HHS Public Access Author manuscript Author Manuscript
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15. Published in final edited form as: Bioorg Med Chem Lett. 2016 January 15; 26(2): 499–504. doi:10.1016/j.bmcl.2015.11.086.
Bromotyrosine-derived Metabolites from an Indonesian Marine Sponge in the Family Aplysinellidae (Order Verongiida) Jingqiu Daia, Stephen M. Parrisha, Wesley Y. Yoshidaa, M. L. Richard Yipb, James Turksonb, Michelle Kellyc, and Philip Williamsa,b a
Author Manuscript
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, 96822 b University of Hawaii Cancer Center, 701 Ilalo Street, Honolulu, Hawaii, 96813 c National Coasts and Oceans Centre, National Institute of Water and Atmospheric Research, Auckland, New Zealand
Abstract Seven new bromotyrosine-derived metabolites, purpuramine M-N (1-2), araplysillin VII-XI (3-7) and six known compounds (8-13) were isolated from an Indonesian sponge belonging to the family Aplysinellidae (Order Verongiida). The structures of the new compounds were determined by extensive NMR experiments and mass spectrometric measurements. These compounds were screened against BACE1 and five cancer cell lines.
Graphical Abstract Author Manuscript Keywords Aplysinellidae; Verongiida; bromotyrosine-derivatives; BACE1 inhibitors; Cell viability
Author Manuscript
Beginning with the work of Morner in 1914 on the skeletal structure of Anthozoa,1 marine organisms and in particular sponges of the Order Verongiida Bergquist, 1978, including
Correspondence Prof. Dr. Philip Williams, Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA, 96822. [email protected] Phone: 808 956 5720 Fax: 808 956 5908. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Supporting Information Available: Copies of the spectroscopic data for all new compounds associated with this article are available as Supporting Information. Conflict of Interest The authors declare no conflict of interest.
Dai et al.
Page 2
Author Manuscript Author Manuscript
Verongula Verrill, Pseudoceratina Carter, Ianthella Gray and Psammaplysilla Keller (now Pseudoceratina), have been intensively studied due to the presence of alkaloids with one or more bromotyrosine residues.2,3 Despite many variations, their structural core generally consists of three building blocks: tyramine, 3-aminopropanol (presumed to be derived from homoserine), and tyrosine. The latter unit is often modified via oxidation of the arene group to form either a spirocyclohexadienyl or a spirooxapenylisoxazole moiety. With these relatively simple building blocks structures have been constructed that display biological activities ranging from antibiotic4 to cytotoxic.5 During our search for biologically active marine natural products for the treatment of neurological disorders and cancers, an extract derived from a sponge from the family Aplysinellidae (Order Verongiida) 6 was identified in our Alzheimer's disease screen as active, and effected cell viability in our counter-screen. Bioassay-guided fractionation of this extract has now led to the isolation of seven new bromotyrosine-derived metabolites, purpuramine M-N (1-2) and araplysillin VII-XI (3-7), in addition to six known compounds. In this paper, we describe that research and the biological activity of these new compounds against BACE1 and five cancer cell lines.
Author Manuscript
The methanolic extract of the sponge was separated by silica flash chromatography and RPHPLC to obtain purpuramine M-N (1-2) and araplysillin VII-XI (3-7) in yields ranging from 0.008 to 0.083%.7 Based on analysis of the spectroscopic and spectrometric data six known compounds were also identified: hexadellin A (8),8 araplysillin II (9),9 araplysillin IV (10),10 purpurealidin I (11),11 aplysamine 4 (12),12 purpuramine G (13).13
Author Manuscript
Purpuramine M (1)14 was isolated as a white amorphous solid that had a molecular formula of C21H2379Br4N3O5 on the basis of its HR ESIMS spectrum. Further support for this molecular formula was obtained from the observed isotope pattern that was consistent with the incorporation of four bromine atoms; a 1:4:6:4:1 ratio at m/z 713, 715, 717, 719, and 721. Analysis of the IR spectrum revealed vibrations indicative of an amide carbonyl (1653 cm−1), hydroxy, and NH (3391 cm−1) groups, while a UV absorption at λmax 280 nm suggested a chromophore based on a substituted benzenoid system. Based on analysis of the 13C NMR spectrum of 1, 14 of the 21 carbons present (Table 1) in the molecule were sp2
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 3
Author Manuscript
hybridized and could be attributed to at least six carbon-carbon and two carbon-heteroatom double bonds. Analysis of the 1H NMR spectroscopic data (Table 2) of 1, in conjunction with the degree of halogenation, revealed several signals suggesting 1 belonged to the purpuramine structural class.11 In particular, signals consistent with 3-amino-1-hydroxypropyl (δH-10 3.57, δH-11 2.09, and δH-12 4.01), and 2-aminoethyl (δH-19 2.85 and δH-20 3.09) moieties, in addition to a symmetrically substituted aromatic ring (δH-15/-17 7.48), were diagnostic of this structural class (Fig. 1). Detailed analyses of the 2D NMR data confirmed the order of connectivity followed the (“tyrosine”)-(3-aminopropanol)-(tyramine) pattern found in purpuramines A-C as opposed to the isomeric order (“tyrosine”)-(tyramine)-(3-aminopropanol) found in purpuramines D-E.13
Author Manuscript Author Manuscript
The final structural constraints required by the molecular formula primarily concerned the placement of heteroatoms. Two of the four bromines were easily assigned to a symmetrical tyramine unit, based on the characteristic singlet observed in the 1H NMR spectrum at δH-15/-17 7.48 as noted above. The nitrogen in the unit derived from tyrosine was present as an oxime, with the more common E-configuration, on the basis of the distinctive carbon chemical shift of C-7 at 25.5 ppm2. HMBC correlations from H-7 of this unit to the quaternary carbons at δC-1 123.0 and δC-2 155.0, and to the methine resonating at δC-6 134.5, indicated that 1 was a rare example of a purpuramine derivative containing oxygenation at C-2. In most cases, both C-6 and C-2 are methine groups, with purealidins M, N, and O being the notable exceptions.15 Oxygenation at this position is typically only found in the spirocycohexadienylisoxazoline derivatives such as the fistularins16, leading to the proposal that a C-2/C-6 oxirane17 is an intermediate in the formation of the spirocyclic system via in the intramolecular cyclization of the hydroxyl group of an oxime with an unstable arene oxide intermediate (Fig. 2). Oxygenation at this position, without cyclization, is therefore interesting. A comparison of the 13C NMR shifts for this ring with purealidin O15 established the substitution pattern of heteroatoms as depicted.
Author Manuscript
The molecular formula of compound 218 was assigned as C22H7979Br4N5O5 based on the HR ESIMS ion at m/z 755.8655 ([M+H]+, Δ -1.5 ppm), and on the basis of the NMR spectroscopic data was clearly structurally related to 1. Comparison of the molecular formulae indicated two additional nitrogens and hydrogens, along with one carbon (δC-21 163.9) indicative of a guanidino moiety. In 2, H-20 was shifted downfield from 3.09 to 3.43 ppm in a manner consistent with the attachment of a guanidino group at the N-terminus of the tyramine unit, and a daughter ion (m/z 713) attributed to the loss of CN2H2 (Fig. 3) was also observed by tandem MS further supporting the proposed structural difference. Therefore, compound 2 was deduced to be purpuramine N (2), which is the first example of guanylation at the N-terminus of the tyramine unit in this class of brominated tyrosine derivatives. Compound 319 was obtained as an optically active amorphous solid ([α]D22 -87.5 (c 0.2, MeOH)), which displayed an isotopic cluster in a ratio of 1:4:6:4:1 consistent with the presence of four bromine atoms. Accurate mass measurement of these ions confirmed a formula of C22H25Br4N5O5 suggesting 3 and 2 were structurally related. The UV spectrum
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 4
Author Manuscript
of 3 was significantly different from 2, suggesting that 3 belonged to either the spirocyclohexadienyl or spirooxapenyl family of brominated metabolites. This conclusion was supported by analysis of the 13C NMR spectrum which showed ten sp2 carbons rather than the 12 sp2 carbons found in 2, and by signals indicative of an oxygenated methine δH-1 4.08 and an AB spin system (δH-7 3.77 and 3.09, J = 18.3 Hz) that were comparable to the 1H NMR data for hexadellin A (8). Thus 3 was the spirocyclohexadienylisooxazoline derivative of 2, possessing the same terminal guanidino functionality. The configuration of this unit was determined by analysis of the 1H NMR data, in which the chemical shifts of H-1 (δH-1 4.08) and H-7 (δH-7 3.09 and 3.77) were indicative of a trans orientation of these stereogenic centers (trans: δH-1 4.2, δH-7 3.1/3.8 vs. cis: δH-1 4.5, δH-7 3.4).20
Author Manuscript Author Manuscript
Araplysillin VIII (4)21 was the largest compounds isolated with a molecular formula of C30H2979Br6N3O8. Given six bromines were present in 4 we initially suspected it might belong to the fistularin or bastadin16 structural classes of polybrominated derivatives, but signals for only one spirocyclohexadienylisooxazole unit were apparent in the NMR thus ruling out this possibility. For example, only one oxygenated methine signal was observed at δH-1 4.08. Three additional signals in the 1H NMR spectrum at δH-22 3.48, δH-28 7.25 and δ 3.81 provided the key HMBC correlations needed to assemble this final unit. Specifically, HMBC correlations were observed from the methylene proton H-22 to the amide C-21, the quaternary sp2 C-23, the oxygenated sp2 C-24, and the sp2 methine carbon C-28. Additionally HMBC correlations observed from H-28 to the quaternary carbons C-24, C-26, and C-27 indicated a substituted phenylacetic acid unit. Finally, HMBC correlations from the remaining methoxy group at δ 3.81 to C-26 established its location, and thus indicated the remaining bromine atoms were attached to C-27 and C-25. Thus, compound 4 appears to be a desguanidino-hybrid between the core of 3 and the known aromatic bromotyrosine derivative 2-hydroxy-3,5-dibromo-4-methoxyphenylacetamide; aeroplysinin I.23 The capping of the traditional core with this latter unit is one of the more unusual aspects of 4 as this modification has not been previously reported for this series. Compound 524 provided MS and NMR data similar to 3 indicative of spirocyclohexadienylisoxazoline and 3-aminopropanol units. No resonances consistent with the ethylamine unit were detected though, and, instead, the 13C NMR spectrum of 5 showed one more carbonyl resonance (δC-19 170.3) that showed an HMBC correlation to the methine protons H-15/H-17 of the aromatic ring indicating that the final structure of 5 was as depicted.
Author Manuscript
Analysis of the APCIMS and NMR data of compound 625 (Tables 3 and 4) indicated it was an acyl derivative of hexadellin A (8).8,9 The presence of two methyl signals (δH 0.84 (t, J = 6.3); δH 0.86 (d, J = 6.6)) suggested derivatization with a branched acyl chain terminating in a methyl group. The alkyl chain of compound 6 was characterized in the same manner as for compound 10; the C-atoms were assigned from comparison to literature, HMBC, COSY, and NMR spectra simulation which suggested C-29 was the branch point. This conclusion was supported by MS2 analysis which provide intense fragments ions at m/z 85 and 514/516/518.
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 5
Author Manuscript
Compound 726 exhibited the same characteristic features as 6 except for a disubstituted olefin (δH 5.32, 2H), implying the presence of an unsaturated acyl chain. The position of the double bond was revealed from daughter ions observed in ESI-MS-MS for fragment ion (m/z 641), corresponding to A (Fig. 4). The intense fragment ions at m/z 488/490/492 indicated that the double bond was located at C-28 (29), while the intense ions observed at m/z 85, 57 and 556/558/560 indicated that the branching Me is on C-33. The Z-configuration of the olefin was deduced from the chemical shift (δC 28.1) of the allylic carbons (C-27 and C-30).15 Thus, the structure of 7 was elucidated as veronidin G (7).
Author Manuscript
Compound 1127 was obtained as a colorless powder and displayed ESIMS and NMR data very similar to 1, suggesting a linear (“tyrosine”)-(3-aminopropanol)-(tyramine) pattern incorporating four bromine atoms. The most obvious difference between the two structures in the 1H NMR data was the addition of one N-methyl group (δH 2.70, 3H) and a sp2hybridized methine H-6 (δH 7,42, 2H) suggesting an N-methyl derivative of 1. However, a thorough analysis of the 2D NMR data indicated 11 more closely resembled aplysamine 4 (12),12 following the (“tyrosine”)-(tyramine)-(3-aminopropanol) pattern, differing only in Nmethylation at C-20. The E-geometry of the oxime was assigned based on the chemical shift of the C-7 (δ 28.8) as the (Z)-oxime is found at >35 ppm.20 We note Tilvi et al11 have previously suggested the planar structure of 11, and named it purpurealidin I, through comparative MS2 analyses of Psammaplysilla purpurea (now Pseudoceratina purpurea (Carter, 1880)) extracts.
Author Manuscript
New compounds 3-7 all possess a spirocycohexadienylisoxazoline core containing two stereogenic centers. In all cases a large negative specific rotation was obtained for the compound consisted with that observed for the 1S,6R configuration of this unit.28 Thus, this configuration is proposed for compounds 3-5. It should be noted that acetylation of the C-6 hydroyxl group appears to reverse the sign, and there is one report of a positive rotation (Aplysinamisine I-III).29 While also recorded in MeOH, the experimental concentrations reported in that manuscript (c = 5.7-7.9) were much higher than typical for this series suggesting intermolecular interactions may explain the difference. Compounds 6 and 7 contain an additional stereogenic center in the lipophilic side chain which potentially complicated the analysis. In general though methyl branched fatty acids display small specific rotations, which decrease as the branch point is moved to the middle of the chain.30 Therefore, based on Van't Hoff's rule of optical superposition, the contribution of the sidechain to the overal specific rotation will be mininal for 6 and 7. Thus, while the configuration of the sidechain remains unassigned, the configuration of spirocycohexadienylisoxazoline cores in 6 and 7 is proposed as 1S,6R as well.
Author Manuscript
In biological screening, 1,3, 5-9, 11-13 showed moderate inhibition of the aspartic protease, BACE1 (memapsin-2), which has a central role in the etiology of Alzheimer's disease. Compounds 2 and 4 were excluded due to concerns about their purity and the quantity that was available after repurification of the NMR samples. Compounds were tested at 30 μg/mL once in triplicate (Table 5) using secretase inhibitor IV from Calbiochem as a positive control (>95% purity by LC-MS), with 6 and 11 the most active.31 The relative inactivity activity of 1 in comparison to 11 is interesting as, with the exception of an additional
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 6
Author Manuscript
hydroxy group, these compounds are essentially isomeric hinting at important structure activity relationships.
Author Manuscript
The isolated compounds were also tested for differential cell viability using the CyQuant proliferation assay. Due to the limited supply of these pure compounds, for cell-based studies we chose to study the biological activities through a two-step process. Initially, the compounds were tested at 50 and 5 μM in five cancer cell lines (ovarian cancer A2780S and cisplatin-resistant variant A2780CP (SCP5), non-small cell lung cancer A549, human breast cancer MCF7 and glioma U251MG cells) and a control cell line (normal mouse fibroblasts, NIH3T3). Table 6 shows the results of the initial screen at 50 μM (triplicates, n=1) at 72h relative to the DMSO control. Five compounds (3, 5, 8, 11 and 12) inhibited the growth of the control cell line and thus were not considered further for differential screening. Of the remaining compounds, five of them (1, 2, 4, 9 and 13) inhibited the growth of some of the cancer cell lines more than 50% at this concentration with little effect on the control cell line. Based on these results and on compound availability, we further examined the biological activities of 1, 2 and 13, in three of the cancer cell lines. Cancer cells were treated with varying doses (maximum 50 μM) of these compounds and compared to cells treated with the solvent alone. IC50 values were then determined (triplicates, n=2) for each of the three compounds and summarized in Table 6. Compound 1 inhibited the growth of A2780S, A2780SCP5, and U251MG with IC50 values equal to 20 μM, 40 μM, and 50 μM, respectively. Compound 2, which is related to 1 with the addition of a guanidino moiety, has reduced biological activities towards the three cancer cell lines (i.e. IC50 >50 μM) which suggests the N-terminus of the tyramine is important for its anti-tumor activity.
Author Manuscript
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgment This work was supported by NIH grant 5R01AG039468 (PW), NIH/NCI grant CA161931 (JT) and University of Hawaii start-up funds (JT). Funds for the upgrades of the NMR instrumentation were provided by the CRIF program of the National Science Foundation (CH E9974921), the Elsa Pardee Foundation, and the University of Hawaii at Manoa. The purchase of the Agilent LC-MS was funded by grant W911NF-04-1-0344 from the Department of Defense.
References Author Manuscript
1. Morner CTZ. Phys. Chem. 1914; 88:138. 2. Peng J, Li J, Hamann MT. Alkaloids Chem. Biol. 2005; 61:59. [PubMed: 16173400] 3. Gotsbacher MP, Karuso P. Mar. Drugs. 2015; 13:1389. [PubMed: 25786066] 4. Rotem M, Carmely S, Kashman Y, Loya Y. Tetrahedron. 1983; 39:667. 5. Rodríguez AD, Piña IC. J. Nat. Prod. 1993; 56:907. [PubMed: 8350091] 6. Sponge Material: The sponge was collected from Manta Point, Sangalaki, Indonesia, on 22 March 1996, at a depth of 21.3 m. The sponge formed a thick to massive encrustation with a softly scalloped and conulose skin-like surface. The surface was lightly encrusted in places with algae, ascidians, and other sessile invertebrates. The color in life was cream with rose pick tinges. The texture was fleshy, rubbery and elastic. The color in preservative was dark nut brown. Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 7
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Histologically, the mesohyl was dense and collagenous with a distinctive cortex delineated lightly by sand particles. The fibres were contorted, knotted, sparse, and set well apart in the choanosome. Fibres were dominated by a thick irregular golden bark surrounding a small granular pith. The sponge is comparable to Aplysinella strongylata Bergquist, 1980 within Family Aplysinellidae, Order Verongida, but it differs in numerous ways: 1) the presence of sandy detritus throughout the sponge; 2) oxidation from creamy pink to a nut brown colouration upon exposure to air; 3) the possession of contort, knotted fibres dominated by bark that surrounds a light granular pith cored with sandy detritus. This material represents a new, undescribed genus within the family Aplysinellidae (Order Verongida) The sponge is thus identified as Aplysinellidae gen. nov. et sp. nov. (Order Verongida, Family Aplysinellidae). A voucher specimen has been deposited at the Natural Museum, London (NHMUK2012.3.27.4). 7. Extraction and Isolation: The freeze-dried sponge (100.0 g) was extracted with MeOH (5 × 1 L) at room temperature to afford 6.0 g of lipophilic extract. The extract was subjected to silica flash chromatography eluting with CH2Cl2 and increasing amounts of MeOH to afford 10 fractions. The residue of fraction 6 (45.0 mg) was further purified by RP-HPLC (Luna C8, 250 × 10 mm, a linear gradient from 10-100% MeCN in water over 40 min and then 100% MeCN over 20 min) to afford 4 (tR 37.4 min, 0.9 mg, 0.015% yield). Fraction 7 (20.0 mg) was separated by RP-HPLC (Jupiter C18, 250 × 10 mm, a linear gradient from 10-100% MeCN in water over 40 min) to afford 3 (tR 29.5 min, 1.1 mg, 0.018% yield) and 2 (tR 33.5 min, 0.5 mg , 0.008% yield). Separation of fraction 8 (35.0 mg) by RP HPLC (Luna C8, 250 × 10 mm, a linear gradient from 10-100% MeCN in water over 40 min) afforded 1 (tR 19.5 min, 1.9 mg, 0.032% yield) and 5 (tR 29.5 min, 0.7 mg, 0.011% yield). The residue of fraction 9 (60.0 mg) was further purified by RP-HPLC (Luna C8, 250 × 10 mm, a linear gradient from 10-100% MeCN in water over 40 min and 100% MeCN over 20 min) afforded 9 (tR 47.6 min, 2.0 mg, 0.033% yield), 6 (tR 49.5 min, 1.5 mg, 0.025% yield), 10 (tR 51.5 min, 1.2 mg, 0.020% yield), 7 (tR 52.0 min, 1.0 mg, 0.016% yield). Fraction 10 (20.0 mg) was separated by RP-HPLC (Jupiter C18, 250 × 10 mm, a linear gradient from 10-100% MeCN in water over 40 min) afforded 13 (tR 12.5 min, 1.1 mg, 0.018 % yield), 8 (tR 15.0 min, 5.0 mg, 0.083% yield), 11 (tR 18.5 min, 0.5 mg, 0.008% yield) and 12 (tR 19.0 min, 2.0 mg, 0.033% yield). 8. Morris SA, Andersen RJ. Can. J. Chem. 1989; 67:677. 9. Longeon A, Guyot M, Vacelet J. Experientia. 1990; 46:548. 10. Mani L, Jullian V, Mourkazel B, Valentin A, Dubois J, Cresteil T, Folcher E, Hooper JNA, Erpenbeck D, Aalbersberg W, Debitus C. Chem. Biodiversity. 2012; 9:1436. 11. Tilvi S, Rodrigues C, Naik CG, Parameswaran PS, Wahidhulla S. Tetrahedron. 2004; 60:10207. 12. Jurek J, Yoshida WY, Scheuer PJ, Kelly-Borges M. J. Nat. Prod. 1993; 56:1609. 13. Yagi H, Matsunaga S, Fusetani N. Tetrahedron. 1993; 49:3749. 14. Purpuramine M (1): white amorphous solid; UV (MeOH) λmax (log ε) 280 (3.50) nm; IR (CaF2) ν −1 max 3391, 2928, 2870, 1653, 1595, 1460 cm ; See Tables 1 and 2 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 713.8448 [M + H]+ (calcd for C21H 79 24 Br4N3O5 713.8448). 15. Kobayashi JI, Honma K, Sasaki T, Tsuda M. Chem. Pharm. Bull. 1995; 43:403. 16. Gopichand Y, Schmitz FJ. Tetrahedron Lett. 1979; 20:3921. 17. James DM, Kunze HB, Faulkner DJ. J. Nat. Prod. 1991; 54:1137. [PubMed: 1791478] 18. Purpuramine N (2): white amorphous solid; UV (MeOH) λmax (log ε) 280 (3.36) nm; IR (CaF2) νmax 3362, 2928, 2847, 1651, 1558, 1456 cm−1; See Tables 1 and 2 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 755.8655 [M ]+ (calcd for C22H 79 26 Br4N5O5 755.8666). 19. Araplysillin VII (3): white amorphous solid; [α]D22 −87.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 282 (3.56), 218 (3.19) nm; IR (CaF2) νmax 3339, 2924, 2848, 1652, 1582, 1459 cm−1; See Tables 1 and 2 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 755.8637 [M]+ (calcd for C22H2679 Br4N5O5 755.8666). 20. Arabshahi L, Schmitz FJ. J. Org. Chem. 1987; 52:3584. 21. Araplysillin VIII (4): white amorphous solid; [α]D22 −25.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 280 (3.98) nm; IR (CaF2) νmax 3320, 2923, 2848, 1650, 1557, 1452 cm−1; See Tables 1 and 2 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 1033.7133 [M + H]+ (calcd for C30H3079 Br6N3O8 1033.7131).
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 8
Author Manuscript Author Manuscript
22. Greve H, Kehraus S, Krick A, Kelter G, Maier A, Fiebig H-H, Wright AD, König GM. J. Nat. Prod. 2008; 71:309. [PubMed: 18298075] 23. Fattorusso E, Minale L, Sodano G. J. Chem. Soc., Perkin Trans. 1. 1972; 1:16. 24. Araplysillin IX (5): white amorphous solid; [α]D22 −75.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 281 (3.90), 218 (3.39) nm; IR (CaF2) νmax 3384, 2954, 2919, 1660, 1589, 1377 cm−1; See Tables 1 and 2 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 714.7920 [M + H]+ (calcd for C20H 79 19 Br4N2O7 714.7924). 25. Araplysillin X (6): colorless oil; [α]D22 −42.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 282 (4.0), 218 (3.38) nm; IR (CaF2) νmax 3319, 2918, 2853, 1714, 1649, 1460 cm−1; See Tables 3 and 4 for tabulated NMR spectral data (MeOH-d4); HR APCIMS m/z 952.0775 [M + H]+ (calcd for C37H 79 54 Br4N3O6 952.0746). 26. Araplysillin XI (7): colorless oil; [α]D22 −33.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 282 (3.9), 218 (3.38) nm; IR (CaF2) νmax 3319, 2923, 2848, 1650, 1557 cm−1; See Tables 3 and 4 for tabulated NMR spectral data (MeOH-d 4); HR APCIMS m/z 978.0916 [M + H]+ (calcd for C39H56Br4N3O6 978.0901). 27. Purpurealidin I (11): white amorphous solid; [α]D22 −2.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 280 (3.36) nm; IR (CaF2) νmax 2922, 2814, 1650, 1583, 1468 cm−1; See Tables 3 and 4 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 711.8645 [M + H]+ (calcd for C22H26Br4N3O4 711.8655). 28. Mani L, Jullian V, Mourkazel B, Valentin A, Dubois J, Cresteil T, Folcher E, Hooper JNA, Erpenbeck D, Aalbersberg W, Debitus C. Chem Biodivers. 2012; 9:1436. [PubMed: 22899605] 29. Rodriguez AD, Piña IC. J. Nat. Prod. 1993; 56:907. [PubMed: 8350091] 30. Bello JE, McElfresh JS, Millar JG. Proc. Natl. Acad. Sci. U.S.A. 2015; 112:1077. [PubMed: 25583471] 31. Naqvi T. J. Biomol. Screen. 2004; 9:398. [PubMed: 15296639]
Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 9
Author Manuscript
Fig. 1.
Fragments of compound 1 and key interfragment HMBC correlations.
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 10
Author Manuscript
Fig. 2.
Proposed biosynthetic pathway from the prearaplysillins to the araplysillins.
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 11
Author Manuscript Fig. 3.
Fragmentation of compound 2 that establish the guanidino group.
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 12
Author Manuscript Fig. 4.
Fragmentation of fragment A
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 13
Table 1
Author Manuscript
13C
NMR Spectroscopic Data (125 MHz, δc) for Compounds 1-5.
Author Manuscript Author Manuscript
1 δc, type
2 δc, type
3 δc, type
4 δc, type
5 δc, type
1
123.0, qC
122.5, qC
75.5, CH
75.5, CH
75.5, CH
2
155.0, qC
154.5, qC
114.2, qC
113.3, qC
114.2, qC
3
108.9, qC
108.9, qC
149.3, qC
149.3, qC
149.3, qC
4
151.7, qC
151.6, qC
122.7, qC
122.8, qC
122.5, qC
5
106.9, qC
106.8, qC
132.2, CH
131.3, CH
132.3, qC
6
134.4, CH
134.6, CH
92.4, qC
92.4, qC
92.4, qC
7
25.5, CH2
25.5, CH2
40.1, CH2
40.2, CH2
40.2, CH2
8
154.8, qC
154.4, qC
155.3, qC
155.1, qC
155.3, qC
9
166.8, qC
166.5, qC
161.6, qC
161.6, qC
161.6, qC
10
37.9, CH2
38.1, CH2
38.0, CH2
38.0, CH2
37.9, CH2
11
30.5, CH2
30.5, CH2
30.6, CH2
30.5, CH2
30.6, CH2
12
72.1, CH2
72.2, CH2
72.1, CH2
72.1, CH2
72.2, CH2
13
153.4, qC
153.6, qC
152.8, qC
152.7, qC
155.7, qC
14/18
119.4, qC
119.0, qC
119.0, qC
118.9, qC
118.4, qC
15/17
134.3, CH
134.3, CH
134.3, CH
134.3, CH
134.9, CH
16
137.7, qC
139.8, qC
139.7, qC
139.8, qC
138.0, qC
19
34.1, CH2
35.0, CH2
35.0, CH2
35.0, CH2
170.3, qC
20
41.8, CH2
40.1, CH2
40.0, CH2
41.5, CH2
---
21
---
163.9, qC
163.9, qC
175.0, qC
---
22
---
---
---
39.7, CH2
---
23
---
---
---
123.0, qC
---
24
---
---
---
156.0, qC
---
25
---
---
---
108.7, qC
---
26
---
---
---
154.2, qC
---
27
---
---
---
106.0, qC
---
28
---
---
---
132.3, CH
---
OCH3
60.8, CH3
60.8, CH3
60.4, CH3
60.4, CH3
---
26-OCH3
---
---
---
60.8, CH3
---
Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 14
Table 2
Author Manuscript
1H
NMR Spectroscopic Data (500 MHz in MeOH-d4) for Compounds 1-5.
Author Manuscript
1 δH, (J in Hz)
2 δH, (J in Hz)
3 δH, (J in Hz)
4 δH, (J in Hz)
5 δH, (J in Hz)
1
---
---
4.08, s
4.08, s
4.09, s
5
---
---
6.41, s
6.42, s
6.41, s
6
7.40, s
7.41, s
---
---
---
7
3.80, s
3.80, s
3.09, d (18.3)
3.10, d (18.3)
3.10, d (18.3)
3.77, d (18.3)
3.77, d (18.3)
3.77, d (18.3)
10
3.57, t (6.7)
3.58, t (6.6)
3.58, t (7.0)
3.58, t (7.0)
3.59, t (7.0)
11
2.09, tt (6.7, 6.0)
2.09, tt (6.6, 6.0)
2.10, tt (7.0, 6.0)
2.09, tt (7.0, 6.1)
2.12, tt (7.0, 6.1)
12
4.01, t (6.0)
4.02, t (6.0)
4.04, t (6.0)
3.99, t (6.1)
4.09, t (6.1)
15/17
7.48, s
7.45, s
7.47, s
7.38, s
8.10, s
19
2.85, t (7.4)
2.75, t (7.0)
2.75, t (7.0)
2.73, t (6.9)
---
20
3.09, t (7.4)
3.43, t (7.0)
3.42, t (7.0)
3.39, t (6.9)
---
22
---
---
---
3.48, s
---
28
---
---
---
7.25, s
---
OCH3
3.78, s
3.79, s
3.71, s
3.72, s
3.72, s
26-OCH3
---
---
---
3.81, s
---
Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 15
Table 3
Author Manuscript
1H
NMR Spectroscopic Data (500 MHz, δH (J in Hz)) for Compounds 11, 6 and 7 11
6
7
1
---
1
4.08, s
1
4.07, s
2
7.42, s
2
---
2
---
3
---
3
---
3
---
4
---
4
---
4
---
5
---
5
6.40, s
5
6.41, s
6
7.42, s
6
---
6
---
7
3.80, s
7
3.08, d (18.1)
7
3.09, d (18.3)
3.78, d (18.1)
3.77, d (18.3)
Author Manuscript Author Manuscript
8
---
8
---
8
---
9
---
9
---
9
---
10
3.43, t (7.1)
10
3.58, t (6.9)
10
3.58, t (7.0)
11
2.75, t (7.1)
11
2.12, tt (6.9, 6.0)
11
2.10, tt (7.0, 6.0)
12
---
12
4.04, t (6.0)
12
4.04, t (6.0)
13
7.47, s
13
---
13
---
15
---
15
7.44, s
15
7.44, s
16
---
16
---
16
---
17
7.47, s
17
7.44, s
17
7.44, s
18
4.04, t (5.7)
18
---
18
---
19
2.18, tt (7.5, 5.7)
19
2.73, t (6.9)
19
2.73, t (6.7)
20
3.26, t (7.5)
20
3.37, t (6.9)
20
3.37, t (6.7)
---
22
2.07-2.16, m
22
2.07-2.16, m
---
23
1.50-1.57, m
23
1.49-1.57, m
---
24
1.20-1.29, m
24
1.20-1.29, m
---
25
1.22-1.38, m
25
1.29-1.38, m
---
26
1.22-1.38, m
26
1.29-1.38, m
---
27
1.22-1.38, m
27
1.99-2.05, m
---
28
1.28-1.39, m
28
5.32, m
1.05-1.17, m
29
5.32, m
-----
29
1.21-1.35, m
30
1.99-2.05, m
---
30
1.05-1.17, m
31
1.29-1.38, m
1.28-1.39, m
32
1.10-1.18, m
---
Author Manuscript
---
31
1.28-1.39, m
1.20-1.29, m
---
32
1.28-1.39, m
33
1.21-1.37, m
---
33
1.28-1.39, m
34
1.10-1.18, m
---
34
1.33-1.40, m
---
35
0.84, t (6.3)
35
1.20-1.29, m
---
36
1.20-1.29, m
0.86, d (6.6)
36
1.32-1.40, m
---
---
37
0.88, t (6.8)
---
---
38
0.85, d (6.6)
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 16
Author Manuscript
11
6
7
NCH3
2.70, s
---
---
OCH3
3.78, s
OCH3
3.72, s
OCH3
3.72, s
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 17
Table 4
Author Manuscript
13C
NMR Spectroscopic Data (125 MHz, δc) for Compounds 11, 6 and 7.
Author Manuscript Author Manuscript Author Manuscript
11 δc, type
6 δc, type
7 δc, type
1
137.4, qC
75.5, CH
75.5, CH
2
134.4, CH
114.2, qC
114.2, qC
3
118.6, qC
149.3, qC
149.3, qC
4
152.0, qC
122.8, qC
122.8, qC
5
118.6, qC
132.2, CH
132.2, CH
6
134.4, CH
92.4, qC
92.4, qC
7
28.8, CH2
40.3, CH2
40.1, CH2
8
153.8, qC
155.2, qC
155.2, qC
9
165.4, qC
161.5, qC
161.5, qC
10
41.4, CH2
38.0, CH2
38.0, CH2
11
35.1, CH2
30.5, CH2
30.5, CH2
12
140.2, qC
72.1, CH2
72.1, CH2
13
134.5, CH
152.8, qC
152.8, qC
14
118.7, qC
118.9, qC
118.9, qC
15
152.2, qC
134.4, CH
134.4, CH
16
118.7, qC
140.0, qC
140.0, qC
17
134.5, CH
134.4, CH
134.4, CH
18
71.7, CH2
118.9, qC
118.9, qC
19
28.1, CH2
35.1, CH2
35.1, CH2
20
48.5, CH2
41.2, CH2
41.2, CH2
21
---
176.4, qC
176.4, qC
22
---
37.2, CH2
37.2, CH2
23
---
26.4, CH2
26.9, CH2
24
---
30.3-31.1, CH2
30.2-31.1, CH2
25
---
30.3-31.1, CH2
30.2-31.1, CH2
26
---
30.3-31.1, CH2
30.2-31.1, CH2
27
---
28.2, CH2
28.1, CH2
28
---
38.2, CH2
130.5, CH
29
---
33.1, CH2
130.5, CH
30
---
38.2, CH2
28.1, CH2
31
---
28.2, CH2
27.1, CH2
32
---
30.3-31.1, CH2
38.2, CH2
33
---
33.9, CH2
33.9, CH
34
---
23.8, CH2
38.2, CH2
35
---
14.5, CH2
30.2-31.1, CH2
36
---
20.1, CH3
23.4, CH2
37
---
---
14.4, CH3
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 18
Author Manuscript
11 δc, type
6 δc, type
7 δc, type
38
---
---
20.1, CH3
OCH3
61.0, CH3
60.4, CH3
60.4, CH3
NCH3
34.1, CH3
---
---
Some 13C chemical shift determined from HMBC experiment.
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 19
Table 5
Author Manuscript
BACE1 Inhibition Comp.
Author Manuscript
Name
μM
% Inhibition
1
Purpuramine M
42.0
36
3
Araplysillin VII
39.6
40
5
Araplysillin IX
41.9
35
6
Araplysillin X
31.4
70
7
Araplysillin XI
30.6
60
8
Hexadellin A
41.7
27
9
Araplysillin II
31.8
57
11
Purpurealidin I
42.0
74
12
Aplysamine 4
42.6
62
13
Purpuramine G
48.3
48
Secretase Inhibitor IV
0.021
80
Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 20
Table 6
Author Manuscript
Effects of compounds on growth of tumor versus NIH3T3 cells a NIH 3T3
Comp.
A2780S
A2780S CP5
U251 MG
A549
MCF7
Observed growth at 50 μM relative to DMSO Control Observed growth at 5 μM relative to DMSO Control
Author Manuscript Author Manuscript
1
104 ± 4 102 ± 4
9±1 60 ± 23
9±2 96 ± 29
20 ± 7 111 ± 5
21 ± 0 96 ± 2
27 ± 3 97 ± 18
3
25 ± 2 100 ± 7
12 ± 1 78 ± 23
8±0 45 ± 9
32 ± 1 94 ± 1
48 ± 6 85 ± 34
24 ± 6 98 ± 6
5
29 ± 1 94 ± 7
21 ± 2 37 ± 11
25 ± 2 33 ± 1
39 ± 15 101 ± 2
68 ± 10 81 ± 10
60 ± 2 86 ± 16
6
95 ± 4 94 ± 6
87 ± 7 85 ± 5
79 ± 27 109 ± 12
97 ± 2 99 ± 2
105 ± 2 89 ± 25
101 ± 2 104 ± 4
7
98 ± 3 100 ± 2
76 ± 18 82 ± 8
66 ± 3 105 ± 12
90 ± 2 98 ± 2
104 ± 2 98 ± 10
104± 2 105± 4
8
7±0 38 ± 14
14 ± 1 8±0
22 ± 1 11 ± 2
30 ± 5 32 ± 1
17 ± 1 16 ± 11
71 ± 5 36 ± 4
9
95 ± 4 88 ± 4
18 ± 1 77 ± 21
48 ± 8 68 ± 15
80 ± 1 98 ± 0
85 ± 10 88 ± 30
64 ± 8 103 ± 3
10
102 ± 8 98 ± 5
88 ± 4 90 ± 1
78 ± 11 100 ± 4
101 ± 3 100 ± 2
106 ± 4 82 ± 33
109 ± 1 106 ± 1
11
3±0 101 ± 5
13 ± 2 11 ± 2
10 ± 1 29 ± 4
30 ± 10 17 ± 1
11 ± 1 42 ± 7
58 ± 1 50 ± 12
12
16 ± 2 105 ± 4
11 ± 2 81 ± 24
12 ± 3 78 ± 26
24 ± 3 107 ± 2
24 ± 6 92 ± 9
26 ± 1 95 ± 15
13
105 ± 3 99 ± 3
26 ± 7 82 ± 26
51 ± 4 94 ± 41
40 ± 16 111 ± 4
64 ± 14 97 ± 4
69 ± 4 100 ± 10
46 ± 1 46 ± 2
23 ± 2 35 ± 8
18 ± 3 33 ± 4
20 ± 2 21 ± 1
51 ± 15 49 ± 7
52 ± 6 46 ± 4
b
SAHA
c
A2780S
A2780S CP5
U251 MG
IC50 μM
IC50 μM
IC50 μM
20
40
50
>50
>50
>50
Etoposide a
Fibroblast cell line.
b
25 μM
c
20 μM
Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15. Published in final edited form as: Bioorg Med Chem Lett. 2016 January 15; 26(2): 499–504. doi:10.1016/j.bmcl.2015.11.086.
Bromotyrosine-derived Metabolites from an Indonesian Marine Sponge in the Family Aplysinellidae (Order Verongiida) Jingqiu Daia, Stephen M. Parrisha, Wesley Y. Yoshidaa, M. L. Richard Yipb, James Turksonb, Michelle Kellyc, and Philip Williamsa,b a
Author Manuscript
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, 96822 b University of Hawaii Cancer Center, 701 Ilalo Street, Honolulu, Hawaii, 96813 c National Coasts and Oceans Centre, National Institute of Water and Atmospheric Research, Auckland, New Zealand
Abstract Seven new bromotyrosine-derived metabolites, purpuramine M-N (1-2), araplysillin VII-XI (3-7) and six known compounds (8-13) were isolated from an Indonesian sponge belonging to the family Aplysinellidae (Order Verongiida). The structures of the new compounds were determined by extensive NMR experiments and mass spectrometric measurements. These compounds were screened against BACE1 and five cancer cell lines.
Graphical Abstract Author Manuscript Keywords Aplysinellidae; Verongiida; bromotyrosine-derivatives; BACE1 inhibitors; Cell viability
Author Manuscript
Beginning with the work of Morner in 1914 on the skeletal structure of Anthozoa,1 marine organisms and in particular sponges of the Order Verongiida Bergquist, 1978, including
Correspondence Prof. Dr. Philip Williams, Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA, 96822. [email protected] Phone: 808 956 5720 Fax: 808 956 5908. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Supporting Information Available: Copies of the spectroscopic data for all new compounds associated with this article are available as Supporting Information. Conflict of Interest The authors declare no conflict of interest.
Dai et al.
Page 2
Author Manuscript Author Manuscript
Verongula Verrill, Pseudoceratina Carter, Ianthella Gray and Psammaplysilla Keller (now Pseudoceratina), have been intensively studied due to the presence of alkaloids with one or more bromotyrosine residues.2,3 Despite many variations, their structural core generally consists of three building blocks: tyramine, 3-aminopropanol (presumed to be derived from homoserine), and tyrosine. The latter unit is often modified via oxidation of the arene group to form either a spirocyclohexadienyl or a spirooxapenylisoxazole moiety. With these relatively simple building blocks structures have been constructed that display biological activities ranging from antibiotic4 to cytotoxic.5 During our search for biologically active marine natural products for the treatment of neurological disorders and cancers, an extract derived from a sponge from the family Aplysinellidae (Order Verongiida) 6 was identified in our Alzheimer's disease screen as active, and effected cell viability in our counter-screen. Bioassay-guided fractionation of this extract has now led to the isolation of seven new bromotyrosine-derived metabolites, purpuramine M-N (1-2) and araplysillin VII-XI (3-7), in addition to six known compounds. In this paper, we describe that research and the biological activity of these new compounds against BACE1 and five cancer cell lines.
Author Manuscript
The methanolic extract of the sponge was separated by silica flash chromatography and RPHPLC to obtain purpuramine M-N (1-2) and araplysillin VII-XI (3-7) in yields ranging from 0.008 to 0.083%.7 Based on analysis of the spectroscopic and spectrometric data six known compounds were also identified: hexadellin A (8),8 araplysillin II (9),9 araplysillin IV (10),10 purpurealidin I (11),11 aplysamine 4 (12),12 purpuramine G (13).13
Author Manuscript
Purpuramine M (1)14 was isolated as a white amorphous solid that had a molecular formula of C21H2379Br4N3O5 on the basis of its HR ESIMS spectrum. Further support for this molecular formula was obtained from the observed isotope pattern that was consistent with the incorporation of four bromine atoms; a 1:4:6:4:1 ratio at m/z 713, 715, 717, 719, and 721. Analysis of the IR spectrum revealed vibrations indicative of an amide carbonyl (1653 cm−1), hydroxy, and NH (3391 cm−1) groups, while a UV absorption at λmax 280 nm suggested a chromophore based on a substituted benzenoid system. Based on analysis of the 13C NMR spectrum of 1, 14 of the 21 carbons present (Table 1) in the molecule were sp2
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 3
Author Manuscript
hybridized and could be attributed to at least six carbon-carbon and two carbon-heteroatom double bonds. Analysis of the 1H NMR spectroscopic data (Table 2) of 1, in conjunction with the degree of halogenation, revealed several signals suggesting 1 belonged to the purpuramine structural class.11 In particular, signals consistent with 3-amino-1-hydroxypropyl (δH-10 3.57, δH-11 2.09, and δH-12 4.01), and 2-aminoethyl (δH-19 2.85 and δH-20 3.09) moieties, in addition to a symmetrically substituted aromatic ring (δH-15/-17 7.48), were diagnostic of this structural class (Fig. 1). Detailed analyses of the 2D NMR data confirmed the order of connectivity followed the (“tyrosine”)-(3-aminopropanol)-(tyramine) pattern found in purpuramines A-C as opposed to the isomeric order (“tyrosine”)-(tyramine)-(3-aminopropanol) found in purpuramines D-E.13
Author Manuscript Author Manuscript
The final structural constraints required by the molecular formula primarily concerned the placement of heteroatoms. Two of the four bromines were easily assigned to a symmetrical tyramine unit, based on the characteristic singlet observed in the 1H NMR spectrum at δH-15/-17 7.48 as noted above. The nitrogen in the unit derived from tyrosine was present as an oxime, with the more common E-configuration, on the basis of the distinctive carbon chemical shift of C-7 at 25.5 ppm2. HMBC correlations from H-7 of this unit to the quaternary carbons at δC-1 123.0 and δC-2 155.0, and to the methine resonating at δC-6 134.5, indicated that 1 was a rare example of a purpuramine derivative containing oxygenation at C-2. In most cases, both C-6 and C-2 are methine groups, with purealidins M, N, and O being the notable exceptions.15 Oxygenation at this position is typically only found in the spirocycohexadienylisoxazoline derivatives such as the fistularins16, leading to the proposal that a C-2/C-6 oxirane17 is an intermediate in the formation of the spirocyclic system via in the intramolecular cyclization of the hydroxyl group of an oxime with an unstable arene oxide intermediate (Fig. 2). Oxygenation at this position, without cyclization, is therefore interesting. A comparison of the 13C NMR shifts for this ring with purealidin O15 established the substitution pattern of heteroatoms as depicted.
Author Manuscript
The molecular formula of compound 218 was assigned as C22H7979Br4N5O5 based on the HR ESIMS ion at m/z 755.8655 ([M+H]+, Δ -1.5 ppm), and on the basis of the NMR spectroscopic data was clearly structurally related to 1. Comparison of the molecular formulae indicated two additional nitrogens and hydrogens, along with one carbon (δC-21 163.9) indicative of a guanidino moiety. In 2, H-20 was shifted downfield from 3.09 to 3.43 ppm in a manner consistent with the attachment of a guanidino group at the N-terminus of the tyramine unit, and a daughter ion (m/z 713) attributed to the loss of CN2H2 (Fig. 3) was also observed by tandem MS further supporting the proposed structural difference. Therefore, compound 2 was deduced to be purpuramine N (2), which is the first example of guanylation at the N-terminus of the tyramine unit in this class of brominated tyrosine derivatives. Compound 319 was obtained as an optically active amorphous solid ([α]D22 -87.5 (c 0.2, MeOH)), which displayed an isotopic cluster in a ratio of 1:4:6:4:1 consistent with the presence of four bromine atoms. Accurate mass measurement of these ions confirmed a formula of C22H25Br4N5O5 suggesting 3 and 2 were structurally related. The UV spectrum
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 4
Author Manuscript
of 3 was significantly different from 2, suggesting that 3 belonged to either the spirocyclohexadienyl or spirooxapenyl family of brominated metabolites. This conclusion was supported by analysis of the 13C NMR spectrum which showed ten sp2 carbons rather than the 12 sp2 carbons found in 2, and by signals indicative of an oxygenated methine δH-1 4.08 and an AB spin system (δH-7 3.77 and 3.09, J = 18.3 Hz) that were comparable to the 1H NMR data for hexadellin A (8). Thus 3 was the spirocyclohexadienylisooxazoline derivative of 2, possessing the same terminal guanidino functionality. The configuration of this unit was determined by analysis of the 1H NMR data, in which the chemical shifts of H-1 (δH-1 4.08) and H-7 (δH-7 3.09 and 3.77) were indicative of a trans orientation of these stereogenic centers (trans: δH-1 4.2, δH-7 3.1/3.8 vs. cis: δH-1 4.5, δH-7 3.4).20
Author Manuscript Author Manuscript
Araplysillin VIII (4)21 was the largest compounds isolated with a molecular formula of C30H2979Br6N3O8. Given six bromines were present in 4 we initially suspected it might belong to the fistularin or bastadin16 structural classes of polybrominated derivatives, but signals for only one spirocyclohexadienylisooxazole unit were apparent in the NMR thus ruling out this possibility. For example, only one oxygenated methine signal was observed at δH-1 4.08. Three additional signals in the 1H NMR spectrum at δH-22 3.48, δH-28 7.25 and δ 3.81 provided the key HMBC correlations needed to assemble this final unit. Specifically, HMBC correlations were observed from the methylene proton H-22 to the amide C-21, the quaternary sp2 C-23, the oxygenated sp2 C-24, and the sp2 methine carbon C-28. Additionally HMBC correlations observed from H-28 to the quaternary carbons C-24, C-26, and C-27 indicated a substituted phenylacetic acid unit. Finally, HMBC correlations from the remaining methoxy group at δ 3.81 to C-26 established its location, and thus indicated the remaining bromine atoms were attached to C-27 and C-25. Thus, compound 4 appears to be a desguanidino-hybrid between the core of 3 and the known aromatic bromotyrosine derivative 2-hydroxy-3,5-dibromo-4-methoxyphenylacetamide; aeroplysinin I.23 The capping of the traditional core with this latter unit is one of the more unusual aspects of 4 as this modification has not been previously reported for this series. Compound 524 provided MS and NMR data similar to 3 indicative of spirocyclohexadienylisoxazoline and 3-aminopropanol units. No resonances consistent with the ethylamine unit were detected though, and, instead, the 13C NMR spectrum of 5 showed one more carbonyl resonance (δC-19 170.3) that showed an HMBC correlation to the methine protons H-15/H-17 of the aromatic ring indicating that the final structure of 5 was as depicted.
Author Manuscript
Analysis of the APCIMS and NMR data of compound 625 (Tables 3 and 4) indicated it was an acyl derivative of hexadellin A (8).8,9 The presence of two methyl signals (δH 0.84 (t, J = 6.3); δH 0.86 (d, J = 6.6)) suggested derivatization with a branched acyl chain terminating in a methyl group. The alkyl chain of compound 6 was characterized in the same manner as for compound 10; the C-atoms were assigned from comparison to literature, HMBC, COSY, and NMR spectra simulation which suggested C-29 was the branch point. This conclusion was supported by MS2 analysis which provide intense fragments ions at m/z 85 and 514/516/518.
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 5
Author Manuscript
Compound 726 exhibited the same characteristic features as 6 except for a disubstituted olefin (δH 5.32, 2H), implying the presence of an unsaturated acyl chain. The position of the double bond was revealed from daughter ions observed in ESI-MS-MS for fragment ion (m/z 641), corresponding to A (Fig. 4). The intense fragment ions at m/z 488/490/492 indicated that the double bond was located at C-28 (29), while the intense ions observed at m/z 85, 57 and 556/558/560 indicated that the branching Me is on C-33. The Z-configuration of the olefin was deduced from the chemical shift (δC 28.1) of the allylic carbons (C-27 and C-30).15 Thus, the structure of 7 was elucidated as veronidin G (7).
Author Manuscript
Compound 1127 was obtained as a colorless powder and displayed ESIMS and NMR data very similar to 1, suggesting a linear (“tyrosine”)-(3-aminopropanol)-(tyramine) pattern incorporating four bromine atoms. The most obvious difference between the two structures in the 1H NMR data was the addition of one N-methyl group (δH 2.70, 3H) and a sp2hybridized methine H-6 (δH 7,42, 2H) suggesting an N-methyl derivative of 1. However, a thorough analysis of the 2D NMR data indicated 11 more closely resembled aplysamine 4 (12),12 following the (“tyrosine”)-(tyramine)-(3-aminopropanol) pattern, differing only in Nmethylation at C-20. The E-geometry of the oxime was assigned based on the chemical shift of the C-7 (δ 28.8) as the (Z)-oxime is found at >35 ppm.20 We note Tilvi et al11 have previously suggested the planar structure of 11, and named it purpurealidin I, through comparative MS2 analyses of Psammaplysilla purpurea (now Pseudoceratina purpurea (Carter, 1880)) extracts.
Author Manuscript
New compounds 3-7 all possess a spirocycohexadienylisoxazoline core containing two stereogenic centers. In all cases a large negative specific rotation was obtained for the compound consisted with that observed for the 1S,6R configuration of this unit.28 Thus, this configuration is proposed for compounds 3-5. It should be noted that acetylation of the C-6 hydroyxl group appears to reverse the sign, and there is one report of a positive rotation (Aplysinamisine I-III).29 While also recorded in MeOH, the experimental concentrations reported in that manuscript (c = 5.7-7.9) were much higher than typical for this series suggesting intermolecular interactions may explain the difference. Compounds 6 and 7 contain an additional stereogenic center in the lipophilic side chain which potentially complicated the analysis. In general though methyl branched fatty acids display small specific rotations, which decrease as the branch point is moved to the middle of the chain.30 Therefore, based on Van't Hoff's rule of optical superposition, the contribution of the sidechain to the overal specific rotation will be mininal for 6 and 7. Thus, while the configuration of the sidechain remains unassigned, the configuration of spirocycohexadienylisoxazoline cores in 6 and 7 is proposed as 1S,6R as well.
Author Manuscript
In biological screening, 1,3, 5-9, 11-13 showed moderate inhibition of the aspartic protease, BACE1 (memapsin-2), which has a central role in the etiology of Alzheimer's disease. Compounds 2 and 4 were excluded due to concerns about their purity and the quantity that was available after repurification of the NMR samples. Compounds were tested at 30 μg/mL once in triplicate (Table 5) using secretase inhibitor IV from Calbiochem as a positive control (>95% purity by LC-MS), with 6 and 11 the most active.31 The relative inactivity activity of 1 in comparison to 11 is interesting as, with the exception of an additional
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 6
Author Manuscript
hydroxy group, these compounds are essentially isomeric hinting at important structure activity relationships.
Author Manuscript
The isolated compounds were also tested for differential cell viability using the CyQuant proliferation assay. Due to the limited supply of these pure compounds, for cell-based studies we chose to study the biological activities through a two-step process. Initially, the compounds were tested at 50 and 5 μM in five cancer cell lines (ovarian cancer A2780S and cisplatin-resistant variant A2780CP (SCP5), non-small cell lung cancer A549, human breast cancer MCF7 and glioma U251MG cells) and a control cell line (normal mouse fibroblasts, NIH3T3). Table 6 shows the results of the initial screen at 50 μM (triplicates, n=1) at 72h relative to the DMSO control. Five compounds (3, 5, 8, 11 and 12) inhibited the growth of the control cell line and thus were not considered further for differential screening. Of the remaining compounds, five of them (1, 2, 4, 9 and 13) inhibited the growth of some of the cancer cell lines more than 50% at this concentration with little effect on the control cell line. Based on these results and on compound availability, we further examined the biological activities of 1, 2 and 13, in three of the cancer cell lines. Cancer cells were treated with varying doses (maximum 50 μM) of these compounds and compared to cells treated with the solvent alone. IC50 values were then determined (triplicates, n=2) for each of the three compounds and summarized in Table 6. Compound 1 inhibited the growth of A2780S, A2780SCP5, and U251MG with IC50 values equal to 20 μM, 40 μM, and 50 μM, respectively. Compound 2, which is related to 1 with the addition of a guanidino moiety, has reduced biological activities towards the three cancer cell lines (i.e. IC50 >50 μM) which suggests the N-terminus of the tyramine is important for its anti-tumor activity.
Author Manuscript
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgment This work was supported by NIH grant 5R01AG039468 (PW), NIH/NCI grant CA161931 (JT) and University of Hawaii start-up funds (JT). Funds for the upgrades of the NMR instrumentation were provided by the CRIF program of the National Science Foundation (CH E9974921), the Elsa Pardee Foundation, and the University of Hawaii at Manoa. The purchase of the Agilent LC-MS was funded by grant W911NF-04-1-0344 from the Department of Defense.
References Author Manuscript
1. Morner CTZ. Phys. Chem. 1914; 88:138. 2. Peng J, Li J, Hamann MT. Alkaloids Chem. Biol. 2005; 61:59. [PubMed: 16173400] 3. Gotsbacher MP, Karuso P. Mar. Drugs. 2015; 13:1389. [PubMed: 25786066] 4. Rotem M, Carmely S, Kashman Y, Loya Y. Tetrahedron. 1983; 39:667. 5. Rodríguez AD, Piña IC. J. Nat. Prod. 1993; 56:907. [PubMed: 8350091] 6. Sponge Material: The sponge was collected from Manta Point, Sangalaki, Indonesia, on 22 March 1996, at a depth of 21.3 m. The sponge formed a thick to massive encrustation with a softly scalloped and conulose skin-like surface. The surface was lightly encrusted in places with algae, ascidians, and other sessile invertebrates. The color in life was cream with rose pick tinges. The texture was fleshy, rubbery and elastic. The color in preservative was dark nut brown. Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 7
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Histologically, the mesohyl was dense and collagenous with a distinctive cortex delineated lightly by sand particles. The fibres were contorted, knotted, sparse, and set well apart in the choanosome. Fibres were dominated by a thick irregular golden bark surrounding a small granular pith. The sponge is comparable to Aplysinella strongylata Bergquist, 1980 within Family Aplysinellidae, Order Verongida, but it differs in numerous ways: 1) the presence of sandy detritus throughout the sponge; 2) oxidation from creamy pink to a nut brown colouration upon exposure to air; 3) the possession of contort, knotted fibres dominated by bark that surrounds a light granular pith cored with sandy detritus. This material represents a new, undescribed genus within the family Aplysinellidae (Order Verongida) The sponge is thus identified as Aplysinellidae gen. nov. et sp. nov. (Order Verongida, Family Aplysinellidae). A voucher specimen has been deposited at the Natural Museum, London (NHMUK2012.3.27.4). 7. Extraction and Isolation: The freeze-dried sponge (100.0 g) was extracted with MeOH (5 × 1 L) at room temperature to afford 6.0 g of lipophilic extract. The extract was subjected to silica flash chromatography eluting with CH2Cl2 and increasing amounts of MeOH to afford 10 fractions. The residue of fraction 6 (45.0 mg) was further purified by RP-HPLC (Luna C8, 250 × 10 mm, a linear gradient from 10-100% MeCN in water over 40 min and then 100% MeCN over 20 min) to afford 4 (tR 37.4 min, 0.9 mg, 0.015% yield). Fraction 7 (20.0 mg) was separated by RP-HPLC (Jupiter C18, 250 × 10 mm, a linear gradient from 10-100% MeCN in water over 40 min) to afford 3 (tR 29.5 min, 1.1 mg, 0.018% yield) and 2 (tR 33.5 min, 0.5 mg , 0.008% yield). Separation of fraction 8 (35.0 mg) by RP HPLC (Luna C8, 250 × 10 mm, a linear gradient from 10-100% MeCN in water over 40 min) afforded 1 (tR 19.5 min, 1.9 mg, 0.032% yield) and 5 (tR 29.5 min, 0.7 mg, 0.011% yield). The residue of fraction 9 (60.0 mg) was further purified by RP-HPLC (Luna C8, 250 × 10 mm, a linear gradient from 10-100% MeCN in water over 40 min and 100% MeCN over 20 min) afforded 9 (tR 47.6 min, 2.0 mg, 0.033% yield), 6 (tR 49.5 min, 1.5 mg, 0.025% yield), 10 (tR 51.5 min, 1.2 mg, 0.020% yield), 7 (tR 52.0 min, 1.0 mg, 0.016% yield). Fraction 10 (20.0 mg) was separated by RP-HPLC (Jupiter C18, 250 × 10 mm, a linear gradient from 10-100% MeCN in water over 40 min) afforded 13 (tR 12.5 min, 1.1 mg, 0.018 % yield), 8 (tR 15.0 min, 5.0 mg, 0.083% yield), 11 (tR 18.5 min, 0.5 mg, 0.008% yield) and 12 (tR 19.0 min, 2.0 mg, 0.033% yield). 8. Morris SA, Andersen RJ. Can. J. Chem. 1989; 67:677. 9. Longeon A, Guyot M, Vacelet J. Experientia. 1990; 46:548. 10. Mani L, Jullian V, Mourkazel B, Valentin A, Dubois J, Cresteil T, Folcher E, Hooper JNA, Erpenbeck D, Aalbersberg W, Debitus C. Chem. Biodiversity. 2012; 9:1436. 11. Tilvi S, Rodrigues C, Naik CG, Parameswaran PS, Wahidhulla S. Tetrahedron. 2004; 60:10207. 12. Jurek J, Yoshida WY, Scheuer PJ, Kelly-Borges M. J. Nat. Prod. 1993; 56:1609. 13. Yagi H, Matsunaga S, Fusetani N. Tetrahedron. 1993; 49:3749. 14. Purpuramine M (1): white amorphous solid; UV (MeOH) λmax (log ε) 280 (3.50) nm; IR (CaF2) ν −1 max 3391, 2928, 2870, 1653, 1595, 1460 cm ; See Tables 1 and 2 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 713.8448 [M + H]+ (calcd for C21H 79 24 Br4N3O5 713.8448). 15. Kobayashi JI, Honma K, Sasaki T, Tsuda M. Chem. Pharm. Bull. 1995; 43:403. 16. Gopichand Y, Schmitz FJ. Tetrahedron Lett. 1979; 20:3921. 17. James DM, Kunze HB, Faulkner DJ. J. Nat. Prod. 1991; 54:1137. [PubMed: 1791478] 18. Purpuramine N (2): white amorphous solid; UV (MeOH) λmax (log ε) 280 (3.36) nm; IR (CaF2) νmax 3362, 2928, 2847, 1651, 1558, 1456 cm−1; See Tables 1 and 2 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 755.8655 [M ]+ (calcd for C22H 79 26 Br4N5O5 755.8666). 19. Araplysillin VII (3): white amorphous solid; [α]D22 −87.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 282 (3.56), 218 (3.19) nm; IR (CaF2) νmax 3339, 2924, 2848, 1652, 1582, 1459 cm−1; See Tables 1 and 2 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 755.8637 [M]+ (calcd for C22H2679 Br4N5O5 755.8666). 20. Arabshahi L, Schmitz FJ. J. Org. Chem. 1987; 52:3584. 21. Araplysillin VIII (4): white amorphous solid; [α]D22 −25.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 280 (3.98) nm; IR (CaF2) νmax 3320, 2923, 2848, 1650, 1557, 1452 cm−1; See Tables 1 and 2 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 1033.7133 [M + H]+ (calcd for C30H3079 Br6N3O8 1033.7131).
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 8
Author Manuscript Author Manuscript
22. Greve H, Kehraus S, Krick A, Kelter G, Maier A, Fiebig H-H, Wright AD, König GM. J. Nat. Prod. 2008; 71:309. [PubMed: 18298075] 23. Fattorusso E, Minale L, Sodano G. J. Chem. Soc., Perkin Trans. 1. 1972; 1:16. 24. Araplysillin IX (5): white amorphous solid; [α]D22 −75.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 281 (3.90), 218 (3.39) nm; IR (CaF2) νmax 3384, 2954, 2919, 1660, 1589, 1377 cm−1; See Tables 1 and 2 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 714.7920 [M + H]+ (calcd for C20H 79 19 Br4N2O7 714.7924). 25. Araplysillin X (6): colorless oil; [α]D22 −42.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 282 (4.0), 218 (3.38) nm; IR (CaF2) νmax 3319, 2918, 2853, 1714, 1649, 1460 cm−1; See Tables 3 and 4 for tabulated NMR spectral data (MeOH-d4); HR APCIMS m/z 952.0775 [M + H]+ (calcd for C37H 79 54 Br4N3O6 952.0746). 26. Araplysillin XI (7): colorless oil; [α]D22 −33.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 282 (3.9), 218 (3.38) nm; IR (CaF2) νmax 3319, 2923, 2848, 1650, 1557 cm−1; See Tables 3 and 4 for tabulated NMR spectral data (MeOH-d 4); HR APCIMS m/z 978.0916 [M + H]+ (calcd for C39H56Br4N3O6 978.0901). 27. Purpurealidin I (11): white amorphous solid; [α]D22 −2.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 280 (3.36) nm; IR (CaF2) νmax 2922, 2814, 1650, 1583, 1468 cm−1; See Tables 3 and 4 for tabulated NMR spectral data (MeOH-d4); HR ESIMS m/z 711.8645 [M + H]+ (calcd for C22H26Br4N3O4 711.8655). 28. Mani L, Jullian V, Mourkazel B, Valentin A, Dubois J, Cresteil T, Folcher E, Hooper JNA, Erpenbeck D, Aalbersberg W, Debitus C. Chem Biodivers. 2012; 9:1436. [PubMed: 22899605] 29. Rodriguez AD, Piña IC. J. Nat. Prod. 1993; 56:907. [PubMed: 8350091] 30. Bello JE, McElfresh JS, Millar JG. Proc. Natl. Acad. Sci. U.S.A. 2015; 112:1077. [PubMed: 25583471] 31. Naqvi T. J. Biomol. Screen. 2004; 9:398. [PubMed: 15296639]
Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 9
Author Manuscript
Fig. 1.
Fragments of compound 1 and key interfragment HMBC correlations.
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 10
Author Manuscript
Fig. 2.
Proposed biosynthetic pathway from the prearaplysillins to the araplysillins.
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 11
Author Manuscript Fig. 3.
Fragmentation of compound 2 that establish the guanidino group.
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 12
Author Manuscript Fig. 4.
Fragmentation of fragment A
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 13
Table 1
Author Manuscript
13C
NMR Spectroscopic Data (125 MHz, δc) for Compounds 1-5.
Author Manuscript Author Manuscript
1 δc, type
2 δc, type
3 δc, type
4 δc, type
5 δc, type
1
123.0, qC
122.5, qC
75.5, CH
75.5, CH
75.5, CH
2
155.0, qC
154.5, qC
114.2, qC
113.3, qC
114.2, qC
3
108.9, qC
108.9, qC
149.3, qC
149.3, qC
149.3, qC
4
151.7, qC
151.6, qC
122.7, qC
122.8, qC
122.5, qC
5
106.9, qC
106.8, qC
132.2, CH
131.3, CH
132.3, qC
6
134.4, CH
134.6, CH
92.4, qC
92.4, qC
92.4, qC
7
25.5, CH2
25.5, CH2
40.1, CH2
40.2, CH2
40.2, CH2
8
154.8, qC
154.4, qC
155.3, qC
155.1, qC
155.3, qC
9
166.8, qC
166.5, qC
161.6, qC
161.6, qC
161.6, qC
10
37.9, CH2
38.1, CH2
38.0, CH2
38.0, CH2
37.9, CH2
11
30.5, CH2
30.5, CH2
30.6, CH2
30.5, CH2
30.6, CH2
12
72.1, CH2
72.2, CH2
72.1, CH2
72.1, CH2
72.2, CH2
13
153.4, qC
153.6, qC
152.8, qC
152.7, qC
155.7, qC
14/18
119.4, qC
119.0, qC
119.0, qC
118.9, qC
118.4, qC
15/17
134.3, CH
134.3, CH
134.3, CH
134.3, CH
134.9, CH
16
137.7, qC
139.8, qC
139.7, qC
139.8, qC
138.0, qC
19
34.1, CH2
35.0, CH2
35.0, CH2
35.0, CH2
170.3, qC
20
41.8, CH2
40.1, CH2
40.0, CH2
41.5, CH2
---
21
---
163.9, qC
163.9, qC
175.0, qC
---
22
---
---
---
39.7, CH2
---
23
---
---
---
123.0, qC
---
24
---
---
---
156.0, qC
---
25
---
---
---
108.7, qC
---
26
---
---
---
154.2, qC
---
27
---
---
---
106.0, qC
---
28
---
---
---
132.3, CH
---
OCH3
60.8, CH3
60.8, CH3
60.4, CH3
60.4, CH3
---
26-OCH3
---
---
---
60.8, CH3
---
Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 14
Table 2
Author Manuscript
1H
NMR Spectroscopic Data (500 MHz in MeOH-d4) for Compounds 1-5.
Author Manuscript
1 δH, (J in Hz)
2 δH, (J in Hz)
3 δH, (J in Hz)
4 δH, (J in Hz)
5 δH, (J in Hz)
1
---
---
4.08, s
4.08, s
4.09, s
5
---
---
6.41, s
6.42, s
6.41, s
6
7.40, s
7.41, s
---
---
---
7
3.80, s
3.80, s
3.09, d (18.3)
3.10, d (18.3)
3.10, d (18.3)
3.77, d (18.3)
3.77, d (18.3)
3.77, d (18.3)
10
3.57, t (6.7)
3.58, t (6.6)
3.58, t (7.0)
3.58, t (7.0)
3.59, t (7.0)
11
2.09, tt (6.7, 6.0)
2.09, tt (6.6, 6.0)
2.10, tt (7.0, 6.0)
2.09, tt (7.0, 6.1)
2.12, tt (7.0, 6.1)
12
4.01, t (6.0)
4.02, t (6.0)
4.04, t (6.0)
3.99, t (6.1)
4.09, t (6.1)
15/17
7.48, s
7.45, s
7.47, s
7.38, s
8.10, s
19
2.85, t (7.4)
2.75, t (7.0)
2.75, t (7.0)
2.73, t (6.9)
---
20
3.09, t (7.4)
3.43, t (7.0)
3.42, t (7.0)
3.39, t (6.9)
---
22
---
---
---
3.48, s
---
28
---
---
---
7.25, s
---
OCH3
3.78, s
3.79, s
3.71, s
3.72, s
3.72, s
26-OCH3
---
---
---
3.81, s
---
Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 15
Table 3
Author Manuscript
1H
NMR Spectroscopic Data (500 MHz, δH (J in Hz)) for Compounds 11, 6 and 7 11
6
7
1
---
1
4.08, s
1
4.07, s
2
7.42, s
2
---
2
---
3
---
3
---
3
---
4
---
4
---
4
---
5
---
5
6.40, s
5
6.41, s
6
7.42, s
6
---
6
---
7
3.80, s
7
3.08, d (18.1)
7
3.09, d (18.3)
3.78, d (18.1)
3.77, d (18.3)
Author Manuscript Author Manuscript
8
---
8
---
8
---
9
---
9
---
9
---
10
3.43, t (7.1)
10
3.58, t (6.9)
10
3.58, t (7.0)
11
2.75, t (7.1)
11
2.12, tt (6.9, 6.0)
11
2.10, tt (7.0, 6.0)
12
---
12
4.04, t (6.0)
12
4.04, t (6.0)
13
7.47, s
13
---
13
---
15
---
15
7.44, s
15
7.44, s
16
---
16
---
16
---
17
7.47, s
17
7.44, s
17
7.44, s
18
4.04, t (5.7)
18
---
18
---
19
2.18, tt (7.5, 5.7)
19
2.73, t (6.9)
19
2.73, t (6.7)
20
3.26, t (7.5)
20
3.37, t (6.9)
20
3.37, t (6.7)
---
22
2.07-2.16, m
22
2.07-2.16, m
---
23
1.50-1.57, m
23
1.49-1.57, m
---
24
1.20-1.29, m
24
1.20-1.29, m
---
25
1.22-1.38, m
25
1.29-1.38, m
---
26
1.22-1.38, m
26
1.29-1.38, m
---
27
1.22-1.38, m
27
1.99-2.05, m
---
28
1.28-1.39, m
28
5.32, m
1.05-1.17, m
29
5.32, m
-----
29
1.21-1.35, m
30
1.99-2.05, m
---
30
1.05-1.17, m
31
1.29-1.38, m
1.28-1.39, m
32
1.10-1.18, m
---
Author Manuscript
---
31
1.28-1.39, m
1.20-1.29, m
---
32
1.28-1.39, m
33
1.21-1.37, m
---
33
1.28-1.39, m
34
1.10-1.18, m
---
34
1.33-1.40, m
---
35
0.84, t (6.3)
35
1.20-1.29, m
---
36
1.20-1.29, m
0.86, d (6.6)
36
1.32-1.40, m
---
---
37
0.88, t (6.8)
---
---
38
0.85, d (6.6)
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 16
Author Manuscript
11
6
7
NCH3
2.70, s
---
---
OCH3
3.78, s
OCH3
3.72, s
OCH3
3.72, s
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 17
Table 4
Author Manuscript
13C
NMR Spectroscopic Data (125 MHz, δc) for Compounds 11, 6 and 7.
Author Manuscript Author Manuscript Author Manuscript
11 δc, type
6 δc, type
7 δc, type
1
137.4, qC
75.5, CH
75.5, CH
2
134.4, CH
114.2, qC
114.2, qC
3
118.6, qC
149.3, qC
149.3, qC
4
152.0, qC
122.8, qC
122.8, qC
5
118.6, qC
132.2, CH
132.2, CH
6
134.4, CH
92.4, qC
92.4, qC
7
28.8, CH2
40.3, CH2
40.1, CH2
8
153.8, qC
155.2, qC
155.2, qC
9
165.4, qC
161.5, qC
161.5, qC
10
41.4, CH2
38.0, CH2
38.0, CH2
11
35.1, CH2
30.5, CH2
30.5, CH2
12
140.2, qC
72.1, CH2
72.1, CH2
13
134.5, CH
152.8, qC
152.8, qC
14
118.7, qC
118.9, qC
118.9, qC
15
152.2, qC
134.4, CH
134.4, CH
16
118.7, qC
140.0, qC
140.0, qC
17
134.5, CH
134.4, CH
134.4, CH
18
71.7, CH2
118.9, qC
118.9, qC
19
28.1, CH2
35.1, CH2
35.1, CH2
20
48.5, CH2
41.2, CH2
41.2, CH2
21
---
176.4, qC
176.4, qC
22
---
37.2, CH2
37.2, CH2
23
---
26.4, CH2
26.9, CH2
24
---
30.3-31.1, CH2
30.2-31.1, CH2
25
---
30.3-31.1, CH2
30.2-31.1, CH2
26
---
30.3-31.1, CH2
30.2-31.1, CH2
27
---
28.2, CH2
28.1, CH2
28
---
38.2, CH2
130.5, CH
29
---
33.1, CH2
130.5, CH
30
---
38.2, CH2
28.1, CH2
31
---
28.2, CH2
27.1, CH2
32
---
30.3-31.1, CH2
38.2, CH2
33
---
33.9, CH2
33.9, CH
34
---
23.8, CH2
38.2, CH2
35
---
14.5, CH2
30.2-31.1, CH2
36
---
20.1, CH3
23.4, CH2
37
---
---
14.4, CH3
Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 18
Author Manuscript
11 δc, type
6 δc, type
7 δc, type
38
---
---
20.1, CH3
OCH3
61.0, CH3
60.4, CH3
60.4, CH3
NCH3
34.1, CH3
---
---
Some 13C chemical shift determined from HMBC experiment.
Author Manuscript Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 19
Table 5
Author Manuscript
BACE1 Inhibition Comp.
Author Manuscript
Name
μM
% Inhibition
1
Purpuramine M
42.0
36
3
Araplysillin VII
39.6
40
5
Araplysillin IX
41.9
35
6
Araplysillin X
31.4
70
7
Araplysillin XI
30.6
60
8
Hexadellin A
41.7
27
9
Araplysillin II
31.8
57
11
Purpurealidin I
42.0
74
12
Aplysamine 4
42.6
62
13
Purpuramine G
48.3
48
Secretase Inhibitor IV
0.021
80
Author Manuscript Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
Dai et al.
Page 20
Table 6
Author Manuscript
Effects of compounds on growth of tumor versus NIH3T3 cells a NIH 3T3
Comp.
A2780S
A2780S CP5
U251 MG
A549
MCF7
Observed growth at 50 μM relative to DMSO Control Observed growth at 5 μM relative to DMSO Control
Author Manuscript Author Manuscript
1
104 ± 4 102 ± 4
9±1 60 ± 23
9±2 96 ± 29
20 ± 7 111 ± 5
21 ± 0 96 ± 2
27 ± 3 97 ± 18
3
25 ± 2 100 ± 7
12 ± 1 78 ± 23
8±0 45 ± 9
32 ± 1 94 ± 1
48 ± 6 85 ± 34
24 ± 6 98 ± 6
5
29 ± 1 94 ± 7
21 ± 2 37 ± 11
25 ± 2 33 ± 1
39 ± 15 101 ± 2
68 ± 10 81 ± 10
60 ± 2 86 ± 16
6
95 ± 4 94 ± 6
87 ± 7 85 ± 5
79 ± 27 109 ± 12
97 ± 2 99 ± 2
105 ± 2 89 ± 25
101 ± 2 104 ± 4
7
98 ± 3 100 ± 2
76 ± 18 82 ± 8
66 ± 3 105 ± 12
90 ± 2 98 ± 2
104 ± 2 98 ± 10
104± 2 105± 4
8
7±0 38 ± 14
14 ± 1 8±0
22 ± 1 11 ± 2
30 ± 5 32 ± 1
17 ± 1 16 ± 11
71 ± 5 36 ± 4
9
95 ± 4 88 ± 4
18 ± 1 77 ± 21
48 ± 8 68 ± 15
80 ± 1 98 ± 0
85 ± 10 88 ± 30
64 ± 8 103 ± 3
10
102 ± 8 98 ± 5
88 ± 4 90 ± 1
78 ± 11 100 ± 4
101 ± 3 100 ± 2
106 ± 4 82 ± 33
109 ± 1 106 ± 1
11
3±0 101 ± 5
13 ± 2 11 ± 2
10 ± 1 29 ± 4
30 ± 10 17 ± 1
11 ± 1 42 ± 7
58 ± 1 50 ± 12
12
16 ± 2 105 ± 4
11 ± 2 81 ± 24
12 ± 3 78 ± 26
24 ± 3 107 ± 2
24 ± 6 92 ± 9
26 ± 1 95 ± 15
13
105 ± 3 99 ± 3
26 ± 7 82 ± 26
51 ± 4 94 ± 41
40 ± 16 111 ± 4
64 ± 14 97 ± 4
69 ± 4 100 ± 10
46 ± 1 46 ± 2
23 ± 2 35 ± 8
18 ± 3 33 ± 4
20 ± 2 21 ± 1
51 ± 15 49 ± 7
52 ± 6 46 ± 4
b
SAHA
c
A2780S
A2780S CP5
U251 MG
IC50 μM
IC50 μM
IC50 μM
20
40
50
>50
>50
>50
Etoposide a
Fibroblast cell line.
b
25 μM
c
20 μM
Author Manuscript Bioorg Med Chem Lett. Author manuscript; available in PMC 2017 January 15.
