Supporting Information Bisebromoamide, a Potent Cytotoxic Peptide from the Marine Cyanobacterium Lyngbya sp.: Isolation, Stereostructure, and Biological Activity Toshiaki Teruya, Hiroaki Sasaki, Hidesuke Fukazawa, and Kiyotake Suenaga * Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, Japan and National Institute of Infections Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan Keio University. National Institute of Infections Diseases. S1
Abbreviation of proteins ERK: extracellular signal regulated protein kinase PDGF: platelet-derived growth factor AKT: protein kinase B PKD: protein kinase D PLC: Phospholipase C MEK: MAP kinase/erk kinase MAP: mitogen-activated protein Experimental Procedures and Spectral Data for All New Compounds. General Methods. Chemicals and solvents were the best grade available and were used as received from commercial sources. Optical rotations were measured with a JASCO DIP-360 polarimeter. 1 H NMR spectra were recorded on a JEOL JNM-EX270 (270 MHz) or a JEOL JNM-A400 (400 MHz), instrument. Chemical shifts are reported δ values in parts per million relative to the residual solvent signal (CHD 2 OD: δ = 3.31 ppm; CHCl 3 : δ = 7.26 ppm; for 1 H) and coupling constants are in hertz (Hz). The following abbreviations are used for spin multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad. 13 C NMR spectra were recorded on a JEOL JNM-A400 (100.4 MHz) instrument using CD 3 OD and CDCl 3 as a solvent, respectively. Chemical shifts are reported in parts per million from the solvent signal (CDCl 3 : δ = 77.2 ppm; CHD 2 OD: δ = 49.0 ppm). The assignments of 1 H NMR and 13 C NMR spectra were determined by H-H COSY, HMQC and HMBC experiments. IR spectra were recorded on a JASCO FT/IR- 410 instrument and are reported in wavenumbers (cm -1 ). ESI mass spectra were recorded on a LCT premier EX spectrometer (Waters). Both TLC analysis and preparative TLC were conducted on E. Merck precoated silical gel 60 F254. Fuji Silysia silica gel BW-820 MH and FL-60D were used for column chromatography unless otherwise noted. Material and method. The marine cyanobacterium Lyngbya sp. was collected at the reef of Bise at 0-1 m depth, Okinawa, Japan (26 4'N, 127 52'E), in April 2007. Rat PDGFR-BB is a product of R&D Systems (Minneapolis, MN, USA). Phospho-Akt (Ser473), phospho-pkd/pkcµ (Ser 916), phospho-erk1/2 (Thr202/Tyr204), phosphop90rsk (Thr359/S363) and phospho-s6 ribosomal protein (Ser 235/236) antibodies were acquired from Cell Signaling Technology, Inc (Danvers, MA, USA). Immunoblotting analysis. To observe effects on PDGF-signaling, NRK (normal rat kidney) cells were seeded in 96-well plates in a volume of 100 µl at a density of 10000 cells per well, cultured for 2 days, and then serum starved for 24 h. Cells were treated with inhibitors for 3 h and stimulated with 50 ng/ml rat PDGF-BB for 10 min. Cells were fixed for 10 min with 10% cold trichloroacetic acid and lysed with 9 M urea, 2% Triton X-100 and 2% lithium dodecyl sulfate. Lysates were neutralized with 2 M Tris and passed through a syringe to reduce viscosity. Proteins were subjected to SDS-PAGE and analyzed by immunoblotting using a cocktail that contained antibodies against phosphorylated forms of the components of the PKC pathway (phospho-pkd), the PI3K-AKT pathway (phospho-akt, phospho-s6r) and the MEK-ERK pathway (phospho-erk and phospho-p90rsk). Extraction and isolation. Approximately 1300 g (wet weight) of cyanobacterium was extracted with methanol (3 L) for one week. The extract was filtered, and the filtrate was concentrated. The residue was partitioned between ethyl acetate (3 0.3 L) and water (0.3 L). The material obtained from the organic S2
layer were partitioned between 90% aqueous methanol and hexane. The aqueous methanol fraction (0.36 g) was first separated by column chromatography on ODS (6 g) using 40% methanol, 60% methanol, 80% methanol, and methanol. The fraction (41 mg) eluted with 80% methanol was subjected to HPLC [Cosmosil 5C 18 -AR-II (φ20 250 mm); flow rate 5mL/min; detection, UV 215 nm; solvent 65% MeCN] to give bisebromoamide (1) (9.6 mg, t R = 36.5 mim): [α] 22 D +17.8 (c 1.00, CHCl 3 ); IR (CHCl 3 ) 3522, 3436, 3328, 1640 cm -1. 1 H NMR Data for bisebromoamide (1; 400 MHz, CDCl 3 ). δ 7.47 (d, J = 6.8 Hz, 1H), 7.31 (d, J = 2.0 Hz, 1H), 7.20 (d, J = 7.3, 1.5 Hz, 2H), 7.13 (dd, J = 7.8, 7.3 Hz, 2H), 7.09 (dd, J = 7.8, 1.5 Hz, 1H), 7.07 (d, J = 8.3, 2.0 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.37 (d, J = 7.8 Hz, 1H), 5.83 (dd, J = 11.2, 5.9 Hz, 1H), 5.59 (dd, J = 9.3, 7.8 Hz, 1H), 4.87 (dd, J = 10.7, 7.3 Hz, 1H), 4.70 (m, 1H), 4.56 (dd, J = 8.8, 3.9 Hz, 1H), 4.50 (m, 1H), 3.54 (m, 2H), 3.52 (m, 1H), 3.39 (d, J = 11.2 Hz, 1H), 3.14 (s, 3H), 3.10 (d, J = 11.2 Hz, 1H), 3.06 (s, 3H), 3.06 (m, 2H), 3.05 (m, 2H), 2.89 (m, 1H), 2.54 (m, 1H), 2.50 (m, 1H), 2.35 (m, 1H), 2.19 (m, 1H), 2.11 (m, 1H), 1.88 (m, 2H), 1.80 (m, 1H), 1.74 (m, 1H), 1.55 (s, 3H), 1.43 (m, 1H), 1.41 (m, 1H), 1.17 (s, 9H), 1.07 (t, J = 7.3 Hz, 3H), 1.03 (d, J = 5.9 Hz, 3H), 0.87 (d, J = 7.3 Hz, 3H), 0.76 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H), 0.54 (m, 1H). Acid hydrolysis of 1. Bisebromoamide 1 (500 µg, 0.49 µmol) and 9 M HCl (0.1 ml) was charged in a reaction tube, and sealed up under reduced pressure. The mixture was heated at 110 for 72 h, diluted with water (1 ml), and evaporated. The acid hydrolysate could be separated into each components except for a mixture of Ala and 2-methylcystine. [Condition for the HPLC separation: column, Cosmosil 5C 18 -PAQ (4.6 250 mm); solvent, MeOH/H 2 O = 5/95; flow rate, 1.0 ml/min; detection at 254 nm. The retention times (min) of components: Ala and 2-methylcystine (3.6), Leu (3.9), N-Me-Tyr (7.0), N-Me-Phe(10.8)]. Conditions for chiral HPLC analyses of Leu, N-Me-Tyr, N-Me-Phe. Leu: column, CHIRALPAK(MA+) (4.6 50 mm); solvent, 2mM CuSO 4 ; flow rate, 1.0 ml/min; detection at 254 nm. t R (min) Authentic samples : D-Leu (7.5), L-Leu (13.2). Leu from natural 1: Leu (7.5). Authentic D,L-Leu Leu from natural 1 S3
N-Me-Tyr: column, CHIRALPAK(MA+) (4.6 50 mm); solvent, MeOH/2 mm CuSO 4 = 95/5; flow rate, 0.5 ml/min; detection at 254 nm. t R (min) Authentic samples : N-Me-D-Tyr (11.3), N-Me-L-Tyr (14.1). N-Me-Tyr from natural 1: N-Me-Tyr (11.3). Authentic N-Me-D,L-Tyr N-Me-Tyr from natural 1 N-Me-Phe: column, CHIRALPAK(MA+) (4.6 50 mm); solvent, MeCN/2 mm CuSO 4 = 90/10; flow rate, 0.5 ml/min; detection at 254 nm. t R (min) Authentic samples : N-Me-D-Phe (11.3), N-Me-L-Phe (12.5). N-Me-Phe from natural 1: N-Me-Phe (12.5). Authentic N-Me-D,L-Phe N-Me-Phe from natural 1 S4
Conditions for HPLC analyses of Marfey derivatives of Ala and 2-methylcystine. Ala: column, Cosmosil 5C 18 -AR-II (4.6 250 mm); solvent, MeOH/0.02 M AcONa =65/35; flow rate, 1.0 ml/min; detection at 340 nm. t R (min) Marfey derivatives of authentic samples : D-Ala (9.2), L-Ala (5.0). Marfey derivatives of Ala from natural 1: Ala (5.0). Marfey derivatives of authentic L-Ala Ala from natural 1 2-Methylcystine: column, Cosmosil 5C 18 -MS-II (4.6 250 mm); solvent, MeOH/0.02 M AcONa =60/40; flow rate, 1.0 ml/min; detection at 340 nm. t R (min) Marfey derivatives of authentic samples : D-2-Methylcystine (5.9), L-2-Methylcystine (8.8). Marfey derivatives of 2-Methylcystine from natural 1: 2-Methylcystine (5.9). Marfey derivatives of authentic D-2-Methylcystine Marfey derivatives of authentic L-2-Methylcystine S5
Marfey derivatives of 2-Methylcystine from natural 1 1 O 3 then aq. HCl 4 N H 2 COOH 7:3 Ozonolysis-acid hydrolysis of 1. Ozone gas was bubbled through a solution of bisebromoamide 1 (4.0 mg, 3.9 µmol) in MeOH (4 ml) for 30 min at -78. Excess ozone was evacuated by bubbling nitrogen, and dimethyl sulfide (0.1 ml) was added. The mixture was warmed to room temperature and concentrated to give an colorless oil. This oil and 9 M HCl (0.1 ml) was charged in a reaction tube, and sealed up under reduced pressure. The mixture was heated at 110 for 48 h, diluted with water (1 ml), and evaporated. The residue was purified by reversed phase HPLC to afford components, Me-Pro [2S : 2R = 7:3 from 1 H NMR and HPLC analysis of Marfey derivatives, see below]. [Condition for the HPLC separation: column, Cosmosil 5C 18 -PAQ (20 250 mm); solvent, MeOH/H 2 O/TFA = 1/99/0.1; flow rate, 5.0 ml/min; detection at 215 nm. The retention time (min) of components: Me-Pro (21.8)]. From 1 H NMR data, the relative stereochemistry of two stereoisomers were established. The major isomer was assigned as cis-4- methylproline and minor isomer was assigned as trans-4-methylproline. + 4 N H 2 COOH S6
Conditions for HPLC analysis of Marfey derivatives of 4-Me-Pro. 4-Me-Pro: column, Cosmosil 5C 18 -MS-II (4.6 250 mm); solvent, MeOH/0.02 M AcONa =70/30; flow rate, 1.0 ml/min; detection at 340 nm. t R (min) Marfey derivatives of authentic samples : (2S, 4S)-4-Me-Pro (4.5), (2R, 4R)-4-Me-Pro (8.0). Marfey derivatives of 4-Me-Pro from natural 1: 4-Me-Pro (4.5, 8.0). The ratio of peak intensity is 7:3. Authentic (2S, 4S)-4-Me-Pro and 4-Me-Pro from natural 1 4-Me-Pro from natural 1 S7
1 H NMR spectrum of Me-Pro from natural 1 [400 MHz, D 2 O]. S8
8 8 1 NaBH 4 then aq. HCl HO HN 2 + HO HN 2 5 1:1 Reduction-acid hydrolysis of 1. To a stirred solution of 1 (3.5 mg, 3.4 µmol) in MeOH (0.5 ml) at room temperature was added sodium borohydride (30 mg, 0.79 mmol). After being stirred at room temperature for 2 h, the mixture was diluted with AcOEt (5 ml) and H 2 O (5 ml), successively. The organic layer was separated, and the aqueous layer was extracted with AcOEt (2 10 ml). The organic layer and the extracts were combined, washed with saturated aqueous NaCl (15 ml), dried (Na 2 SO 4 ), and concentrated. The residual oil (3.3 mg, 3.2 µmol) and 9 M HCl (0.1 ml) was charged in a reaction tube, and sealed up under reduced pressure. The mixture was heated at 110 for 48 h, diluted with water (1 ml), and evaporated. The residue was purified by reversed phase HPLC to afford components, 2-(1-hydroxypropyl)- piperidine [6S : 6R = 1:1]. [Condition for the HPLC separation: column, Cosmosil 5C 18 -PAQ (20 250 mm); solvent, MeOH/H 2 O/TFA = 1/99/0.1; flow rate, 5.0 ml/min; detection at 215 nm. The retention times (min) of components: 2-(1-hydroxypropyl)-piperidine (33.9)]. The absolute stereochemistry of C6 was not determined. Conditions for HPLC analyses of Marfey derivatives of 2-(1-hydroxypropyl)-piperidine. 2-(1-hydroxypropyl)-piperidine: Condition 1. column, Cosmosil 5C 18 -MS-II (4.6 250 mm); solvent, MeOH/0.02 M AcONa =70/30; flow rate, 1.0 ml/min; detection at 340 nm. t R (min) Marfey derivatives of authentic samples : 2(R)-(1-hydroxypropyl)-piperidine [6S : 6R = 1:1] (9.7, 3.3), 2(S)- (1-hydroxypropyl)-piperidine [6S : 6R = 1:1] (15.3, 3.3). Marfey derivatives of 2-(1-hydroxypropyl)-piperidine from natural 1: 2-(1-hydroxypropyl)-piperidine (15.3, 3.3). 5 2-(1-hydroxypropyl)-piperidine from natural 1 Authentic 2(S) and from natural 1 S9
Although the retention time of one diastereomer of Marfey derivatives of authentic samples was identical to that from natural 1 (retention time : 15.3 min), two diastereomers of Marfey derivatives of authentic samples could not be separated above condition (retention time : 3.3 min). These diastereomers could be separated under the following conditions. Condition 2. column, Cosmosil 5C 18 -MS-II (4.6 250 mm); solvent, MeCN/0.02 M AcONa =40/60; flow rate, 1.0 ml/min; detection at 340 nm. t R (min) Marfey derivatives of authentic samples : 2(R)-(1-hydroxypropyl)-piperidine [6S : 6R = 1:1] (33.4), 2(S)-(1- hydroxypropyl)-piperidine [6S : 6R = 1:1] (30.4). Marfey derivatives of 2-(1-hydroxypropyl)-piperidine from natural 1: 2-(1-hydroxypropyl)-piperidine (30.4). Authentic 2(R) and from natural 1 Authentic 2(S) and from natural 1 Although one diastereomer of Marfey derivatives of authentic samples could not be detected under the condition 2, the retention time of one diastereomer of Marfey derivative of authentic sample was identical to that from natural 1 (retention time : 30.4 min). S10
1 H NMR spectrum of 1 [400 MHz, CD 3 OD]. S11
1 H NMR spectrum of 1 [400 MHz, CDCl 3 ]. S12
13 C NMR spectrum of 1 [100 MHz, CD 3 OD]. S13
COSY spectrum of 1 [400 MHz, CD 3 OD]. S14
HMQC spectrum of 1 [400 MHz, CD 3 OD]. S15
HMBC spectrum of 1 [400 MHz, CD 3 OD]. S16
GI 50 values of bisebromoamide (1) against 39 human cancer cell lines Type of cancer Cell line GI 50 a,b (nm) Breast HBC-4 97 BSY-1 24 HBC-5 27 MCF-7 35 MDA-MB-23 38 Central nervous system U251 34 SF-268 38 SF-295 33 SF-539 28 SNB-75 30 SNB-78 36 Colon HCC2998 22 KM-12 100 HT-29 33 HCT-15 100 HCT-116 93 Lung NCI-H23 92 NCI-H226 33 NCI-H522 27 NCI-H460 35 A549 39 DMS273 29 DMS273 37 Melanoma LOX-IMVI 26 Ovary OVCAR-3 28 OVCAR-4 27 OVCAR-5 100 OVCAR-8 100 SK-OV-3 86 Kidney RXF-631L 36 ACHN 32 Stomach St-4 19 MKN1 24 MKN7 13 MKN28 MKN45 47 37 MKN74 34 Prostate DU-145 61 PC-3 100 MG-MID c 40 Delta d 0.49 Range e 0.89 a Concentrations for the inhibition of cell growth at 50% relative to control. b Cell growth was determined according to the sulforhodamine B assay. c Mean GI 50 value in all of the cell lines tested. d Difference in the GI 50 value between the most-sensitive cells and the MG-MID value. e Difference in the log GI 50 value between the most- and least-sensitive cells. S17