.50, 52.73, 51. 90, 36. 21, 36 .11 , 29.68, 28.03, 25.93, 24. 62, 22.98, 21. 58, 21. 00, 20.96, 17 . 14 ) (Note: inseparable mixture of diastreomers) 1, 2,3 ,4, 4a, 9b-hexahydrodibenzo[b,d]furan -4- ol... (s, 1H), 6.96 (s, 2H), 6.57 (s, 1H), 6 .48 (s, 1H), 6 .47 (s, 1H), 6 .47 (s, 1H), 4. 47 (t, J = 5.5 Hz, 1H), 2.76 (m, 1H), 2.69 (m, 1H), 2.66 (s, 3H), 2.65 (s, 6H), 2.36 (m, 1H), 2.35 (s, 3H), 2.32 (m, 1H); 13 C-NMR (12 5 MHz, CDCl3) δ 2 04. 2, 2 04. 2, 2 04 .1, 2 04. 0, 16 1.5, 16 1.5, 16 1 .4, 16 1.3, 15 9.8, 15 9.7, 15 9.7, 15 9.6, 15 8.7, 15 8.2, 15 7.3, 15 3.7, 14 4. 7, 12 3.9, 12 3.9, 12 3.6, 12 3.3, 12 1.2, 12 0.8, 12 0.7, 11 9.5,... 5 -18 in 89% yield (1. 42 g, 2. 61 mmol) as a white solid IR (NaCl): νmax = 3062, 29 24, 15 97, 14 65, 13 81, 11 88, 11 11, 10 26, 9 64 cm -1; 1H-NMR (500 MHz, CDCl3) δ 7.73 (dd, J = 8.2, 8.2 Hz, 4H), 7.67 (d, J = 8.2 Hz, 1H), 7. 34 (dd, J = 8.2, 8.2 Hz, 4H), 6.97 (d, J = 2.5 Hz, 1H), 6. 74 (dd, J = 2.5, 8.2 Hz, 1H), 2 .46 (s, 6H); 13 CNMR (12 5 MHz, CDCl3) δ 15 0.30, 14 9.98, 14 6.08, 14 6.03, 14 0 .11 , 13 2. 41 , 13 1. 74, 13 0.06, . combined organic layers were washed with 50 mL of 1M HCl, saturated NaHCO 3 , water and brine. The organic layer was dried over Na 2 SO 4 and the solvent was removed on a rotary evaporator. The. of a saturated solution of sodium thiosulfate. The aqueous layer was extracted 4 times and the combined organic layers were washed four times with water, dried over Na 2 SO 4 and evaporated. of 5% NaHCO 3 , water and brine. The organic layer was dried over Na 2 SO 4 and the solvent was removed on a rotary evaporator. After silica gel chromatography 18.0 mg of a yellow powder (32%,