Isolation and structure elucidation of cytoxotic natural products with potential anticancer activity

Isolation and Structure Elucidation of Cytotoxic Natural Products with Potential Anticancer Activity Trong Duc Tran - 2010 - Isolation and Structure Elucidation of Cytotoxic Natural Products with Potential Anticancer Activity Trong Duc Tran (B.Eng) Eskitis Institute for Cell and Molecular Therapies Science, Environment, Engineering and Technology Griffith University Submitted in fulfilment of the requirements of the degree of Master of Philosophy July 2010 Declaration This work has not previously been submitted for a degree or diploma in any university To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself ……………………………… …………………… Trong Duc Tran Date i ii Acknowledgements First of all, I would like to express my sincere appreciation to my supervisors, Professor Ronald J Quinn and Dr Ngoc B Pham, for their patience, guidance and support My skills as a scientist have definitely matured under the supervision of Prof Quinn and Dr Pham and for that I am most grateful I would like to thank Dr Gregory Fechner for his kind supervision in training for the bioassay screening I also wish to thank Assoc Prof Anthony Carroll, Dr Rohan Davis, Dr Yun Feng, Dr Phuc Le, Dr Hoan Vu, Dr Harish Holla, Dr Xinzhou Yang, Dr Sheng Yin and Ms Lekha Suraweera for all academic and technical discussions; Assoc Prof Andreas Hoffmann for the circular dichroism measurements; Ms B Aldred for the provision of cancer cell lines; Dr John Hooper of the Queensland Museum, Dr Paul Forster and Dr G Guymer of the Queensland Herbarium for the collection and identification of biota samples I thank all my friends Emma Barnes, Michelle Liberio and Asiah Osman for many scientific and non-scientific conversations I acknowledge Education Australia Ltd for the provision of the “EAL Postgraduate Research Student Mobility Scholarships” which allowed me to pursue my full-time research iii iv Abstract This project presented a strategy to select a subset of prefractionated fractions for screening Marine and plant biota samples were chosen based on their rare taxonomies and mass spectroscopic data A method to reduce 1155 fractions generated from 105 selected samples to 330 UV active fractions was developed Cancer cell-based screening of 330 prefractionated fractions against four cancer cell lines (A549, HeLa, LNCaP and PC3) and non-cancer cells (HEK) resulted in nineteen active fractions belonging to fourteen biota samples (two plants and twelve marine organisms) One plant and six marine animals were chosen for further investigation Subsequent mass-guided isolation led to the identification of forty-four secondary metabolites, nine of which were not previously reported Structures of these compounds were elucidated by spectroscopic methods (1D and 2D-NMR, MS, CD and specific optical rotation) and chemical methods In the plant sample Neolitsea dealbata, a total of nine alkaloids (55-63) were isolated A new aporphine, normecambroline (55), showed selective activity against HeLa cells with an IC50 of 4.0 μM while its analogue, roemerine (56), displayed nonselective activity against all four cancer cell lines (A549, HeLa, LNCaP and PC3) and two non cancer cell lines (HEK, NFF) One of the marine samples, a specimen of the Potter Reef marine sponge Diacarnus sp., showed the presence of terpene peroxides Three known peroxide compounds, sigmosceptrellin (27), deacarperoxide (28) and methyldiacarnoate (29), were identified Compound 27 inhibited cytotoxicity against all six cell lines with IC50 values ranging from 0.4 to 3.1 μM while the other two compounds were not active Chemical investigation of a marine specimen from Houghton Reef, Neopetrosia exigua, resulted in the isolation of two cytotoxic compounds, mortuporamine C (42) and a new 3-alkylpyridinium alkaloid, dehydrocyclostellettamine A (43) These two compounds displayed activity against four cancer cell lines with IC50 values in micromolar concentrations (3.0-13.7 μM) v Three new cyclodepsipeptides, neamphamide B (86), neamphamide C (87) and neamphamide D (88), were found as constituents of a rare marine sponge Neamphius huxleyi collected at Milln Reef, off Cape Grafton Their structures were elucidated by NMR spectroscopy and multiple stages of accurate mass measurements (ESI-FTICRMSn) Stereochemistry of residues of the peptides was determined by the Marfey amino acid method and J-based configurational analysis These compounds inhibited the cell growth with IC50 values ranging from 91.3 to 366.1 nM Two previously unreported milnamide E (116) and hemiasterlin D (117) together with nine known small peptides were isolated from a new sponge genus, Pipestela candelabra Compound 117 was identified as the first peptide skeleton discovered in nature with a side chain containing 2-hydroxyacetic acid, tert-leucine and N-methylvinylogous valine residues attached to the indole nitrogen This compound exhibited activity against HeLa cells with an IC50 of 1.8 nM Cytotoxic results indicated all hemiasterlin derivatives were approximately 100 fold more active against cancer cell lines than the milnamide family A series of sixteen bromotyrosine alkaloids were identified from two Australian sponges Suberea clavate and Pseudoceratina sp Two new bromotyrosine derivatives, pseudoceralidinone A (148) and aplysamine (149) were isolated from a specimen of the Hook Reef lagoon sponge, Pseudoceratina sp Their absolute stereostructures were determined by synthetic methods Compound 149 inhibited moderate cytotoxicity (an IC50 of 4.9 μM against PC3 cells) while compound 148 displayed no activity All isolated compounds were evaluated for their physico-chemical properties Results showed that thirty-six out of forty-four compounds (81.8%) passed Lipinski’s rule and twenty-nine compounds (65.9%) displayed no violation against the requirements of both Lipinski’s and Veber’s rule vi Abbreviations HPLC High pressure liquid chromatography RP Reverse phase 18 C octadecyl bonded silica PAG polyamide gel PDA Photo diode array UV ultra violet IR Infra red CD circular dichroism [α]D specific rotation MS mass spectrometry MSn multi stage mass spectrometry ESI electrospray ionization LRESIMS low resolution electrospray mass spectrum HRESIMS high resolution electrospray mass spectrum FTICR Fourier transform ion cyclotron resonance MW molecular weight m/z mass-ion ratio (z = 1) amu atomic mass unit 2D two dimensional NMR nuclear magnetic resonance COSY correlation spectroscopy HSQC heteronuclear single quantum coherence HMBC heteronuclear multiple bond correlations ROESY rotating frame overhauser effect spectroscopy TOCSY total correlation spectroscopy HSQMBC heteronuclear single quantum multiple bond correlation HSQC-TOCSY heteronuclear multiple bond correlation total correlation spectroscopy 2,3 JCH or bond hydrogen to carbon correlation s singlet d doublet t triplet vii m multiplet br broad ppm parts per million MHz megahertz DMSO dimethylsulfoxide DCM dichloromethane EtOH ethanol MeOH methanol CDCl3 deuterated chloroform DMSO-d6 deuterated dimethylsylfoxide CD3OD-d4 deuterated methanol CD3OH-d3 deuterated methanol TFA trifluoroacetic acid FA formic acid CE Cotton effect Ro5 the Rule of Five IC50 concentration of a compound required to inhibit 50% of the receptor population SAR structure-activity relationship (Boc)2O di-tert-butyl dicarbonate EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HOBt Hydroxybenzotriazole DMF Dimethylformamide viii Appendix Chapter 5: Experimental Neamphamide B (86) Colourless amorphous powder; [α]24D -5.5 (c 0.08, MeOH); UV (MeOH) λmax (logε) 233 (3.8), 274 (3.1) nm; IR (film) νmax 3315, 1740, 1659, 1510 cm-1; 1H (600 MHz) and 13C (150 MHz) NMR data are summarized in Table 5.4; (+) HRESIMS m/z 1574.8935 ([M+H]+) (calcd (+) m/z 1574.8901, Δ 2.2 ppm) O H2N * NH * * O OH N O NH H N H2N * O O * NH OH * O O H N * O HN NH O O O OH O OH N H O O HN H2N OH * N HN NH2 O O HN H2N NH Neamphamide C (87) Colourless amorphous powder; [α]24D -8.1 (c 0.08, MeOH); UV (MeOH) λmax (logε) 233 (3.8), 275 (3.1) nm; IR (film) νmax 3315, 1740, 1660, 1512 cm-1; 1H (600 MHz) and 13C (150 MHz) NMR data are summarized in Table 5.5; (+) HRESIMS m/z 1575.8726 ([M+H]+) (calcd 1575.8741, Δ -0.9 ppm) O H2 N * NH * * O OH N O H N H2 N NH * O O NH * OH * OH O O H N * O O OH N H HN O HN H2 N NH O O O OH * O N HN OH O O HN H2N NH Neamphamide D (88) Colourless amorphous powder; [α]24D -18.9 (c 0.08, MeOH); UV (MeOH) λmax (logε) 233 (3.7), 275 (3.0) nm; IR (film) νmax 3318, 1739, 1659, 1518 cm-1; 1H (600 MHz) and 13 C (150 MHz) NMR data are summarized in Table 5.5; (+) HRESIMS m/z 1588.9052 ([M+H]+) (calculated 1588.9057, Δ -0.3 ppm) 168 O H2 N * NH * * O OH N O H N H2 N NH * O O NH * OH * OH O O H N * O O OH N H HN O HN H2 N NH O O O OH * O N HN NH2 O O HN H2N NH Peptide Hydrolysis Peptide samples (200μg) were dissolved in degassed 6N HCl (500μl) and heated at 120oC for 16h The solvent was removed under dry nitrogen and the resulting material was subjected to further derivatization for stereochemical assignment LC/MS Analysis of Marfey’s Derivatives A portion of the hydrolysate mixture (100μg) or the amino acid standard was added a solution of L-FDAA 1% (w/w) in acetone and 100μl of a 1N NaHCO3 solution The vial was heated at 50oC for 3h and the contents were neutralized with 0.2N HCl (50μl) after cooling to room temperature An aliquot of the L-FDAA derivative was dried under dry nitrogen, diluted in DMSO and loaded on a Phenomenex Luna column (C18, 3μm, 2.0 mm x 150 mm) using a linear gradient from 100% water (0.1% formic acid) to 100% acetonitrile (0.1% formic acid) in 50 minutes and an isocratic at 100% acetonitrile (0.1% formic acid) in the next ten minutes FDAA derivatives were detected by absorption at 340nm and assignment was secured by ion-selective monitoring Retention times of authentic FDAA-amino acids are given in parenthesis: L-Arg (16.18), D-Arg (16.32), L-Asn (20.71), D-Asn (21.08), L-Hpr (27.03), D-Hpr (26.35), L-Leu (28.95), D-Leu (30.09), L-NMeGln (25.16), D-NMeGln (25.89), L-NMeGlu (25.71), D-NMeGlu (26.52), L-Thr (21.68), D-Thr (23.27), L-aThr (21.24) and D-aThr (22.54) The hydrolysate of neamphamides B (2) contained D-Arg (16.35), L-Asn (20.74), L-Hpr (27.08), L-Leu (28.93), L-NMeGln (25.19) and D-aThr (22.58) The hydrolysate of neamphamides C (3) contained D-Arg (16.31), L-Asn (20.73), L-Hpr (27.03), L-Leu (28.94), L-NMeGlu (25.75) and D-aThr (22.55) The hydrolysate of neamphamides D (4) contained D-Arg (16.34), L-Asn (20.75), L-Hpr (27.06), L-Leu (28.95), L-NMeGln (25.20) and D-aThr (22.59) 169 ESI-FTICR-MSn Analysis Mass spectral data was obtained in the positive ion mode on a Bruker Apex III 4.7 Tesla, which was equipped with an ESI Apollo source Samples were directly infused by a Cole-Parmer syringe pump with a flow rate of μL per minute The end plate or counter electrode voltage was biased at 3900 V and the capillary voltage at 4400 V relative to the ESI needle N2 gas was used as nebulizing gas with a pressure of 50 psi and as counter-current drying N2 gas with a flow of 50 L/min The drying gas temperature was maintained at 125ºC The capillary exit voltage was tuned at 120 V ESI mass spectra were recorded in the mass range m/z 50-3000 Da SORI-CID was used for fragmentation in FTMSn experiments Data acquisition and processing were performed using Xmass software Parameters for MS2 were correlated sweep pulse length, 1000 μs; correlated sweep attenuation, 21.4 dB; ejection safety belt, Hz; user pulse length, 40000 μs; ion activation pulse length, 250000 μs; ion activation attenuation, 42.0 dB; frequency offset from activation mass, 500 Hz; user delay length, 10 s Parameters for MS3 were correlated sweep pulse length, 600 μs; correlated sweep attenuation, 42.5 dB; ejection safety belt, Hz; user pulse length, 40000 μs; ion activation pulse length, 250000 μs; ion activation attenuation, 42.0 dB; frequency offset from activation mass, 500 Hz; user delay length, 10 s 170 Appendix Chapter 6: Experimental Hemiasterlin (89) Colourless amorphous powder; (+) LRESIMS m/z O O 527 ([M+H]+); N N H NH H, 13 C-NMR data and optical OH rotation value were identical with those reported in O N the literature (reference in chapter 6) Hemiasterlin A (91) Colourless amorphous powder; (+) LRESIMS m/z O 537 ([M+H]+); 1H, O N N H NH OH O N H 13 C-NMR data and optical rotation value were identical with those reported in the literature (reference in chapter 6) Milnamide A (90) Colourless amorphous powder; (+) LRESIMS m/z O 539 ([M+H]+); O N N H N H, 13 C-NMR data and optical OH rotation value were identical with those reported in O N the literature (reference in chapter 6) Milnamide C (94) Colourless amorphous powder; (+) LRESIMS m/z O O 553 ([M+H]+); 1H, N N H N 13 C-NMR data and optical OH rotation value were identical with those reported O N in the literature (reference in chapter 6) O Milnamide D (95) Light yellow powder; (+) LRESIMS m/z 537 O O N N H N N OH O ([M+H]+); 1H, 13 C-NMR data and optical rotation value were identical with those reported in the literature (reference in chapter 6) 171 Geodiamolide D (101) Colourless amorphous powder; (+) LRESIMS m/z 628 I OH 13 C-NMR data and optical rotation value were identical with those reported in the literature (reference 16 in H N N chapter 6) O O ([M+H]+); 1H, O O O NH Geodiamolide E (102) Colourless amorphous powder; (+) LRESIMS m/z 580 Br OH 13 C-NMR data and optical rotation value were identical with those reported in the literature (reference 16 in H N N chapter 6) O O ([M+H]+); 1H, O O O NH Geodiamolide E (103) Colourless amorphous powder; (+) LRESIMS m/z 536 Cl OH 13 C-NMR data and optical rotation value were identical with those reported in the literature (reference 16 in H N N chapter 6) O O ([M+H]+); 1H, O O O NH Jaspamide (115) White amorphous powder; (+) LRESIMS m/z 709 ([M+H]+); HO Br NH H, 13 C-NMR data and optical rotation value were identical with those reported in the literature (reference 19 in chapter H N N 6) O O O O O NH Minamide E (116) Colourless amorphous powder; [α]25D +10.8 (c 0.02, O O MeOH); CD (MeOH) λmax (Δε) 222 (-5.5), 235 (- N N H N N H OH O 3.5), 270 (+0.3) nm; UV (MeOH) λmax (logε) 227 (4.3), 284 (3.5), 290 (3.4) nm; IR (film) νmax 3398, 2960, 1682, 1650, 1480 cm-1; 1H (600 MHz) and 13C (150 MHz) NMR data are summarized in Table 6.2; 172 (+) HRESIMS m/z 525.3418 ([M+H]+) (calcd 525.3435, Δ -3.3 ppm) Hemiasterlin D (30) Colourless amorphous solid; [α]24D -52.3 (c 0.02, O O N N H HN OH (3.3) nm; IR (film) νmax 3410, 2965, 1689, 1659, O N O HO MeOH); UV (MeOH) λmax (logε) 225 (4.3), 270 1418 cm-1; 1H (600 MHz) and O HN 13 C (150 MHz) OH N O NMR data are summarized in Table 6.4; (+) HRESIMS m/z 853.5470 ([M+H]+) (calcd 853.5434, Δ 4.2 ppm) Peptide Hydrolysis Peptide samples (150μg) were dissolved in degassed 6N HCl (500μl) and heated at 120oC for 8h The solvent was removed under dry nitrogen and the resulting material was subjected to further derivatization for stereochemical assignment LC/MS Analysis of Marfey’s Derivatives The hydrolysate mixture or the amino acid standard was added a solution of LFDAA 1% (w/w) in acetone and 100μl of a 1N NaHCO3 solution The vial was heated at 50oC for 3h and the contents were neutralized with 0.2N HCl (50μl) after cooling to room temperature An aliquot of the L-FDAA derivative was dried under dry nitrogen, diluted in DMSO and loaded on a Phenomenex Luna column (C18, 3μm, 2.0 mm x 150 mm) using a linear gradient from 100% water (0.1% formic acid) to 100% acetonitrile (0.1% formic acid) in 50 minutes and an isocratic at 100% acetonitrile (0.1% formic acid) in the next ten minutes FDAA derivatives were detected by absorption at 340nm and assignment was secured by ion-selective monitoring Retention times of authentic FDAA-amino acids are given in parenthesis: L-tertLeu (33.49) and D-tert-Leu (35.12) The hydrolysate of hemiasterlin D (1) and milnamide E (2) contained L-tert-Leu eluted at 33.54 and 33.51 min, respectively 173 Appendix Chapter 7: Experimental Purealidin L (134) Light yellow amorphous powder; (+) LRESIMS m/z 494, OCH3 Br Br 496, 498 (1:2:1); 1H, HO O N H C-NMR data and optical rotation value were identical with those reported in the literature NH H N N 13 NH2 (reference 27 in chapter 7) O N,N,N-trimethyl-3,5-dibromotyrosine (139) Light yellow amorphous powder; (+) LRESIMS m/z 336, 338, 340 (1:2:1); OH Br Br H and 13 C-NMR data were identical with those reported in the literature (reference 74 in chapter 7) N Purealidin O (140) Light yellow amorphous powder; (+) LRESIMS m/z 494, OCH3 Br Br HO O 496, 498 (1:2:1); 1H and H N NH2 N H N 13 C-NMR data were identical with those reported in the literature (reference 27 in chapter 7) NH HO Aerophobins (141) Yellow amorphous powder; (+) LRESIMS m/z 504, 506, 508 OCH3 Br Br NH2 HO O N H N N NH (1:2:1); 1H, 13 C-NMR data and optical rotation value were identical with those reported in the literature (reference 62 in chapter 7) O Aplysinamisine (142) Light yellow amorphous powder; (+) LRESIMS m/z OCH3 Br Br 508, 510, 512 (1:2:1); 1H, 13 C-NMR data and optical HO O H N N O H N NH2 NH rotation value were identical with those reported in the literature (reference 75 in chapter 7) 174 11,19-dideoxyfistularin (143) Br Light yellow amorphous powder; OCH3 (-) LRESIMS m/z 1080 (cluster); Br Br H3CO H N Br O O OH N O Br O OH N H N H, 13 C-NMR data and optical rotation value were identical with O Br those reported in the literature (reference 29 in chapter 7) 11-hydroxyaerothionin (144) Light yellow amorphous powder; (-) LRESIMS OCH3 Br m/z 830, 832, 834, 836, 838; 1H, 13C-NMR data Br HO O N O OH and optical rotation value were identical with Br O H N those reported in the literature (reference 29 in O N H N O HO chapter 7) Br Aerothionin (145) Light yellow amorphous powder; (-) LRESIMS OCH3 Br m/z 814, 816, 818, 820, 822; 1H, 13C-NMR data Br Br HO and optical rotation value were identical with O O H N N O those reported in the literature (reference 76 in N H O N O Br chapter 7) HO Homoaerothionin (146) Light yellow amorphous powder; (-) LRESIMS OCH3 OCH3 Br Br Br HO O m/z 828, 830, 832, 834, 836; 1H, OH and optical rotation value were identical with O H N H N N Br N C-NMR data those reported in the literature (reference 76 in O O 13 chapter 7) Fistularin (147) Br OCH3 Br Br OH H3CO H N Br O O OH N O Br O OH N N H LRESIMS m/z 1114 (cluster); 1H, 13 C-NMR data and optical rotation value were identical with those O OH Light yellow amorphous powder; (-) Br 175 reported in the literature (reference 77 in chapter 7) Pseudoceralidinone A (148) Colourless amorphous solid; [α]25D +3.5 (c 0.1, MeOH); UV Br N O Br (MeOH) λmax (logε) 257 (3.4), 362 (2.7) nm; IR (film) νmax NH 3444, 1748, 1682, 1203 cm-1; 1H (600 MHz) and O O 13 C (150 MHz) NMR data are summarized in Table 7.3; (+) HRESIMS m/z 420.9746 (calcd (+) m/z 420.9757, Δ -2.6 ppm) Aplysamine (149) Colourless amorphous solid; [α]24D +8.1 (c Br N 0.08, MeOH); UV (MeOH) λmax (logε) 282 O O Br Br (3.5) nm; IR (film) νmax 3405, 1673, 1204 cm- O N H OH N OH ; 1H (600 MHz) and 13 C (150 MHz) NMR data are summarized in Table 2; (+) HRESIMS m/z 663.9662 (calcd (+)-m/z 663.9652, Δ 1.5 ppm) 3-methylmaleimide-5-oxime (150) Light yellow amorphous powder; (+) LRESIMS m/z 126; 1H and N N H O OH 13 C- NMR data were identical with those reported in the literature (reference 79 in chapter 7) 5-[3,5-dibromo-4-[(2-oxo-5-oxazolidinyl)methoxy]phenyl]-2-oxazolidinone (151) HN Light yellow amorphous powder; (+) LRESIMS m/z 435, Br O O 437, 439 (1:2:1); 1H, 13C-NMR data and optical rotation value O Br NH O were identical with those reported in the literature (reference O 41 in chapter 7) Fistularin (152) Light yellow amorphous powder; (+) LRESIMS m/z OCH3 Br Br 772, 774, 776, 778, 780; 1H, HO O C-NMR data and optical rotation value were identical with those OH H N N 13 Br reported in the literature (reference 77 in chapter 7) O O Br NH O O 176 (3,5-dibromo-2-hydroxy-4-methoxyphenyl)-acetic acid (153) OCH3 Br Br Colourless amorphous powder; (-) LRESIMS m/z 320, 322, 324, 326, 328; OH H and 13 C-NMR data were identical with those reported in the literature (reference 12 in chapter 7) O Hydrolysis and Boc-protection of Pseudoceralidinone A A solution of 148 (7.0 mg, 0.016 mmol) in HCl 6N (1.5 mL) was heated at 140oC in 10 under microwave irradiation The reaction mass was dried in vacuo before it was basified by NaOH 10% (1 mL) and added Boc2O (5 mg, 0.02 mmol) with stirring at r.t in 30 The solvent was removed in vacuo and the residue was subjected on RP-HPLC column to give 154 (3.8 mg, 46% yield in two steps) Compound 154: colourless amorphous solid; [α]25D +3.1 (c 0.1, MeOH); 1H-NMR (600MHz, DMSO-d6) δ 7.51 (2H, s), 6.67 (1H, t, J=5.4 Hz), 4.54 (1H, t, J=6.0 Hz), 3.95 (2H, t, J=6.6 Hz), 3.08 (2H, m), 2.41 (2H, t, J=6.6 Hz), 2.14 (6H, s), 1.91 (2H, m), 1.32 (9H, s); 13C-NMR (150MHz, DMSO-d6) δ 155.7 (C), 151.5 (C), 143.2 (C), 130.3 (2 x CH), 117.3 (2 x C), 77.7 (C), 71.5 (CH2), 69.8 (CH), 55.4 (CH2), 47.0 (CH2), 44.9 (2 x N-CH3), 27.8 (3 x CH3), 27.4 (CH2); (+)-LRESIMS m/z 495.0, 497.0, 499.0 Preparation of MTPA Esters of 154 (155 and 156) (R) or (S)-MTPA-Cl (2μL, 0.01mmol) was added to 154 (0.5mg, 0.001mmol) in anhydrous pyridine (150μL) and stirred at room temperature Reactions were monitored by LC/MS and stopped after 24h An aliquot was then dried under dry nitrogen The residue was partitioned with the solvent system 1:1 (H2O-CH2Cl2) The CH2Cl2 fraction was evaporated to dryness to yield the Mosher ester The 1H and COSY NMR were performed on the Mosher esters (155 and 156) to obtain the δ S and δ R values, which were used to determined the absolute stereochemistry at C-5 (S)-MTPA ester of pseudoceralidinone A (155): 1H-NMR (500MHz, DMSO-d6) δ 7.62 (2H, s, H-7 and H-11), 7.49 (2H, m, MTPA-ArH), 7.46 (3H, m, MTPA-ArH), 7.13 (1H, t, J=5.5 Hz, NH-3), 5.96 (1H, t, J=6.0 Hz, H-5), 4.01 (2H, t, J=6.0 Hz, H-13), 3.49 (3H, s, MTPA-OCH3), 3.45 (1H, m, H-4b), 3.32 (1H, m, H-4a), 3.31 (2H, m, H-15), 2.79 (6H, s, x N-CH3), 2.24 (2H, m, H-14), 1.32 (9H, s, Boc-H); (+)-LRESIMS m/z 711.1, 713.1, 715.1 177 (R)-MTPA ester of pseudoceralidinone A (156): 1H-NMR (500MHz, DMSO-d6) δ 7.63 (2H, s, H-7 and H-11), 7.50 (2H, m, MTPA-ArH), 7.46 (3H, m, MTPA-ArH), 7.12 (1H, t, J=5.5 Hz, NH-3), 5.95 (1H, t, J=6.0 Hz, H-5), 4.01 (2H, t, J=6.0 Hz, H-13), 3.49 (3H, s, MTPA-OCH3), 3.44 (1H, m, H-4b), 3.30 (1H, m, H-4a), 3.33 (2H, m, H-15), 2.83 (6H, s, x N-CH3), 2.24 (2H, m, H-14), 1.32 (9H, s, Boc-H); (+)-LRESIMS m/z 711.1, 713.1, 715.1 3-bromo-O-methyltyrosine (158): To a cooled solution (5oC) of O-methyl-L-tyrosine (157, 261mg, 1.3 mmol) in glacial acetic acid (8.0 mL, 0.14 mol), Br2 (0.12 mL, 2.3 mmol) was added and stirred for 3h at r.t After completion of the reaction, the reaction was quenched with saturated Na2S2O3 solution and the solvent was removed under vacuum pressure The reaction mass was then extracted with ethyl acetate The organic layer was dried prior to being purified by RP-HPLC column to give 158 as a colorless amorphous solid (285 mg, 80%); IR (film) νmax 3411, 1625, 1255, 1024 cm-1, ; 1H-NMR (600MHz, DMSO-d6) δ 8.31 (3H, br.s), 7.47 (1H, d, J=1.8 Hz), 7.23 (1H, dd, J=9.0, 1.8 Hz), 7.07 (1H, d, J=8.4 Hz), 4.17 (1H, br.s), 3.83 (3H, s), 3.05 (2H, m); 13 C-NMR (150MHz, DMSO-d6) δ 170.3 (C), 154.7 (C), 133.8 (CH), 130.1 (CH), 128.5 (C), 112.7 (CH), 110.6 (C), 56.2 (OCH3), 53.1 (CH), 34.5 (CH2); (+)-LRESIMS m/z 274.0, 276.0 3-bromo-4-methoxyphenylpyruvic acid (159): A solution of 158 (161 mg, 0.6 mmol) in (CF3CO)2O (3.0 mL, 21.2 mmol) was heated at 90oC for 18h The solvent was removed under reduced pressure The residue was again dissolved in 70% aqueous TFA and allowed to stand 16h at r.t The product was chromatographed on RP-HPLC column to yield 159 (74 mg, 46%); IR (film) νmax 3410, 1650, 1254, 1054 cm-1; 1H-NMR (600MHz, DMSO-d6) δ 9.28 (1H, br.s), 8.10 (1H, d, J=1.8 Hz), 7.66 (1H, dd, J=8.4, 1.8 Hz), 7.10 (1H, d, J=8.4 Hz), 3.85 (3H, s); 13C-NMR (150MHz, DMSO-d6) δ 166.2 (C), 154.3 (C), 141.1 (C), 133.2 (CH), 130.2 (CH), 129.2 (C), 112.5 (CH), 110.5 (C), 108.2 (CH), 56.2 (OCH3); (-)-LRESIMS m/z 271.0, 273.0 2-(benzyloxyimino)-3-(3-bromo-4-methoxyphenyl)propanoic acid (160): To a sulution of 159 (33 mg, 0.12 mmol) in EtOH (2 mL), O-benxylhydroxylamine (56 mg, 0.35 mmol) was added and refluxed for 4h The crude product was purified by RPHPLC to afford 160 (29 mg, 64%); IR (film) νmax 3420, 2940, 1625, 1599, 1541, 1397, 178 1255, 1022, 806 cm -1; 1H-NMR (600MHz, DMSO-d6) δ 7.42 (1H, d, J=1.8 Hz); 7.32 (2H, d, J=7.8 Hz), 7.29 (1H, d, J=7.2 Hz), 7.25 (2H, d, J=7.2 Hz), 7.19 (1H, dd, J=8.4, 1.8 Hz), 6.96 (1H, d, J=8.4 Hz), 5.05 (2H, s), 3.79 (3H, s), 3.72 (2H, s); 13 C-NMR (150MHz, DMSO-d6) δ 164.7 (C), 158.2 (C), 153.5 (C), 138.2 (C), 133.1 (CH), 131.5 (C), 129.6 (CH), 128.2 (2 x CH), 127.54 (2 x CH), 127.50 (CH), 112.3 (CH), 110.0 (C), 74.8 (CH2), 56.2 (OCH3), 30.8 (CH2); (+)-LRESIMS m/z 378.0, 380.0 tert-butyl-2-(3,5-dibromo-4-hydroxyphenyl)-2-hydroxyethylcarbamate (162): To a solution of racemic octopamine (384 mg, 2.0 mmol) in distilled water (3 mL), 6N HCl (3 mL) was added and the mixture was cooled to 5oC Bromine (0.35 mL, 6.8 mmol) was then injected into the stirred solution The reaction mass was dried in vacuo before it was basified by NaOH 10% (5 mL) and added Boc2O (500 mg, 2.2 mmol) with stirring at r.t in 30 The solvent was removed in vacuo and the residue was subjected on RP-HPLC column to give 162 (654 mg, 80% yield in two steps); IR (film) νmax 3411, 2977, 2932, 1693, 1468, 1250, 1170, 1055 cm-1; 1H-NMR (600MHz, DMSO-d6) δ 7.10 (2H, s), 6.64 (1H, t, J=5.4 Hz), 4.29 (1H, dd, J=7.2, 5.4 Hz), 3.00 (1H, m), 2.92 (1H, m), 1.36 (9H, s); 13C-NMR (150MHz, DMSO-d6) δ 160.8 (C), 155.7 (C), 128.8 (2 x CH), 123.9 (C), 114.4 (2 x C), 77.5 (C), 70.7 (CH), 48.3 (CH2), 28.3 (3 x CH3); (+)-LRESIMS m/z 410.0, 412.0, 414.0 tert-butyl-2-(3,5-dibromo-4-(3-(dimethylamino)propoxy)phenyl)-2-hydroxyethyl carbamate (163): To a mixture of compound 162 (327 mg, 0.8 mmol), K2CO3 (0.90 g, 6.5 mmol) and KI (1.08 g, 6.5 mmol) in dry 50% acetone – 50 % acetonitrile (5.0 mL), 3-dimethylamino-1-propyl chloride hydrochloride (406 mg, 2.5 mmol) was added and refluxed for 16h The solvent was evaporated in vacuo and extracted with ethyl acetate The organic layer was concentrated and purified by RP-HPLC column to obtain a racemate of 163 (198 mg, 50%); IR (film) νmax 3336, 2974, 2823, 1698, 1456, 1253, 1168, 1042 cm-1; 1H-NMR (500MHz, DMSO-d6) δ 7.52 (2H, s), 6.76 (1H, br.s), 4.55 (1H, t, J=6.0 Hz), 3.95 (2H, t, J=6.5 Hz), 4.00 (2H, t, J=6.5 Hz), 2.41 (2H, t, J=7.0 Hz), 2.14 (6H, s), 1.91 (2H, m), 1.32 (9H, s); 13 C-NMR (125MHz, DMSO-d6) δ 155.5 (C), 151.3 (C), 142.7 (C), 130.4 (2 x CH), 117.0 (2 x C), 77.6 (C), 71.7 (CH2), 70.0 (CH), 55.6 (CH2), 47.3 (CH2), 45.1 (2 x N-CH3), 28.1 (3 x CH3), 27.7 (CH2); (+)-LRESIMS m/z 495.0, 497.0, 499.0 179 2-amino-1-(3,5-dibromo-4-(3-(dimethylamino)propoxy)phenyl)ethanol (164): Compound 164 (158 mg, 0.3 mmol) was taken in mL of 50% DCM – 50% TFA and stirred for 30 After the solvent was removed, the crude product was purified by RP-HPLC column to get free amine 164 (114 mg, 90%); IR (film) νmax 3408, 3033, 2739, 1676, 1202, 1133 cm-1; 1H-NMR (600MHz, DMSO-d6) δ 8.10 (2H, br.s), 7.68 (2H, s), 4.82 (1H, dd, J=9.0, 3.0 Hz), 4.00 (2H, t, J=6.6 Hz), 3.36 (2H, t, J=7.2 Hz), 3.10 (1H, d, J=12.0 Hz), 2.89 (1H, t, J=11.6 Hz), 2.84 (6H, s), 2.19 (2H, m); 13C-NMR (150MHz, DMSO-d6) δ 151.4 (C), 141.4 (C), 130.5 (2 x CH), 117.6 (2 x C), 70.3 (CH2), 67.6 (CH), 54.3 (CH2), 45.1 (CH2), 42.3 (2 x N-CH3), 24.8 (CH2); (+)-LRESIMS m/z 395.0, 397.0, 399.0 2-(benzyloxyimino)-3-(3-bromo-4-methoxyphenyl)-N-(2-(3,5-dibromo-4-(3(dimethylamino)propoxy)phenyl)-2-hydroxyethyl)propanamide (165): To a solution of 164 (30 mg, 0.076 mmol) in dry dimethylformamide (3.5 mL), Nhydroxybenzotriazole (HOBt, 20 mg, 0.15 mmol) was added and the reaction mixture was stirred for 15 at r.t The reaction mixture was then cooled to 0oC and N-(3Dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI, 28.7 mg, 0.15 mmol) was added and stirring continued for 30 at 0oC To this mixture, compound 160 (28 mg, 0.076 mmol) was then added and stirred for 2h at r.t The crude product was concentrated in vacuo and separated on RP-HPLC column (MeOH, H2O, 0.1% TFA) to get a racemic mixture of 165 (34 mg, 60%); IR (film) νmax 3395, 2940, 1673, 1496, 1204 cm-1; 1HNMR (600MHz, DMSO-d6) δ 9.47 (1H, br.s), 7.96 (1H, t, J=6.0 Hz), 7.57 (2H, s), 7.36 (3H, m), 7.33 (2H, d, J=7.2 Hz), 7.08 (1H, d, J=8.4 Hz), 6.97 (1H, d, J=8.4 Hz), 5.25 (2H, s), 4.67 (1H, t, J=6.0 Hz), 3.98 (2H, t, J=6.0 Hz), 3.80 (3H, s), 3.72 (2H, s), 3.36 (1H, m), 3.35 (2H, m), 3.27 (1H, m), 2.84 (6H, d, J=4.2 Hz), 2.16 (2H, m); 13C-NMR (150MHz, DMSO-d6) δ 162.2 (C), 154.0 (C), 152.2 (C), 150.8 (C), 143.1 (C), 136.8 (C), 133.1 (CH), 130.4 (2 x CH), 129.6 (C), 129.2 (CH), 128.5 (2 x CH), 128.1 (CH), 128.0 (2 x CH), 117.2 (2 x C), 112.6 (CH), 110.3 (CH), 76.6 (CH2), 70.2 (CH2), 69.4 (CH), 56.2 (OCH3), 54.4 (CH2), 46.3 (CH2), 42.4 (N-CH3), 28.6 (CH2), 24.8 (CH2); (+)LRESIMS m/z 754.0, 756.0, 758.0, 760.0 Isolation of two enantiomers in a racemic oxime-protected aplysamin (165): Compound 165 was further purified by HPLC on a chiral HPLC column (Phenomenex, Lux 5μm, Amylose-2, 250 x 4.6 mm) with an isocratic condition of 30% acetonitrile 180 (0.1% TFA)-70% water (0.1% TFA) in 15 minutes, flow rate 0.8 ml/min Two enantiomers (165a and 165b) were eluted at 7.5 and 9.8 min, respectively Compound 165a: colourless amorphous solid; [α]24D +5.2 (c 0.08, MeOH); 1H-NMR (600MHz, DMSO-d6) δ 9.47 (1H, br.s), 7.96 (1H, t, J=6.0 Hz), 7.57 (2H, s), 7.36 (3H, m), 7.33 (2H, d, J=7.2 Hz), 7.08 (1H, d, J=8.4 Hz), 6.97 (1H, d, J=8.4 Hz), 5.25 (2H, s), 4.67 (1H, t, J=6.0 Hz), 3.98 (2H, t, J=6.0 Hz), 3.80 (3H, s), 3.72 (2H, s), 3.36 (1H, m), 3.35 (2H, m), 3.27 (1H, m), 2.84 (6H, d, J=4.2 Hz), 2.16 (2H, m); (+)-LRESIMS m/z 754.0, 756.0, 758.0, 760.0 Compound 165b: colourless amorphous solid; [α]24D -6.7 (c 0.08, MeOH); 1H-NMR (600MHz, DMSO-d6) δ 9.47 (1H, br.s), 7.96 (1H, t, J=6.0 Hz), 7.57 (2H, s), 7.36 (3H, m), 7.33 (2H, d, J=7.2 Hz), 7.08 (1H, d, J=8.4 Hz), 6.97 (1H, d, J=8.4 Hz), 5.25 (2H, s), 4.67 (1H, t, J=6.0 Hz), 3.98 (2H, t, J=6.0 Hz), 3.80 (3H, s), 3.72 (2H, s), 3.36 (1H, m), 3.35 (2H, m), 3.27 (1H, m), 2.84 (6H, d, J=4.2 Hz), 2.16 (2H, m); (+)-LRESIMS m/z 754.0, 756.0, 758.0, 760.0 Preparation of MTPA Esters of 165a (166a and 167a) (R) or (S)-MTPA-Cl (2μL, 0.01mmol) was added to 165a (0.5mg, 0.001mmol) in anhydrous pyridine (150μL) and stirred at room temperature Reactions were monitored by LC/MS and stopped after 24h An aliquot was then dried under dry nitrogen The residue was partitioned with the solvent system 1:1 (H2O-CH2Cl2) The CH2Cl2 fraction was evaporated to dryness to yield the Mosher ester The 1H and COSY NMR were performed on the Mosher esters (166a and 167a) to obtain the δ S and δ R values, which were used to determined the absolute stereochemistry at C-5 (S)-MTPA ester of 165a (166a): 1H-NMR (600MHz, DMSO-d6) δ 7.99 (1H, t, J=6.0 Hz, NH-10), 7.541 (2H, s, H-14 and H-18), 5.259 (2H, s, H-24), 4.715 (1H, t, J=6.0 Hz, H-12), 3.26 (1H, m, H-11), 2.79 (6H, s, N-CH3); (+)-LRESIMS m/z 970.0, 972.0, 974.0, 976.0 (R)-MTPA ester of 165a (167a): 1H-NMR (600MHz, DMSO-d6) δ 7.97 (1H, t, J=6.0 Hz, NH-10), 7.551 (2H, s, H-14 and H-18), 5.254 (2H, s, H-24), 4.710 (1H, t, J=6.0 Hz, 181 H-12), 3.25 (1H, m, H-11), 2.80 (6H, s, N-CH3) ; (+)-LRESIMS m/z 970.0, 972.0, 974.0, 976.0 Preparation of MTPA Esters of 165b (166b and 167b) Carried out as same procedure with MTPA Esters of 165a (S)-MTPA ester of 165b (166b): 1H-NMR (600MHz, DMSO-d6) δ 7.97 (1H, t, J=6.0 Hz, NH-10), 7.543 (2H, s, H-14 and H-18), 5.257 (2H, s, H-24), 4.71 (1H, t, J=6.0 Hz, H-12), 3.24 (1H, m, H-11), 2.80 (6H, s, N-CH3) ; (+)-LRESIMS m/z 970.0, 972.0, 974.0, 976.0 (R)-MTPA ester of 165b (167b): 1H-NMR (600MHz, DMSO-d6) δ 7.98 (1H, t, J=6.0 Hz, NH-10), 7.535 (2H, s, H-14 and H-18), 5.261 (2H, s, H-24), 4.71 (1H, t, J=6.0 Hz, H-12), 3.25 (1H, m, H-11), 2.79 (6H, s, N-CH3) ; (+)-LRESIMS m/z 970.0, 972.0, 974.0, 976.0 182 .. .Isolation and Structure Elucidation of Cytotoxic Natural Products with Potential Anticancer Activity Trong Duc Tran (B.Eng) Eskitis Institute for Cell and Molecular Therapies... contribution of natural products to drug discovery 1.2 Natural products as potential anticancer agents 1.3 The application of “drug-like” properties into natural product discovery 1.3.1 The decline of. .. Details are discussed in chapter Aim 2: Isolation and structure elucidation of active components and their analogues Large scale isolation and structure elucidation are performed for biota samples