Two biotinylated derivatives of the fungal metabolite galiellalactone (1) were synthesized in order to facilitate the investigation of the molecular mechanism of action of the galiellalactonoids. Galiellalactone is a STAT3-signaling inhibitor that inhibits growth in vitro as well as in vivo of prostate cancer cells expressing activated STAT3. To provide a suitable point of attachment for biotin, the 8-hydroxymethyl derivative (3) and its 7-phenyl analogue 4 were synthesized by a modified tandem Pd-catalysed carbonylation and intramolecular vinyl allene Diels-Alder procedure previously developed. The two primary alcohols obtained, 3 and 4, were coupled to biotin as the 6-aminohexanoic acid amide, activated as the acid chloride, yielding the derivatives 5 and 6.
The protein STAT3 (Signal Transducer and Activator of Transcription 3) is a transcription factor that is involved in different cellular processes. It has been shown to be constitutively activated in malignancies and is involved in the proliferation of several types of cancer cells [
1 contains an α, β-unsaturated lactone moiety, and it has been demonstrated to react with sulfur nucleophiles to produce inactive adducts [
Reagents and solvents were used from commercial sources without purification, except THF and CH2Cl2 that were passed through a MBraun SPS-800 solvent system. All reactions were carried out in standard dry glassware and atmospheric surroundings. Analytical thin layer chromatography (TLC) was performed on Kiselgel 60 F254 plates (Merck) and visualized by spraying with vanillin/H2SO4 and heating. Silica gel column chromatography was performed on SiO2 (Matrex LC-gel: 60A, 35-70 MY, Grace). 1H and 13C NMR spectra were rec-
orded with a Bruker DRX 400 spectrometer (at 400 MHz for 1H and 100 MHz for 13C) and a Bruker DRX 500 spectrometer (at 500 and 125 MHz, respectively). The spectra were recorded in CDCl3 and CD3OD, and the solvent signals (7.27/77.0 and 3.31/49.0 ppm) were used as reference. The data for the 1H signals are given as chemical shifts in ppm and (number of protons, multiplicity, and coupling constants (J) in Hz), while the 13C data are given as chemical shifts in ppm. HR-ESIMS spectra were recorded with a Waters Q-TOF Micro system.
Hept-6-en-1-yn-3-ol(8a): To a solution of ethynyl magnesium bromide (100 ml, 0.5 M in THF, 50 mmol) was slowly added to a solution of 4-pentenal (7a, 3.70 g, 44.0 mmol) in 54 ml of dried THF at 0˚C. After the addition was complete, the solution was allowed to reach room temperature and stirred overnight. The reaction mixture was quenched by the addition of 50 ml NH4Cl (sat.), the mixture was extracted 3 times with 80 ml EtOAc and the organic phase was dried with MgSO4 and concentrated under reduced pressure. 8a was obtainedas a yellowish oil (4.75 g, quantitative yield), which was used without further purification in the next step. 1H NMR (400 MHz, CDCl3) 5.69 (1 H, ddt, 17.1, 10.2, 6.7), 4.93 (1 H, ddd, 17.1, 3.4, 1.5), 4.85 (1 H, ddd, 10.2, 3.4, 1.5), 4.25 (1 H, td, 6.7, 2.1), 2.37 (1 H, d, 2.1), 2.10 (2 H, m), 1.67 (2 H, m). 13C NMR (100 MHz, CDCl3) 137.5, 115.4, 84.6, 73.2, 61.7, 36.6, 29.2. HRMS calcd for C7H11O [M + H]: 111.0810, found: 111.0834.
4-Phenylhept-6-en-1-yn-3-ol(8b): Phenylacetic acid (30.0 g, 220 mmol) was dissolved in 100 ml MeOH and SOCl2 (28.8 g, 242 mmol) was added dropwise at 0˚C under stirring. The reaction mixture was allowed to reach room temperature overnight and the volatiles were removed under reduced pressure. Phenylacetic acid methyl ester was obtained as a yellowish oil (32.8 g, quantitative yield) and used directly in the next step. 1H NMR (400 MHz, CDCl3) 7.34 (5 H, m), 3.72 (3 H, s), 3.67 (2 H, s). 13C NMR (100 MHz, CDCl3) 171.0, 133.6, 128.6, 127.8, 126.3, 51.0, 40.2. HRMS calcd for C9H10O2Na [M + Na]: 173.0578, found: 173.0558. Phenylacetic acid methyl ester (14.0 g, 93 mmol) was dissolved in 150 ml dry THF and added slowly at −78˚C over 30 min to a freshly prepared LDA solution [
(Z)-11-((Triisopropylsilyl)oxy)undeca-1,8-dien-6-yn-5-ol(10a): TEA (55 ml, 400 mmol) was added to a mixture of PdCl2(PPh3)2 (0.70 g, 0.99 mmol) and CuI (0.38 g, 1.99 mmol) at 0˚C under nitrogen atmosphere and the solution was stirred for 30 minutes. To this solution 9 (7.25 g, 20.5 mmol), prepared according to reference 12 and identical in all aspects with the reported compound, in 40 ml dry THF was added dropwise followed by 8a (2.19 g, 20.0 mmol) in 40 ml dry THF at 0˚C. The reaction mixture was stirred overnight at room temperature, quenched with 50 ml NaHCO3 (sat.), extracted 3 times with 60 ml EtOAc. The combined organic phases were dried with Na2SO4 and concentrated by evaporation. The yield was quantitative and 10a was used directly in the next step without purification. 1H NMR (400 MHz, CDCl3) 6.04 (1 H, dt, 10.8, 7.0), 5.85 (1 H, ddt, 17.0, 10.2, 7.2), 5.56 (1 H, ddd, 10.8, 3.1, 1.4), 5.08 (1 H, ddd, 17.0, 3.2, 1.5), 5.00 (1 H, dd, 10.2, 3.2), 4.54 (1 H, td, 6.5, 3.1), 3.76 (2 H, t, 7.0), 2.56 (2 H, qd, J 7.0, 1.4), 2.26 (2 H, td, J 7.2, 1.5), 1.83 (2 H, m), 1.13 - 1.03 (21 H, m).13C NMR (100 MHz, CDCl3) 141.1, 137.9, 115.5, 110.0, 94.4, 82.1, 62.7, 62.5, 37.1, 34.4, 29.7, 18.2, 12.2. HRMS calcd for C20H37O2Si [M + H]: 337.2563, found: 337.2560.
(Z)-Methyl (11-((triisopropylsilyl)oxy)undeca-1,8-dien-6-yn-5-yl) carbonate(11a): 10a (6.70 g, 20.0 mmol) was dissolved in 130 ml of dry CH2Cl2 at 0˚C. DMAP (4.86 g, 39.8 mmol) was added and the mixture was stirred for 20 min. Methyl chloroformate (5.6 g, 59.7 mmol) was added dropwise, and the reaction was brought to room temperature overnight before 50 ml NaHCO3 (sat.) was added and the mixture was extracted 2 times with 80 ml CH2Cl2. The combined organic phases were dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography with CH2Cl2/heptane 1:1 as eluent, giving 3.59 g (46%) of 11a as a yellowish oil. 1H NMR (400 MHz, CDCl3) 6.09 (1 H, dt, 10.8, 7.3), 5.82 (1 H, ddt, 17.0, 10.2, 6.6), 5.56 (1 H, ddd, 10.8, 3.3, 1.7), 5.40 (1 H, td, 6.6, 1.7), 5.07 (1 H, ddd, 17.0, 3.2, 1.3), 5.02 (1 H, ddd, 10.2, 3.2, 1.3), 3.81 (3 H, s), 3.76 (2 H, t, 6.5), 2.55 (2 H, qd, 6.6, 1.3), 2.25 (2 H, m), 1.95 (2 H, m), 1.09 - 1.03 (21 H, m). 13C NMR (100 MHz, CDCl3) 155.2, 142.3, 137.0, 115.9, 109.6, 89.9, 83.7, 68.5, 62.5, 55.1, 34.4, 34.3, 29.4, 18.2, 12.2. HRMS calcd for C22H39O4NaSi [M + H]: 395.2618, found: 395.2647.
(Z)-4-Phenyl-11-((triisopropylsilyl)oxy)undeca-1,8-dien-6-yn-5-ol(10b):10b was prepared in the same way as 10a, from 8b, and 10b was obtained in a quantitative yield as a 1:0.2 mixture of diastereomers. Diastereomer a) 1H NMR (400 MHz, CDCl3) 7.30 (5 H, m), 6.05 (1 H, dt, 10.8, 7.4), 5.71 (1 H, ddt, 17.1, 10.2, 6.3), 5.55 (1 H, ddd, 10.8, 3.3, 1.5), 5.08 (1 H, ddd, 17.1, 3.2, 1.6), 4.99 (1 H, ddd, 10.2, 3.2, 1.6), 4.74 (1 H, ddd, 7.5, 5.7, 1.5), 3.74 (2 H, t, 6.5), 3.04 (1 H, m), 2.65 (2 H m), 2.51 (2 H, m), 1.77 (1 H, d, 7.5), 1.17 - 1.04 (21 H, m). 13C NMR (100 MHz, CDCl3) 141.2, 139.4, 136.1, 129.2, 128.2, 127.2, 116.7, 109.6, 92.4, 83.8, 66.4, 62.4, 51.4, 35.6, 34.1, 18.0, 12.0. Diastereomer b) 1H NMR (400 MHz, CDCl3) 7.30 (5 H, m), 6.03 (1 H, dt, 10.8, 7.4), 5.71 (1 H, ddt, 17.1, 10.2, 6.3), 5.57 (1 H, ddd, 10.8, 3.3, 1.5), 5.06 (1 H, ddd, 17.1, 3.2, 1.6), 4.99 (1 H, ddd, 10.2, 3.2, 1.6), 4.77 (1 H, ddd, 7.5, 5.7, 1.5), 3.78 (2 H, t, 6.5), 3.01 (1 H, m), 2.70 (2 H m), 2.51 (2 H, m), 1.78 (1 H, d, 7.5), 1.17 - 1.04 (21 H, m). 13C NMR (100 MHz, CDCl3) 141.3, 139.6, 136.2, 128.9, 128.4 127.1, 116.8, 109.7, 92.8, 83.5, 66.5, 62.4, 52.1, 35.6, 34.2, 18.0, 12.0.HRMS calcd for C26H40O2NaSi [M + Na]: 435.2695, found: 435.2663.
(Z)-Methyl (4-phenyl-11-((triisopropylsilyl)oxy)undeca-1,8-dien-6-yn-5-yl) carbonate(11b):11b was prepared in the same way as 11a, from 10b, and 11b was obtained in a 75% yield as a 1:0.2 mixture of diastereomers. Diastereomer a) 1H NMR (400 MHz, CDCl3) 7.28 (5 H, m), 6.04 (1 H, dt, 11.5, 6.9), 5.65 (1 H, ddd, 17.1, 10.9, 6.7), 5.58 (1 H, dd, 4.8, 2.1), 5.48 (1 H, ddd, 11.5, 2.1, 1.6), 5.04 (1 H, ddd, 17.1, 3.2, 1.5), 4.96 (1 H, ddd, 10.9, 3.2, 1.5), 3.78 (3 H, s), 3.68 (2 H, t, 6.4), 3.15 (1 H, m), 2.71 (1 H, m), 2.56 (1 H, m), 2.37 (2 H, dtd, 6.9, 6.4, 1.6), 1.17 - 0.95 (21 H, m). 13C NMR (100 MHz, CDCl3) 154.9, 142.1, 139.2, 135.4, 128.8, 128.2, 127.1, 117.0, 109.2, 88.5, 84.6, 71.7, 62.3, 54.9, 49.4, 35.5, 34.0, 18.0, 11.9. Diastereomer b) 1H NMR (400 MHz, CDCl3) 7.28 (5 H, m), 6.06 (1 H, dt, 11.5, 6.9), 5.66 (1 H, ddd, 17.1, 10.9, 6.7), 5.58 (1 H, dd, 4.8, 2.1), 5.50 (1 H, ddd, 11.5, 2.1, 1.6), 5.05 (1 H, ddd, 17.1, 3.2, 1.5), 4.97 (1 H, ddd, 10.9, 3.2, 1.5), 3.76 (3 H, s), 3.70 (2 H, t, 6.4), 3.15 (1 H, m), 2.73 (1 H, m), 2.57 (1 H, m), 2.50 (2 H, dtd, 6.9, 6.4, 1.6), 1.17 - 0.95 (21 H, m). 13C NMR (100 MHz, CDCl3) 154.8, 142.3, 138.9, 135.2, 128.8, 128.2, 127.1, 116.9, 109.3, 88.46, 84.7, 71.8, 62.3, 54.8, 49.7, 34.9, 34.1, 18.0, 11.9. HRMS calcd for C28H42O4NaSi [M + Na]: 493.2750, found: 493.2773.
rac-(6R,7aR)-Methyl 6-(2-((triisopropylsilyl)oxy)ethyl)-2,6,7,7a-tetrahydro-1H-indene-4-carboxylate(12a): To a dried autoclave flask was added Pd(OAc)2 (0.41 g, 1.8 mmol) and DPPP (0.75 g, 1.8 mmol) under a nitrogen atmosphere, followed by 9 ml dry toluene and 11a (3.59 g, 9.1 mmol) dissolved in 9 ml toluene/MeOH (1:1). CO was bubbled though the solution before the autoclave was pressurized with CO to 5 bar. The reaction mixture was stirred for 48 h at 5 bar at room temperature before being diluted with 15 ml EtOAc and filtered through a celite plug. The filtered solution was concentrated and purified by silica gel chromatography with heptane/Et2O (94:6) to afford 0.55 g (16%) of 12a as a colourless oil. 1H NMR (400 MHz, CDCl3) 6.82 (1 H, d, 2.1), 6.27 (1 H, dd, 6.1, 2.6), 3.82 (2 H, m), 3.78 (3 H, s), 2.73 (1 H, m), 2.72 (1 H, dddd, 16.4, 8.8, 5.2, 2.1), 2.44 (2 H, ddd, 11.5, 4.5, 2.6), 2.14 (1 H, m), 2.12 (1 H, m), 1.77 (1 H, td, 13.5, 6.6), 1.60 (1 H, td, 13.5, 5.2), 1.37 (1 H, ddd, 12.5, 11.5, 3, 6.1), 1.10 (1 H, m), 1.08 - 1.04 (21 H, m). 13C NMR (100 MHz, CDCl3) 167.2, 145.7, 137.8, 127.0, 126.9, 61.2, 51.7, 44.7, 38.6, 36.4, 35.2, 32.4, 31.2, 18.2, 12.2. HRMS calcd for C22H38O3NaSi [M + Na]: 401.2518, found: 401.2488.
Methyl 2-phenyl-6-(2-((triisopropylsilyl)oxy)ethyl)-2,6,7,7a-tetrahydro-1H-indene-4-carboxylate(12b):12b was prepared in the same way as 12a, starting from 11b (6.55 g, 13.9 mmol). 12b was obtained as colourless oil as a 1:0.8 inseparable mixture of two diastereomers (total yield 2.64 g, 42%). Diastereomer a) 1H NMR (400 MHz, CDCl3) 7.24 (5 H, m), 6.94 (1 H, t, 2.5), 6.32 (1 H, m), 4.02 (1 H, m), 3.85 (2 H, m), 3.79 (3 H, s), 2.86 (1 H, m), 2.75 (1 H, m), 2.60 (1 H, dt, 12.3, 7.0), 2.16 (1 H, dd, 10.2, 2.5), 2.00 (1 H, dt, 10.2, 9.2), 1.81 (1 H, tt, 13.2, 6.5), 1.65 (1 H, dt, 13.2, 6.5), 1.37 (1 H, dt, 12.3, 10.2), 1.21 - 0.91 (21 H, m). 13C NMR (100 MHz, CDCl3) 166.9, 146.8, 146.0, 138.3, 130.4, 129.4, 128.4, 127.5, 126.6, 126.1, 60.9, 51.6, 51.3, 50.3, 44.6, 43.2, 42.2, 39.8, 38.4, 36.2, 35.2, 18.0, 12.0. Diastereomer b) 1H NMR (400 MHz, CDCl3) 7.24 (5 H, m), 6.40 (1 H, m), 6.32 (1 H, m), 4.02 (1 H, m), 3.85 (2 H, m), 3.78 (3 H, s), 2.96 (1 H, m), 2.75 (1 H, m), 2.60 (1 H, dt, 12.3, 7.0), 2.16 (1 H, dd, 10.2, 2.5), 2.00 (1 H, dt, 10.2, 9.2), 1.81 (1 H, tt, 13.2, 6.5), 1.61 (1 H, m), 1.37 (1 H, dt, 12.3, 10.2), 1.21 - 0.91 (21 H, m). 13C NMR (100 MHz, CDCl3) 166.9, 146.7, 145.6, 138.4, 130.4, 129.3, 128.4, 127.2, 126.6, 126.0, 60.9, 51.6, 51.3, 50.3, 44.6, 43.2, 42.2, 39.8, 38.3, 36.1, 35.0, 18.0, 12.0. HRMS calcd for C28H43O3Si [M + H]: 455.2981, found: 455.2956.
rac-(1aS,3aR,5R,7aS)-Methyl 5-(2-((triisopropylsilyl)oxy)ethyl)-1a,2,3,3a,4,5-hexahydroindeno [1,7a-b]oxir- ene-7-carboxylate(13a): 12a (0.55 g, 1.4 mmol) was dissolved in 30 ml dry CH2Cl2 under nitrogen atmosphere and m-CPBA (0.32 g, 1.9 mmol) was added at 0˚C. The reaction mixture was stirred at this temperature for 1 h before it was quenched by the addition of 10 ml Na2S2O3 (sat.). The phases were separated and organic phase was washed with NaHCO3 (sat.). The water phases were extracted with 3 times with 20 ml dry CH2Cl2 and the combined organic phases were dried over Na2SO4, filtered and evaporated. Flash chromatography (SiO2, heptane/Et2O 85:15) afforded the required isomer 13a as a colourless oil in 79% (0.45 g) yield. 1H NMR (400 MHz, CDCl3) 7.29 (1 H, d, 2.5), 4.41 (1 H, s), 3.84 (2 H, m), 3.72 (3 H, s), 2.70 (1 H, dddd, 16.5, 8.3, 6.1, 2.5), 2.05 (1 H, dd, 13.9, 7.2), 1.97 (1 dddd, 16.5, 13.2, 6.8, 3.3), 1.89 (1 H, m), 1.83 (1 H, ddd, 13.2, 7.2, 5.3), 1.66 (1 H, m), 1.62 (1 H, s), 1.58 (1 H, m), 1.30 (1 H, td, 13.2, 8.3), 1.12 (1 H, m), 1.08 - 1.04 (21 H, m). 13C NMR (100 MHz, CDCl3) 165.4, 153.9, 126.3, 63.6, 63.3, 60.7, 51.5, 39.7, 38.0, 35.8, 30.1, 27.5, 23.2, 18.0, 11.9. HRMS calcd for C22H38O4NaSi [M + Na]: 417.2437, found: 417.2413.
rac-(1aS,2S,3aS,5R,7aS)-Methyl 2-phenyl-5-(2-((triisopropylsilyl)oxy)ethyl)-1a,2,3,3a,4,5-hexahydroindeno [1,7a-b]oxirene-7-carboxylate(13b): 13b was prepared in the same way as 13a, starting from 12b (2.64 g, 5.8 mmol). The crude product (2.71 g) was purified by flash chromatography (SiO2, heptane/Et2O 85:15). 13b was obtained as a colourless oil (0.43 g, 16%). 1H NMR (400 MHz, CDCl3) 7.42 (2 H, dd, 8.2, 1.3), 7.34 (1 H, d, 0.9), 7.31 (2 H, td, 8.2, 7.3), 7.24 (1 H, td, 7.3, 1.3), 4.57 (1 H, s), 3.85 (2 H, m), 3.72 (3 H, s), 3.16 (1 H, dd, 12.1, 7.2), 2.75 (1 H, m), 2.12 (1 H, m), 2.04 (1 H, m), 1.92 (1 H, dt, 12.1, 7.2), 1.86 (1 H, dt, 15.2, 6.8), 1.67 (1 H, ddd, 15.2, 10.8, 6.8), 1.42 (1 H, m), 1.32 (1 H, dd, 12.1, 11.5), 1.17 - 1.03 (21 H, m). 13C NMR (100 MHz, CDCl3) 165.4, 154.1, 141.6, 128.4, 127.7, 126.6, 126.1, 66.9, 62.3, 60.6, 51.6, 45.9, 40.2, 38.0, 35.6, 32.3, 30.0, 18.0, 12.0. HRMS calcd for C28H42O4SiNa [M + Na]: 493.2750, found: 493.2773.
rac-(2aS,4R,5aR,7aR)-2a-Hydroxy-4-(2-hydroxyethyl)-4,5,5a,6,7,7a-hexahydroindeno [1,7-bc]furan-2(2aH)- one(3): 13a (0.45 g, 1.2 mmol) was dissolved in 5 ml THF and a solution of LiOH·H2O (0.12 g, 2.8 mmol) in 5 ml H2O was added at room temperature. The reaction mixture was stirred for 3 days until a TLC analysis revealed that the starting material was consumed. After the addition of 5 ml THF and 5 ml of H2SO4 (10%), the mixture was stirred at room temperaturefor 3 days. The reaction mixture was quenched by the addition of 3 ml NaHCO3 (sat.) and extracted 3 times with 20 ml EtOAc, the combined organic phases were dried with Na2SO4 and concentrated by evaporation. The crude product was purified by chromatography (SiO2, CHCl3/MeOH 9:1) to afford 0.13 g (50%) of 3 as a colourless oil. 1H NMR (500 MHz, CD3OD) 7.11 (1 H, d, 3.1), 4.69 (1 H, dd, 7.5, 2.7), 3.71 (2 H, t, 6.4), 2.70 (1 H, m), 2.43 (1 H, dtd, 10.5, 7.9, 5.0), 2.27 (1 H, m), 2.10 (1 H, ddt, 14.3, 10.5, 7.5), 1.87 (1 H, m), 1.76 (1 H, td, 13.2, 6.4), 1.69 (1 H, dddd, 14.3, 7.5, 5.0, 2.7), 1.65 (1 H, m), 1.18 (1 H, m), 1.10 (1 H, m). 13C NMR (125 MHz, CD3OD) δ 172.2, 149.7, 132.9, 91.9, 83.0, 60.9, 44.4, 39.1, 32.7, 32.6, 32.5, 32.4. HRMS calcd for C12H16O4Na [M + Na]: 247.0946, found: 247.0932.
rac-(2aS,4R,5aS,7S,7aR)-2a-Hydroxy-4-(2-hydroxyethyl)-7-phenyl-4,5,5a,6,7,7a-hexahydroindeno [1,7-bc] furan- 2(2aH)-one(4): 4 was prepared in the same way as 3, starting from 13b (0.13 g, 0.4 mmol). The crude product was purified by chromatography (SiO2,CHCl3/MeOH 9:1) to afford 3 (60 mg, 73%) as a colourless oil. 1H (500 MHz, CDCl3) 7.24 (2 H, t, 7.4), 7.17 (1 H, td, 7.4, 2.3), 7.12 (1 H, d, 3.4), 7.09 (2 H, dt, 7.4, 2.3), 4.82 (1 H, dd, 6.3, 1.0), 3.72 (2 H, m), 3.47 (1 H, dt, 13.2, 6.3), 2.78 (1 H, m), 2.56 (1 H, dddd, 12.0, 8.1, 6.4, 3.8), 2.32 (1 H, ddd, 13.5, 8.1, 7.8), 2.06 (1 H, dddd, 13.2, 6.4, 6.3, 1.0), 1.72 (2 H, m), 1.36 (1 H, td, 13.2, 12.0), 1.04 (1 H, ddd, 13.5, 12.0, 3.8). 13C NMR (125 MHz, CDCl3) 170.3, 148.4, 136.8, 131.4, 128.3, 128.1, 126.7, 90.9, 81.2, 59.8, 48.2, 42.2, 37.7, 37.4, 31.2, 30.1. HRMS calcd for C18H20O4Na [M + Na]: 323.1259, found: 323.1283.
2-(-2a-Hydroxy-2-oxo-2,2a,4,5,5a,6,7,7a-octahydroindeno [1,7-bc]furan-4-yl)ethyl 6-(5-((3aS,4S,6aR)-2-oxo- hexahydro-1H-thieno [3,4-d]imidazol-4-yl)pentanamido)hexanoate(5): To a dried flask containing the N-(+)- biotinyl-6-aminohexanoic acid (50 mg, 0.13 mmol) was added thionyl chloride (0.50 ml, 6.3 mmol) under a nitrogen atmosphere and the mixture was stirred for 40 min at room temperature. The excess thionyl chloride was subsequently removed under reduced pressure and the crude product was used directly in the next step. 3 (19.2 mg, 0.09 mmol) was dissolved in 1.5 ml of freshly distilled MeCN and the previously prepared acid chloride dissolved in 1.5 ml MeCN was added dropwise under a nitrogen atmosphere. The reaction mixture was stirred for 5 h at room temperature and then concentrated under reduced pressure. The crude product was purified by chromatography (SiO2, CHCl3/MeOH 97:3) to afford 5 (9 mg, 19%). The two diastereomers have identical NMR data. 1H (500 MHz, CD3OD) 7.11 (1 H, d, 3.2), 4.70 (1 H, dd, 7.3, 2.4), 4.52 (1 H, dd, 7.8, 4.5), 4.33 (1 H, dd, 7.8, 4.5), 4.24 (2 H, m), 3.23 (1 H, m), 3.17 (2 H, t, 6.9), 2.94 (1 H, dd, 12.8, 4.5), 2.72 (1 H, d, 12.8), 2.66 (1 H, tdd, 12.2, 7.1, 3.2), 2.44 (1 H, dtd, 10.5, 7.5, 2.4), 2.35 (2 H, t, 7.8), 2.29 (1 H, m), 2.20 (2 H, t, 7.4), 2.11 (1 H, ddt, 14.5, 10.5, 7.3), 1.89 (1 H, td, 14.5, 7.5), 1.81 (1 H, m), 1.73 (1 H, m), 1.77 - 1.55 (7 H, m), 1.52 (2 H, dt, 12.2, 6.9), 1.44 (2 H, m), 1.35 (2 H, m), 1.16 (1 H, m), 1.09 (1 H, m). 13C NMR (125 MHz, CD3OD) δ 176.2, 175.4, 172.0, 166.2, 148.9, 133.4, 91.9, 83.0, 63.7, 63.6, 62.0, 57.1, 44.5, 41.1, 40.4, 36.9, 35.1, 35.0, 34.8, 32.9, 32.9, 32.7, 32.5, 30.2, 29.9, 29.6, 27.6, 27.1. HRMS calcd for C28H42N3O7S [M + H]: 564.2743, found: 564.2744.
2-(-2a-Hydroxy-2-oxo-7-phenyl-2,2a,4,5,5a,6,7,7a-octahydroindeno [1,7-bc]furan-4-yl)ethyl 6-(5-((3aS,4S, 6aR)-2-oxohexahydro-1H-thieno [3,4-d]imidazol-4-yl)pentanamido)hexanoate(6): 6 was prepared and purified in the same way as 5, starting from 4 (10 mg, 0.033 mmol) to afford 6 (9.7 mg, 45%) as a colourless oil. The two diastereomers have identical NMR data. 1H (500 MHz, CD3OD) 7.27 (2 H, m), 7.20 (1 H, m), 7.17 (2 H, dd, 6.1, 2.7), 7.15 (1 H, s), 4.86 (1 H, d, 6.5), 4.47 (1 H, dd, 7.9, 4.8), 4.28 (1 H, m), 4.28 (2 H, dd, 13.1, 5.3), 3.58 (1 H, dt, 13.5, 6.5), 3.21 (1 H, m), 3.16 (2 H, dd, 8.9, 7.4), 2.92 (1 H, dt, 12.8, 4.8), 2.79 (1 H, m), 2.70 (1 H, dd, 12.8, 3.8), 2.62 (1 H, dddd, 10.0, 8.1, 6.5, 3.6), 2.36 (3 H, m), 2.19 (2 H, td, 7.4, 2.1), 2.09 (1 H, ddd, 13.5, 6.5, 5.5), 1.95 (1 H, td, 13.1, 6.2), 1.86 (1 H, m), 1.73 (1 H, m), 1.69 - 1.55 (6 H, m), 1.51 (2 H, m), 1.44 (2 H, m), 1.36 (2 H, m), 1.20 (1 H, ddd, 13.2, 8.1, 3.9). 13C NMR (125 MHz, CD3OD) 176.1, 175.4, 172.0, 166.3, 148.9, 138.9, 133.2, 129.7, 129.3, 127.8, 92.6, 82.7, 63.7, 63.5, 61.7, 57.2, 49.6, 43.6, 41.2, 40.3, 38.7, 37.0, 35.2, 35.0, 33.1, 30.82, 30.2, 29.9, 29.6, 27.6, 27.1, 25.9. HRMS calcd for C34H46N3O7S [M + H]: 640.3056, found: 640.3051.
(S)-N-Methoxy-N-methyl-2-phenylpent-4-enamide(17):To a solution of (S)-2-phenylpent-4-enoic acid (16) (6.8 g, 38 mmol), prepared from phenylacetic acid (14) according to reference 16 and identical in all aspects with the reported compound, in dry CH2Cl2 (4 ml) was added freshly distilled SOCl2 (11.4 g, 96 mmol) at 0˚C. The resulting brown mixture was stirred overnight at room temperature, where after the volatiles were removed under reduced pressure. The acid chloride, obtained as a black solid (7.8 g), was then dissolved in dry CH2Cl2 (40 ml) and cooled to 0˚C before the addition of N-methyl-O-methylhydroxylamine hydrochloride (4.3 g, 44 mmol) under N2. After stirring at 0˚C for 2 h, pyridine (6.7 g, 84 mmol) was added and the mixture was stirred at room temperature overnight. The solvent was removed under vacuum, and the remaining pale yellow solid was dissolved in a 1:1mixture of Et2O/CH2Cl2 (50 mL) and washed with brine (50 ml).The organic layer was dried over MgSO4, and the solvents were removed under vacuum. The crude product was purified using flash chromatography (SiO2, heptane/EtOAc 8:2) to afford 3.06 g (37% over two steps from 14 via 16) of 17 as a colourless oil. [α]D 20 +75.0 (c 0.2 in CDCl3). 1H (400 MHz, CDCl3) 7.37 - 7.21 (5 H, m), 5.75 (1 H, ddt, J 17.0, 10.2, 6.9), 5.06 (1 H, ddd, J 17.0, 3.3, 1.5), 4.99 (1 H, ddd, J 10.2, 3.3, 1.0), 4.09 (1 H, m), 3.47 (3 H, s), 3.17 (3 H, s), 2.85 (1 H, m), 2.47 (1 H, m). 13C (100 MHz, CDCl3) 177.60, 139.64, 136.12, 128.56, 128.17, 126.99, 116.56, 61.29, 51.20, 38.21, 32.26. HRMS calcd for C13H18NO2 [M + H]: 220.1338, found: 220.1343
(S)-4-Phenylhept-6-en-1-yn-3-one(18):To a solution of 17 (3.1 g, 14 mmol) in 20 ml of dry THF at –78˚C was added dropwise a solution of ethynylmagnesium bromide (110 ml, 0.5 M in THF, 55 mmol) under N2. The mixture was stirred at −78˚C for 30 min and then allowed to warm to room temperature overnight. The reaction was quenched with 70 ml of NH4Cl (sat). The layers were separated and the aqueous phase was extracted 3 times with Et2O. The combined organic extracts were dried over MgSO4 and concentrated under reduced pressure. Flash chromatography (SiO2, heptane/EtOAc 7:3) afford 2.6 g (quantitative yield) of the corresponding ynone 18. [α]D 20 +73.3 (c 0.3 in CDCl3). 1H (400 MHz, CDCl3) 7.37 (2 H, td, J 8.0, 1.9), 7.32 (1 H, tt, J 8.0, 1.9), 7.27 (2 H, dd, J 8.0, 1.9), 5.70 (1 H, ddt, J 17.0, 10.2, 7.1), 5.08 (1 H, ddd, J 17.0, 3.1, 1.5), 5.02 (1 H, ddd, J 10.2, 3.1, 1.1), 3.86 (1 H, t, J 7.1), 3.19 (1 H, s), 2.93 (1 H, dtd, J 14.4, 7.1, 1.1), 2.56 (1 H, dtd, J 14.4, 7.1, 1.5). 13C (100 MHz, CDCl3) 184.61, 134.74, 128.90, 128.69, 127.81, 117.33, 80.17, 77.20, 60.39, 35.38.HRMS calcd for C13H13O [M + H]: 185.0961, found: 185.0960.
(S)-4-Phenylhept-6-en-1-yn-3-ol(19):18 (3.4 g, 18 mmol) was dissolved in dry THF (100 ml) and cooled to −78˚C before the addition of DIBAL (46 ml, 1M in THF) under N2. The reaction mixture was stirred for 3 h at –78˚C and then quenched by the addition of 3 ml of MeOH and 20 ml of a solution of Na/K tartrate (sat), extracted with EtOAc (40 ml x 3), dried over Na2SO4 and concentrated under reduced pressure. 19 (3.2 g, 92%) was obtained as a 1:0.24 diasteromeric mixture and used in the next step without further purification. Diastereomer a). 1H (400 MHz, CDCl3) 7.39 - 7.28 (5 H, m), 5.69 (1 H, ddd, 17.1, 10.1, 5.8), 5.07 (1 H, ddd, J 17.1, 3.3, 1.5), 4.98 (1 H, ddt, J 10.1, 3.3, 1.1), 4.55 (1 H, dd, J 5.8, 2.2), 2.98 (1 H, dt, J 9.2, 5.8), 2.78 (1 H, ddd, J 14.4, 5.8, 1.5), 2.57 (1 H, dd, 14.4, 9.2, 1.1), 2.53 (1 H, d, J 2.2). 13C (100 MHz, CDCl3) 139.17, 135.99, 128.86, 128.41, 127.22, 116.97, 83.25, 74.73, 65.72, 51.66, 35.35. Diastereomer b). 1H (400 MHz, CDCl3) 7.39 - 7.28 (5 H, m), 5.69 (1 H, ddd, 17.1, 10.1, 5.8), 5.09 (1 H, ddd, J 17.1, 3.3, 1.5), 4.98 (1 H, ddt, J 10.1, 3.3, 1.1), 4.59 (1 H, dd, J 5.8, 2.2), 3.03 (1 H, dt, J 9.2, 5.8), 2.78 (1 H, ddd, J 14.4, 5.8, 1.5), 2.57 (1 H, dd, 14.4, 9.2, 1.1), 2.49 (1 H, d, J 2.2). 13C (100 MHz, CDCl3) 139.02, 135.84, 129.19, 128.27, 128.00, 116.92, 83.25, 75.05, 65.58, 50.90, 35.29. HRMS calcd for C13H14ONa [M + Na]: 209.0942, found: 209.0946.
Methyl ((4S,Z)-4-phenyldeca-1,8-dien-6-yn-5-yl) carbonate(20): The Sonogashira reaction was performed in the corresponding way as when 8a/8b was transformed to 10a/10b, starting from 19 (3.4 g, 18 mmol) and cis-1- bromo-prop-1-ene (6.6 g, 55 mmol), and the crude product was used directly for the preparation of the dienyne carbonate 20 as described for 11a/11b. 20 (0.70 g, 13%) was obtained as a 1:0.15 diastereomeric mixture after purification by flash chromatography (Si2O, heptane/CH2Cl2 1:1). Diasteromer a. 1H (400 MHz, CDCl3) 7.36 - 7.22 (5 H, m), 6.03 (1 H, dq, J 10.7, 6.9), 5.66 (1 H, ddt, J 17.2, 10.2, 7.0), 5.59 (1 H, dd, J 6.0, 1.9), 5.50 (1 H, ddd, 10.7, 1.7, 1.5), 5.03 (1 H, ddd, J 17.2, 3.4, 1.4), 4.96 (1 H, ddt, J 10.2, 3.4, 1.1), 3.76 (3 H, s), 3.14 (1 H, dt, J 6.0, 5.1), 2.77 (1 H, m), 2.65 (1 H, m), 1.82 (3 H, dd, J 6.9, 1.7). δ C (100 MHz, CDCl3) 154.87, 140.19, 138.92, 135.46, 128.89, 128.25, 127.15, 116.99, 109.12, 88.69, 84.65, 71.89, 54.89, 49.70, 35.08, 16.05. Diasteromer b. 1H (400 MHz, CDCl3) 7.36 - 7.22 (5 H, m), 6.00 (1 H, dq, J 10.7, 6.9), 5.66 (1 H, ddt, J 17.2, 10.2, 7.0), 5.59 (1 H, dd, J 6.0, 1.9), 5.45 (1 H, ddd, 10.7, 1.7, 1.5), 5.01 (1 H, ddd, J 17.2, 3.4, 1.4), 4.96 (1 H, ddt, J 10.2, 3.4, 1.1), 3.80 (3 H, s), 3.14 (1 H, dt, J 6.0, 5.1), 2.77 (1 H, m), 2.65 (1 H, m), 1.74 (3 H, dd, J 6.9, 1.7). 13C (100 MHz, CDCl3) 154.87, 139.97, 139.17, 135.46, 128.78, 128.19, 127.11, 116.96, 109.09, 88.69, 84.65, 71.89, 54.89, 49.41, 35.47, 15.95. HRMS calcd for C18H20O3Na [M + Na]: 307.1310, found: 307.1331
Methyl 6-methyl-(2R)-phenyl-2,6,7,7a-tetrahydro-1H-indene-4-carboxylate(21): The diene 21 was obtained following the same procedure as when 12a/12b was prepared from 11a/11b, starting with 20 (0.70 g, 2.4 mmol). 21 (0.32 g, 48%) was obtained as a 1:0.7 mixture of two diasteromers as a yellow oil, after flash chromatography (SiO2, heptane/Et2O 95:5). Diasteromer a).1H (400 MHz, CDCl3) 7.35 - 7.17 (5 H, m), 6.81 (1 H, t, J 2.8), 6.33 (1 H, t, J 2.7), 4.04 (1 H, dd, J 3.0, 1.6), 3.79 (3H, s), 2.98 (1 H, m), 2.65 (1 H, m), 2.12 (1 H, m), 2.06 (1 H, t, J 5.4), 1.99 (1 H, dt, J 12.7, 9.6), 1.14 (3 H, dd, J 7.3, 5.7), 1.07 (1 H, m). 13C (100 MHz, CDCl3) 166.93, 166.91, 147.73, 145.90, 138.18, 130.52, 128.34, 127.19, 125.96, 51.59, 50.32, 43.34, 39.69, 38.45, 32.92, 20.74. Diasteromer b). 1H (400 MHz, CDCl3) 7.35 - 7.17 (5 H, m), 6.81 (1 H, t, J 2.8), 6.41 (1 H, t, J 2.8), 4.00 (1 H, ddt, J 10.5, 3.0, 1.6), 3.80 (3 H, s), 2.86 (1 H, ddt, J 13.0, 9.5, 2.8), 2.65 (1 H, m), 2.59 (1 H, dt, J 12.2, 7.1), 2.06 (1 H, t, J 5.4), 1.37 (1 H, dt, J 12.3, 10.3), 1.16 (3 H, dd, J 7.3, 5.7), 1.07 (1 H, m). 13C (100 MHz, CDCl3) 166.93, 166.91, 147.60, 145.50, 138.00, 129.53, 128.36, 127.43, 126.11, 51.59, 51.27,44.66, 42.09, 38.34, 33.14, 20.83. HRMS calcd for C18H21O2 [M + H]: 269.1542, found: 269.1550.
(1aS,2S,3aS,5S,7aS)-Methyl 5-methyl-2-phenyl-1a,2,3,3a,4,5-hexahydroindeno [1,7a-b]oxirene-7-carboxylate (22):22 was prepared following the same procedure as when 13a/13b was prepared from 12a/12b, starting from 21 (0.31 g, 1.17 mmol). 22 (0.14 g, 42%) was obtained after flash chromatography (SiO2, heptane/Et2O 85:15). [α]D 20 + 8.5 (c 4.1 in CDCl3). 1H (400 MHz, CDCl3) 7.42 (1 H, m), 7.32 (2 H, m), 7.23 (2 H, m), 7.17 (1 H, dd, J 7.4, 2.2), 4.57 (1 H, s), 3.75 (3 H, s), 3.52 (1 H, m), 2.56 (1 H, m), 2.14 (1 H, m), 1.94 (1 H, m), 1.90 (1 H, m), 1.64 (1 H, m), 1.40 (1 H, ddd, J 14.8, 9.7, 7.5), 1.20 (3 H, dd, J 7.3). 13C (100 MHz, CDCl3) 165.40, 155.17, 141.93, 128.61, 127.49, 126.58, 125.84, 66.40, 64.14, 62.15, 51.71, 44.67, 37.81, 33.76, 32.19, 20.40. HRMS calcd for C18H21O3 [M + H]: 285.1491, found: 285.1472.
(2aS,4S,5aS,7S,7aR)-2a-Hydroxy-4-methyl-7-phenyl-4,5,5a,6,7,7a-hexahydroindeno [1,7-bc]furan-2(2aH)-one(2):2 was prepared in the same way as when 13a/13b was transformed to 3/4, starting from 22 (0.14 g, 0.5 mmol). The crude product was purified by chromatography (SiO2, heptane/EtOAc 60:40.) to afford 2 (30 mg, 22%) as a colourless oil. [α]D20 +59 (c 12.5 in CDCl3). 1H (400 MHz, CDCl3) 7.30 (2 H, m), 7.23 (1 H, m), 7.13 (2 H, m), 7.10 (1 H, d, J 3.2), 4.90 (1 H, dd, J 6.7, 1.4), 3.52 (1 H, dt, J 13.4, 6.7), 2.77 (1 H, m), 2.62 (1 H, m), 2.36 (1 H, dt, J 14.0, 7.6), 2.12 (1 H, dtd, J 12.7, 6.7, 1.4), 1.45 (1 H, dd, J 13.4, 12.7), 1.25 (3 H, d, J 7.3), 1.12 (1 H, m). 13C (100 MHz, CDCl3) 170.0, 150.3, 136.8, 130.7, 128.4, 128.2, 126.8, 90.5, 81.3, 48.2, 42.2, 37.6, 31.6, 29.1, 20.6. HRMS calcd for C17H19O3 [M + H]: 271.1334, found: 271.1351
WST-1 cell proliferation assay
The functional activity of 1, 2, 3 and 4 were evaluated using a WST-1 proliferation assay with DU145 cells (ATCC, American Type Culture Collection, LGC Standards AB, Borås, Sweden) which expresses constitutively activated STAT3, as previously described [
Our aim was to introduce a suitable functional group on the C-4 methyl group, as a handle to attach biotin via a linker. Initial attempts to introduce an alkyne or an azide to introduce the biotin group by “click chemistry” [
Scheme 1. Synthesis of 8-hydroxymethyl galiellalactone biotin 6-aminohexanoic acid amide ester (5) and 8-hydroxyme- thyl-7-phenyl galiellalactone biotin 6-aminohexanoic acid amide ester (6). *Intermediates 8b, 10b, 11b and 12b as well as the end products 5 and 6 were obtained as mixtures of diastereomers.
Ethynyl magnesium bromide was added to 5-pentenal (7a) to give the propargylic alcohol 8a in a quantative yield. A Sonogashira coupling of the terminal alkyne of 8a with the known cis-iodoalkene 9 [
N-(+)-biotinyl-6-aminohexanoic acid had previously been described as a suitable reagent for making biotin conjugates of a biologically active compound [
The corresponding biotin labeled 7-phenyl substituted derivative 6 was synthesized in an analogous manner. Starting from aldehyde 7b, prepared by a DIBAL-H reduction of the corresponding 2-phenyl pent-4-ene acid ester, the key intermediate 11b could be obtained in three steps as a mixture of diastereomers. As we previously have observed, the tandem carbonylation/intramolecular Diels-Alder reaction gives a higher yield when a substituent in position 7 is present [
To investigate the effect of a phenyl group in position 7, an asymmetric synthesis of 7-phenyl galiellalactone (2) was devised. The key objective was to obtain the substituted propargylic alcohol 19 with the absolute stereochemistry of C-4 set as shown in Scheme 2, as this would influence the stereochemistry of the subsequent stereocenters generated in the cyclization step. By applying a procedure developed for the enantioselective direct alkylation of arylacetic acids [
To determine how the structural changes in 2, 3 and 4 affected their ability to block STAT3 signaling compared to galiellalactone (1), they were assayed for their ability to inhibit proliferation of DU145 prostate cancer cells which express active STAT3 in vitro, using a WST-1 assay (see
The biotinylated analogues 5 and 6 will be used in various biological studies to elucidate the mechanism of action of galiellalactone, starting with its effect on STAT3 signaling [
ability to inhibit the proliferation of DU145 cells expressing constitutively active STAT3 (with the GIC50 values 6.6 and 14 μM, respectively, in the same WST-1 assay [
This work was supported by the Swedish Science Research Council.