Improved Biomimetic Total Synthesis of d,l-stephacidin A Thomas J. Greshock 1 and Robert M. Williams 1,2 * 1 Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523 2 University of Colorado Cancer Center, Aurora, Colorado 80045 General thods. Unless otherwise noted, all materials were obtained from commercial sources and used without purification. All reactions requiring anhydrous conditions were performed under a positive pressure of argon using flame-dried glassware. Dichloromethane, acetonitrile, toluene, and tetrahydrofuran were degassed with argon and dried through a solvent purification system (J.C. yer of Glass Contour). Flash chromatography was performed on standard grade silica gel (230 x 400 mesh) from Sorbent Technologies with the indicated solvent. 1 MR and 13 C MR spectra were recorded on Varian 300 or 400 Mz spectrometers as indicated. Infrared spectra were recorded on a icolet Avatar 320-FT IR spectrometer. Mass spectra were obtained at the Colorado State University CIF on a Fisons VG Autospec. lting points were obtained on a l-temp Laboratory Device. S1
13 stephacidin A (1) + 6 6-epi-stephacidin A Stephacidin A (1). To a solution of alcohols 13 (79 mg, 0.18 mmol) in C 2 Cl 2 (18 ml) at rt was added DEAD (166 µl, 1.06 mmol). The mixture was stirred at rt for 5 min and PBu 3 (264 µl, 1.06 mmol) was then added. The solution was heated to 40 ºC for 20 h. The resulting mixture was concentrated. Purification by silica gel chromatography (ethyl acetate : hexane, 1 : 1, then methanol : C 2 Cl 2, 5 : 95) afforded stephacidin A (1) and 6- epi-stephacidin A as a 2.4 : 1 crude mixture of diastereomers, respectively. This mixture was triturated with methanol to afford stephacidin A (1) as a white solid (34.7 mg, 46%). The methanol triturate was then concentrated to afford 6-epi-stephacidin A (13.5 mg, 18%); All spectral data for stephacidin A (1) was identical to that previously reported; 1 6-epi-stephacidin A: 1 MR (400 Mz, CDCl 3 : CD 3 D, 20 : 1) δ 1.21 (s, 3 ), 1.28 (s, 3 ), 1.41 (s, 3 ), 1.42 (s, 3 ), 1.81 (dt, J = 7.4, 14.6 z, 1 ), 1.88 2.09 (m, 4 ), 2.24 (dd, J = 4.1, 9.9 z, 1 ), 2.71 (m, 1 ), 2.82 (d, J = 17.9 z, 1 ), 3.42 3.52 (m, 2 ), 3.78 (d, J = 17.9 z, 1 ), 5.62 (d, J = 9.7 z, 1 ), 6.61 (d, J = 8.3 z, 1 ), 6.64 (d, J = 9.7 z, 1 ), 7.19 (d, J = 8.3 z, 1 ), 8.54 (s, 1 ); 13 C MR (100 Mz, CDCl 3 : CD 3 D, 20 : 1) δ 23.6, 24.5, 24.9, 27.3, 27.5, 28.8, 29.1, 32.7, 34.7, 44.2, 45.9, 61.6, 67.1, 75.7, 103.8, 105.3, 110.1, 117.4, 118.3, 122.1, 129.7, 133.1, 138.7, 148.7, 1 Greshock, T. J.; Grubbs, A. W.; Tsukamoto, S.; Williams, R. M. Angew. Chem. Int. Ed. 2007, 46, 2262. S2
169.5, 173.3; IR (neat) 3291, 2917, 1693, 1666 cm -1 ; ESI/APCI-RMS (M + ) calcd for C 26 30 3 3 432.2282, found 432.2267. S3
Improved Stephacidin A Synthesis 88% C 2 Fmoc Et 2 C Cl ATU, i-pr 2 Et C 3 C Et 2 C Fmoc 1. (Boc) 2 DMAP 86% 2. CS DMF, rt 80% Boc Cl 1. Et 3, 82% 2. TFA, 97% 9-BB 2 C CuCl 85% 95% C 6 4 Cl 2!, 17h 1. FmocCl a 2 C 3 dioxane 2. 3 Sn C 2 4 Cl 2 74% 2 steps C 2 Et 2 Et 2 C 1. Ph Ph Bu 3 P, C 3 C reflux 2. 1 M Cl, C 2 Cl 2 93% 2 steps 2 C 2 95% morpholine Bu 3 P, DEAD TF C 2 Cl 2 95% 40 ºC, 20 h 64% (46% (1) + 18% epi) 13 stephacidin A (1) + epi 2.4 : 1 14 steps, 11.1% overall yield S4
Et 2 C Et Et 2 C Boc Boc -Boc-3-ketoproline ethyl ester. To a solution of LIMDS (1.0 M in TF, 7.91 ml, 7.91 mmol) in TF (60 ml) at -78 ºC was added ethyl 3-(tert-butoxycarbonyl(2-ethoxy- 2-oxoethyl)-amino)propanoate 2 in TF (6 ml) dropwise over 5 min. The mixture was stirred at -78 ºC for 4 h. The resulting solution was poured onto 1 M aqueous Cl and extracted 3 times with ether. The combined extracts were washed 2 times with 10% aqueous a 2 C 3, dried (a 2 S 4 ), and concentrated to afford -Boc-3-ketoproline ethyl ester as a colorless oil (1.20 g, 71%); All spectral data was identical to that previously reported. 2 Et 2 C Et 2 C Et 2 C Boc Boc CF 3 C - 2 + 15 cis-3-ydroxyproline ethyl ester trifluoroacetic acid salt (15). To a solution of - Boc-3-ketoproline ethyl ester (450 mg, 1.75 mmol) in (9 ml) at 0 ºC was added ab 4 (66 mg, 1.75 mmol) in one portion. The mixture was stirred at 0 ºC for 25 min. The resulting solution was quenched with 1 M aqueous Cl and extracted with ether. The combined extracts were dried (a 2 S 4 ) and concentrated to afford the crude cis-- Boc-3-hydroxyproline ethyl ester along with a small amount of the trans diastereomer (424 mg, 93%) as a mixture of amide rotamers, which was carried on without further 2 Williams, R. M.; Cao, J.; Tsujishima,.; Cox, R. J. J. Am. Chem. Soc. 2003, 125, 12172. S5
purification. 1 MR (300 Mz, CDCl 3 ) δ 1.25 (m, 3 ), 1.37 and 1.42 (s, 9 ), 1.91 2.11 (m, 2 ), 2.80 (br s, 1 ), 3.42 (m, 1 ), 3.60 (m, 1 ), 4.10 4.39 (m, 3 ), 4.57 (m, 1 ). To a solution of the crude alcohol (276 mg, 1.06 mmol) in C 2 Cl 2 (5 ml) at 0 ºC was added TFA (1 ml). The mixture was warmed to rt and stirred 3 h. The resulting solution was concentrated directly to afford the crude amine TFA salt 15, which was used directly in the next reaction without further purification. 14 Boc + Et 2 C CF 3 C - 2 + 15 Et 2 C 16 Boc Peptide (16). To a solution of acid 14 3 (330 mg, 0.89 mmol) in C 3 C (4.5 ml) was added ATU (505 mg, 1.33 mmol), ipr 2 Et (617 µl, 3.54 mmol), and cis-3- hydroxyproline ethyl ester 15 successively at rt. The mixture was stirred at rt for 3 h. The resulting solution was quenched with 2 M aqueous Cl and extracted with C 2 Cl 2. The combined extracts were dried (a 2 S 4 ) and concentrated. Purification by silica gel chromatography (ethyl acetate : hexane, 3 : 1) afforded amide 16 as a mixture of diastereomers and amide rotamers (440 mg, 97%); 1 MR (400 Mz, CDCl 3 ) δ 1.21 and 1.25 (t, J = 7.1 z, 3 ), 1.41 (s, 9 ), 1.58 (s, 3 ), 1.60 (s, 3 ), 2.12 2.21 (m, 2 ), 3.00 3.40 (m, 4 ), 3.59 and 3.90 (m, 1 ), 4.06 (m, 1 ), 4.14 and 4.21 (q, J = 7.1 3 Stocking, E. M.; Sanz-Cervera, J. F.; Williams, R. M. J. Am. Chem. Soc. 2000, 122, 1675. S6
z, 2 ), 4.61 (m, 1 ), 5.16 (d, J = 10.6 z, 1 ), 5.20 (d, J = 17.6 z, 1 ), 5.58 and 5.60 (br s, 1 ), 6.09 (dd, J = 10.6, 17.6 z, 1 ), 6.95 7.11 (m, 2 ), 7.21 (m, 1 ), 7.40 (m, 1 ), 8.00 and 8.12 (s, 1 ); 13 C MR (100 Mz, CDCl 3 ) δ 27.4, 27.9, 28.3, 28.5, 30.5, 30.8, 31.1, 32.4, 33.8, 39.2, 44.0, 44.3, 45.4, 52.8, 53.4, 61.6, 62.1, 62.9, 63.9, 65.1, 70.0, 70.3, 79.7, 106.1, 110.5, 112.7, 118.7, 119.5, 121.9, 129.7, 133.8, 140.8, 145.4, 154.6, 155.1, 167.8, 169.3, 171.9; IR (neat) 3383, 2977, 1740, 1694, 1635 cm -1 ; ESI/APCI-RMS (M + ) calcd for C 28 40 3 6 514.2912, found 514.2899. Et 2 C 16 Boc 17 Alcohol (17). To a solution of amide 16 (439 mg, 0.85 mmol) in C 2 Cl 2 (17 ml) at 0 ºC was added TFA (1.7 ml). The mixture was warmed to rt and stirred 2 h. The resulting solution was quenched slowly with saturated aqueous ac 3 and extracted with C 2 Cl 2. The combined extracts were dried (a 2 S 4 ) and concentrated to afford the crude primary amine. To a solution of the crude primary amine in toluene (17 ml) was added 2-hydroxypyridine (16.3 mg, 0.17 mmol). The mixture was heated to reflux for 16 h and then concentrated. The crude residue was diluted with C 2 Cl 2, washed twice with 1 M Cl, dried (a 2 S 4 ) and concentrated to afford alcohol 17 as an inseparable mixture of diastereomers (292 mg, 93%); 1 MR (400 Mz, CDCl 3 ) δ 1.48 (s, 3 ), 1.52 (s, 3 ), 1.92 2.18 (m, 2 ), 3.21 (m, 1 ), 3.48 (m, 1 ), 3.62 3.95 (m, 3 ), S7
4.20 (m, 1 ), 4.58 (m, 1 ), 5.09 5.18 (m, 2 ), 5.90 and 6.40 (s, 1 ), 6.10 (m, 1 ), 7.00 7.35 (m, 3 ), 7.47 (m, 1 ), 8.24 and 8.33 (s, 1 ); 13 C MR (100 Mz, CDCl 3 ) δ 27.8, 28.0, 29.8, 30.0, 30.4, 33.5, 39.0, 44.0, 44.2, 45.2, 54.9, 58.4, 61.8, 63.9, 64.6, 65.1, 69.8, 70.8, 104.4, 104.9, 110.6, 111.0, 112.0, 112.8, 117.9, 118.5, 119.7, 120.1, 121.8, 122.1, 125.4, 128.3, 129.0, 134.3, 134.5, 141.8, 145.6, 146.0, 165.8, 166.4, 167.4; IR (neat) 3358, 2969, 1742, 1655 cm -1 ; ESI/APCI-RMS (M + ) calcd for C 21 26 3 3 368.1969, found 368.1957. 17 19 + 20 Cycloadducts (19 and 20). To a solution of alcohols 17 (100 mg, 0.27 mmol) in C 2 Cl 2 (27 ml) at rt was added DEAD (257 µl, 1.63 mmol). The mixture was stirred at rt for 5 min and PBu 3 (408 µl, 1.63 mmol) was then added. The solution was heated to 40 ºC for 20 h. The resulting mixture was concentrated. Purification by silica gel chromatography (ethyl acetate : hexane, 1 : 1, then methanol : C 2 Cl 2, 5 : 95) afforded cycloadducts 19 and 20 as a 2.1 : 1 crude mixture of diastereomers, respectively. This mixture was further separated by preparative TLC ( : C 2 Cl 2, 3 : 97) to afford syn cycloadduct 19 as a white solid (43.6 mg, 46%) along with anti cycloadduct 20 as a S8
white solid (20.1 mg, 21%); Both cycloadducts 19 4 and 20 5 have been previously synthesized. All data was identical to that previously reported. 17 18 Enamide (18). To a solution of alcohols 17 (120 mg, 0.33 mmol) in C 2 Cl 2 (6.5 ml) at rt was added DEAD (40% wt. in toluene, 446 µl, 0.98 mmol). The mixture was stirred at rt for 5 min and PBu 3 (245 µl, 0.98 mmol) was then added. The solution was stirred at rt for 3 h. The resulting mixture was then concentrated. Purification by silica gel chromatography (ethyl acetate : hexane, 2 : 3 to 1 : 0) afforded enamide 18 as a white foam (98.2 mg, 86%); 1 MR (400 Mz, CDCl 3 ) δ 1.52 (s, 3 ), 1.53 (s, 3 ), 2.76 (m, 2 ), 3.22 (dd, J = 11.3, 14.5 z, 1 ), 3.70 (dd, J = 3.5, 14.5 z, 1 ), 4.05 (m, 2 ), 4.50 (br d, J = 10.3 z, 1 ), 5.12 (d, J = 10.5 z, 1 ), 5.14 (d, J = 17.5 z, 1 ), 5.73 (s, 1 ), 6.10 (dd, J = 10.5, 17.5 z, 1 ), 6.13 (t, J = 2.9 z, 1 ), 7.08 (t, J = 7.7 z, 1 ), 7.14 (t, J = 7.7 z, 1 ), 7.30 (d, J = 7.7 z, 1 ), 7.50 (d, J = 7.7 z, 1 ), 8.39 (s, 1 ); 13 C MR (100 Mz, CDCl 3 ) δ 27.8, 27.9, 28.0, 30.9, 39.1, 45.6, 57.5, 104.4, 110.9, 112.3, 118.2, 119.0, 120.0, 122.0, 128.8, 133.1, 134.4, 141.8, 145.8, 156.5, 162.6; IR 4 (a) Williams, R. M.; Glinka, T.; Kwast, E.; Coffman,.; Stille, J. K. J. Am. Chem. Soc. 1990, 112, 808. (b) Jin, S.; Wessig, P.; Liebscher, J. J. rg. Chem. 2001, 66, 3984. (c) Baran, P. S.; afensteiner, B. D.; Ambhaikar,. B.; Guerrero, C. A.; Gallagher, J. D. J. Am. Chem. Soc. 2006, 128, 8678. 5 Adams, L. A.; Valente, M. W..; Williams, R. M. Tetrahedron 2006, 62, 5195. S9
(neat) 3341, 2967, 1677, 1644 cm -1 ; ESI/APCI-RMS (M + ) calcd for C 21 24 3 2 350.1863, found 350.1842. 18 19 + 20 Cycloadducts (19 and 20). To a solution of enamide 18 (92 mg, 0.26 mmol) in (20 ml) at 0 o C was added 20% aqueous K (5 ml). The mixture was slowly warmed to rt over 1 h and stirred for 18 h. The resulting solution was quenched with saturated aqueous 4 Cl and extracted with C 2 Cl 2. The combined extracts were dried (a 2 S 4 ) and concentrated. Purification by preparative TLC ( : C 2 Cl 2, 3 : 97) afforded syn cycloadduct 19 as a white solid (55.2 mg, 60%) along with anti cycloadduct 20 as a white solid (26.9 mg, 29%). 20 (±)-brevianamide B (9) Brevianamide B (9). To a solution of indole 20 (10.0 mg, 0.029 mmol) in TF (1 ml) at rt was added mcpba (6.4 mg, 0.037 mmol). The mixture was stirred at rt for 1.5 h. The resulting solution was quenched with two drops of dimethyl sulfide and S10
concentrated to dryness to afford the crude 3-hydroxyindolinene, which was used without further purification. To a solution of the crude 3-hydroxyindolinene in (1 ml) was added 0.5 M aqueous a (2 ml). The mixture was stirred at rt for 16 h and then warmed to 70 ºC for 2 h. The resulting solution was cooled to rt, quenched with 1 M aqueous Cl, and extracted with C 2 Cl 2. The combined extracts were dried (a 2 S 4 ) and concentrated. Purification by preparative TLC ( : C 2 Cl 2, 5 : 95) afforded brevianamide B (9) as a yellow film (4.7 mg, 45%); All spectral data was identical to the natural sample as well as those synthetic samples previously prepared in our group. 5 See attached 1 MR spectrum. 20 23 xindole (23). To a solution of indole 20 (16 mg, 0.046 mmol) in C 2 Cl 2 (1.8 ml) at rt was added oxaziridine 22 (33 mg, 0.14 mmol). The mixture was stirred at rt for 16 h. The resulting solution was placed directly on a column of silica gel and eluted with C 2 Cl 2, followed by : C 2 Cl 2 (5 : 95) to afford oxindole 23 as a thin film (11.8 mg, 71%); 1 MR (400 Mz, CDCl 3 ) δ 1.33 (s, 3 ), 1.45 (s, 3 ), 1.84 (dt, J = 7.3, 13.1 z, 1 ), 1.96 2.10 (m, 5 ), 2.75 (dt, J = 6.7, 13.1 z, 1 ), 2.82 (d, J = 15.8 z, 1 ), 3.42 (t, J = 6.7 z, 2 ), 3.49 (m, 1 ), 5.30 (s, 1 ), 7.13 (s, 1 ), 7.23 (t, J = 7.5 z, 1 ), 7.37 (t, J = 7.5 z, 1 ), 7.43 (d, J = 7.5 z, 1 ), 7.53 (d, J = 7.5 z, 1 ); 13 C S11
MR (100 Mz, CDCl 3 ) δ 20.2, 24.5, 27.5, 29.2, 32.7, 38.0, 40.5, 44.2, 50.5, 62.1, 67.3, 82.5, 121.3, 122.4, 126.7, 130.3, 140.5, 152.1, 168.4, 172.5, 188.2; IR (neat) 3365, 2968, 1688 cm -1 ; ESI/APCI-RMS (M + ) calcd for C 21 24 3 3 366.1812, found 366.1800. S12
400 Mz, CDCl 3 : CD 3 D (20 : 1) 6 S13 6-epi-stephacidin A 1 ppm 8ppm 78 67 56 45 34 23 12
173.341 169.547 148.721 138.705 133.135 129.728 122.116 118.312 117.381 110.126 105.273 103.792 75.670 67.143 61.608 45.873 44.237 34.673 32.688 29.125 28.808 27.498 27.264 24.871 24.512 23.583 100 Mz, CDCl 3 : CD 3 D (20 : 1) 6 S14 6-epi-stephacidin A 180ppm-298.349 20 170180 160170 150160 140150 130140 120130 110120 100110 90100 8090 7080 6070 5060 4050 3040 2030
300 Mz, CDCl 3 Et 2 C S15 Boc 18ppm 78 67 56 45 34 23 12
400 Mz, CDCl 3 Et 2 C S16 16 Boc 1 ppm 8ppm 78 67 56 45 34 23 12
171.938 169.281 167.786 155.074 154.565 145.360 140.799 133.807 129.691 121.912 119.526 118.720 112.738 110.483 106.056 79.683 70.255 70.019 65.135 63.888 62.899 62.042 61.552 53.374 52.793 45.379 44.320 44.030 39.211 33.781 32.436 31.080 30.758 30.475 28.536 28.245 27.907 27.405 100 Mz, CDCl 3 Et 2 C Boc S17 16 20 ppm-298.349 170ppm 160170 150160 140150 130140 120130 110120 100110 90100 8090 7080 6070 5060 4050 3040 2030
400 Mz, CDCl 3 S18 17 1 ppm 8ppm 78 67 56 45 34 23 12
30 3040 4050 5060 6070 7080 8090 90100 100110 110120 120130 130140 140150 150160 160170 170ppm ppm-303.855 27.782 27.963 29.818 29.989 30.394 33.535 39.039 44.046 44.212 45.236 54.957 58.353 61.798 63.909 64.578 65.064 69.827 70.807 104.353 104.852 110.589 111.034 111.983 112.828 117.857 118.471 119.692 120.077 121.756 122.113 125.353 128.282 129.094 134.275 134.456 141.617 145.588 145.996 165.788 166.377 167.391 17 100 Mz, CDCl 3 S19
400 Mz, CDCl 3 S20 18 2 ppm 8ppm 78 67 56 45 34 23
162.628 156.515 145.789 141.819 134.441 133.096 128.798 122.008 120.002 119.003 118.159 112.301 110.899 104.364 57.484 45.633 39.080 30.850 27.989 27.940 27.848 100 Mz, CDCl 3 S21 18 20 170ppm-298.349 160170 150160 140150 130140 120130 110120 100110 90100 8090 7080 6070 5060 4050 3040 2030
400 Mz, CDCl 3 S22 23 1 8ppm 78 67 56 45 34 23 12
188.171 172.547 168.355 152.114 140.528 130.351 126.738 122.429 121.265 82.532 67.293 62.085 50.482 44.220 40.548 38.033 32.691 29.176 27.459 24.536 20.196 100 Mz, CDCl 3 S23 23 10 190ppm-298.349 180190170180160170150160140150130140120130110120100110 90100 8090 7080 6070 5060 4050 3040 2030 10
S24