Synthesis and Assignment of the Absolute Configuration of an Indenotryptoline Bisindole Alkaloid, BE-54017

Transcription

1 Supporting Information Synthesis and Assignment of the Absolute Configuration of an Indenotryptoline Bisindole Alkaloid, BE Tomoyuki Kimura, Shuhei Kanagaki, Yusuke Matsui, Masaya Imoto, Takumi Watanabe*,, and Masakatsu Shibasaki*, Institute of Microbial Chemistry, Tokyo, Japan Faculty of Science and Technology, Department of Biosciences and Informatics, Keio University, Japan Procedure for Chemical Experiments S2 1. General S2 2. Instrumentation S2 3. Materials S2 4. Synthetic procedure for BE (1) S3-S9 Procedure for Biological Experiments S10 1. Materials S10 2. Cell culture S10 3. Cell viability assay S10 4. Staining of intracellular acidic organelles S10 5. Isolation and identification of BE S10 6. Activity profiles of synthetic BE (( )-1) and the antipode ((+)-1) S10-S11 7. V- ATPase inhibition and cell death in EGF-stimulated A431 cells S11-S12 NMR Data S13-S28 S1

2 Procedure for Chemical Experiments 1. General All the reactions were performed in flame-dried glass pear shaped flasks or test tubes with a Teflon -coated magnetic stirring bar. The flasks or test tubes were fitted with a 3-way glass stopcock and reactions were run under argon atmosphere. Air- and moisture-sensitive liquids were transferred via a gas-tight syringe and a stainless-steel needle. All work-up and purification procedures were carried out with reagent-grade solvents under ambient atmosphere. Column chromatography was performed using silica gel 60N ( m) purchased from KANTO chemical co., inc. 2. Instrumentation Infrared (IR) spectra were recorded on a JASCO FT/IR 4100 Fourier transform infrared spectrophotometer. NMR was recorded on JEOL ECS-400 and ECA-600 spectrometers. Chemical shifts for proton are reported in parts per million downfield from tetramethylsilane and are referenced to residual protium in the NMR solvent (CDCl 3 : δ 7.26 ppm, CD 3 OD: δ 3.30 ppm, acetone-d 6 : 2.04 ppm, DMSO-d 6 : 2.49). For 13 C NMR, chemical shifts were reported in the scale relative to NMR solvent (CDCl 3 : 77.0 ppm, CD 3 OD: δ 49.0 ppm, Acetone-d 6 : 29.8 ppm, DMSO-d 6 : 39.7 ppm) as an internal reference. NMR data are reported as follows: chemical shifts, multiplicity (s: singlet, d: doublet, dd: doublet of doublets, t: triplet, m: multiplet), coupling constant (Hz), and integration. Optical rotation was measured using a 2 ml cell with a 1.0 dm path length on a JASCO polarimeter P High-resolution mass spectra (ESI TOF (+)) were measured on Thermo Fisher Scientific LTQ Orbitrap XL. 3. Materials Unless otherwise noted, materials were purchased from commercial suppliers and were used without purification. Toluene, THF, diethyl ether, and CH 2 Cl 2 were purified by passing through a solvent purification system (Glass Contour). 4. Synthetic procedure for BE (1) 11-Chloro-3a,12c-dihydroxy-2-methyl-8,12c-dihydropyrrolo[3',4':5,6]indolizino[8,7-b]indole-1,3(2 H,3aH)-dione (11) S2

3 11-Chloro-2-methylpyrrolo[3',4':5,6]indolizino[8,7-b]indole-1,3(2H,8H)-dione 10 1 (8.3 mg, 25.6 mol) was dissolved in 0.1 M OsO 4 in pyridine (0.28 ml, 28.2 mol) and stirred for 12 h at 40 C. The saturated aqueous NaHSO 3 was added to the reaction mixture and stirred for 6 h at 50 C. The mixture was extracted with AcOEt (3 10 ml). The combined extracts were washed with brine (30 ml), dried (Na 2 SO 4 ), and concentrated. The residual oil was purified by column chromatography on silica gel (500 mg, CHCl 3 / MeOH = 20 / 1) to give 11 (5.1 mg, 14.3 mol, 56%) as a colorless solid; IR (KBr) 3345, 2928, 1726, 1597, 1449, 1254, 1176, 753 cm -1 ; HRMS (ESI) Anal. calcd. for C 18 H 11 ClN 3 O 2 m / z [M H], found ; 1 H NMR (DMSO-d 6 ) (s, 1H), 8.11 (s, 1H), 7.80 (d, J = 2.1 Hz, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.29 (dd, J = 2.8, 1.6 Hz, 1H), 7.08 (dd, J = 8.7, 2.1 Hz, 1H), 6.92 (s, 1H), 6.59 (dd, J = 3.4, 1.6 Hz, 1H), 6.30 (t, J = 3.43 Hz, 1H), 2.86 (s, 3H); 13 C NMR (DMSO-d 6 ) 174.6, 171.3, 135.4, 130.6, 126.8, 124.4, 121.9, 121.4, 120.7, 119.1, 105.9, 101.5, 85.3, 74.5, (5-Chloro-1H-indol-2-yl)-2-methoxyethanone (8) To a stirred mixture of 1-carboxy-2-(tributylstannyl)indole 12 2 (12.8 g, 26.4 mmol) and methoxyacetyl chloride (3.6 ml, 39.6 mmol) in THF / DMF (1 / 1) (120 ml) was added Pd 2 (dba) 3 (2.50 g, 2.64 mmol) under the argon. The mixture was stirred at 40 C for 24 h. The reaction mixture was filtered through a pad of Celite and concentrated in vacuo. The residual oil was purified by column chromatography on silica gel (150 g, AcOEt / n-hexane = 1 / 4) to give methyl ether 8 (4.30 g, 19.5 mmol, 74%) as a colorless solid; IR (KBr) 3314, 1671, 803, 701 cm -1 ; HRMS (ESI) Anal. calcd. for C 11 H 9 ClNO 2 m / z [M H], found ; 1 H NMR (CDCl 3 ); 9.16 (s 1H), 7.69 (d, J = 1.8 Hz, 1H), 7.34 (m, 2H), 7.25 (m, 1H),4.62 (s 2H), 3.55 (s, 3H); 13 C NMR (CDCl 3 ); 189.5, 135.4, 133.7, 128.3, 127.2, 126.8, 113.4, 108.9, 75.2, '-Chloro-3-methoxy-1H,1'H-2,2'-biindole (7) The mixture of 8 (416 mg, 1.86 mmol) and phenylhydrazine (0.37 ml, 3.72 mmol) were dissolved 1 (a) Hugon, B.; Pfeiffer, B.; Renard, P.; Prudhomme, M. Tetrahedron Lett. 2003, 44, (b) Hugon, B.; Anizon, F.; Bailly, C.; Golsteyn, R. M.; Pierré, A.; Léonce, S.; Hickman, J.; Pfeiffer, B.; Prudhomme, M. Bioorg. Med. Chem. 2007, 15, Hudkins, R. L.; Diebold, J. L.; Marsh, F. D. J. Org. Chem. 1995, 60, S3

4 in AcOH (5 ml) at reflux condition (118 C) for 1 h. The reaction mixture was diluted with AcOEt (20 ml) and quenched with saturated aqueous NaHCO 3 (300 ml). The mixture was extracted with AcOEt (3 10 ml). The combined extracts were washed with saturated aqueous NaHCO 3 (50 ml), H 2 O (50 ml), and brine (30 ml) then dried (Na 2 SO 4 ), and concentrated to give crude 5 (593 mg) which was used for the next step without further purification; IR (KBr) 3426, 1606, 1518, 743 cm -1 ; HRMS (ESI) Anal. calcd. for C 17 H 12 ClN 2 O m / z [M H], found ; 1 H NMR (CDCl 3 ) 9.43 (s, 1H), 7.83 (s, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.57 (d J = 2.1 Hz, 1H), (m, 2H), (m, 1H), (m, 2H), 6.55 (d, J=1.7 Hz, 1H), 4.19 (s, 3H); 13 C NMR (CDCl 3 ) 137.5, 134.8, 134.6, 131.5, 129.4, 125.8, 123.4, 122.5, 121.1, 120.1, 119.5, 118.3, 117.1, 111.8, 111.6, 96.5, (5-Chloro-3'-methoxy-1H,1'H-[2,2'-biindol]-3-yl)-1-methylpyrrolidine-2,5-dione (6) To a solution of 7 (593 mg, 2.00 mmol) and N-metylmareimide (413 mg, 3.72 mmol) in toluene was added SnCl 2 (70.5 mg, 0.37 mmol). The reaction mixture was refluxed (110 C) for 24 h. After evaporation of toluene, the residual oil was purified by column chromatography on silica gel (25 g, AcOEt / n-hexane = 1 / 5 to 1 / 2) to give 6 (230 mg, 0.56 mmol, 31%) as a red-violet color solid; IR (KBr) 3320, 1774, 1693, 747 cm -1 ; HRMS (ESI) Anal. calcd. for C 22 H 17 ClN 3 O 3 m / z [M H], found ; 1 H (CDCl 3 ) 9.54 (s, 1H), 9.04 (s, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.23 (m, 2H), 7.15 (m, 2H), 4.68 (dd, J = 9.6, 3.7 Hz, 1H), 4.06 (s, 3H), 3.46 (dd, J = 18.9, 3.7 Hz, 1H), 3.19 (s, 3H), 3.18 (m, 1H); 13 C NMR (DMSO-d 6 ) 178.1, 176.5, 137.7, 134.9, 134.2, 129.2, 127.3, 123.9, 122.5, 121.9, 120.1, 119.0, 117.9, 117.1, 115.6, 113.2, 111.9, 109.2, 79.1, 61.1, 37.7, 35.6, Chloro-14-methoxy-7-methylpyrrolo[3',4':5,6]pyrido[1,2-a:3,4-b']diindole-6,8(7H,13H)-dione (13) 6 (39.2 mg, 96.1 µmol) was dissolved in nitrobenzene (20 ml) and treated with Pd black (10.2 mg, S4

5 96.1 µmol). The reaction mixture was stirred at 200 C. Cyclohexane was added to the solution and the mixture was filtered over silica gel pad (25 g). Nitrobenzene was eluted out with a mixture of cyclohexane / CHCl 3 (95 / 5 to 50 / 1). Then, 13 was eluted with a mixture of CHCl 3 / MeOH / TFA (10 / 1 / 0.05). The filtrate was concentrated and the residue was dissolved in AcOEt (20 ml). The solution was washed with saturated aqueous NaHCO 3 (20 ml), H 2 O (20 ml), and brine (20 ml), then dried (Na 2 SO 4 ), and concentrated. The residual oil was purified by column chromatography on silica gel (8 g, CHCl 3 / MeOH = 100 / 1) to give 13 (18.8 mg, 46.1 µmol, 48%) as a purple solid; IR (KBr) 3269, 3056, 1702, 753 cm -1, HRMS (ESI) Anal. calcd. for C 22 H 13 ClN 3 O 3 m / z [M H], found ; 1 H (CDCl 3 ) 12.3 (s, 1H), 9.35 (d, J = 8.6 Hz, 1H), 8.49 (d, J = 1.7 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 7.2 Hz, 1H), 7.47 (m, 1H), 7.35 (dd, J = 8.59, 2.06 Hz, 1H), 4.20 (s, 3H), 3.07 (s, 3H); 13 C NMR (CDCl 3 ) 167.7, 164.6, 137.9, 132.8, 132.2, 128.8, 126.3, 125.5, 124.3, 123.3, 122.2, , 121.4, 121.2, 117.6, 117.4, 116.8, 115.9, 114.0, 103.0, 62.6, Chloro-14-methoxy-7,13-dimethylpyrrolo[3',4':5,6]pyrido[1,2-a:3,4-b']diindole-6,8(7H,13H)-dio ne (5) To a stirred solution of 3 (9.7 mg, 24.0 mol) in DMF (2 ml) at 0 C was added MeI (10.6 µl, µmol) and KOH (4.0 mg, 72.1 µmol) under argon. The mixture was stirred at room temperature for 3 h, and the reaction was added AcOEt (10 ml) and H 2 O (10 ml). The mixture was extracted with AcOEt (3 10 ml). The combined extracts were washed with brine (30 ml), dried (Na 2 SO 4 ), and concentrated. The residual oil was purified by column chromatography on silica gel (4 g, AcOEt / n-hexane = 1 / 4) to give 7 (7.0 mg, 16.8 µmol, 70%) as a brown solid; IR (KBr) 3114, 2929, 1762, 1447, 740 cm -1 ; HRMS (ESI) Anal. calcd. for C 23 H 16 ClN 3 NaO 3 m / z [M+Na] +, found ; 1 H NMR (CDCl 3 ) 9.5 (d, J = 7.4 Hz, 1H), 8.92 (d, J = 1.7 Hz, 1H), 7.91 (d, J = 6.6 Hz, 1H), 7.50 (m, 2H), 7.40 (m, 2H), 4.38 (s, 3H), 4.13 (s, 3H), 3.22 (s, 3H); 13 C NMR (CDCl 3 ) 167.8, 164.8, 139.8, 134.8, , 129.9, 127.7, 126.4, 125.3, 123.6, 123.2, 123.1, 122.8, 122.7, 118.2, 117.3, 117.3, 116.2, 116.8, 105.2, 62.7, 34.1, BE (1) S5

6 7 (10.3 mg, 24.7 mol) was dissolved in 0.1 M OsO 4 in pyridine (0.27 ml, 27.2 mol) and stirred for 10 h at 40 C. The saturated aqueous NaHSO 3 was added to the reaction mixture and stirred for 6 h at 50 C. The mixture was extracted with AcOEt (3 10 ml). The combined extracts were washed with brine (30 ml), dried (Na 2 SO 4 ), and concentrated. The residual oil was purified by column chromatography on silica gel (500 mg, CHCl 3 / MeOH = 20 / 1) to give 1 (4.1 mg, 9.14 mol, 37%) as a pearl crystal (needle); IR (KBr) 3345, 2928, 1726, 1597, 1449, 1254, 1176, 753 cm -1 ; HRMS (ESI) Anal. calcd. for C 23 H 18 ClN 3 NaO 5 m / z [M+Na] +, found ; 1 H NMR (acetone-d 6 ) 8.21 (d, J = 8.5 Hz, 1H), 8.08 (d, J = 2.1 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.7 Hz, 1H), (m, 1H), 7.14 (t, J = 7.1 Hz, 1H), 6.67 (s, 1H), 6.39 (s, 1H), 4.17 (s, 3H), 4.08 (s, 3H), 2.96 (s, 3H); 13 C NMR (acetone-d 6 ) 174.6, 171.6, 139.2, 138.1, 137.8, 132.9, 127.2, 127.0, 124.9, 123.7, 123.4, 121.3, 120.9, 118.9, 116.7, 114.7, 112.4, 104.6, 87.6, 75.5, 62.7, 34.1, Daicel CHIRALPAC IC ( mm), AcOEt / n-hexane = 20 / 80, 2.0 ml / min, detected at 350 nm, t R = 8.0 min (1), t R = 10.0 min (ent-1). 1; [ ] 24 D 347 (c = 0.075, DMSO) (lit. [ ] 20 D 428 (c = 0.5, DMSO)), m.p C. ent-1; [ ] 24 D +332 (c = 0.065, DMSO), m.p C. Caldoniamide A (2) S6

7 13 (16.2 mg, 40.0 mol) was dissolved in 0.1 M OsO 4 in pyridine (0.4 ml, 40.0 mol) and stirred for 10 h at 40 C. The saturated aqueous sodium hydrogen sulfite was added to the reaction mixture and stirred for 6 h at 50 C. The mixture was extracted with AcOEt (3 10 ml). The combined extracts were washed with brine (10 ml), dried (Na 2 SO 4 ), and concentrated. The residual oil was purified by column chromatography on silica gel (200 mg, CHCl 3 / MeOH = 20 / 1) to give 2 (2.8 mg, 6.4 mol, 16%) as light green crystals; IR (KBr) 3320, 1774, 1440, 1283, 747 cm -1 ; HRMS (ESI) Anal. calcd. for C 22 H 15 ClN 3 O 5 m / z [M H], found ; 1 H NMR (DMSO-d 6 ) (s, 1H), 8.11 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 1.6 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), (m, 3H), 4.09 (s, 3H), 2.85 (s, 3H); 13 C NMR ((DMSO-d 6 ) 174.4, 171.5, 136.7, 136.7, 136.5, 128.3, 126.1, 127.8, 123.4, 122.3, 121.6, 120.4, 119.4, 117.9, 115.6, 114.4, 113.6, 103.6, 87.4, 75.0, 61.8, Daicel CHIRALPAC IC ( mm), AcOEt / n-hexane = 20 / 80, 2.0 ml / min, detected at 350 nm, t R = 8.3 min (2), t R = 9.4 min (ent-2). 2; [ ] 22 D 256 (c = 0.080, MeOH) (lit. [ ] 22.5 D 390 (c = 1.88, MeOH)), m.p C (lit C). ent-2; [ ] 22 D +227 (c = 0.075, MeOH), m.p C. BE (( )-1) S7

8 To a stirred solution of 2 (0.5 mg, 1.1 mol) in DMF (1 ml) cooled at 0 C was added methyl iodide (0.36 L, 5.7 mol) and KOH (0.2 mg, 3.4 mol) under argon. The mixture was stirred at room temperature for 3 h, and the reaction was added AcOEt and H 2 O (5 ml). The mixture was extracted with AcOEt (3 10 ml). The combined extracts were washed with brine (10 ml), dried (Na 2 SO 4 ), and concentrated. The residual oil was purified by preparative TLC (100 mm 100 mm, CHCl 3 / MeOH = 20 / 1) to give 1 (0.2 mg, 0.4 mol, 40%) as a pearl crystal (needle); Daicel CHIRALPAC IC ( mm), AcOEt / n-hexane = 20 / 80, 2.0 ml / min, detected at 350 nm, t R = 8.0 min. BE (1) from 5 BE (1) from 2 Mixture (from 5 + from 2) ent-be54017 ((+)-1) To a stirred solution of ent-2 (0.5 mg, 1.1 mol) in DMF (1 ml) cooled at 0 C was added methyl iodide (0.36 L, 5.7 mol) and KOH (0.2 mg, 3.4 mol) under argon. The mixture was stirred at room temperature for 3 h, and the reaction was added AcOEt and H 2 O (5 ml). The mixture was extracted with AcOEt (3 10 ml). The combined extracts were washed with brine (10 ml), dried (Na 2 SO 4 ), and concentrated. The residual oil was purified by preparative TLC (100 mm 100 mm, CHCl 3 / MeOH = 20 / 1) to give ent-1 (0.3 mg, 0.6 mol, 60%) as a pearl crystal (needle); Daicel CHIRALPAC IC ( mm), AcOEt / n-hexane = 20 / 80, 2.0 ml / min, detected at 350 nm, t R = 10.0 min. S8

9 (ent)-be54017 ((ent)-1) from 5 (ent)-be54017 ((ent)-1) from 2 Mixture (from 5 + from 2) S9

10 Procedure for Biological Experiments 1. Materials Natural BE54017 was isolated from fermentation broth of Streptomyces strain Acridine orange were from KANTO chemical Co.. 2. Cell culture Human epidermal carcinoma A431 cells were grown in Dulbecco's modified Eagle's medium (Nissui, Ohita, Japan) supplemented with 5% calf serum (Gibco BRL, Gaithersburg, MD), 100 U / ml of penicillin G, and 0.1 mg / ml of kanamycin at 37 C in a 5% CO 2-95% air atmosphere. 3. Cell viability assay Cells were seeded at per well in 48-wells plate and cultured overnight. After the culture supernatant was replaced with medium containing 0.2% serum, the cells were treated with drug in the presence or absence of 30 ng/ml EGF (Funakoshi, Tokyo, Japan) for 48 h. After adherent and detached cells were combined together, the cells were stained with trypan blue, and the percentage of viable cells was determined using a hemocytometer. Cell viability [%] means the ratio of number of trypan blue-impermeable cells in total cell counts (Trypan blue-impermeable cell number / Total cell number). 4. Staining of intracellular acidic organelles Cells grown on cover slips were incubated at 37 for 4 h with BE or Bafilomycin A1. They were then incubated for 30 min with 3 M acridine orange. After three washes with Hank's buffered solution, the cover slips were examined with a laser-scanning confocal microscope. 5. Isolation and identification of BE The cultivation of strain was carried out in ninety 500-ml Erlenmeyer flasks. After fermentation, the culture was extracted with MeOH (6 L), filtrated and obtained suspension was further extracted with EtOAc (3 L) twice, and the organic layer was concentrated to give a brown oily residue (911 mg). Then, the obtained crude active oil was subsequently subjected to centrifugal liquid-liquid partition chromatography (CPC) using hexane-acetonitrile (1 : 1, descending mode) elution. Active fraction was further purified by preparative octadecyl silyl (ODS) HPLC (Sun Fire, 10 μm, mm; Waters, Milford, MA, USA) with 70% aqueous MeOH to give pure BE (3.3 mg) 6. Activity profiles of synthetic BE (( )-1) and the antipode ((+)-1) The activity profiles of synthetic BE (( )-1) or the antipode ((+)-1) as selective inducers of cell death in A431 cells overexpressing EGFR are shown in Fig.S1. A431 cells stimulated with or S10

11 without EGF were incubated with various concentrations of samples for 48 h, and their viability was assessed by trypan blue dye exclusion assay. Synthetic BE (( )-1) as well as natural BE did not affect the viability of unstimulated cells up to 1 g/ml, whereas it induced cell death at 0.1 g/ml when the cells were stimulated with EGF. On the other hand, the antipode ((+)-1) did not induce significant cell death up to 1 g / ml even when the cells were stimulated with EGF, as shown in Figure S1 Furthermore, natural or synthetic BE did not induce EGF-dependent cell death in other types of human tumor cells, which do not overexpress EGFR as shown in Figure S2. Figure S1: Effect of BE on cell death in EGFR-overexpressing A431 cells. Figure S2: Effect of BE on cell death in lung and colon carcinoma cell lines. 7. V- ATPase inhibition and cell death in EGF-stimulated A431 cells Previously we reported that V-ATPase inhibitors induced apoptosis in EGF-stimulated A431 cells, with the same dose range inhibiting cellular V-ATPase activity, as determined by the disappearance of acidic organelles. 3 Therefore, we examined whether the selective induction of cell death by BE54017 in EGF-stimulated A431 cells was due to the inhibition of V-ATPase. The low ph of intracellular acidic organelles including lysosomes is known to be maintained by V-ATPases, and this acidification is detected as an orange fluorescence when the cells are stained with acridine 3 Yoshimoto, Y.; Imoto, M. Exp. Cell Res. 2002, 279, 118. S11

12 orange, a weak base fluorescent reagent. As shown in Figure S3, acidic organelles in A431 cells were stained with acridine orange, but treatment with 0.1 g / ml of BE (( )-1) caused a complete disappearance of the fluorescence, indicating that BE (( )-1) at 0.1 g / ml inhibited V-ATPase in cultured A431 cells. On the other hand, the antipode ((+)-1) did not show such activity up to 1 g / ml. Thus selective induction of apoptosis by BE in EGF-stimulated A431 cells was due to the inhibition of V-ATPase. ctrl BE ((-)-1) BE ((+)-1) ) 0.1 mg/ml 1 mg/ml Figure S3: Effect of BE on acidification of intracellular acidic organelles in A431 cells. S12

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